Interleukin-13 receptor α2 peptide brain cancer vaccine

Abstract

To provide interleukin-13 receptor α2 peptide-based brain cancer vaccines and methods for treating and vaccinating against brain cancer comprising administering to patients in need thereof interleukin-13 receptor α2 peptide-based brain cancer vaccines.SOLUTION: A pharmaceutical composition comprises an interleukin-13 receptor α2 peptide, an EphA2 peptide, a survivin peptide, and a WT1 peptide.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 61 / 376,582, filed August 24, 2010, which is incorporated herein by reference.

[0002] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with government support under Grant Nos. NS40923 and CA117152 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0003] (Incorporation by reference of electronically submitted materials) The entire contents of the computer-readable nucleotide / amino acid sequence listing identified as One 3.00 Kilobyte ASCII (Text) file name "708849_ST25.TXT", dated August 22, 2011, which was submitted concurrently with this specification, are incorporated herein by reference.

[0004] 1. Introduction The present invention provides interleukin-13 receptor α2 peptide brain cancer vaccines and methods of treating and vaccinating against brain cancer comprising administering the interleukin-13 receptor α2 peptide brain cancer vaccine to a patient in need thereof. The present invention also provides vaccine regimens comprising an interleukin-13 receptor α2 peptide and at least one additional peptide and / or immunostimulant. [Background technology]

[0005] 2.Background technology Brain tumors are particularly difficult to treat using existing methods such as surgery, radiation therapy, and chemotherapy. Factors such as an infiltrative growth pattern and the blood-brain barrier make the treatment of malignant gliomas more problematic than other tumors. The lack of effective treatment options for patients has led to the development of alternative treatments, such as immunotherapy.

[0006] Immunotherapy is a promising new approach for the treatment of malignant gliomas. The efficacy of peripheral immunization with autologous glioma cells or dendritic cells pulsed with synthetic peptides against tumor antigen-specific T cell epitopes has been demonstrated in preclinical mouse models (Okada et al., 2001; Okada et al., 1998). Specific T cell epitope vaccines are likely safer than whole glioma cell vaccines because they theoretically lack autoimmune responses against normal brain components. Such antigen-specific approaches may be more effective than bulk tumor antigen approaches because the use of specific antigenic peptides rather than bulk tumor antigens results in more efficient presentation of immunogenic T cell epitopes and stimulation of antigen-specific T cell precursors.

[0007] Identification of T cell immune epitopes in human glioma-associated antigens is necessary for the development of such vaccines against human gliomas. Few cytotoxic T lymphocyte (CTL) immune epitopes for human malignant gliomas have been identified. However, an HLA (human lymphocyte antigen)-A2-restricted cytotoxic T lymphocyte (CTL) derived from interleukin (IL)-13 receptor (R) α2 has been identified in recent years (Okano et al., 2002). IL-13Rα2 is known to be expressed in most human malignant gliomas but not in normal tissues (Debinski et al., 2000). The identified epitope (IL-13Rα23) 45-353 ) is an attractive component of peptide vaccines against glioma. 345-353 CD8 with peptides + Stimulating cells to generate unique CTL lines The peptide vaccines efficiently lysed IL-13Rα2-positive, HLA-A2-positive glioma cells in an antigen-specific manner. However, it remains unclear how efficiently such peptide vaccines can induce specific CTLs and whether peptide analogs can be used to optimally expand and activate IL-13Rα2-specific, HLA-A2-restricted CTLs.

[0008] Substitution of certain amino acids in a peptide identified as a CTL epitope has been demonstrated to greatly increase the binding affinity of the peptide to the HLA (human lymphocyte antigen) complex, increasing the immunogenicity of the peptide (Bownds et al., 2001; Chen et al., 2000). 345-353 Increasing the immunogenicity of peptides and other such epitopes could lead to the development of potent, tumor-specific peptide vaccines, which would significantly improve current treatment regimes for malignant gliomas. However, there remains a need for improved polypeptides for HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes.

[0009] As mentioned above, few cytotoxic T lymphocyte (CTL) immune epitopes have been identified in human malignant gliomas. Given the remarkable antigenic heterogeneity of gliomas, immunotherapy with a single tumor-specific T cell epitope may only promote temporary stabilization of the disease before progression to antigen-deficient variants. EphA2 is a member of the Eph family of receptor tyrosine kinases, which consists of two major classes (EphA2 and EphB), distinguished by their specificity for their ligands (ephrin-A and ephrin-B, respectively). EphA2 is frequently overexpressed and often functionally dysregulated in advanced cancers, including metastases (Kinch et al., 2003). Due to the aggressive and invasive nature of malignant gliomas, EphA2 may be expressed in these tumor entities and could be a potential target for glioma vaccines. T cell immune epitopes in EphA2 have been identified and characterized as potential targets and surrogate markers for other forms of cancer immunotherapy (Alves et al., 2003, and Tatsumi et al., 2003). Identifying additional CTL epitopes is a necessary step in the development of multiepitope vaccines for gliomas, which would provide significant improvements in current treatment regimens for malignant gliomas.

[0010] 3. Overview In one embodiment, the present invention provides IL-13α2-derived peptides that serve as HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes. IL-13α2 peptides can comprise, consist of, or consist essentially of substitution mutant variants of WLPFGFILI (SEQ ID NO: 1), in which at least one of the amino acid residues can be substituted with an amino acid other than the indicated residue. Additionally, IL-13α2 peptides can comprise, consist of, or essentially of any of the following sequences: WLPFGFILV (SEQ ID NO: 2), ALPFGFILV (SEQ ID NO: 3), or ELPFGFILV (SEQ ID NO: 4).

[0011] The present invention also provides the use of any of the above-described IL-13Rα2 peptides as a vaccine against glioma. Additionally, the present invention provides a method for vaccinating a patient against glioma, comprising introducing the peptide into the patient under conditions sufficient to induce a CTL response. Furthermore, the present invention provides the use of an EphA2 peptide having the sequence TLADFDPRV (SEQ ID NO: 6), or a composition comprising the peptide and a physiologically acceptable carrier, as a vaccine against glioma. The present invention also provides a method for vaccinating a patient against glioma, comprising introducing into the patient an EphA2 peptide having the sequence TLADFDPRV (SEQ ID NO: 6) or a composition comprising the peptide and a physiologically acceptable carrier under conditions sufficient to induce a CTL response in the patient.

[0012] In another aspect, the present invention provides an IL-13Rα2 peptide-based vaccine comprising an IL-13Rα2 peptide and one, two, three, or more additional peptides associated with brain cancer. In certain embodiments, the IL-13Rα2 peptide-based vaccine described herein is administered simultaneously with one or more helper T cell epitopes and / or immune response modifiers. In such embodiments, the one or more helper T cell epitopes and / or one or more immune response modifiers may be administered as part of the vaccine (e.g., a solution containing the IL-13Rα2 peptide and one, two, three, or more additional peptides associated with brain cancer) or separately from the vaccine (i.e., the helper T cell epitopes and / or immune response modifiers may be administered as a formulation that is not part of the vaccine formulation). In some embodiments, the IL-13Rα2 peptide-based vaccine described herein is administered as a cell-free vaccine. In other embodiments, the IL-13Rα2 peptide-based vaccine described herein is administered with an adjuvant. In a preferred embodiment, the IL-13Rα2 peptide-based vaccine is administered in conjunction with an additional peptide. In another embodiment, the IL-13Rα2 peptide-based vaccine is administered in conjunction with an immunomodulatory agent. In another embodiment, the IL-13Rα2 peptide-based vaccine is administered as an emulsion in Montanide ISA-51 as a component of a regimen that includes injections with an immunomodulatory agent. In one embodiment, the immunostimulatory agent is poly-ICLC.In another embodiment, the IL-13Rα2 peptide-based vaccines described herein are administered as dendritic cell vaccines.

[0013] In one aspect, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide, an EphA2 peptide, a YKL-40 peptide, and GP100. In a specific embodiment, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide set forth in any of SEQ ID NOs: 1-4, an EphA2 peptide set forth in SEQ ID NO: 6, a YKL-40 peptide set forth in SEQ ID NO: 10, and a GP100 peptide set forth in SEQ ID NO: 11. In another specific embodiment, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide set forth in SEQ ID NO: 3, an EphA2 peptide set forth in SEQ ID NO: 6, a YKL-40 peptide set forth in SEQ ID NO: 10, and a GP100 peptide set forth in SEQ ID NO: 11. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with one or more helper T cell epitopes. In certain embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with one or more helper T cell epitopes, wherein the helper T cell epitope is a PADRE peptide. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with an immune response modifier. In some embodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine. In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cell vaccine.

[0014] In other embodiments, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide, an EphA2 peptide, a survivin peptide, and a WT1 peptide. In specific embodiments, the IL-13Rα2 peptide comprises any of the IL-13Rα2 peptides set forth in SEQ ID NOs: 1-4, an EphA2 peptide set forth in SEQ ID NO: 6, a survivin peptide set forth in SEQ ID NO: 7, and a WT1 peptide set forth in SEQ ID NO: 8. In other specific embodiments, the IL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptide set forth in SEQ ID NO: 3, an EphA2 peptide set forth in SEQ ID NO: 6, a survivin peptide set forth in SEQ ID NO: 7, and a WT1 peptide set forth in SEQ ID NO: 8. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with one or more helper T cell epitopes. In specific embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with a helper T cell epitope that is tetanus toxoid. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered concurrently with one or more immune response modifiers. In a specific embodiment, the IL-13Rα2 peptide-based vaccine is administered concurrently with an immune response modifier, where the immune response modifier is poly-ICLC. In this state, the IL-13Rα2 peptide vaccine contains Montanide as an immune response modifier. The IL-13Rα2 peptide-based vaccine is administered simultaneously with an immune response modifier, ISA-51. In some embodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine. In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cell vaccine.

[0015] In other embodiments, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide, an EphA2 peptide, and a survivin peptide. In specific embodiments, the IL-13Rα2 peptide-based vaccine comprises any of the IL-13Rα2 peptides represented by SEQ ID NOs: 1-4, the EphA2 peptide represented by SEQ ID NO: 6, and the survivin peptide represented by SEQ ID NO: 7. In other specific embodiments, the IL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptide represented by SEQ ID NO: 3, the EphA2 peptide represented by SEQ ID NO: 6, and the survivin peptide represented by SEQ ID NO: 7. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with one or more helper T cell epitopes. In specific embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with a helper T cell epitope where the helper T cell epitope is tetanus toxoid. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with one or more immune response modifiers. In certain embodiments, the IL-13Rα2 peptide-based vaccine is administered simultaneously with an immune response modifier, wherein the immune response modifier is poly-ICLC. In certain embodiments, the IL-13Rα2 peptide-based vaccine is administered with an immune response modifier, wherein the immune response modifier is Montanide ISA-51. In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cell vaccine.

[0016] 4.Definition As used herein, the terms "about" or "approximately," when used in conjunction with a number, mean any number within 1, 5, or 10% of the referenced number.

[0017] As used herein, the term "agent" refers to any molecule, compound, and / or substance used in or in combination with the interleukin-13 receptor α2 peptide brain cancer vaccines described herein, including, but not limited to, proteins, immunoglobulins (e.g., multi-specific Ig, single-chain Ig, Ig fragments, polyclonal antibodies and fragments thereof, monoclonal antibodies and fragments thereof), peptides (e.g., peptide receptors, selectins), binding proteins, biologics, chemospecific agents, chemotoxic agents, anti-angiogenic agents, and small molecule drugs.

[0018] As used herein, the term "amino acid sequence identity" refers to the degree of identity or similarity between a pair of aligned amino acid sequences, usually expressed as a percentage. As used herein, the terms "percent identical," "percent identical," "% identical," and "% identical," when referring to amino acid sequences, refer to the percentage of amino acid residues in a candidate sequence that are identical to the corresponding amino acid residues in the peptide (i.e., the amino acid residues at specified positions in the alignment are the same residues), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. As used herein, the terms "percent similarity," "percent similarity," "% similarity," and "% similarity," when referring to amino acid sequences, refer to the percentage of amino acid residues in a candidate sequence that are similar to the corresponding amino acid residues in the peptide (i.e., substitutions of amino acid residues at specified positions in the alignment are conservative substitutions, as described below), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Sequence homology, including percent sequence identity and similarity, is determined using sequence alignment techniques well known in the art, including computer algorithms designed for this purpose, with the default parameters of such algorithms, or software packages that include them.

[0019] As used herein, the term "conservative substitution" refers to the substitution of an amino acid of one class with another amino acid of the same class. In certain embodiments, a conservative substitution does not alter the structure or function of the peptide, or both. Classes of amino acids for conservative substitutions include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformation disrupters; (Gly, Pro), aromatic (Trp , Tyr, Phe).

[0020] As used herein, the term "peptide," as known to those of skill in the art, refers to a polymer of amino acids linked by amide bonds. A peptide can be a polymer of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids linked by covalent amide bonds. In some embodiments, a peptide is a polymer of 6-8, 8-10, 10-15, 10-20, 10-25, 10-30, 10-40, 10-50, or 25-25 amino acids linked by covalent amide bonds. In certain embodiments, a peptide is a polymer of 50-65, 50-75, 50-85, 50-95, 50-100, or 75-100 amino acids linked by covalent amide bonds. As used herein, the term can refer to a single peptide chain linked by covalent amide bonds. The term can also refer to multiple peptide chains linked by non-covalent bonds, such as ionic bonds, hydrogen bonds, van der Waals forces, hydrophobic bonds, etc. One of skill in the art will recognize that the term also includes modified peptides, for example, post-translational modifications such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage, and lipid modification (e.g., S-palmitoylation).

[0021] As used herein, the terms "purified" and "isolated," when used in connection with peptides obtained from a natural source, e.g., cells, mean that the peptide is substantially free of contaminants of the natural source, such as soil particles, minerals, chemicals from the environment, and / or intracellular material from the natural source, including, but not limited to, cell debris, cell wall material, membranes, organelles, and abundant nucleic acids, carbohydrates, proteins, and / or lipids present within the cell. Thus, an isolated peptide includes preparations of polypeptides that contain less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of intracellular material and / or contaminants. As used herein, the terms "purified" and "isolated," when used in connection with chemically synthesized peptides, mean that the peptide is substantially free of chemical precursors or other chemicals involved in the synthesis of the polypeptide.

[0022] As used herein, the term "nucleic acid" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), and DNA or RNA analogs produced using nucleic acid analogs. Nucleic acids can be single-stranded or double-stranded.

[0023] As used herein, the term "prophylactic vaccine" refers to a vaccine described herein that is used to prevent cancer.

[0024] As used herein, the term "prophylactically effective regimen" refers to a regimen effective in dosing, timing, frequency, and duration of one or more therapeutic actions for the prevention of brain cancer or its symptoms.

[0025] As used herein, the term "therapeutic vaccine" refers to a vaccine described herein that is used to treat and / or manage brain cancer.

[0026] As used herein, the term "therapeutically effective regimen" refers to a regimen for the dosing, timing, frequency, and duration of one or more therapeutic actions to treat and / or manage brain cancer or its symptoms.

[0027] As used herein, the terms "subject" and "patient" are used interchangeably and refer to animals (e.g., birds, amphibians, and mammals). In certain embodiments, the subject is a bird. In other embodiments, the subject is an animal, which is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, and mice), and primates (e.g., monkeys, chimpanzees, and humans). In certain embodiments, the subject is a non-human animal. In some embodiments, the subject is a livestock or pet. In other embodiments, the subject is a human. In other embodiments, the subject is a human infant. In other embodiments, the subject is a human toddler. In other embodiments, the subject is a child. In other embodiments, the subject is an adult. In other embodiments, the subject is an elderly person.

[0028] As used herein, the term "human infant" refers to a human from birth to one year of age.

[0029] As used herein, the term "human infant" means a human between the ages of 1 and 3.

[0030] As used herein, the term "child" refers to a human between the ages of 1 and 18 years.

[0031] As used herein, the term "adult" refers to a human being 18 years of age or older.

[0032] As used herein, the term "elderly" refers to people aged 65 and over.

[0033] As used herein, the term "brain cancer" refers to tumors located within the intracranial sphere or central spinal canal. Brain cancer refers to both primary tumors (i.e., tumors that originate in the intracranial sphere or central spinal canal) and secondary tumors (i.e., tumors that invade the intracranial sphere or central spinal canal after arising from tumors originally located in other tissues).

[0035] As used herein, the term "effective amount" means an amount of a treatment sufficient to prevent the development, recurrence, or onset of brain cancer and / or one or more symptoms of brain cancer, enhance or improve the preventative efficacy of other therapies, reduce the severity or persistence of brain cancer, ameliorate one or more symptoms, prevent the progression of brain cancer, cause regression of brain cancer, and / or enhance or improve the therapeutic efficacy of other therapies.

[0036] As used herein, the term "in combination" when referring to the administration of a therapy to a subject refers to the use of two or more therapies (e.g., prophylactic and / or therapeutic). The use of the term "in combination" is not limited to the order in which the therapies (e.g., primary and secondary therapies) are administered to a subject. The treatment may be administered to a subject who has had, has, or may have brain cancer prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), the secondary treatment .... Administration of the treatments can occur after 8 weeks, or 12 weeks. The treatments can be administered to a subject in any order and within any time interval such that they work together. In certain embodiments, the treatments are administered in any order and within any time interval such that they provide increased benefit when administered separately. Any additional treatments can be administered in any order with other additional treatments.

[0037] As used herein, the terms "manage," "managing," and "management," in the context of administering a treatment to a subject, refer to a beneficial effect obtained from a treatment (e.g., a prophylactic or therapeutic vaccine) or combination of treatments that does not result in a cure for brain cancer. In certain embodiments, a subject is administered one or more therapies (e.g., one or more prophylactic or therapeutic vaccines) to manage brain cancer to prevent progression or worsening of the condition.

[0038] As used herein, the terms "prevent," "preventing," and "prevention," in the context of administration of a therapy to a subject, refer to the prevention or inhibition of brain cancer recurrence, onset, and / or symptoms associated therewith resulting from the administration of a therapy (e.g., a prophylactic or therapeutic agent) or a combination of therapies (e.g., a combination of prophylactic or therapeutic agents).

[0039] As used herein, the term "concurrently" means sufficiently close in time to produce a combined effect (i.e., two or more events that may occur simultaneously, perhaps simultaneously, or within a period before or after each other). When administered with other agents, the IL-13Rα2 peptide-based vaccines provided herein may be administered with other active agents. In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein and one or more other agents (e.g., helper T cell epitopes, adjuvants, and / or immune response modifiers) are administered to a subject simultaneously, where the IL-13Rα2 peptide-based vaccines provided herein and one or more other agents administered are in the same composition. In other embodiments, the IL-13Rα2 peptide-based vaccines provided herein and one or more other agents (e.g., helper T cell epitopes, adjuvants, and / or immune response modifiers) are administered to a subject simultaneously, where the IL-13Rα2 peptide-based vaccines provided herein and one or more other agents are not in the same composition. In one embodiment, the agents administered together with the IL-13Rα2 peptide-based vaccine are injected separately. In a specific embodiment, the IL-13Rα2 peptide-based vaccine provided herein and one or more other reagents (e.g., helper T cell epitopes, adjuvants, and / or immune response modifiers) are administered to a subject simultaneously, and the combined administration is separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks.

[0040] As used herein, the term "brain cancer-associated peptide" refers to a peptide associated with one or more brain cancers and serving as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In some embodiments, the brain cancer-associated peptide is a glioma-associated peptide, i.e., the brain cancer with which the peptide is associated is glioma. In preferred embodiments, the cancer-associated peptide is expressed by glioma cells. Exemplary brain cancer-associated peptides include, but are not limited to, IL-13Rα2 peptide, EphA2 peptide, YKL-40 peptide, GP100 peptide, survivin peptide, and WT1 peptide.

[0041] As used herein, the term "IL-13Rα2 peptide" refers to a peptide that is derived from the IL-13Rα2 protein and serves as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In certain embodiments, the IL-13Rα2 protein from which the IL-13Rα2 peptide is derived is a human IL-13Rα2 protein. In other specific embodiments, the IL-13Rα2 peptide has the sequence of any of SEQ ID NOs: 1-4. In some embodiments, the IL-13Rα2 peptide is a peptide that is specific to the IL-13Rα2 peptide as it exists in the native (e.g., wild-type) form of the IL-13Rα2 protein. and includes one, two, three or more amino acid mutations (e.g., additions, substitutions, deletions).

[0042] As used herein, the term "EphA2 peptide" refers to a peptide derived from the EphA2 protein and serving as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In specific embodiments, the EphA2 protein from which the EphA2 peptide is derived is a human EphA2 protein. In other specific embodiments, the EphA2 peptide has the sequence of SEQ ID NO:6. In some embodiments, the EphA2 peptide contains one, two, three, or more amino acid mutations (e.g., additions, substitutions, deletions) relative to the EphA2 peptide as present in the native (e.g., wild-type) form of the EphA2 protein.

[0043] As used herein, the term "YKL-40 peptide" refers to a peptide derived from the YKL-40 protein and serving as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In a specific embodiment, the YKL-40 protein from which the YKL-40 peptide is derived is human YKL-40 protein. In another specific embodiment, the YKL-40 peptide has the sequence of SEQ ID NO: 10. In some embodiments, the YKL-40 peptide contains one, two, three or more amino acid mutations (e.g., additions, substitutions, deletions) relative to the YKL-40 peptide as present in the native (e.g., wild-type) form of the YKL-40 protein.

[0044] As used herein, the term "GP100 peptide" refers to a peptide derived from the GP100 protein and serving as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In certain embodiments, the GP100 protein from which the GP100 peptide is derived is a human GP100 protein. In other specific embodiments, the GP100 peptide has the sequence of SEQ ID NO: 11. In some embodiments, the GP100 peptide contains one, two, three or more amino acid mutations (e.g., additions, substitutions, deletions) relative to the GP100 peptide as present in the native (e.g., wild-type) form of the GP100 protein.

[0045] As used herein, the term "survivin peptide" refers to a peptide derived from the survivin protein and serving as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In certain embodiments, the survivin protein from which the survivin peptide is derived is a human survivin protein. In other specific embodiments, the survivin peptide has the sequence of SEQ ID NO: 7. In some embodiments, the survivin peptide contains one, two, three, or more amino acid mutations (e.g., additions, substitutions, deletions) relative to the survivin peptide as present in the native (e.g., wild-type) form of the survivin protein.

[0046] As used herein, the term "WT1 peptide" refers to a peptide derived from the WT1 protein and serving as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. In certain embodiments, the WT1 protein from which the WT1 peptide is derived is a human WT1 protein. In other specific embodiments, the WT1 peptide has the sequence of SEQ ID NO: 7. In some embodiments, the WT1 peptide contains one, two, three, or more amino acid mutations (e.g., additions, substitutions, deletions) relative to the WT1 peptide as present in the native (e.g., wild-type) form of the WT1 protein.

[0047] As used herein, the term "acellular vaccine" refers to a vaccine comprising IL-13Rα2 peptides in which the IL-13Rα2 peptides are not loaded onto cells (e.g., dendritic cells) in the vaccine (e.g., peptides derived from IL-13Rα2 are present in solution). In a preferred embodiment, the peptides are emulsified with an adjuvant. Other preferred embodiments include In an embodiment, the adjuvant is Montanide ISA 51.

[0048] As used herein, the term "dendritic cell vaccine" refers to a vaccine comprising an IL-13Rα2 peptide, in which the IL-13Rα2 peptide is loaded into dendritic cells in the vaccine. [Brief explanation of the drawings]

[0049] [Figure 1]Figure 1 shows data demonstrating that IL-13Rα2-V9 and IL-13Rα2-A 1V9 elicited higher CTL reactivity than native IL-13Rα2 or IL-13Rα2-E 1V9 in T2 cells loaded with various concentrations of native IL-13Rα2. CD8 T cells from an HLA-A2 glioma patient were stimulated for 10 days with dendritic cells loaded with native IL-13Rα2 (●), IL-13Rα2-V9 (○), IL-13Rα2-A 1V9 (△), IL-13Rα2-E 1V9 (X), influenza M158-66 peptide (▼), or no peptide (□). The T cells were then loaded with the indicated concentrations of IL-13Rα2 345-353 or no peptide and tested for lytic activity against T2 cells in a 4-hour 51Cr-release assay. The E / T ratio was 12.5. P<0.01 for native vs. IL-13Rα2-A 1V9 at 0.1 and 1 nM, as well as native vs. IL-13Rα2-V9, by two-tailed Student's t test. These data represent one of three independent experiments with similar results. [Figure 2] Figure 2 illustrates data demonstrating that CTL lines induced by the V9 peptide (open circles) exhibited increased lytic activity against T2 cells loaded with various concentrations of wild-type IL-13Rα2 peptide. The CTL lines induced by each of the three agonist analogs (V9 (open circles), A1V9 (triangles); E1V9 (X)) or the wild-type peptide (filled circles) were tested for CTL reactivity against lower concentrations of the target IL-13Rα2 peptide (E / T ratio = 50) in a 4-hour 51Cr-release assay, along with T2 cells loaded with various concentrations (1-100 nM) of IL-13Rα2. [Figure 3]Figure 3 illustrates data demonstrating that modified peptides induced higher CTL reactivity against human glioma cell lines compared with native IL-13Rα2345-353. CD8+ cells from an HLA-A2+ glioma patient were stimulated with native IL-13Rα2345-353 (●), IL-13Rα2-V9 (○), IL-13Rα2-A1V9 (△), or IL-13Rα2-E1V9 (X). On day 10, the cells were tested for their ability to lyse human glioma cells SNB19 (A) and U-251 (B) (both IL-13Rα+ / HLA-A2+) in a 4-hour 51Cr-release assay. For SNB19 glioma cells, P<0.05 at all E / T ratios for IL-13Rα2-A1V9 vs. native IL-13Rα2345-353, as well as for IL-13Rα2-V9 vs. native IL-13Rα2345-353, by two-tailed Student's t test. For U251 glioma cells, P<0.05 at E / T ratios of 10 and 40 for IL-13Rα2-A1V9 vs. native IL-13Rα2345-353, as well as for IL-13Rα2-V9 vs. native IL-13Rα2345-353, by two-tailed Student's t test. Data represent one of three experiments with different donors showing similar results. [Figure 4] Figure 4 illustrates data demonstrating that the addition of "cold" T2 cells pulsed with IL-13Rα2345-353 inhibited the antigen-specific CTL activity of the CTL line. CTL lines induced with each peptide (control (A); Flu (B); IL-13Rα2345-353 (C); IL-13Rα2345-9V (D)) were cultured with 51Cr-labeled human glioma cell line SNB19 at the indicated E:T ratio for 4 hours to assess specific lysis (●). For cold target inhibition assays, 51Cr-labeled target SNB19 cells (1 x 103 cells / well) pulsed with (△) or without (○) peptide IL-13Rα2345-353 and cold T2 cells (1 x 104 cells / well) were cultured with CTLs. [Figure 5]Figure 5 illustrates data demonstrating that the addition of anti-HLA-A2 antibody inhibited CTL activity, indicating HLA-A2-restricted recognition of the CTL line. CTL lines induced with each peptide (control (A); Flu (B); IL-13Rα2345-353 (C); IL-13Rα2345-9V (D)) were cultured for 4 hours with 51Cr-labeled human glioma cell line SNB19 at the indicated E:T ratios for assessment of specific lytic activity (●). Anti-HLA-A2 antibody (W6 / 32; 10 μg / ml) was added to block HLA-A2-mediated recognition by T cells (○). [Figure 6] Figure 6 illustrates data demonstrating that the modified peptides induced higher CTL reactivity than native IL-13Rα2 against EL4-HHD loaded with native IL-13Rα2. SPCs obtained from HHD mice immunized with either control MART-127-35 (●), native IL-13Rα2 (○), IL-13Rα2-V9 (△), or IL-13Rα2-A1V9 (X) were tested for specific lytic activity against EL4-HHD pulsed with native IL-13Rα2 in a standard 4-hour 51Cr-release assay. [Figure 7] Figure 7 depicts data demonstrating that the modified peptides induced higher CTL reactivity to EL4-HHD-IL-13Rα than native IL-13Rα2. SPCs obtained from HHD mice immunized with either control MART-127-35 (A), native IL-13Rα2 (B), IL-13Rα2-V9 (C), or IL-13Rα2-A1V9 (D) were tested for specific lytic activity against EL4-HHD-IL-13Rα2 (○) or control EL4-HHD (●) in a standard 4-hour 51Cr-release assay. [Figure 8]Figure 8 shows the expression of EphA2 protein in glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA). Paraffin-embedded sections of surgical specimens obtained from patients with GBM (AC) or AA (D) were deparaffinized and stained with anti-EphA2 polyclonal antibody (C-20: Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) or control rabbit IgG (the upper right corner window represents each sample). Relatively dense staining was observed in the endothelium and tumor cells surrounding blood vessels (D). Nine of the 14 GBMs and six of the 9 AA cases examined were EphA2 positive (data not shown). Magnification: Original ×20 [Figure 9] Figure 9 illustrates data demonstrating that CD8+ cells stimulated with EphA2883-891 induced CTL responses against human glioma cells expressing HLA-A2 and EphA2 proteins. CD8+ T cells from an HLA-A2+ glioma patient were stimulated with DCs loaded with EphA2883-891 for 10 days. These T cells were tested for lytic activity against the human glioma cells SNB19 (HLA-A2+, EphA2+) (▲), U251 (HLA-A2+, EphA2+) (■), and A172 (HLA-A2-, EphA2+) (▼) in a 4-hour 51Cr-release assay. [Figure 10] Figure 10: IL-12 production levels were positively correlated with TTP. P=0.0255 based on Cox regression followed by a likelihood ratio test. Filled circles indicate patients who had already progressed, and filled diamonds represent patients who have not relapsed to date. [Figure 11]Figure 11: T-cell responses to IL-13Rα2 (A), PADRE (B), EphA2 (C), YKL-40 (D), or gp100 (E) assessed by IFN-γ ELISPOT. Box plots show the time course of IFN-γ ELISPOT assays for all evaluated patients (boxes = 25th-75th percentiles; vertical lines = min-max). Numbers below each time point in the YKL-40 (D) panel indicate the number of patients evaluable at the indicated time. These numbers relate to other GAAs and PADRE. [Figure 12] Figure 12: T-cell responses to IL-13Rα2 (■), PADRE (*), EphA2 (▲), YKL-40 (◆) or gp100 (●) in patient 10 assessed by IFN-γ ELISPOT. [Figure 13] Figure 13: Patient 6 was analyzed up to 33 weeks and showed durable tetramer responses (IL-13Rα2 tetramer+ cells (■); EphA2 tetramer+ cells (▲); gp100 tetramer+ cells (●)) (C). An example dot plot of a positive tetramer response against the IL-13Rα2-epitope is shown (AB). [Figure 14] Figure 14: Induction of type 1 cytokine and chemokine responses. Line graphs show paired gene expression by RT-PCR for IFNα1 (A), CXCL10 (B), CCL5 (C), IL-12α (D), TLR3 (E), or CCL22 (F) on the day before the first vaccination compared with 24 hours after the first vaccination for case numbers 10 (■), 11 (|), 16 (△), 19 (□), or 22 (?). The Y-axis indicates cytokine / chemokine concentration in pg / ml. Numbers in each (A)-(F) panel indicate P values ​​based on a paired Student t-test using the mean ΔΔCT values ​​for each patient. [Figure 15]Figure 15: Induction of type 1 cytokine and chemokine responses. Line graphs show the paired relative gene expression of IFNα1 (A), CXCL10 (B), IFNγ (C), and TLR3 (D) by RT-PCR on the day before the first vaccination compared with 9 weeks after the first vaccination for case numbers 9 (●), 10 (■), 12 (no symbol), 16 (△), 18 (○), 19 (□), or 20 (▲). The Y-axis indicates cytokine / chemokine concentration in pg / ml. Numbers in each (A)-(D) panel indicate P values ​​based on a paired Student t-test using the mean ΔΔCT values ​​for each patient. [Figure 16] Figure 16: Luminex analysis was performed on pre-1 and post-4 vaccine serum samples. The Y-axis shows cytokine / chemokine concentration in pg / ml. Numbers in each (A)-(E) panel indicate P values ​​based on paired Student's t-test using the mean concentrations. [Figure 17] Figure 17: Patient 1 showed an increase in the size of Gd-enhanced lesions following two booster vaccinations, leading to surgical resection of the lesions. In situ hybridization detected CXCL10 (dark spots) mRNA in post-vaccination tissue (B) but not in the initially resected tumor (pre-vaccination) (A) and control (C). The other two pre-vaccination tissues showed no CXCL10 messenger positivity. Scale bar corresponds to 100 μm. Hematoxylin and eosin staining was performed for background. [Figure 18] Figure 18: Patients with clinical response. Patient 20 demonstrated a complete radiological response with Gd-enhancing tumors on MRI at 17 and 33 weeks (three consecutive sections shown at 0 (AC), 17 (DF), and 33 weeks (GI)). Following two booster vaccinations, patient 1 showed an increase in Gd-enhancing foci. Resected tissue revealed no evidence of mitotically active tumor (J), but a prominent infiltration of CD68+ macrophages (K) and CD8+ T cells (L). Magnification of JL; original size ×20. [Figure 19]Figure 19: Flow diagram for the study. See Section 7.7 for treatment details. The second phase of booster vaccinations could start anytime after 37 weeks, as long as the patient did not experience major SE or disease progression, and was administered every 3 months from the first vaccination for up to 3 years. The αDC1 vaccine was administered using ultrasound guidance to the inguinal lymph nodes (right and left representing the first and second vaccinations, respectively) and axillary lymph nodes (right and left representing the third and fourth vaccinations, respectively). The sites were rotated in the same order as the booster vaccinations to minimize the potential impact of injection trauma on the lymph node microenvironment due to repeated injections within a short period of time. [Figure 20] Figure 20: Time to progression (A) and overall survival (B) for GBM (■) and AG (◆). Median TTP was 4 and 13 months for GBM and AG, respectively. [Figure 21] Figure 21: IFA peptide vaccine induces superior CTL activity relative to dendritic cell vaccine combined with intramuscular (im) injection of poly-ICLC. C57BL / 6 mice received two injections (on days 0 and 7): 1) ovalbumin 257-264 peptide emulsified in subcutaneous (sc) IFA (IFA-OVA) plus im poly-ICLC (50 μg / injection); 2) scIFA-OVA plus im saline; 3) ovalbumin 257-264 peptide-loaded bone marrow-derived dendritic cells (DC-OVA) plus im poly-ICLC; or 4) DC-OVA plus im saline. Other control groups included mice injected with IFA or dendritic cells alone without OVA-peptide. The IFA-OVA vaccine combined with poly-ICLC showed the highest OVA-specific CTL levels in vivo. The use of unmutated self-GAA peptide and poly-ICLC emulsified in IFA improved mouse survival without inducing autoimmunity. These data demonstrate that poly-ICLC-adjuvanted IFA-peptide vaccine represents an effective and safe vaccine strategy. Detailed Description of the Invention

[0050] The present specification provides an interleukin-13 receptor alpha 2 (IL-13Rα2) peptide vaccine comprising an IL-13Rα2 peptide. The IL-13Rα2 peptide vaccine provided herein comprises an IL-13Rα2 peptide and at least one additional brain cancer-associated peptide.

[0051] In one embodiment, the IL-13Rα2 peptide-based vaccines provided herein comprise an IL-13Rα2 peptide and one, two, three, or more additional brain cancer-associated peptides. In certain embodiments, the IL-13Rα2 peptide-based vaccines described herein are administered in conjunction with one or more helper T cell epitopes and / or one or more immune response modifiers. Depending on the embodiment, the one or more helper T cell epitopes and / or one or more immune response modifiers may be administered as part of the vaccine (e.g., a solution of the IL-13Rα2 peptide and one, two, three, or more additional brain cancer-associated peptides) or separately from the vaccine (i.e., the helper T cell epitopes and / or immune response modifiers may be administered in a formulation that is not part of the vaccine formulation). In some embodiments, the IL-13Rα2 peptide-based vaccines described herein are administered as a cell-free vaccine. In other embodiments, the IL-13Rα2 peptide-based vaccines described herein are administered as a dendritic cell vaccine.

[0052] In one embodiment, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide, an EphA2 peptide, a YKL-40 peptide, and a GP100 peptide. In a specific embodiment, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide of any one of SEQ ID NOs: 1-4, an EphA2 peptide of SEQ ID NO: 6, a YKL-40 peptide of SEQ ID NO: 10, and a GP100 peptide of SEQ ID NO: 11. In other embodiments, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide of SEQ ID NO: 3, an EphA2 peptide of SEQ ID NO: 6, a YKL-40 peptide of SEQ ID NO: 10, and a GP100 peptide of SEQ ID NO: 11. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with one or more helper T cell epitopes. In a specific embodiment, the IL-13Rα2 peptide-based vaccine is administered in conjunction with a helper T cell epitope, wherein the helper T cell epitope is the PADRE peptide. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered in combination with one or more immune response modifiers. In some embodiments, the 13Rα2 peptide-based vaccine is a cell-free vaccine. In other embodiments, the 13Rα2 peptide-based vaccine is a dendritic cell vaccine.

[0053] In another embodiment, the IL-13Rα2 peptide vaccine comprises an IL-13Rα2 peptide, an EphA2 peptide, a survivin peptide, and a WT1 peptide. In a specific embodiment, the IL-13Rα2 peptide vaccine comprises an IL-13Rα2 peptide of any one of SEQ ID NOs: 1-4, an EphA2 peptide of SEQ ID NO: 6, a survivin peptide of SEQ ID NO: 7, and a WT1 peptide. In another embodiment, the IL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptide of SEQ ID NO: 3, the EphA2 peptide of SEQ ID NO: 6, the survivin peptide of SEQ ID NO: 7, and the WT1 peptide of SEQ ID NO: 8. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with one or more helper T cell epitopes. In a specific embodiment, the IL-13Rα2 peptide-based vaccine is administered in conjunction with a helper T cell epitope that is tetanus toxoid. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with one or more immune response modifiers. In a specific embodiment, the IL-13Rα2 peptide-based vaccine is administered in conjunction with an immune response modifier that is poly-ICLC. In a specific embodiment, the IL-13Rα2 peptide-based vaccine is administered in conjunction with an immune response modifier that is Montanide ISA-51. In some embodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine. In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cell vaccine.

[0054] In other embodiments, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide, an EphA2 peptide, and a survivin peptide. In specific embodiments, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide of any of SEQ ID NOs: 1-4, an EphA2 peptide of SEQ ID NO: 6, and a survivin peptide of SEQ ID NO: 7. In other embodiments, the IL-13Rα2 peptide-based vaccine comprises an IL-13Rα2 peptide of SEQ ID NO: 3, an EphA2 peptide of SEQ ID NO: 6, and a survivin peptide of SEQ ID NO: 7. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with one or more helper T cell epitopes. In specific embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with a helper T cell epitope that is tetanus toxoid. In some embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with one or more immune response modifiers. In certain embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with an immune response modifier, where the immune response modifier is poly-ICLC. In certain embodiments, the IL-13Rα2 peptide-based vaccine is administered in conjunction with an immune response modifier, where the immune response modifier is Montanide ISA-51. In some embodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine. In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cell vaccine.

[0055] 6.1 Peptides 6.1.1 IL-13Rα2 peptides IL-13Rα2 is a membrane glycoprotein that binds as a component of a heterodimer with IL-13, a Th2 cytokine that induces monocytes and macrophages to produce TGFβ (see, e.g., Fichtner-Feigl et al., Nat. Med., 12: 99-106, 2006).

[0056] The IL-13Rα2 peptide-based vaccines provided herein comprise an IL-13Rα2 peptide. Any IL-13Rα2 peptide that can serve as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope may be used in the vaccines described herein. In some embodiments, the IL-13Rα2 peptide used in the vaccines described herein comprises any of SEQ ID NOs: 1-4. In a specific embodiment, the IL-13Rα2 peptide used in the vaccines described herein comprises SEQ ID NO: 3.

[0057] In some embodiments, the IL-13Rα2 peptide used in the vaccines described herein comprises a variant of SEQ ID NO: 1. A variant of SEQ ID NO: 1 comprises at least one, at least two, or at least three amino acid substitutions (e.g., conservative substitutions), additions, or deletions. nothing.

[0058] In some embodiments, an IL-13Rα2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO: 1. In other embodiments, an IL-13Rα2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50%-60%, 50%-70%, 60%-70%, 70%-80%, 70%-90%, or 80%-90% identical to SEQ ID NO: 1. In some embodiments, an IL-13Rα2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% similar to SEQ ID NO: 1. In other embodiments, the IL-13Rα2 peptide used in the vaccines described herein comprises an amino acid sequence that is at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% similar to SEQ ID NO:1.

[0059] 6.1.2 EphA2 Peptides EphA2 is a tyrosine kinase receptor that interacts with ephrinA1 and is involved in notochord formation (see, for example, Naruse-Nakajima et al., Mech. Dev., 102: 95-105, 2001).

[0060] In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein comprise an EphA2 peptide. Any EphA2 peptide that can serve as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope may be used in the vaccines described herein. In some embodiments, the EphA2 peptide used in the vaccines described herein comprises SEQ ID NO: 6. In other embodiments, the EphA2 peptide used in the vaccines described herein comprises the EphA2 peptide disclosed in U.S. Patent No. 7,297,337. It is an EphA2 peptide.

[0061] In some embodiments, the EphA2 peptides used in the vaccines described herein comprise variants of SEQ ID NO:6, which variants of SEQ ID NO:6 comprise at least one, at least two, or at least three amino acid substitutions (e.g., conservative substitutions), additions, or deletions.

[0062] In some embodiments, an EphA2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO:6. In other embodiments, an EphA2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% identical to SEQ ID NO:6. In some embodiments, an EphA2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% similar to SEQ ID NO:6. In other embodiments, an EphA2 peptide used in a vaccine described herein comprises an amino acid sequence at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% similar to SEQ ID NO:6.

[0063] 6.1.3 Survivin peptide Survivin is an apoptosis-inhibiting protein that is overexpressed in most human cancers, and inhibition of its function increases apoptosis (see, for example, Blanc-Brude et al., Nat. Med., 8: 987-994, 2002).

[0064] In some embodiments, the IL-13Rα2 peptide-based vaccine provided herein comprises a survivin peptide. Any survivin peptide that can function as a IL-13Rα2 epitope may be used in the vaccines described herein. In some embodiments, the survivin peptide used in the vaccines described herein comprises SEQ ID NO: 7. In specific embodiments, the IL-13Rα2 peptide used in the vaccines described herein comprises SEQ ID NO: 7. In other embodiments, the survivin peptide used in the vaccines described herein is a survivin peptide described in U.S. Patent Application Publication No. 2009 / 0041732 or Ciesielski et al., Cancer Immunol. Immunother., 59:1211-1221, 2010.

[0065] In some embodiments, the survivin peptide used in the vaccines described herein comprises a variant of SEQ ID NO: 7, which variant of SEQ ID NO: 7 comprises at least one, at least two, or at least three amino acid substitutions (e.g., conservative substitutions), additions, or deletions.

[0066] In some embodiments, the survivin peptides used in the vaccines described herein comprise an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO:7. In other embodiments, the survivin peptides used in the vaccines described herein comprise an amino acid sequence at least 50%-60%, 50%-70%, 60%-70%, 70%-80%, 70%-90%, or 80%-90% identical to SEQ ID NO:7. In some embodiments, the survivin peptides used in the vaccines described herein comprise an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% similar to SEQ ID NO:7. In other embodiments, the survivin peptides used in the vaccines described herein comprise an amino acid sequence at least 50%-60%, 50%-70%, 60%-70%, 70%-80%, 70%-90%, or 80%-90% similar to SEQ ID NO:7.

[0067] 6.1.4 WT1 peptide WT1 is a transcription factor that is expressed during kidney development and regulates the caudal mesonephric tubules (see, for example, Sainio, Development, 124: 1293-1299, 1997).

[0068] In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein comprise a WT1 peptide. Any WT1 peptide that can serve as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope may be used in the vaccines described herein. In some embodiments, the WT1 peptide used in the vaccines described herein comprises SEQ ID NO:8.

[0069] In some embodiments, the WT1 peptide used in the vaccines described herein comprises a variant of SEQ ID NO:8, which variant of SEQ ID NO:8 comprises at least one, at least two, or at least three amino acid substitutions (e.g., conservative substitutions), additions, or deletions.

[0070] In some embodiments, the WT1 peptide used in the vaccines described herein comprises an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO: 8. In other embodiments, the WT1 peptide used in the vaccines described herein comprises an amino acid sequence at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% identical to SEQ ID NO: 8. In some embodiments, the WT1 peptide used in the vaccines described herein comprises an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% similar to SEQ ID NO: 8. In other embodiments, the WT1 peptide used in the vaccines described herein comprises an amino acid sequence at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% similar to SEQ ID NO: 8.

[0071] 6.1.5 GP100 peptide The human melanoma-associated antigen GP100 is a melanin differentiation antigen expressed in nucleated mammalian cells (see, for example, Koch et al., FEBS Lett., 179: 294-298, 1985).

[0072] In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein comprise a GP100 peptide. Any GP100 peptide that can serve as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope may be used in the vaccines described herein. In some embodiments, the GP100 peptide used in the vaccines described herein comprises SEQ ID NO: 11.

[0073] In some embodiments, the GP100 peptide used in the vaccines described herein comprises a variant of SEQ ID NO:11, which variant of SEQ ID NO:11 comprises at least one, at least two, or at least three amino acid substitutions (e.g., conservative substitutions), additions, or deletions.

[0074] In some embodiments, the GP100 peptides used in the vaccines described herein comprise an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO:11. In other embodiments, the GP100 peptides used in the vaccines described herein comprise an amino acid sequence at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% identical to SEQ ID NO:11. In some embodiments, the GP100 peptides used in the vaccines described herein comprise an amino acid sequence at least 50%, 60%, 70%, 80%, or 90% similar to SEQ ID NO:11. In other embodiments, the GP100 peptides used in the vaccines described herein comprise an amino acid sequence at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% similar to SEQ ID NO:11.

[0075] 6.1.6 YKL-40 peptide YKL-40 is a secreted glycoprotein known to be involved in extracellular matrix degradation and / or angiogenesis, for example, in liver fibrosis, rheumatoid arthritis, and severe osteoarthritis (see, for example, Bigg et al., (2006), J. Biol. Chem. 281, 21082-95).

[0076] In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein comprise a YKL-40 peptide. Any YKL-40 peptide that can serve as an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope may be used in the vaccines described herein. In some embodiments, the YKL-40 peptide used in the vaccines described herein comprises SEQ ID NO: 10.

[0077] In some embodiments, the YKL-40 peptide used in the vaccines described herein comprises a variant of SEQ ID NO: 10, which variant of SEQ ID NO: 10 comprises at least one, at least two, or at least three amino acid substitutions (e.g., conservative substitutions), additions, or deletions.

[0078] In some embodiments, the YKL-40 peptides used in the vaccines described herein comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO: 10. In other embodiments, the YKL-40 peptides used in the vaccines described herein comprise an amino acid sequence that is at least 50%-60%, 50%-70%, 60%-70%, 70%-80%, 70%-90%, or 80%-90% identical to SEQ ID NO: 10. In some embodiments, the YKL-40 peptides used in the vaccines described herein comprise an amino acid sequence that is at least 50%-60%, 50%-70%, 60%-70%, 70%-80%, 70%-90%, or 80%-90% identical to SEQ ID NO: 10. The YKL-40 peptides used in the vaccines described herein comprise an amino acid sequence that is at least 50%, 60%, 70%, 80% or 90% similar to SEQ ID NO: 10. In another embodiment, the YKL-40 peptides used in the vaccines described herein comprise an amino acid sequence that is at least 50%-60%, 50%-70%, 60%-70%, 70%-80%, 70%-90% or 80%-90% similar to SEQ ID NO: 10.

[0079] 6.2 Immune response modifiers In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein are administered with an immune response modifier. An immune response modifier includes an agent capable of modulating a subject's immune response. In some embodiments, the immune response modifier biases the subject's immune response toward a Th1 response. In other embodiments, the immune response modifier biases the subject's immune response toward a Th2 response. In preferred embodiments, the modulated immune response binds to a toll-like receptor, such as TLR3, also known as a TLR. Examples of immune response modifiers that can be administered with the IL-13Rα2 peptide-based vaccines provided herein include, but are not limited to, poly-ICLC, imiquimod (Aldara®; Beselna®), and MIS-416 (Innate Therapeutics).

[0080] 6.2.1 Poly-ICLC Polyinosinic acid polycytidylic acid (poly-ICLC), stabilized with polylysine and carboxymethylcellulose, is a synthetic nucleic acid that functions as a Toll-like receptor 3 (TLR3) ligand. Poly-ICLC is also known as Hiltonol.

[0081] 6.3 Adjuvants In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein are administered with an adjuvant. In some embodiments, the term "adjuvant" refers to an agent that, when administered together with or in the same composition as an IL-13Rα2 peptide-based vaccine described herein, enhances, accelerates, prolongs, strengthens, and / or boosts the immune response to the IL-13Rα2 peptide-based vaccine. In some embodiments, the adjuvant elicits an immune response to the IL-13Rα2 peptide-based vaccine without eliciting an allergic or other adverse reaction. Adjuvants can enhance the immune response through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T cells, stimulation of dendritic cells, and stimulation of macrophages.

[0082] Specific examples of adjuvants include Montanide ISA-51, Montanide ISA 50V, Montanide ISA 206, Montanide IMS 1312, VaxImmune® (CpG7909; Coley Pharmaceuticals), aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-O-deacylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as WO2007 / 109812), imidazoquinoline compounds (see International Application No. PCT / US2007 / 064857, published as WO2007 / 109813), Adjuvants include, but are not limited to, adjuvants such as monophosphoryl lipid A (see, e.g., PCT Publication No. PCT / US2007 / 064858) and saponins, such as QS21 (Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds., Powell & Newman, Plenum Press, NY, 1995); see U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants include, for example, monophosphoryl lipid A (Stoute et al., ., N. Engl. J. Med. 336, 86-91 (1997)). A good adjuvant is an oil-in-water emulsion (e.g., squalene or peanut oil). Another adjuvant is CpG (Bioworld Today, Nov. 15, 1998). The IL-13Rα2 peptide-based vaccine may be used with or without other specific immunostimulatory or immunopotentiating agents, such as MPL or 3-DMP, QS-21, or polymeric or monomeric amino acids such as polyglutamic acid or polylysine. It should be understood that different formulations of the IL-13Rα2 peptide-based vaccine may contain different adjuvants or the same adjuvant.

[0083] 6.4 Helper T Cell Epitopes In some embodiments, the IL-13Rα2 peptide-based vaccines provided herein are administered in conjunction with a helper T cell epitope. The helper T cell epitope comprises an agent capable of inducing a helper T cell response by the immune system. Helper T cells are CD4 + In some embodiments, the helper T cell epitope is presented by a Class II MHC molecule and is expressed by a helper T cell (CD4 + T cells) and can be recognized by the T cell receptor (TCR) of CD4 +They activate T cells, cause them to proliferate, secrete cytokines such as IL2, and activate professional antigen-presenting cells. Through various mechanisms, activated helper T cells can transform into killer T cells (CD8 + also stimulates CD8 + Exemplary helper T cell epitopes that can be administered with the IL-13Rα2 peptide-based vaccines provided herein include HBV core 128-140 and tetanus toxoid.

[0084] 6.4.1 PADRE Peptides PADRE is a non-natural epitope optimized for both HLA-DR binding and T cell receptor stimulation (see, eg, Alexander et al, Immunity, 1:751-761, 1994).

[0085] 6.4.2 Tetanus toxoid A well-characterized Th epitope (SEQ ID NO: 9) from the tetanus toxoid (TT) protein to which the majority of the population has been sensitized is known to function as a helper T cell epitope.

[0086] 6.4.2.1 HBV Core 128-140 A well-characterized Th epitope (SEQ ID NO: 5) derived from an HBV protein is known to function as a helper T cell epitope.

[0087] 6.5 Peptide production and purification The peptides described herein can be produced by any method known in the art for peptide synthesis, particularly chemical synthesis or recombinant expression techniques. The methods provided herein include, unless otherwise specified, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described and fully explained in the literature cited herein. See, for example, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2009. & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.)(1991) Oligonucleotides and Analogues : A Practical Approach, IRL Press; see Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0088] 6.5.1.1 Synthetic production of peptides The peptides described herein can be prepared using conventional stepwise solution or solid phase synthesis methods (e.g., Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., 1997, CRC Press, Boca Raton Fla., and references cited therein; Solid Phase Peptide Synthesis: A Practical Approach, Atherton & Sheppard, Eds., 1989, IRL Press, Oxford, England, and references cited therein).

[0089] Alternatively, the peptides described herein may be prepared by the methods described, for example, in Liu et al., 1996, Tetrahedron Lett. 37(7):933-936; Baca, et al., 1995, J. Am. Chem. Soc. 117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res. 45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and Tam, 1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc. Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J. Peptide Protein Res. 31:322-334. Other methods useful for synthesizing the peptides described herein are described in Nakagawa et al., 1985, J. Am. Chem. Soc. 107:7087-7092.

[0090] Formation of disulfide bonds is typically carried out in the presence of a mild oxidizing agent, if necessary. Chemical oxidizing agents may be used, or the compounds may simply be exposed to atmospheric oxygen, which results in these bonds. Various methods are known in the art and include: See, e.g., Tam et al., 1979, Synthesis 955-957; Stewart et al., 1984, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, Ill.; Ahmed et al., 1975, J. Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides 1990 164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. Additional alternatives include those described by Kamber et al., 1980, Helv. Chim. Acta 63:899-915. Methods performed on solid supports are described in Albericio, 1985, Int. J. Peptide Protein Res. 26:92-97, all of which are incorporated by reference in their entireties.

[0091] 6.5.1.2 Recombinant Expression of Peptides Recombinant expression of a peptide requires the construction of an expression vector containing a polynucleotide encoding the peptide. Once a polynucleotide encoding the peptide is obtained, a vector for producing the peptide can be constructed using recombinant DNA techniques well known in the art. Methods for preparing a peptide by expressing a polynucleotide containing a polynucleotide sequence encoding the peptide are described herein. Expression vectors containing a peptide-encoding sequence and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, provided herein are replicable expression vectors containing a nucleic acid sequence encoding the peptide operably linked to a promoter.

[0092] An expression vector comprises a nucleic acid encoding a peptide in a form suitable for expression of the nucleic acid in a host cell. In certain embodiments, the host cell is an isolated host cell. In certain embodiments, the expression vector comprises one or more regulatory sequences operably linked to the nucleic acid to be expressed, selected based on the host cell to be used for expression. In an expression vector, "operably linked" is intended to mean linked to a regulatory sequence in a manner that allows the nucleic acid of interest to be expressed (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). Regulatory sequences include promoters, enhancers, and and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleic acid in many types of host cells, those that direct expression of a nucleic acid only in certain host cells (e.g., tissue-specific regulatory sequences), and those that direct expression of a nucleic acid upon stimulation by a particular agent (e.g., inducible regulatory sequences). It will be understood by those of skill in the art that the design of an expression vector depends on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. The term "host cell" is intended to include the particular subject cell transformed or transfected with a nucleic acid and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transformed or transfected with the nucleic acid due to mutations or environmental influences that may occur during successful production or insertion of the nucleic acid into the host cell genome. In certain embodiments, the host cell is isolated.

[0093] Expression vectors can be introduced into host cells by conventional transformation or transfection techniques. Such techniques include, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation. Suitable techniques for transforming or transfecting host cells are disclosed in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, New York, and other laboratory manuals. In certain embodiments, host cells are transiently transfected with an expression vector comprising a nucleic acid encoding the peptide. In other embodiments, host cells are stably transfected with an expression vector comprising a nucleic acid encoding the peptide. Thus, the host cells provided herein are host cells comprising a polynucleotide encoding a peptide described herein or produced according to the methods provided herein.

[0094] A variety of host-expression vector systems can be used to express peptides, which represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequence, can express the peptide in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the peptide coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the peptide coding sequences; insect cell systems transformed with recombinant viral expression vectors (e.g., baculovirus) containing the peptide coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the peptide coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells or mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as E. coli, and more preferably, eukaryotic cells, are used for peptide expression. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective peptide expression systems (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2). In certain embodiments, expression of nucleic acid sequences encoding peptides described herein or produced according to the methods provided herein is controlled by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0095] In bacterial systems, a number of expression vectors are advantageously selected depending on the use of the expressed peptide. For example, when large quantities of a peptide must be produced for pharmaceutical purposes, a vector that directs high-level expression of a fusion protein product that is easily purified may be desirable. Such a vector is the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the peptide-encoding sequence is expressed as a fusion protein. To generate a protein, the lacZ-encoding region can be ligated separately into a vector in frame with the lacZ-encoding region, such as the pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509). pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites to allow release of the cloned target gene product from the GST moiety.

[0096] In insects, the Autographa californica nuclear polyhedrosis virus (AcNPV) is an exogenous The virus can be used as a gene expression vector. The virus grows in Spodoptera frugiperda cells. The peptide encoding sequence is then transfected into the virus. Non-essential regions (for example, the polyhedrin gene) of the vector may be cloned individually into the vector and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).

[0097] Numerous viral expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the peptide encoding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene is then inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., the El or E3 region) can produce recombinant virus that is viable and capable of expressing the peptide in infected hosts (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of the inserted peptide-coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0098] In addition, a host cell strain can be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the peptide. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for correct processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, Vero, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells.

[0099] For long-term, high-yield production of recombinant peptides, stable expression is preferred. By using this method, cell lines that stably express peptide molecules can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following introduction of the foreign DNA, the engineered cells may be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to the selection and allows the plasmid to stably integrate into the cell chromosome, allowing it to grow and form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express peptides. Cell lines engineered in this way may be particularly useful for screening and evaluating compositions that interact directly or indirectly with the peptide. Generally known recombinant DNA technology methods can be applied to select the desired recombinant clones, and are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties.

[0100] Peptide expression levels can be increased by vector amplification (for review, see Bebbington and Hentschel, "DNA cloning in mammalian cells"). "The use of gene amplification-based vectors to express transcriptional genes," Vol. 3 (Academic Press, New York, 1987). If the marker is amplifiable in the peptide expression vector system, Increasing the level of inhibitor present in the host cell culture medium increases the copy number of the marker gene, and because the amplified region is associated with the peptide, peptide production also increases (see Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0101] As an alternative to recombinant expression of peptides using host cells, an expression vector containing a nucleic acid encoding the peptide can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. In certain embodiments, coupled transcription / translation systems such as Promega TNT® or cell lysates or cell extracts containing the necessary components for transcription and translation can be used to produce the peptide.

[0102] Thus, provided herein are methods for producing a peptide. In one embodiment, the method comprises culturing a host cell comprising nucleic acid encoding the peptide in a medium suitable for producing the peptide. In some embodiments, the method further comprises isolating the peptide from the medium or the host cell.

[0103] In certain embodiments, plants (e.g., plants of the genus Nicotiana) can be engineered to express the peptides described herein. In certain embodiments, plants can be engineered using known methods. Plants can be engineered to express the peptides described herein via agroinfiltration, for example, by introducing a nucleic acid encoding a gene of interest, e.g., a gene encoding a peptide described herein, into a strain of Agrobacterium, followed by growing the strain in liquid medium and washing the resulting bacteria to express the peptides. The peptides described herein are then coated onto Agrobacterium, which transforms a portion of plant cells with the gene of interest. Expose the plant to Agrobacterium containing the nucleic acid to be transduced (e.g., by injection) or via immersion in water). The plants then transiently express the peptide, and the peptide can be isolated using methods known in the art and described herein (see, for example, Shoji et al., 2008, Vaccine, 26(23):2930-2934; and D'Aoust et al., 2008, J. Plant Biotechnology, 6(9):930-940). In certain embodiments, the plant is a tobacco plant. In other specific embodiments, the plant is a tobacco plant (e.g., Nicotiana benthamiana).

[0104] In other embodiments, algae (e.g., Chlamydomonas reinhardtii) can be engineered to express the peptides described herein. (See, for example, Rasala et al., 2010, Plant Biotechnology Journal (Published online March 7, 2010)).

[0105] 6.5.1.3 Peptide purification The peptides described herein and peptides produced using the methods described herein can be purified by any method known in the art for purifying peptides, such as chromatography (e.g., ion exchange, affinity, Protein A followed by affinity for a specific antigen, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Additionally, the peptides may be fused to heterologous peptides described herein or other known in the art to facilitate purification. The actual conditions used to purify a particular peptide will depend, in part, on the synthesis strategy (e.g., synthetic versus recombinant production), the net charge, hydrophobicity, and / or hydrophilicity of the peptide, and will be apparent to one of skill in the art.

[0106] 6.6 Pharmaceutical Compositions and Routes of Administration Pharmaceutical compositions are also provided herein. In some embodiments, the compositions provided herein comprise an interleukin-13 receptor α2 peptide-based brain cancer vaccine. In other embodiments, the compositions provided herein comprise an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier. In other embodiments, the compositions provided herein comprise an immune response modifier. The pharmaceutical compositions provided herein are suitable for veterinary and / or human administration.

[0107] The pharmaceutical compositions provided herein (e.g., compositions comprising an IL-13Rα2 peptide-based vaccine, compositions comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or compositions comprising an immune response modifier) ​​may be in any form suitable for administration to a subject, which is preferably an animal, including but not limited to humans, mammals or non-human animals such as cows, horses, sheep, pigs, poultry, cats, dogs, mice, rats, rabbits, and guinea pigs, more preferably mammals, and most preferably humans.

[0108] In certain embodiments, the compositions provided herein (e.g., a composition comprising an IL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or a composition comprising an immune response modifier) ​​are in the form of a liquid (e.g., an elixir, syrup, solution, emulsion, or suspension). Typical routes of administration of the liquid compositions provided herein include, but are not limited to, parenteral, intradermal, intratumoral, intracerebral, and intrathecal. Parenteral administration includes, but is not limited to, subcutaneous, intranodal, intravenous, intramuscular, intraperitoneal, and intrapleural administration techniques. In certain embodiments, the compositions are administered parenterally. Compositions for administration by injection can contain one or more surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents. In certain embodiments, a pump can be used to deliver the vaccine (see, e.g., Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14, 201; Buchwald et al., Surgery 1980, 88: 507; Saudek et al., N. Engl. J. Med. 1989, 321: 574). In certain embodiments, the pump is an insulin-like pump, but This is not limited to this.

[0109] Materials used to prepare the pharmaceutical compositions provided herein (e.g., compositions comprising an IL-13Rα2 peptide-based vaccine, compositions comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or compositions comprising an immune response modifier) ​​can be non-toxic in the amounts used. Those skilled in the art will recognize that the optimal dose of the active ingredient in a pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of subject (e.g., human), the overall health of the subject, the type of brain cancer in the subject requiring treatment, use of the composition as part of a multi-drug regimen, the specific form of the vaccine administered, the method of administration, and the composition utilized.

[0110] Liquid compositions of the present invention, whether in solution, suspension, or other form, may contain one or more of the following: a sterile diluent such as water for injection; saline, preferably physiological saline, Ringer's solution, isotonic saline; fixed oils such as synthetic mono- or diglycerides that can be used as solvents or suspending media; polyethylene glycol, glycerin, cyclodextrin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral compositions can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass, plastic, or other materials. Injectable compositions are preferably sterilized.

[0111] The compositions provided herein (e.g., compositions comprising an IL-13Rα2 peptide-based vaccine, compositions comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or compositions comprising an immune response modifier) ​​may comprise a pharmaceutically acceptable carrier or vehicle. As used herein, "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopeia or other commonly used pharmacopeia. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Saline, aqueous dextrose, and glycerol solutions may be utilized as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The formulation should conform to the formula.

[0112] In one embodiment, the compositions provided herein (e.g., a composition comprising an IL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or a composition comprising an immune response modifier) ​​can be prepared according to routine procedures as pharmaceutical compositions adapted for parenteral administration to animals, particularly humans. Generally, the components of the compositions are supplied individually or mixed together in unit-dose form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the compositions described herein are administered by injection, an ampoule of sterile water for injection or saline can be provided, if necessary, to allow the components to be mixed prior to administration.

[0113] Compositions provided herein (e.g., compositions comprising an IL-13Rα2 peptide-based vaccine, compositions comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or compositions comprising an immune response modifier) These include, but are not limited to, additional active agents selected from additional prophylactic agents, additional therapeutic agents, antiemetic agents, hematopoietic colony stimulating factors, adjuvant therapy, antibody / antibody fragment agents, antidepressants, and analgesics.

[0114] Pharmaceutical compositions provided herein (e.g., compositions comprising an IL-13Rα2 peptide-based vaccine, compositions comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or compositions comprising an immune response modifier) ​​can be prepared using methods well known in the pharmaceutical art. For example, compositions intended to be administered by injection can be prepared by combining the vaccine peptides described herein with water and / or other liquid components to form an aqueous solution. A surfactant can be added to promote the formation of a homogeneous solution or suspension.

[0115] The pharmaceutical compositions described herein can be included in a container, pack, or dispenser together with instructions for administration.

[0116] 6.7 Prophylactic and Therapeutic Use In one aspect, the present specification provides a method for preventing, treating, and / or managing brain cancer in a subject in need thereof by administering an effective amount of the IL-13Rα2 peptide-based vaccine described herein.

[0117] In another aspect, the present specification provides a method for preventing, treating, and / or managing brain cancer in a patient (e.g., a human patient), comprising administering to the patient a prophylactically effective regimen or a therapeutically effective regimen, the method comprising administering to the patient diagnosed with brain cancer an IL-13Rα2 peptide-based vaccine described herein or a pharmaceutical composition described herein.

[0118] In another aspect, the present specification provides a method for preventing, treating, and / or managing brain cancer in a patient (e.g., a human patient), comprising administering to the patient a prophylactically effective regimen or a therapeutically effective regimen, and comprising administering to the patient who has experienced a recurrence of brain cancer an IL-13Rα2 peptide-based vaccine described herein or a pharmaceutical composition described herein.

[0119] In another aspect, the present specification provides methods for preventing, treating, and / or managing brain cancer in a patient (e.g., a human patient), comprising administering to the patient a prophylactically effective regimen or a therapeutically effective regimen, the method comprising administering an IL-13Rα2 peptide-based vaccine described herein or a pharmaceutical composition described herein to a patient who has failed or is failing a brain cancer treatment that does not include a vaccine described herein.

[0120] In another aspect, the present specification provides a method for preventing, treating, and / or managing brain cancer in a patient (e.g., a human patient), comprising administering to the patient a prophylactically effective regimen or a therapeutically effective regimen, wherein the method comprises administering to a patient in remission of brain cancer an IL-13Rα2 peptide-based vaccine described herein or a pharmaceutical composition described herein.

[0121] In another aspect, provided herein are methods for preventing, treating, and / or managing brain cancer in a patient (e.g., a human patient), comprising administering to the patient a prophylactically effective regimen or a therapeutically effective regimen, the method comprising administering an IL-13Rα2 peptide-based vaccine described herein or a pharmaceutical composition described herein to a patient who is refractory to a brain cancer treatment that does not include a vaccine described herein. In one embodiment of this aspect, the patient has been or is receiving a brain cancer treatment that does not include a vaccine described herein. In another embodiment of this aspect, the patient is receiving a brain cancer treatment that does not include a vaccine described herein for the prevention, treatment, and / or management of brain cancer. Therefore, they have not received any previous brain cancer treatment that does not include the vaccines described herein.

[0122] In another aspect, the present specification provides methods for preventing, treating, and / or managing brain cancer in a patient (e.g., a human patient), the method comprising administering to the patient a prophylactically or therapeutically effective regimen comprising administering an IL-13Rα2 peptide-based vaccine described herein or a pharmaceutical composition described herein to the patient who has received another brain cancer treatment. In some embodiments, the previous brain cancer treatment is, for example, chemotherapy, radiation therapy, surgery, small molecule therapy, biological therapy, antibody therapy, hormone therapy, immunotherapy, antiangiogenic therapy, or any combination thereof. In some embodiments, the patient's previous brain cancer treatment has failed. In some embodiments, the therapeutically effective regimen comprising administration of an IL-13Rα2 peptide-based vaccine described herein is administered to the patient immediately after receiving the previous treatment. For example, in certain embodiments, the results of the previous treatment will not be known before the patient is administered the IL-13Rα2 peptide-based vaccine. In one embodiment, the previous chemotherapy is temozolomide. In some embodiments, the previous therapy is radiation therapy. In other embodiments, the previous therapy is a combination of temozolomide and radiation therapy. In a preferred embodiment, the combination of temozolomide and radiation therapy is administered using the Stupp regimen. In other embodiments, the previous therapy is surgical therapy. In some embodiments, the patient undergoes surgical therapy before the initiation of the combination therapy. In some embodiments, the patient undergoes surgical therapy before temozolomide. In some embodiments, the patient undergoes surgical therapy before the initiation of radiation therapy. In each of these embodiments described herein, the combination therapy can involve administering an IL-13Rα2 peptide-based vaccine to the patient before, during, or after the combined therapy.

[0123] In some embodiments, the IL-13Rα2 peptide-based vaccines described herein can be administered as monotherapy for the prevention, treatment, and / or management of brain cancer. In other embodiments, the methods provided herein include administering to a subject in need thereof an IL-13Rα2 peptide-based vaccine described herein and one or more other agents, other than the IL-13Rα2 peptide-based vaccine described herein, that are currently being used, have been used, are known to be useful, or may be useful for the prevention, treatment, and / or management of brain cancer or one or more symptoms thereof. In certain embodiments, the combination therapy functions with the IL-13Rα2 peptide-based vaccine described herein to improve the prophylactic or therapeutic efficacy of the IL-13Rα2 peptide-based vaccine described herein, with additive or synergistic effects. In some embodiments, the combination therapy can be administered before, during, or after the administration of a composition described herein.

[0124] In other embodiments, methods are provided for inducing an immune response in a subject with brain cancer, comprising administering an effective amount of an IL-13Rα2 peptide-based vaccine described herein. In some embodiments, the immune response induced by the IL-13Rα2 peptide-based vaccine described herein or a composition described herein is effective in preventing, treating, and / or managing brain cancer in the subject. In some embodiments, the immune response induced in a subject by the IL-13Rα2 peptide-based vaccine described herein or a composition described herein is effective in alleviating brain cancer symptoms in the subject.

[0125] A physician can diagnose a patient using any conventional brain cancer screening methodology, including, but not limited to, neurological examination, imaging methods (e.g., computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, X-ray imaging, positron emission tomography (PET) scan), and biopsy (e.g., sterotactic biopsy).

[0126] 6.7.1 Dosage and frequency of administration Compositions described herein that are effective in the treatment, prevention, and / or management of brain cancer (e.g., I The amount of a composition comprising an IL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or a composition comprising an immune response modifier will depend on the state of the brain cancer, the patient to whom the composition is administered, the route of administration, and / or the type of brain cancer. Such amounts can be determined by standard clinical techniques and according to the judgment of a physician.

[0127] For example, the effective amount may vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight, and health), whether the patient is a human or an animal, whether other drugs are being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Therapeutic doses are titrated to optimize optimal safety and efficacy.

[0128] In certain embodiments, in vitro assays are employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0129] In certain embodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine, and the cell-free vaccine comprises an IL-13Rα2 peptide and one, two, three, or more additional brain cancer-associated peptides. In some embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines comprise about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, or 800 μg of each brain cancer-associated peptide per dose. In other embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines comprise about 25-50, 25-75, 25-100, 50-100, 50-150, 50-200, 100-150, 100-200, 100-250, 100-300, 150-200, 150-250, 150-300, 200-250, 2 Each of the brain cancer-associated peptides contains 50-300, 250-350, 250-400, 300-350, 300-400, 300-450, 300-500, 350-400, 350-450, 400-500, 400-600, 500-600, 500-700, 600-700, 600-800, or 700-800 μg. In other embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines comprise about 5 μg-100 mg, 15 μg-50 mg, 15 μg-25 mg, 15 μg-10 mg, 15 μg-5 mg, 15 μg-1 mg, 5 μg-100 μg, 15 μg-75 μg, 5 μg-50 μg, 10 μg-50 μg, 15 μg-45 μg, 20 μg-40 μg, or 25-35 μg of each brain cancer-associated peptide per kilogram of patient.

[0130] In certain embodiments, the cell-free IL-13Rα2 peptide-based vaccine is administered in conjunction with a helper T cell epitope. In some embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines are administered in conjunction with about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, or 600 μg of a helper T cell epitope. In other embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines are administered with about 25-50, 25-75, 25-100, 50-100, 50-150, 50-200, 100-150, 100-200, 100-250, 100-300, 150-200, 150-250, 150-300, 200-250, 250-300, 250-350, 250-400, 300-350, 300-400, 300-450, 300-500, 350-400, 350-450, 400-500, 400-600, or 500-600 μg of helper T cell epitopes.

[0131] In certain embodiments, the cell-free IL-13Rα2 peptide-based vaccine comprises an immune response modifier. In some embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines are administered with about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 μg of an immune response modifier. In other embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines are administered with about 100-300, 200-400, 400-800, 600-800, 800-1000, 800-1200, 1000-1200, 1000-1400, 1200-1400, 1200-1600, 1400-1600, 1400-1800, or 1600-1800 μg of immune response modifier. In other embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines are administered with about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 μg of immune response modifier per kilogram of patient. In other embodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines are administered with about 1-5, 1-10, 5-10, 5-15, 10-15, 10-20, 15-20, 15-25, 15-30, 20-25, 20-30, 20-35, 25-30, 25-35, 25-40, 30-35, 30-40, 35-40, 35-45, 40-45, 40-50, 45-50, 50-55, or 50-60 μg of immune response modifier per kilogram of patient.

[0132] In certain embodiments, the cell-free IL-13Rα2 peptide-based vaccine is administered with an adjuvant. In some embodiments, a composition comprising the cell-free IL-13Rα2 peptide-based vaccine is mixed with a 0.5:1, 1:0.5, 1:1, 1:2, 1:3, 2:1, or 3:1 adjuvant.

[0133] In certain embodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cell vaccine, which comprises dendritic cells loaded with an IL-13Rα2 peptide and dendritic cells loaded with one, two, three, or more additional brain cancer-associated peptides. In some embodiments, an exemplary dendritic cell IL-13Rα2 peptide-based vaccine comprises approximately 10 of the dendritic cells loaded with the brain cancer-associated peptide per dose. 3 , 5x10 3 , 10 4 , 5x10 4 , 10 5 , 5x10 5 , 10 6 , 5x10 6 , 10 7 , 3x10 7 , 5x10 7 , 7x10 7 , 10 8 , 5x10 8 , 1x10 9 , 5x10 9 , 1x10 10 , 5x10 10 , 1x10 11 , 5x10 11 or 10 12 In another embodiment, an exemplary dendritic cell-based IL-13Rα2 peptide-based vaccine comprises about 10 of dendritic cells loaded with a brain cancer-associated peptide per dose. 3 -10 4 , 10 3 -10 5 , 10 4 -10 5 , 10 4 -10 6 , 10 5 -10 6 , 10 5 -10 7 , 10 6 -10 7 , 10 6 -10 8 , 10 7 -10 8 , 10 7 -10 9 , 10 8 -10 9 , 10 9 -10 10 , 1010 -10 11 or 10 11 -10 12 Includes:

[0134] In certain embodiments, dendritic cell-mediated IL-13Rα2 peptide-based vaccines are administered in conjunction with a helper T cell epitope. In some embodiments, exemplary dendritic cell-mediated IL-13Rα2 peptide-based vaccines are administered in conjunction with about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, or 600 μg of a helper T cell epitope. In other embodiments, exemplary dendritic cell IL-13Rα2 peptide-based vaccines are administered with about 25-50, 25-75, 25-100, 50-100, 50-150, 50-200, 100-150, 100-200, 100-250, 100-300, 150-200, 150-250, 150-300, 200-250, 250-300, 250-350, 250-400, 300-350, 300-400, 300-450, 300-500, 350-400, 350-450, 400-500, 400-600, or 500-600 μg of helper T cell epitopes.

[0135] In certain embodiments, the dendritic cell-mediated IL-13Rα2 peptide-based vaccine comprises an immune response modifier. In some embodiments, exemplary dendritic cell-mediated IL-13Rα2 peptide-based vaccines are administered with about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 μg of an immune response modifier. In other embodiments, exemplary dendritic cell-mediated IL-13Rα2 peptide-based vaccines are administered with about 100-300, 200-400, 400-800, 600-800, 800-1000, 800-1200, 1000-1200, 1000-1400, 1200-1400, 1200-1600, 1400-1600, 1400-1800, or 1600-1800 μg of an immune response modifier. In other embodiments, exemplary dendritic cell-mediated IL-13Rα2 peptide-based vaccines are administered with about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 μg of an immune response modifier per kilogram of patient. In other embodiments, exemplary dendritic cell-IL-13Rα2 peptide-based vaccines are administered with about 1-5, 1-10, 5-10, 5-15, 10-15, 10-20, 15-20, 15-25, 15-30, 20-25, 20-30, 20-35, 25-30, 25-35, 25-40, 30-35, 30-40, 35-40, 35-45, 40-45, 40-50, 45-50, 50-55, or 50-60 μg of immune response modifier per kilogram of patient.

[0136] In certain embodiments, the dendritic cell-based IL-13Rα2 peptide-based vaccine is administered with an adjuvant. In some embodiments, a composition comprising the dendritic cell-based IL-13Rα2 peptide-based vaccine is mixed with an adjuvant at a ratio of 0.5:1, 1:0.5, 1:1, 1:2, 1:3, 2:1, or 3:1.

[0137] In certain embodiments, a composition described herein (e.g., a composition comprising an IL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or a composition comprising an immune response modifier) ​​is administered to a subject once as a single dose. In some embodiments, a composition described herein (e.g., a composition comprising an IL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2 peptide-based vaccine and a helper T cell epitope, an adjuvant, and / or an immune response modifier, or a composition comprising an immune response modifier) ​​is administered multiple times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times), and the doses may be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 30 days. In a specific embodiment, the IL-13Rα2 peptide-based vaccine is administered intranodally or subcutaneously and the immune response modifier is administered intramuscularly.

[0138] In some embodiments, when the composition described herein comprises a cell-free IL-13Rα2 peptide-based vaccine, it can be administered at weeks 0, 3, 6, 9, 12, 15, 18, and 21, over a 21-week period. In certain embodiments, the composition comprising the cell-free IL-13Rα2 peptide-based vaccine is administered together with a helper T cell epitope, an adjuvant, and / or an immune response modifier. In certain embodiments, the composition described herein comprising a cell-free IL-13Rα2 peptide-based vaccine can be administered at weeks 0, 3, 6, 9, 12, 15, 18, and 21, over a 21-week period, and the composition is administered together with an immune response modifier, which is administered on each administration day of the cell-free IL-13Rα2 peptide-based vaccine and four days after each administration of the cell-free IL-13Rα2 peptide-based vaccine. In another specific embodiment, a composition described herein comprising a cell-free IL-13Rα2 peptide-based vaccine is administered at weeks 0, 3, 6, 9, 12, 15, 18, and 21, and the composition can be administered over a 21-week period. The composition is then administered with an immune response modifier, and the immune response modifier is administered on each day the cell-free IL-13Rα2 peptide-based vaccine is administered. In a specific embodiment, the cell-free IL-13Rα2 peptide-based vaccine is administered at weeks 0, 3, 6, 9, 12, 15, 18, and 21. The -13Rα2 peptide vaccine is administered subcutaneously, and the immune response modifier is administered intramuscularly.

[0139] In some embodiments, when the composition described herein comprises a dendritic cell-based IL-13Rα2 peptide-based vaccine, the composition can be administered at weeks 0, 2, 4, and 6 for a period of 6 weeks. In a specific embodiment, the composition comprising a cell-free IL-13Rα2 peptide-based vaccine is administered together with a helper T cell epitope, an adjuvant, and / or an immune response modifier. In a specific embodiment, the composition described herein comprising a dendritic cell-based IL-13Rα2 peptide-based vaccine can be administered at weeks 0, 2, 4, and 6 for a period of 6 weeks, and the composition is administered together with an immune response modifier, with administration of the immune response modifier beginning on the first day of administration of the dendritic cell-based IL-13Rα2 peptide-based vaccine and administered twice a week. In a specific embodiment, the dendritic cell-based IL-13Rα2 peptide-based vaccine is administered intranodally, and the immune response modifier is administered intramuscularly.

[0140] In some embodiments, when the composition described herein comprises a dendritic cell-based IL-13Rα2 peptide-based vaccine, it can be administered at weeks 0, 2, 4, 6, 10, 14, 18, 22, and 26, for a total of 26 weeks. In certain embodiments, the composition comprising a cell-free IL-13Rα2 peptide-based vaccine is administered together with a helper T cell epitope, an adjuvant, and / or an immune response modifier. In certain embodiments, the composition described herein comprising a dendritic cell-based IL-13Rα2 peptide-based vaccine can be administered at weeks 0, 2, 4, 6, 10, 14, 18, 22, and 26, for a total of 26 weeks, and the composition is administered together with an immune response modifier, with the immune response modifier administered twice weekly starting on the first day of administration of the dendritic cell-based IL-13Rα2 peptide-based vaccine. In a specific embodiment, the dendritic cell-IL-13Rα2 peptide-based vaccine is administered intranodally and the immune response modifier is administered intramuscularly.

[0141] 6.7.2 Brain cancer The IL-13Rα2 peptide-based vaccines described herein can be used to prevent, treat, and / or manage brain cancer. Any type of brain cancer can be treated with the IL-13Rα2 peptide-based vaccines described herein according to the methods described herein. Exemplary brain cancers include gliomas (astrocytomas, including pilocytic astrocytomas, diffuse astrocytomas, and anaplastic astrocytomas, glioblastomas, oligodendrogliomas, brainstem gliomas, non-brainstem gliomas, ependymomas, and mixed tumors containing more than one glial cell type), acoustic neuroma, craniopharyngioma, meningioma, medulloblastoma, primary central nervous system tumors, tumors of the pineal gland (e.g., pineal astrocytomas and pineal parenchymal tumors), and tumors of the pituitary gland. Gliomas further include, but are not limited to, recurrent malignant glioma, high-risk WHO Grade II astrocytoma, oligoastrocytoma, recurrent WHO Grade II glioma, newly diagnosed malignant or intrinsic brainstem glioma, incompletely resected non-brainstem glioma, and recurrent unresectable low-grade glioma. Additional types of brain cancers that may be treated with the IL-13Rα2 peptide-based vaccines described herein according to the methods described herein include adult low-grade invasive supratentorial astrocytoma / oligodendroglioma, adult low-grade invasive supratentorial astrocytoma, adult low-grade invasive supratentorial oligodendroglioma, adult low-grade invasive supratentorial astrocytoma / oligodendroglioma (excluding pilocytic astrocytoma), adult low-grade invasive supratentorial astrocytoma (excluding pilocytic astrocytoma), and adult low-grade invasive supratentorial astrocytoma (excluding pilocytic astrocytoma). ), adult low-grade invasive supratentorial oligodendroglioma (excluding pilocytic astrocytoma), adult intracranial ependymoma, adult intracranial ependymoma (excluding subependymoma and myxopapillary), adult intracranial anaplastic ependymoma, anaplastic glioma, anaplastic glioblastoma, pilocytic astrocytoma, subependymoma, myxopapillary, 1-3 limited metastatic lesions (intraparenchymal), 3 or more metastatic lesions (intraparenchymal), leptomeningeal metastasis (neoplastic meningitis), primary central nervous system, metastatic spinal tumor, or meningioma.

[0142] In one embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccine described herein according to the methods described herein is a glioma. In a specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccine described herein according to the methods described herein is a recurrent malignant glioma. In another specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccine described herein according to the methods described herein is a recurrent WHO grade II glioma. In another specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccine described herein according to the methods described herein is a newly diagnosed malignant or invasive brainstem glioma. In another specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccine described herein according to the methods described herein is an incompletely resected non-brainstem glioma. In another specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccine described herein according to the methods described herein is a recurrent unresectable low-grade glioma. In one embodiment, the patient is an adult with recurrent malignant glioma, recurrent glioblastoma, anaplastic astrocytoma, anaplastic oligodendroglioma, or mixed oligoastrocytoma. In another specific embodiment, the patient is an adult with newly diagnosed high-risk low-grade glioma. In another specific embodiment, the patient is an adult with newly diagnosed high-risk low-grade astrocytoma. In another specific embodiment, the patient is an adult with newly diagnosed high-risk low-grade oligoastrocytoma. In another specific embodiment, the patient is an adult with recurrent high-risk low-grade oligoastrocytoma. In another specific embodiment, the patient is an adult with recurrent high-risk low-grade oligoastrocytoma. In another specific embodiment, the patient is an adult with recurrent high-risk low-grade oligodendroglioma. In another specific embodiment, the patient is a child with newly diagnosed malignant glioma. In another specific embodiment, the patient is a child with intrinsic brainstem glioma. In another specific embodiment, the patient is a child with incompletely resected non-brainstem high-grade glioma. In another particular embodiment, the patient is a child with recurrent unresectable low-grade glioma.In another specific embodiment, the patient is a child with newly diagnosed diffuse intrinsic pontine glioma. In another specific embodiment, the patient is a child with any high-grade glioma involving the brainstem that has been treated with RT or without chemotherapy during RT. In another specific embodiment, the patient is a child with newly diagnosed non-brainstem high-grade glioma that has been treated with RT and chemotherapy. In another specific embodiment, the patient is a child with recurrent non-brainstem high-grade glioma that has recurred after treatment.

[0143] In other embodiments, the brain cancer treated with the IL-13Rα2 peptide-based vaccines described herein as described herein is an astrocytoma. In a specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccines described herein as described herein is a high-risk WHO grade II astrocytoma. In another specific embodiment, the brain cancer treated with the IL-13Rα2 peptide-based vaccines described herein as described herein is an oligoastrocytoma.

[0144] 6.7.3 Patient groups In one embodiment, the IL-13Rα2 peptide-based vaccine or composition described herein can be administered to a naive subject, i.e., a subject not suffering from brain cancer. In one embodiment, the IL-13Rα2 peptide-based vaccine or composition described herein can be administered to a subject at risk for brain cancer.

[0145] In certain embodiments, the IL-13Rα2 peptide-based vaccines or compositions described herein can be administered to patients diagnosed with brain cancer. In some embodiments, the IL-13Rα2 peptide-based vaccines or compositions described herein are administered to patients before they exhibit symptoms of brain cancer or before their symptoms worsen. In a preferred embodiment, the brain cancer is glioma.

[0146] In certain embodiments, the IL-13Rα2 peptide-based vaccines or compositions described herein can be administered to patients in need of treatment, prevention, and / or management of brain cancer. The subject may or may not have previously been treated for cancer. Alternatively, the subject may be in remission, relapse, or have undergone treatment failure. The patient may have abnormal cytogenetics. The 13Rα2 peptide-based vaccines and compositions described herein can be used in any series of brain cancer treatments, for example, as the first, second, or third line of brain cancer treatment. In certain embodiments, the subject receiving or receiving the vaccine or pharmaceutical composition described herein is receiving or has received other brain cancer treatments. In other embodiments, the subject receiving or receiving the vaccine or pharmaceutical composition described herein is not receiving or has not received other brain cancer treatments.

[0147] In certain embodiments, the subject has been diagnosed with brain cancer by methods known to those of skill in the art, including neurological examination, imaging methods (e.g., computed tomography (CT), magnetic resonance imaging (MRI), or the like). Magnetic resonance imaging (MRI), ultrasound, fluid-attenuated inversion-recovery (FLAIR) sequences, T2-weighted images, and positron emission tomography (PET) scans) These include, but are not limited to, stereotactic biopsy and biopsy. Tumor response to treatment can be assessed using the McDonald criteria or RANO (Response Assessment in Neuro-Oncology) criteria. Tumor size or response to treatment can be assessed using various magnetic resonance imaging techniques: diffusion-weighted imaging, perfusion-weighted imaging, dynamic contrast-enhanced T1 permeability imaging, dynamic susceptibility contrast, diffusion tensor imaging, magnetic resonance spectroscopy, anatomical MRI T2-weighted imaging, fluid-attenuated inversion recovery (FLAIR) T2-weighted imaging, and gadolinium-enhanced T1-enhanced imaging. These imaging techniques can be used to assess tumor cellularity, white matter infiltration, metabolic abnormalities including hypoxia and necrosis, angiogenesis, capillary blood volume, or magnetic permeability. 18F-fluoromisonidazole PET and 3'-deoxy-3'-18F-fluorothymidine PET, etc. Positron emission tomography (PET) technology also images tumor response. can be used for

[0148] In one embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein can be administered to a subject undergoing or who has undergone radiation therapy to treat a brain cancer tumor. In a specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein can be administered to a subject simultaneously with or after radiation therapy to treat a brain cancer tumor. In other embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein can be administered to a subject prior to, and in some embodiments, during and / or after, radiation therapy to treat a brain cancer tumor. In some preferred embodiments, the radiation therapy is fractionated external beam radiotherapy, limited-field fractionated external beam radiotherapy, whole brain radiotherapy, stereotactic radiosurgery, or craniospinal radiotherapy.

[0149] In one embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject undergoing or who has undergone chemotherapy to treat a brain cancer tumor. In a specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject simultaneously with or following chemotherapy to treat a brain cancer tumor. In other embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject prior to, and in some embodiments during and / or after, chemotherapy to treat a brain cancer tumor. In some preferred embodiments, the chemotherapy is administered with or without temozolomide (Temodar®), nitrosoureas, platinum-based regimens, etoposide, or other anti-cancer drugs. cisplatin, bevacizumab (Avastin®), irinotecan, cyclophosphamide, These include phenidate, BCNU (carmustine), capecitabine, high-dose methotrexate, topotecan, high-dose ARA-C, hydroxyurea, alpha-interferon, somatostatin analogues, and intra-CSF chemotherapy (liposomal cytarabine, methotrexate, cytarabine, thiotepa, or rituximab (Rituxan®)).

[0150] In one embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject who has failed, is undergoing treatment, or has previously been treated with a therapeutic strategy including chemotherapy, radiation therapy, or surgery to treat one or more brain cancer tumors. In a preferred embodiment, the brain cancer is a glioma. For example, the patient may have failed, undergone, or previously undergone both chemotherapy and surgery. Alternatively, the patient may have undergone, or previously undergone, radiation and surgery. Furthermore, the patient may have undergone, or previously undergone, chemotherapy and radiation. In some preferred embodiments, the combination of treatments that the patient has failed, is undergoing, or previously undergone is resection and temozolomide (Temodar®) (150- 200 mg / m 2 ) 5 / 28 schedule, excision and BCNU wafer (Gliadel® )), bevacizumab (Avastin®) and chemotherapy, PCV combination (CCNU (lomustine) and procarbazine and vincristine), high-dose methotrexate and and vincristine, procarbazine, cytaribine, or rituximab, high-dose chemotherapy with stem cell rescue or rituximab ®) and temozolomide (Temodar®).

[0151] In one embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject undergoing or who has previously undergone surgery to remove a brain cancer tumor. In certain embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject at the time of or after surgery to remove a brain cancer tumor. In other embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject prior to surgery to remove a brain cancer tumor, and in some embodiments, during and / or after surgery.

[0152] In certain embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject as an alternative to other treatments, e.g., chemotherapy, radiation therapy, hormone therapy, surgery, small molecule therapy, antiangiogenic therapy, and / or biological therapy, including immunotherapy, that have proven or may prove to be too toxic, i.e., cause side effects that are intolerable or intolerable to the subject.

[0153] In certain embodiments, the IL-13Rα2 peptide-based vaccines or compositions described herein are administered to subjects who may be undergoing, are undergoing, or have undergone radiation therapy, including chemotherapy, hormone therapy, small molecule therapy, antiangiogenic therapy, and / or biologic therapy, including immunotherapy, as well as surgery.

[0154] In another embodiment, the IL-13Rα2 peptide-based vaccines or compositions described herein are administered to subjects who are undergoing, are undergoing, or have undergone biological therapy, including hormonal therapy and / or immunotherapy, including chemotherapy, small molecule therapy, antiangiogenic therapy, and / or radiation therapy, as well as surgery.

[0155] In certain embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject who is refractory to one or more therapies. A cancer that is refractory means that at least a significant portion of the cancer cells are not killed or do not stop dividing. Whether cancer cells are refractory, in this context, can be determined by any method for analyzing the effectiveness of treating cancer cells, either in vivo or in vitro, using the art-accepted meaning of "refractory." In various embodiments, a cancer is refractory if the amount of cancer cells is not significantly reduced or is increasing.

[0156] In some embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject in remission from brain cancer. In certain embodiments, the subject is free of detectable brain cancer, i.e., brain cancer cannot be detected using conventional methods described herein (e.g., MRI) or methods known to those skilled in the art.

[0157] In one embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a glioma. In a specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with an astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, and anaplastic astrocytoma). In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a glioblastoma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with an oligodendroglioma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a brainstem glioma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with an ependymoma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a mixed tumor comprising one or more glial cell types.

[0158] In certain embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a recurrent malignant glioma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a high-risk WHO grade II astrocytoma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with an oligoastrocytoma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a recurrent WHO grade II glioma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject with a newly diagnosed malignant or intrinsic brainstem glioma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with an incompletely resected non-brainstem glioma. In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with unresectable recurrent low-grade glioma.

[0159] In certain embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with acoustic neuroma. In other specific embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with craniopharyngioma. In other specific embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with meningioma. In other specific embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with medulloblastoma. In other specific embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with primary central nervous system lymphoma. In other specific embodiments, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a pineal gland tumor (e.g., a pineal astrocytic tumor or a pineal parenchymal tumor). In another specific embodiment, an IL-13Rα2 peptide-based vaccine or composition described herein is administered to a subject diagnosed with a pituitary tumor.

[0160] In certain embodiments, the subject to which an IL-13Rα2 peptide-based vaccine or composition described herein is administered is a human adult. In certain embodiments, the subject to which an IL-13Rα2 peptide-based vaccine or composition described herein is administered is an elderly person. In certain embodiments, the subject to which an IL-13Rα2 peptide-based vaccine or composition described herein is administered is a human child. In certain embodiments, the subject to which an IL-13Rα2 peptide-based vaccine or composition described herein is administered is a human fetus. In certain embodiments, the subject to which an IL-13Rα2 peptide-based vaccine or composition described herein is administered is a human infant.

[0161] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein is HLA-A2 positive, for example, as determined by flow cytometry.

[0162] In certain embodiments, subjects receiving the IL-13Rα2 peptide-based vaccines or compositions described herein have a Karnofsky performance status (KPS) of >60. KPS is used as a variable stratification and selection in randomized trials of chemotherapy drugs and ranges from 0 to 100. Patients with a score >60 are unable to work but are able to stay at home, require varying degrees of assistance, and are able to care for most of their personal needs. Patients with a score >70 are able to perform normal activities with effort but exhibit some signs and symptoms of illness. Patients with a score >80 are able to perform normal activities and exhibit only mild signs and symptoms of illness. Patients with a score >90 are normal, have no health complaints, and exhibit no signs or symptoms of illness.

[0163] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a white blood cell count of about 1000 / mm 3 , 1500 / mm 3 , 2000 / mm 3 , 2500 / mm 3 , 3000 / mm 3 or 3500 / mm 3 , or about 1000 / mm 3 -1500 / mm 3 , 1000 / mm 3 -2000 / mm 3 , 1500 / mm 3 -2500 / mm 3 , 1500 / mm 3 -3000 / mm 3 , 2000 / mm 3 -3500 / mm 3 or 2500 / mm 3 -3500 / mm 3 In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a white blood cell count of 2500 / mm 3 Greater than or equal to.

[0164] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a lymphocyte count of about 100 / mm 3 , 200 / mm 3 , 300 / mm 3 , 400 / mm 3 , 500 / mm 3 or 600 / mm 3 , or about 100 / mm 3 -400 / mm 3 , 200 / mm 3 -400 / mm 3 , 300 / mm 3 -500 / mm 3 , 300 / mm 3 -600 / mm 3 , 400 / mm 3 -500 / mm 3 , or 400 / mm 3 -600 / mm 3In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a lymphocyte count of 400 / mm 3 Greater than or equal to.

[0165] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a platelet count of about 25,000 / mm 3 , 50,000 / mm 3 , 75,000 / mm 3 , 100,000 / mm 3 , 200,000 / mm 3 , 300,000 / mm 3 , or approximately 25,000 / mm 3 -100,000 / mm 3 , 50,000 / mm 3 -100,000 / mm 3 , 75,000 / mm 3 -100,000 / mm 3 , 100,000 / mm 3 -200,000 / mm 3 , 100,000 / mm 3 -300,0 00 / mm 3 or 200,000 / mm 3 -300,000 / mm 3 In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a platelet count of 100,000 / mm 3 Greater than or equal to.

[0166] In specific embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a hemoglobin count of about 5 g / dL, 10 g / dL, 15 g / dL, or 20 g / dL, or about 5-10 g / dL, 5-15 g / dL, 10-15 g / dL, or 10-20 g / dL. In specific embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a hemoglobin count of greater than or equal to 10 g / dL.

[0167] In specific embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has AST, ALT, GGT, LDH, and alkaline phosphatase levels within 1, 1.5, 2, 2.5, or 3 times the upper limit of normal. In specific embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has AST, ALT, GGT, LDH, and alkaline phosphatase levels within 2.5 times the upper limit of normal.

[0168] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a total bilirubin of about 1 mg / dL, 1.5 mg / dL, 2 mg / dL, 2.5 mg / dL, or 3 mg / dL, or about 1.5-2.5 mg / dL, 1.5-3 mg / dL, 2-2.5 mg / dL, or 2-3 mg / dL. In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a total bilirubin of greater than or equal to 2 mg / dL.

[0169] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a serum quencher level within 0.5, 1, 1.5, 2, 2.5, or 3 times the upper limit of normal. In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has a serum quencher level within 1.5 times the upper limit of normal.

[0170] In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has coagulation tests PT and PTT within 0.5, 1, 1.5, 2, 2.5, or 3 times the upper limit of normal. In certain embodiments, a subject to be administered an IL-13Rα2 peptide-based vaccine or composition described herein has coagulation tests PT and PTT within the upper limit of normal.

[0171] In some embodiments, the IL-13Rα2 peptide-based vaccines or compositions described herein are administered to one or more of the following patient groups: elderly patients; infants 6 months of age or younger; pregnant women; infants 1 year of age or younger; children 2 years of age or younger; children 3 years of age or younger; children 4 years of age or younger; children 5 years of age or younger; adults 20 years of age or younger; adults 25 years of age or younger; adults 30 years of age or younger; adults 35 years of age or younger; adults 40 years of age or younger; adults 45 years of age or younger; adults 50 years of age or younger; elderly patients 70 years of age or older; elderly patients 80 years of age or older; elderly patients 85 years of age or older; elderly patients 90 years of age or older; elderly patients 95 years of age or older; subjects receiving chemotherapy; subjects receiving radiation therapy; subjects receiving biological therapy. subjects receiving interferon therapy; subjects receiving allergy desensitization injections; subjects receiving illicit drugs; subjects receiving growth factor (e.g., Procrit®, Aranesp®, Neulasta®) therapy; subjects receiving interleukin (e.g., Proleukin®) therapy; subjects with metastatic disease; lactating women; subjects with an active viral, bacterial, or fungal infection; subjects with a history of autoimmune disease; subjects with HIV; subjects being treated with an investigational drug, not including the vaccines described herein; neurological It is recommended that the drug not be administered to subjects with gliomatosis, cranial or spinal leptomeningeal metastatic disease; and / or subjects receiving immunosuppressive therapy.

[0172] 6.7.4 Combination therapy In certain embodiments, the methods provided herein for the prevention, treatment, and / or management of brain cancer comprise administering to a patient (e.g., a human patient) in need thereof a prophylactically and / or therapeutically effective regimen, wherein the patient is administered an IL-13Rα2 peptide-based vaccine or composition described herein and one or more additional therapies, where the additional therapies are not the IL-13Rα2 peptide-based vaccines or compositions described herein. The IL-13Rα2 peptide-based vaccines or compositions described herein and the additional therapies may be administered separately, simultaneously, or sequentially. The combined therapies may act additively or synergistically.

[0173] The combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the combination therapies can be administered to a subject simultaneously in separate pharmaceutical compositions. The combination therapies can be administered to a subject by the same or different routes of administration.

[0174] Any therapy (e.g., therapeutic or prophylactic agent) useful, used, or currently used in the prevention, treatment, and / or management of cancer (e.g., brain cancer) can be used in the methods described herein in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein. Therapies include, but are not limited to, peptides, polypeptides, antibodies, conjugates, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and / or organic molecules. Non-limiting examples of cancer therapies include chemotherapy, radiation therapy, hormone therapy, surgery, small molecule therapy, antiangiogenic therapy, differentiation therapy, epigenetic therapy, immunotherapy, targeted therapy, and / or biological therapy, including immunotherapy. In certain embodiments, the prophylactically and / or therapeutically effective regimens of the present invention comprise the administration of a combination of therapies.

[0175] In one embodiment, the previous chemotherapy is temozolomide. In an embodiment, the previous therapy is radiation therapy. In another embodiment, the previous therapy is a combination of temozolomide and radiation therapy. In a preferred embodiment, a combination of temozolomide and radiation therapy using the Stupp regimen is administered. In another embodiment, the previous therapy is surgical therapy. In some embodiments, the patient undergoes surgical therapy before the initiation of the combination therapy. In some embodiments, the patient undergoes surgical therapy before temozolomide. In some embodiments, the patient undergoes surgical therapy before the initiation of radiation therapy. In each of these embodiments describing the use of combination therapy, the IL-13Rα2 peptide-based vaccine may be administered before, during, or after the patient is treated with the combined therapy.

[0176] Examples of cancer treatments that may be used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein include acivicin; aclarubicin; acodazole hydrochloride; acronine; adzeresi Adozelesin; Aldesleukin; Altretamine; Ambomycin; Amethantrone acetate; Aminoglutethimine Aminoglutethimide; Amsacrine; Anastrozole; Anthracycline; Anthramycin; Asthma Paraginase; Asperlin; Azacitidine (vidaza); Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene hydrochloride; Bisnafide dimesylate; Bisphosphonates ( For example, pamidronate (Aredria), sodium clondronate (Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate, ib and / or nate, cimadronic acid, risedromate, and tiludromate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin Carboplatin; Carmustine; Carubicin hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Ciloremycin; Cisplatin; Cladribine; Crisnatol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dacarbazine; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Demethylating agents; Dexorubicin Platin (dexormaplatin); dezaguanine; dezaguanine ifesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomay duazomycin; edatrexate; eflornithine hydrochloride; EphA2 inhibitor; elsamitrucin; Enlop enloplatin; enpromate; epipropidine ); epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanida Etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; 5-fdump; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; histone deacetylase inhibitors (HDACs) gemcitabine hydrochloride Hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Ilmofosine; Meth Imatinib (Gleevec, Glivec); interleukin II (including recombinant interleukin II or RIL2), interferon alpha 2a; interferon alpha 2b; interferon alpha n1; interferon alpha n3; interferon beta i a; interferon gamma i b; iproplatin; irinotecan hydrochloride; lanreotide acetate; lenalidomide (Revlimid); letrozole; leuprolide acetate; liarozole liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine salts anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; WO 02 / 098370, all of which are incorporated by reference in their entireties)); megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitochromin; mitogillin; mitomalcin; mitomycin Itomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nokoda Nocodazole; Nogalamycin; Ormaplatin ); oxaliplatin; Oxisuran; Paclitaxel; Pegaspargase; Periomycin; Pentamastine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium ;Porfiromycin;Prednimustine;Proca Procarbazine hydrochloride; Puromycin; Pi Puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol ;Safingol hydrochloride;Semustine;Shim Simtrazene; Sparfosate sodium; Spar Sparsomycin; spirogermanium hydrochloride );spiromustine;spiroplatin;strepton Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan sodium; Tegafur; teloxantrone hydrochloride; temo temoporfin; teniposide; teroxirone ;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin;Tirapazamine;Toremifene citrate;Trestolone acetate;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubulozole hydrochloride;U Uracil mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate ); Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vindesine These include, but are not limited to, vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin hydrochloride.

[0177] Examples of other cancer treatments that may be used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein include 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; and adecipenoside. adecypenol; adozelesin; aldesleukin ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; ana Anagrelide; anastrozole; andrographolide; angiogenesis inhibitor; antagonist D; antagonist G; antarelli antarelix; anti-dorsalizing morphogenetic protein-1; anti-androgens, anti-prostate cancer agents; anti-estrogens; anti-neoplastic agents; antisense oligonucleotides; aphidicolin glycinate; Apoptosis gene modulator; Apoptosis regulator; Apurinic acid ); ara-CDP-DL-PTBA (ara-CDP-DL-PTBA); arginine deaminase (arginine deaminase); asulacrine (asulacrine); atamestane (atamestane); Atrimustine; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azasetron; Azatoxin; Azatyrosine; Baccatin III Inducer Baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; β-betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; b Bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; Capecitabine; Carboxamide-amino-triazole; Carboxamidotriazole; CaRestM3; CARN 700; Cartilage-derived inhibitor; Carze Carzelesin; Casein kinase inhibitor (ICOS); Castanospermine; Cecropin B; Cetrorelix; Chloride Chlorins; chloroquinoxaline sulfonamide ; cicaprost; cis-porphyrin; cladribine; clomiphene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogues; conagenin; crambecidin 816; crisnatol; cryptophycin 8; crimson Cryptophycin A derivative; Curacin A; Cyclopentasiloxane cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolysis Factors; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; Dexamethasone; dexifosfamide; dexrazoxane; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didem didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dioxamycin Samycin (dioxamycin); Diphenyl spiromustine (diphenyl spiromustine); Cetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen ); ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefa Epirubicin; Epristeride; Estrogen agonist; estramustine analogue; Genitourinary antagonists; etanidazole; etoposide phosphate phosphate); exemestane; fadrozole; Faza Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Flezelastine; Fluasterone; Fludarabine; Fluorodaunorunicin hydrochloride; Forfenimex; Formestane; Fostriecin; Fotemustine; Gadolinium texaphyrin; Gallium nitrate; Galocitabine; Ganire Ganirex; gelatinase inhibitors; gemcitabine; glutathione inhibitors; HMG-CoA reductase inhibitors (e.g., atorvastatin) cin; cerivastatin; fluvastatin; lescol; lupitor; lovastatin; rosuvastatin simvastatin; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitors; interferon Interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; 4-Ilop 4-Iroplac (4-iroplact); Irsogladine; Isobengazole; Isohomohalichondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellar lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; Leukemia inhibitory factor; leukocyte alpha-interferon; leuprolide + estrogen + pro Gesterone; leuprorelin; levamisole; LFA-3TIP (Biogen, Cambridge, MA; WO 93 / 0686 and U.S. Pat. No. 6,162,432); liarozole; linear polyamine analogues; Lipophilic disaccharide peptides; lipid-soluble platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; cytolytic peptides; maytansine; mannostatin; marimastat; masoprocol; maspin; matrilysin inhibitors Matrilysin inhibitors; matrix metalloproteinase inhibitors; Menogalli Menogaril; Merbarone; Meterelin; Methionin Methioninase; Metoclopramide; MIF inhibitor; Mifepristone; Miltefosine; Mirimostim ); mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; Molgramostim; monoclonal antibody; human chorionic gonadotropin; monophosphoryl lipid A + myobacterial cell wall sk; mopidamol; poly Drug resistance gene inhibitors; multiple tumor suppressor 1-based therapy; mustard anticancer drugs; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline (N-acetyldinaline); N-substituted benzamide; Nafarelin; Nagress Tip (nagrestip); naloxone + pentazocine (naloxone + pentazocine); Napa Napavin; Naphterpin; Nartograstim; Nedaplatin; Nemorubicin; Neridronic acid; Neutral endopeptidase; Nilutamide; Nisamycin; Nitric oxide modulators; Nitroxide antioxidants; Nitrullin; O6-benzylguanine; Octreotide; Oxenone; Oligonucleotides; Onapristone; Ondansetron; Oracin; Oral cytokine inducer; Oral cytokine inducer Oxaliplatin (oxaliplatin); Oxaunomycin (oxaunomycin); Paclitaxel (paclitaxel); Paclitaxel paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; Pamidronic acid; Panaxytriol; Panomifene; Parabactin; Pazelliptine; Pegaspargase; Peldesine; Pentosan polysulfate sodium Pentosan polysulfate sodium Pentostatin; Pentrozole; Perflubron perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase Inhibitors; Picibanil; Pilocarpine hydrochloride; Pirarubicin; Piritrexim; Placetin A; Placetin B; Plasminogen activator inhibitors; Platinum complexes (platinum complex); platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulators; protein kinase C inhibitors; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyro Pyridoxylated hemoglobin polyoxyethylene conjugate ;raf antagonist;raltitrexed;ramosetron;RAS farnesyl protein transferase inhibitor;ras inhibitor;ras-GAP inhibitor;demethylated leterlipid;rhenium Re 186 etidronate;rhizoxin; Ribozyme; RII retinamide; rogletimide; Rohitukine; Romurtide; Roquinimex ; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen binding proteins; sizofiran; sobuzoxane; borocaptan nate sodium borocaptate; sodium phenylacetate; sorberol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stem cell inhibitor Stipiamide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal peptide antagonist; sladis Suradista; Suramin; Swainsonin; Synthetic glycosaminoglycan; Tallimustine; 5-fluorouracil; Leucovorin; Tamoxifen methiodide ); Tauromustine; Tazarotene; Tecogalan sodium; Tegafur; Tellurapyrylium; Telomerase inhibitors; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Talicarpine; Thiocoraline; Thrombopoietin; Thrombopoietin mimics; Thymalfasin; Thymopoietin Thyrotropin receptor agonist; Thymotrin; Thyroid-stimulating hormone; Ethyl etiopurpurin tin; Tirapazamine; Titanocene dichloride; Topsentin; Toremifene; Totipotent stem cell factor ;Translation inhibitors;Tretinoin;Triacetyluridine;Triciribine;Tri;Triptorelin;Tropisetron;Turosteride;Tyrosine kinase inhibitors;Tyrphostin;UBC inhibitors Antineoplastic agents; Ubenimex; Urogenital sinus-derived growth inhibitor; Urokinase receptor antagonist; Vapreotide; Variolin B; Vector systems, red blood cell gene therapy; Thalidomide; Verareso velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin TM (See U.S. Patent Application Publication No. 2002 / 0168360 A1, published November 14, 2002, titled "Treating Inflammatory or Autoimmune Disorders by Administering Integrin αvβ3 Antagonists in Combination With Other Prophylactic or Therapeutic Agents"); Vorozole; Zanoterone; Zenipla Examples of antihistamines that may be used include, but are not limited to, zeniplatin; zilascorb; zinostatin stimalamer.

[0178] In some embodiments, a therapy used in combination with an IL-13Rα2 peptide-based vaccine or composition described herein is an immunomodulatory agent. Non-limiting examples of immunomodulatory agents used in combination with an IL-13Rα2 peptide-based vaccine or composition described herein include, for example, cytokines, peptidomimetics, and proteinaceous reagents of antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or epitope-binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules and triple helices), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulatory agents include methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine, minocycline, azathioprine, antibiotics, and the like. Substances (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin ( Examples of immunomodulatory agents include, but are not limited to, sirolimus, mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), T cell receptor modulators, cytokine receptor modulators, and mast cell modulators. Other examples of immunomodulatory agents are disclosed, for example, in U.S. Patent Application Publication No. 2005 / 0002934, paragraphs 259-275, which include, Both are incorporated herein by reference in their entireties. In one embodiment, the immunomodulatory agent is a chemotherapeutic agent. In alternative embodiments, the immunomodulatory agent is an immunomodulatory agent other than a chemotherapeutic agent. In some embodiments, the therapy used in accordance with the present invention is not an immunomodulatory agent.

[0179] In some embodiments, the therapy used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein is an anti-angiogenic agent. Non-limiting examples of anti-angiogenic agents that can be used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein include proteins, polypeptides, peptides, conjugates, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or antigen-binding fragments thereof), antibodies that specifically bind to TNF-α, nucleic acid molecules (e.g., antisense nucleic acid molecules and triple helices), organic molecules, inorganic molecules, and the like. Antiangiogenic agents include small molecules that reduce or inhibit angiogenesis. Other examples of antiangiogenic agents are disclosed, for example, in U.S. Patent Application Publication No. 2005 / 0002934, paragraphs 277-282, all of which are incorporated herein by reference. In a preferred embodiment, the anti-angiogenic therapy is bevacizumab (Avastin®). In another embodiment, the anti-angiogenic therapy according to the present invention is The therapy used is not anti-angiogenic.

[0180] In some embodiments, a therapy used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein is an anti-inflammatory agent. Non-limiting examples of anti-inflammatory agents used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein include any anti-inflammatory agent, including agents useful in the treatment of inflammatory diseases known to those of skill in the art. Non-limiting examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory agents, anticholinergics (e.g., atropine sulfate, atropine methyl nitrate, and ipratropium bromide (ATROVENT)), and the like. TM )), β2-agonists (e.g., abuterol (VENTOLIN) TM and PROVENTIL TM ), Bitolterol (TORNALATE TM ), levalbuterol (XOPONEX TM ), metaproterenol (ALUPENTTM ), Pirbuterol (MAXAIR TM ), terbutlaine (BRETHAIRE TM and BRETHINE TM ) , albuterol (PROVENTIL TM , REPETABS TM and VOLMAX TM ), formoterol (FORADIL AEROLIZER TM ), and salmeterol (SEREVENT TM and SEREVENT DISKUS TM ) and methyl Xanthines (e.g., theophylline (UNIPHYL TM , THEO-DUR TM , SLO-BID TM , and TEHO-42 TM Examples of NSAIDs are aspirin, ibuprofen, and celecoxib. Kisib (CELEBREX TM ), diclofenac (VOLTAREN TM ), Etodrak (LODINE TM ), fenoprofen (NALFON TM ), indomethacin (INDOCIN TM ), ketorolac (TORADOL TM ), Oxaprozin (DAYPRO TM ), nabumentone (RELAFEN TM ), sulindac (CLINORIL TM ), Tolmentin (TOLECTIN TM ), rofecoxib (VIOXX TM ), Naprox Sen (ALEVE TM , NAPROSYN TM ), ketoprofen (ACTRON TM ) and nabumetone (RELAFEN TMNSAIDs function by inhibiting cyclooxygenase enzymes (e.g., COX-1 and / or COX-2). Examples of steroidal anti-inflammatory agents include, but are not limited to, glucocorticoids, dexamethasone (DECADRON), and steroids. TM ), corticosteroids (e.g., methylprednisolone (MEDROL TM )), cortisone, hydrocortisone (hydrocortisone), prednisone TM and DELTASONE TM ), Prednisolone TM and PEDIAPRED TM Other examples of anti-inflammatory agents include, but are not limited to, benzodiazepines ... The bodies of which are incorporated herein by reference. In other embodiments, the therapy used in accordance with the present invention is not anti-angiogenic.

[0181] In certain embodiments, therapies used in combination with the IL-13Rα2 peptide-based vaccines or compositions described herein are alkylating agents, nitrosoureas, antimetabolites, and anthracyclines, topoisomerase II inhibitors, or mitotic inhibitors. Alkylating agents include, but are not limited to, busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, vasodilator, mechlorethamine, melphalan, and temozolomide. Nitrosoureas include, but are not limited to, carmustine (BCNU) and lomustine (CCNU). Antimetabolites include, but are not limited to, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine, and fludarabine. Anthracyclines include, but are not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone. Topoisomerase II inhibitors include, but are not limited to, topotecan, irinotecan, etoposide (VP-16), and teniposide. Mitotic inhibitors include, but are not limited to, taxanes (paclitaxel, docetaxel) and vinca alkaloids (vinblastine, vincristine, and vinorelbine).

[0182] Currently available cancer therapies and dosages, routes of administration, and recommended uses are known in the art and described in such publications as the Physician's Desk Reference (60th ed., 2006). In accordance with the present invention, the dosage and frequency of administration of chemotherapeutic agents are as described above.

[0183] 6.7.5 Biological Assays The IL-13Rα2 peptide-based vaccines and compositions described herein can be tested for their ability to treat, prevent, or manage brain cancer.

[0184] 6.7.5.1 In Vivo Assays The IL-13Rα2 peptide-based vaccines and components described herein can be tested in suitable animal model systems before use in humans. Such animal model systems include, but are not limited to, rats, mice, chickens, cattle, monkeys, pigs, dogs, rabbits, etc. Any animal model known in the art can be used. Several aspects of the procedure can be varied, including, but not limited to, the temporal regime of administering the vaccine components, whether the vaccine compositions are administered separately or as a mixture, and the frequency of administration of the vaccine components.

[0185] Animal cancer models can be used to evaluate the efficacy of the IL-13Rα2 peptide-based vaccines or compositions described herein, or the combination therapies described herein. Exemplary animal models for brain cancer include, but are not limited to, xenograft studies using IL-13Rα2-expressing brain cancer cell lines or IL-13Rα2-expressing primary human tumor cells. In these models, mice are immunized to induce an IL-13Rα2-specific T cell response and assessed for its ability to inhibit tumor growth. In one embodiment, prior to immunization, the tumor xenograft forms a test for the ability of the IL-13Rα2-specific T cell response to inhibit the growth of an existing tumor. In another embodiment, an IL-13Rα2-specific T cell response is induced prior to tumor cell injection to evaluate the ability of the immune response to prevent tumor formation.

[0186] 6.7.5.2 Cytotoxicity analysis The toxicity and / or efficacy of the IL-13Rα2 peptide-based vaccines and compositions described herein can be determined in cell cultures or experimental animals using standard pharmaceutical methods, such as determining the LD50 (lethal dose in 50% of the population) and ED50 (therapeutically effective dose in 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Treatment regimens that exhibit large therapeutic indices are preferred. While treatment regimens that exhibit toxic side effects are acceptable, care should be taken to design a delivery system that targets the reagent to affected tissues to minimize potential damage to uninfected cells and reduce side effects.

[0187] 6.8 Products The present invention also includes packaged and labeled pharmaceutical products. The products may be packaged in suitable unit doses in a suitable vessel or container, such as a sealed glass vial or other container. Pharmaceutical products may contain, for example, the components of the IL-13Rα2 peptide-based vaccine described herein in unit dosage forms.

[0188] In certain embodiments, the unit dosage form is suitable for oral, intravenous, intramuscular, intranasal, or subcutaneous delivery. Thus, encompassed herein are solutions, preferably sterile, suitable for each delivery route.

[0189] The packaging materials and containers for any pharmaceutical product protect the stability of the product during storage and shipping. Additionally, the articles of manufacture provided herein include instructions for use or other informational material that advises a physician, technician, or patient on how to properly prevent or treat the brain cancer in question. In other words, the articles of manufacture include instructional means that indicate or suggest a dosing regimen, including, but not limited to, actual dosages, monitoring procedures, and other information.

[0190] Specifically, provided herein are articles of manufacture that include packaging material, such as, for example, a box, bottle, tube, vial, container, sprayer, syringe, intravenous (iv) bag, or medicine pouch, wherein at least one unit dose form of a vaccine or pharmaceutical composition described herein contained within the packaging material includes instructions that the vaccine or pharmaceutical composition described herein comprises an IL-13Rα2 peptide-based vaccine described herein, and that the packaging material comprises an IL-13Rα2 peptide-based vaccine described herein that can be administered in a specific dosage and using a specific dosing regimen described herein to prevent, manage, and / or treat brain cancer or one or more symptoms thereof.

[0191] 7. Working Example The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0192] 7.1 Example 1 This example shows the effect of natural IL-13Rα2 345-353 IL-13Rα2 enhances the induction of CTL responses against 345-353 The modified peptide identification is shown.

[0193] Three modified peptides shown in Table 1 were synthesized. The binding ability of these modified peptides was evaluated using an HLA-A2 transfected T2 cell line. Aliquots of T2 cells were treated with 1 nM of the modified peptide or IL-13Rα2. 345-353 The cells were cultured overnight with the peptide epitope and the cell surface HLA-A2 expression level on T2 cells was examined by flow cytometry. Stable binding of HLA-A2 to the peptide epitope further stabilizes the surface expression of HLA-A2 (Francini et al., 2002; Alves et al., 2003). The mean fluorescence intensity (MFI) in Table 1 shows the quantitative expression level of HLA-A2, which correlates with the binding affinity of the peptide epitope co-cultured with T2 cells. The modified peptides V9 and A1V9 bind to the natural IL-13Rα2. 345-353These modified peptides have higher affinity for HLA-A2 compared to IL-13Rα2 (Table 1). 345-353 It may be more immunogenic than

[0194] [Table 1]

[0195] 7.2 Example 2 This example shows that CTLs induced by V9 analog agonists bind to HLA-A more efficiently than CTLs induced by wild-type peptides. * Peptide IL-13Rα2 presented on 0201 345-353 indicates that the user has recognized the

[0196] HLA-A * Dendritic cells (DCs) from O201+ glioma patients were pulsed with V9, A1V9, E1V9, control influenza (flu), or wild-type peptide (10 μg / mL), respectively, and autologous CD8 + On day 7, individual responder cell cultures were restimulated once with autologous dendritic cells loaded with the corresponding peptide used in the primary stimulation. On day 10, the specific CTL reactivity of the induced T cell lines was analyzed using wild-type IL-13Rα2 345-353 Alternatively, T2 cells loaded with no peptide were tested first.

[0197] As shown in Figure 1, T cells stimulated with each wild-type (IL-13R) or agonist analog (V9, A1V9, and E1V9) showed a significant increase in IL-13Rα2 at 100 ng / ml. 345-353 The agonist analog-induced CTL lines efficiently lysed wild-type IL-13Rα2 target cells pulsed with flu-peptide. In contrast, only low background lysis was observed on T2 cells lacking the peptide. T cells stimulated with control flu-peptide or no peptide (control) did not show any lytic activity above background levels. The results indicate that wild-type or agonist analog-induced CTL lines were able to lyse wild-type IL-13Rα2 target cells. 345-353In particular, the V9 peptide specifically recognized and lysed wild-type IL-13Rα2 at various effector / target (E / T) ratios (p=0.018, 0.020, and 0.011, respectively, for E / T ratios of 50, 25, and 12.5). 345-353 The same set of experiments was performed with at least three individual HLA-A2 + Repeatedly in glioma patients, V9 peptide inhibited natural IL-13Rα2 in all four donors tested. 345-353 The CTL reactivity was consistently higher than that of the control group (data not shown).

[0198] The sensitivity of the CTL cell lines induced with the agonist analogs or wild-type peptide was then assessed by 4-hour incubation. 51 IL-13Rα2 at various concentrations (1-100 nM) was detected in a Cr-release assay. 345-353 We investigated the effect of peptide-loaded T2 cells (Fig. 2). All CTL cell lines showed peptide dose-dependent lytic activity against peptide-loaded T2 cells. At all concentrations tested (P = 0.029, 0.039, and 0.018, 1, 10, and 100 nM, respectively), CTL lines induced with agonist analog V9 inhibited wild-type IL-13Rα2 345-353 However, due to the large standard deviation, the difference was not statistically significant. 345-353 It is noteworthy that the mean percent lysis achieved by V9-induced CTLs at 100 nM peptide was higher than that shown by wild-type peptide-induced CTLs at 100 nM peptide. However, due to the large standard deviation, the difference was not statistically significant. The results were consistent with those obtained at low concentrations of target wild-type IL-13Rα2. 345-353 We show that the V9 peptide is more effective than the wild-type peptide in inducing CTLs capable of recognizing the peptide. This is because human tumor cells express only low levels of target CTL epitopes on HLA-molecules (Bakker et al., 1995; Lupetti et al., 2000). et al., 1998), this ability is important.

[0199] 7.3 Example 3 This example demonstrates that CTLs induced by modified peptides express IL-13Rα2 more efficiently than CTLs induced by native peptides. + This indicates that the glioma was dissolved.

[0200] HLA-A2 endogenously expresses and presents IL-13Rα2-derived epitopes + We investigated the ability of modified peptides, such as IL-13Rα2-V9, to enhance CTL activity against human glioma cells. The human glioma cell lines U251 and SNB19 express HLA-A2 and IL-13Rα2, whereas the human glioma cell line A172 expresses IL-13Rα2 but not HLA-A2 (Okano et al., 2002). Therefore, U251 and SNB19 were used as relative target glioma cells, and A172 was used as a negative control to demonstrate the HLA-A2 restriction of the response.

[0201] The lytic ability of peptide-induced CTL lines against these glioma cells was examined for 4 hours. 51 As shown in Figure 3, U-251 and SNB19 cell lines expressed IL-13Rα2. 345-353 The modified peptides showed high reactivity to the cytotoxic activity of all CTL lines induced with the modified peptides. In contrast, A172 cells were not lysed above background levels (<10%) by any of the CTL lines tested, indicating that IL-13Rα2 345-353 These results suggest that the modified peptide-induced CTL line lysed SNB19 and U-251 glioma cells in an HLA-A2-restricted manner (data not shown). 27-35 T cells stimulated with and without peptide showed background levels of lysis (<10%) at all effector / target (E / T) ratios tested. In this particular patient, both IL-13Rα2-V9 and -A1V9 suppressed the natural IL-13Rα2 345-353Compared with the peptide, each E / T ratio induced higher levels of lysis of SNB19 and U-251.

[0202] To determine the specificity of the lytic activity, 51 In the Cr-release assay, IL-13Rα2 345-353 Cold target competition experiments were performed by adding non-radiolabeled (cold) T2 cells pulsed with the peptide (Figure 4). 345-353 The anti-SNB19 glioma cell lytic activity of the CTL line induced by IL-13Rα2-V9 was also confirmed by IL-13Rα2 345-353 However, the CTL activity was not inhibited by the addition of non-peptide-pulsed cold T2 cells, suggesting that the lytic activity of CTLs was mediated by the epitope IL-13Rα2. 345-353 This indicates that it was specific.

[0203] Furthermore, to block HLA-A2-mediated signaling in CTL reactivity, anti-HLA-A2 antibody (W6 / 32) was used. As shown in Figure 5, the addition of this antibody inhibited CTL-mediated lysis and suppressed the anti-glioma CTL induced by these peptides. TL reactivity was confirmed to be HLA-A2 restricted.

[0204] 7.4 Example 4 This example demonstrates vaccination of HLA-A2 transgenic (HHD) mice with IL-13Rα2-derived CTL epitopes.

[0205] IL-13Rα2 345-353 and / or its modified peptides can induce CTL responses in vivo, and whether the induced CTL responses are related to IL-13Rα2 345-353 To investigate whether IFN-γ-glucan can mediate therapeutic antitumor responses in brain tumors expressing IFN-γ-glucan, we received a grant from Dr. Francois A. Lemonnier (Pasteur Institute, Paris) for the treatment of H We received HD mice. HHD mice lack Db×β2 microglobulin (β2M) (null) and express modified HLA-A2.1-β2 microglobulin single chain (HHD). In vivo studies have shown that HHD mice display HLA-A2-restricted responses to multiple epitopes, including intact influenza virus (Pascolo et al., 1997), novel cancer-associated antigens such as EphA2 (Alves et al., 2003), HER-2 / neu and hTERT (Scardino et al., 2002), MAGE (Graff-Dubois et al., 2002), and novel breast cancer-associated BA46 (Carmon et al., 2002). Therefore, these mice are useful tools for identifying and characterizing potential tumor-derived HLA-A2-restricted CTL epitopes.

[0206] To generate a syngeneic tumor cell line expressing IL-13Rα2 in HHD mice, we obtained HHD gene-transfected EL4 lymphoma cells (EL4-HHD). EL4-HHD cells were generated by deleting Dbxβ2M and inserting a modified HLA-A2.1-β2M single chain (Pascolo et al., 1997), thereby enabling syngeneic transplantation in HHD mice. EL4-HHD cells were stably transfected with an expression plasmid encoding IL-13Rα2. The cell line (EL4-HHD-IL-13Rα2) expressed the IL-13Rα2 protein and formed tumors in the subcutaneous (sc) and intracranial (ic) spaces after injection into syngeneic HHD mice.

[0207] 7.5 Example 5 This example demonstrates that in vivo immunization of HHD mice with modified peptides enhances IL-13Rα2 345-353 The results show that the peptide induced a higher CTL response against target cells expressing the peptide than the native peptide.

[0208] HHD mice were treated with CD4 +Stimulating helper T cell responses + IA promotes CTL stimulation b -Restricted HBV core 128-140 100 μg of IL-13Rα2-V9, -A1V9, IL-13Rα2 (TPPAYRPPNAPIL) (SEQ ID NO: 5) emulsified in incomplete Freund's adjuvant (IFA) in the presence of 140 μg of T-helper epitope 345-353 The peptides were injected subcutaneously (sc) (on days 7 and 14). Control animals were injected with IFA containing only HBV helper peptides. Seven days after the last immunization, these animals were sacrificed and challenged with 5 × 10 HBV helper peptides (10 μM) of the same peptides used for in vivo stimulation. 6 Spleen cells (SPCs) were stimulated in vitro. On day 6 of culture, the mixed population was stimulated with IL-13Rα2. 345-353 The specific cytotoxicity of IL-13Rα2- or EL4-HHD-expressing EL4-HHD cells pulsed with IL-13Rα2 was tested.

[0209] EL4-HHD-IL-13Rα2 and EL4-HHD were administered at 100 μCi 51 Label with Cr for 60 min and plate in a 96-well V-bottom plate (3 × 10 3 The labeled EL4-HHD was plated onto IL-13Rα2 345-353 Control target cells were pulsed with peptide (1 μM) for 2 hours at 37°C. Control target cells were not pulsed with peptide. Stimulated splenocytes were The cells were added as effector cells and cultured for 4 hours at 37° C. 100 μL of the supernatant was collected and the radioactivity was measured using a gamma counter.

[0210] Figure 6 shows that the CTL responses induced by the modified peptides were significantly higher than those induced by the native IL-13Rα 345-353 The control, non-pulsed EL4-HHD was not lysed by CTLs above background levels (Figure 7). Furthermore, immunization with IL-13Rα2-V9 significantly reduced the cytotoxicity of native IL-13Rα2 compared to the other peptides tested. 345-353There was a tendency for the level of CTL reactivity to peptide-pulsed EL4-HHD cells to be higher, but this difference was not statistically significant due to variation within triplicate samples. These data suggest that the modified peptides were more potent than the native peptides in anti-IL-13Rα2 activity. 345-353 Human HLA-A2 induced higher levels of CTL + This confirms previous data on patient-derived T cells.

[0211] The ability of CTLs from the same HHD mice used in Figure 6 to lyse EL4-HHD-IL-13Rα2 cells was compared with that of IL-13Rα2 naturally processed by cells that endogenously express IL-13Rα2. 345-353 The ability of CTLs to recognize the peptide was evaluated. 345-353 These results show that immunization with IL-13Rα2-V9 or -A1V9 induced specific CTL reactivity against EL4-HHD-IL-13Rα2 cells. The control EL4-HHD cells were not lysed above background levels, so the CTL reactivity was antigen-specific. The modified peptides IL-13Rα2-V9 and -A1V9 inhibited the natural IL-13Rα2 response against EL4-HHD-IL-13Rα cells. 345-353 IL-13Rα2 in HHD mice bearing EL4-HHD-IL-13Rα2 tumors. 345-353 The in vivo antitumor effects of vaccination with modified IL-13Rα2 peptides are currently being evaluated.

[0212] 7.6 Example 6 This example demonstrates that EphA2 can be used as an HLA-A2-restricted CTL epitope.

[0213] As five HLA-A2 and three DR4 T cell epitopes have been previously identified (Tatsumi et al., 2003), EphA2 is an attractive tumor-associated antigen and a target for tumor vaccines. As shown in Figure 8, 9 of 14 human glioblastoma multiforme (GBM) cases and 6 of 9 anaplastic astrocytoma (AA) cases express high levels of EphA2. In addition, EphA2 883-891 Epitope-stimulated HLA-A2 + CD8 obtained from glioma patients + Anti-glioma CTL reactivity was induced in the cells (Figure 9). 883-891 Parallel assays using T2 cells loaded with EphA2 demonstrated a peptide-specific response compared with control unloaded T2 targets, suggesting that this response was due to EphA2 883-891 The data were epitope-specific (not shown). 883-891 This strongly suggests that β-glucan may serve as a CTL epitope.

[0214] 7.7 Example 7 In this example, human leukocyte antigen (HLA)-A2 + We describe a phase I / II study conducted to evaluate the safety and immunogenicity of vaccination with novel α1-polarized dendritic cells (αDC1) loaded with synthetic peptides directed against glioma-associated antigen (GAA) epitopes and poly-ICLC administration in patients with recurrent malignant glioma. The GAA epitopes for these peptides are EphA2, interleukin-13 receptor (IL-13R) α2, YKL-40, and gp100.

[0215] 7.7.1 Patients and Methods 7.7.1.1 Patient Patients with recurrent malignant glioma were enrolled after obtaining informed consent and approval from the Institutional Review Board (IRB) and the U.S. Food and Drug Administration (FDA) (BB-IND#12415). Patient clinical characteristics are summarized in Table 2 and Table 3A. Enrollment criteria included glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA); anaplastic glioma (AG), including anaplastic oligodendroglioma (AO) or anaplastic oligoastrocytoma (AOA); up to two previous recurrences; age >18 years; Karnofsky performance status >60; adequate liver function, renal function, and HLA-A2 status. + The lowest dose of corticosteroids (maximum 4 mg / day of dexamethasone) was tolerated. 22 patients were enrolled and received at least one vaccination. 19 of the 22 patients completed the first four scheduled immunizations; three patients (patients 4, 11, and 13) withdrew from the protocol due to early tumor progression. Nine patients completed the booster vaccination. Immunogenicity and safety data are presented for patients who received at least four vaccinations (n=19) and at least one vaccination (n=22).

[0216] [Table 2]

[0217] [Table 3A-1]

[0218] [Table 3A-2] [Table 3A-3]

[0219] [Table 3B-1]

[0220] [Table 3B-2]

[0221] 7.7.1.2 Clinical Trial Design This study was designed to evaluate the toxicity of GAA-loaded αDC1 vaccination and poly-ICLC (Hiltonol®, Oncovir, Inc.) administration, as well as immune induction and preliminary clinical responses. The initial vaccine course was administered every 2 weeks with 1 or 3 × 10 vaccinated mice, with rotation of each inguinal and axillary lymph node cluster to minimize the potential impact of injection-induced trauma on the lymph node microenvironment due to repeated injections within a short period of time. 7 The first 10 evaluable patients received 1 x 10 7 αDC1 / injection (dose level 1), and then 9 received 3 × 10 7 All patients received poly-ICLC (20 μg / kg) intramuscular (im) injections twice a week (twice / week) for 8 weeks from day 1 onwards. After four vaccinations, patients who showed stable disease or regression of disease without major adverse events (AD) were considered for further vaccination. Starting at week 13, these patients received vaccine injections and intramuscular poly-ICLC injections in the same dose as the booster vaccinations every four weeks for up to five times, twice a week starting with the first booster vaccination (first booster phase). After the first booster phase, patients without major adverse events or tumor progression received the same dose of booster vaccine (every three months) and poly-ICLC (each week) for up to three years from the first vaccination (second booster phase).

[0222] 7.7.1.3 Toxicity assessment and stopping rules The study was continuously monitored for treatment-related adverse events (AEs) using the National Cancer Institute Common Toxicity Criteria version 3.0. The following were considered dose-limiting toxicities (DLTs) if they were judged to be probably, probably, or definitely related to treatment: ≥ Grade 2 hypersensitivity; ≥ Grade 3 nonhematologic / metabolic toxicity; and ≥ Grade 3 hematologic (excluding lymphopenia) or metabolic toxicity that persisted 4 weeks after temporary discontinuation of poly-ICLC. Stopping rules were implemented if the DLT rate observed at any time point was ≥ 33% ​​and at least two DLTs had been observed, such that the dose level was deemed overly toxic and warranted stopping its accrual.

[0223] 7.7.1.4 Peptides The HLA-A2 restricted peptide used in this study was: ALPFGFILV (SEQ ID NO: 3; IL-13Rα2 345-353 :1A9V); TLADFDPRV (SEQ ID NO:6; EphA2 883-891 );IMDQVPFSV(SEQ ID NO:11;GP100 209-217 :M2); and SIMTYDFHGA (SEQ ID NO:10; YKL-40 20 1-210 ) αDC1 was also loaded with the pan-DR epitope (PADRE), a non-natural epitope optimized for helper T cell responses (see, e.g., Alexander et al., Immunity, 1:751-761, 1994). Peptides were synthesized by automated solid-phase peptide synthesis. Peptides were tested for numerous quality assurance checks, including purity, sterility, identity, potency, pyrogenicity, and stability.

[0224] 7.7.1.5 Vaccine Preparation For dendritic cell culture, monocytes were obtained from leukapheresis transfusion products and purified using the Elutra™ system. Using the Aastrom RepliCell system, monocytes were cultured in sterile cartridges in CellGenix antibiotic-free medium supplemented with 1000 U / mL GM-CSF and 1000 U / mL IL-4. Immature dendritic cells (iDCs) were harvested on day 6 and cryopreserved. Before each vaccination, frozen iDCs were thawed and further matured and polarized with clinical-grade IL-1β (10 ng / mL), TNF-α (10 ng / mL), IFN-α (3000 U / mL), IFN-γ (1000 U / mL), and poly-I:C (20 μg / mL) for 48 hours at 37°C in 5% CO2, and then loaded with GAA peptide for 4–6 hours. PADRE peptide was added to the culture medium 2 hours before harvest. The criteria for αDC1 release were sterility on Gram staining and bacterial culture; mycoplasma negativity; endotoxin <5.0 EU / kg body weight; and 70% or more expression of both CD86 and HLA-DR on αDC1.

[0225] 7.7.1.6 Peripheral Blood (PBMS) Collection Peripheral blood (50-60 ml) was collected at weeks 0, 9, and 33, as well as at each visit for vaccination (pre-vaccination). Ficoll-isolated PBMS were cryopreserved in 10% dimethyl sulfoxide / 90% FBS.

[0226] 7.7.1.7 ELISPOT Assay Enzyme-linked immunosorbent spot (ELISPOT) assays were performed as previously described (see, e.g., Kirkwood et al., Clin. Cancer Res., 15:1443-1451, 2009) with minor modifications. Briefly, wild-type IL-13Rα 345-353 , EphA2 883-891 , GP100 209-217 and YKL-40 202-211Batch PBMC samples were assessed simultaneously following in vitro stimulation with autologous irradiated PBMCs loaded for 1 week with IgG. A positive ELISPOT response was defined as a 2-fold increase in spot-forming T cells relative to prevaccination levels and at least 10 spots / 20,000 cells at at least two consecutive postvaccination time points to any antigen.

[0227] 7.7.1.8 Tetramer Assay Phycoerythrin (PE)-conjugated HLA-A * 0201 / ALPFGFILV (SEQ ID NO: 3) (IL-13Rα2-tetramer), HLA-A * 0201 / IMDQVPFSV (SEQ ID NO: 11) (gp100-tetramer) and HLA-A * 0201 / TLADFDPRV (SEQ ID NO: 6) (EphA2-tetramer) was produced at the Emory University Vaccine Center (Atlanta, GA) using peptides synthesized at the University of Pittsburgh Peptide Manufacturing Facility. Tetramer antibodies were produced at the National Institute of Allergy and Infectious Disease tetramer facility at the University of California, San Diego, CA. Fluorescein isothiocyanate (FITC)-conjugated anti-human CD8 was obtained from BD Biosciences. Total CD8 antibodies were measured by tetramer assay (see, e.g., Weber et al., J Immunother., 31:215-23, 2008; and Celis, Cancer, 110:203-14, 2007). + (0.1+B)% of cells were positive A positive response at one time point for a given peptide could be defined as B, where B is the percent positive at baseline, which was less than 0.01% in all cases. A patient was considered to have responded if he / she showed two consecutive single-time point responses to any peptide.

[0228] 7.7.1.9 Cytokine and Chemokine Assays Total RNA samples were obtained from PBMCs using the PAXgene Blood RNA System (PreAnalytix, Switzerland). RT-PCR was performed in triplicate, values ​​were normalized to GAPDH, and relative mRNA expression was calculated using the ΔΔC method (see, e.g., Livak and Schmittgen, Methods, 25:402-8, 2001). As previously described (see, e.g., Zczepanski et al., Cancer Res., 69:3105-3113, 2009), A Luminex-based assay was performed on serum samples. Pre-tested multiplex plates (Invitrogen) contained calibration curves and cytokine standards (R&D Systems). A radiolabeled cRNA probe for CXCL10 was used, as described in in situ hybridization (see, e.g., Fallert and Reinhart, J Virol Methods, 99:23-32, 2002). Autoradiography exposure was performed over a 14-day period as described.

[0229] 7.7.1.10 Radiation response monitoring Tumor size was assessed using contrast-enhanced MRI scans at weeks 9, 17, 25, and 33, and every 3 months thereafter. Response was assessed based on pretreatment MRI appearance, McDonald criteria, and gadolinium-enhanced T1-weighted images, signal extension areas on T2-weighted images, or a combination of both.

[0230] 7.7.1.11 Other Clinical Endpoints Overall survival was defined as the interval from study entry to the date of death. MRI scans were used to assess time to progression (TTP).

[0231] 7.7.2 Results 7.7.2.1 Summary of clinical toxicology Table 4 shows the treatment-related adverse events for all 22 patients. The study described AEs (adverse events). There were no grade 3 or 4 toxicities, on-study deaths, or DLTs at any dose during the first booster phase. There were no autoimmune events. The toxicity profile was comparable across dose levels. Grade 1 or 2 injection site reactions were fairly common (82%). Grade 1 flu-like symptoms, including fatigue (73%), myalgia (32%), fever (23%), chills / chills (18%), and headache (32%), were common and usually limited to 24 hours after each vaccination. Grade 2 lymphopenia was recorded in one patient (5%).

[0232] [Table 4]

[0233] 7.7.2.2 IL-12 production by αDC1 As shown in Tables 3A-3B, the level of CD40L-induced IL-12 p70 production by αDC1 varied substantially among patients and was positively correlated with TTP, but not with IFN-γ ELISPOT response, patient age, or tumor type.

[0234] 7.7.2.3 Induction of epitope-specific immune responses to GAA All 19 patients who completed the first course of four vaccinations were on immunosurveillance. All patients had available PBMCs for IFN-γ ELISA (immunization). PBMCs from patients 17, 21, and 22 were insufficient to perform both ELISPOT and tetramer assays, so functional ELISPOT assays were prioritized. The first four scheduled vaccinations induced immune responses to at least one vaccine target GAA in 6 of 10 and 5 of 9 patients at dose levels 1 and 2, respectively, by IFN-γ ELISPOT or tetramer assay. In patients 6, 7, 8, 16, 19, 20, and 22, some indications reached the criteria for a positive response following a booster vaccination. In summary, 11 of 19 evaluable patients (58%) had a positive response after the first four vaccinations, and 3 of 19 (patients 8, 19, and 20; 16%) had a positive response only after a booster vaccination.

[0235] The positive response rates (by either tetramer or ELISPOT) did not differ significantly across the two αDC1 doses per Fisher's exact test. Furthermore, the magnitude of the ELISPOT response, based on the sum of positive spots from weeks 3 to 9, was comparable across the two αDC1 dose levels (Wilcoxon test). Therefore, the time course of the IFN-γ ELISPOT response represents the combined results from both dose levels (Fig. 11). The gp100 epitope showed the highest reactivity among the GAA peptides tested (p = 0.0001, 0.0003, and 0.0005 for IL-13Rα2-, EphA2-, and YKL-40-derived peptides, respectively; Wilcoxon test). For other epitopes, booster vaccination appeared to improve the induction of specific responses. A transient decrease in response was generally observed at week 13. This may reflect the fact that some patients who had positive responses by week 9 did not participate in the booster phase due to tumor progression (patients 2, 9, 18, and 21) or lymphopenia (patient 10), resulting in an overall decreased response when all data are pooled for all patients. Patient 10 demonstrated the highest IFN-γ ELISPOT response to PADRE as well as IL-13Rα2 and gp100-derived epitopes (Figure 12). However, tetramer analysis of this patient revealed no response at all (Table 3A). Patient 6, who demonstrated stable disease for more than 30 months, demonstrated durable and high-level responses in both tetramer (Figure 13) and ELISPOT assays.

[0236] 7.7.2.4 Induction of Type 1 Cytokine and Chemokine Responses RT-PCR analysis of PBMCs (Figures 14 and 15) revealed increased mRNA expression of several type 1 cytokines and chemokines, specifically IFN-α1, CXCL10, and TLR3, both after the first and fourth vaccinations. IFN-γ expression was increased after the fourth vaccination but not after the first vaccination. This suggests that increased IFN-γ expression may be associated with the induction of adaptive rather than innate immune responses. CCL22, which is known to attract regulatory T cells (see, e.g., Muthuswamy et al., Cancer Res. 68:5972-5978, 2008), is also known to increase the expression of IFN-γ. (control) and CCL5 levels were decreased in paired analyses of samples after the first vaccination. Perforin, Granzyme B, COX-2, and Foxp3 levels did not change significantly.

[0237] A panel of cytokines and chemokines was evaluated at the protein level in available serum samples from five patients before and after vaccination (Figure 16). Among them, IFN-α, CXCL10, IL-15, MCP-1, and MIP-1β were significantly upregulated in post-vaccination serum. IL-17 was below the detectable range by both RT-PCR and serum analysis.

[0238] In addition, three of five available tumors resected due to radiation progression after vaccination showed CD8 + CXCL10, a chemokine important for the efficient trafficking of T cells (e.g., Nishimura et al., Cancer Res 66:4478-4487, 2006; and Fuj Ita et al., Cancer Res 69:1587-1595, 2009) mRNA (representative These data demonstrate that this regimen induces a systemic, polyfunctional immune response in patients with malignant glioma who are generally immunocompromised.

[0239] 7.7.2.5 Immunohistochemistry Data Immunohistochemistry data for the seven GAA cases are summarized in Table 5. The data indicate that gp100 expression may be very low in primary high-grade gliomas. For immunohistochemistry, the following polyclonal antibodies (Abs) and corresponding secondary antibodies were used: anti-human (h)IL-13Rα2 (goat IgG; R&D Systems); anti-human EphA2 (H-77) (rabbit IgG; Santa Cruz Biotechnology); anti-human YKL-40 (rabbit IgG; Quidel); and anti-human gp100 (goat IgG; Santa Cruz Biotechnology).

[0240] [Table 5]

[0241] 7.7.2.6 Clinical results Two patients (patients 1 and 20) experienced objective clinical tumor regression (response rate = 9%). Both patients were non-responders by ELISPOT but tetramer responders. Patient 20 with recurrent GBM demonstrated a complete response at week 17 after vaccination based on disappearance of Gd-enhancing masses compared with baseline MRI. This was durable and continued for at least 13 months since treatment initiation (Figure 18A-I). Patient 1 with recurrent GBM demonstrated a partial response at week 9. Following two booster vaccinations, the Gd-enhancing lesions enlarged. However, biopsies of the lesions revealed CD8 + A strong infiltration of T cells and CD68+ macrophages and no evidence of mitotically active tumor were evident (Figure 18J-L). Therefore, the patient received one booster vaccination 7 months after initiation of vaccination before relapse. Nine patients (41%; 4 and 5 for GBM and AG, respectively) remained progression-free for at least 12 months. Five patients remained progression-free (Table 3A) and continued to receive booster vaccinations. The median TTP was 4 and 13 months for GBM and AG, respectively (Figure 20).

[0242] 7.7.3 Conclusion The study described in this example evaluated an αDC1 vaccine loaded with a novel GAA-derived peptide in combination with poly-ICLC. The findings demonstrate the preliminary efficacy of this approach, as well as its safety and immunogenicity.

[0243] 7.8 Example 8 This example describes a safety and efficacy study of a treatment regimen for adults with recurrent WHO grade II glioma that included HLA-A2-restricted glioma antigen peptide vaccination combined with poly-ICLC.

[0244] 7.8.1 Rational This example describes a vaccine regime designed to efficiently induce antitumor T cell responses in patients with recurrent WHO grade II glioma. The regime combines subcutaneous injection of a glioma-associated antigen (GAA)-derived cytotoxic T lymphocyte (CTL) epitope-peptide with simultaneous intramuscular (im) administration of poly-ICLC.

[0245] Adults with supratentorial low-grade gliomas (LGGs) are at significant risk of tumor progression 2 years after treatment with radiation therapy (RT) followed by surgery or endoscopic surgery (24%). The study described in this example has immunoprophylactic and immunotherapeutic potential to reduce the risk of tumor recurrence, potentially leading to improved survival. Therapeutically, immunotherapeutic approaches may inhibit the indolent expansion of neoplastic low-grade II tumor cells. Preventively, such approaches may prevent anaplastic transformation, which occurs in approximately half of recurrent LGGs. The slower growth rate of LGGs (compared to malignant gliomas) should allow sufficient time for multiple immunizations, potentially resulting in the induction of high levels of GAA-specific immunity. In addition, poly-ICLCs have been shown to promote vaccine efficacy in preclinical brain tumor models (see, e.g., Zhu et al., J.Transl.Med., 5:10, 2007) and are safe in patients with malignant gliomas (see, e.g., (See Salazar et al., Neurosurgery, 38: 1096-1103, 1996). Therefore, we hypothesize that this form of vaccine combined with poly-ICLC will induce a strong anti-glioma immune response and be safe.

[0246] 7.8.2 Purpose This example describes a vaccine study in adults with recurrent WHO grade II glioma. The objectives of this example include collecting immunological and safety data that can be used in further studies. Patients in the study described in this example will be followed for a minimum of 2 years. Therefore, actual 2-year overall survival (OS) rates, as well as 6-month and 2-year progression-free survival (PFS) rates, can be determined in a preliminary manner.

[0247] 7.8.2.1 Induction of GAA-specific T cell responses The response rate and magnitude of immune response in post-vaccination peripheral blood mononuclear cells (PBMCs) to GAA peptides in response to this form of vaccine can be determined using IFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

[0248] 7.8.2.2 Safety The incidence and severity of adverse events associated with the vaccine regime can be assessed, along with early stopping rules based on the frequency of regimen-defined toxicities (RLTs).

[0249] 7.8.2.3 Clinical response Standard WHO response criteria can be used to determine radiation response. Progression-free survival (PFS) can be assessed in a preliminary manner based on serial magnetic resonance imaging (MRI) scans.

[0250] 7.8.2.4 Tumor tissue for biological correlation For patients with progression, biopsy / resection is recommended. When post-vaccination tumor tissue is available, GAA expression status and GAA-specific T cell infiltration can be analyzed.

[0251] 7.8.3 Patient Selection 7.8.3.1 Good Practice Pathological Criteria - Patients have histologically confirmed recurrent supratentorial WHO grade II astrocytoma, oligoastrocytoma, or oligodendroglioma either at a previous biopsy or resection, or at the time of reoperation (reoperation before entry into the study is acceptable; however, postoperative dexamethasone (Decadron) must be discontinued for at least 4 weeks before the first vaccine administration). Patients in this study must have received prior external beam radiation therapy and / or chemotherapy. Regarding previous treatment, patients in this study must have been treated for recurrence no more than twice before. Recurrence is defined as progression after the first treatment (i.e., radiation + / - chemotherapy if used as initial treatment). Therefore, it is intended that patients in this study must have been treated three times previously. If a patient has undergone surgical resection for recurrent disease and anticancer treatment has not been initiated until up to 12 weeks, and the patient undergoes another surgical resection, this will be considered one resection.

[0252] Patients in this study should be HLA-A2 positive based on flow cytometry.

[0253] Patients in this study should have recovered from the toxic effects of previous treatments, including: 4 weeks after any investigational drug; 4 weeks after previous cytotoxic therapy; and / or at least 2 weeks after vincristine, 4 weeks after nitrosoureas, 3 weeks after procarbazine, and other therapies such as interferon, tamoxifen, thalidomide, and cis-retinoic acid. 1 week after a non-cytotoxic agent (radiosensitizers do not count). For prior radiation therapy (RT), at least 6 months must have passed since the completion of RT (or radiosurgery).

[0254] Patients in this study should be >18 years of age.

[0255] Patients in this study should have a Karnofsky Performance Status (Appendix I) of >60.

[0256] Female patients in this study who are of childbearing potential should undergo a serum β-HCG pregnancy test.

[0257] Patients should be free of systemic infection. Patients with active infection (whether or not they required antibiotic therapy) may be eligible after complete resolution of the infection. Patients on antibiotic therapy should be off antibiotics for at least 7 days before initiating treatment.

[0258] Patients in this study had a white blood cell count ≥ 2500 / mm 3 lymphocytes ≥ 400 / mm 3 ;platelets ≧100,000 / mm 3 Patients should have adequate organ function as measured by hemoglobin ≥ 10.0 g / dL, AST, ALT, GGT, LDH, alkaline phosphatase, and total bilirubin ≤ 2.0 mg / dL within 2.5x the upper limit of normal, and creatinine within 1.5x the upper serum normal limit. Patients in this study should have coagulation tests and PT and PTT within the upper limits of normal.

[0259] 7.8.3.2 Exclusion criteria Patients in this study should be excluded if gliomatosis, cerebral, cranial, or spinal leptomeningeal metastases are present.

[0260] Even if the initial diagnosis was WHO grade II glioma, patients should be excluded from the study if the pathological diagnosis for recurrent disease indicates transformation to a more malignant glioma (i.e., WHO grade III or IV).

[0261] Patients in this study should be excluded if they are receiving concurrent treatments or medications, including radiation therapy; chemotherapy; interferon (e.g., Intron-A®); allergy desensitization injections; growth factors (e.g., Procrit®, Aranesp®, Neulasta®); interleukins (e.g., Proleukin®); and / or any investigational therapeutic agent.

[0262] Patients in this study should not have had a previous autoimmune disease requiring cytotoxic or immunosuppressive treatment, or an autoimmune disease associated with visceral damage. Patients in this study with active autoimmune disease requiring these treatments should also be excluded. Mild arthritis requiring NSAID medication should not be excluded.

[0263] Patients in this study should be excluded if they have used or are scheduled to use immunosuppressants within 4 weeks prior to study entry. If dexamethasone or other corticosteroids are being used perioperatively and / or during radiation therapy, they should be tapered and discontinued by the patient for at least 4 weeks prior to the first study vaccine administration. Topical corticosteroids and inhaled steroids (e.g., Advair®, Flovent®, Azmacort®) should be tolerated.

[0264] Patients in this study should be excluded if they have a diagnosis of another cancer, except for the following diagnoses: squamous cell carcinoma of the skin without known metastases; basal cell carcinoma of the skin without known metastases; non-invasive breast cancer (DCIS or LCIS); non-invasive cervical cancer; and / or any cancer without distant metastases that has been successfully treated with no signs of recurrence or metastasis for more than 5 years.

[0265] Patients in this study should exclude those with known alcoholism or illicit drug use.

[0266] Immunocompromised patients are unlikely to respond to this treatment, and HIV-positive patients should be excluded from this study.

[0267] 7.8.4 Peptide vaccines 7.8.4.1 Peptides The following peptides: IL-13Rα2 345-353 1A9V (ALPFGFILV; SEQ ID NO: 3); EphA2 883-891 (TLADFDPRV; SEQ ID NO: 6); Survivin 96-104 :M2 (LMLGEFLKL; SEQ ID NO: 7); WT1 126 -134 :Y1 (YMFPNAPYL; SEQ ID NO:8); and tetanus toxoid (TetA830) (AQYIKANSKFIGITEL; SEQ ID NO:9) may be included in the vaccine formulation.

[0268] All peptides can be synthesized and purified by HPLC. The identity of the synthetic peptides can be confirmed by mass and amino acid sequence verification by mass spectrometry. Each peptide lot can be assessed for identity, purity, sterility, and pyrogenicity by FDA.

[0269] Peptides can be stored in vials under GMP conditions at -70°C. The stability of lyophilized peptides can be tested annually by mass spectrometry.

[0270] 7.8.4.2 Other Reagents Montanide ISA-51 (SEPPIC Inc., Fairfield, NJ) can be used as an additional agent in peptide vaccines.

[0271] 7.8.4.3 Dosage and Preparation To form one oil-in-water emulsion (i.e., 1 mL volume / injection), aqueous solutions (500 μL) each containing four HLA-A2-restricted GAA peptides (300 μg / peptide) and tetanus peptide (peptide-tet; 200 μg) can be mixed 1 / 1 with Montanide ISA-51.

[0272] 7.8.4.4 Administration Patients in this study will be vaccinated subcutaneously in the right or left upper arm into intact draining axillary lymph nodes. In the case of patients who do not have intact axillary lymph nodes as draining nodes, the vaccine may be administered into the intact inguinal lymph nodes on the same side of the upper thigh.

[0273] The vaccine can be administered at weeks 0, 3, 6, 9, 12, 15, 18 and 21.

[0274] 7.8.5 Poly-ICLC Poly-ICLC was prepared in a GMP facility at Bioserv, Corporation (San Diego, California). It can be prepared and packaged in-house. Poly-ICLC can be supplied in vials containing 1 cc of a translucent solution at a concentration of 2 mg / cc. Poly-ICLC is stable at room temperature for several days but can be stored frozen at approximately 4°C.

[0275] 7.8.5.1 Dosage and Administration Poly-ICLC can be administered intramuscularly at doses ranging from 20 μg / kg to 1640 μg / injection, given as two injections on days 0 and 4 after each vaccination.

[0276] The first course of poly-ICLC (20 μg / kg im and up to 1640 μg / injection) can be administered on the day of the first GAA / TT vaccination and on day 4 after the vaccination. After each repeat vaccination (3, 6, 9, 12, 15, 18, and 21 weeks), poly-ICLC (20 μg / kg im and up to 1640 μg / injection) can be administered on the day of vaccination and on day 4 after the vaccination.

[0277] Regarding the injection site, poly-ICLC should be administered intramuscularly (i.m.) close to the site of the previous peptide injection (e.g., less than 3 cm from the center of the previous peptide injection site) because it is believed to enhance the antigen presentation process in the draining axillary lymph nodes.

[0278] Poly-ICLC should be administered intramuscularly (i.m.) using aseptic technique when supplied from a vial at the prescribed dose per patient weight (maximum 1640 μg / injection). Vital signs can be monitored before the first treatment and for at least 20 minutes after treatment.

[0279] 7.8.6 Action Plan The study described in this example demonstrates the role of HLA-A2 in recurrent WHO grade II gliomas. + Two cohorts of patients can be used to evaluate the immunogenicity, safety, and clinical efficacy of the GAA / TT peptide vaccine and poly-ICLC in patients. Because the vaccine is locally isolated and the immune response occurs primarily locally in the draining lymph nodes, the vaccine dose does not need to be scaled up proportionally to the recipient's size (by weight or body surface area). As with any drug, its effectiveness is related to its distribution within body fluids. Regarding the dose of poly-ICLC, a fixed dose (20 μg / kg / injection and up to 1640 μg / injection) can be used, which has been shown to be safe and induce a biological response in patients with malignant gliomas (e.g., Salazar et al., Neurosurgery, 38: 1096-1103, 1996).

[0280] 7.8.6.1 Schedule Patients can be treated with subcutaneous injections of the GAA / TT vaccine at weeks 0, 3, 6, 9, 12, 15, 18, and 21. Poly-ICLC can be administered intramuscularly (Im) (20 μg / kg / injection and a maximum of 1640 μg / injection) on the day and 4 days after each vaccination (e.g., if the vaccine is administered on a Thursday, poly-ICLC can be administered on the day of vaccination and the following Monday). Each vaccine can be administered within 2 hours before or after poly-ICLC administration.

[0281] Patients can be evaluated for clinical / radiological response as well as any possible adverse events, regimen-defining toxicities (RLTs) at clinic visits or MRI scans. MRI scans can be performed at weeks 0, 12, and 24. If the scan at week 12 shows clear tumor progression, patients can be withdrawn.

[0282] Peripheral blood mononuclear cells (PBMCs) obtained before the first vaccination can be used as a baseline sample. If the patient shows positive responses in both immunological assays (ELISPOT or tetramer) without RLT or tumor progression, the patient can be offered additional GAA / TT vaccines (see, e.g., Section 7.8.6.2) starting any time between 34-40 weeks and every 3 months thereafter until the patient shows tumor progression, loss of immune response, or RLT.

[0283] 7.8.6.2 Add-on Therapies Patients' PBMCs can be assessed for the presence of GAA-specific T cell responses to GAA peptides at weeks 0 (baseline), 12, 15, 18, 21, and 24. If such responses are observed with any of the GAA peptides, patients can receive poly-ICLC starting at weeks 34-40 after the initial vaccination and any time thereafter every 12 months for up to two years, as well as booster vaccinations with GAA, which indicates a durable response. Additional PBMC samples can be obtained every 12 weeks (at the same visit for vaccine administration) for immunological monitoring. Booster vaccinations can be terminated upon any of the following: 1) tumor progression, 2) RLT, or 3) negative immunological responses at two consecutive time points.

[0284] 7.8.6.3 Dose Modification 7.8.6.3.1 Dose Modification of Poly-ICLC For grade 2 or greater flu-like symptoms, including fever and fatigue, poly-ICLC can be interrupted until symptoms return to grade 0. If grade 2 or greater flu-like symptoms occur on the day of vaccination and do not return to grade 0 by day 4 after vaccination, the next poly-ICLC dose on day 4 after vaccination can be skipped. If the patient is asymptomatic (grade 0) on day 4, poly-ICLC can be resumed at 50% of the initial dose. If grade 2 or greater flu-like symptoms occur after poly-ICLC administration on day 4 after vaccination, the next vaccine cycle can include two poly-ICLC doses (on days 0 and 4 after vaccination) at 50% of the initial dose. Pretreatment with acetaminophen 650-1000 mg or any NSAID can be administered. If further medication is well tolerated, If so, the original dose can be resumed.

[0285] In the event of liver enzyme assessment >4x baseline or other unpredictable intolerable side effects of grade 2 or greater, poly-ICLC can be discontinued until toxicity has decreased to grade 1 or less. Poly-ICLC can then be re-administered at half the original dose, and patients can be closely observed. If poly-ICLC cannot be resumed at the time of the next vaccine cycle, patients may be withdrawn by RLT.

[0286] For patients with grade 3 or less lymphopenia at study entry (our eligibility criteria require 400 cells / μL), the occurrence or continued presence of grade 3 lymphopenia during the study does not mandate discontinuation of poly-ICLC. However, poly-ICLC may be temporarily suspended in the event of grade 4 lymphopenia. Furthermore, poly-ICLC administration may be temporarily suspended even in the presence of grade 3 lymphopenia if the attribution of poly-ICLC is strongly suspected. In such cases, poly-ICLC may be re-administered at half the initial dose once the total lymphocyte count returns to at least 400 cells / μL.

[0287] Patients may remain on the initial dose for Grade 1 toxicity. However, the dose may be reduced by 50% for Grade 2 hematologic or non-hematologic toxicity (excluding transient fever or fatigue as outlined earlier in this section). If there is no toxicity for at least 2 weeks at the 50% dose level, the dose may be increased to the starting dose. Subsequent toxicities, if they occur, may require a dose reduction of 50%, with no further increases permitted. If toxicity reoccurs at the reduced dose, the patient may be discontinued.

[0288] 7.8.6.3.2 Delayed Dosing for Peptide Vaccines In situations where poly-ICLC administration is suspended, if the event is not attributable to the peptide / ISA-51 vaccine, vaccination should continue as scheduled. If the event is attributable to both the poly-ICLC and peptide vaccine, both may be suspended. If the event is thought to be attributable to the peptide / ISA-51 vaccine alone and not to the poly-ICLC, vaccination and poly-ICLC administration may be suspended. In situations where adverse event assessment is limited, such as when intercurrent illness or laboratory evaluation is required for other causes of toxicity, the vaccination schedule may be interrupted for up to 4 weeks. A delay in administration of one vaccine by up to 4 weeks due to an adverse event, regardless of cause, will not be considered a protocol violation. If one or more vaccinations are delayed by up to 4 weeks due to an adverse event, regardless of cause, treatment should be interrupted.

[0289] Patients may be monitored for regimen-defined toxicities (RLTs) throughout the study. The following are considered RLTs if they are judged to be probably, probably, or definitely related to treatment. If they occur, the individual patient will be removed from the study and will not receive further injections.

[0290] ≥ Grade 2 or higher: Bronchospasm or generalized urticaria (hypersensitivity)

[0291] Grade 2 or higher: Allergic reactions such as exfoliative erythroderma, anaphylaxis, or vascular collapse

[0292] ≥ Grade 2 or higher Autoimmune disease (e.g., hypothyroidism, autoimmune encephalitis)

[0293] Any ≥ Grade 3 toxicity possibly, probably, or definitely related to vaccination with particular attention to the following adverse events:

[0294] ≥ Grade 3 injection site reaction due to peptide vaccine or poly-ICLC administration

[0295] ≥ Grade 3 hematologic or hepatotoxicity

[0296] ≥ Grade 3 neurotoxicity: Signs and symptoms indicating either tumor progression requiring biopsy or resection with pathological findings of inflammatory / lymphoid infiltrate or an inflammatory immune response (i.e., pseudotumor progression)

[0297] ≥ Grade 3 Nausea and vomiting without adequate antiemetic prophylaxis is not considered RLT

[0298] Delayed dosing >4 weeks for each poly-ICLC or peptide vaccine

[0299] Treatment may be discontinued for the following reasons: (i) regimen-limiting toxicity as defined above; (ii) disease progression—an increase of at least 25% in the sum of the longest diameters on MRI scans or the appearance of contrast enhancement in previously non-enhancing tumors. However, if pseudotumor progression is suspected, patients may be given dexamethasone up to 4 mg / day and re-examined 4–8 weeks later. If >4 mg / day dexamethasone is required or if criteria for disease progression are met on repeat imaging studies, the patient may be removed from the study and further study treatment may be discontinued. However, if the steroid dose is <4 mg / day and criteria for disease progression are not met on repeat imaging studies, the patient may continue in the study and receive study treatment as defined herein. Any cases of suspected tumor progression or pseudotumor progression should be reviewed to determine whether the subject should remain in the study. (iii) Intercurrent illness that prevents further vaccination or poly-ICLC administration. (iv) Pregnancy: Pregnant patients will continue to be followed throughout the pregnancy.

[0300] 7.8.6.4 Treatment duration If there are no treatment delays due to adverse events, treatment may continue for 21 weeks or until one of the following criteria applies (8 vaccinations): regimen-defined toxicity (RLT); disease progression and / or intercurrent illness that prevents further treatment administration.

[0301] 7.8.6.5 Concurrent Treatment 7.8.6.5.1 Tolerance Acetaminophen can be used for fever (325 mg tablets, 1 or 2 orally every 4 hours). Patient pretreatment with acetaminophen is initiated as a natural consequence of poly-ICLC side effects. Fever lasting more than 8 hours after treatment is evaluated for potential infection.

[0302] For mild localized pain, oral opiates (oxycodone, 5-10 mg orally every 3-4 hours) can be planned. Pain of mild-moderate or higher grade can be investigated for nonmedical causes and managed accordingly.

[0303] Dexamethasone (or similar corticosteroids) should not be used for at least 4 weeks before starting vaccine / poly-ICLC treatment (week 0). Dexamethasone (up to 4 mg / day) may be used in the setting of pseudotumor progression and tapered / interrupted as soon as possible.

[0304] Anticonvulsants should be used as indicated.

[0305] Antiemetics can be administered if necessary.

[0306] Other acceptable medications may include topical steroids; nonsteroidal anti-inflammatory drugs; antihistamines (e.g., Claritin®, Allegra®); chronic medications except those listed in Section 7.8.6.5.2; influenza vaccines (which should be administered at least 2 weeks before the start of study vaccinations or at least 2 weeks after the 8th vaccination); and / or corticosteroid medications administered orally or by inhalation (e.g., Advair®, Flovent®, Azmacort®).

[0307] 7.8.6.5.2 Unacceptable Patients in this study should be excluded if they are receiving any concurrent treatments or medications, including radiation therapy; chemotherapy; interferon (e.g., Intron-A®); allergy desensitization injections; growth factors (e.g., Procrit®, Aranesp®, Neulasta®); interleukins (e.g., Proleukin®); and / or any investigational therapeutic agent.

[0308] 7.8.7 Correlation / Special Studies 7.8.7.1 Immunological surveillance 7.8.7.1.1 Enzyme-Linked Immuno-SPOT (ELISPOT) Assay The frequency of glioma-associated antigen (GAA)-reactive T lymphocyte precursors in peripheral blood mononuclear cells (PBMCs) before and after GAA vaccination can be measured by ELISPOT assay. The biological response measured by ELISPOT should be performed on at least the same time point in an individual to avoid inter-assay variability. Successful vaccination stimulates a clonal population of T cells capable of secreting cytokines in an antigen-specific, MHC-restricted manner. ELISPOT assays are used to measure CD8 + CD4 T cell populations reacting to the helper TT peptide as well as GAA-specific immune responses + It can be used to assess T cells. IFN-γ production can be assessed by assessing type 1 T cell responses.

[0309] A subject is considered to have responded if, at any time point after two consecutive vaccinations against the same antigen (weeks 12, 15, 18, 21, and 24), the number of spots doubles at baseline, there are at least 10 spots / 20,000 cells, and the number of spots after vaccination is at least three standard deviations above the pre-vaccination value. The response can be to any one of the antigens.

[0310] 7.8.7.1.2 Tetramer Analysis of GAA-Reactive T Cells in Patient PBMCs Tetramer analysis allows for the sensitive assessment of the presence of GAA-specific CD8+ T cells in peripheral blood without in vitro restimulation of the cells. Based on previous data available from patients with malignant glioma, we predict that a significant (log or greater) increase in the frequency of peptide-responsive CD8+ T cells may be observed in some, but not all, patients immunized with tumor antigen vaccines. In a preliminary approach, the PBMCs may also be assessed for surface expression of the integrin receptor very late antigen (VLA) 4, which has been implicated in T cell homing to CNS tumors and chemokine receptors (e.g., CXCR3 and CCR5) (see, e.g., Zhu et al., J. Transl. Med., 5:10, 2007). Procedures for tetramer analysis are well established.

[0311] Tetramer assays can be performed at baseline and at five time points after vaccination (12, 15, 18, 21, and 24 weeks). Total CD8+ by tetramer assay + cell A positive response at a single time point for a peptide can be defined as (1 + B)% positive, where B is the percent positive at baseline, which is usually considered to be less than 0.1%. Similar to the definition of an ELISPOT response, a patient is considered to have responded if he / she shows two consecutive single time point responses to any peptide.

[0312] 7.8.7.1.3 Flow cytometric analysis of lymphocyte populations At consecutive time points before and after vaccination, CD4 + and CD8 + Like T cell counts, CD4 + / Foxp3 + The number of T regulatory cells can be assessed.

[0313] 7.8.7.1.4 Serum Autoimmune Assays Stored serum can be assessed for the presence of autoantibodies.

[0314] 7.8.7.2 Evaluation of Primary and Recurrent Tumor Tissue GAA expression in patients' available tumor tissue can be assessed by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) (pre-vaccination or post-vaccination, respectively, or both).

[0315] If tumors recur after vaccination, it may be important to assess how the tumor escaped the vaccine effect. To this end, the following specific aspects can be assessed, as well as tissue availability: (i) Antigen loss: IHC and RT-PCR can be used to assess whether recurrent tumors express target GAAs, HLA-A2, and antigen processing machinery components such as transporters involved in antigen processing. (ii) Anti-apoptotic up-regulated molecules: Survivin may be targeted, but other anti-apoptotic molecules may be up-regulated, such as cFLIP (intracellular FLICE (Fas-associated death domain-like IL-1β-converting enzyme) inhibitory protein). (iii) Immune cell infiltration: One reason tumors may escape vaccine-induced immune responses is the failure of reactive T cells to infiltrate the tumor. To investigate this, when freshly excised tumor tissue (not fixed or frozen) is available, tumor-infiltrating lymphocytes (TILs) can be isolated and characterized for their number, phenotype, and antigen specificity using HLA-A2 tetramers for each GAA. Multicolor flow cytometry can be used to characterize the tetramers. + The function and survival of TILs can be determined by staining for perforin / IFN-γ and annexin V, respectively. Control tissues can include pre-vaccine tumors (if available) and recurrent tumors from patients not in this study. This study will assess whether vaccine-induced T cells can efficiently traffic to the brain tumor site and maintain their function and survival.

[0316] 7.8.8 Study parameters This study will be conducted on an outpatient basis, with patients scheduled to be evaluated at weeks 0, 3, 6, 9, 12, 15, 18, 21, and 24. After that period, if the patient does not receive a booster vaccination, they can be removed from the study and clinically followed every 2-4 months thereafter, as is typically done for patients with similar tumor types. If the patient is found to have a progressive tumor, they can receive other treatments, such as chemotherapy or resection. If the patient receives a booster vaccination, the booster vaccination will be administered every 12 weeks, and clinical, immunological, and radiological (MRI) monitoring can be performed at every visit (q12 weeks) until the patient withdraws. Subjects in remission will be re-treated with two additional vaccinations at 12-week intervals and followed thereafter. Vaccination can be discontinued for any patient with progression or unacceptable toxicity at any time during the scheduled vaccination.

[0317] 7.8.8.1 Pretreatment (Screening and Baseline Data) The following procedures may be performed before proceeding with treatment: informed consent must be obtained before initiating screening; HLA typing (flow cytometer assessment for HLA-A2 positivity); documentation of diagnosis (pathology); complete medical history and physical examination (along with vital signs and weight) including neurological examination and performance status; vaccine sites will be designed with confirmation of intact draining lymph nodes; demographic information should be recorded; CBC and platelets should be assessed; PT / PTT should be assessed; comprehensive metabolic panel including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK, phosphorus, total bilirubin, LDH, calcium, and albumin should be assessed; GGT, phosphorus, and magnesium should be assessed; blood should be drawn for in vitro assays; HGBA1C should be performed in patients with diabetic symptoms; ECG and echocardiogram should be performed in patients with cardiac symptoms, history, or current disease; urinalysis should be performed; brain MRI should be taken to assess baseline status of disease; and / or a pregnancy test for serum beta-HCG should be performed in women of childbearing potential.

[0318] 7.8.8.2 Intraprocedural evaluation The following procedures may be performed as treatment progresses; pre-dose (weeks 0, 3, 6, 9, 12, 15, 18, and 21 prior to vaccine administration on the day of vaccination): history and physical, including vital signs, weight, performance status, and neurological function; blood should be drawn for in vitro assays; chemistries, including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK phosphorus, total bilirubin, LDH, calcium, and albumin, should be assessed (except week 0); AED levels should be assessed if clinically indicated; patients should be screened for adverse events from the previous dose, to include a neurological evaluation and skin exam (injection site); and / or an MRI should be performed (at week 12 only during the vaccine injection visit).

[0319] After vaccination, all patients should be closely observed for adverse events for at least 20 minutes after each GAA peptide vaccine administration. On the same day, poly-ICLC (im 30 mg / kg) can be administered after each vaccination, and patients can be monitored for at least 20 minutes after poly-ICLC injection.

[0320] 7.8.8.3 24-week (after 8 vaccinations) evaluation After the vaccine cycle is completed, the following procedures can be performed: history and physical including vital signs, weight, Karnofsky performance status, and neurological function; blood should be drawn for in vitro assays (except for weeks 3, 6, and 9); CBC with differential and platelets should be assessed (except for week 0); chemistries including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK phosphorus, total bilirubin, LDH, calcium, and albumin should be assessed (except for week 0); AED levels should be assessed if clinically indicated; patients should be screened for adverse events from the previous dose, including a neurological evaluation and skin exam (injection site); and / or an MRI should be performed.

[0321] 7.8.8.4 Evaluation with Booster Vaccines (Case of Booster Vaccines) Prior to administering a booster vaccine, the following procedures may be performed: history and physical including vital signs, weight, Karnofsky performance status, and neurological function; blood should be drawn for in vitro assays (except at weeks 3, 6, and 9); CBC with differential and platelets should be assessed (except at week 0); chemistries including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK phosphorus, total bilirubin, LDH, calcium, and albumin should be assessed (except at week 0); AED levels should be assessed if clinically indicated; patients should be screened for adverse events from the previous dose, including neurological evaluation and skin exam (injection site); and / or an MRI should be performed. be.

[0322] After booster vaccinations, all patients should be closely observed for adverse events for at least 20 minutes after each vaccine administration. Booster vaccinations may be terminated by any of the following conditions: 1) tumor progression; 2) RLT; or 3) negative immune responses at two consecutive time points after initiation of booster vaccinations.

[0323] [Table 6-1]

[0324] [Table 6-2]

[0325] 7.8.9 Measuring effectiveness 7.8.9.1 Purpose 7.8.9.1.1 Immunogenicity CD8 response to GAA peptide in PBMCs after vaccination + The response rate and magnitude of the T cell response can be assessed using IFN-γ ELISPOT and flow cytometric tetramer analysis as a secondary assay.

[0326] ELISPOT assays indicate the functional status of antigen-specific T cells as expressed cytokines. Flow cytometry analysis using tetramers allows for relatively accurate estimation of the frequency of antigen-binding T cells without in vitro manipulation of primary patient-derived PBMCs or phenotypic analysis of integrin expression on antigen-specific T cells.

[0327] Biological assays measuring responses in peripheral blood can be performed at the same time point to avoid inter-assay variability.

[0328] Using flow cytometry, CD4 + T cells, CD4 + / Foxp3 + The number of lymphocyte subpopulations, such as regulatory T cells, can also be assessed. In addition, in patients undergoing debulking surgery for advanced tumors, if tumor tissue is available, the infiltration of antigen-specific CTLs can be assessed by flow cytometry of tumor-infiltrating lymphocytes along with epitope-specific MHV tetramers.

[0329] 7.8.9.1.2 Safety To determine the safety of administering class II MHC-restricted tetanus toxoid (TT)-derived helper T cells and im poly-ICLC in combination with four HLA-A2-restricted glioma-associated antigen (GAA) epitope peptides in patients with recurrent grade II glioma.

[0330] Endpoints can include the incidence and severity of adverse events, using close clinical follow-up and standard criteria, as is typically done in post-vaccination patient populations. A regimen is considered unacceptably toxic if >33% of patients in a given cohort progress to RLT.

[0331] 7.8.9.1.3 Response and Progression-Free Survival Contrast-enhanced MRI scans can be used to assess tumor recurrence at a minimum, at 12 weeks, 24 weeks, and every 3 months thereafter. Because low-grade gliomas are invasive tumors that generally do not enhance with contrast administration, tumors (i.e., target lesions) can be measured with T2 or FLAIR MRI images to assess response and progression-free survival. If there is an enhancing lesion at baseline, careful discussion can be held regarding whether pathology information for WHO grade II tumors truly represents the tumor's status. If the enhancing tumor is considered grade II, the size of the enhancing lesion can be assessed. In addition, as described below, the appearance of enhancement in a previously non-enhancing tumor is considered progressive disease (PD).

[0332] (A) Response (according to RECIST criteria) Complete remission (CR): Disappearance of all target lesions

[0333] Partial response (PR): At least a 30% reduction in the sum of the longest diameters (LD) of target lesions, referenced to baseline sum LD.

[0334] Progression (PD): At least a 20% increase in the sum of LD of target lesions, referring to the smallest sum of LD reported since treatment initiation or the appearance of contrast enhancement in a previously non-enhancing tumor. For pseudotumor progression, change to low-dose steroids and re-image before being declared to have PD.

[0335] No change (SD): No contraction to qualify for PR or sufficient increase to qualify for PD, referring to the smallest total LD ​​since the start of treatment

[0336] (B) Overall survival (OS) and progression-free survival (PFS) PFS is defined as the time from treatment initiation to the time of progression or death. All patients will be followed for a minimum of 2 years, allowing for the determination of actual 2-year OS and PFS.

[0337] 7.8.9.1.4 Post-Vaccination Tumor Tissue Analysis Although tumor tissues were not available from all patients in this study, the following characteristics can be assessed in a preliminary manner in all tumor tissues obtained before and / or after vaccination: (i) antigen loss, (ii) upregulation of anti-apoptotic molecules, and (iii) immune cell infiltration.

[0338] 7.8.10 Statistical considerations 7.8.10.1 Assessment of immunological responses Immune responses can be assessed using IFN-γ ELISPOT and tetramer assays.

[0339] Responders were matched by either IFN-γ ELISPOT or tetramer assays. A cohort can be defined as a patient with at least 4 responses out of 9 subjects. If there are at least 4 responses out of 9 subjects, the cohort can be considered worthy of further investigation. This criterion has the property that if the true response rate is <17%, there is a <5% chance of observing 4 or more responses, and if the true response rate is >66%, there is a <5% chance of observing 3 or few responses.

[0340] 7.8.10.2 Safety demonstration and evaluation The NCI Common Terminology Criteria for Adverse Events (AEs) (CTCAE 3.0) can be used to assess toxicity. Toxicity can be considered as an adverse event that is probably, probably, or definitely related to treatment. A maximum rating of toxicity for each category of interest is recorded for each patient, and aggregate results can be summarized by category and grade.

[0341] For safety, a regimen can be considered excessively toxic whenever a regimen-defined toxicity (RLT) ≥ 33% ​​and at least 2 RLTs are observed.

[0342] This study design has the following properties: if the true RLT ratio for the patient group is ≥ 45%, there is at least a 90% chance that accrual will cease; if the true RLT ratio is ≤ 9%, there is at least a 90% chance that accrual will not cease and the regimen is considered safe.

[0343] 7.8.10.3 Clinical Endpoint Assessment All patients will be followed for at least 2 years, allowing actual 2-year overall survival (OS), progression-free survival (PFS), and response rate to be compiled as exploratory endpoints. PFS will be defined as the time interval from treatment initiation to progression based on serial MRI scans. Exploratory analyses, where appropriate, will explore the relationship of imaging response and immune response to OS / PFS (using Fisher's exact test and log-rank test, respectively).

[0344] 7.8.10.4 Demographic Data Baseline descriptive statistics for all evaluable patients can be defined by demographic variables (age, sex, race / ethnicity), Karnofsky or Lansky performance status, disease stage and status at enrollment (stable disease, progressive disease), and previous treatment regimens used (for stratum C).

[0345] 7.9 Example 9 This example describes a study to evaluate the efficacy of vaccination with HLA-A2-restricted glioma antigen peptides in combination with poly-ICLC administration in patients with high-risk WHO grade II astrocytoma and oligoastrocytoma.

[0346] 7.9.1 Rationality This example describes a vaccine regime designed to efficiently induce antitumor T cell responses in patients with recurrent WHO grade II astrocytomas and oligoastrocytomas, i.e., patients with a 50% chance of progression after surgery alone or surgery plus postoperative radiation therapy. The regime described in the study presented here combines subcutaneous injection of a glioma-associated antigen (GAA)-derived cytotoxic T lymphocyte (CTL) epitope peptide with simultaneous intramuscular (im) administration of poly-ICLC.

[0347] Adults with supratentorial low-grade gliomas (LGG) are at significant risk of tumor progression (24%) two years after treatment with radiation therapy (RT) followed by surgery or surgery. For an unfavorable subset of patients, the two-year risk of progression is 40-50%. Research has demonstrated immunoprophylactic and immunotherapeutic potential to reduce the risk of tumor recurrence, potentially leading to improved survival. Therapeutically, immunotherapeutic approaches may inhibit the indolent expansion of neoplastic low-grade II tumor cells. Prophylactically, such approaches may prevent anaplastic transformation, which occurs in approximately half of recurrent LGGs. The slower growth rate of LGGs (compared to malignant gliomas) should allow sufficient time for multiple immunizations, potentially resulting in the induction of high levels of GAA-specific immunity. Additionally, poly-ICLC has been shown to promote vaccine efficacy in preclinical brain tumor models (see, e.g., Zhu et al., J.Transl.Med., 5: 10, 2007) and to be safe in patients with malignant gliomas (see, e.g., Salazar et al., Neurosurgery, 38: 1096-1103, 1996). Therefore, Therefore, we hypothesize that this form of vaccine combined with poly-ICLC will induce a strong anti-glioma immune response and be safe.

[0348] 7.9.2 Purpose This example describes a vaccine study in adults with WHO Grade II astrocytoma and oligoastrocytoma. The purpose of this example includes collecting immunological safety data that may be used in further studies. Patients in the study described in this example will be followed for a minimum of 2 years. Therefore, actual 2-year overall survival (OS) rates and 6-month and 2-year progression-free survival (PFS) rates can be determined in a preliminary manner.

[0349] 7.9.2.1 Induction of GAA-specific T cell responses The response rate and magnitude of immune response in post-vaccination peripheral blood mononuclear cells (PBMCs) to GAA peptides in response to this form of vaccine can be determined using IFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

[0350] 7.9.2.2 Safety The incidence and severity of adverse events associated with the vaccine regime can be assessed, along with early stopping rules based on the frequency of regimen-defined toxicities (RLTs).

[0351] 7.9.2.3 Cytotoxicity Radiologic response can be determined using standard WHO response criteria. Two-year progression-free survival (PFS) can be assessed in an exploratory manner based on serial magnetic resonance imaging (MRI) scans.

[0352] 7.9.2.4 Tumor tissue for biological correlation For patients with progression, biopsy / resection is recommended. When post-vaccination tumor tissue is available, GAA expression status and GAA-specific T cell infiltration can be analyzed.

[0353] 7.9.2.5 Effect of RT on the induction of GAA-specific T cell responses The response rate and magnitude of the GAA-specific immune response in the two cohorts can be determined using IFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

[0354] 7.9.3 Patient Selection 7.9.3.1 Good Practice Pathologic criteria—Patients should have a pathologic diagnosis of supratentorial WHO grade II astrocytoma or oligoastrocytoma.

[0355] Patients in this study should be HLA-A2 positive based on flow cytometry.

[0356] Patients in this study should have recovered from the toxic effects of previous treatments: 4 weeks after any investigational drug; 4 weeks after previous cytotoxic therapy and / or at least 2 weeks after vincristine, 4 weeks after nitrosourea compounds, 3 weeks after procarbazine, and 1 week after non-cytotoxic agents such as interferon, tamoxifen, thalidomide, or cis-retinoic acid (radiosensitizers do not count). For previous radiation therapy (RT), at least 6 months must have passed since the completion of RT (or radiosurgery).

[0357] Two cohorts of patients should be analyzed based on whether they have received prior RT. Cohort 1: Patients should have undergone surgery or biopsy only (no postoperative radiation or chemotherapy) and a baseline MRI scan (within 4 weeks of first vaccination) showing stable or progressive disease (no progression since first surgery / biopsy). Cohort 2: Patients should have undergone surgery or biopsy and radiation therapy (RT) (including external fractionated radiotherapy and / or stereotactic radiotherapy) completed ≥ 6 months prior to enrollment and a baseline MRI scan within 4 weeks prior to first vaccination showing stable or progressive disease.

[0358] Patients in this study were (i) aged ≥40 years with any extent of resection; (ii) aged 18-39 years Complete resection (postoperative MRI appearance >1cm residual disease, either from the edge of the postoperative cavity) laterally, anterior-posteriorly, or superior-laterally-inferiorly, based on the largest dimension of residual T2 or FLAIR abnormality) or (iii) age 18-39 years, neurosurgeon-defined GTR, but tumor size ≥ 4 cm (maximum preoperative tumor diameter based on axial and / or coronal T2 or FLAIR MR images). All patients should be ≥ 18 years of age.

[0359] Patients in this study should have a Karnofsky Performance Status (Appendix I) of >60.

[0360] Female patients in this study who are of childbearing potential should undergo a serum β-HCG pregnancy test.

[0361] Patients should be free of systemic infection. Patients with active infection (whether or not they required antibiotic therapy) may be eligible after complete resolution of the infection. Patients on antibiotic therapy should be off antibiotics for at least 7 days before initiating treatment.

[0362] Patients in this study had a white blood cell count ≥ 2500 / mm 3 lymphocytes ≥ 400 / mm 3 ;platelets ≧100,000 / mm 3 Patients should have adequate organ function as measured by hemoglobin ≥ 10.0 g / dL, AST, ALT, GGT, LDH, alkaline phosphatase, and total bilirubin ≤ 2.0 mg / dL within 2.5x the upper limit of normal, and creatinine within 1.5x the upper serum normal limit. Patients in this study should have coagulation tests and PT and PTT within the upper limits of normal.

[0363] 7.9.3.2 Exclusion criteria Patients in this study should be excluded if gliomatosis, cerebral, cranial, or spinal leptomeningeal metastases are present.

[0364] Patients in this study should be excluded if they had previously received any type of anti-glioma treatment other than chemotherapy or radiotherapy.

[0365] Patients in this study should be excluded if they are receiving concurrent treatments or medications, including radiation therapy; chemotherapy; interferon (e.g., Intron-A®); allergy desensitization injections; growth factors (e.g., Procrit®, Aranesp®, Neulasta®); interleukins (e.g., Proleukin®); and / or any investigational therapeutic agent.

[0366] Patients in this study should not have had a previous autoimmune disease requiring cytotoxic or immunosuppressive treatment, or an autoimmune disease associated with visceral damage. Patients in this study with active autoimmune disease requiring these treatments should also be excluded. Mild arthritis requiring NSAID medication should not be excluded.

[0367] Patients in this study should be excluded if they have been using or are scheduled to use immunosuppressants within 4 weeks prior to study entry. If dexamethasone or other corticosteroids are being used perioperatively and / or during radiation therapy, they should be reduced and discontinued by the patient for at least 4 weeks prior to the first vaccine administration in this study. Topical corticosteroids and inhaled steroids (e.g., Advair®, Flovent®, Azmacort®) should be tolerated.

[0368] Patients in this study should be excluded if they have a diagnosis of another cancer, except for the following diagnoses: squamous cell carcinoma of the skin without known metastases; basal cell carcinoma of the skin without known metastases; non-invasive breast cancer (DCIS or LCIS); non-invasive cervical cancer; and / or any cancer without distant metastases that has been successfully treated with no signs of recurrence or metastasis for more than 5 years.

[0369] Patients in this study should exclude those with known alcoholism or illicit drug use.

[0370] Immunocompromised patients are unlikely to respond to this treatment, and HIV-positive patients should be excluded from this study.

[0371] 7.9.4 Peptide vaccines 7.9.4.1 Peptides The following peptides bind to IL-13Rα2 345-353 1A9V (ALPFGFILV; SEQ ID NO: 3); EphA2 883-891 (TLADFDPRV; SEQ ID NO: 6); Survivin 96-104 :M2 (LMLGEFLKL; SEQ ID NO: 7); WT1 12 6-134 :Y1 (YMFPNAPYL; SEQ ID NO:8); and tetanus toxoid (TetA830) (AQYIKANSKFIGITEL; SEQ ID NO:9) may be included in the vaccine formulation.

[0372] All peptides can be synthesized and purified by HPLC. The identity of the synthetic peptides can be confirmed by mass and amino acid sequence verification by mass spectrometry. Each peptide lot can be assessed for identity, purity, sterility, and pyrogenicity by FDA.

[0373] Peptides can be stored in vials under GMP conditions at -70°C. The stability of lyophilized peptides can be tested annually by mass spectrometry.

[0374] 7.9.4.2 Other Reagents Montanide ISA-51 (SEPPIC Inc., Fairfield, NJ) can be used as an additional agent in peptide vaccines.

[0375] 7.9.4.3 Dosage and Preparation Aqueous solutions (500 μL) containing each of the four HLA-A2-restricted GAA peptides (300 μg / peptide) and tetanus peptide (peptide-tet; 200 μg) can be mixed 1 / 1 with Montanide ISA-51 to form an oil-in-water emulsion (i.e., total volume / injection 1 mL).

[0376] 7.9.4.4 Administration Patients in this study will be vaccinated subcutaneously in the right or left upper arm into intact draining axillary lymph nodes. In the case of patients who do not have intact axillary lymph nodes as draining nodes, the vaccine will be administered into the intact inguinal lymph nodes on the same side of the upper thigh.

[0377] The vaccine can be administered at weeks 0, 3, 6, 9, 12, 15, 18 and 21.

[0378] 7.9.5 Poly-ICLC Poly-ICLC was prepared in a GMP facility at Bioserv, Corporation (San Diego, California). It can be prepared and packaged in-house. Poly-ICLC can be supplied in vials containing 1 cc of a translucent solution at a concentration of 2 mg / cc. Poly-ICLC is stable at room temperature for several days but can be stored frozen at approximately 4°C.

[0379] 7.9.5.1 Dosage and Administration Poly-ICLC can be administered intramuscularly at doses ranging from 20 μg / kg to 1640 μg / injection, given as two injections on days 0 and 4 after each vaccination.

[0380] The first course of poly-ICLC (20 μg / kg im and up to 1640 μg / injection) can be administered on the day of the first GAA / TT vaccination and on day 4 after the vaccination. After each repeat vaccination (3, 6, 9, 12, 15, 18, and 21 weeks), poly-ICLC (20 μg / kg im and up to 1640 μg / injection) can be administered on the day of vaccination and on day 4 after the vaccination.

[0381] Regarding the injection site, poly-ICLC should be administered im close to the site of the previous peptide injection (e.g., less than 3 cm from the center of the previous peptide injection site) because it is believed to enhance the antigen presentation process in the draining axillary lymph nodes.

[0382] Poly-ICLC should be administered intramuscularly (i.m.) using aseptic technique when supplied from a vial at the prescribed dose per patient weight (maximum 1640 μg / injection). Vital signs can be monitored before the first treatment and for at least 20 minutes after treatment.

[0383] 7.9.6 Action Plan The study described in this example demonstrated that HLA-A2 is a poor prognostic factor for WHO grade II astrocytoma or oligoastrocytoma. + Two patient cohorts can be used to evaluate the immunogenicity, safety, and clinical efficacy of the GAA / TT peptide vaccine and poly-ICLC in patients. Because peptide vaccines are locally isolated and the immune response occurs primarily locally in the draining lymph nodes, vaccine doses do not need to be scaled up proportionally to the recipient's size (by weight or body surface area). As with any drug, its efficacy is related to its distribution within body fluids. Regarding the dose of poly-ICLC, a fixed dose (20 μg / kg / injection and up to 1640 μg / injection) can be used, which has been shown to be safe and induce a biological response in patients with malignant glioma (e.g., Salazar et al., Neurosurgery, 38: 1096-1103, 1996).

[0384] 7.9.6.1 Schedule Eligible patients in Cohort 1 should have undergone surgery or biopsy only (no postoperative radiation or chemotherapy) and a baseline MRI scan (within 4 weeks of first vaccination) showing stable or regression (no progression since initial surgery / biopsy); patients in Cohort 2 should have completed RT ≥ 6 months prior to enrollment and a baseline MRI scan (within 4 weeks of first vaccination) showing stable or regression. All patients must be off dexamethasone or similar corticosteroids for at least 4 weeks before their first vaccination.

[0385] Patients can be treated with subcutaneous injections of the GAA / TT vaccine at weeks 0, 3, 6, 9, 12, 15, 18, and 21. Poly-ICLC can be administered intramuscularly (Im) (20 μg / kg / injection and a maximum of 1640 μg / injection) on the day and 4 days after each vaccination (e.g., if the vaccine is administered on a Thursday, poly-ICLC can be administered on the day of vaccination and the following Monday). Each vaccine can be administered within 2 hours before or after poly-ICLC administration.

[0386] Patients can be evaluated for clinical / radiological response as well as any possible adverse events, regimen-defining toxicities (RLTs) at clinical visits or MRI scans.

[0387] Peripheral blood mononuclear cells (PBMCs) obtained before the first vaccination can be used as a baseline sample. If the patient shows positive responses in both immunological assays (ELISPOT or tetramer) without RLT or tumor progression, the patient can be offered additional GAA / TT vaccines (see, e.g., Section 7.9.6.2) starting any time between 34-40 weeks and every 3 months thereafter until the patient shows tumor progression, loss of immune response, or RLT.

[0388] 7.9.6.2 Add-on Therapies The presence of GAA-specific T cell responses to GAA peptides can be assessed in patient PBMCs at weeks 0 (baseline), 12, 15, 18, 21, and 24. If such responses are observed with any of the GAA peptides, patients can receive poly-ICLC starting at 34-40 weeks after the initial vaccination and any time thereafter every 12 months for up to two years, as well as booster vaccinations with GAA, which indicates a durable response. Additional PBMC samples can be obtained every 12 weeks (at the same visit for vaccine administration) for immunological monitoring. Booster vaccinations can be terminated upon any of the following: 1) tumor progression, 2) RLT, or 3) negative immunological responses at two consecutive time points.

[0389] 7.9.6.3 Dose Modification 7.9.6.3.1 Dose Modifications for Poly-ICLC For grade 2 or higher flu-like symptoms, including fever and fatigue, poly-ICLC can be interrupted until symptoms return to grade 0. If grade 2 or higher flu-like symptoms occur on the day of vaccination and do not return to grade 0 by day 4 after vaccination, the next poly-ICLC dose on day 4 after vaccination can be skipped. If the patient is asymptomatic (grade 0) on day 4, poly-ICLC can be resumed at 50% of the initial dose. If grade 2 or higher flu-like symptoms occur after poly-ICLC administration on day 4 after vaccination, two poly-ICLC doses (on days 0 and 4 after vaccination) can be administered at 50% of the initial dose in the next vaccine cycle. Pretreatment with acetaminophen 650-1000 mg or any NSAID can be administered. If the additional doses are well tolerated, the initial dose can be resumed.

[0390] In the event of liver enzyme assessment >4x baseline or other unpredictable intolerable side effects of grade 2 or greater, poly-ICLC may be discontinued until toxicity has decreased to grade 1 or less. Poly-ICLC may then be re-administered at half the original dose, and patients may be closely observed. If poly-ICLC cannot be resumed at the time of the next vaccine cycle, patients may be withdrawn by RLT.

[0391] Patients may remain on the initial dose for Grade 1 or 2 hematologic toxicity or Grade 1 non-hematologic toxicity. If there is no toxicity for at least 2 weeks at the 50% dose level, the dose may be increased to the starting dose at the investigator's discretion. Subsequent toxicities, if they occur, may require a dose reduction by 50%, with no further increases permitted. If toxicity reoccurs at the reduced dose, the patient may be withdrawn from treatment.

[0392] 7.9.6.3.2 Delayed Dosing for Peptide Vaccines In situations where poly-ICLC administration is suspended, if the event is not attributable to the peptide / ISA-51 vaccine, vaccination should continue as scheduled. In situations where adverse event assessment is limited, such as when intercurrent illness or laboratory evaluation for other causes of toxicity is required, the vaccination schedule may be interrupted for up to 4 weeks. If vaccination is delayed for more than 4 weeks due to an adverse event other than pseudotumor progression, regardless of the cause, treatment should be interrupted.

[0393] Patients may be monitored for regimen-defined toxicities (RLTs) throughout the study. The following are considered RLTs if they are judged to be probably, probably, or definitely related to treatment. If they occur, the individual patient will be removed from the study and will not receive further injections.

[0394] ≥ Grade 2 or higher: Bronchospasm or generalized urticaria (hypersensitivity)

[0395] ≥ Grade 2 or higher: allergic reactions such as exfoliative erythroderma, anaphylaxis, or vascular collapse

[0396] ≥ Grade 2 or higher Autoimmune disease (e.g., hypothyroidism, autoimmune encephalitis)

[0397] Any ≥ Grade 3 toxicity possibly, probably, or definitely related to vaccination with particular attention to the following adverse events:

[0398] ≥ Grade 3 injection site reaction due to peptide vaccine or poly-ICLC administration

[0399] ≥ Grade 3 hematologic or hepatotoxicity

[0400] ≥ Grade 3 neurotoxicity: Signs and symptoms indicating either tumor progression requiring biopsy or resection with pathological findings of inflammatory / lymphoid infiltrate or an inflammatory immune response (i.e., pseudotumor progression)

[0401] ≥ Grade 3 Nausea and vomiting without adequate antiemetic prophylaxis is not considered RLT

[0402] Delayed dosing > 4 weeks for each poly-ICLC or peptide vaccine

[0403] Treatment may be discontinued for the following reasons: (i) regimen-limiting toxicity as defined above; (ii) disease progression - an increase of at least 25% in the sum of the longest diameters on an MRI scan or the appearance of contrast enhancement in a previously non-enhancing tumor. However, if pseudotumor progression is suspected, patients may be given dexamethasone up to 4 mg / day and re-examined in 4-8 weeks. If >4 mg / day dexamethasone is required or if criteria for disease progression are met on repeat imaging studies, the patient may be removed from the study and further study may be discontinued. However, if the steroid dose is <4 mg / day and criteria for disease progression are not met on repeat imaging studies, the patient may continue on the study and continue treatment as defined herein. Subjects who are pregnant will be able to receive the study treatment prescribed by the FDA. Any cases of suspected tumor progression or pseudotumor progression should be reviewed to determine whether the subject will remain in the study. (iii) Concomitant illness that prevents further vaccine or poly-ICLC administration. (iv) Pregnancy: Patients who become pregnant will continue to be followed for the duration of the pregnancy.

[0404] 7.9.6.4 Treatment duration If there are no treatment delays due to adverse events, treatment may continue for 21 weeks or until one of the following criteria applies (8 vaccinations): regimen-defined toxicity (RLT); disease progression and / or intercurrent illness that prevents further treatment administration.

[0405] 7.9.6.5 Concurrent Actions 7.9.6.5.1 Tolerance Acetaminophen can be used for fever (325 mg tablets, 1 or 2 orally every 4 hours). Patient pretreatment with acetaminophen is initiated as a natural consequence of poly-ICLC side effects. Fever lasting more than 8 hours after treatment is evaluated for potential infection.

[0406] For mild localized pain, oral opiates (oxycodone, 5-10 mg orally every 3-4 hours) can be planned. Pain of mild-moderate or higher grade can be investigated for nonmedical causes and managed accordingly.

[0407] Dexamethasone (or similar corticosteroids) should not be used for at least 4 weeks before starting vaccine / poly-ICLC treatment (week 0). Dexamethasone (up to 4 mg / day) may be used in the setting of pseudotumor progression and tapered / interrupted as soon as possible.

[0408] Anticonvulsants should be used as indicated.

[0409] Antiemetics can be administered if necessary.

[0410] Other acceptable medications may include topical steroids; nonsteroidal anti-inflammatory drugs; antihistamines (e.g., Claritin®, Allegra®); chronic medications except those listed in Section 7.8.6.5.2; influenza vaccines (which should be administered at least 2 weeks before the start of study vaccinations or at least 2 weeks after the 8th vaccination); and / or corticosteroid medications administered orally or by inhalation (e.g., Advair®, Flovent®, Azmacort®).

[0411] 7.9.6.5.2 Unacceptable Unacceptable medications include interferon therapy (e.g., Intron-A®); chemotherapy; allergy desensitization injections; growth factors (e.g., Procrit®, Aranesp®, Neulasta®); interleukins (e.g., Proleukin®); any investigational therapeutic agent and / or illicit drug.

[0412] 7.9.7 Correlation / Special Studies 7.9.7.1 Immunological surveillance 7.9.7.1.1 ELISPOT Assay The frequency of glioma-associated antigen (GAA)-reactive T lymphocyte precursors in peripheral blood mononuclear cells (PBMCs) before and after GAA vaccination can be measured by ELISPOT assay. The biological response measured by ELISPOT should be performed on at least one individual at the same time point to avoid inter-assay variability. Successful vaccination stimulates a clonal population of T cells capable of secreting cytokines in an antigen-specific, MHC-restricted manner. ELISPOT assays measure CD8 + CD4 T cell populations reacting to the helper TT peptide as well as GAA-specific immune responses + It can be used to assess T cells. IFN-γ production can be assessed by assessing type 1 T cell responses.

[0413] A subject is considered to have responded if, at any time point after two consecutive vaccinations against the same antigen (weeks 12, 15, 18, 21, and 24), the number of spots doubles at baseline, there are at least 10 spots / 20,000 cells, and the number of spots after vaccination is at least three standard deviations above the pre-vaccination value. The response can be to any one of the antigens.

[0414] 7.9.7.1.2 Tetramer Analysis of GAA-Responsive T Cells in Patient PBMCs The frequency of glioma-associated antigen (GAA)-reactive T lymphocyte precursors in peripheral blood mononuclear cells (PBMCs) before and after GAA vaccination can be measured by ELISPOT assay. The biological response measured by ELISPOT should be performed on at least the same time point in an individual to avoid inter-assay variability. Successful vaccination stimulates a clonal population of T cells capable of secreting cytokines in an antigen-specific, MHC-restricted manner. ELISPOT assays are used to measure CD8 + CD4 T cell populations reacting to the helper TT peptide as well as GAA-specific immune responses + It can be used to assess T cells. IFN-γ production can be assessed by assessing type 1 T cell responses.

[0415] Tetramer assays can be performed at baseline and at five time points after vaccination (12, 15, 18, 21, and 24 weeks). Total CD8+ by tetramer assay + A single-time point positive response for a peptide can be defined as (1 + B)% of cells positive, where B is the percent positive at baseline, which is usually considered to be less than 0.1%. Similar to the definition of an ELISPOT response, a patient is considered to have responded if he / she has two consecutive single-time point responses to any peptide.

[0416] 7.9.7.1.3 Flow Cytometric Analysis of Lymphocyte Populations At consecutive time points before and after vaccination, CD4 + and CD8 +Like T cell counts, CD4 + / Foxp3 + The number of T regulatory cells can be assessed.

[0417] 7.9.7.1.4 Serum Autoimmune Assays Stored serum can be assessed for the presence of autoantibodies.

[0418] 7.9.7.2 Evaluation of Primary and Recurrent Tumor Tissue GAA expression in patients' available tumor tissue can be assessed by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) (pre-vaccination or post-vaccination, respectively, or both).

[0419] When tumors recur after vaccination, it may be important to assess how the tumor escaped the effects of the vaccine. To this end, as well as assessing tissue availability, the following specific aspects can be assessed: (i) antigen loss: IHC and RT-PCR can be used to assess whether recurrent tumors express antigen processing machinery components such as target GAA, HLA-A2, and transporters involved in antigen processing; (ii) anti-apoptotic up-regulated molecules: Survivin may be targeted, but other anti-apoptotic molecules may be up-regulated, such as cFLIP (intracellular FLICE (Fas-associated death domain-like IL-1)), and (iii) anti-apoptotic up-regulated molecules: Survivin may be targeted, but other anti-apoptotic molecules may be up-regulated, such as cFLIP (intracellular FLICE (Fas-associated death domain-like IL-1)). (1β-converting enzyme) inhibitory proteins); and (iii) immune cell infiltration: One reason tumors may escape vaccine-induced immune responses is the failure of reactive T cells to infiltrate the tumor. To investigate this, when freshly excised tumor tissue (not fixed or frozen) is available, tumor-infiltrating lymphocytes (TILs) can be isolated and characterized for their number, phenotype, and antigen specificity using HLA-A2 tetramers for each GAA. Multicolor flow cytometry can be used to characterize the tetramers. +The function and survival of TILs can be determined by staining for perforin / IFN-γ and annexin V, respectively. Control tissues can include pre-vaccine tumors (if available) and recurrent tumors from patients not in this study. This study will assess whether vaccine-induced T cells can efficiently traffic to the brain tumor site and maintain their function and survival.

[0420] 7.9.8 Study parameters This study can be conducted on an outpatient basis, with patients scheduled to be evaluated at weeks 0, 3, 6, 9, 12, 15, 18, 21, and 24. After that period, if the patient does not receive a booster vaccination, they can be removed from the study and clinically followed every 2-4 months thereafter, as is typically done for patients with similar tumor types. If the patient is found to have a progressive tumor, they can receive other treatments, such as chemotherapy or resection. If the patient receives a booster vaccination, the booster vaccination will be administered every 12 weeks, and clinical, immunological, and radiological (MRI) monitoring can be performed at every visit (q12 weeks) until the patient withdraws. Subjects in remission will be re-treated with two additional vaccinations at 12-week intervals and followed thereafter. Vaccination can be discontinued for any patient with progression or unacceptable toxicity at any time during the scheduled vaccination.

[0421] 7.9.8.1 Pretreatment (Screening and Baseline Data) The following procedures may be performed before proceeding with treatment: informed consent must be obtained before initiating screening; HLA typing (flow cytometer assessment for HLA-A2 positivity); documentation of diagnosis (pathology); complete medical history and physical examination (along with vital signs and weight) including neurological examination and performance status; vaccine sites will be designed with confirmation of intact draining lymph nodes; demographic information should be recorded; CBC and platelets should be assessed; PT / PTT should be assessed; comprehensive metabolic panel including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK, phosphorus, total bilirubin, LDH, calcium, and albumin should be assessed; GGT, phosphorus, and magnesium should be assessed; blood should be drawn for in vitro assays; HGBA1C should be performed in patients with diabetic symptoms; ECG and echocardiogram should be performed in patients with cardiac symptoms, history, or current disease; urinalysis should be performed; brain MRI should be taken to assess baseline status of disease; and / or a pregnancy test for serum beta-HCG should be performed in women of childbearing potential.

[0422] 7.9.8.2 Intraprocedural evaluation The following procedures may be performed as treatment progresses; pre-dose (weeks 0, 3, 6, 9, 12, 15, 18, and 21 prior to vaccine administration on the day of vaccination): history and physical, including vital signs, weight, performance status, and neurological function; blood should be drawn for in vitro assays; chemistries, including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK phosphorus, total bilirubin, LDH, calcium, and albumin, should be assessed (except week 0); AED levels should be assessed if clinically indicated; patients should be screened for adverse events from the previous dose, to include a neurological evaluation and skin exam (injection site); and / or an MRI should be performed (at week 12 only during the vaccine injection visit).

[0423] After vaccination, patients were monitored for adverse events for at least 20 minutes after each GAA peptide vaccine administration. On the same day after each vaccination, poly-ICLC (im 30 mg / kg) can be administered, and patients can be monitored for at least 20 minutes after poly-ICLC injection.

[0424] 7.9.8.3 Week 24 (after 8 vaccinations) evaluation After the vaccine cycle is completed, the following procedures can be performed: history and physical including vital signs, weight, Karnofsky performance status, and neurological function; blood should be drawn for in vitro assays (except for weeks 3, 6, and 9); CBC with differential and platelets should be assessed (except for week 0); chemistries including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK phosphorus, total bilirubin, LDH, calcium, and albumin should be assessed (except for week 0); AED levels should be assessed if clinically indicated; patients should be screened for adverse events from the previous dose, including a neurological evaluation and skin exam (injection site); and / or an MRI should be performed.

[0425] 7.9.8.4 Evaluation with Booster Vaccine (in case of booster vaccine) Prior to administering a booster vaccine, the following procedures may be performed: history and physical including vital signs, weight, Karnofsky performance status, and neurological function; blood should be drawn for in vitro assays (except 3, 6, and 9 weeks); CBC with differential and platelets should be assessed (except 0 week); chemistries including electrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, ALK phosphorus, total bilirubin, LDH, calcium, and albumin should be assessed (except 0 week); AED levels should be assessed if clinically indicated; patients should be screened for adverse events from the previous dose, including a neurological evaluation and skin exam (injection site); and / or an MRI should be performed.

[0426] After booster vaccinations, all patients should be closely observed for adverse events for at least 20 minutes after each vaccine administration. Booster vaccinations may be terminated by any of the following conditions: 1) tumor progression; 2) RLT; or 3) negative immune responses at two consecutive time points after initiation of booster vaccinations.

[0427] [Table 7-1]

[0428] [Table 7-2]

[0429] 7.9.9 Measuring effectiveness 7.9.9.1 Purpose 7.9.9.1.1 Immunogenicity CD8 response to GAA peptide in PBMCs after vaccination + The response rate and magnitude of the T cell response can be assessed using IFN-γ ELISPOT and flow cytometric tetramer analysis as a secondary assay.

[0430] ELISPOT assays indicate the functional status of antigen-specific T cells as expressed cytokines. Flow cytometry analysis using tetramers allows for relatively accurate estimation of the frequency of antigen-binding T cells without in vitro manipulation of primary patient-derived PBMCs or phenotypic analysis of integrin expression on antigen-specific T cells.

[0431] Biological assays measuring responses in peripheral blood can be performed at the same time point to avoid inter-assay variability.

[0432] Using flow cytometry, CD4 + T cells, CD4 + / Foxp3 +The number of lymphocyte subpopulations, such as regulatory T cells, can also be assessed. In addition, in patients undergoing debulking surgery for advanced tumors, if tumor tissue is available, the infiltration of antigen-specific CTLs can be assessed by flow cytometry of tumor-infiltrating lymphocytes along with epitope-specific MHV tetramers.

[0433] 7.9.9.1.2 Safety Class II MHC-restricted cytoplasmic leukemia in patients with grade II astrocytoma and oligoastrocytoma The safety of administering four HLA-A2-restricted glioma-associated antigen (GAA) epitope peptides in combination with tanner toxoid (TT)-derived helper T cells and im poly-ICLC can be determined.

[0434] Endpoints can include the incidence and severity of adverse events, using close clinical follow-up and standard criteria, as is typically done in post-vaccination patient populations. A regimen is considered unacceptably toxic if >33% of patients in a given cohort progress to RLT.

[0435] 7.9.9.1.3 Response and Progression-Free Survival Contrast-enhanced MRI scans can be used to assess tumor recurrence at a minimum, at 12 weeks, 24 weeks, and every 3 months thereafter. Because low-grade gliomas are invasive tumors that generally do not enhance with contrast administration, tumors (i.e., target lesions) can be measured with T2 or FLAIR MRI images to assess response and progression-free survival. If there is an enhancing lesion at baseline, careful discussion can be held regarding whether pathology information for WHO grade II tumors truly represents the tumor's status. If the enhancing tumor is considered grade II, the size of the enhancing lesion can be assessed. In addition, as described below, the appearance of enhancement in a previously non-enhancing tumor is considered progressive disease (PD).

[0436] (A) Response (according to RECIST criteria) Complete remission (CR): Disappearance of all target lesions

[0437] Partial response (PR): A reduction of at least 30% in the sum of the longest diameters (LD) of target lesions, taken as the reference baseline sum LD.

[0438] Progression (PD): At least a 20% increase in the sum of LD of target lesions, referring to the smallest sum of LD reported since treatment initiation or the appearance of contrast enhancement in a previously non-enhancing tumor. For pseudotumor progression, change to low-dose steroids and re-image before being declared to have PD.

[0439] No change (SD): neither contraction to qualify for PR nor sufficient increase to qualify for PD, referring to the smallest total LD ​​since the start of treatment.

[0440] (B) Overall survival (OS) and progression-free survival (PFS) PFS is defined as the time from treatment initiation to the time of progression or death. All patients will be followed for a minimum of 2 years, allowing for the determination of actual 2-year OS and PFS.

[0441] 7.9.9.1.4 Post-Vaccination Tumor Tissue Analysis Although tumor tissues were not available from all patients in this study, the following characteristics can be assessed in a preliminary manner in all tumor tissues obtained before and / or after vaccination: (i) antigen loss, (ii) upregulation of anti-apoptotic molecules, and (iii) immune cell infiltration.

[0442] 7.9.10 Statistical considerations 7.9.10.1 Assessment of immunological responses Immune responses can be assessed using IFN-γ ELISPOT and tetramer assays.

[0443] Responders can be defined as patients who respond by either the IFN-γ ELISPOT or tetramer assay. If there are at least 4 responses among 9 subjects, the cohort will be further recruited. The criterion has the property that if its true response rate is <17%, there is a <5% chance of observing 4 or more responses, and if its true response rate is >66%, there is a <5% chance of observing 3 or very few responses.

[0444] 7.9.10.2 Safety demonstration and evaluation The NCI Common Terminology Criteria for Adverse Events (AEs) (CTCAE 3.0) can be used to assess toxicity. Toxicity can be considered as an adverse event that is probably, probably, or definitely related to treatment. A maximum rating of toxicity for each category of interest is recorded for each patient, and aggregate results can be summarized by category and grade.

[0445] For safety, a regimen can be considered excessively toxic whenever a regimen-defined toxicity (RLT) ≥ 33% ​​and at least 2 RLTs are observed.

[0446] This study design has the following properties: if the true RLT ratio for the patient group is ≥ 45%, there is at least a 90% chance that accrual will cease; if the true RLT ratio is ≤ 9%, there is at least a 90% chance that accrual will not cease and the regimen is considered safe.

[0447] 7.9.10.3 Clinical Endpoint Assessment All patients will be followed for at least 2 years, allowing actual 2-year overall survival (OS), progression-free survival (PFS), and response rate to be compiled as exploratory endpoints. PFS will be defined as the time interval from pathological diagnosis to progression in patients with WHO grade II astrocytoma or oligoastrocytoma based on serial MRI scans. Exploratory analyses, where appropriate, will explore the relationship of imaging response and immune response to OS / PFS (using Fisher's exact test and log-rank test, respectively).

[0448] 7.9.10.4 Demographic Data Baseline descriptive statistics for all evaluable patients can be defined by demographic variables (age, sex, race / ethnicity), Karnofsky or Lansky performance status, disease stage and status at enrollment (stable disease, progressive disease), and / or previously used treatment regimens.

[0449] 7.10 Example 10 This example describes a study to evaluate the effect of vaccination with HLA-A2-restricted glioma antigen peptides in combination with poly-ICLC in children with newly diagnosed malignant or intrinsic brainstem glioma (BSG), incompletely resected non-brainstem malignant glioma (HGG), or recurrent unresectable low-grade glioma (LGG).

[0450] 7.10.1 Rationality Currently, there are no effective treatments for pediatric malignant gliomas. Immunotherapy, particularly active vaccination, has the potential to develop as an effective and safe method. Vaccines using GAA-specific peptides offer greater potential than whole-glioma-derived antigens because they can induce glioma-specific immune responses without the theoretical risk of autoimmune encephalitis. Recent evidence suggests that pediatric gliomas and intrinsic brainstem gliomas share similar glioma-associated antigen (GAA) expression patterns and may be targeted by vaccine therapy. Given the poor prognosis in children with incompletely resected intrinsic brainstem gliomas and malignant gliomas, it is appropriate to evaluate the activity and safety of immunotherapy after radiation therapy for these tumors. Similarly, deep-seated low-grade gliomas also express a similar spectrum of GAA. Because the disease is generally refractory to conventional treatments, with increased morbidity and mortality, it is appropriate to evaluate the potential efficacy of vaccine therapy in patients whose disease has progressed after at least two chemotherapy or biologic therapy regimens or irradiation.

[0451] Combined administration of GAA peptides with poly-ICLC significantly enhanced the induction of anti-GAA CTL responses and antigen-specific T cell trafficking to brain tumor sites. In the study described here, pediatric patients newly diagnosed with malignant gliomas or refractory low-grade gliomas were vaccinated with multiple novel GAA-derived HLA-A2-restricted CTL epitopes in combination with intramuscular administration of poly-ICLC.

[0452] 7.10.2 Purpose This example describes a vaccine study in children newly diagnosed with malignant or refractory brainstem glioma (BSG) or incompletely resected non-brainstem high-grade glioma (HGG) or recurrent unresectable low-grade glioma (LGG).

[0453] 7.10.2.1 Induction of GAA-specific T cell responses The response rate and magnitude of immune response in post-vaccination peripheral blood mononuclear cells (PBMCs) to GAA peptides in response to this form of vaccine can be determined using IFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

[0454] 7.10.2.2 Safety To assess the incidence and severity of adverse events associated with vaccine regimens with early stopping rules based on the frequency of regimen-defined toxicity (RLT) in children with newly diagnosed malignant brainstem gliomas (BSG) and newly diagnosed incompletely resected non-brainstem malignant gliomas (HGG). To evaluate the incidence and severity of adverse events associated with vaccine regimens in patients with treatment-refractory, unresectable low-grade gliomas that have progressed after two chemotherapy or biotherapy regimens or irradiation. The incidence and severity of adverse events associated with the treatment can also be assessed.

[0455] 7.10.2.3 Clinical response Radiologic response can be determined using standard WHO response criteria. Two-year progression-free survival (PFS) can be assessed in an exploratory manner based on serial magnetic resonance imaging (MRI) scans.

[0456] 7.10.2.4 Tumor Tissue for Biological Correlation For patients with advanced non-brainstem tumors, biopsy / tumor debulking may be encouraged. When post-vaccination tumor tissue is available, GAA expression status and GAA-specific T cell infiltration can be analyzed.

[0457] 7.10.3 Patient Selection 7.10.3.1 Suitability criteria Pathological Criteria—Patients will have glioma. In some embodiments, glioma patients are in one of the following strata: (i) Stratum A: newly diagnosed diffuse intrinsic pontine glioma or any biopsy-diagnosed high-grade glioma involving the brainstem; (ii) Stratum B: newly diagnosed incompletely resected, non-brainstem high-grade glioma (i.e., residual tumor visible on imaging); or (iii) Stratum C: unresectable, progressive low-grade glioma of any subtype that has recurred despite two prior chemotherapy or biologic therapy regimens and / or radiation therapy; (iv) Stratum D: newly diagnosed diffuse intrinsic pontine glioma (DIPG) or any biopsy-diagnosed high-grade glioma involving the brainstem that has been treated with radiation therapy or without chemotherapy during radiation therapy. * , (v) Hierarchy E : Newly diagnosed non-brainstem high-grade gliomas treated without radiotherapy or intraradiotherapy chemotherapy * (HGG), (vi) Tier F: Recurrent non-brainstem high-grade glioma that recurred after treatment * Patients must have recovered from the toxic effects of previous treatment. Eligible histologies of high-grade gliomas include glioblastoma multiforme (GBM), anaplastic astrocytoma (AA), or gliosarcoma. Patients with any oligodendroglioma component may not be suitable for the specific protocol described in this example.

[0458] Patients in this study should be HLA-A2 positive based on flow cytometry.

[0459] Patients in strata A and B should receive standard-field RT, defined as fractionated external beam radiation therapy at a total dose between 5000-6000 cGy. Patients in these strata should be enrolled within 4-12 weeks of completing RT.

[0460] Patients in this study should be clinically stable and should be off low-dose (no more than 0.1 mg / kg / day, maximum 4 mg / day dexamethasone) corticosteroids at least 1 week before study enrollment.

[0461] Patients in this study should be aged 3 years and under 21 years at the time of the study.

[0462] Patients in this study should have a performance status (ps) of ≥ 50; (Karnofsky > 16 years of age, Lansky < 16 years of age or younger).

[0463] Postmenarcheal female patients in this study should have negative serum beta-HCG.

[0464] Patients in this study should be free of systemic infection. Patients on antibody therapy should not be taking antibiotics for at least 7 days before starting treatment.

[0465] Patients in this study should have adequate organ function as measured by: (i) bone marrow: ANC >1,000 / μL; platelets >100,000 / μL (transfusion independent); hemoglobin >8 g / dL (transfusion capable); (ii) liver: bilirubin ≤1.5 x institutional normal for age; SGPT (ALT) <3 x institutional normal and albumin ≥2 g / dL; (iii) kidney: serum creatinine or creatinine clearance based on age or radioisotope GFR ≥70 ml / min / 1.73 m 2 . Patients in this study should be within the normal range for their age and undergo coagulation tests as well as PT and PTT.

[0466] Patients in this study should be free of overt cardiac, gastrointestinal, pulmonary, or psychiatric disease.

[0467] For tier C patients, reversal of the effects of previous chemotherapy may be necessary.

[0468] 7.10.3.2 Exclusion criteria Patients in stratum A and stratum B of this study should be excluded if there is presence of leptomeningeal metastatic disease.

[0469] Patients in this study should be excluded if they had a whole, completely resected tumor, i.e., no residual disease visible on MRI scans at the time of the study.

[0470] Patients in stratum A and stratum B of this study should be excluded if they had received any type of antiglioma treatment other than previous chemotherapy or radiotherapy. The patient has previously received at least two chemotherapy or biotherapy regimens and / or radiation should be receiving therapy.)

[0471] Patients in this study should be excluded if they are receiving any concurrent treatments or medications, including radiation therapy; chemotherapy; interferon (e.g., Intron-A®); allergy desensitization injections; growth factors (e.g., Procrit®, Aranesp®, Neulasta®); interleukins (e.g., Proleukin®); and / or any investigational therapeutic agent.

[0472] Patients in this study should not have had a previous autoimmune disease requiring cytotoxic or immunosuppressive treatment, or an autoimmune disease associated with visceral damage. Patients in this study with active autoimmune disease requiring these treatments should also be excluded. Mild arthritis requiring NSAID medication should not be excluded.

[0473] Patients in this study should be excluded if they have been using or are scheduled to use immunosuppressants within 4 weeks prior to study entry. If dexamethasone or other corticosteroids are being used perioperatively and / or during radiation therapy (as little as 0.1 mg / kg / day, up to a maximum of 4 mg / day dexamethasone), they should be reduced by the patient at least 1 week prior to study enrollment. Topical corticosteroids should be tolerated.

[0474] Patients in this study should exclude those with known alcoholism or illicit drug use.

[0475] Immunocompromised patients are unlikely to respond to this treatment, and HIV-positive patients should be excluded from this study.

[0476] 7.10.4 Peptide vaccines 7.10.4.1 Peptides The following peptides: IL-13Rα2 345-353 1A9V (ALPFGFILV; SEQ ID NO: 3); EphA2 883-891 (TLADFDPRV; SEQ ID NO: 6); Survivin 96-104 :M2 (LMLGEFLKL; SEQ ID NO: 7); and tetanus TetA830 (AQYIKANSKFIGITEL; SEQ ID NO: 9) may be included in the vaccine formulation.

[0477] All peptides can be synthesized and purified by HPLC. The identity of the synthetic peptides can be confirmed by mass and amino acid sequence verification by mass spectrometry. Each peptide lot can be assessed for identity, purity, sterility, and pyrogenicity by FDA.

[0478] Peptides can be stored in vials under GMP conditions at -70°C. The stability of lyophilized peptides can be tested annually by mass spectrometry.

[0479] 7.10.4.2 Other Reagents Montanide ISA-51 (SEPPIC Inc., Fairfield, NJ) can be used as an additional agent in peptide vaccines.

[0480] 7.10.4.3 Dosage and Preparation Aqueous solutions (400 μL) containing all four HLA-A2-restricted GAA peptides (300 μg / peptide) and tetanus peptide (peptide-tet; 200 μg) each can be mixed with Montanide ISA-51 to form an oil-in-water emulsion (i.e., total volume / injection 800 μL).

[0481] 7.10.4.4 Administration Patients in this study will be vaccinated subcutaneously in the upper arm or thigh.

[0482] The vaccine was administered Q3Wk starting from 4-12 weeks after the end of RT (1 week).

[0483] 7.10.5 Poly-ICLC Poly-ICLC was prepared in a GMP facility at Bioserv, Corporation (San Diego, California). It can be prepared and packaged in-house. Poly-ICLC can be supplied in vials containing 1 cc of a translucent solution at a concentration of 2 mg / cc. Poly-ICLC is stable at room temperature for several days but can be stored frozen at approximately 4°C.

[0484] 7.10.5.1 Dosage and Administration The first period of poly-ICLC administration (30 μg / kg im) can be administered on the day of the first GAA / TT vaccination. After each repeat vaccination (Q3 weeks), poly-ICLC (30 μg / kg im) can be administered on the day after vaccination.

[0485] Regarding the injection site, poly-ICLC should be administered im close to the site of the previous peptide injection (e.g., less than 3 cm from the center of the previous peptide injection site) because it is believed to enhance the antigen presentation process in the draining axillary lymph nodes.

[0486] Poly-ICLC should be administered intramuscularly (i.m.) using aseptic technique when supplied from a vial at the prescribed dose per patient weight (maximum 1640 μg / injection). Vital signs can be monitored before the first treatment and for at least 20 minutes after treatment.

[0487] 7.10.6 Action Plan The study described in this example examined HLA-A2 expression in newly diagnosed intrinsic brainstem glioma (BSG) or biopsy-diagnosed GBM. + Three strata can be used to evaluate the immunogenicity, safety, and preliminary clinical efficacy of GAA / TT peptide vaccine and poly-ICLC in children with AA or brainstem-involving gliosarcoma (Strata A); incompletely resected non-brainstem GBM, AA, or gliosarcoma (Strata B); and recurrent progressive low-grade glioma (Strata C).

[0488] 7.10.6.1 Schedule Potentially eligible patients can be discussed for the study described in this example after diagnosis (stratum A and B) or disease progression (stratum C). All patients in stratum A and B can receive fractionated external beam radiation therapy (FEBRT). Patients can be assessed for HLA-A2 status. Eligibility screening and baseline MRI and laboratory studies should be completed within two weeks of study enrollment and within three weeks of the first vaccination. Patients in stratum A and B should enroll for study participation within 4-12 weeks after completing FEBRT. The timing of study enrollment for such patients depends on whether the post-RT MRI (typically performed at week 4) shows increased enhancement or signs of mass effect and the patient is clinically symptomatic / worsening. In that case, study enrollment will occur if the patient is clinically stable / improved and on low-dose (0.1 mg / kg / day up to 4 mg Decadron) or steroid-free for one week.

[0489] Patients can be treated with subcutaneous injections of the GAA / TT vaccine every 3 weeks for up to 8 cycles. On the same day as the vaccine, patients can be treated with intramuscular (im) poly-ICLC (30 μg / kg Each vaccine can be administered immediately prior to the administration of im poly-ICLC. Regarding the injection site, the poly-ICLC should be administered im close to the site of the previous peptide injection (e.g., less than 3 cm from the center of the previous peptide injection site).

[0490] Patients will be evaluated for any possible adverse events, RLT, as well as clinical / radiological response with clinic visits and MRI scans. Follow-up MRIs can be performed every 9 weeks starting at week 7 (weeks 7, 16, and 25).

[0491] Baseline samples can be PBMCs obtained before the first vaccination. Post-vaccination samples can be obtained at weeks 7, 16, and 25. Immunological assays can be performed on all PBMC samples obtained from at least one participant at a given time point, which will avoid inter-assay variability.

[0492] 7.10.6.2 Additional “sequential” treatments If, after the scheduled eighth vaccination, the patient shows stable disease without a radiological response (i.e., complete or partial response) or RLT, the patient can receive additional peptide vaccinations in combination with poly-ICLC starting six weeks after the eighth vaccination and every six weeks thereafter for up to two years from the first vaccination, barring tumor progression and RLT. Additional PBMC samples can be obtained at each vaccine administration visit for immunological monitoring. The booster vaccinations can be terminated in the event of 1) tumor progression, 2) RLT, or 3) patient dropout.

[0493] 7.10.6.3 Dose Modification 7.10.6.3.1 Dose Modifications for Poly-ICLC Pretreatment with acetaminophen or any NSAID should precede each poly-ICLC dose. For grade 2 or greater symptoms lasting more than 48 hours after injection, the next poly-ICLC dose should be 67% of the initial dose (i.e., 20 μg / kg). If further administration is discontinued, the initial dose can be subsequently resumed. If, despite a dose reduction, grade 2 or greater symptoms recur and persist for >48 hours, the patient may be withdrawn for RLT.

[0494] Liver enzyme assessment >5x baseline (grade 3) or any intolerable grade 2 non-hematologic toxicity lasting ≥7 days may result in poly-ICLC being withheld until the toxicity has decreased to grade 1 or below. Poly-ICLC may then be re-administered at 2 / 3 of the original dose (i.e., 20 μg / kg) and the patient may be closely observed. If the same dose-limiting toxicity reoccurs despite the reduced dose, the participant may be withdrawn by RLT.

[0495] For Grade 3 or higher hematologic toxicity, the next dose should be reduced by 67% (i.e., 20 μg / kg) unless the toxicity has resolved to Grade 1 or lower by the time the next dose is given. If the toxicity has not resolved by the time the next dose is given, the patient will not be treated. If the same dose-limiting hematologic toxicity reoccurs despite the dose reduction, the patient may be discontinued with RLT.

[0496] 7.10.6.3.2 Delayed Dosing for Peptide Vaccines In situations where poly-ICLC administration is suspended, if the event is not attributable to the peptide / ISA-51 vaccine, vaccination should continue as scheduled. In situations where adverse event assessment is limited, such as when intercurrent illness or laboratory evaluation is required for other causes of toxicity, the vaccination schedule may be interrupted for up to 6 weeks. If vaccination is delayed for more than 6 weeks due to an adverse event other than pseudotumor progression, regardless of cause, vaccination should be discontinued. If this occurs, treatment should be discontinued.

[0497] Patients may be monitored for regimen-defined toxicities (RLTs) throughout the study. The following are considered RLTs if they are judged to be probably, probably, or definitely related to treatment. If they occur, the individual patient will be removed from the study and will not receive further injections.

[0498] ≥ Grade 2 or higher bronchospasm or generalized urticaria (hypersensitivity)

[0499] Grade 2 or higher: Allergic reactions such as exfoliative erythroderma, anaphylaxis, or vascular collapse

[0500] ≥ Grade 3 Any ≥ Grade 3 possibly, probably, or definitely related to the treatment regimen, including injection site reactions due to peptide vaccine or poly-ICLC administration Non-hematologic toxicity (excluding hepatotoxicity)

[0501] ≥ Grade 3 Hematologic or hepatic toxicity that recurs despite a 33% dose reduction or does not resolve to Grade 1 or below by the next due date

[0502] Intolerable Grade 2 non-hematologic toxicity lasting >7 days despite a 33% dose reduction or not resolving to Grade 1 or below by the next due date

[0503] Grade 2 or greater systemic symptoms persisting for >48 hours despite dose reduction

[0504] ≥ Grade 3 neurotoxicity due to regimen-related inflammatory immune response (i.e., pseudotumor progression unresponsive to a 7-day trial of 0.3 mg / kg / day Decadron (maximum 12 mg / day) and / or requiring debulking surgery, if possible)

[0505] ≥ Grade 3 Nausea and vomiting despite adequate antiemetic prophylaxis

[0506] Delayed dosing >6 weeks for poly-ICLC or peptide vaccine due to toxicity other than PTP

[0507] Treatment may be interrupted for the following reasons: (i) regimen-limiting toxicity other than PTP as defined above; (ii) disease progression - at least a 25% increase in the product of the largest tumor diameter and its perpendicular diameter on MRI scan; (iii) intercurrent illness of 6 weeks or more that prevents further vaccination or poly-ICLC administration; (iv) pregnancy: pregnant patients will continue to be followed for the duration of the pregnancy.

[0508] 7.10.6.4 Treatment duration If there are no treatment delays due to adverse events, treatment may continue for 25 weeks (8 vaccinations followed by a visit at 25 weeks) until one of the Off-Treatment events in section 7.10.6.3.2 occurs.

[0509] 7.10.6.5 Concurrent Actions 7.10.6.5.1 Tolerance Patients should receive a dose of acetaminophen (15 mg / kg up to a maximum of 1000 mg) 30-60 minutes before each poly-ICLC injection. For fever after injection, acetaminophen (15 mg / kg up to a maximum of 1000 mg every 4-6 hours, as needed, not to exceed 4 g / day) can be administered. Patients with fever lasting more than 48 hours should be considered for possible infection. This should be evaluated.

[0510] For mild localized pain, oral opiates (Tylenol and Codey 0.5 mg / kg) Oral, every 4 hours) can be used. Pain of mild-moderate or higher grade will be investigated for non-treatment related causes and managed accordingly.

[0511] Dexamethasone - as little as 0.1 mg / kg / day, up to a maximum of 4 mg / day, for at least 1 week before starting vaccine / poly-ICLC treatment (week 0). The dexamethasone dose may be increased in the event of pseudotumor progression and reduced / interrupted as soon as possible.

[0512] Anticonvulsants should be used as indicated.

[0513] Antiemetics can be administered if necessary.

[0514] Other acceptable medications include topical steroids; nonsteroidal anti-inflammatory drugs; antihistamines (e.g., Claritin®, Allegra®); chronic medications except those listed in Section 7.10.6.5.2; and / or influenza vaccines (which should be administered at least 2 weeks before the start of study vaccinations or at least 2 weeks after the 8th vaccination).

[0515] 7.10.6.5.2 Unacceptable Unacceptable treatments include interferon treatment (e.g., Intron-A®); chemotherapy; allergy desensitization injections; corticosteroids administered orally or by inhalation (e.g., Advair®, Flovent®, Azmacort®); growth factors (e.g., Procrit®, Aranesp®, Neulasta®); interleukins (e.g., Proleukin®); other investigational drugs and / or illicit drugs.

[0516] 7.10.7 Correlation / Special Studies 7.10.7.1 Immunological surveillance 7.10.7.1.1 ELISPOT Assay The frequency of glioma-associated antigen (GAA)-reactive T lymphocyte precursors in peripheral blood mononuclear cells (PBMCs) before and after GAA vaccination can be measured by ELISPOT assay. The biological response measured by ELISPOT should be performed on at least the same time point in an individual to avoid inter-assay variability. Successful vaccination stimulates a clonal population of T cells capable of secreting cytokines in an antigen-specific, MHC-restricted manner. ELISPOT assays are used to measure CD8 + CD4 T cell populations reacting to the helper TT peptide as well as GAA-specific immune responses + It can be used to assess T cells. IFN-γ production can be assessed by assessing type 1 T cell responses.

[0517] A subject is considered to have responded if, at any time point after two consecutive vaccinations against the same antigen (weeks 12, 15, 18, 21, and 24), the number of spots doubles at baseline, there are at least 10 spots / 20,000 cells, and the number of spots after vaccination is at least three standard deviations above the pre-vaccination value. The response can be to any one of the antigens.

[0518] 7.10.7.1.2 Tetramer Analysis of GAA-Responsive T Cells in Patient PBMCs Tetramer analysis revealed that GAA-specific CD8 + It allows the assessment of the presence of T cells with high sensitivity and without in vitro restimulation of the cells. Available for use in patients with malignant glioma. Based on previous data, peptide-responsive CD8 T cells may be observed in some, but not all, patients immunized with tumor antigen vaccines. + A significant (log or greater) increase in T cell frequency is predicted. Preliminary methods also assess the surface expression of the integrin receptor very late antigen (VLA) 4, which has been implicated in T cell homing to CNS tumors, and chemokine receptors (e.g., CXCR3 and CCR5) in the PBMCs (see, e.g., Zhu et al., J. Transl. Med., 5: 10, 2007). Tetramer analysis procedures are well established.

[0519] Tetramer assays can be performed at baseline and at five time points after vaccination (12, 15, 18, 21, and 24 weeks). Total CD8+ by tetramer assay + A single-time point positive response for a peptide can be defined as (1 + B)% of cells positive, where B is the percent positive at baseline, which is usually considered to be less than 0.1%. Similar to the definition of an ELISPOT response, a patient is considered to have responded if he / she has two consecutive single-time point responses to any peptide.

[0520] 7.10.7.1.1 Flow Cytometric Analysis of Lymphocyte Populations At consecutive time points before and after vaccination, CD4 + and CD8 + Like T cell counts, CD4 + / Foxp3 + The number of T regulatory cells can be assessed.

[0521] 7.10.7.2 Evaluation of Primary and Recurrent Tumor Tissue GAA expression in patients' available tumor tissue can be assessed by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) (pre-vaccination or post-vaccination, respectively, or both).

[0522] When tumors recur after vaccination, it may be important to assess how the tumor escapes the vaccine's effects. To achieve this goal, as well as assessing tissue availability, the following specific aspects can be evaluated: (i) antigen loss: IHC and RT-PCR can be used to assess whether recurrent tumors express target GAAs, HLA-A2, and antigen-processing machinery components, such as transporters involved in antigen processing; (ii) anti-apoptotic up-regulated molecules: Survivin may be targeted, but other anti-apoptotic molecules may be up-regulated, such as cFLIP (intracellular FLICE (Fas-associated death domain-like IL-1β-converting enzyme) inhibitory protein); and (iii) immune cell infiltration: One reason tumors may escape vaccine-induced immune responses is the failure of reactive T cells to infiltrate the tumor. To address this issue, when freshly resected tumor tissue (not fixed or frozen) is available, tumor-infiltrating lymphocytes (TILs) can be isolated and characterized for their number, phenotype, and antigen specificity using HLA-A2 tetramers for each GAA. Using multicolor flow cytometry, tetramer +The function and survival of TILs can be determined by staining for perforin / IFN-γ and annexin V, respectively. Control tissues can include pre-vaccine tumors (if available) and recurrent tumors from patients not in this study. This study will assess whether vaccine-induced T cells can efficiently traffic to the brain tumor site and maintain their function and survival.

[0523] 7.10.8 Study parameters The study can be conducted as an outpatient setting, with patients scheduled to be evaluated every 3 weeks for up to eight vaccinations. If patients receive additional vaccinations every 6 weeks as part of a continuation phase, clinical, immunological, and radiological (MRI) monitoring may be performed at every visit (Q6Wk) until one of the criteria for ending treatment is met. Vaccinations may be stopped at any time during the schedule for any patient with progressive disease or unacceptable toxicity. ...

Claims

1. 1. A combination pharmaceutical for treating or preventing brain cancer in a subject in need thereof, comprising: The pharmaceutical is a combination pharmaceutical comprising (i) a cell-free pharmaceutical composition comprising an IL-13Rα2 peptide, an EphA2 peptide, a YKL-40 peptide, and a GP100 peptide, in combination with (ii) bevacizumab.

2. The combined pharmaceutical composition of claim 1, wherein the IL-13Rα2 peptide comprises any one of SEQ ID NOs: 1-4, the EphA2 peptide comprises SEQ ID NO: 6, the YKL-40 peptide comprises SEQ ID NO: 10, and the GP100 peptide comprises SEQ ID NO:

11.

3. The pharmaceutical combination of claim 1 or 2, further comprising an adjuvant.

4. 4. The pharmaceutical combination of claim 3, wherein the adjuvant is Montanide ISA-51.

5. The pharmaceutical combination according to any one of claims 1 to 4, further comprising a helper T cell epitope.

6. The combined pharmaceutical composition according to claim 5, wherein the helper T cell epitope is a pan-DR epitope peptide, a tetanus toxoid peptide, or an HBV128-140 core peptide.

7. The pharmaceutical combination of any one of claims 1 to 6, formulated for subcutaneous or intranodal administration.

8. The pharmaceutical combination of any one of claims 1 to 7, comprising 250-400 μg of an IL-13Rα2 peptide and 250-400 μg of an EphA2 peptide.

9. 1. A combination pharmaceutical for treating or preventing brain cancer in a subject in need thereof, comprising:

10. A pharmaceutical combination comprising: (i) a first pharmaceutical composition comprising the pharmaceutical composition of any one of claims 1-8; (ii) a second pharmaceutical composition comprising an immune response modifier; and (iii) bevacizumab.

10. The combination pharmaceutical of claim 9, wherein the immune response modifier is poly-ICLC or imiquimod.

11. The combined pharmaceutical composition of any one of claims 1 to 10, wherein the subject is a human.

12. The combined pharmaceutical composition of any one of claims 1 to 11, wherein the brain cancer is glioblastoma.

13. The pharmaceutical combination of any one of claims 1-12, wherein the subject has failed previous temozolomide, radiation therapy, and surgery.

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