WO2015167748A1 - Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers - Google Patents

Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers Download PDF

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WO2015167748A1
WO2015167748A1 PCT/US2015/024048 US2015024048W WO2015167748A1 WO 2015167748 A1 WO2015167748 A1 WO 2015167748A1 US 2015024048 W US2015024048 W US 2015024048W WO 2015167748 A1 WO2015167748 A1 WO 2015167748A1
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another embodiment
neu
recombinant
recombinant attenuated
radiation therapy
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PCT/US2015/024048
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French (fr)
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Yvonne Paterson
Nicola Mason
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The Trustees Of The University Of Pennsylvania
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Priority claimed from US14/268,436 external-priority patent/US20140234370A1/en
Priority claimed from PCT/US2015/017559 external-priority patent/WO2015130810A2/en
Priority claimed from US14/669,629 external-priority patent/US10016617B2/en
Priority to EP15786740.9A priority Critical patent/EP3137107A4/en
Priority to SG11201609135VA priority patent/SG11201609135VA/en
Priority to KR1020167033949A priority patent/KR20170002552A/en
Application filed by The Trustees Of The University Of Pennsylvania filed Critical The Trustees Of The University Of Pennsylvania
Priority to CA2947677A priority patent/CA2947677A1/en
Priority to CN201580036168.0A priority patent/CN106794234A/en
Priority to AU2015253737A priority patent/AU2015253737A1/en
Priority to JP2017509588A priority patent/JP2017514904A/en
Priority to MX2016014367A priority patent/MX2016014367A/en
Publication of WO2015167748A1 publication Critical patent/WO2015167748A1/en
Priority to TW105109639A priority patent/TW201707715A/en
Priority to IL248704A priority patent/IL248704A0/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Definitions

  • This invention provides methods for inducing an immune response against a Her- 2/neu antigen-expressing tumor and for treating the same and vaccinating against the same in human and canine subjects using a combination of radiation therapy and a recombinant attenuated Listeria strain vaccine.
  • Her-2/neu (referred to henceforth as "Her-2") is a 185 kDa glycoprotein that is a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinases, and is overexpressed in 25 to 40% of all breast cancers and in many cancers of the bone (osteosarcoma - OSA), ovaries, lung, pancreas, brain, and gastrointestinal tract. Patients with cancers that overexpress Her-2 exhibit tolerance even with detectable humoral, CD8 + T cell, and CD4 + T cell responses directed against Her-2.
  • EGFR epidermal growth factor receptor
  • RT Radiation therapy
  • RT may synergize with targeted immune therapy.
  • RT induces immunogenic cell death wherein tumor cells die slowly over time from apoptosis, necrosis and/or mitotic catastrophe, leading to the clearance of the dying cells by the immune system.
  • This serves as a potential source of tumor antigens for immune therapy.
  • RT also modulates tumor cell surface expression of cell death receptors, tumor-associated antigens and adhesion molecules, which render the tumor cells more susceptible to immune-mediated killing.
  • the present invention meets the needs of subjects suffering from OSA with surprising findings that radiation therapy when combined with a recombinant Listeria-Her- 2/neu vaccine (ADXS31-164) that was generated using the LmddA vaccine vector which has a well-defined attenuation mechanism and is devoid of antibiotic selection markers is particularly effective against osteosarcoma and pulmonary metastasis.
  • the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the subject is a human. In one embodiment, the human subject is a child. In another embodiment, the human subject is an adult. In another embodiment, the subject is a canine.
  • administering said fusion polypeptide to said subject prevents escape mutations within said tumor.
  • said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes. In another embodiment, said Her- 2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the canine MHC-class I epitopes.
  • the nucleic acid molecule is integrated into the Listeria genome.
  • the nucleic acid molecule is in a plasmid in said recombinant Listeria vaccine strain and the plasmid is stably maintained in the recombinant Listeria vaccine strain in the absence of antibiotic selection.
  • the recombinant Listeria lacks the actA virulence gene.
  • the additional polypeptide is selected from the group consisting of: a) nonhemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment.
  • the metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.
  • a recombinant attenuated Listeria strain is ADXS31-164.
  • the recombinant attenuated Listeria strain is administered with an independent adjuvant, which, in one embodiment, comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
  • an independent adjuvant which, in one embodiment, comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • the cancer is osteosarcoma (OSA).
  • the cancer or tumor is pulmonary metastatic disease.
  • administration comprises at least two administrations of said recombinant attenuated Listeria strain.
  • the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • provided herein is a combination therapy comprising a radiation therapy and administration of ADXS31-164 provided herein.
  • the radiation therapy is administered prior to administration of the recombinant attenuated Listeria strain.
  • the subject does not undergo amputation prior to administration of said radiation therapy and said recombinant attenuated Listeria strain.
  • the method further comprises administering said radiation therapy and said recombinant attenuated Listeria strain following a relapse or metastasis in said subject, which in one embodiment, is pulmonary metastatic disease.
  • the method results in increased overall survival of said subject. In another embodiment, the method results in a delay of metastatic disease in a subject. In another embodiment, the method results in an increased Her-2/neu specific T cell response.
  • said elicitation of an enhanced immune response results in increased overall survival of said subject. In another embodiment, said elicitation of an enhanced immune response results in a delay of metastatic disease in a subject. In one embodiment, the metastatic disease is pulmonary metastatic disease. In another embodiment, said elicitation of an enhanced immune response results in an increased Her-2/neu specific T cell response.
  • Figure 1 Construction of ADXS31-164.
  • A Plasmid map of pAdvl64, which harbors bacillus subtilis dal gene under the control of constitutive Listeria p60 promoter for complementation of the chromosomal dal-dat deletion in LmddA strain. It also contains the fusion of truncated LLO ( i_44i) to the chimeric human HER2/neu gene, which was constructed by the direct fusion of 3 fragments the HER2/neu: ECl (aa 40-170), EC2 (aa 359-518) and ICI (aa 679-808).
  • the vector schematic on the right shows details pAdvl64 expressing a chimeric HER2/neu fusion protein consisting of 2 extracellular domains and one intracellular domain of human HER2/neu fused to truncated LLO.
  • the plasmid is maintained within the recombinant dal/dat/ actA " listeria strain (LmddA) by means of auxotrophic complementation of the dal gene (See Examples).
  • ChHer2 The endogenous LLO is detected as a 58 KD band. Listeria control lacked ChHer2 expression.
  • FIG. 1 Immunogenic properties of ADXS31-164
  • A Cytotoxic T cell responses elicited by HER2/neu Listeria-b&sed vaccines in splenocytes from immunized mice were tested using NT-2 cells as stimulators and 3T3/neu cells as targets. Lm-control was based on the LmddA background that was identical in all ways but expressed an irrelevant antigen (HPV16-E7).
  • B IFN- ⁇ secreted by the splenocytes from immunized FVB/N mice into the cell culture medium, measured by ELISA, after 24 hours of in vitro stimulation with mitomycin C treated NT-2 cells.
  • C IFN- ⁇ secretion by splenocytes from HLA-A2 transgenic mice immunized with the chimeric vaccine, in response to in vitro incubation with peptides from different regions of the protein.
  • a recombinant ChHer2 protein was used as positive control and an irrelevant peptide or no peptide groups constituted the negative controls as listed in the figure legend.
  • IFN- ⁇ secretion was detected by an ELISA assay using cell culture supernatants harvested after 72 hours of co-incubation. Each data point was an average of triplicate data +/- standard error. * P value ⁇ 0.001.
  • FIG. 4 Effect of immunization with ADXS31-164 on the % of Tregs in Spleens.
  • FVB/N mice were inoculated s.c. with 1 x 10 6 NT-2 cells and immunized three times with each vaccine at one week intervals. Spleens were harvested 7 days after the second immunization. After isolation of the immune cells, they were stained for detection of Tregs by anti CD3, CD4, CD25 and FoxP3 antibodies, dot-plots of the Tregs from a representative experiment showing the frequency of CD25 + /FoxP3 + T cells, expressed as percentages of the total CD3 + or CD3 + CD4 + T cells across the different treatment groups.
  • FIG. 5 Effect of immunization with ADXS31-164 on the % of tumor infiltrating Tregs in NT-2 tumors.
  • FVB/N mice were inoculated s.c. with 1 x 10 6 NT-2 cells and immunized three times with each vaccine at one week intervals. Tumors were harvested 7 days after the second immunization. After isolation of the immune cells, they were stained for detection of Tregs by anti CD3, CD4, CD25 and FoxP3 antibodies.
  • A dot- plots of the Tregs from a representative experiment.
  • B dot- plots of the Tregs from a representative experiment.
  • Frequency of CD25 + /FoxP3 + T cells expressed as percentages of the total CD3 + or CD3 + CD4 + T cells (left panel) and intratumoral CD8/Tregs ratio (right panel) across the different treatment groups. Data is shown as mean+SEM obtained from 2 independent experiments.
  • FIG. 6 Vaccination with ADXS31-164 can delay the growth of a breast cancer cell line in the brain.
  • Balb/c mice were immunized thrice with ADXS31-164 or a control Listeria vaccine.
  • EMT6-Luc cells (5,000) were injected intracranially in anesthetized mice.
  • A Ex vivo imaging of the mice was performed on the indicated days using a Xenogen X-
  • Figure 7 Shows the first 18 canine osteosarcoma patients vaccinated with ADXS31- 164, following amputation and chemotherapy.
  • FIG. 8 Shows that ADXS31-164 administration does not cause A) early evidence of dilated cardiomyopathy.
  • Figure 9 Shows ADXS31-164 associated changes in A) body temperature and B) systolic blood pressure. Body temperature and systolic blood pressure were recorded at baseline and every 2 hours post ADXS31-164 administration. Parameters for each dog at each vaccination are displayed. Horizontal bars represent median values for all dogs in each dose group at each time point. *p ⁇ 0.05, ** p ⁇ 0.005 [0031] Figure 10. Shows treatment schedule of combination ADXS31-164 and palliative radiation therapy (RT) in the context of primary appendicular osteosarcoma without amputation and chemotherapy.
  • RT palliative radiation therapy
  • Top panel Radiographs showing the presence of a fracture of the proximal humerus associated with osteosarcoma and those taken after fracture fixation using two bone plates and an intramedullary pin.
  • Bottom panel A CT scan of the chest shoing no evidence of metastatic disease at enrollment. Radiographs were also taken at baseline and after 8 ADXS31-164 administrations. These radiographs show no evidence of pulmonary metastatic disease and the presence of boney callus surrounding the fracture site indicating fracture healing despite the presence of osteosarcoma.
  • Figure 12 Timeline of a pilot phase I clinical trial to evaluate the safety and efficacy of a L. monocytogenes recombinant expressing ADXS31-164 to elicit therapeutically effective anti-tumor immunity in dogs with appendicular osteosarcoma, that undergo limb amputation and follow up chemotherapy.
  • FIG. 13A shows treatment-related adverse events.
  • Figure 13B shows all dogs without metastatic disease at the time of trial enrollment. Dogs in the control group underwent limb amputation followed by either carboplatin alone or carboplatin plus Adriamycin. 2 dogs have been censored from the vaccine arm as they died of unrelated causes (1 dog died from aspiration pneumonia, the other died from nephroblastoma).
  • FIG. 14 Radiographic images of primary and metastatic osteosarcoma (OSA) in a human (A) and canine (B) patient. In both species, primary lesions are characterized by areas of proliferation and lysis in the bone metaphysis (arrows in A).
  • OSA primary and metastatic osteosarcoma
  • Figure 15 Schematic of the phase I, 3+3 clinical trial to evaluate the safety and efficacy of ADXS31-164 in dogs with HER2+ osteosarcoma (OSA).
  • OSA HER2+ osteosarcoma
  • Privately owned dogs with spontaneous HER2+ appendicular OSA underwent standard of care amputation and follow up carboplatin chemotherapy.
  • FIG. 16 HER2/neu expression in canine primary osteosarcoma.
  • A H&E stain of primary OSA from a dog showing nests of malignant osteoblasts and osteoid deposition.
  • B Immunohistochemical evaluation of canine primary OSA showing HER2/neu expression within malignant osteoblasts.
  • C Western blot of primary OSA samples from 5 privately owned dogs showing variable expression of HER2/neu. Positive controls are: MCF-7 human mammary carcinoma cell line andCAMAC2 a canine mammary carcinoma cell line.
  • FIG. Hematological values at baseline (Pre) and at 24 hours after (Post) ADXS31-164 administration. Pre and Post values from all dogs within each dose group at each vaccination were averaged. *p ⁇ 0.05, ** p ⁇ 0.005. Shows a transient, but statistically significant increase in white blood cell and neutrophil counts (A-B) that occurred 24 hours after ADXS31-164 administration and that were accompanied by a transient decrease in platelets and lymphocytes (C-D).
  • FIG. ADXS31-164 induced increases in white blood cells (WBC), neutrophil and monocyte counts correlate with survival. WBC, neutrophil and monocyte counts were measured at baseline and 24 hours after vaccination. The percent increase was calculated following each vaccination and averaged for each dog.
  • A Results are displayed according to survival (dead or alive).
  • B Results are displayed according to ADXS31-164 dose received. Horizontal bars represent median values of the group.
  • Figure 19 Shows the results of evaluation of Her-2 specific T cell responses induced by ADXS31-164 by IFN- ⁇ ELISpot.
  • Figure 20 Shows repeat "booster” vaccinations Stimulate Her-2 specific immunity.
  • A Shows the results for patient 289-003.
  • B Shows the results for patient 289-004.
  • ECl, EC2 and IC1 represent the peptide fragments of the HER2/neu polypeptide.
  • FIG. 21 Kaplan Meier estimates for (A) Time To Metastasis (TTM) and (B) OS A
  • FIG. 22 Shows that ADXS31-164 prevents development of metastatic disease.
  • a and B Thoracic radiographs taken 3 weeks after carboplatin therapy (A) and 3 weeks after the third ADXS31-164 vaccine (B) showing an increase in size of the pre-existing metastatic nodule in the right cranial lung lobe but lack of further metastatic disease development in remaining lung lobes.
  • C and D Pulmonary nodule identified on thoracoscopy that fluoresces under near infra-red light following administration of ICG (C). Grossly normal appearing pulmonary tissue removed at the time of metastatectomy showing fluorescence under near infra-red light (inset) (D).
  • E and F H&E stained histopathology of (E) pulmonary nodule and (F) fluorescing normal pulmonary tissue showing significant hemorrhage and necrosis of encapsulated pulmonary nodule (E) and focal area of inflammation in grossly normal appearing pulmonary tissue (F).
  • G and H Immunohistochemistry of pulmonary nodule at low (G) and high (H) magnification showing CD3+ T cells surrounding the pulmonary nodule with minimal CD3+ T cells within the neoplastic tissue.
  • I and J Immunohistochemistry of normal appearing pulmonary tissue at low (I) and high (J) magnification showing focal accumulations of CD3+ T cells.
  • FIG. 23 ADXS31-164 delays/prevents metastatic disease and prolongs overall survival in dogs with spontaneous HER2+ osteosarcoma. Shown is a Kaplan-Meier survival curve of vaccinated dogs compared with a historical control group. The control group consisted of dogs with HER2+ appendicular OSA, treated with amputation and follow-up chemotherapy but who did not receive ADXS31-164. P ⁇ 0.0001. Vaccinated group Red line; Control group Black line.
  • FIG. 24 Shows that ADXS31-164 breaks tolerance to HER2/neu.
  • PBMCs were collected at baseline, 3 weeks after the 3 rd vaccine (9 weeks) and 2 months later (17 weeks) and analyzed by IFN- ⁇ ELISpot for responses to the highly conserved ICl domain of HER2/neu. Results presented divided dogs into early responders, late responders and apparent non-responders. NA indicates that the 17 week sample for these dogs was not yet evaluated.
  • FIG 25A-D Shows that ADXS31-164 does not adversely affect cardiac function.
  • Cardiac parameters LVID (diastole) ( Figure 25A), LVH) (systole) ( Figure 25B) and fractional shortening (Figure 25C) were evaluated for each dog at baseline, the time of vaccination and every 2 months thereafter.
  • Cardiac troponin I levels were evaluated at the same time points (Figure 25D).
  • Figure 26 Shows that ADXS31-164 breaks immune tolerance to the highly conserved intracellular domain of HER2/neu.
  • FIG 27A-D Shows that radiation therapy in conjunction with ADXS31-164 therapy delays progression of primary osteosarcoma (OSA) in subject 386-002 (Figure 27 A), subject 385-005 (Figure 27B), subject 386-003 (Figure 27C), and subject 386-007 (Figure 27D).
  • OSA primary osteosarcoma
  • Figure 28 A-C Shows the results of a pain questionnaire in subjects with pain interfering with general activity (Figure 28 A), in subjects with pain interfering with the ability to walk ( Figure 28B), and in subjects with pain interfering with the enjoyment of life
  • Figure 29A-D Shows that palliative radiation therapy in conjunction with ADXS31- 164 therapy reduces lysis, promotes tumor consolidation, and prolongs survival of subjects (Figure 29 A-D).
  • FIG 30A-B Shows that radiation therapy in conjunction with ADXS31-164 prolongs survival in dogs with osteosarcoma (OSA).
  • the present invention provides a method of treating a tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the present invention provides a method of preventing a tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the present invention provides a method of eliciting an enhanced immune response against a tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the present invention provides a method of prolonging survival in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the present invention provides a method of delaying metastatic disease in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the present invention provides a method of breaking tolerance to a tumor specific antigen in a subject suffering from a tumor growth or cancer expressing said tumor specific antigen comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the tumor specific antigen is Her-2/neu.
  • the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
  • the present invention provides a method of preventing a Her- 2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
  • the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
  • the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
  • the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
  • the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant.
  • the recombinant attenuated Listeria strain further comprises a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of preventing a Her- 2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
  • the subject is a human. In one embodiment, the human subject is a child. In another embodiment, the human subject is an adult.
  • the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a human subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of preventing a Her-
  • 2/neu-expressing tumor growth or cancer in a human subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a human subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of prolonging survival in a human subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of delaying metastatic disease in a human subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of breaking tolerance to Her-2/neu in a human subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the subject is a canine.
  • the canine is a dog.
  • the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a canine subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of preventing a Her-
  • 2/neu-expressing tumor growth or cancer in a canine subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a canine subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of prolonging survival in a canine subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of delaying metastatic disease in a canine subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of breaking tolerance to Her-2/neu in a canine subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the present invention provides a method of delaying metastatic disease or treating metastatic disease in a subject.
  • the metastatic disease is pulmonary metastatic disease.
  • the present invention provides a method of delaying pulmonary metastatic disease in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • the present invention provides a method of treating pulmonary metastatic disease in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
  • provided herein are methods for preventing, treating, prolonging survival, delaying metastatic disease, breaking tolerance to Her-2/neu, vaccinating against a Her2-neu antigen-expressing tumor, inducing an immune response, eliciting an enhanced immune response against sub-dominant epitopes of the Her2-neu antigen, while circumventing mutation avoidance.
  • the administration of the fusion polypeptide of the present invention to the subject prevents escape mutations within said tumor.
  • circumventing mutation avoidance is due to epitope spreading.
  • mutation avoidance is due to the chimeric nature of the antigen.
  • an immunogenic composition for use in the claimed methods comprising a fusion polypeptide, wherein said fusion polypeptide comprises a Her-2/neu chimeric antigen fused to an additional polypeptide, and wherein administering the fusion protein to a subject having an Her-2/neu-expressing tumor prevents escape mutations within said tumor.
  • a recombinant Listeria vaccine strain for use in the claimed methods comprising the immunogenic composition.
  • the recombinant attenuated Listeria strain is a vaccine strain.
  • the nucleic acid referred to herein is a nucleic acid molecule.
  • the recombinant attenuated Listeria strain for use in the methods of the present invention further comprises a nucleic acid molecule comprising a third open reading frame encoding a metabolic enzyme, and wherein the metabolic enzyme complements an endogenous gene that is mutated in the chromosome of the recombinant Listeria strain.
  • a recombinant attenuated Listeria strain comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her-2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second and a third open reading frame, each encoding a metabolic enzyme, and wherein the metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant Listeria strain.
  • the nucleic acid molecule is integrated into the Listeria genome.
  • the nucleic acid molecule is in a plasmid in the recombinant Listeria vaccine strain.
  • the plasmid is stably maintained in the recombinant Listeria vaccine strain in the absence of antibiotic selection.
  • the plasmid does not confer antibiotic resistance upon the recombinant Listeria.
  • the recombinant Listeria strain is attenuated.
  • the recombinant Listeria is an attenuated auxotrophic strain.
  • the high metabolic burden that the expression of a foreign antigen exerts on a bacterium such as one of the present invention is also an important mechanism of attenuation.
  • the attenuated strain is LmddA.
  • this strain exerts a strong adjuvant effect, which is an inherent property of Listeria-b&sed vaccines.
  • One manifestation of this adjuvant effect is the 5-fold decrease in the number of the intratumoral Tregs caused by either Listeria expressing an antigen other than a human chimeric Her-2/neu or the ADXS-31-164 (expressing a human chimeric Her-2/neu) vaccines (see Figure 5 herein).
  • the LmddA vector expressing a different antigen HPV16 E7 is also associated with a significant decrease in the frequency of Tregs in the tumors, likely as a consequence of innate immunity responses.
  • the LmddA vector expresses a prostate-specific antigen (PSA), a human papilloma virus (HPV) antigen (E6, E7).
  • PSA prostate-specific antigen
  • HPV human papilloma virus
  • the HPV strain is HPV16, HPV18, or any strain known in the art.
  • the attenuated auxotrophic Listeria vaccine strain is the ADXS- 31-164 strain.
  • ADXS-31-164 is based on a Listeria vaccine vector which is attenuated due to the deletion of virulence gene actA and retains the plasmid for Her-2/neu expression in vivo and in vitro by complementation of dal gene.
  • ADXS31-164 expresses and secretes the chimeric Her-2/neu protein fused to the first 441 amino acids of listeriolysin
  • ADXS31-164 exerts strong and antigen specific antitumor responses with ability to break tolerance toward Her-2/neu in transgenic animals (see Examples).
  • the ADXS31-164 strain is highly attenuated and has a better safety profile than previous Listeria vaccine generations, as it is more rapidly cleared from the spleens of the immunized mice.
  • the ADXS31-164 results in a longer delay of tumor onset in transgenic animals than Lm-LLO-ChHer2, the antibiotic resistant and more virulent version of this vaccine (see Figure 3).
  • the Zm-LLO-ChHer2 strain is Lm-LLO-138.
  • ADXS31-164 strain is highly immunogenic, able to break tolerance toward the Her-2/neu self-antigen and prevent tumor formation in Her-2/neu transgenic animals.
  • ADXS31-164 causes a significant decrease in intra- tumoral T regulatory cells (Tregs).
  • Tregs intra- tumoral T regulatory cells
  • the lower frequency of Tregs in tumors treated with LmddA vaccines resulted in an increased intratumoral CD8/Tregs ratio, suggesting that a more favorable tumor microenvironment can be obtained after immunization with LmddA vaccines.
  • this chimeric antigen does not result in escape mutations indicating that tumors do not mutate away from a therapeutic efficacious response to treatment with this novel antigen (see Example 6).
  • peripheral immunization with ADXS31-164 delays the growth of a metastatic breast cancer cell line in the brain (see Example 7).
  • canine subjects suffering from osteosarcoma and provided treatment including amputation, chemotherapy, and vaccination with ADXS31-164 have prolonged survival compared with control subjects not receiving the vaccination with ADXS31-164 (see Examples 9 and 10).
  • canine subjects suffering from osteosarcoma and provided treatment including amputation, chemotherapy, and vaccination with ADXS31-164 show reduced metastasis compared with control subjects not receiving the vaccination with ADXS31-164 (see Example 10).
  • canine subjects suffering from osteosarcoma and provided treatment including amputation, chemotherapy, and vaccination with ADXS31-164 show increased specific T cell response induced compared with control subjects not receiving the vaccination with ADXS31-164 (see Example 10).
  • canine subjects suffering from osteosarcoma and provided radiation therapy prior to vaccination with ADXS31-164 have prolonged survival compared with control subjects receiving either only radiation therapy or only vaccination with ADXS31-164 (see Example 11).
  • canine subjects suffering from osteosarcoma and provided radiation therapy prior to vaccination with ADXS31-164 show reduced metastasis compared with control subjects receiving either only radiation therapy or only vaccination with ADXS31-164 (see Example 11).
  • ADXS31-164 Lm-human chimeric Her- 2/neu
  • Zm-huHer2-neu Zm-hucHer-2/neu
  • osteosarcoma cells are not easily killed by radiation, so radiation therapy is rarely used to treat osteosarcoma.
  • recombinant attenuated, antibiotic -free L/siena-expressing chimeric antigens are useful for preventing, and treating a cancer or solid tumors, as exemplified herein.
  • the tumor is a Her-2/neu positive tumor.
  • the cancer is a Her-2/neu- expressing cancer.
  • the cancer is breast cancer, a central nervous system (CNS) cancer, a head and neck cancer, an osteosarcoma (OSA), a canine OSA,
  • Ewing's sarcoma ES
  • a canine osteosarcoma is an appendicular osteosarcoma.
  • the tumor is an osteosarcoma tumor, a breast tumor, a head and neck tumor, or any other antigen-expressing tumor known in the art.
  • said cancer or solid tumor is a result of relapse or metastatic disease.
  • the metastatic disease is pulmonary metastatic disease.
  • the present invention provides methods of treating, preventing, or delaying metastases. In one embodiment, the present invention provides methods of treating, preventing, or delaying metastases of OSA. In one embodiment, the metastases are in the lung. In another embodiment, the metastases are in another tissue. In another embodiment, the metastases are in bone, which in one embodiment is proximal to the site of the initial OSA, and in another embodiment, is distal to the site of the initial OSA. In another embodiment, the metastases are in the kidney. In another embodiment, the metastases are in the heart. In another embodiment, the metastases are isolated. In another embodiment, the metastases are an isolated local recurrence. In another embodiment, the metastases are multi- site metastases.
  • recombinant Listeria expressing a chimeric Her-2/neu are useful as a therapeutic vaccine for the treatment of Her-2/neu overexpressing solid tumors.
  • the Her-2/neu chimeric antigen provided herein is useful for treating Her-2/neu-expressing tumors and preventing escape mutations of the same.
  • the term "escape mutation" refers to a tumor mutating away from a therapeutic efficacious response to treatment.
  • nucleic acid molecule comprising a first open reading frame encoding a recombinant polypeptide provided herein, wherein the nucleic acid molecule resides within the recombinant Listeria vaccine strain.
  • nucleic acid molecule provided herein is used to transform the Listeria in order to arrive at a recombinant Listeria.
  • nucleic acid provided herein lacks a virulence gene.
  • nucleic acid molecule integrated into the Listeria genome carries a non-functional virulence gene.
  • the virulence gene is mutated in the genome of the recombinant Listeria.
  • the nucleic acid molecule is used to inactivate the endogenous gene present in the Listeria genome.
  • the virulence gene is an actA gene.
  • the virulence gene is a prfA gene.
  • the virulence gene is an MB gene.
  • the virulence gene can be any gene known in the art to be associated with virulence in the recombinant Listeria.
  • the metabolic gene, the virulence gene, or both is lacking in a chromosome of the Listeria strain. In another embodiment, the metabolic gene, the virulence gene, or both is lacking in the chromosome and in any episomal genetic element of the Listeria strain. It will be appreciated by a skilled artisan that the term "episome,” “episomal,” etc. refer to a plasmid vector or use thereof that does not integrate into the chromosome of the Listeria provided herein. In another embodiment, the term refers to plasmid vectors that integrate into the chromosome of the Listeria provided herein. In another embodiment, the metabolic gene, the virulence gene, or both is lacking in the genome of the Listeria strain.
  • the metabolic gene, the virulence gene, or both is mutated in the chromosome. In another embodiment, the metabolic gene, the virulence gene, or both is deleted from the chromosome. In another embodiment, the metabolic gene, the virulence gene, or both is inactivated in the chromosome.
  • nucleic acids and plasmids provided herein do not confer antibiotic resistance upon the recombinant Listeria.
  • Nucleic acid molecule refers, in one embodiment, to a plasmid.
  • the term refers to an integration vector.
  • the term refers to a non-integration vector.
  • the term refers to a plasmid comprising an integration vector.
  • the integration vector is a site-specific integration vector.
  • a nucleic acid molecule of methods and compositions of the present invention are composed of any type of nucleotide known in the art. Each possibility represents a separate embodiment of the present invention.
  • Metal enzyme refers, in another embodiment, to an enzyme involved in synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme required for synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient utilized by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient required for sustained growth of the host bacteria. In another embodiment, the enzyme is required for synthesis of the nutrient. Each possibility represents a separate embodiment of the present invention.
  • “Stably maintained” refers, in another embodiment, to maintenance of a nucleic acid molecule or plasmid in the absence of selection (e.g. antibiotic selection) for 10 generations, without detectable loss.
  • the period is 15 generations.
  • the period is 20 generations.
  • the period is 25 generations.
  • the period is 30 generations.
  • the period is 40 generations.
  • the period is 50 generations.
  • the period is 60 generations.
  • the period is 80 generations.
  • the period is 100 generations.
  • the period is 150 generations.
  • the period is 200 generations.
  • the period is 300 generations.
  • the period is 500 generations.
  • the period is more than 500 generations.
  • the nucleic acid molecule or plasmid is maintained stably in vitro (e.g. in culture). In another embodiment, the nucleic acid molecule or plasmid is maintained stably in vivo. In another embodiment, the nucleic acid molecule or plasmid is maintained stably both in vitro and in vitro. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a recombinant Listeria strain expressing the antigen.
  • the present invention also provides recombinant polypeptides comprising a listeriolysin (LLO) protein fragment fused to a Her-2 chimeric protein or fragment thereof, vaccines and immunogenic compositions comprising same, and methods of inducing an anti-Her-2 immune response and treating and vaccinating against a Her-2- expressing tumor, comprising the same.
  • LLO listeriolysin
  • a recombinant Listeria strain of the present invention has been passaged through an animal host.
  • the passaging maximizes efficacy of the strain as a vaccine vector.
  • the passaging stabilizes the immunogenicity of the Listeria strain.
  • the passaging stabilizes the virulence of the Listeria strain.
  • the passaging increases the immunogenicity of the Listeria strain.
  • the passaging increases the virulence of the Listeria strain.
  • the passaging removes unstable substrains of the Listeria strain.
  • the passaging reduces the prevalence of unstable sub-strains of the Listeria strain.
  • the Listeria strain contains a genomic insertion of the gene encoding the antigen-containing recombinant peptide.
  • the Listeria strain carries a plasmid comprising the gene encoding the antigen-containing recombinant peptide.
  • the passaging is performed by any other method known in the art.
  • the polypeptide provided herein is a fusion protein comprising an additional polypeptide selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment, and further wherein said additional polypeptide is fused to the Her-2/neu chimeric antigen.
  • the additional polypeptide is functional.
  • a fragment of the additional polypeptide is immunogenic.
  • the additional polypeptide is immunogenic.
  • the polypeptide provided herein is a fusion protein comprising a non-hemolytic LLO protein or N-terminal fragment fused to the Her-2/neu chimeric antigen.
  • a fusion protein of methods and compositions of the present invention comprises an ActA sequence from a Listeria organism.
  • ActA proteins and fragments thereof augment antigen presentation and immunity in a similar fashion to LLO.
  • the fusion protein comprises the Her-2/neu antigen and an additional polypeptide.
  • the additional polypeptide fused to Her-2/neu antigen is referred to as an additional adjuvant polypeptide.
  • the additional polypeptide is a nonhemolytic LLO protein or fragment thereof (Examples herein).
  • the additional polypeptide is a PEST sequence.
  • the additional polypeptide is an ActA protein or a fragment thereof.
  • the additional polypeptide of methods and compositions of the present invention is, in another embodiment, a listeriolysin (LLO) peptide.
  • the additional polypeptide is an ActA peptide.
  • the additional polypeptide is a PEST sequence peptide.
  • the additional polypeptide is any other peptide capable of enhancing the immunogenicity of an antigen peptide. Each possibility represents a separate embodiment of the present invention.
  • Fusion proteins comprising the Her-2/neu chimeric antigen may be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods discussed below. Alternatively, subsequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence. In one embodiment,
  • DNA encoding the antigen can be produced using DNA amplification methods, for example polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the segments of the native DNA on either side of the new terminus are amplified separately.
  • the 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction.
  • the amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence).
  • the antigen is ligated into a plasmid. Each method represents a separate embodiment of the present invention.
  • compositions of the present invention has utility for inducing formation of antigen-specific T cells (e.g. cytotoxic T cells) that recognize and kill tumor cells (Examples herein).
  • antigen-specific T cells e.g. cytotoxic T cells
  • the present invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof. In one embodiment, the present invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof.
  • the Her-2 chimeric protein of the methods and compositions of the present invention is a human Her-2 chimeric protein.
  • the Her-2 protein is a mouse Her-2 chimeric protein.
  • the Her-2 protein is a rat Her-2 chimeric protein.
  • the Her-2 protein is a primate Her-2 chimeric protein.
  • the Her-2 protein is a canine Her-2 chimeric protein.
  • the Her-2 protein is a Her-2 chimeric protein of human or any other animal species or combinations thereof known in the art. Each possibility represents a separate embodiment of the present invention.
  • a Her-2 protein is a protein referred to as "Her-2/neu,” “Erbb2,” “v-erb-b2,” “c-erb-b2,” “neu,” or “cNeu.”
  • Her2/neu, or grammatical equivalents thereof is also referred to herein as "Her-2,” “Her-2 protein,” “HER2 protein,” or “HER2”).
  • Her-2 protein HER2 protein
  • HER2 HER2 protein
  • the Her2-neu chimeric protein harbors two of the extracellular and one intracellular fragments of Her-2/neu antigen showing clusters of MHC-class I epitopes of the oncogene, where, in another embodiment, the chimeric protein, harbors 3
  • the chimeric protein harbors at least 13 of the mapped human MHC-class I epitopes (fragments EC2 and ICl). In another embodiment, the chimeric protein harbors at least 14 of the mapped human MHC-class I epitopes (fragments ECl and ICl). In another embodiment, the chimeric protein harbors at least 9 of the mapped human MHC-class I epitopes (fragments ECl and IC2).
  • the Her2-neu chimeric protein is fused to a non-hemolytic listeriolysin O (LLO). In another embodiment, the Her2-neu chimeric protein is fused to truncated listeriolysin O (tLLO). In another embodiment, the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and expressed and secreted by the Listeria monocytogenes attenuated auxotrophic strain LmddA.
  • LLO Listeria-monocytogenes listeriolysin O
  • the expression and secretion of the fusion protein tLLO-ChHer2 from the attenuated auxotrophic strain provided herein that expresses a chimeric Her-2/neu antigen/LLO fusion protein is comparable to that of the Lm-LLO- ChHer2 in TCA precipitated cell culture supernatants after 8 hours of in vitro growth (See
  • no CTL activity is detected in naive animals or mice injected with an irrelevant Listeria vaccine (See Figure 2A). While in another embodiment, the attenuated auxotrophic strain (ADXS31-164) provided herein is able to stimulate the secretion of IFN- ⁇ by the splenocytes from wild type FVB/N mice ( Figure 2B).
  • the metabolic enzyme of the methods and compositions provided herein is an amino acid metabolism enzyme, where, in another embodiment, the metabolic enzyme is an alanine racemase enzyme. In another embodiment, the metabolic enzyme is a D-amino acid transferase enzyme. In another embodiment, the metabolic enzyme catalyzes a formation of an amino acid used for a cell wall synthesis in the recombinant Listeria strain, where in another embodiment, the metabolic enzyme is an alanine racemase enzyme.
  • the gene encoding the metabolic enzyme is expressed under the control of the Listeria p60 promoter.
  • the inlA (encodes internalin) promoter is used.
  • the hly promoter is used.
  • the ActA promoter is used.
  • the integrase gene is expressed under the control of any other gram positive promoter.
  • the gene encoding the metabolic enzyme is expressed under the control of any other promoter that functions in Listeria.
  • promoters or polycistronic expression cassettes may be used to drive the expression of the gene. Each possibility represents a separate embodiment of the present invention.
  • Her-2 chimeric protein is encoded by the following nucleic acid sequence set forth in SEQ ID NO: 1
  • the Her-2 chimeric protein has the sequence:
  • Table 1 shows the percent ( ) identity between the amino acid sequences of human and canine Her-2 EC and IC fragments, respectively.
  • an amino acid sequence encoding a human Her-2/neu ECl fragment is set forth in (SEQ ID NO: 69):
  • an amino acid sequence encoding a canine Her-2/neu ECl fragment is set forth in (SEQ ID NO: 70):
  • an amino acid sequence encoding a human Her-2/neu EC2 fragment is set forth in (SEQ ID NO: 71):
  • an amino acid sequence encoding a canine Her-2/neu EC2 fragment is set forth in (SEQ ID NO: 72):
  • TAPLQPEQLRVFEALEEITGYLYISAWPDSLPNLSVFQNLRVIRGRVLHDGA YSLTLQGLGISWLGLRSLRELGS (SEQ ID NO: 72).
  • an amino acid sequence encoding a human Her-2/neu IC1 fragment is set forth in (SEQ ID NO: 73):
  • an amino acid sequence encoding a canine Her-2/neu IC1 fragment is set forth in (SEQ ID NO: 74):
  • the human amino acid sequence of Her-2 ECl fragment (SEQ ID NO: 69) has 89% identity with that of a canine Her-2 ECl fragment (SEQ ID NO: 70).
  • the human amino acid sequence of Her-2 EC2 fragment (SEQ ID NO: 71) has 93% identity with that of a canine Her-2 EC2 fragment (SEQ ID NO: 72).
  • the human amino acid sequence of Her-2 IC1 fragment (SEQ ID NO: 73) has
  • the Her2 chimeric protein or fragment thereof of the methods and compositions provided herein does not include a signal sequence thereof.
  • omission of the signal sequence enables the Her2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the signal sequence.
  • the fragment of a Her2 chimeric protein of methods and compositions of the present invention does not include a transmembrane domain (TM) thereof.
  • TM transmembrane domain
  • omission of the TM enables the Her-2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the TM.
  • nucleic acid sequence of rat-Her-2/neu gene is
  • the nucleic acid sequence encoding the rat/Her-2/neu ECl fragment is
  • nucleic acid sequence encoding the rat Her-2/neu EC2 fragment is:
  • nucleic acid sequence encoding the rat Her-2/neu IC1 fragment is:
  • nucleic acid sequence of human-Her-2/neu gene is:
  • nucleic acid sequence encoding the human Her-2/neu ECl fragment implemented into the chimera spans from 120-510 bp of the human ECl region and is set forth in (SEQ ID NO: 50).
  • the complete ECl human Her-2/neu fragment spans from (58-
  • nucleic acid sequence encoding the human Her-2/neu EC2 fragment implemented into the chimera spans from 1077-1554 bp of the human Her- 2/neu EC2 fragment and includes a 50 bp extension, and is set forth in (SEQ ID NO: 51).
  • ATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAA GTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGG GCTGGGCATCAGCTGGCTGGCTGCGCTCACTGAGGGAACTGGGCAGTGGAC TGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGG ACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAG AGGACGAGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCCCGAGGG
  • complete EC2 human Her-2/neu fragment spans from 907- 1504 bp of the human Her-2/neu gene and is set forth in (SEQ ID NO: 55).
  • IC1 fragment implemented into the chimera is set forth in (SEQ ID NO: 52).
  • CAGCAGAAGATCCGGAAGTAC ACGATGCGGAGACTGCTGCAGGAAAC GGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGA TGCGGATCCTGAAAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGC GCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAA AATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACA AAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCT CCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTA TGCCCTATGGCTGCCTCTTAGACT (SEQ ID NO:52).
  • nucleic acid sequence encoding the complete human Her-2/neu ICl fragment spans from 2034-3243 of the human Her-2/neu gene and is set forth in (SEQ ID NO: 56).
  • the LLO utilized in the methods and compositions provided herein is, in one embodiment, a Listeria LLO.
  • the Listeria from which the LLO is derived is Listeria monocytogenes (LM).
  • the Listeria is Listeria ivanovii.
  • the Listeria is Listeria welshimeri.
  • the Listeria is Listeria seeligeri.
  • the LLO protein is a non-Listerial LLO protein.
  • the LLO protein is a synthetic LLO protein. In another embodiment it is a recombinant LLO protein.
  • the LLO protein is encoded by the following nucleic acid sequence set forth in (SEQ ID NO: 3)
  • the LLO protein comprises the sequence SEQ ID NO: 4 [00175] M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S M A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N N A V N T L V E R W N E K Y A Q A Y P N V S A K I D Y D D E M A Y S E S Q L I A K
  • the first 25 amino acids of the proprotein corresponding to this sequence are the signal sequence and are cleaved from LLO when it is secreted by the bacterium.
  • the full length active LLO protein is 504 residues long.
  • the LLO protein has a sequence set forth in GenBank Accession No. DQ054588, DQ054589, AY878649, U25452, or U25452.
  • the LLO protein is a variant of an LLO protein.
  • the LLO protein is a homologue of an LLO protein. Each possibility represents a separate embodiment of the present invention.
  • truncated LLO or "tLLO” refers to a fragment of LLO that comprises the PEST domain.
  • the terms refer to an LLO fragment that does not contain the activation domain at the amino terminus and does not include cystine 484.
  • the LLO fragment consists of a PEST sequence.
  • the LLO fragment comprises a PEST sequence.
  • the LLO fragment consists of about the first 400 to 441 amino acids of the 529 amino acid full-length LLO protein.
  • the LLO fragment is a nonhemolytic form of the LLO protein.
  • a polypeptide encoded by a nucleic acid sequence of methods and compositions of the present invention is a fusion protein comprising the chimeric Her-2/neu antigen and an additional polypeptide, where in another embodiment, the fusion protein comprises, inter alia, a Listeria
  • the LLO fragment consists of about residues 1-25. In another embodiment, the LLO fragment consists of about residues 1-50. In another embodiment, the LLO fragment consists of about residues 1-75. In another embodiment, the LLO fragment consists of about residues 1-100. In another embodiment, the LLO fragment consists of about residues 1-125. In another embodiment, the LLO fragment consists of about residues 1-150. In another embodiment, the LLO fragment consists of about residues 1175. In another embodiment, the LLO fragment consists of about residues 1-200. In another embodiment, the LLO fragment consists of about residues 1-225. In another embodiment, the LLO fragment consists of about residues 1-250.
  • the LLO fragment consists of about residues 1-275. In another embodiment, the LLO fragment consists of about residues 1-300. In another embodiment, the LLO fragment consists of about residues 1-325. In another embodiment, the LLO fragment consists of about residues 1-350. In another embodiment, the LLO fragment consists of about residues 1-375. In another embodiment, the LLO fragment consists of about residues 1-400. In another embodiment, the LLO fragment consists of about residues 1-425. Each possibility represents a separate embodiment of the present invention.
  • a fusion protein of methods and compositions of the present invention comprises a PEST sequence, either from an LLO protein or from another organism, e.g. a prokaryotic organism.
  • the PEST amino acid sequence has, in another embodiment, a sequence selected from SEQ ID NO: 5-9.
  • the PEST sequence is a PEST sequence from the Listeria Monocytogenes ActA protein.
  • the PEST sequence is KTEEQPSEVNTGPR (SEQ ID NO: 5), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 6), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 7), or
  • the PEST sequence is from Streptolysin O protein of Streptococcus sp. In another embodiment, the PEST sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 9) at amino acids 35-51. In another embodiment, the PEST sequence is from Streptococcus equisimilis Streptolysin O, e.g.
  • the PEST sequence is another PEST amino acid sequence derived from a prokaryotic organism.
  • the PEST sequence is any other PEST sequence known in the art. Each possibility represents a separate embodiment of the present invention.
  • Monocytogenes enhanced cell mediated and anti-tumor immunity of the antigen.
  • fusion of an antigen to other PEST sequences derived from other prokaryotic organisms will also enhance immunogenicity of the antigen.
  • PEST sequence of other prokaryotic organism can be identified in accordance with methods such as described by, for example Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21 :267-271) for Listeria Monocytogenes.
  • PEST amino acid sequences from other prokaryotic organisms can also be identified based by this method.
  • the PEST sequence is embedded within the antigenic protein.
  • "fusion" refers to an antigenic protein comprising both the antigen and the PEST amino acid sequence either linked at one end of the antigen or embedded within the antigen.
  • a vaccine comprising a recombinant polypeptide of the present invention.
  • a vaccine consisting of a recombinant polypeptide of the present invention.
  • nucleotide molecule encoding a recombinant polypeptide of the present invention.
  • a vaccine comprising the nucleotide molecule.
  • nucleotide molecule encoding a recombinant polypeptide of the present invention.
  • a recombinant polypeptide encoded by the nucleotide molecule of the present invention is provided herein.
  • a vaccine comprising a nucleotide molecule or recombinant polypeptide of the present invention.
  • an immunogenic composition comprising a nucleotide molecule or recombinant polypeptide of the present invention.
  • a vector comprising a nucleotide molecule or recombinant polypeptide of the present invention.
  • a recombinant form of Listeria comprising a nucleotide molecule of the present invention.
  • a vaccine comprising a recombinant form of Listeria of the present invention.
  • a vaccine or composition for use in the methods of the present invention comprises a recombinant Listeria monocytogenes, in any form or embodiment as described herein.
  • the vaccine or composition for use in the present invention consists of a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein.
  • the vaccine or composition for use in the methods of the present invention consists essentially of a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein.
  • the term “comprise” refers to the inclusion of a recombinant Listeria monocytogenes in the vaccine or composition, as well as inclusion of other vaccines, compositions or treatments that may be known in the art.
  • the term “consisting essentially of” refers to a vaccine, whose functional component is the recombinant Listeria monocytogenes, however, other components of the vaccine may be included that are not involved directly in the therapeutic effect of the vaccine and may, for example, refer to components which facilitate the effect of the recombinant Listeria monocytogenes (e.g. stabilizing, preserving, etc.).
  • the term “consisting” refers to a vaccine, which contains the recombinant Listeria monocytogenes.
  • a method of impeding or delaying metastatic disease origination from a HER2-expressing tumor in a subject comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein.
  • the methods of the present invention comprise the step of administering a recombinant Listeria monocytogenes, in any form or embodiment as described herein.
  • the methods of the present invention consist of the step of administering a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein.
  • the methods of the present invention consist essentially of the step of administering a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein.
  • the term “comprise” refers to the inclusion of the step of administering a recombinant Listeria monocytogenes in the methods, as well as inclusion of other methods or treatments that may be known in the art.
  • the term “consisting essentially of” refers to a methods, whose functional component is the administration of recombinant Listeria monocytogenes, however, other steps of the methods may be included that are not involved directly in the therapeutic effect of the methods and may, for example, refer to steps which facilitate the effect of the administration of recombinant Listeria monocytogenes.
  • the term “consisting” refers to a method of administering recombinant Listeria monocytogenes with no additional steps.
  • the Listeria of methods and compositions of the present invention is Listeria monocytogenes.
  • the Listeria is Listeria ivanovii.
  • the Listeria is Listeria welshimeri.
  • the Listeria is Listeria seeligeri.
  • Each type of Listeria represents a separate embodiment of the present invention.
  • the Listeria strain of the methods and compositions of the present invention is the ADXS31-164 strain.
  • ADXS31-164 stimulates the secretion of IFN- ⁇ by the splenocytes from wild type FVB/N mice. Further, the data presented herein show that ADXS31-164 is able to elicit anti-Her-2/neu specific immune responses to human epitopes that are located at different domains of the targeted antigen.
  • the present invention provides a recombinant form of Listeria comprising a nucleotide molecule encoding a Her-2 chimeric protein or a fragment thereof.
  • the two molecules of the fusion protein are joined directly.
  • the two molecules are joined by a short spacer peptide, consisting of one or more amino acids.
  • the spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them.
  • the constituent amino acids of the spacer are selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
  • the two molecules of the protein (the LLO fragment and the antigen) are synthesized separately or unfused.
  • the two molecules of the protein are synthesized separately from the same nucleic acid.
  • the two molecules are individually synthesized from separate nucleic acids. Each possibility represents a separate embodiment of the present invention.
  • nucleic acids encoding the recombinant polypeptides provided herein also encode a signal peptide or sequence.
  • the fusion protein of methods and compositions of the present invention comprises an LLO signal sequence from LLO.
  • a heterologous antigen may be expressed through the use of a signal sequence, such as a Listerial signal sequence, for example, the hemolysin signal sequence or the actA signal sequence.
  • foreign genes can be expressed downstream from a L. monocytogenes promoter without creating a fusion protein.
  • the signal peptide is bacterial (Listerial or non-Listerial).
  • the signal peptide is native to the bacterium.
  • the signal peptide is foreign to the bacterium.
  • the signal peptide is a signal peptide from Listeria monocytogenes, such as a secAl signal peptide.
  • the signal peptide is a Usp45 signal peptide from Lactococcus lactis, or a Protective Antigen signal peptide from Bacillus anthracis.
  • the signal peptide is a secA2 signal peptide, such the p60 signal peptide from Listeria monocytogenes.
  • the recombinant nucleic acid molecule optionally comprises a third polynucleotide sequence encoding p60, or a fragment thereof.
  • the signal peptide is a Tat signal peptide, such as a B. subtilis Tat signal peptide (e.g., PhoD).
  • the signal peptide is in the same translational reading frame encoding the recombinant polypeptide.
  • a method of inducing an anti-Her-2 immune response in a subject comprising administering to the subject a recombinant nucleotide encoding a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof, thereby inducing an anti-Her-2 immune response in a subject.
  • provided herein is a method of eliciting an enhanced immune response to a Her-2/neu-expressing tumor in a subject, where in another embodiment, the method comprises administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • the immune response against the Her-2-expressing tumor comprises an immune response to a subdominant epitope of the Her-2 protein.
  • the immune response against the Her-2-expressing tumor comprises an immune response to several subdominant epitopes of the Her-2 protein.
  • the immune response against the Her-2-expressing tumor comprises an immune response to at least 1-5 subdominant epitopes of the Her-2 protein.
  • the immune response against the Her-2-expressing tumor comprises an immune response to at least 1-10 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to at least 1-17 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to at least 17 subdominant epitopes of the Her-2 protein.
  • Point mutations or amino-acid deletions in the oncogenic protein Her-2/neu have been reported to mediate treatment of resistant tumor cells, when these tumors have been targeted by small fragment Listeria-b&sed vaccines or trastuzumab (a monoclonal antibody against an epitope located at the extracellular domain of the Her-2/neu antigen).
  • trastuzumab a monoclonal antibody against an epitope located at the extracellular domain of the Her-2/neu antigen.
  • Described herein is a chimeric Her-2/neu based composition which harbors two of the extracellular and one intracellular fragments of Her-2/neu antigen showing clusters of MHC-class I epitopes of the oncogene.
  • This chimeric protein which harbors 3 H2Dq and at least 17 of the mapped human MHC-class I epitopes of the Her-2/neu antigen was fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O protein and expressed and secreted by the Listeria monocytogenes attenuated strain LmddA.
  • nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her-2/neu chimeric antigen.
  • nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, and wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of the recombinant
  • Listeria strain thereby engineering a Listeria vaccine strain to express a Her-2 chimeric protein.
  • the methods and compositions provided herein further comprise an adjuvant, which in one embodiment, is an independent adjuvant, where in another embodiment, the adjuvant or independent adjuvant comprises a granulocyte/macrophage colony- stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
  • GM-CSF granulocyte/macrophage colony- stimulating factor
  • the adjuvant is an aluminum adjuvant, Freund's adjuvant, MPL, emulsion, SBAS2, a nucleotide molecule encoding an immune-stimulating cytokine, a bacterial mitogen, or a bacterial toxin.
  • an "adjuvant” is a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune responses.
  • the adjuvant is an immunologic adjuvant which in one embodiment is a substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.
  • an "independent" adjuvant is an adjuvant that is independent, which in one embodiment, is not identical to the "additional adjuvant polypeptide" of the present invention which is present in a fusion polypeptide with a tumor specific antigen, which in one embodiment, is Her-2/neu.
  • Attenuated Listeria strains such as Listeria Monocytogenes delta-actA mutant (Brundage et al, 1993, Proc. Natl. Acad. Sci., USA, 90: 11890-11894), L. monocytogenes delta-plcA (Camilli et al, 1991, J. Exp. Med., 173:751-754), or delta-ActA, delta INL-b (Brockstedt et 5 al, 2004, PNAS, 101 : 13832-13837) are used in the present invention.
  • Attenuated Listeria strains are constructed by introducing one or more attenuating mutations, as will be understood by one of ordinary skill in the art when equipped with the disclosure herein.
  • examples of such strains include, but are not limited to Listeria strains auxotrophic for aromatic amino acids (Alexander et al, 1993, Infection and Immunity 10 61 :2245-2248) and mutant for the formation of lipoteichoic acids (Abachin et al, 2002, Mol. Microbiol. 43: 1-14) and those attenuated by a lack of a virulence gene (see examples herein).
  • nucleic acid molecule of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence.
  • first open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence.
  • second open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence.
  • third open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence.
  • each of the open reading frames are operably linked to a promoter/regulatory sequence. Each possibility represents a separate embodiment of the present invention.
  • transcriptional promoters, terminators, carrier vectors or specific gene sequences can be used successfully in methods and compositions of the present invention.
  • these functionalities are provided in, for example, the commercially available vectors known as the pUC series.
  • nonessential DNA sequences e.g. antibiotic resistance genes
  • plasmids are available from a variety of sources, for example, Invitrogen (La Jolla, CA), Stratagene (La Jolla, CA),
  • Clontech (Palo Alto, CA), or can be constructed using methods well known in the art.
  • a plasmid such as pCR2.1 (Invitrogen, La Jolla, CA), which is a prokaryotic expression vector with a prokaryotic origin of replication and promoter/regulatory elements is used to facilitate expression of a polypeptide of the present invention in a prokaryotic organism.
  • extraneous nucleotide sequences are removed to decrease the size of the plasmid and increase the size of the cassette that can be placed therein.
  • Antibiotic resistance genes are used in the conventional selection and cloning processes commonly employed in molecular biology and vaccine preparation.
  • Antibiotic resistance genes contemplated in the present invention include, but are not limited to, gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, cloramphenicol (CAT), neomycin, hygromycin, gentamicin and others well known in the art. Each gene represents a separate embodiment of the present invention.
  • Methods for transforming bacteria are well known in the art, and include calcium- chloride competent cell-based methods, electroporation methods, bacteriophage-mediated transduction, chemical, and physical transformation techniques (de Boer et al, 1989, Cell 56:641-649; Miller et al, 1995, FASEB J., 9: 190-199; Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for
  • the Listeria vaccine strain of the present invention is transformed by electroporation. Each method represents a separate embodiment of the present invention.
  • conjugation is used to introduce genetic material and/or plasmids into bacteria.
  • Methods for conjugation are well known in the art, and are described, for example, in Nikodinovic J et al. (A second generation snp-derived Escherichia coli- Streptomyces shuttle expression vector that is generally transferable by conjugation. Plasmid. 2006 Nov;56(3):223-7) and Auchtung JM et al (Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl
  • Transforming in one embodiment, is used identically with the term “transfecting,” and refers to engineering a bacterial cell to take up a plasmid or other heterologous DNA molecule.
  • transforming refers to engineering a bacterial cell to express a gene of a plasmid or other heterologous DNA molecule.
  • Plasmids and other expression vectors useful in the present invention are described elsewhere herein, and can include such features as a promoter/regulatory sequence, an origin of replication for gram negative and gram positive bacteria, an isolated nucleic acid encoding a fusion protein and an isolated nucleic acid encoding an amino acid metabolism gene. Further, an isolated nucleic acid encoding a fusion protein and an amino acid metabolism gene will have a promoter suitable for driving expression of such an isolated nucleic acid.
  • Promoters useful for driving expression in a bacterial system include bacteriophage lambda, the bla promoter of the beta-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325.
  • prokaryotic promoters include the major right and left promoters of 5 bacteriophage lambda (PL and PR), the trp, recA, lacZ, lad, and gal promoters of E. coli, the alpha-amylase (Ulmanen et al, 1985. J. Bacterid. 162: 176-182) and the S28-specific promoters of B.
  • subtilis (Oilman et al, 1984 Gene 32:11- 20), the promoters of the bacteriophages of Bacillus (Gryczan, 1982, In: The Molecular Biology of the Bacilli, Academic Press, Inc., New York), and Streptomyces promoters (Ward et al, 1986, Mol. Gen. Genet. 203:468-478). Additional prokaryotic promoters contemplated in the present invention are reviewed in, for example, Glick (1987, J. Ind. Microbiol. 1 :277-282); Cenatiempo, (1986, Biochimie, 68:505-516); and Gottesman, (1984, Ann. Rev. Genet.
  • promoter/regulatory elements contemplated in the present invention include, but are not limited to the Listerial prfA promoter, the Listerial hly promoter, the Listerial p60 promoter and the Listerial ActA promoter (GenBank Acc. No. NC_003210) or fragments thereof.
  • a plasmid of methods and compositions of the present invention comprises a gene encoding a fusion protein.
  • subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then, in another embodiment, ligated to produce the desired DNA sequence.
  • DNA encoding the antigen is produced using DNA amplification methods, for example polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction.
  • the amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence).
  • the antigen is ligated into a plasmid.
  • the present invention further comprises a phage based chromosomal integration system for clinical applications.
  • a host strain that is auxotrophic for essential enzymes including, but not limited to, d-alanine racemase will be used, for example Lmdal(-)dat(-).
  • a phage integration system based on PSA is used (Lauer, et al., 2002 J Bacterid, 184:4177-4186). This requires, in another embodiment, continuous selection by antibiotics to maintain the integrated gene.
  • the current invention enables the establishment of a phage based chromosomal integration system that does not require selection with antibiotics. Instead, an auxotrophic host strain will be complemented.
  • the recombinant proteins of the present invention are synthesized, in another embodiment, using recombinant DNA methodology. This involves, in one embodiment, creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette, such as the plasmid of the present invention, under the control of a particular promoter/regulatory element, and expressing the protein.
  • DNA encoding the fusion protein (e.g. non-hemolytic LLO/antigen) of the present invention is prepared, in another embodiment, by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979, Meth. Enzymol.
  • chemical synthesis is used to produce a single stranded oligonucleotide.
  • This single stranded oligonucleotide is converted, in various embodiments, into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill in the art would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences can be obtained by the ligation of shorter sequences.
  • subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated to produce the desired DNA sequence.
  • DNA encoding the fusion protein or the recombinant protein of the present invention is cloned using DNA amplification methods such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the gene for non-hemolytic LLO is PCR amplified, using a sense primer comprising a suitable restriction site and an antisense primer comprising another restriction site, e.g. a non-identical restriction site to facilitate cloning.
  • a sense primer comprising a suitable restriction site
  • an antisense primer comprising another restriction site, e.g. a non-identical restriction site to facilitate cloning.
  • Another restriction site e.g. a non-identical restriction site
  • LLO and antigen sequences and insertion into a plasmid or vector produces a vector encoding non-hemolytic LLO joined to a terminus of the antigen.
  • the two molecules are joined either directly or by a short spacer introduced by the restriction site.
  • the molecules are separated by a peptide spacer consisting of one or more amino acids, generally the spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them.
  • the constituent amino acids of the spacer are selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
  • the nucleic acid sequences encoding the fusion or recombinant proteins are transformed into a variety of host cells, including E. coli, other bacterial hosts, such as Listeria, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
  • the recombinant fusion protein gene will be operably linked to appropriate expression control sequences for each host.
  • Promoter/ regulatory sequences are described in detail elsewhere herein.
  • the plasmid further comprises additional promoter regulatory elements, as well as a ribosome binding site and a transcription termination signal.
  • the control sequences will include a promoter and an enhancer derived from e.g. immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence.
  • the sequences include splice donor and acceptor sequences.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • transformed auxotrophic bacteria are grown on a media that will select for expression of the amino acid metabolism gene.
  • a bacteria auxotrophic for D-glutamic acid synthesis is transformed with a plasmid comprising a gene for D-glutamic acid synthesis, and the auxotrophic bacteria will grow in the absence of D- glutamic acid, whereas auxotrophic bacteria that have not been transformed with the plasmid, or are not expressing the plasmid encoding a protein for D-glutamic acid synthesis, will not grow.
  • a bacterium auxotrophic for D-alanine synthesis will grow in the absence of D-alanine when transformed and expressing the plasmid of the present invention if the plasmid comprises an isolated nucleic acid encoding an amino acid metabolism enzyme for D-alanine synthesis.
  • Such methods for making appropriate media comprising or lacking necessary growth factors, supplements, amino acids, vitamins, antibiotics, and the like are well known in the art, and are available commercially (Becton- Dickinson, Franklin Lakes, NJ). Each method represents a separate embodiment of the present invention.
  • the bacteria are propagated in the presence of a selective pressure. Such propagation comprises growing the bacteria in media without the auxotrophic factor.
  • the presence of the plasmid expressing an amino acid metabolism enzyme in the auxotrophic bacteria ensures that the plasmid will replicate along with the bacteria, thus continually selecting for bacteria harboring the plasmid.
  • the skilled artisan when equipped with the present disclosure and methods herein will be readily able to scale-up the production of the Listeria vaccine vector by adjusting the volume of the media in which the auxotrophic bacteria comprising the plasmid are growing.
  • a method of impeding the growth of a Her- 2-expressing tumor in a subject comprising the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein.
  • a method of eliciting an enhanced immune response to a Her-2/neu-expressing tumor in a subject comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein.
  • the immune response against the Her-2/neu-expressing tumor comprises an immune response to at least one subdominant epitope of the Her-2/neu protein.
  • provided herein is a method of preventing an escape mutation in the treatment of Her-2/neu over-expressing tumors, wherein and in another embodiment, the method comprises the step of administering to said subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • Her-2/neu antigen-expressing tumor in a subject wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • provided herein is a method of decreasing the frequency of intra-tumoral T regulatory cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • provided herein is a method of decreasing the frequency of intra-tumoral myeloid derived suppressor cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • a method of decreasing the frequency of myeloid derived suppressor cells comprising the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • a method of preventing the development of a Her-2/neu-expressing tumor in a subject comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • provided herein is a method of preventing the formation of a metastatic disease coming from an Her-2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain the provided herein.
  • a method of treating a metastatic disease originating from a Her-2/neu-expressing tumor in a subject comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • provided herein is a method of administering the composition of the present invention. In another embodiment, provided herein is a method of administering the vaccine of the present invention. In another embodiment, provided herein is a method of administering the recombinant polypeptide or recombinant nucleotide of the present invention. In another embodiment, the step of administering the composition, vaccine, recombinant polypeptide or recombinant nucleotide of the present invention is performed with an attenuated recombinant form of Listeria comprising the composition, vaccine, recombinant nucleotide or expressing the recombinant polypeptide, each in its own discrete embodiment.
  • the administering is performed with a different attenuated bacterial vector.
  • the administering is performed with a DNA vaccine (e.g. a naked DNA vaccine).
  • administration of a recombinant polypeptide of the present invention is performed by producing the protein recombinantly, then administering the recombinant protein to a subject. Each possibility represents a separate embodiment of the present invention.
  • the immune response elicited by methods and compositions of the present invention comprises a CD8 + T cell-mediated response.
  • the immune response consists primarily of a CD8 + T cell-mediated response.
  • the only detectable component of the immune response is a CD8 + T cell-mediated response.
  • the immune response elicited by methods and compositions provided herein comprises a CD4 + T cell-mediated response.
  • the immune response consists primarily of a CD4 + T cell-mediated response.
  • the only detectable component of the immune response is a CD4 + T cell-mediated response.
  • the CD4 + T cell-mediated response is accompanied by a measurable antibody response against the antigen.
  • the CD4 + T cell-mediated response is not accompanied by a measurable antibody response against the antigen.
  • the present invention provides a method of inducing a
  • CD8 + T cell-mediated immune response in a subject against a subdominant CD8 + T cell epitope of an antigen comprising the steps of (a) fusing a nucleotide molecule encoding the Her2-neu chimeric antigen or a fragment thereof to a nucleotide molecule encoding an N- terminal fragment of a LLO protein, thereby creating a recombinant nucleotide encoding an LLO-antigen fusion protein; and (b) administering the recombinant nucleotide or the LLO- antigen fusion to the subject; thereby inducing a CD8 + T cell-mediated immune response against a subdominant CD8 + T cell epitope of an antigen.
  • a method of increasing intratumoral ratio of CD8+/T regulatory cells comprising the step of administering to the subject a composition comprising the recombinant polypeptide, recombinant Listeria, or recombinant vector of the present invention.
  • a method of decreasing the frequency of intra- tumoral T regulatory cells comprising the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • the immune response elicited by the methods and compositions provided herein comprises an immune response to at least one subdominant epitope of the antigen.
  • the immune response does not comprise an immune response to a subdominant epitope.
  • the immune response consists primarily of an immune response to at least one subdominant epitope.
  • the only measurable component of the immune response is an immune response to at least one subdominant epitope.
  • Each type of immune response represents a separate embodiment of the present invention.
  • methods of this invention break tolerance in a subject to a Her- 2/neu expressing tumor or cancer in said subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
  • Methods of measuring immune responses include, e.g. measuring suppression of tumor growth, flow cytometry, target cell lysis assays (e.g. chromium release assay), the use of tetramers, and others. Each method represents a separate embodiment of the present invention.
  • the present invention provides a method of impeding the growth of a Her-2-expressing tumor in a subject, wherein and in another embodiment, the method comprises administering to the subject a combination of radiation therapy and a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to the
  • Her-2 chimeric protein or a fragment thereof or a recombinant nucleotide encoding the recombinant polypeptide wherein the subject mounts an immune response against the Her-2- expressing tumor, thereby impeding the growth of a Her-2-expressing tumor in a subject.
  • the present invention provides a method of delaying or inhibiting a metastatic disease emanating from a Her-2-expressing tumor in a subject, wherein and in another embodiment, the method comprises administering to the subject a combination of radiation therapy and a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to the Her-2 chimeric protein or a fragment thereof or a recombinant nucleotide encoding the recombinant polypeptide, wherein the subject mounts an immune response against the Her-2-expressing tumor, thereby delaying or inhibiting the metastatic disease emanating from a Her-2-expressing tumor in a subject.
  • the present invention provides a method of improving the antigenicity of a Her-2 chimeric protein, wherein and in another embodiment, the method comprises the step of fusing a nucleotide encoding an N-terminal fragment of a LLO protein to a nucleotide encoding the Her-2 protein or a fragment thereof to create a recombinant nucleotide, thereby improving the antigenicity of a Her-2 chimeric protein.
  • a method of improving the antigenicity of a Her-2 chimeric protein comprising engineering a Listeria strain to express the recombinant nucleotide.
  • a different bacterial vector is used to express the recombinant nucleotide.
  • the bacterial vector is attenuated.
  • a DNA vaccine e.g. a naked DNA vaccine
  • administration of the LLO-Her-2 chimera fusion peptide encoded by the nucleotide is performed by producing the protein recombinantly, then administering the recombinant protein to a subject.
  • the present invention provides a method that induces "epitope spreading" of a tumor.
  • the immunization using the compositions and methods provided herein induce epitope spreading onto other tumors bearing antigens other than the antigen carried in the vaccine of the present invention.
  • the dominant epitope or subdominant epitope is dominant or subdominant, respectively, in the subject being treated. In another embodiment, the dominant epitope or subdominant epitope is dominant or subdominant in a population being treated.
  • provided herein is a method of preventing, treating, suppressing, inhibiting, inducing an immune response against, or eliciting an enhanced immune response against sub-dominant epitopes against a cancer or a tumor growth in a subject by epitope spreading wherein and in another embodiment, said cancer is associated with expression of an antigen or fragment thereof comprised in the composition of the present invention.
  • the method comprises administering to said subject a composition comprising the recombinant polypeptide, recombinant Listeria, or recombinant vector of the present invention.
  • the subject mounts an immune response against the antigen-expressing cancer or the antigen-expressing tumor, thereby treating, suppressing, or inhibiting a cancer or a tumor growth in a subject.
  • Dominant CD8 + T cell epitope refers to an epitope that is recognized by over 30% of the antigen-specific CD8 + T cells that are elicited by vaccination, infection, or a malignant growth with a protein or a pathogen or cancer cell containing the protein. In another embodiment, the term refers to an epitope recognized by over 35% of the antigen-specific CD8 + T cells that are elicited thereby. In another embodiment, the term refers to an epitope recognized by over 40% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 45% of the antigen-specific CD8 + T cells.
  • the term refers to an epitope recognized by over 50% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 55% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 60% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 65% of the antigen- specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 70% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 75% of the antigen-specific CD8 + T cells.
  • the term refers to an epitope recognized by over 80% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 85% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 90% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 95% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 96% of the antigen- specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 97% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 98% of the antigen-specific CD8 + T cells.
  • Subdominant CD8 + T cell epitope refers to an epitope recognized by fewer than 30% of the antigen-specific CD8 + T cells that are elicited by vaccination, infection, or a malignant growth with a protein or a pathogen or cancer cell containing the protein.
  • the term refers to an epitope recognized by fewer than 28% of the antigen-specific CD8 + T cells.
  • the term refers to an epitope recognized by over 26% of the antigen-specific CD8 + T cells.
  • the term refers to an epitope recognized by fewer than 24% of the antigen- specific CD8 + T cells.
  • the term refers to an epitope recognized by over 22% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 20% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 18% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than
  • the term refers to an epitope recognized by over 14% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 12% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 10% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 8% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 6% of the antigen-specific CD8 + T cells.
  • the term refers to an epitope recognized by fewer than 5% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 4% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 3% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 2% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 1% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 0.5% of the antigen-specific CD8 + T cells.
  • Each type of the dominant epitope and subdominant epitope represents a separate embodiment of the present invention.
  • the antigen in methods and compositions of the present invention is, in one embodiment, expressed at a detectable level on a non-tumor cell of the subject. In another embodiment, the antigen is expressed at a detectable level on at least a certain percentage (e.g. 0.01%, 0.03%, 0.1%, 0.3%, 1%, 2%, 3%, or 5%) of non-tumor cells of the subject.
  • “non-tumor cell” refers to a cell outside the body of the tumor.
  • non-tumor cell refers to a non-malignant cell.
  • non- tumor cell refers to a non-transformed cell.
  • the non-tumor cell is a somatic cell.
  • the non-tumor cell is a germ cell.
  • Detectable level refers, in one embodiment, to a level that is detectable when using a standard assay.
  • the assay is an immunological assay.
  • the assay is enzyme-linked immunoassay (ELISA).
  • the assay is Western blot.
  • the assay is FACS. It is to be understood by a skilled artisan that any other assay available in the art can be used in the methods provided herein.
  • a detectable level is determined relative to the background level of a particular assay. Methods for performing each of these techniques are well known to those skilled in the art, and each technique represents a separate embodiment of the present invention.
  • vaccination with recombinant antigen-expressing Listeria Monocytogenes induces epitope spreading.
  • vaccination with LLO- antigen fusions, even outside the context of Her2, induces epitope spreading as well.
  • the present invention provides a method of impeding the growth of an Her-2-expressing tumor in a subject, comprising administering to the subject a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to a Her-2 chimeric antigen, wherein the antigen has one or more subdominant CD8 + T cell epitopes, wherein the subject mounts an immune response against the antigen-expressing tumor, thereby impeding the growth of an Her-2-expressing tumor in a subject.
  • the antigen does not contain any of the dominant CD8 + T cell epitopes.
  • provided herein is a method of impeding the growth on a Her-2- expressing tumor in a subject, comprising administering to the subject a recombinant form of Listeria comprising a recombinant nucleotide encoding the recombinant polypeptide provided herein.
  • the present invention provides a method for inducing formation of cytotoxic T cells in a host having cancer, comprising administering to the host a composition of the present invention, thereby inducing formation of cytotoxic T cells in a host having cancer.
  • the present invention provides a method of reducing an incidence of cancer, comprising administering a composition of the present invention.
  • the present invention provides a method of ameliorating cancer, comprising administering a composition of the present invention.
  • Each possibility represents a separate embodiment of the present invention.
  • the composition is administered to the cells of the subject ex vivo; in another embodiment, the composition is administered to the cells of a donor ex vivo; in another embodiment, the composition is administered to the cells of a donor in vivo, then is transferred to the subject.
  • the composition is administered to the cells of the subject ex vivo; in another embodiment, the composition is administered to the cells of a donor ex vivo, then is transferred to the subject.
  • the cancer treated by a method of the present invention is breast cancer.
  • the cancer is a Her2 containing cancer.
  • the cancer is a melanoma.
  • the cancer is pancreatic cancer.
  • the cancer is ovarian cancer.
  • the cancer is gastric cancer.
  • the cancer is a carcinomatous lesion of the pancreas.
  • the cancer is pulmonary adenocarcinoma.
  • the cancer is colorectal adenocarcinoma.
  • the cancer is pulmonary squamous adenocarcinoma.
  • the cancer is gastric adenocarcinoma.
  • the cancer is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof).
  • the cancer is an oral squamous cell carcinoma.
  • the cancer is non small-cell lung carcinoma.
  • the cancer is a CNS carcinoma.
  • the cancer is an endometrial carcinoma.
  • the cancer is a bladder cancer.
  • the cancer is mesothelioma.
  • the cancer is malignant mesothelioma (MM).
  • the cancer is a head and neck cancer.
  • the cancer is a prostate carcinoma.
  • the cancer is an osteosarcoma, which in one embodiment is a cancerous bone tumor.
  • the osteosarcoma is any one of the following subtypes: osteoblastic, chondroblastic, fibroblastic OSA, telangiectatic OSA, small cell OSA, low-grade central OSA, periosteal OSA, paraosteal OSA, secondary OSA, high-grade periosteal OSA, or extraskeletal OSA.
  • the cancer is a Her-2/neu expressing osteosarcoma.
  • the osteosarcoma is canine osteosarcoma.
  • the osteosarcoma is localized osteosarcoma.
  • the osteosarcoma is metastatic osteosarcoma.
  • the osteosarcoma is high grade osteosarcoma.
  • the osteosarcoma is canine appendicular osteosarcoma.
  • the cancer is pulmonary metastatic disease. Each possibility represents a separate embodiment of the present invention.
  • the subject mounts an immune response against the antigen-expressing tumor or target antigen, thereby mediating the anti-tumor effects.
  • the present invention provides an immunogenic composition for treating cancer, the composition comprising a fusion of a truncated LLO to a Her-2 chimeric protein.
  • the immunogenic composition further comprises a Listeria strain expressing the fusion.
  • Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an immunogenic composition for treating cancer, the composition comprising a Listeria strain expressing a Her-2 chimeric protein.
  • an immunogenic composition comprising a recombinant form of Listeria of the present invention.
  • a treatment protocol of the present invention is therapeutic.
  • the protocol is prophylactic.
  • the vaccines of the present invention are used to protect people at risk for cancer such as breast cancer or other types of Her2-containing tumors because of familial genetics or other circumstances that predispose them to these types of ailments as will be understood by a skilled artisan.
  • the vaccines are used as a cancer immunotherapy after debulking of tumor growth by surgery, conventional chemotherapy or radiation treatment.
  • the vaccines are combined with radiation treatment and either surgery, conventional chemotherapy or both. Following such treatments, the vaccines of the present invention are administered so that the CTL response to the tumor antigen of the vaccine destroys remaining metastases and prolongs remission from the cancer.
  • vaccines are used as a cancer immunotherapy in combination with surgery, conventional chemotherapy, radiation treatment, or any combination thereof.
  • such combination treatment is used in subjects that cannot undergo amputation.
  • such combination treatment is used in subjects with primary osteosarcoma that cannot undergo amputation.
  • vaccines of the present invention are used to affect the growth of previously established tumors and to kill existing tumor cells. Each possibility represents a separate embodiment of the present invention.
  • the vaccines and immunogenic compositions utilized in any of the methods described above have any of the characteristics of vaccines and immunogenic compositions of the present invention. Each characteristic represents a separate embodiment of the present invention. It is to be understood that compositions described in the context of the compositions and uses of the present invention may be referred to as immunogenic compositions and vice versa.
  • the dosage in the range of 0.4 LDso/dose. In another embodiment, the dosage is from about 0.4-4.9 LDso/dose. In another embodiment the dosage is from about 0.5-0.59 LDso/dose. In another embodiment the dosage is from about 0.6-0.69 LDso/dose. In another embodiment the dosage is from about 0.7-0.79 LDso/dose. In another embodiment the dosage is about 0.8 LDso/dose. In another embodiment, the dosage is 0.4 LD 50 /dose to 0.8 of the LD 50 /dose.
  • the dosage is 10 7 bacteria/dose. In another embodiment, the dosage is 1.5 x 10 7 bacteria/dose. In another embodiment, the dosage is 2 x 10 7 bacteria/dose. In another embodiment, the dosage is 3 x 10 7 bacteria/dose. In another embodiment, the dosage is 4 x 10 7 bacteria/dose. In another embodiment, the dosage is 6 x 10 7 bacteria/dose. In another embodiment, the dosage is 8 x 10 7 bacteria/dose. In another embodiment, the dosage is 1 x 10 8 bacteria/dose. In another embodiment, the dosage is 1.5 x 10 8 bacteria/dose. In another embodiment, the dosage is 2 x 10 8 bacteria/dose. In another embodiment, the dosage is 3 x 10 8 bacteria/dose. In another embodiment, the dosage is 4 x 10 8 bacteria/dose.
  • the dosage is 6 x 10 8 bacteria/dose. In another embodiment, the dosage is 8 x 10 8 bacteria/dose. In another embodiment, the dosage is 1 x 10 9 bacteria/dose. In another embodiment, the dosage is 1.5 x 10 9 bacteria/dose. In another embodiment, the dosage is 2 x 10 9 bacteria/dose. In another embodiment, the dosage is 3 x 10 9 bacteria/dose. In another embodiment, the dosage is 5 x 10 9 bacteria/dose. In another embodiment, the dosage is 6 x 10 9 bacteria/dose. In another embodiment, the dosage is 8 x 10 9 bacteria/dose. In another embodiment, the dosage is 1 x 10 10 bacteria/dose. In another embodiment, the dosage is 1.5 x 10 10 bacteria/dose. In another embodiment, the dosage is 2 x 10 10 bacteria/dose.
  • the dosage is 3 x 10 10 bacteria/dose. In another embodiment, the dosage is 5 x 10 10 bacteria/dose. In another embodiment, the dosage is 6 x 10 bacteria/dose. In another embodiment, the dosage is 8 x 10 bacteria/dose. In another embodiment, the dosage is 8 x 10 9 bacteria/dose. In another embodiment, the dosage is 1 x 10 11 bacteria/dose. In another embodiment, the dosage is 1.5 x 10 11 bacteria/dose. In another embodiment, the dosage is 2 x 10 11 bacteria/dose. In another embodiment, the dosage is 3 x 10 11 bacteria/dose. In another embodiment, the dosage is 5 x 10 11 bacteria/dose. In another embodiment, the dosage is 6 x 10 11 bacteria/dose. In another embodiment, the dosage is 8 x 10 11 bacteria/dose.
  • the dosage is 5.0 x 10 8 bacteria/dose. In another embodiment, the dosage is 3.3 x 10 9 bacteria/dose. In another embodiment, a composition for the use in the methods provided herein comprises 3.3 x 10 9 Listeria/dose. Each possibility represents a separate embodiment of the present invention.
  • a vaccine or immunogenic composition of the present invention is administered alone to a subject.
  • the vaccine or immunogenic composition is administered together with another cancer therapy, which in one embodiment is radiation therapy.
  • another cancer therapy which in one embodiment is radiation therapy.
  • the recombinant Listeria of methods and compositions of the present invention is, in one embodiment, stably transformed with a construct encoding a Her-2 chimeric antigen or an LLO-Her-2 chimeric antigen fusion.
  • the construct contains a polylinker to facilitate further subcloning.
  • the construct or nucleic acid molecule is integrated into the Listerial chromosome using homologous recombination.
  • homologous recombination techniques for homologous recombination are well known in the art, and are described, for example, in Baloglu S, Boyle SM, et al. (Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein.
  • a recombinant Listeria Monocytogenes strain that expresses E7 was made by chromosomal integration of the E7 gene under the control of the hly promoter and with the inclusion of the hly signal sequence to ensure secretion of the gene product, yielding the recombinant referred to as Lm-AZ/E7.
  • a temperature sensitive plasmid is used to select the recombinants.
  • the construct or nucleic acid molecule is integrated into the Listerial chromosome using transposon insertion. Techniques for transposon insertion are well known in the art, and are described, inter alia, by Sun et al.
  • Transposon mutagenesis has the advantage, in another embodiment, that a stable genomic insertion mutant can be formed but the disadvantage that the position in the genome where the foreign gene has been inserted is unknown.
  • the construct or nucleic acid molecule is integrated into the Listerial chromosome using phage integration sites (Lauer P, Chow MY et al, Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J Bacterid 2002;184(15): 4177-86).
  • an integrase gene and attachment site of a bacteriophage e.g. U153 or PSA listeriophage
  • endogenous prophages are cured from the attachment site utilized prior to integration of the construct or heterologous gene.
  • this method results in single-copy integrants. Each possibility represents a separate embodiment of the present invention.
  • one of various promoters is used to express the antigen or fusion protein containing same.
  • a Listeria monocytogenes promoter is used, e.g. promoters for the genes hly, actA, pica, plcB and mpl, which encode the Listerial proteins hemolysin, actA, phosphotidylinositol-specific phospholipase, phospholipase C, and metalloprotease, respectively.
  • a Listeria monocytogenes promoter is used, e.g. promoters for the genes hly, actA, pica, plcB and mpl, which encode the Listerial proteins hemolysin, actA, phosphotidylinositol-specific phospholipase, phospholipase C, and metalloprotease, respectively.
  • methods and compositions of the present invention utilize a homologue of a Her-2 chimeric protein or LLO sequence of the present invention.
  • the methods and compositions of the present invention utilize a Her-2 chimeric protein from a non-human mammal.
  • the terms "homology,” “homologous,” etc, when in reference to any protein or peptide, refer in one embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
  • the present invention provides an isolated nucleic acid encoding a signal peptide or a recombinant polypeptide or fusion protein of the present invention.
  • the isolated nucleic acid comprises a sequence sharing at least
  • the isolated nucleic acid comprises a sequence sharing at least 75% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 85% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention.
  • the isolated nucleic acid comprises a sequence sharing at least 90% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 95% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 97% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 99% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention.
  • Homology is, in one embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art.
  • computer algorithm analysis of nucleic acid sequence homology may include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
  • identity refers to identity to a sequence selected from a sequence (nucleic acid or amino acid sequence) provided herein of greater than 65%.
  • identity refers to identity to a sequence selected from a sequence provided herein of greater than 70%.
  • the identity is greater than 75%.
  • the identity is greater than 78%.
  • the identity is greater than 80%.
  • the identity is greater than 82%.
  • the identity is greater than 83%.
  • the identity is greater than 85%.
  • the identity is greater than 87%.
  • identity is greater than 88%.
  • identity is greater than 90%.
  • the identity is greater than 92%. In another embodiment, the identity is greater than 93%. In another embodiment, the identity is greater than 95%. In another embodiment, the identity is greater than 96%. In another embodiment, the identity is greater than 97%. In another embodiment, the identity is greater than 98%. In another embodiment, the identity is greater than 99%. In another embodiment, the identity is 100%. Each possibility represents a separate embodiment of the present invention.
  • homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al.,
  • hybridization may be carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide.
  • Hybridization conditions being, for example, overnight incubation at 42 °C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • nucleic acids refers to a string of at least two base-sugar-phosphate combinations.
  • the term includes, in one embodiment, DNA and RNA.
  • Nucleotides refers, in one embodiment, to the monomelic units of nucleic acid polymers.
  • RNA may be, in one embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes.
  • DNA may be in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups.
  • these forms of DNA and RNA may be single, double, triple, or quadruple stranded.
  • the term also includes, in another embodiment, artificial nucleic acids that may contain other types of backbones but the same bases.
  • the artificial nucleic acid is a PNA (peptide nucleic acid).
  • PNA contain peptide backbones and nucleotide bases and are able to bind, in one embodiment, to both DNA and RNA molecules.
  • the nucleotide is oxetane modified. In another embodiment, the nucleotide is modified by replacement of one or more phosphodiester bonds with a phosphorothioate bond.
  • the artificial nucleic acid contains any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353- 57; and Raz NK et al Biochem Biophys Res Commun. 297:1075-84.
  • nucleic acid derivative represents a separate embodiment of the present invention.
  • Protein and/or peptide homology for any amino acid sequence listed herein is determined, in one embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via established methods. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example. Each method of determining homology represents a separate embodiment of the present invention.
  • the present invention provides a kit comprising a reagent utilized in performing a method of the present invention.
  • the present invention provides a kit comprising a composition, tool, or instrument of the present invention.
  • contacting refers to directly contacting the cancer cell or tumor with a composition of the present invention.
  • the terms refer to indirectly contacting the cancer cell or tumor with a composition of the present invention.
  • methods of the present invention include methods in which the subject is contacted with a composition of the present invention after which the composition is brought in contact with the cancer cell or tumor by diffusion or any other active transport or passive transport process known in the art by which compounds circulate within the body.
  • methods of this invention may include at least a single administration of a composition of this invention, wherein in another embodiment, methods of this invention may include multiple administrations of a composition of this invention.
  • the present invention provides methods in which recombinant Listeria is administered only once.
  • Listeria is administered twice.
  • Listeria is administered three times.
  • Listeria is administered four times.
  • Listeria is administered more than four times.
  • Listeria is administered multiple times.
  • Listeria is administered at regular intervals, which in one embodiment, may be daily, weekly, every two weeks, every three weeks, or every month. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides methods in which radiation therapy is administered only once.
  • radiation therapy is administered twice.
  • radiation therapy is administered three times.
  • radiation therapy is administered four times.
  • radiation therapy is administered more than four times.
  • radiation therapy is administered multiple times.
  • radiation therapy is administered at regular intervals, which in one embodiment, may be daily, weekly, every two weeks, every three weeks, or every month. Each possibility represents a separate embodiment of the present invention.
  • the radiation therapy is administered prior to the administration of the recombinant attenuated Listeria. In another embodiment, the radiation therapy is administered twice prior to the first administration of the recombinant attenuated Listeria. In another embodiment, the radiation therapy is administered three times prior to the first administration of the recombinant attenuated Listeria.
  • the recombinant attenuated Listeria is administered prior to the administration of the radiation therapy. In another embodiment, the recombinant attenuated Listeria is administered twice prior to the first administration of the radiation therapy. In another embodiment, the recombinant attenuated Listeria is administered three times prior to the first administration of the radiation therapy.
  • the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention.
  • allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
  • Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals or organisms. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals or organisms. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
  • compositions may be used interchangeably. It is also to be understood that administration of such compositions enhances an immune response, or increase a T effector cell to regulatory T cell ratio or elicit an anti-tumor immune response, as further provided herein.
  • the immunogenic composition provided herein comprises a recombinant Listeria provided herein.
  • a "combination therapy” refers to the combination of radiation therapy described herein administered in conjunction with, or prior to administration of a composition comprising the recombinant Listeria provided herein.
  • compositions containing vaccines and compositions of the present invention are, in another embodiment, administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra- peritonealy, intra- ventricularly, intra-cranially, intra-vaginally or intra-tumorally.
  • the vaccines or compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation.
  • suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the active ingredient is formulated in a capsule.
  • the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelating capsule.
  • the vaccines or compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation.
  • suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration.
  • the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration.
  • the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.
  • repeat administrations (booster doses) of compositions of this invention may be undertaken immediately following the first course of treatment or after an interval of days, weeks or months to achieve tumor regression.
  • repeat doses may be undertaken immediately following the first course of treatment or after an interval of days, weeks or months to achieve suppression of tumor growth.
  • Assessment may be determined by any of the techniques known in the art, including diagnostic methods such as imaging techniques, analysis of serum tumor markers, biopsy, or the presence, absence or amelioration of tumor associated symptoms.
  • a subject is administered a booster dose every 1-2 weeks, every 2-3 weeks, every 3-4 weeks, every 4-5 weeks, every 6-7 weeks, every 7-8 weeks, or every 9-
  • a subject is administered a booster dose every 1-2 months, every 2-3 months, every 3-4 months, every 4-5 months, every 6-7 months, every 7-8 months, or every 9-10 months in order to achieve the intended anti-tumor response.
  • the term “treating” refers to curing a disease. In another embodiment, “treating” refers to preventing a disease. In another embodiment, “treating” refers to reducing the incidence of a disease. In another embodiment, “treating” refers to ameliorating symptoms of a disease. In another embodiment, “treating” refers to increasing performance free survival or overall survival of a patient. In another embodiment, “treating” refers to stabilizing the progression of a disease. In another embodiment, “treating” refers to inducing remission. In another embodiment, “treating” refers to slowing the progression of a disease. The terms “reducing”, “suppressing” and “inhibiting” refer in another embodiment to lessening or decreasing. Each possibility represents a separate embodiment of the present invention.
  • the term “subject” can encompass a mammal including an adult human or a human child, teenager or adolescent in need of therapy for, or susceptible to, a condition or its sequelae, and also may include non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. It will also be appreciated that the term may encompass livestock. The term “subject” does not exclude an individual that is normal in all respects.
  • the term “subject” also encompasses dogs that cannot undergo amputation. In another embodiment, the term “subject” also encompasses humans that cannot undergo surgery. In another embodiment, the term “subject” also encompasses humans that cannot undergo amputation.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals, such as canines, including dogs, and horses, cats, cattle, pigs, sheep, etc.
  • a “therapeutically effective amount”, in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder.
  • a “therapeutically effective amount" of a vaccine provided herein for purposes of treatment of tumor may be determined empirically and in a routine manner.
  • compositions for use in the methods of the present invention comprise a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
  • the metabolic enzyme complements an endogenous gene that is lacking in the chromosome of said recombinant attenuated Listeria strain.
  • mutated or “mutant” describes a deletion. In another embodiment, “mutated” or “mutant” describes an inactivation. In another embodiment,
  • mutated or “mutant” describes a truncation. In another embodiment, “mutated” or “mutant” describes an addition. In another embodiment, “mutated” or “mutant” describes a substitution. In another embodiment, “mutated” or “mutant” describes insertion of a premature stop codon. In another embodiment, “mutated” or “mutant” describes a change to one or more nucleic acids within a gene which disrupts expression of the gene.
  • radiotherapy refers to the medical use of ionizing radiation as part of cancer treatment to control or eradicate malignant cells. Radiotherapy may be used for curative, adjuvant, or palliative treatment. Suitable types of radiotherapy include conventional external beam radiotherapy, stereotactic radiation therapy (e.g., Axesse, Cyberknife, Gamma Knife, Novalis, Primatom, Synergy, X-Knife,
  • radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells.
  • X-rays, gamma rays, and charged particles are types of radiation that may be used for cancer treatment.
  • radiation therapy kills cancer cells by damaging their DNA either directly or by creating free radicals within the cells that can in turn damage the DNA.
  • the radiation may be delivered by a machine outside the body (external-beam radiation therapy), or in another embodiment, it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachy therapy).
  • systemic radiation therapy uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells.
  • radioactive substances such as radioactive iodine
  • the present invention provides a method for concomitantly treating radiation insensitive cancers such as osteosarcomas with standard radiation in combination with immunotherapy, such as administration of recombinant Listeria in a regimen which requires shorter radiation treatment times, thus ameliorating side effects ordinarily associated with radiation treatment.
  • the radiation is administered according to this invention by standard techniques with standard megavoltage equipment, such as AECL Theratron 80, Varian Clinac 4 or Varian Clinac.
  • the maximum size of the radiation portal should be no greater than 300 cm2.
  • a suitable does is between about 15 Gy and 35 Gy, with the specific dose dependent on the area of the body treated.
  • a dose to the spinal cord would be about 35 Gy
  • a dose to the bilateral kidneys would be about 15 Gy and to the whole liver 20 Gy. Breaks in the therapy are at the discretion of the clinician taking into consideration the patients tolerance for radiation therapy.
  • radiation dosages in the combination therapy are administered in sequence.
  • radiation dosages in the combination therapy are administered in on consecutive days as exemplified herein (see Example 11).
  • radiation doses of 8Gy in the combination therapy are on consecutive days for a total dose of 16 Gy prior to administration of Multiple doses (up to 8) of ADXS31-
  • radiation doses of 8Gy in the combination therapy are on consecutive days for a total dose of 16 Gy prior to administration of Multiple doses (up to 8) of up to 3.3xl0 9 CFU ADXS31-164 were given once every 3 weeks as demonstrated in Examples herein (see Example 11).
  • radiation doses of 8Gy in the combination therapy are on consecutive days for a total dose of 16 Gy prior to administration of Multiple doses (up to 8) of 3.3xl0 9 CFU ADXS31-164 were given once every 3 weeks, as demonstrated in Examples herein (see Example 11).
  • the multiple doses range of ADXS31-164 provided herein is from 1-8, 8-15, 15-25, or as many as required to achieve an intended therapeutic goal.
  • total radiation doses administered for a combination therapy provide herein range from 70-80 Gy.
  • total radiation doses administered for a combination therapy provided herein range from 10-26 GY.
  • radiation doses ranging from 10-26 GY are administered in sequence. In one embodiment, the sequence may be hourly, daily, weekly or bi-weekly.
  • radiation doses ranging from 70-80 Gy are administered in sequence.
  • the radiation therapy described in the present invention as part of a combination therapy is palliative radiation therapy.
  • radiation therapy may be given with palliative intent.
  • palliative treatments are intended to relieve symptoms and reduce the suffering caused by cancer or a tumor.
  • the radiation therapy described herein as part of a combination therapy is meant to cure the cancer or tumor.
  • Oligonucleotides were synthesized by Invitrogen (Carlsbad, CA) and DNA sequencing was done by Genewiz Inc, South Plainfield, NJ.
  • Flow cytometry reagents were purchased from Becton Dickinson Biosciences (BD, San Diego, CA). Cell culture media, supplements and all other reagents, unless indicated, were from Sigma (St. Louise, MO).
  • Her-2/neu HLA-A2 peptides were synthesized by EZbiolabs (Westfield, IN). Complete
  • RPMI 1640 (C-RPMI) medium contained 2mM glutamine, 0.1 mM non-essential amino acids, and ImM sodium pyruvate, 10% fetal bovine serum, penicillin/streptomycin, Hepes (25mM).
  • the polyclonal anti-LLO antibody was described previously and anti-Her-2/neu antibody was purchased from Sigma.
  • mice and Cell Lines [00326] All animal experiments were performed according to approved protocols by IACUC at the University of Pennsylvania or Rutgers University.
  • FVB/N mice were purchased from Jackson laboratories (Bar Harbor, ME).
  • the FVB/N Her-2/neu transgenic mice, which overexpress the rat Her-2/neu onco-protein were housed and bred at the animal core facility at the University of Pennsylvania.
  • the NT-2 tumor cell line expresses high levels of rat Her-2/neu protein, was derived from a spontaneous mammary tumor in these mice and grown as described previously.
  • DHFR-G8 (3T3/neu) cells were obtained from ATCC and were grown according to the ATCC recommendations.
  • the EMT6-Luc cell line was a generous gift from Dr. John Ohlfest (University of Minnesota, MN) and was grown in complete C-RPMI medium. Bioluminescent work was conducted under guidance by the Small Animal Imaging Facility (SAIF) at the University of Pennsylvania (Philadelphia, PA).
  • SAIF Small Animal Imaging
  • Her-2/neu-pGEM7Z was kindly provided by Dr. Mark Greene at the University of Pennsylvania and contained the full-length human Her-2/neu (hHer2) gene cloned into the pGEM7Z plasmid (Promega, Madison WI). This plasmid was used as a template to amplify three segments of hHer-2/neu, namely, ECl, EC2, and IC1, by PCR using pfx DNA polymerase (Invitrogen) and the oligos indicated in Table 1.
  • Her-2/neu chimera construct was generated by direct fusion by the SOEing PCR method and each separate hHer-2/neu segment as templates. Primers are shown in Table 3.
  • ChHer2 gene was excised from pAdvl38 using Xhol and Spel restriction enzymes, and cloned in frame with a truncated, non-hemolytic fragment of LLO in the Lmdd shuttle vector, pAdvl34.
  • the sequences of the insert, LLO and hly promoter were confirmed by DNA sequencing analysis.
  • This plasmid was electroporated into electro-competent act A, dal, dat mutant Listeria monocytogenes strain, LmddA and positive clones were selected on Brain Heart infusion (BHI) agar plates containing streptomycin (250 ⁇ g/ml).
  • mice Groups of 3-5 FVB/N mice were immunized three times with one week intervals with 1 x 10 8 colony forming units (CFU) of Lm-LLO-ChHer2, ADXS31-164, Lm-hHer2 ICI or Lm-control (expressing an irrelevant antigen) or were left naive.
  • CFU colony forming units
  • NT-2 cells were grown in vitro, detached by trypsin and treated with mitomycin C (250 ⁇ g/ml in serum free C-RPMI medium) at 37°C for 45 minutes.
  • splenocytes harvested from immunized or naive animals at a ratio of 1 :5 (Stimulator: Responder) for 5 days at 37°C and 5% C0 2 .
  • a standard cytotoxicity assay was performed using europium labeled 3T3/neu (DHFR-G8) cells as targets according to the method previously described. Released europium from killed target cells was measured after 4 hour incubation using a spectrophotometer (Perkin Elmer, Victor 2 ) at 590 nm. Percent specific lysis was defined as (lysis in experimental group-spontaneous lysis)/(Maximum lysis-spontaneous lysis).
  • FVB/N or HLA-A2 transgenic mice were immunized three times with one week intervals with 1 x 10 8 CFU of ADXS31-164, a negative Listeria control (expressing an irrelevant antigen) or were left naive.
  • Splenocytes from FVB/N mice were isolated one week after the last immunization and co-cultured in 24 well plates at 5 x 10 6 cells/well in the presence of mitomycin C treated NT-2 cells in C-RPMI medium.
  • Splenocytes from the HLA-A2 transgenic mice were incubated in the presence of ⁇ of HLA-A2 specific peptides or ⁇ g/ml of a recombinant His-tagged ChHer2 protein, produced in E. coli and purified by a nickel based affinity chromatography system. Samples from supematants were obtained 24 or 72 hours later and tested for the presence of interferon- ⁇ (IFN- ⁇ ) using mouse IFN- ⁇ Enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer's recommendations.
  • IFN- ⁇ interferon- ⁇
  • ELISA Enzyme-linked immunosorbent assay
  • Cells were harvested, washed twice in 1 x PBS and counted.
  • IFN- ⁇ ELISpot assays were performed according to the manufacturer's protocol using a commercial canine IFN- ⁇ ELISpot assay kit (R&D Systems, Minneapolis, MN). Briefly, 0.8 - 2 x 105 stimulated cells were incubated with 2.5 uM of ECl, EC2 or ICI peptide pools plus IL-2 or IL-2 alone (to determine background counts). All assays were performed in duplicates. Plates were developed according to the manufacturer's instructions. Spots were counted using a CTL-Immunospot analyzer (C.T.L, Shaker Heights, OH). Number of spots were normalized by subtracting twice the number of spots counted in non- stimulated wells.
  • mice were implanted subcutaneously (s.c.) with 1 x 10 6 NT-2 cells. On days 7, 14 and 21, they were immunized with 1 x 10 8 CFUs of ADXS31-164, Lm ⁇ M -control or left naive. Tumors and spleens were extracted on day 28 and tested for the presence of CD3 + /CD4 + /FoxP3 + Tregs by FACS analysis. Briefly, splenocytes were isolated by homogenizing the spleens between two glass slides in C-RPMI medium.
  • Tumors were minced using a sterile razor blade and digested with a buffer containing DNase (12U/ml), and collagenase (2mg/ml) in PBS. After 60 min incubation at RT with agitation, cells were separated by vigorous pipetting. Red blood cells were lysed by RBC lysis buffer followed by several washes with complete RPMI-1640 medium containing 10% FBS. After filtration through a nylon mesh, tumor cells and splenocytes were resuspended in FACS buffer (2% FBS/PBS) and stained with anti-CD3-PerCP-Cy5.5, CD4-FITC, CD25-APC antibodies followed by permeabilization and staining with anti-Foxp3-PE. Flow cytometry analysis was performed using 4-color FACS calibur (BD) and data were analyzed using cell quest software (BD).
  • BD 4-color FACS calibur
  • ChHer2 gene was generated by direct fusion of two extracellular (aa 40-170 and aa 359-433) and one intracellular fragment (aa 678-808) of the Her-2/neu protein by SOEing PCR method.
  • the chimeric protein harbors most of the known human MHC class I epitopes of the protein.
  • ChHer2 gene was excised from the plasmid, pAdvl38 (which was used to construct Lm-LLO-ChHer2) and cloned into LmddA shuttle plasmid, resulting in the plasmid pAdvl64 ( Figure 1A).
  • pAdvl38 uses the chloramphenicol resistance marker (cat) for in vitro selection of recombinant bacteria
  • pAdvl64 harbors the D-alanine racemase gene (dot) from bacillus subtilis, which uses a metabolic complementation pathway for in vitro selection and in vivo plasmid retention in LmddA strain which lacks the dal-dat genes.
  • This vaccine platform was designed and developed to address FDA concerns about the antibiotic resistance of the engineered Listeria vaccine strains.
  • pAdvl64 does not harbor a copy of the prfA gene in the plasmid (see sequence below and Figure 1A), as this is not necessary for in vivo complementation of the Lmdd strain.
  • the LmddA vaccine strain also lacks the actA gene (responsible for the intracellular movement and cell-to-cell spread of Listeria) so the recombinant vaccine strains derived from this backbone are 100 times less virulent than those derived from the Lmdd, its parent strain.
  • LmddA -based vaccines are also cleared much faster (in less than 48 hours) than the Lmdd-b&sed vaccines from the spleens of the immunized mice.
  • ADXS31-164 IS AS IMMUNOGENIC AS LM-LLO-ChHER2.
  • mice immunized with a Listeria expressing only an intracellular fragment of Her2-fused to LLO showed lower lytic activity than the chimeras which contain more MHC class I epitopes.
  • ADXS31-164 was also able to stimulate the secretion of IFN- ⁇ by the splenocytes from wild type FVB/N mice ( Figure 2B). This was detected in the culture supernatants of these cells that were co-cultured with mitomycin C treated NT-2 cells, which express high levels of Her-2/neu antigen (Figure 5C).
  • ADXS31-164 WAS MORE EFFICACIOUS THAN LM-LLO-ChHER2 IN
  • ADXS31-164 Anti-tumor effects of ADXS31-164 were compared to those of Lm-LLO-ChHer2 in Her-2/neu transgenic animals which develop slow growing, spontaneous mammary tumors at 20-25 weeks of age. All animals immunized with the irrelevant Listeria-control vaccine developed breast tumors within weeks 21-25 and were sacrificed before week 33. In contrast, Listeria-Her-2/neu recombinant vaccines caused a significant delay in the formation of the mammary tumors. On week 45, more than 50% of ADXS31-164 vaccinated mice (5 out of 9) were still tumor free, as compared to 25% of mice immunized with Lm-LLO-ChHer2. At week 52, 2 out of 8 mice immunized with ADXS31-164 still remained tumor free, whereas all mice from other experimental groups had already succumbed to their disease (Figure 3).
  • ADXS31-164 is more efficacious than Lm-LLO-ChHer2 in preventing the onset of spontaneous mammary tumors in Her-2/neu transgenic animals.
  • mice were implanted with NT-2 tumor cells.
  • Splenocytes and intra- tumoral lymphocytes were isolated after three immunizations and stained for Tregs, which were defined as CD3 + /CD4 + /CD25 + /FoxP3 + cells, although comparable results were obtained with either FoxP3 or CD25 markers when analyzed separately.
  • the lower frequency of Tregs in tumors treated with LmddA vaccines resulted in an increased intratumoral CD8/Tregs ratio, suggesting that a more favorable tumor microenvironment can be obtained after immunization with LmddA vaccines.
  • the vaccine expressing the target antigen Her-2/neu was able to reduce tumor growth, indicating that the decrease in Tregs has an effect only in the presence on antigen-specific responses in the tumor.
  • Tumor samples of the mice immunized with different vaccines such as Lm-LLO- 138, LmddA164 and irrelevant vaccine Lm-LLO-NY were harvested.
  • the DNA was purified from these samples and the DNA fragments corresponding to Her-2/neu regions
  • ICl, ECl and EC2 were amplified and were sequenced to determine if there were any immune escape mutations.
  • the alignment of sequence from each DNA was performed using CLUSTALW. The results of the analysis indicated that there were no mutations in the DNA sequences harvested from tumors.
  • the reference sequences are listed below:
  • GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGC CCGGTGCAAGGG SEQ ID NO: 43
  • mice were immunized IP with ADXS31-164 or irrelevant Lm-control vaccines and then implanted intra-cranially with 5,000 EMT6-Luc tumor cells, expressing lucif erase and low levels of Her-2/neu (Figure 6C). Tumors were monitored at different times post- inoculation by ex vivo imaging of anesthetized mice. On day 8 post-tumor inoculation, tumors were detected in all control animals, but none of the mice in ADXS31-164 group showed any detectable tumors ( Figure 6A and B).
  • ADXS31-164 could clearly delay the onset of these tumors, as on day 11 post- tumor inoculation, all mice in the negative control group had already succumbed to their tumors, but all mice in ADXS31-164 group were still alive and only showed small signs of tumor growth.
  • Canine Osteosarcoma is a cancer of long (leg) bones that is a leading killer of large dogs over the age of 10 years. Standard treatment is amputation immediately after diagnosis, followed by chemotherapy. Invariably, however, the cancer metastasizes to the lungs. With chemotherapy, dogs survive about 18 months compared to 6-12 months, without treatment. The HER2 antigen is believed to be present in up to 50% of osteosarcoma.
  • ADXS31-164 creates an immune attack on cells expressing this antigen and has been developed to treat human breast cancer.
  • Dogs with a histological diagnosis of osteosarcoma and evidence of expression of HER2/neu by malignant cells are eligible for enrollment.
  • Group 1 (3 dogs) receive lxlO 8 CFU per dose
  • Group 2 (3 dogs) each receive 5xl0 8
  • Group 3 (3 dogs) receives lxlO 9 CFU per dose. Additional dogs are added to a Group to gather more data should if a potentially dose limiting toxicities, be observed.
  • a pilot phase I dose escalation study was performed to determine the dose of a L. monocytogenes expressing human Her-2/neu recombinant vaccine that can safely and effectively stimulate tumor-specific immunity in dogs with osteosarcoma.
  • the tumors of all dogs presenting to PennVet for limb amputation due to suspected or confirmed OSA were routinely harvested and evaluated histopathologically to confirm the diagnosis of OSA.
  • tumor sections from all dogs were evaluated by IHC and Western blot analysis to determine whether the tumor expresses Her-2/neu. Only dogs with a histological diagnosis of OSA and evidence of expression of Her-2/neu by malignant cells were eligible for enrollment. Single cell suspensions of tumor tissue taken at surgery were cryopreserved and used as autologous tumor targets in chromium release assays to determine anti-tumor immunity.
  • Group 1 (3 dogs) received the ADXS31-164 (Lm-hucHer-2/neu) vaccine at lxl 0 8 CFU per dose, Group 2 (3 dogs) each received 5xl0 8 CFU per dose, Group 3 (3 dogs) receive lxlO 9 CFU per dose, and 3.3 x 10 9 CFU per dose (1 dog).
  • Recombinant Lm was administered as a slow intravenous infusion over 30 minutes.
  • the dose chosen for Group 1 is the established safe dose for the chimeric huHer-2/neu recombinant in mice. In humans, the non-toxic dose for Lm-LLO-E7 is only one log higher than that established in mice, and this dose is the dose evaluated in Group 3 in this pilot trial.
  • PBMCs are taken at this time for baseline evaluation of anti-tumor immunity. Repeat immune assessment is performed at the time of each vaccination and three weeks after the last vaccination. PBMCs are analyzed for Her-2/neu specific T cell responses by CFSE proliferation, cytokine production (ELISpot and qRT-PCR) and CTL assay against autologous tumor targets as outlined below ( Figure 12).
  • ADXS31-164 dose has ranged from 1 x 10 8 , 5 x 10 8 , 1 x 10 9 and 3.3 x 10 9 CFU.
  • ADXS31- 164 A standard operating procedure was developed for the administration of ADXS31- 164.
  • patients receive 2 mg/kg diphenhydramine via intramuscular injection and 0.2mg/kg ondansetron as a slow intravenous push.
  • the vaccine was kept at -80°C and thawed patient-side. It was administered in 200mls of 0.9% NaCl over 30 mins.
  • the infusion line is then flushed with 30 mis of Plasmalyte. Dogs are sent home with a three day course of amoxicillin (to start 72 hours post vaccination) and a 7 day course of liver supplement (S-adenosyl-methionine) that aids in cellular growth and repair.
  • HER2/neu staining 5 micron thick serial sections of formalin fixed, decalcified, paraffin embedded tissues were mounted on negatively charged glass slides. Sections were heated at 80°C for 20 minutes, immersed in Pro Par (clearant) and rehydrated in ethanol.
  • Antigen retrieval was performed by boiling sections in sodium citrate buffer (pH -9.0).
  • Endogenous peroxidase was blocked using 3% hydrogen peroxide. Staining was performed with a rabbit anti-human HER2/neu antibody (Neu(c-18):sc-284, Santa Cruz Biotecnology) or a rabbit IgG isotype (Universal Negative Control serum, NC498, Biocare Medical). Bound antibody was detected using the Universal Streptavadin-Biotin2 System (DAKO/LSAB2, HRP). Tissues were stained with 3,3'-diaminobenzidine solution (DAKO) and counterstained with hematoxylin. Slides were viewed using a Nikon E600 infinity corrected upright microscope.
  • CBC Complete Blood Count
  • CS Chemistry Screen
  • U Urinalysis
  • PBMCs Peripheral Blood Mononuclear Cells
  • PBMCs Peripheral Blood Mononuclear Cells
  • T cell subset analysis PBMCs taken at baseline, prior to each vaccination, at re- stage and at every 2 months thereafter were analyzed for CD4 and CD8 T cell subsets. Briefly, cryopreserved cells were thawed and washed twice in FACS buffer (lx PBS, 0.2%
  • ADXS31-164 was administered at the following doses; Group 1 (2 x 10 8 CFU) , Group 2 (5 x 10 8 CFU), Group 3 (1 x 10 9 CFU) and Group 4 (3.3 x 10 9 CFU). ADXS31-164 was diluted in lOOmls 0.9% NaCl (Groups 1 and 2) and 200mls 0.9% NaCl (Groups 3 and 4) and administered intravenously over 30 minutes. Temperature, pulse, respiratory rate, heart rate and rhythm (by EKG) and blood pressure were monitored every hour following infusion.
  • Toxicity was graded according to the Veterinary Co-operative Oncology Group- Common Terminology Criteria for Adverse Events (VCOG-CTCAE). Assessment of cardiac toxicity was performed through serial electrocardiograms, echocardiograms and serum cardiac troponin I levels at baseline, at the time of each vaccination, 3 weeks after the last vaccination and every 2 months thereafter until death. Parameters assessed included Left Ventricular Fractional Shortening (LVFS) and Left Ventricular Internal Dimension in diastole (LVIDd) and Left Ventricular Internal Dimension in systole (LVIDs). LVIDd and
  • LVIDs were normalized to body weight to account for the wide range of body size amongst dogs.
  • Time To Metastasis was calculated as the time between amputation and development of metastatic disease.
  • OSA Specific Survival was calculated as the time between amputation and death. Patients that died of unrelated causes were censored at the time of their death.
  • Five additional dogs with pre-existing pulmonary metastatic disease, identified at the time of screening also received ADXS31-164 on a compassionate care basis (Table 4).
  • Dogs with strong HER2/neu staining in >50 of neoplastic cells from their primary tumor had strong HER2/neu staining in >50 of neoplastic cells from their primary tumor.
  • the two dogs with single nodules received the full course of three vaccines each.
  • Figure 15 shows a schematic of the time-line of the phase 1 clinical trial, wherein three vaccinations were administered following amputation and follow-up chemotherapy.
  • Table 5 Signalment and tumor characteristics of dogs with pre-existing metastatic disease treated on a compassionate care basis.
  • transient single or bigeminal ventricular premature contractions were identified shortly after vaccination.
  • One dog with pre-existing metastatic disease developed ventricular tachycardia within 2 hours of vaccination.
  • Treatment with lidocaine, procainamide, sotalol and corticosteroids had little effect however, the arrhythmia resolved within 72 hours.
  • HER2/neu targeted monoclonal antibodies cause cardio toxicity we evaluated biomarkers of cardiac damage and echocardiographic measures of dysfunction including cardiac troponin I, fractional shortening ( ), LVIDd and LVIDs at baseline, prior to each vaccination and every 2 months thereafter. No significant, sustained changes in cardiac troponin I, fractional shortening, LVIDd or LVIDs were identified in any of the vaccinated dogs (Fig. 26 A-D).
  • One dog in Group 3 showed a stepwise increase in serum cardiac troponin I at the time of each vaccination however, this was not accompanied by echocardiographic signs of dysfunction. Values returned to baseline following the last vaccination and were not elevated on repeat assessments.
  • Table 6 Treatment Related Adverse Events occurring at or within 48 hours of ADXS31-164 vaccination.
  • ADXS31-164 The ability of ADXS31-164 to induce and maintain an immune response, and in particular to induce HER2/Neu specific T cell immunity was assessed during the clinical trial. In order to evaluate the immune response and to determine if a HER2/Neu specific T cell response was induced by ADXS31-164, HER2/Neu specific T cell numbers were assessed by IFN- ⁇ ELISpot. Samples were taken at baseline (3 weeks post carboplatin), at every vaccination and every 2 months thereafter. Figure 19 shows the results of the ELISpot assay.
  • HER2/neu Specific Immune Responses Immunological responses against the human ECl, EC2 and IC1 domains of HER2/neu (sharing 89%, 93% and 98% identity with canine HER2/neu respectively) were detected at baseline in 4/18, 6/18 and 1/18 dogs respectively. Induced IFN- ⁇ responses against one or more of the HER2/neu domains were detected in 7 dogs 3 weeks after the third ADXS31-164 vaccination (Table 7). Five of these dogs developed immune responses against the highly conserved IC1 domain. Five additional dogs developed IFN- ⁇ responses against the IC1 domain 2 months later.
  • Booster vaccinations Ten of the 18 do gs without metastatic disease at enrollment were administered a single booster vaccine between 5 and 10 months after the initial vaccine series. Four of these dogs received additional booster vaccines given between 4 and 15 months after the first booster vaccine. Similar low grade, transient side effects were noted at the time of booster vaccination as with the initial vaccination series.
  • Figure 20 show that repeat booster vaccinations also stimulated HER2 specific immunity. Repeat booster vaccinations were administered at 6 and 10 months for animal 289-003, and at 8 months for animal 289-004.
  • the 4 dogs with bone metastases were treated with analgesics only (1 dog), palliative radiation alone (1 dog) or in combination with chemotherapy (2 dogs).
  • Median OSA specific survival for vaccinated dogs has not yet been reached.
  • Kaplan-Meier survival curves for TTM and OS A Specific Survival are shown in Fig. 21.
  • Overall survival rates at 1 and 2 years for vaccinated dogs are 71.4% and 57% respectively.
  • 9 are still alive (3 dogs > 900 days, 1 dog>700 days, 3 dogs > 400 days and 2 dogs > 300 days and 7 remain tumor free to date (Table 7)).
  • the results presented in Figure 24 demonstrate that ADXS31-164 breaks the tolerance to HER2/Neu. This may be significant for the treatment of OSA as well as other HER2/Neu tumors and/or cancers.
  • Necropsy findings 6/18 dogs died during the study period and necropsies were performed on 4 of these dogs. Three dogs were found to have multifocal grade II and III metastatic osteosarcoma involving the lungs (3 dogs), bone (2 dogs), mediastinum (1 dog) and kidney (1 dog). One dog, euthanized on account of a large progressive renal mass was found to have nephroblastoma. This dog also had a single pulmonary nodule but this was unfortunately not evaluated by histopathology.
  • ICG dye indocyanine green
  • the dog recovered well from surgery and remained free of pulmonary metastatic disease for 5 months before developing widespread aggressive, HER2/neu+ metastatic disease in the subcutaneous tissue (osteoblastic, grade II and chondroblastic, grade III), mediastinum (osteoblastic, grade II) and diaphragm (osteoblastic, grade III).
  • Results show that despite induction of HER2/neu specific T cell responses, off-tumor side effects were not identified, hence induction of HER2/neu specific T cells is responsible for elimination of HER2/neu positive metastatic cells and long term protection from disease recurrence. This is supported by the timing of HER2/Neu-specific T cell expansion which in 5 dogs occurred approximately 8 months post diagnosis, when many dogs will develop metastatic disease and by the histopathological findings of focal T cell responses within the pulmonary parenchyma of one dog following vaccination and metastatectomy.
  • ADXS31-164 prevents pulmonary metastatic disease through its ability to induce potent innate immune responses that may sensitize metastatic OSA cells to FAS/FASL mediated apoptosis and adaptive immune responses in the form of HER2/Neu specific T cells that eliminate micrometastatic pulmonary disease.
  • ADXS31-164 in doses up to 3.3 x 10 ⁇ 9 CFU are safe in the dog and despite inducing HER2/neu specific immunity, do not lead to short or long term cardio toxicity.
  • off tumor side effects including cardio toxicity has been associated with the administration of large numbers of HER2/neu specific T cells or when trastuzumab has been used concurrently with anthracyclines.
  • ADXS31-164 can prevent pulmonary metastatic disease in dogs with OSA. These results demonstrate safety and unprecedented survival times in dogs with OSA and pave the way to investigate the ability of ADXS31-164 to prevent metastatic disease in patients with HER2/neu expressing tumors including pediatric osteosarcoma and mammary carcinoma.
  • ADXS31-164 vaccine The details of the construction of ADXS31-164 vaccine have been described above.
  • the ADXS31-164 vaccine stocks were prepared and stored as 1 ml aliquots in freezer at -70 °C. Before injection, vaccine stocks were thawed at 37 °C for 2-3 min and then washed twice with phosphate-buffer saline (PBS) and resuspended in PBS at a final concentration of PBS.
  • PBS phosphate-buffer saline
  • HER2 + appendicular OSA, and no evidence of cardiac or metastatic disease were enrolled.
  • a bone biopsy to confirm the diagnosis of osteosarcoma was performed at the time of enrollment.
  • cTnl and radiographs of the affected limb and the thorax were performed on Days 0, 70, and 133 and every 2 months thereafter until euthanasia.
  • CBC Complete Blood Count
  • CS Chemistry Screen
  • U Urinary Analysis
  • Immune analysis electrocardiogram (EKG)/Echocardiogram/ serum concentration of cardiac troponin I (cTnl); and necropsy were performed.
  • ELISpot assayCryopreserved PBMC from each indicated time point were thawed, rested overnight at 37 °C and then counted.
  • Cells were stimulated with 2.0 uM pools of overlapping human HER2/Neu peptides (l lmers overlapping by 5 amino acids) that represent the ECl, EC2 and ICl domains of HER2/Neu present in the chimeric vaccine, and recombinant human IL-2 (Invitrogen, Fredrick, MD) for 5 days. Cells were harvested, washed twice in 1 x PBS and counted.
  • IFN- ⁇ ELISpot assays were performed according to the manufacturer's protocol using a commercial canine IFN- ⁇ ELISpot assay kit (R&D Systems, Minneapolis, MN). Briefly, 0.1 - 3 x 10 5 stimulated cells were incubated with 2.5 uM of ECl, EC2 or IC1 peptide pools or none (to determine background counts). All assays were performed in duplicates. Plates were developed according to the manufacturer's instructions. Spots were counted using a CTL-Immunospot analyzer (C.T.L, Shaker Heights, OH).
  • CTL-Immunospot analyzer C.T.L, Shaker Heights, OH.
  • ADXS31-164 was administered to dogs with spontaneous appendicular OS A following 16 Gy RT administered on two consecutive days. Up to 8 doses of ADXS31-164 were administered. This work showed repeat administrations of 3.3 x 10 9 CFU of ADXS31-164 to be safe.
  • Figure 10 describes the treatment protocol. Dogs were screened on day 0 for enrollment in the trial. Screening included evaluation of baseline blood tests, urinalysis, cardiac evaluation, thoracic and affected limb radiographs and bone biopsy to confirm the diagnosis of osteosarcoma. Palliative radiation was given on 2 consecutive days following enrollment. Multiple doses (up to 8) of ADXS31-164 were given once every 3 weeks following palliative radiation therapy with only transient, low-grade, side effects (data not shown). Thoracic and limb radiographs were repeated at day 70 and 154. Lameness scores were assigned by two board certified veterinary orthopedic surgeons to each dog at each time point based on their evaluation of videos taken of each dog at each time point.
  • results show that palliative radiation therapy in conjunction with ADXS31-164 therapy reduces lysis, promotes tumor consolidation, and prolongs survival of subjects (Figure 29 A-Dand Figure 30-A-B).

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Abstract

This invention provides methods for inducing an immune response against a Her-2/neu antigen-expressing tumor and for treating the same and vaccinating against the same in human and canine subjects using a combination of radiation therapy and a recombinant attenuated Listeria strain vaccine.

Description

COMBINATION IMMUNO THERAPY AND RADIOTHERAPY FOR THE TREATMENT OF HER-2-POSITIVE CANCERS
FIELD OF INVENTION
[001] This invention provides methods for inducing an immune response against a Her- 2/neu antigen-expressing tumor and for treating the same and vaccinating against the same in human and canine subjects using a combination of radiation therapy and a recombinant attenuated Listeria strain vaccine.
BACKGROUND OF THE INVENTION
[002] Her-2/neu (referred to henceforth as "Her-2") is a 185 kDa glycoprotein that is a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinases, and is overexpressed in 25 to 40% of all breast cancers and in many cancers of the bone (osteosarcoma - OSA), ovaries, lung, pancreas, brain, and gastrointestinal tract. Patients with cancers that overexpress Her-2 exhibit tolerance even with detectable humoral, CD8+ T cell, and CD4+ T cell responses directed against Her-2.
[003] Large breed dogs spontaneously develop OSA that recapitulates many aspects of human pediatric OSA including histologic heterogeneity, aggressive local disease and early metastases. At diagnosis, 95% of dogs have micrometastatic disease and despite amputation and chemotherapy, the median survival time is 10 months with most dogs euthanized due to progressive metastatic disease. The overall survival of human patients with metastatic osteosarcoma ranges from 10-50%, depending on the location and the number of metastatic foci.
[004] Radiation therapy (RT), which is used to destroy tumor cells or to alter tumor/stroma architecture, is an integral part of treatment of many types of cancer. However, because OSA is radioresistant to standard dose of radiotherapy, it is not used for treating OSA.
[005] Recently there has been evidence that RT may synergize with targeted immune therapy. For example, RT induces immunogenic cell death wherein tumor cells die slowly over time from apoptosis, necrosis and/or mitotic catastrophe, leading to the clearance of the dying cells by the immune system. This in turn serves as a potential source of tumor antigens for immune therapy. RT also modulates tumor cell surface expression of cell death receptors, tumor-associated antigens and adhesion molecules, which render the tumor cells more susceptible to immune-mediated killing.
[006] The present invention meets the needs of subjects suffering from OSA with surprising findings that radiation therapy when combined with a recombinant Listeria-Her- 2/neu vaccine (ADXS31-164) that was generated using the LmddA vaccine vector which has a well-defined attenuation mechanism and is devoid of antibiotic selection markers is particularly effective against osteosarcoma and pulmonary metastasis.
SUMMARY OF THE INVENTION
[007] In one embodiment, the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[008] In another embodiment, the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[009] In another embodiment, the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0010] In another embodiment, the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0011] In another embodiment, the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0012] In one embodiment, the subject is a human. In one embodiment, the human subject is a child. In another embodiment, the human subject is an adult. In another embodiment, the subject is a canine.
[0013] In another embodiment, administering said fusion polypeptide to said subject prevents escape mutations within said tumor.
[0014] In another embodiment, said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes. In another embodiment, said Her- 2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the canine MHC-class I epitopes.
[0015] In one embodiment, the nucleic acid molecule is integrated into the Listeria genome.
In another embodiment, the nucleic acid molecule is in a plasmid in said recombinant Listeria vaccine strain and the plasmid is stably maintained in the recombinant Listeria vaccine strain in the absence of antibiotic selection.
[0016] In one embodiment, the recombinant Listeria lacks the actA virulence gene. In one embodiment, the additional polypeptide is selected from the group consisting of: a) nonhemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment. In one embodiment, the metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme. In some embodiments of this invention, a recombinant attenuated Listeria strain is ADXS31-164.
[0017] In one embodiment, the recombinant attenuated Listeria strain is administered with an independent adjuvant, which, in one embodiment, comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
[0018] In one embodiment, the cancer is osteosarcoma (OSA). In another embodiment, the cancer or tumor is pulmonary metastatic disease. In one embodiment, administration comprises at least two administrations of said recombinant attenuated Listeria strain. In one embodiment, the administration of said radiation therapy comprises at least two administrations of said radiation therapy. In one embodiment, provided herein is a combination therapy comprising a radiation therapy and administration of ADXS31-164 provided herein. In one embodiment, the radiation therapy is administered prior to administration of the recombinant attenuated Listeria strain.
[0019] In another embodiment, the subject does not undergo amputation prior to administration of said radiation therapy and said recombinant attenuated Listeria strain. In another embodiment, the method further comprises administering said radiation therapy and said recombinant attenuated Listeria strain following a relapse or metastasis in said subject, which in one embodiment, is pulmonary metastatic disease.
[0020] In one embodiment, the method results in increased overall survival of said subject. In another embodiment, the method results in a delay of metastatic disease in a subject. In another embodiment, the method results in an increased Her-2/neu specific T cell response.
In another embodiment, said elicitation of an enhanced immune response results in increased overall survival of said subject. In another embodiment, said elicitation of an enhanced immune response results in a delay of metastatic disease in a subject. In one embodiment, the metastatic disease is pulmonary metastatic disease. In another embodiment, said elicitation of an enhanced immune response results in an increased Her-2/neu specific T cell response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:
[0022]Figure 1. Construction of ADXS31-164. (A) Plasmid map of pAdvl64, which harbors bacillus subtilis dal gene under the control of constitutive Listeria p60 promoter for complementation of the chromosomal dal-dat deletion in LmddA strain. It also contains the fusion of truncated LLO(i_44i) to the chimeric human HER2/neu gene, which was constructed by the direct fusion of 3 fragments the HER2/neu: ECl (aa 40-170), EC2 (aa 359-518) and ICI (aa 679-808). The vector schematic on the right shows details pAdvl64 expressing a chimeric HER2/neu fusion protein consisting of 2 extracellular domains and one intracellular domain of human HER2/neu fused to truncated LLO. The plasmid is maintained within the recombinant dal/dat/ actA" listeria strain (LmddA) by means of auxotrophic complementation of the dal gene (See Examples). (B) Expression and secretion of tLLO- ChHer2 was detected in Lm-LLO-ChHer2 (Lm-LLO-138) and Lmi«A-LLO-ChHer2 (ADXS31-164) by western blot analysis of the TCA precipitated cell culture supernatants blotted with anti-LLO antibody. A differential band of -104 KD corresponds to tLLO-
ChHer2. The endogenous LLO is detected as a 58 KD band. Listeria control lacked ChHer2 expression.
[0023] Figure 2. Immunogenic properties of ADXS31-164 (A) Cytotoxic T cell responses elicited by HER2/neu Listeria-b&sed vaccines in splenocytes from immunized mice were tested using NT-2 cells as stimulators and 3T3/neu cells as targets. Lm-control was based on the LmddA background that was identical in all ways but expressed an irrelevant antigen (HPV16-E7). (B) IFN-γ secreted by the splenocytes from immunized FVB/N mice into the cell culture medium, measured by ELISA, after 24 hours of in vitro stimulation with mitomycin C treated NT-2 cells. (C) IFN-γ secretion by splenocytes from HLA-A2 transgenic mice immunized with the chimeric vaccine, in response to in vitro incubation with peptides from different regions of the protein. A recombinant ChHer2 protein was used as positive control and an irrelevant peptide or no peptide groups constituted the negative controls as listed in the figure legend. IFN-γ secretion was detected by an ELISA assay using cell culture supernatants harvested after 72 hours of co-incubation. Each data point was an average of triplicate data +/- standard error. * P value < 0.001.
[0024] Figure 3. Tumor Prevention Studies for Listeria -ChHER2/neu Vaccines HER2/neu transgenic mice were injected six times with each recombinant Listeria-C Rer2 or a control Listeria vaccine. Immunizations started at 6 weeks of age and continued every three weeks until week 21. Appearance of tumors was monitored on a weekly basis and expressed as percentage of tumor free mice. *p<0.05, N = 9 per group.
[0025] Figure 4. Effect of immunization with ADXS31-164 on the % of Tregs in Spleens. FVB/N mice were inoculated s.c. with 1 x 106 NT-2 cells and immunized three times with each vaccine at one week intervals. Spleens were harvested 7 days after the second immunization. After isolation of the immune cells, they were stained for detection of Tregs by anti CD3, CD4, CD25 and FoxP3 antibodies, dot-plots of the Tregs from a representative experiment showing the frequency of CD25+/FoxP3+ T cells, expressed as percentages of the total CD3+ or CD3+CD4+ T cells across the different treatment groups.
[0026] Figure 5. Effect of immunization with ADXS31-164 on the % of tumor infiltrating Tregs in NT-2 tumors. FVB/N mice were inoculated s.c. with 1 x 106 NT-2 cells and immunized three times with each vaccine at one week intervals. Tumors were harvested 7 days after the second immunization. After isolation of the immune cells, they were stained for detection of Tregs by anti CD3, CD4, CD25 and FoxP3 antibodies. (A), dot- plots of the Tregs from a representative experiment. (B). Frequency of CD25+/FoxP3+ T cells, expressed as percentages of the total CD3+ or CD3+CD4+ T cells (left panel) and intratumoral CD8/Tregs ratio (right panel) across the different treatment groups. Data is shown as mean+SEM obtained from 2 independent experiments.
[0027] Figure 6. Vaccination with ADXS31-164 can delay the growth of a breast cancer cell line in the brain. Balb/c mice were immunized thrice with ADXS31-164 or a control Listeria vaccine. EMT6-Luc cells (5,000) were injected intracranially in anesthetized mice. (A) Ex vivo imaging of the mice was performed on the indicated days using a Xenogen X-
100 CCD camera. (B) Pixel intensity was graphed as number of photons per second per cm2 of surface area; this is shown as average radiance. (C) Expression of HER2/neu by EMT6- Luc cells, 4T1-Luc and NT-2 cell lines was detected by Western blots, using an anti- HER2/neu antibody. J774.A2 cells, a murine macrophage like cell line was used as a negative control.
[0028] Figure 7. Shows the first 18 canine osteosarcoma patients vaccinated with ADXS31- 164, following amputation and chemotherapy.
[0029] Figure 8. Shows that ADXS31-164 administration does not cause A) early evidence of dilated cardiomyopathy. B) Sequential cardiac troponin I levels evaluated over the course of the study showing that the levels stay within the normal range throughout the study period for the majority of dogs. It should be noted that the one dog with a temporarily increased cardiac troponin I level had unremarkable echocardiograms at the time these values were mildly increased (see also Fig 26D).
[0030] Figure 9. Shows ADXS31-164 associated changes in A) body temperature and B) systolic blood pressure. Body temperature and systolic blood pressure were recorded at baseline and every 2 hours post ADXS31-164 administration. Parameters for each dog at each vaccination are displayed. Horizontal bars represent median values for all dogs in each dose group at each time point. *p<0.05, ** p<0.005 [0031] Figure 10. Shows treatment schedule of combination ADXS31-164 and palliative radiation therapy (RT) in the context of primary appendicular osteosarcoma without amputation and chemotherapy.
[0032]Figure 11. Top panel: Radiographs showing the presence of a fracture of the proximal humerus associated with osteosarcoma and those taken after fracture fixation using two bone plates and an intramedullary pin. Bottom panel: A CT scan of the chest shoing no evidence of metastatic disease at enrollment. Radiographs were also taken at baseline and after 8 ADXS31-164 administrations. These radiographs show no evidence of pulmonary metastatic disease and the presence of boney callus surrounding the fracture site indicating fracture healing despite the presence of osteosarcoma.
[0033] Figure 12. Timeline of a pilot phase I clinical trial to evaluate the safety and efficacy of a L. monocytogenes recombinant expressing ADXS31-164 to elicit therapeutically effective anti-tumor immunity in dogs with appendicular osteosarcoma, that undergo limb amputation and follow up chemotherapy.
[0034]Figure 13 A-B. Treatment-related adverse events and survival curves following
ADXS-31-164 administration. Figure 13A shows treatment-related adverse events. Figure 13B shows all dogs without metastatic disease at the time of trial enrollment. Dogs in the control group underwent limb amputation followed by either carboplatin alone or carboplatin plus Adriamycin. 2 dogs have been censored from the vaccine arm as they died of unrelated causes (1 dog died from aspiration pneumonia, the other died from nephroblastoma).
Vaccinated group Red line; Control group Black line.
[0035] Figure 14. Radiographic images of primary and metastatic osteosarcoma (OSA) in a human (A) and canine (B) patient. In both species, primary lesions are characterized by areas of proliferation and lysis in the bone metaphysis (arrows in A).
[0036]Figure 15. Schematic of the phase I, 3+3 clinical trial to evaluate the safety and efficacy of ADXS31-164 in dogs with HER2+ osteosarcoma (OSA). Privately owned dogs with spontaneous HER2+ appendicular OSA underwent standard of care amputation and follow up carboplatin chemotherapy. Three weeks after the last carboplatin dose, dogs were vaccinated with either 2xl08, 5xl08, lxlO9 or 3.3xl09 CFU of ADXS31-164 intravenously (three vaccinations given three weeks apart). Dogs were re-staged every 2 months until death to determine vaccine efficacy in preventing metastatic disease.
[0037] Figure 16. HER2/neu expression in canine primary osteosarcoma. (A) H&E stain of primary OSA from a dog showing nests of malignant osteoblasts and osteoid deposition. (B) Immunohistochemical evaluation of canine primary OSA showing HER2/neu expression within malignant osteoblasts. (C) Western blot of primary OSA samples from 5 privately owned dogs showing variable expression of HER2/neu. Positive controls are: MCF-7 human mammary carcinoma cell line andCAMAC2 a canine mammary carcinoma cell line.
[0038] Figure 17. Hematological values at baseline (Pre) and at 24 hours after (Post) ADXS31-164 administration. Pre and Post values from all dogs within each dose group at each vaccination were averaged. *p<0.05, ** p<0.005. Shows a transient, but statistically significant increase in white blood cell and neutrophil counts (A-B) that occurred 24 hours after ADXS31-164 administration and that were accompanied by a transient decrease in platelets and lymphocytes (C-D).
[0039] Figure 18. ADXS31-164 induced increases in white blood cells (WBC), neutrophil and monocyte counts correlate with survival. WBC, neutrophil and monocyte counts were measured at baseline and 24 hours after vaccination. The percent increase was calculated following each vaccination and averaged for each dog. (A) Results are displayed according to survival (dead or alive). (B) Results are displayed according to ADXS31-164 dose received. Horizontal bars represent median values of the group.
[0040] Figure 19. Shows the results of evaluation of Her-2 specific T cell responses induced by ADXS31-164 by IFN-γ ELISpot.
[0041] Figure 20. Shows repeat "booster" vaccinations Stimulate Her-2 specific immunity. (A) Shows the results for patient 289-003. (B) Shows the results for patient 289-004. ECl, EC2 and IC1 represent the peptide fragments of the HER2/neu polypeptide.
[0042] Figure 21. Kaplan Meier estimates for (A) Time To Metastasis (TTM) and (B) OS A
Specific Survival.
[0043] Figure 22. Shows that ADXS31-164 prevents development of metastatic disease. (A and B) Thoracic radiographs taken 3 weeks after carboplatin therapy (A) and 3 weeks after the third ADXS31-164 vaccine (B) showing an increase in size of the pre-existing metastatic nodule in the right cranial lung lobe but lack of further metastatic disease development in remaining lung lobes. (C and D) Pulmonary nodule identified on thoracoscopy that fluoresces under near infra-red light following administration of ICG (C). Grossly normal appearing pulmonary tissue removed at the time of metastatectomy showing fluorescence under near infra-red light (inset) (D). (E and F) H&E stained histopathology of (E) pulmonary nodule and (F) fluorescing normal pulmonary tissue showing significant hemorrhage and necrosis of encapsulated pulmonary nodule (E) and focal area of inflammation in grossly normal appearing pulmonary tissue (F). (G and H) Immunohistochemistry of pulmonary nodule at low (G) and high (H) magnification showing CD3+ T cells surrounding the pulmonary nodule with minimal CD3+ T cells within the neoplastic tissue. (I and J) Immunohistochemistry of normal appearing pulmonary tissue at low (I) and high (J) magnification showing focal accumulations of CD3+ T cells. (K) High magnification H&E stain of focal pneumonia showing large abnormal cells with mitotic figures surrounded by lymphocytes. (L) Vimentin stain of pneumonic region showing a large vimentin positive cell, with prominent mitotic figures surrounded by mononuclear cells.
[0044] Figure 23. ADXS31-164 delays/prevents metastatic disease and prolongs overall survival in dogs with spontaneous HER2+ osteosarcoma. Shown is a Kaplan-Meier survival curve of vaccinated dogs compared with a historical control group. The control group consisted of dogs with HER2+ appendicular OSA, treated with amputation and follow-up chemotherapy but who did not receive ADXS31-164. P<0.0001. Vaccinated group Red line; Control group Black line.
[0045] Figure 24. Shows that ADXS31-164 breaks tolerance to HER2/neu. PBMCs were collected at baseline, 3 weeks after the 3rd vaccine (9 weeks) and 2 months later (17 weeks) and analyzed by IFN-γ ELISpot for responses to the highly conserved ICl domain of HER2/neu. Results presented divided dogs into early responders, late responders and apparent non-responders. NA indicates that the 17 week sample for these dogs was not yet evaluated.
[0046] Figure 25A-D. Shows that ADXS31-164 does not adversely affect cardiac function. Cardiac parameters LVID (diastole) (Figure 25A), LVH) (systole) (Figure 25B) and fractional shortening (Figure 25C) were evaluated for each dog at baseline, the time of vaccination and every 2 months thereafter. Cardiac troponin I levels were evaluated at the same time points (Figure 25D).
[0047] Figure 26. Shows that ADXS31-164 breaks immune tolerance to the highly conserved intracellular domain of HER2/neu.
[0048] Figure 27A-D. Shows that radiation therapy in conjunction with ADXS31-164 therapy delays progression of primary osteosarcoma (OSA) in subject 386-002 (Figure 27 A), subject 385-005 (Figure 27B), subject 386-003 (Figure 27C), and subject 386-007 (Figure 27D).
[0049] Figure 28 A-C. Shows the results of a pain questionnaire in subjects with pain interfering with general activity (Figure 28 A), in subjects with pain interfering with the ability to walk (Figure 28B), and in subjects with pain interfering with the enjoyment of life
(Figure 28C).
[0050] Figure 29A-D. Shows that palliative radiation therapy in conjunction with ADXS31- 164 therapy reduces lysis, promotes tumor consolidation, and prolongs survival of subjects (Figure 29 A-D). Lateral (Figure 29 A) and AP (Figure 29C) radiographic views of a distal tibial osteosarcoma lesion demonstrating marked cortical bone remodeling, and reduction in lysis, following 16Gy radiation (given as 8Gy on 2 consecutive days starting on 9.13.2014) and 3 doses of ADXS31-164 (11.25.2014). Lateral (Figure 29B) and AP (Figure 29D) radiographic views of a distal radial osteosarcoma lesion treated with 16Gy radiation (given as 8Gy on 2 consecutive days starting on 7.16.2014) and 3 doses of ADXS31-164 (10.13.2014). Note the significant reduction in swelling and bony lysis within the distal portion of the radius (compare radiographs dated 10.13.2014 with 7.16.2014 in Figure 29B). There is increased bone density on the medial aspect of the distal tibia (compare radiographs dated 10.13.2014 with 7.16.2014 in Figure 29D). There is a small minimally displaced bone fracture of the medial aspect of the distal radius seen on the 10.13.2014 radiographs in Figure 29D.
[0051] Figure 30A-B. Shows that radiation therapy in conjunction with ADXS31-164 prolongs survival in dogs with osteosarcoma (OSA).
[0052] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0053] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.
[0054] In one embodiment, the present invention provides a method of treating a tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0055] In one embodiment, the present invention provides a method of preventing a tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0056] In one embodiment, the present invention provides a method of eliciting an enhanced immune response against a tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0057] In one embodiment, the present invention provides a method of prolonging survival in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0058] In one embodiment, the present invention provides a method of delaying metastatic disease in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0059] In one embodiment, the present invention provides a method of breaking tolerance to a tumor specific antigen in a subject suffering from a tumor growth or cancer expressing said tumor specific antigen comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0060] In one embodiment, the tumor specific antigen is Her-2/neu.
[0061] In one embodiment, the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
[0062] In one embodiment, the present invention provides a method of preventing a Her- 2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
[0063] In another embodiment, the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
[0064] In another embodiment, the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
[0065] In another embodiment, the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide.
[0066] In another embodiment, the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant.
[0067] In one embodiment, the recombinant attenuated Listeria strain further comprises a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0068] In one embodiment, the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0069] In one embodiment, the present invention provides a method of preventing a Her- 2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0070] In another embodiment, the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0071] In another embodiment, the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0072] In another embodiment, the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0073] In another embodiment, the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0074] In one embodiment, the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0075] In one embodiment, the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0076] In another embodiment, the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0077] In another embodiment, the present invention provides a method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0078] In another embodiment, the present invention provides a method of delaying metastatic disease in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0079] In another embodiment, the present invention provides a method of breaking tolerance to Her-2/neu in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
[0080] In one embodiment, the subject is a human. In one embodiment, the human subject is a child. In another embodiment, the human subject is an adult.
[0081] In one embodiment, the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a human subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0082] In one embodiment, the present invention provides a method of preventing a Her-
2/neu-expressing tumor growth or cancer in a human subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0083] In another embodiment, the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a human subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0084] In another embodiment, the present invention provides a method of prolonging survival in a human subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0085] In another embodiment, the present invention provides a method of delaying metastatic disease in a human subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0086] In another embodiment, the present invention provides a method of breaking tolerance to Her-2/neu in a human subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0087] In another embodiment, the subject is a canine. In one embodiment, the canine is a dog.
[0088] In one embodiment, the present invention provides a method of treating a Her-2/neu- expressing tumor growth or cancer in a canine subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0089] In one embodiment, the present invention provides a method of preventing a Her-
2/neu-expressing tumor growth or cancer in a canine subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0090] In another embodiment, the present invention provides a method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a canine subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0091] In another embodiment, the present invention provides a method of prolonging survival in a canine subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0092] In another embodiment, the present invention provides a method of delaying metastatic disease in a canine subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0093] In another embodiment, the present invention provides a method of breaking tolerance to Her-2/neu in a canine subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional adjuvant and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain.
[0094] In one embodiment, the present invention provides a method of delaying metastatic disease or treating metastatic disease in a subject. In one embodiment, the metastatic disease is pulmonary metastatic disease.
[0095] Thus, in one embodiment, the present invention provides a method of delaying pulmonary metastatic disease in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0096] In another embodiment, the present invention provides a method of treating pulmonary metastatic disease in a subject suffering from a tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a tumor specific antigen fused to an additional polypeptide.
[0097] In one embodiment, provided herein are methods for preventing, treating, prolonging survival, delaying metastatic disease, breaking tolerance to Her-2/neu, vaccinating against a Her2-neu antigen-expressing tumor, inducing an immune response, eliciting an enhanced immune response against sub-dominant epitopes of the Her2-neu antigen, while circumventing mutation avoidance. In another embodiment, the administration of the fusion polypeptide of the present invention to the subject prevents escape mutations within said tumor. In another embodiment, circumventing mutation avoidance is due to epitope spreading. In yet another embodiment, mutation avoidance is due to the chimeric nature of the antigen.
[0098] In another embodiment, provided herein is an immunogenic composition for use in the claimed methods comprising a fusion polypeptide, wherein said fusion polypeptide comprises a Her-2/neu chimeric antigen fused to an additional polypeptide, and wherein administering the fusion protein to a subject having an Her-2/neu-expressing tumor prevents escape mutations within said tumor. In another embodiment, provided herein is a recombinant Listeria vaccine strain for use in the claimed methods comprising the immunogenic composition.
[0099] In one embodiment, the recombinant attenuated Listeria strain is a vaccine strain. In one embodiment, the nucleic acid referred to herein is a nucleic acid molecule.
[00100] In one embodiment, the recombinant attenuated Listeria strain for use in the methods of the present invention further comprises a nucleic acid molecule comprising a third open reading frame encoding a metabolic enzyme, and wherein the metabolic enzyme complements an endogenous gene that is mutated in the chromosome of the recombinant Listeria strain.
[00101] In another embodiment, provided herein is a recombinant attenuated Listeria strain comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her-2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second and a third open reading frame, each encoding a metabolic enzyme, and wherein the metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant Listeria strain.
[00102] In one embodiment, the nucleic acid molecule is integrated into the Listeria genome. In another embodiment, the nucleic acid molecule is in a plasmid in the recombinant Listeria vaccine strain. In yet another embodiment, the plasmid is stably maintained in the recombinant Listeria vaccine strain in the absence of antibiotic selection. In another embodiment, the plasmid does not confer antibiotic resistance upon the recombinant Listeria. In another embodiment, the recombinant Listeria strain is attenuated. In another embodiment, the recombinant Listeria is an attenuated auxotrophic strain. In another embodiment, the high metabolic burden that the expression of a foreign antigen exerts on a bacterium such as one of the present invention is also an important mechanism of attenuation.
[00103] In one embodiment the attenuated strain is LmddA. In another embodiment, this strain exerts a strong adjuvant effect, which is an inherent property of Listeria-b&sed vaccines. One manifestation of this adjuvant effect is the 5-fold decrease in the number of the intratumoral Tregs caused by either Listeria expressing an antigen other than a human chimeric Her-2/neu or the ADXS-31-164 (expressing a human chimeric Her-2/neu) vaccines (see Figure 5 herein). In another embodiment, the LmddA vector expressing a different antigen (HPV16 E7) is also associated with a significant decrease in the frequency of Tregs in the tumors, likely as a consequence of innate immunity responses. In another embodiment, the LmddA vector expresses a prostate-specific antigen (PSA), a human papilloma virus (HPV) antigen (E6, E7). In another embodiment, the HPV strain is HPV16, HPV18, or any strain known in the art.
[00104] In one embodiment, the attenuated auxotrophic Listeria vaccine strain is the ADXS- 31-164 strain. ADXS-31-164 is based on a Listeria vaccine vector which is attenuated due to the deletion of virulence gene actA and retains the plasmid for Her-2/neu expression in vivo and in vitro by complementation of dal gene. In one embodiment, ADXS31-164 expresses and secretes the chimeric Her-2/neu protein fused to the first 441 amino acids of listeriolysin
O (LLO). In another embodiment, ADXS31-164 exerts strong and antigen specific antitumor responses with ability to break tolerance toward Her-2/neu in transgenic animals (see Examples). In another embodiment, the ADXS31-164 strain is highly attenuated and has a better safety profile than previous Listeria vaccine generations, as it is more rapidly cleared from the spleens of the immunized mice. In another embodiment, the ADXS31-164 results in a longer delay of tumor onset in transgenic animals than Lm-LLO-ChHer2, the antibiotic resistant and more virulent version of this vaccine (see Figure 3). In one embodiment, the Zm-LLO-ChHer2 strain is Lm-LLO-138.
[00105] In another embodiment, ADXS31-164 strain is highly immunogenic, able to break tolerance toward the Her-2/neu self-antigen and prevent tumor formation in Her-2/neu transgenic animals. In another embodiment, ADXS31-164 causes a significant decrease in intra- tumoral T regulatory cells (Tregs). In another embodiment, the lower frequency of Tregs in tumors treated with LmddA vaccines resulted in an increased intratumoral CD8/Tregs ratio, suggesting that a more favorable tumor microenvironment can be obtained after immunization with LmddA vaccines. In another embodiment, the use of this chimeric antigen does not result in escape mutations indicating that tumors do not mutate away from a therapeutic efficacious response to treatment with this novel antigen (see Example 6). In another embodiment, peripheral immunization with ADXS31-164 delays the growth of a metastatic breast cancer cell line in the brain (see Example 7).
[00106] In another embodiment, canine subjects suffering from osteosarcoma and provided treatment including amputation, chemotherapy, and vaccination with ADXS31-164, have prolonged survival compared with control subjects not receiving the vaccination with ADXS31-164 (see Examples 9 and 10). In another embodiment, canine subjects suffering from osteosarcoma and provided treatment including amputation, chemotherapy, and vaccination with ADXS31-164, show reduced metastasis compared with control subjects not receiving the vaccination with ADXS31-164 (see Example 10). In another embodiment, canine subjects suffering from osteosarcoma and provided treatment including amputation, chemotherapy, and vaccination with ADXS31-164, show increased specific T cell response induced compared with control subjects not receiving the vaccination with ADXS31-164 (see Example 10). In another embodiment, canine subjects suffering from osteosarcoma and provided radiation therapy prior to vaccination with ADXS31-164, have prolonged survival compared with control subjects receiving either only radiation therapy or only vaccination with ADXS31-164 (see Example 11). In another embodiment, canine subjects suffering from osteosarcoma and provided radiation therapy prior to vaccination with ADXS31-164 show reduced metastasis compared with control subjects receiving either only radiation therapy or only vaccination with ADXS31-164 (see Example 11).
[00107] In another embodiment, the terms "ADXS31-164," "Lm-human chimeric Her- 2/neu," "Zm-huHer2-neu," and "Lm-hucHer-2/neu," are used interchangeably herein.
[00108] In one embodiment, osteosarcoma cells are not easily killed by radiation, so radiation therapy is rarely used to treat osteosarcoma. In one embodiment, recombinant attenuated, antibiotic -free L/siena-expressing chimeric antigens are useful for preventing, and treating a cancer or solid tumors, as exemplified herein. In another embodiment, the tumor is a Her-2/neu positive tumor. In another embodiment, the cancer is a Her-2/neu- expressing cancer. In another embodiment, the cancer is breast cancer, a central nervous system (CNS) cancer, a head and neck cancer, an osteosarcoma (OSA), a canine OSA,
Ewing's sarcoma (ES), or any Her-2/neu-expressing cancer known in the art. In another embodiment, a canine osteosarcoma is an appendicular osteosarcoma. In another embodiment, the tumor is an osteosarcoma tumor, a breast tumor, a head and neck tumor, or any other antigen-expressing tumor known in the art. In another embodiment, said cancer or solid tumor is a result of relapse or metastatic disease. In one embodiment, the metastatic disease is pulmonary metastatic disease.
[00109] In one embodiment, the present invention provides methods of treating, preventing, or delaying metastases. In one embodiment, the present invention provides methods of treating, preventing, or delaying metastases of OSA. In one embodiment, the metastases are in the lung. In another embodiment, the metastases are in another tissue. In another embodiment, the metastases are in bone, which in one embodiment is proximal to the site of the initial OSA, and in another embodiment, is distal to the site of the initial OSA. In another embodiment, the metastases are in the kidney. In another embodiment, the metastases are in the heart. In another embodiment, the metastases are isolated. In another embodiment, the metastases are an isolated local recurrence. In another embodiment, the metastases are multi- site metastases.
[00110] In another embodiment, recombinant Listeria expressing a chimeric Her-2/neu are useful as a therapeutic vaccine for the treatment of Her-2/neu overexpressing solid tumors. In another embodiment, the Her-2/neu chimeric antigen provided herein is useful for treating Her-2/neu-expressing tumors and preventing escape mutations of the same. In another embodiment, the term "escape mutation" refers to a tumor mutating away from a therapeutic efficacious response to treatment.
[00111] In one embodiment, provided herein is a nucleic acid molecule comprising a first open reading frame encoding a recombinant polypeptide provided herein, wherein the nucleic acid molecule resides within the recombinant Listeria vaccine strain. In another embodiment, the nucleic acid molecule provided herein is used to transform the Listeria in order to arrive at a recombinant Listeria. In another embodiment, the nucleic acid provided herein lacks a virulence gene. In another embodiment, the nucleic acid molecule integrated into the Listeria genome carries a non-functional virulence gene. In another embodiment, the virulence gene is mutated in the genome of the recombinant Listeria. In yet another embodiment, the nucleic acid molecule is used to inactivate the endogenous gene present in the Listeria genome. In yet another embodiment, the virulence gene is an actA gene. In another embodiment, the virulence gene is a prfA gene. In another embodiment, the virulence gene is an MB gene. As will be understood by a skilled artisan, the virulence gene can be any gene known in the art to be associated with virulence in the recombinant Listeria.
[00112] In one embodiment, the metabolic gene, the virulence gene, or both is lacking in a chromosome of the Listeria strain. In another embodiment, the metabolic gene, the virulence gene, or both is lacking in the chromosome and in any episomal genetic element of the Listeria strain. It will be appreciated by a skilled artisan that the term "episome," "episomal," etc. refer to a plasmid vector or use thereof that does not integrate into the chromosome of the Listeria provided herein. In another embodiment, the term refers to plasmid vectors that integrate into the chromosome of the Listeria provided herein. In another embodiment, the metabolic gene, the virulence gene, or both is lacking in the genome of the Listeria strain. In one embodiment, the metabolic gene, the virulence gene, or both is mutated in the chromosome. In another embodiment, the metabolic gene, the virulence gene, or both is deleted from the chromosome. In another embodiment, the metabolic gene, the virulence gene, or both is inactivated in the chromosome.
[00113] In another embodiment, the nucleic acids and plasmids provided herein do not confer antibiotic resistance upon the recombinant Listeria.
[00114] "Nucleic acid molecule" refers, in one embodiment, to a plasmid. In another embodiment, the term refers to an integration vector. In another embodiment, the term refers to a non-integration vector. In another embodiment, the term refers to a plasmid comprising an integration vector. In another embodiment, the integration vector is a site-specific integration vector. In another embodiment, a nucleic acid molecule of methods and compositions of the present invention are composed of any type of nucleotide known in the art. Each possibility represents a separate embodiment of the present invention.
[00115] "Metabolic enzyme" refers, in another embodiment, to an enzyme involved in synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme required for synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient utilized by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient required for sustained growth of the host bacteria. In another embodiment, the enzyme is required for synthesis of the nutrient. Each possibility represents a separate embodiment of the present invention.
[00116] "Stably maintained" refers, in another embodiment, to maintenance of a nucleic acid molecule or plasmid in the absence of selection (e.g. antibiotic selection) for 10 generations, without detectable loss. In another embodiment, the period is 15 generations. In another embodiment, the period is 20 generations. In another embodiment, the period is 25 generations. In another embodiment, the period is 30 generations. In another embodiment, the period is 40 generations. In another embodiment, the period is 50 generations. In another embodiment, the period is 60 generations. In another embodiment, the period is 80 generations. In another embodiment, the period is 100 generations. In another embodiment, the period is 150 generations. In another embodiment, the period is 200 generations. In another embodiment, the period is 300 generations. In another embodiment, the period is 500 generations. In another embodiment, the period is more than 500 generations. In another embodiment, the nucleic acid molecule or plasmid is maintained stably in vitro (e.g. in culture). In another embodiment, the nucleic acid molecule or plasmid is maintained stably in vivo. In another embodiment, the nucleic acid molecule or plasmid is maintained stably both in vitro and in vitro. Each possibility represents a separate embodiment of the present invention.
[00117] In one embodiment, the present invention provides a recombinant Listeria strain expressing the antigen. The present invention also provides recombinant polypeptides comprising a listeriolysin (LLO) protein fragment fused to a Her-2 chimeric protein or fragment thereof, vaccines and immunogenic compositions comprising same, and methods of inducing an anti-Her-2 immune response and treating and vaccinating against a Her-2- expressing tumor, comprising the same.
[00118] In another embodiment, a recombinant Listeria strain of the present invention has been passaged through an animal host. In another embodiment, the passaging maximizes efficacy of the strain as a vaccine vector. In another embodiment, the passaging stabilizes the immunogenicity of the Listeria strain. In another embodiment, the passaging stabilizes the virulence of the Listeria strain. In another embodiment, the passaging increases the immunogenicity of the Listeria strain. In another embodiment, the passaging increases the virulence of the Listeria strain. In another embodiment, the passaging removes unstable substrains of the Listeria strain. In another embodiment, the passaging reduces the prevalence of unstable sub-strains of the Listeria strain. In another embodiment, the Listeria strain contains a genomic insertion of the gene encoding the antigen-containing recombinant peptide. In another embodiment, the Listeria strain carries a plasmid comprising the gene encoding the antigen-containing recombinant peptide. In another embodiment, the passaging is performed by any other method known in the art.
[00119] In one embodiment, the polypeptide provided herein is a fusion protein comprising an additional polypeptide selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment, and further wherein said additional polypeptide is fused to the Her-2/neu chimeric antigen. In another embodiment, the additional polypeptide is functional. In another embodiment, a fragment of the additional polypeptide is immunogenic. In another embodiment, the additional polypeptide is immunogenic.
[00120] In another embodiment, the polypeptide provided herein is a fusion protein comprising a non-hemolytic LLO protein or N-terminal fragment fused to the Her-2/neu chimeric antigen. In another embodiment, a fusion protein of methods and compositions of the present invention comprises an ActA sequence from a Listeria organism. In one embodiment, ActA proteins and fragments thereof augment antigen presentation and immunity in a similar fashion to LLO.
[00121] In one embodiment of methods and compositions of the present invention, the fusion protein comprises the Her-2/neu antigen and an additional polypeptide. In another embodiment, the additional polypeptide fused to Her-2/neu antigen is referred to as an additional adjuvant polypeptide. In one embodiment, the additional polypeptide is a nonhemolytic LLO protein or fragment thereof (Examples herein). In another embodiment, the additional polypeptide is a PEST sequence. In another embodiment, the additional polypeptide is an ActA protein or a fragment thereof.
[00122] The additional polypeptide of methods and compositions of the present invention is, in another embodiment, a listeriolysin (LLO) peptide. In another embodiment, the additional polypeptide is an ActA peptide. In another embodiment, the additional polypeptide is a PEST sequence peptide. In another embodiment, the additional polypeptide is any other peptide capable of enhancing the immunogenicity of an antigen peptide. Each possibility represents a separate embodiment of the present invention.
[00123] Fusion proteins comprising the Her-2/neu chimeric antigen may be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods discussed below. Alternatively, subsequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence. In one embodiment,
DNA encoding the antigen can be produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately. The 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction. The amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence). The antigen is ligated into a plasmid. Each method represents a separate embodiment of the present invention.
[00124] The results of the present invention demonstrate that administration of compositions of the present invention has utility for inducing formation of antigen-specific T cells (e.g. cytotoxic T cells) that recognize and kill tumor cells (Examples herein).
[00125] In one embodiment, the present invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof. In one embodiment, the present invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof.
[00126] In another embodiment, the Her-2 chimeric protein of the methods and compositions of the present invention is a human Her-2 chimeric protein. In another embodiment, the Her-2 protein is a mouse Her-2 chimeric protein. In another embodiment, the Her-2 protein is a rat Her-2 chimeric protein. In another embodiment, the Her-2 protein is a primate Her-2 chimeric protein. In another embodiment, the Her-2 protein is a canine Her-2 chimeric protein. In another embodiment, the Her-2 protein is a Her-2 chimeric protein of human or any other animal species or combinations thereof known in the art. Each possibility represents a separate embodiment of the present invention.
[00127] In another embodiment, a Her-2 protein is a protein referred to as "Her-2/neu," "Erbb2," "v-erb-b2," "c-erb-b2," "neu," or "cNeu." In another embodiment, the term Her2/neu, or grammatical equivalents thereof, is also referred to herein as "Her-2," "Her-2 protein," "HER2 protein," or "HER2"). Each possibility represents a separate embodiment of the present invention.
[00128] In one embodiment, the Her2-neu chimeric protein, harbors two of the extracellular and one intracellular fragments of Her-2/neu antigen showing clusters of MHC-class I epitopes of the oncogene, where, in another embodiment, the chimeric protein, harbors 3
H2Dq and at least 17 of the mapped human MHC-class I epitopes of the Her-2/neu antigen (fragments ECl, EC2, and ICl) (See Figure 1A). In another embodiment, the chimeric protein harbors at least 13 of the mapped human MHC-class I epitopes (fragments EC2 and ICl). In another embodiment, the chimeric protein harbors at least 14 of the mapped human MHC-class I epitopes (fragments ECl and ICl). In another embodiment, the chimeric protein harbors at least 9 of the mapped human MHC-class I epitopes (fragments ECl and IC2). In another embodiment, the Her2-neu chimeric protein is fused to a non-hemolytic listeriolysin O (LLO). In another embodiment, the Her2-neu chimeric protein is fused to truncated listeriolysin O (tLLO). In another embodiment, the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and expressed and secreted by the Listeria monocytogenes attenuated auxotrophic strain LmddA. In another embodiment, the expression and secretion of the fusion protein tLLO-ChHer2 from the attenuated auxotrophic strain provided herein that expresses a chimeric Her-2/neu antigen/LLO fusion protein is comparable to that of the Lm-LLO- ChHer2 in TCA precipitated cell culture supernatants after 8 hours of in vitro growth (See
Figure IB).
[00129] In one embodiment, no CTL activity is detected in naive animals or mice injected with an irrelevant Listeria vaccine (See Figure 2A). While in another embodiment, the attenuated auxotrophic strain (ADXS31-164) provided herein is able to stimulate the secretion of IFN-γ by the splenocytes from wild type FVB/N mice (Figure 2B).
[00130] In another embodiment, the metabolic enzyme of the methods and compositions provided herein is an amino acid metabolism enzyme, where, in another embodiment, the metabolic enzyme is an alanine racemase enzyme. In another embodiment, the metabolic enzyme is a D-amino acid transferase enzyme. In another embodiment, the metabolic enzyme catalyzes a formation of an amino acid used for a cell wall synthesis in the recombinant Listeria strain, where in another embodiment, the metabolic enzyme is an alanine racemase enzyme.
[00131] In another embodiment, the gene encoding the metabolic enzyme is expressed under the control of the Listeria p60 promoter. In another embodiment, the inlA (encodes internalin) promoter is used. In another embodiment, the hly promoter is used. In another embodiment, the ActA promoter is used. In another embodiment, the integrase gene is expressed under the control of any other gram positive promoter. In another embodiment, the gene encoding the metabolic enzyme is expressed under the control of any other promoter that functions in Listeria. The skilled artisan will appreciate that other promoters or polycistronic expression cassettes may be used to drive the expression of the gene. Each possibility represents a separate embodiment of the present invention.
[00132] In another embodiment, the Her-2 chimeric protein is encoded by the following nucleic acid sequence set forth in SEQ ID NO: 1
[00133] gagacccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacct gcccaccaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcaggt cccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgc tgaacaataccacccctgtcacaggggcctccccaggaggcctgcgggagctgcagcttcgaagcctcacagagatcttgaaagga ggggtcttgatccagcggaacccccagctctgctaccaggacacgattttgtggaagaatatccaggagtttgctggctgcaagaaga tctttgggagcctggcatttctgccggagagctttgatggggacccagcctccaacactgccccgctccagccagagcagctccaag tgtttgagactctggaagagatcacaggttacctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctg caagtaatccggggacgaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctggggctgcgctc actgagggaactgggcagtggactggccctcatccaccataacacccacctctgcttcgtgcacacggtgccctgggaccagctcttt cggaacccgcaccaagctctgctccacactgccaaccggccagaggacgagtgtgtgggcgagggcctggcctgccaccagctg tgcgcccgagggcagcagaagatccggaagtacacgatgcggagactgctgcaggaaacggagctggtggagccgctgacacc tagcggagcgatgcccaaccaggcgcagatgcggatcctgaaagagacggagctgaggaaggtgaaggtgcttggatctggcgc ttttggcacagtctacaagggcatctggatccctgatggggagaatgtgaaaattccagtggccatcaaagtgttgagggaaaacaca tcccccaaagccaacaaagaaatcttagacgaagcatacgtgatggctggtgtgggctccccatatgtctcccgccttctgggcatctg cctgacatccacggtgcagctggtgacacagcttatgccctatggctgcctcttagactaa (SEQ ID NO: 1).
[00134] In another embodiment, the Her-2 chimeric protein has the sequence:
[00135] E T H L D M L R H L Y Q G C Q V V Q G N L E L T Y L P T N
A S L S F L Q D I Q E V Q G Y V L I A H N Q V R Q V P L Q R L R I V R G T Q L F E D N Y A L A V L D N G D P L N N T T P V T G A S P G G L R E L Q L R S L T E I L K G GV L I QR N P Q L C Y Q D T I L W K N I Q E F A G C KK I F G S L A F L P E S F D G D P A S N T A P L Q P E Q L Q V F E T L E E I T G Y L Y I S A W P D S L P D L S V F QN L Q V I R G R I L H N G A Y S L T L Q G L G I S W L G L R S L R EL G S G L A L I H H N T H L C F V H T V P W D Q L F R N P H Q A L L H T A N R P E D E C V G E G L A C H Q L C A R G Q Q K I R K Y T M R R L L Q E T E L V E P L T P S G A M P N Q A Q M R I L K E T E L R K V K V L G S G A F G T V Y K G I W I P D G E N V K I P V A I KV L R E N T S P K A NKEI LDE A YV M A G V G S PY V S R LLG I C L T S T V Q L V T Q L M P Y G C L L D (SEQ ID NO: 2).
[00136] Table 1 below shows the percent ( ) identity between the amino acid sequences of human and canine Her-2 EC and IC fragments, respectively.
Table 1 x χχ. x t «ίί > Λ « χΧ-χ χ· ί x* χχ:χχχχχχχ· χχ;χχ;,χ f «¾>:«Μ¾^
S: ¾il^A ; < :: t Ϊ }:i i< ^Λι Χ ϊ ^ ^ΐΛ^ ^
Figure imgf000031_0001
XXXAXSX Χή ΧΧΧΧΧΐ ΧΧΧ *i'C?x O: X' ; 1
■m^&xxsxxift;^ ftft
<■:··:'.<«« X ¾ x ixx* χχ«, ¾: x & *x, x ? xx > xx t xftx x- ft x. x χ t,xv ¾ *·£>* xx .χχχ; .v> x x tx x ft ft
::x;x:xxx::: :x : A:x xxs HSQ » ;; a' , ixL^HH'
:££ft£*ft!fti¾ X
χχχχχ s xx sxx-x. ¾ x*x xx x; xxxxx χχχχ x x X· ; ίίχχχχχχχ ·. ;¾¾ ; xx xftxsT Xftftxx s * §
¾ :« xx Χ«ΑΪ xxsx X· -X :x xx ,xx; ; ϊ : XXX Ks; x x
xxxxxft sxx x ΧΛΥΧ xs-xx xxx x x x x x χ,χχ x xxxxxxxyxx Ϊ ¾ '> X¾ -
[00137] In another embodiment, an amino acid sequence encoding a human Her-2/neu ECl fragment is set forth in (SEQ ID NO: 69):
[00138] SLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDN GDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKN NQLALTLIDTNRSRACHPCSPMCK (SEQ ID NO: 69).
[00139] In another embodiment, an amino acid sequence encoding a canine Her-2/neu ECl fragment is set forth in (SEQ ID NO: 70):
[00140] SLSFLQDIQEVQGYVLIAHSQVRQIPLQRLRIVRGTQLFEDNYALAVLDNG DPLEGGIPAPGAAPGGLRELQLRSLTEILKGGVLIQRSPQLCHQDTILWKDVFHKNN QLALTLIDTNRSRACPPCSPACK (SEQ ID NO: 70).
[00141] In another embodiment, an amino acid sequence encoding a human Her-2/neu EC2 fragment is set forth in (SEQ ID NO: 71):
[00142] TAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGA YSLTLQGLGISWLGLRSLRELGS (SEQ ID NO: 71).
[00143] In another embodiment, an amino acid sequence encoding a canine Her-2/neu EC2 fragment is set forth in (SEQ ID NO: 72):
[00144] TAPLQPEQLRVFEALEEITGYLYISAWPDSLPNLSVFQNLRVIRGRVLHDGA YSLTLQGLGISWLGLRSLRELGS (SEQ ID NO: 72). [00145] In another embodiment, an amino acid sequence encoding a human Her-2/neu IC1 fragment is set forth in (SEQ ID NO: 73):
[00146] NQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLREN TSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENR GRLGSQDLLNWCMQIAKGMSYLED(SEQ ID NO: 73).
[00147] In another embodiment, an amino acid sequence encoding a canine Her-2/neu IC1 fragment is set forth in (SEQ ID NO: 74):
[00148] NQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLREN TSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENR GRLGSQDLLNWCMQIAKGMSYLED(SEQ ID NO: 74).
[00149] In one embodiment, the human amino acid sequence of Her-2 ECl fragment (SEQ ID NO: 69) has 89% identity with that of a canine Her-2 ECl fragment (SEQ ID NO: 70). In another embodiment, the human amino acid sequence of Her-2 EC2 fragment (SEQ ID NO: 71) has 93% identity with that of a canine Her-2 EC2 fragment (SEQ ID NO: 72). In another embodiment, the human amino acid sequence of Her-2 IC1 fragment (SEQ ID NO: 73) has
98% identity with that of a canine Her-2 IC1 fragment (SEQ ID NO: 74).
[00150] In one embodiment, the Her2 chimeric protein or fragment thereof of the methods and compositions provided herein does not include a signal sequence thereof. In another embodiment, omission of the signal sequence enables the Her2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the signal sequence. Each possibility represents a separate embodiment of the present invention.
[00151] In another embodiment, the fragment of a Her2 chimeric protein of methods and compositions of the present invention does not include a transmembrane domain (TM) thereof. In one embodiment, omission of the TM enables the Her-2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the TM. Each possibility represents a separate embodiment of the present invention.
[00152] In one embodiment, the nucleic acid sequence of rat-Her-2/neu gene is
CCGGAATCGCGGGCACCCAAGTGTGTACCGGCACAGACATGAAGTTGCGGCTC
CCTGCCAGTCCTGAGACCCACCTGGACATGCTCCGCCACCTGTACCAGGGCTGT CAGGTAGTGCAGGGCAACTTGGAGCTTACCTACGTGCCTGCCAATGCCAGCCTC TCATTCCTGCAGGACATCCAGGAAGTTCAGGGTTACATGCTCATCGCTCACAAC CAGGTGAAGCGCGTCCCACTGCAAAGGCTGCGCATCGTGAGAGGGACCCAGCT CTTTGAGGACAAGTATGCCCTGGCTGTGCTAGACAACCGAGATCCTCAGGACA ATGTCGCCGCCTCCACCCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAG CTTCGAAGTCTCACAGAGATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCT CAACTGGCTCCTGTCGATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGT GCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCCGGAAGACTGTCA GATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCT GCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGC ATTCTGACTGCCTGGCCTGCCTCCACTTCAATCATAGTGGTATCTGTGAGCTGCA CTGCCCAGCCCTCGTCACCTACAACACAGACACCTTTGAGTCCATGCACAACCC TGAGGGTCGCTACACCTTTGGTGCCAGCTGCGTGACCACCTGCCCCTACAACTA CCTGTCTACGGAAGTGGGATCCTGCACTCTGGTGTGTCCCCCGAATAACCAAGA GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTG CTCGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATC ACCAGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCT
GAGGCCTGAGCAGCTCCAAGTGTTCGAAACCCTGGAGGAGATCACAGGTTACC TGTACATCTCAGCATGGCCAGACAGTCTCCGTGACCTCAGTGTCTTCCAGAACC TTCGAATCATTCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGC AAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGGGAGCTGGGCAGT GGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACTGTACCTT GGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCAC GGGCACTGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTT CGGGGCCAGGAGTGTGTGGAGGAGTGCCGAGTATGGAAGGGGCTCCCCCGGGA GTATGTGAGTGACAAGCGCTGTCTGCCGTGTCACCCCGAGTGTCAGCCTCAAAA CAGCTCAGAGACCTGCTTTGGATCGGAGGCTGATCAGTGTGCAGCCTGCGCCCA CTACAAGGACTCGTCCTCCTGTGTGGCTCGCTGCCCCAGTGGTGTGAAACCGGA CCTCTCCTACATGCCCATCTGGAAGTACCCGGATGAGGAGGGCATATGCCAGCC GTGCCCCATCAACTGCACCCACTCCTGTGTGGATCTGGATGAACGAGGCTGCCC AGCAGAGCAGAGAGCCAGCCCGGTGACATTCATCATTGCAACTGTAGTGGGCG TCCTGCTGTTCCTGATCTTAGTGGTGGTCGTTGGAATCCTAATCAAACGAAGGA GACAGAAGATCCGGAAGTATACGATGCGTAGGCTGCTGCAGGAAACTGAGTTA GTGGAGCCGCTGACGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGAT
GCACTGTCTACAAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCC GTGGCTATCAAGGTGTTGAGAGAAAACACATCTCCTAAAGCCAACAAAGAAAT TCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCT CCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTA CGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCCAGGA CCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGT GCGGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCA ACCACGTCAAGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGA CAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAA TCTATTCTCAGACGCCGGTTCACCCATCAGAGTGATGTGTGGAGCTATGGAGTG ACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCC CGGGAGATCCCTGATTTGCTGGAGAAGGGAGAACGCCTACCTCAGCCTCCAAT CTGCACCATTGATGTCTACATGATTATGGTCAAATGTTGGATGATTGACTCTGA ATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGA
GGACAGTACCTTCTACCGTTCACTGCTGGAAGATGATGACATGGGTGACCTGGT AGACGCTGAAGAGTATCTGGTGCCCCAGCAGGGATTCTTCTCCCCGGACCCTAC CCCAGGCACTGGGAGCACAGCCCATAGAAGGCACCGCAGCTCGTCCACCAGGA GTGGAGGTGGTGAGCTGACACTGGGCCTGGAGCCCTCGGAAGAAGGGCCCCCC AGATCTCCACTGGCTCCCTCGGAAGGGGCTGGCTCCGATGTGTTTGATGGTGAC CTGGCAATGGGGGTAACCAAAGGGCTGCAGAGCCTCTCTCCACATGACCTCAG CCCTCTACAGCGGTACAGCGAGGACCCCACATTACCTCTGCCCCCCGAGACTGA TGGCTATGTTGCTCCCCTGGCCTGCAGCCCCCAGCCCGAGTATGTGAACCAATC AGAGGTTCAGCCTCAGCCTCCTTTAACCCCAGAGGGTCCTCTGCCTCCTGTCCG GCCTGCTGGTGCTACTCTAGAAAGACCCAAGACTCTCTCTCCTGGGAAGAATGG GGTTGTCAAAGACGTTTTTGCCTTCGGGGGTGCTGTGGAGAACCCTGAATACTT AGTACCGAGAGAAGGCACTGCCTCTCCGCCCCACCCTTCTCCTGCCTTCAGCCC AGCCTTTGACAACCTCTATTACTGGGACCAGAACTCATCGGAGCAGGGGCCTCC ACCAAGTAACTTTGAAGGGACCCCCACTGCAGAGAACCCTGAGTACCTAGGCC TGGATGTACCTGTA (SEQ ID NO: 45).
[00153] In one embodiment, the nucleic acid sequence encoding the rat/Her-2/neu ECl fragment is
Figure imgf000034_0001
ATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAG ACAATCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACC ATCTGTACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGC CATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA (SEQ ID NO: 46).
[00154] In another embodiment, the nucleic acid sequence encoding the rat Her-2/neu EC2 fragment is:
GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTG CTCGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATC ACCAGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCT
GAGGCCTGAGCAGCTCCAAGTGTTCGAAACCCTGGAGGAGATCACAGGTTACC TGTACATCTCAGCATGGCCAGACAGTCTCCGTGACCTCAGTGTCTTCCAGAACC TTCGAATCATTCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGC AAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGGGAGCTGGGCAGT GGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACTGTACCTT GGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCAC GGGCACTGCTGGGGGCCAGGGCCCACCCA (SEQ ID NO: 47).
[00155] In another embodiment, the nucleic acid sequence encoding the rat Her-2/neu IC1 fragment is:
[00156] CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAG TACAAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTAT CAAGGTGTTGAGAGAAAACACATCTCCTAAAGCCAACAAAGAAATTCTAGATG AAGCGTATGTGATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTCCTGGGCA TCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTACGGCTGCC TTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCA ACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGCGGCTT GTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGT CAAGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGT ACCATGCAGATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATT CTCAGACGCCGGTTCACCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTG TGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCCCGGGA GATCCCTGATTTGCTGGAGAAGGGAGAACGCCTACCTCAGCCTCCAATCTGCAC CATTGATGTCTACATGATTATGGTCAAATGTTGGATGATTGACTCTGAATGTCG CCCGAGATTCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGACCCCCA
TACCTTCTACCGTTCACTGCTGGAA (SEQ ID NO: 48). [00157] In one embodiment, the nucleic acid sequence of human-Her-2/neu gene is:
[00158] ATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGC CCCCCGGAGCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGG CTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGC TGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAG CCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCA CAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCC AGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGA ACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAG CTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCC
CCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTG TTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCA GAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACT GCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGC ACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGC ACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATC CCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACT ACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAG AGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGT GCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGT TACCAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCT GGCATTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCT CCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACC TATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACC TGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGC AAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGT GGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCC TGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGG CCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCG AGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCT TCGGGGCCAGGAGTGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGG AGTATGTGAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGA ACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTG ACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGCGCATGCCAGC CTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGGCTGCC CCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCGTCTCTGCGGTGGTTGGCA TTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATCAAGCGACGGC AGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCT GGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGA TCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTT GGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCC AGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAA TCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCC TTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCT ATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAG GACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGA TGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCC CAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGA GACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGG AGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTG TGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAG CCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCC ATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCT GAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGG GACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCC TTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTG GTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCT GCCCCGGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCAG GAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCCTCTGAAGAGGAGGCCC CCAGGTCTCCACTGGCACCCTCCGAAGGGGCTGGCTCCGATGTATTTGATGGTG ACCTGGGAATGGGGGCAGCCAAGGGGCTGCAAAGCCTCCCCACACATGACCCC AGCCCTCTACAGCGGTACAGTGAGGACCCCACAGTACCCCTGCCCTCTGAGACT GATGGCTACGTTGCCCCCCTGACCTGCAGCCCCCAGCCTGAATATGTGAACCAG CCAGATGTTCGGCCCCAGCCCCCTTCGCCCCGAGAGGGCCCTCTGCCTGCTGCC CGACCTGCTGGTGCCACTCTGGAAAGGGCCAAGACTCTCTCCCCAGGGAAGAA
CTTGACACCCCAGGGAGGAGCTGCCCCTCAGCCCCACCCTCCTCCTGCCTTCAG CCCAGCCTTCGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGG CTCCACCCAGCACCTTCAAAGGGACACCTACGGCAGAGAACCCAGAGTACCTG GGTCTGGACGTGCCAGTGTGAACCAGAAGGCCAAGTCCGCAGAAGCCCTGA
(SEQ ID NO: 49).
[00159] In another embodiment, the nucleic acid sequence encoding the human Her-2/neu ECl fragment implemented into the chimera spans from 120-510 bp of the human ECl region and is set forth in (SEQ ID NO: 50).
[00160] GAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTG GTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTC CTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGT GAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTG AGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACC ACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAG CCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCT GCTACCAGGACACGATTTTGTGGAAG (SEQ ID NO: 50).
[00161] In one embodiment, the complete ECl human Her-2/neu fragment spans from (58-
979 bp of the human Her-2/neu gene and is set forth in (SEQ ID NO: 54).
[00162] GCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTC CCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGC CAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCT GTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAA CCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGC TCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACA ATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTC GAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAG CTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAG CTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCT CCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAG CCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCC CACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTC TGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTG CCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGA GGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCT TTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGT GACAGCAGAGGAT (SEQ ID NO: 54).
[00163] In another embodiment, the nucleic acid sequence encoding the human Her-2/neu EC2 fragment implemented into the chimera spans from 1077-1554 bp of the human Her- 2/neu EC2 fragment and includes a 50 bp extension, and is set forth in (SEQ ID NO: 51).
[00164] AATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCA TTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAG CCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATAC ATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAA GTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGG GCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGAC TGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGG ACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAG AGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCGAGGG
(SEQ ID NO:51).
[00165] In one embodiment, complete EC2 human Her-2/neu fragment spans from 907- 1504 bp of the human Her-2/neu gene and is set forth in (SEQ ID NO: 55).
[00166] TACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACA
ACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAA GCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGA GGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTG GGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTG CCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACA GGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTC CAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCT GACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAAC TGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACA CGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTG CCAACCGGCCAGAG (SEQ ID NO: 55).
[00167] In another embodiment, the nucleic acid sequence encoding the human Her-2/neu
IC1 fragment implemented into the chimera is set forth in (SEQ ID NO: 52).
[00168] CAGCAGAAGATCCGGAAGTAC ACGATGCGGAGACTGCTGCAGGAAAC GGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGA TGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGC GCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAA AATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACA AAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCT CCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTA TGCCCTATGGCTGCCTCTTAGACT (SEQ ID NO:52).
[00169] In another embodiment, the nucleic acid sequence encoding the complete human Her-2/neu ICl fragment spans from 2034-3243 of the human Her-2/neu gene and is set forth in (SEQ ID NO: 56).
[00170] CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAAC
GGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGA TGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGC GCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAA AATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACA AAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCT CCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTA TGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCT CCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTG GAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAA GAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACAT TGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGG CGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTT ATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGA TCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAG CCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATT GACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATG GCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGC CAGTCCCTTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGG GGACCTGGTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCC AGACCCTGCCCCGGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCAT CTACCAGGAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCCTCTGAAGAG GAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGCT (SEQ ID NO: 56).
[00171] The LLO utilized in the methods and compositions provided herein is, in one embodiment, a Listeria LLO. In one embodiment, the Listeria from which the LLO is derived is Listeria monocytogenes (LM). In another embodiment, the Listeria is Listeria ivanovii. In another embodiment, the Listeria is Listeria welshimeri. In another embodiment, the Listeria is Listeria seeligeri. In another embodiment, the LLO protein is a non-Listerial LLO protein. In another embodiment, the LLO protein is a synthetic LLO protein. In another embodiment it is a recombinant LLO protein.
[00172] In one embodiment, the LLO protein is encoded by the following nucleic acid sequence set forth in (SEQ ID NO: 3)
[00173] atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatct gcattcaataaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacg cggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccg ccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagt tgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgta aaacgtgattcattaacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacg ttaacaacgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatga cgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgca atcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagat ttttcggcaaagctgttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatg gccgtcaagtttatttgaaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcag gtgatgtagaactaacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcga cggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaa cttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaaca tcgatcactctggaggatacgttgctcaattcaacatttcttgggatgaagtaaattatgat (SEQ ID NO: 3).
[00174] In another embodiment, the LLO protein comprises the sequence SEQ ID NO: 4 [00175] M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S M A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N N A V N T L V E R W N E K Y A Q A Y P N V S A K I D Y D D E M A Y S E S Q L I A K F G T A F K A V N N S L N V N F G A I S E G K M Q E E V I S F K Q I Y Y N V N V N E P T R P S R F F G K A V T K E Q L Q A L G V N A E N P P A Y I S S V A Y G R Q V Y L K L S T N S H S T K V K A A F D A A V S G K S V S G D V E L T N I I K N S S F K A V I Y G G S A K D E V Q I I D G N L G D L R D I L K K G A T F N R E T P G V P I A Y T T N F L K D N E L A V I K N N S E Y I E T T S K A Y T D G K I N I D H S G G Y V A Q F N I S W D E V N Y D (SEQ ID NO: 4)
[00176] The first 25 amino acids of the proprotein corresponding to this sequence are the signal sequence and are cleaved from LLO when it is secreted by the bacterium. Thus, in this embodiment, the full length active LLO protein is 504 residues long. In another embodiment, the LLO protein has a sequence set forth in GenBank Accession No. DQ054588, DQ054589, AY878649, U25452, or U25452. In another embodiment, the LLO protein is a variant of an LLO protein. In another embodiment, the LLO protein is a homologue of an LLO protein. Each possibility represents a separate embodiment of the present invention.
[00177] In another embodiment, "truncated LLO" or "tLLO" refers to a fragment of LLO that comprises the PEST domain. In another embodiment, the terms refer to an LLO fragment that does not contain the activation domain at the amino terminus and does not include cystine 484. In another embodiment, the LLO fragment consists of a PEST sequence. In another embodiment, the LLO fragment comprises a PEST sequence. In another embodiment, the LLO fragment consists of about the first 400 to 441 amino acids of the 529 amino acid full-length LLO protein. In another embodiment, the LLO fragment is a nonhemolytic form of the LLO protein.
[00178] In another embodiment of methods and compositions of the present invention, a polypeptide encoded by a nucleic acid sequence of methods and compositions of the present invention is a fusion protein comprising the chimeric Her-2/neu antigen and an additional polypeptide, where in another embodiment, the fusion protein comprises, inter alia, a Listeria
Monocytogenes non-hemolytic LLO protein (Examples herein).
[00179] In one embodiment, the LLO fragment consists of about residues 1-25. In another embodiment, the LLO fragment consists of about residues 1-50. In another embodiment, the LLO fragment consists of about residues 1-75. In another embodiment, the LLO fragment consists of about residues 1-100. In another embodiment, the LLO fragment consists of about residues 1-125. In another embodiment, the LLO fragment consists of about residues 1-150. In another embodiment, the LLO fragment consists of about residues 1175. In another embodiment, the LLO fragment consists of about residues 1-200. In another embodiment, the LLO fragment consists of about residues 1-225. In another embodiment, the LLO fragment consists of about residues 1-250. In another embodiment, the LLO fragment consists of about residues 1-275. In another embodiment, the LLO fragment consists of about residues 1-300. In another embodiment, the LLO fragment consists of about residues 1-325. In another embodiment, the LLO fragment consists of about residues 1-350. In another embodiment, the LLO fragment consists of about residues 1-375. In another embodiment, the LLO fragment consists of about residues 1-400. In another embodiment, the LLO fragment consists of about residues 1-425. Each possibility represents a separate embodiment of the present invention.
[00180] In another embodiment, a fusion protein of methods and compositions of the present invention comprises a PEST sequence, either from an LLO protein or from another organism, e.g. a prokaryotic organism. [00181] The PEST amino acid sequence has, in another embodiment, a sequence selected from SEQ ID NO: 5-9. In another embodiment, the PEST sequence is a PEST sequence from the Listeria Monocytogenes ActA protein. In another embodiment, the PEST sequence is KTEEQPSEVNTGPR (SEQ ID NO: 5), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 6), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 7), or
RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 8). In another embodiment, the PEST sequence is from Streptolysin O protein of Streptococcus sp. In another embodiment, the PEST sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 9) at amino acids 35-51. In another embodiment, the PEST sequence is from Streptococcus equisimilis Streptolysin O, e.g.
KQNTANTETTTTNEQPK (SEQ ID NO: 10) at amino acids 38-54. In another embodiment, the PEST sequence is another PEST amino acid sequence derived from a prokaryotic organism. In another embodiment, the PEST sequence is any other PEST sequence known in the art. Each possibility represents a separate embodiment of the present invention.
[00182] In one embodiment, fusion of an antigen to the PEST sequence of Listeria
Monocytogenes enhanced cell mediated and anti-tumor immunity of the antigen. Thus, fusion of an antigen to other PEST sequences derived from other prokaryotic organisms will also enhance immunogenicity of the antigen. PEST sequence of other prokaryotic organism can be identified in accordance with methods such as described by, for example Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21 :267-271) for Listeria Monocytogenes.
Alternatively, PEST amino acid sequences from other prokaryotic organisms can also be identified based by this method. Other prokaryotic organisms wherein PEST amino acid sequences would be expected to include, but are not limited to, other Listeria species. In another embodiment, the PEST sequence is embedded within the antigenic protein. Thus, in another embodiment, "fusion" refers to an antigenic protein comprising both the antigen and the PEST amino acid sequence either linked at one end of the antigen or embedded within the antigen.
[00183] In another embodiment, provided herein is a vaccine comprising a recombinant polypeptide of the present invention. In another embodiment, provided herein is a vaccine consisting of a recombinant polypeptide of the present invention.
[00184] In another embodiment, provided herein is a nucleotide molecule encoding a recombinant polypeptide of the present invention. In another embodiment, provided herein is a vaccine comprising the nucleotide molecule.
[00185] In another embodiment, provided herein is a nucleotide molecule encoding a recombinant polypeptide of the present invention. [00186] In another embodiment, provided herein is a recombinant polypeptide encoded by the nucleotide molecule of the present invention.
[00187] In another embodiment, provided herein is a vaccine comprising a nucleotide molecule or recombinant polypeptide of the present invention.
[00188] In another embodiment, provided herein is an immunogenic composition comprising a nucleotide molecule or recombinant polypeptide of the present invention.
[00189] In another embodiment, provided herein is a vector comprising a nucleotide molecule or recombinant polypeptide of the present invention.
[00190] In another embodiment, provided herein is a recombinant form of Listeria comprising a nucleotide molecule of the present invention.
[00191] In another embodiment, provided herein is a vaccine comprising a recombinant form of Listeria of the present invention.
[00192] In another embodiment, provided herein is a culture of a recombinant form of Listeria of the present invention.
[00193] In one embodiment, a vaccine or composition for use in the methods of the present invention comprises a recombinant Listeria monocytogenes, in any form or embodiment as described herein. In one embodiment, the vaccine or composition for use in the present invention consists of a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein. In another embodiment, the vaccine or composition for use in the methods of the present invention consists essentially of a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein. In one embodiment, the term "comprise" refers to the inclusion of a recombinant Listeria monocytogenes in the vaccine or composition, as well as inclusion of other vaccines, compositions or treatments that may be known in the art. In another embodiment, the term "consisting essentially of refers to a vaccine, whose functional component is the recombinant Listeria monocytogenes, however, other components of the vaccine may be included that are not involved directly in the therapeutic effect of the vaccine and may, for example, refer to components which facilitate the effect of the recombinant Listeria monocytogenes (e.g. stabilizing, preserving, etc.). In another embodiment, the term "consisting" refers to a vaccine, which contains the recombinant Listeria monocytogenes.
[00194] In another embodiment, provided herein is a method of impeding or delaying metastatic disease origination from a HER2-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein.
[00195] In another embodiment, the methods of the present invention comprise the step of administering a recombinant Listeria monocytogenes, in any form or embodiment as described herein. In one embodiment, the methods of the present invention consist of the step of administering a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein. In another embodiment, the methods of the present invention consist essentially of the step of administering a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein. In one embodiment, the term "comprise" refers to the inclusion of the step of administering a recombinant Listeria monocytogenes in the methods, as well as inclusion of other methods or treatments that may be known in the art. In another embodiment, the term "consisting essentially of refers to a methods, whose functional component is the administration of recombinant Listeria monocytogenes, however, other steps of the methods may be included that are not involved directly in the therapeutic effect of the methods and may, for example, refer to steps which facilitate the effect of the administration of recombinant Listeria monocytogenes. In one embodiment, the term "consisting" refers to a method of administering recombinant Listeria monocytogenes with no additional steps.
[00196] In another embodiment, the Listeria of methods and compositions of the present invention is Listeria monocytogenes. In another embodiment, the Listeria is Listeria ivanovii. In another embodiment, the Listeria is Listeria welshimeri. In another embodiment, the Listeria is Listeria seeligeri. Each type of Listeria represents a separate embodiment of the present invention.
[00197] In one embodiment, the Listeria strain of the methods and compositions of the present invention is the ADXS31-164 strain. In another embodiment, ADXS31-164 stimulates the secretion of IFN-γ by the splenocytes from wild type FVB/N mice. Further, the data presented herein show that ADXS31-164 is able to elicit anti-Her-2/neu specific immune responses to human epitopes that are located at different domains of the targeted antigen.
[00198] In another embodiment, the present invention provides a recombinant form of Listeria comprising a nucleotide molecule encoding a Her-2 chimeric protein or a fragment thereof.
[00199] In one embodiment, the two molecules of the fusion protein (the LLO, ActA fragment or PEST sequence and the antigen) are joined directly. In another embodiment, the two molecules are joined by a short spacer peptide, consisting of one or more amino acids. In one embodiment, the spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. In another embodiment, the constituent amino acids of the spacer are selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. In another embodiment, the two molecules of the protein (the LLO fragment and the antigen) are synthesized separately or unfused. In another embodiment, the two molecules of the protein are synthesized separately from the same nucleic acid. In yet another embodiment, the two molecules are individually synthesized from separate nucleic acids. Each possibility represents a separate embodiment of the present invention.
[00200] In one embodiment, nucleic acids encoding the recombinant polypeptides provided herein also encode a signal peptide or sequence. In another embodiment, the fusion protein of methods and compositions of the present invention comprises an LLO signal sequence from LLO. In one embodiment, a heterologous antigen may be expressed through the use of a signal sequence, such as a Listerial signal sequence, for example, the hemolysin signal sequence or the actA signal sequence. Alternatively, for example, foreign genes can be expressed downstream from a L. monocytogenes promoter without creating a fusion protein. In another embodiment, the signal peptide is bacterial (Listerial or non-Listerial). In one embodiment, the signal peptide is native to the bacterium. In another embodiment, the signal peptide is foreign to the bacterium. In another embodiment, the signal peptide is a signal peptide from Listeria monocytogenes, such as a secAl signal peptide. In another embodiment, the signal peptide is a Usp45 signal peptide from Lactococcus lactis, or a Protective Antigen signal peptide from Bacillus anthracis. In another embodiment, the signal peptide is a secA2 signal peptide, such the p60 signal peptide from Listeria monocytogenes.
In addition, the recombinant nucleic acid molecule optionally comprises a third polynucleotide sequence encoding p60, or a fragment thereof. In another embodiment, the signal peptide is a Tat signal peptide, such as a B. subtilis Tat signal peptide (e.g., PhoD). In one embodiment, the signal peptide is in the same translational reading frame encoding the recombinant polypeptide.
[00201] In another embodiment, provided herein is a method of inducing an anti-Her-2 immune response in a subject, comprising administering to the subject a recombinant nucleotide encoding a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof, thereby inducing an anti-Her-2 immune response in a subject.
[00202] In one embodiment, provided herein is a method of eliciting an enhanced immune response to a Her-2/neu-expressing tumor in a subject, where in another embodiment, the method comprises administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to a subdominant epitope of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to several subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to at least 1-5 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to at least 1-10 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to at least 1-17 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against the Her-2-expressing tumor comprises an immune response to at least 17 subdominant epitopes of the Her-2 protein.
[00203] Point mutations or amino-acid deletions in the oncogenic protein Her-2/neu, have been reported to mediate treatment of resistant tumor cells, when these tumors have been targeted by small fragment Listeria-b&sed vaccines or trastuzumab (a monoclonal antibody against an epitope located at the extracellular domain of the Her-2/neu antigen). Described herein is a chimeric Her-2/neu based composition which harbors two of the extracellular and one intracellular fragments of Her-2/neu antigen showing clusters of MHC-class I epitopes of the oncogene. This chimeric protein, which harbors 3 H2Dq and at least 17 of the mapped human MHC-class I epitopes of the Her-2/neu antigen was fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O protein and expressed and secreted by the Listeria monocytogenes attenuated strain LmddA.
[00204] Previous reports have shown that when Her-2/neu transgenic mice were immunized with Listeria-b&sed vaccines expressing and secreting small fragments of the Her-2/neu antigen separately (each of which harbored only one H2Dq epitope of the Her-2/neu oncogene), Her-2/neu over-expressing tumors could escape due to mutations in those epitopes of the Her-2/neu antigen targeted by each vaccine (see Singh R, Paterson Y.
Immunoediting sculpts tumor epitopes during immunotherapy. Cancer Res 2007;67: 1887- 92). Demonstrated herein is the unexpected result that when three or more epitopes of the Her-2/neu protein are incorporated in a chimeric vaccine, it can eliminate the selection and escape of these tumors by escape mutations. Immunization with the novel Her-2/neu chimeric Listeria vaccines did not result in any escape mutations that could be associated with point mutations or amino acid deletions in the Her-2/neu antigen (see Example 4 herein).
[00205] In one embodiment, provided herein is a method of engineering a Listeria vaccine strain to express a Her-2 chimeric protein or recombinant polypeptide expressing the chimeric protein, the method comprising transforming a Listeria strain with a nucleic acid molecule. In another embodiment, the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her-2/neu chimeric antigen. In another embodiment, the nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, and wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of the recombinant
Listeria strain, thereby engineering a Listeria vaccine strain to express a Her-2 chimeric protein.
[00206] In one embodiment, the methods and compositions provided herein further comprise an adjuvant, which in one embodiment, is an independent adjuvant, where in another embodiment, the adjuvant or independent adjuvant comprises a granulocyte/macrophage colony- stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide. In another embodiment, the adjuvant is an aluminum adjuvant, Freund's adjuvant, MPL, emulsion, SBAS2, a nucleotide molecule encoding an immune-stimulating cytokine, a bacterial mitogen, or a bacterial toxin.
[00207] In one embodiment, an "adjuvant" is a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune responses. In one embodiment, the adjuvant is an immunologic adjuvant which in one embodiment is a substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.
[00208] In one embodiment, an "independent" adjuvant is an adjuvant that is independent, which in one embodiment, is not identical to the "additional adjuvant polypeptide" of the present invention which is present in a fusion polypeptide with a tumor specific antigen, which in one embodiment, is Her-2/neu.
[00209] In one embodiment, attenuated Listeria strains, such as Listeria Monocytogenes delta-actA mutant (Brundage et al, 1993, Proc. Natl. Acad. Sci., USA, 90: 11890-11894), L. monocytogenes delta-plcA (Camilli et al, 1991, J. Exp. Med., 173:751-754), or delta-ActA, delta INL-b (Brockstedt et 5 al, 2004, PNAS, 101 : 13832-13837) are used in the present invention. In another embodiment, attenuated Listeria strains are constructed by introducing one or more attenuating mutations, as will be understood by one of ordinary skill in the art when equipped with the disclosure herein. Examples of such strains include, but are not limited to Listeria strains auxotrophic for aromatic amino acids (Alexander et al, 1993, Infection and Immunity 10 61 :2245-2248) and mutant for the formation of lipoteichoic acids (Abachin et al, 2002, Mol. Microbiol. 43: 1-14) and those attenuated by a lack of a virulence gene (see examples herein). [00210] In another embodiment, the nucleic acid molecule of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence. In another embodiment, the first open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence. In another embodiment, the second open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence. In another embodiment, the third open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence. In another embodiment, each of the open reading frames are operably linked to a promoter/regulatory sequence. Each possibility represents a separate embodiment of the present invention.
[00211] The skilled artisan, when equipped with the present disclosure and the methods provided herein, will readily understand that different transcriptional promoters, terminators, carrier vectors or specific gene sequences (e.g. those in commercially available cloning vectors) can be used successfully in methods and compositions of the present invention. As is contemplated in the present invention, these functionalities are provided in, for example, the commercially available vectors known as the pUC series. In another embodiment, nonessential DNA sequences (e.g. antibiotic resistance genes) are removed. Each possibility represents a separate embodiment of the present invention. In another embodiment, a commercially available plasmid is used in the present invention. Such plasmids are available from a variety of sources, for example, Invitrogen (La Jolla, CA), Stratagene (La Jolla, CA),
Clontech (Palo Alto, CA), or can be constructed using methods well known in the art.
[00212] In another embodiment, a plasmid such as pCR2.1 (Invitrogen, La Jolla, CA), which is a prokaryotic expression vector with a prokaryotic origin of replication and promoter/regulatory elements is used to facilitate expression of a polypeptide of the present invention in a prokaryotic organism. In another embodiment, extraneous nucleotide sequences are removed to decrease the size of the plasmid and increase the size of the cassette that can be placed therein.
[00213] Such methods are well known in the art, and are described in, for example, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubei et al. (1997, Current Protocols in Molecular
Biology, Green & Wiley, New York).
[00214] Antibiotic resistance genes are used in the conventional selection and cloning processes commonly employed in molecular biology and vaccine preparation. Antibiotic resistance genes contemplated in the present invention include, but are not limited to, gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, cloramphenicol (CAT), neomycin, hygromycin, gentamicin and others well known in the art. Each gene represents a separate embodiment of the present invention.
[00215] Methods for transforming bacteria are well known in the art, and include calcium- chloride competent cell-based methods, electroporation methods, bacteriophage-mediated transduction, chemical, and physical transformation techniques (de Boer et al, 1989, Cell 56:641-649; Miller et al, 1995, FASEB J., 9: 190-199; Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for
Microbiology, Washington, DC; Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) In another embodiment, the Listeria vaccine strain of the present invention is transformed by electroporation. Each method represents a separate embodiment of the present invention.
[00216] In another embodiment, conjugation is used to introduce genetic material and/or plasmids into bacteria. Methods for conjugation are well known in the art, and are described, for example, in Nikodinovic J et al. (A second generation snp-derived Escherichia coli- Streptomyces shuttle expression vector that is generally transferable by conjugation. Plasmid. 2006 Nov;56(3):223-7) and Auchtung JM et al (Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl
Acad Sci U S A. 2005 Aug 30; 102 (35): 12554-9). Each method represents a separate embodiment of the present invention.
[00217] "Transforming," in one embodiment, is used identically with the term "transfecting," and refers to engineering a bacterial cell to take up a plasmid or other heterologous DNA molecule. In another embodiment, "transforming" refers to engineering a bacterial cell to express a gene of a plasmid or other heterologous DNA molecule. Each possibility represents a separate embodiment of the present invention.
[00218] Plasmids and other expression vectors useful in the present invention are described elsewhere herein, and can include such features as a promoter/regulatory sequence, an origin of replication for gram negative and gram positive bacteria, an isolated nucleic acid encoding a fusion protein and an isolated nucleic acid encoding an amino acid metabolism gene. Further, an isolated nucleic acid encoding a fusion protein and an amino acid metabolism gene will have a promoter suitable for driving expression of such an isolated nucleic acid. Promoters useful for driving expression in a bacterial system are well known in the art, and include bacteriophage lambda, the bla promoter of the beta-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325. Further examples of prokaryotic promoters include the major right and left promoters of 5 bacteriophage lambda (PL and PR), the trp, recA, lacZ, lad, and gal promoters of E. coli, the alpha-amylase (Ulmanen et al, 1985. J. Bacterid. 162: 176-182) and the S28-specific promoters of B. subtilis (Oilman et al, 1984 Gene 32:11- 20), the promoters of the bacteriophages of Bacillus (Gryczan, 1982, In: The Molecular Biology of the Bacilli, Academic Press, Inc., New York), and Streptomyces promoters (Ward et al, 1986, Mol. Gen. Genet. 203:468-478). Additional prokaryotic promoters contemplated in the present invention are reviewed in, for example, Glick (1987, J. Ind. Microbiol. 1 :277-282); Cenatiempo, (1986, Biochimie, 68:505-516); and Gottesman, (1984, Ann. Rev. Genet.
18:415-442). Further examples of promoter/regulatory elements contemplated in the present invention include, but are not limited to the Listerial prfA promoter, the Listerial hly promoter, the Listerial p60 promoter and the Listerial ActA promoter (GenBank Acc. No. NC_003210) or fragments thereof.
[00219] In another embodiment, a plasmid of methods and compositions of the present invention comprises a gene encoding a fusion protein. In another embodiment, subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then, in another embodiment, ligated to produce the desired DNA sequence. In another embodiment, DNA encoding the antigen is produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately. The 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction. The amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence). The antigen is ligated into a plasmid. Each method represents a separate embodiment of the present invention.
[00220] In another embodiment, the present invention further comprises a phage based chromosomal integration system for clinical applications. A host strain that is auxotrophic for essential enzymes, including, but not limited to, d-alanine racemase will be used, for example Lmdal(-)dat(-). In another embodiment, in order to avoid a "phage curing step," a phage integration system based on PSA is used (Lauer, et al., 2002 J Bacterid, 184:4177-4186). This requires, in another embodiment, continuous selection by antibiotics to maintain the integrated gene. Thus, in another embodiment, the current invention enables the establishment of a phage based chromosomal integration system that does not require selection with antibiotics. Instead, an auxotrophic host strain will be complemented.
[00221] The recombinant proteins of the present invention are synthesized, in another embodiment, using recombinant DNA methodology. This involves, in one embodiment, creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette, such as the plasmid of the present invention, under the control of a particular promoter/regulatory element, and expressing the protein. DNA encoding the fusion protein (e.g. non-hemolytic LLO/antigen) of the present invention is prepared, in another embodiment, by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979, Meth. Enzymol. 68: 90-99); the phosphodiester method of Brown et al. (1979, Meth. Enzymol 68: 109-151); the diethylphosphoramidite method of Beaucage et al. (1981, Tetra. Lett., 22: 15 1859-1862); and the solid support method of U.S. Pat. No. 4,458,066.
[00222] In another embodiment, chemical synthesis is used to produce a single stranded oligonucleotide. This single stranded oligonucleotide is converted, in various embodiments, into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill in the art would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences can be obtained by the ligation of shorter sequences. In another embodiment, subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated to produce the desired DNA sequence.
[00223] In another embodiment, DNA encoding the fusion protein or the recombinant protein of the present invention is cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, the gene for non-hemolytic LLO is PCR amplified, using a sense primer comprising a suitable restriction site and an antisense primer comprising another restriction site, e.g. a non-identical restriction site to facilitate cloning. The same is repeated for the isolated nucleic acid encoding an antigen. Ligation of the non-hemolytic
LLO and antigen sequences and insertion into a plasmid or vector produces a vector encoding non-hemolytic LLO joined to a terminus of the antigen. The two molecules are joined either directly or by a short spacer introduced by the restriction site.
[00224] In another embodiment, the molecules are separated by a peptide spacer consisting of one or more amino acids, generally the spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. In another embodiment, the constituent amino acids of the spacer are selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. In another embodiment, the nucleic acid sequences encoding the fusion or recombinant proteins are transformed into a variety of host cells, including E. coli, other bacterial hosts, such as Listeria, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant fusion protein gene will be operably linked to appropriate expression control sequences for each host. Promoter/ regulatory sequences are described in detail elsewhere herein. In another embodiment, the plasmid further comprises additional promoter regulatory elements, as well as a ribosome binding site and a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and an enhancer derived from e.g. immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence. In another embodiment, the sequences include splice donor and acceptor sequences.
[00225] In one embodiment, the term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
[00226] In another embodiment, in order to select for an auxotrophic bacterium comprising the plasmid, transformed auxotrophic bacteria are grown on a media that will select for expression of the amino acid metabolism gene. In another embodiment, a bacteria auxotrophic for D-glutamic acid synthesis is transformed with a plasmid comprising a gene for D-glutamic acid synthesis, and the auxotrophic bacteria will grow in the absence of D- glutamic acid, whereas auxotrophic bacteria that have not been transformed with the plasmid, or are not expressing the plasmid encoding a protein for D-glutamic acid synthesis, will not grow. In another embodiment, a bacterium auxotrophic for D-alanine synthesis will grow in the absence of D-alanine when transformed and expressing the plasmid of the present invention if the plasmid comprises an isolated nucleic acid encoding an amino acid metabolism enzyme for D-alanine synthesis. Such methods for making appropriate media comprising or lacking necessary growth factors, supplements, amino acids, vitamins, antibiotics, and the like are well known in the art, and are available commercially (Becton- Dickinson, Franklin Lakes, NJ). Each method represents a separate embodiment of the present invention.
[00227] In another embodiment, once the auxotrophic bacteria comprising the plasmid of the present invention have been selected on appropriate media, the bacteria are propagated in the presence of a selective pressure. Such propagation comprises growing the bacteria in media without the auxotrophic factor. The presence of the plasmid expressing an amino acid metabolism enzyme in the auxotrophic bacteria ensures that the plasmid will replicate along with the bacteria, thus continually selecting for bacteria harboring the plasmid. The skilled artisan, when equipped with the present disclosure and methods herein will be readily able to scale-up the production of the Listeria vaccine vector by adjusting the volume of the media in which the auxotrophic bacteria comprising the plasmid are growing.
[00228] The skilled artisan will appreciate that, in another embodiment, other auxotroph strains and complementation systems are adopted for the use with this invention.
[00229] In one embodiment, provided herein is a method of impeding the growth of a Her- 2-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein.
[00230] In another embodiment, provided herein is a method of eliciting an enhanced immune response to a Her-2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein. In yet another embodiment, the immune response against the Her-2/neu-expressing tumor comprises an immune response to at least one subdominant epitope of the Her-2/neu protein.
[00231] In one embodiment, provided herein is a method of preventing an escape mutation in the treatment of Her-2/neu over-expressing tumors, wherein and in another embodiment, the method comprises the step of administering to said subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00232] In another embodiment, provided herein is a method of preventing the onset of a
Her-2/neu antigen-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00233] In one embodiment, provided herein is a method of decreasing the frequency of intra-tumoral T regulatory cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00234] In one embodiment, provided herein is a method of decreasing the frequency of intra-tumoral myeloid derived suppressor cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00235] In another embodiment, provided herein is a method of decreasing the frequency of myeloid derived suppressor cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00236] In one embodiment, provided herein a method of preventing the development of a Her-2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00237] In another embodiment, provided herein is a method of preventing the formation of a metastatic disease coming from an Her-2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain the provided herein.
[00238] In another embodiment, provided herein is a method of treating a metastatic disease originating from a Her-2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00239] In one embodiment, provided herein is a method of administering the composition of the present invention. In another embodiment, provided herein is a method of administering the vaccine of the present invention. In another embodiment, provided herein is a method of administering the recombinant polypeptide or recombinant nucleotide of the present invention. In another embodiment, the step of administering the composition, vaccine, recombinant polypeptide or recombinant nucleotide of the present invention is performed with an attenuated recombinant form of Listeria comprising the composition, vaccine, recombinant nucleotide or expressing the recombinant polypeptide, each in its own discrete embodiment. In another embodiment, the administering is performed with a different attenuated bacterial vector. In another embodiment, the administering is performed with a DNA vaccine (e.g. a naked DNA vaccine). In another embodiment, administration of a recombinant polypeptide of the present invention is performed by producing the protein recombinantly, then administering the recombinant protein to a subject. Each possibility represents a separate embodiment of the present invention.
[00240] In another embodiment, the immune response elicited by methods and compositions of the present invention comprises a CD8+ T cell-mediated response. In another embodiment, the immune response consists primarily of a CD8+ T cell-mediated response. In another embodiment, the only detectable component of the immune response is a CD8+ T cell-mediated response.
[00241] In another embodiment, the immune response elicited by methods and compositions provided herein comprises a CD4+ T cell-mediated response. In another embodiment, the immune response consists primarily of a CD4+ T cell-mediated response. In another embodiment, the only detectable component of the immune response is a CD4+ T cell-mediated response. In another embodiment, the CD4+ T cell-mediated response is accompanied by a measurable antibody response against the antigen. In another embodiment, the CD4+ T cell-mediated response is not accompanied by a measurable antibody response against the antigen.
[00242] In another embodiment, the present invention provides a method of inducing a
CD8+ T cell-mediated immune response in a subject against a subdominant CD8+ T cell epitope of an antigen, comprising the steps of (a) fusing a nucleotide molecule encoding the Her2-neu chimeric antigen or a fragment thereof to a nucleotide molecule encoding an N- terminal fragment of a LLO protein, thereby creating a recombinant nucleotide encoding an LLO-antigen fusion protein; and (b) administering the recombinant nucleotide or the LLO- antigen fusion to the subject; thereby inducing a CD8+ T cell-mediated immune response against a subdominant CD8+ T cell epitope of an antigen.
[00243] In one embodiment, provided herein is a method of increasing intratumoral ratio of CD8+/T regulatory cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant polypeptide, recombinant Listeria, or recombinant vector of the present invention.
[00244] In another embodiment, provided herein is a method of decreasing the frequency of intra- tumoral T regulatory cells, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00245] In another embodiment, the immune response elicited by the methods and compositions provided herein comprises an immune response to at least one subdominant epitope of the antigen. In another embodiment, the immune response does not comprise an immune response to a subdominant epitope. In another embodiment, the immune response consists primarily of an immune response to at least one subdominant epitope. In another embodiment, the only measurable component of the immune response is an immune response to at least one subdominant epitope. Each type of immune response represents a separate embodiment of the present invention.
[00246] In one embodiment, methods of this invention break tolerance in a subject to a Her- 2/neu expressing tumor or cancer in said subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
[00247] Methods of measuring immune responses are well known in the art, and include, e.g. measuring suppression of tumor growth, flow cytometry, target cell lysis assays (e.g. chromium release assay), the use of tetramers, and others. Each method represents a separate embodiment of the present invention.
[00248] In another embodiment, the present invention provides a method of impeding the growth of a Her-2-expressing tumor in a subject, wherein and in another embodiment, the method comprises administering to the subject a combination of radiation therapy and a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to the
Her-2 chimeric protein or a fragment thereof or a recombinant nucleotide encoding the recombinant polypeptide, wherein the subject mounts an immune response against the Her-2- expressing tumor, thereby impeding the growth of a Her-2-expressing tumor in a subject.
[00249] In another embodiment, the present invention provides a method of delaying or inhibiting a metastatic disease emanating from a Her-2-expressing tumor in a subject, wherein and in another embodiment, the method comprises administering to the subject a combination of radiation therapy and a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to the Her-2 chimeric protein or a fragment thereof or a recombinant nucleotide encoding the recombinant polypeptide, wherein the subject mounts an immune response against the Her-2-expressing tumor, thereby delaying or inhibiting the metastatic disease emanating from a Her-2-expressing tumor in a subject.
[00250] In another embodiment, the present invention provides a method of improving the antigenicity of a Her-2 chimeric protein, wherein and in another embodiment, the method comprises the step of fusing a nucleotide encoding an N-terminal fragment of a LLO protein to a nucleotide encoding the Her-2 protein or a fragment thereof to create a recombinant nucleotide, thereby improving the antigenicity of a Her-2 chimeric protein.
[00251] In another embodiment, provided herein is a method of improving the antigenicity of a Her-2 chimeric protein, wherein and in another embodiment, the method comprises engineering a Listeria strain to express the recombinant nucleotide. In another embodiment, a different bacterial vector is used to express the recombinant nucleotide. In another embodiment, the bacterial vector is attenuated. In another embodiment, a DNA vaccine (e.g. a naked DNA vaccine) is used to express the recombinant nucleotide. In another embodiment, administration of the LLO-Her-2 chimera fusion peptide encoded by the nucleotide is performed by producing the protein recombinantly, then administering the recombinant protein to a subject. Each possibility represents a separate embodiment of the present invention.
[00252] In one embodiment, the present invention provides a method that induces "epitope spreading" of a tumor. In another embodiment, the immunization using the compositions and methods provided herein induce epitope spreading onto other tumors bearing antigens other than the antigen carried in the vaccine of the present invention.
[00253] In another embodiment, the dominant epitope or subdominant epitope is dominant or subdominant, respectively, in the subject being treated. In another embodiment, the dominant epitope or subdominant epitope is dominant or subdominant in a population being treated.
[00254] In one embodiment, provided herein is a method of preventing, treating, suppressing, inhibiting, inducing an immune response against, or eliciting an enhanced immune response against sub-dominant epitopes against a cancer or a tumor growth in a subject by epitope spreading wherein and in another embodiment, said cancer is associated with expression of an antigen or fragment thereof comprised in the composition of the present invention. In another embodiment, the method comprises administering to said subject a composition comprising the recombinant polypeptide, recombinant Listeria, or recombinant vector of the present invention. In yet another embodiment, the subject mounts an immune response against the antigen-expressing cancer or the antigen-expressing tumor, thereby treating, suppressing, or inhibiting a cancer or a tumor growth in a subject.
[00255] "Dominant CD8+ T cell epitope," in one embodiment, refers to an epitope that is recognized by over 30% of the antigen-specific CD8+ T cells that are elicited by vaccination, infection, or a malignant growth with a protein or a pathogen or cancer cell containing the protein. In another embodiment, the term refers to an epitope recognized by over 35% of the antigen-specific CD8+ T cells that are elicited thereby. In another embodiment, the term refers to an epitope recognized by over 40% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 45% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 50% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 55% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 60% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 65% of the antigen- specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 70% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 75% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 80% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 85% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 90% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 95% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 96% of the antigen- specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 97% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 98% of the antigen-specific CD8+ T cells.
[00256] "Subdominant CD8+ T cell epitope," in one embodiment, refers to an epitope recognized by fewer than 30% of the antigen-specific CD8+ T cells that are elicited by vaccination, infection, or a malignant growth with a protein or a pathogen or cancer cell containing the protein. In another embodiment, the term refers to an epitope recognized by fewer than 28% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 26% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 24% of the antigen- specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 22% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 20% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 18% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than
16% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 14% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 12% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 10% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 8% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 6% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 5% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by over 4% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 3% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 2% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 1% of the antigen-specific CD8+ T cells. In another embodiment, the term refers to an epitope recognized by fewer than 0.5% of the antigen-specific CD8+ T cells.
[00257] Each type of the dominant epitope and subdominant epitope represents a separate embodiment of the present invention.
[00258] The antigen in methods and compositions of the present invention is, in one embodiment, expressed at a detectable level on a non-tumor cell of the subject. In another embodiment, the antigen is expressed at a detectable level on at least a certain percentage (e.g. 0.01%, 0.03%, 0.1%, 0.3%, 1%, 2%, 3%, or 5%) of non-tumor cells of the subject. In one embodiment, "non-tumor cell" refers to a cell outside the body of the tumor. In another embodiment, "non-tumor cell" refers to a non-malignant cell. In another embodiment, "non- tumor cell" refers to a non-transformed cell. In another embodiment, the non-tumor cell is a somatic cell. In another embodiment, the non-tumor cell is a germ cell. Each possibility represents a separate embodiment of the present invention.
[00259] "Detectable level" refers, in one embodiment, to a level that is detectable when using a standard assay. In one embodiment, the assay is an immunological assay. In one embodiment, the assay is enzyme-linked immunoassay (ELISA). In another embodiment, the assay is Western blot. In another embodiment, the assay is FACS. It is to be understood by a skilled artisan that any other assay available in the art can be used in the methods provided herein. In another embodiment, a detectable level is determined relative to the background level of a particular assay. Methods for performing each of these techniques are well known to those skilled in the art, and each technique represents a separate embodiment of the present invention.
[00260] In one embodiment, vaccination with recombinant antigen-expressing Listeria Monocytogenes induces epitope spreading. In another embodiment, vaccination with LLO- antigen fusions, even outside the context of Her2, induces epitope spreading as well. Each possibility represents a separate embodiment of the present invention.
[00261] In another embodiment, the present invention provides a method of impeding the growth of an Her-2-expressing tumor in a subject, comprising administering to the subject a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to a Her-2 chimeric antigen, wherein the antigen has one or more subdominant CD8+ T cell epitopes, wherein the subject mounts an immune response against the antigen-expressing tumor, thereby impeding the growth of an Her-2-expressing tumor in a subject. In another embodiment, the antigen does not contain any of the dominant CD8+ T cell epitopes. In another embodiment, provided herein is a method of impeding the growth on a Her-2- expressing tumor in a subject, comprising administering to the subject a recombinant form of Listeria comprising a recombinant nucleotide encoding the recombinant polypeptide provided herein.
[00262] In another embodiment, the present invention provides a method for inducing formation of cytotoxic T cells in a host having cancer, comprising administering to the host a composition of the present invention, thereby inducing formation of cytotoxic T cells in a host having cancer.
[00263] In another embodiment, the present invention provides a method of reducing an incidence of cancer, comprising administering a composition of the present invention. In another embodiment, the present invention provides a method of ameliorating cancer, comprising administering a composition of the present invention. Each possibility represents a separate embodiment of the present invention.
[00264] In one embodiment, the composition is administered to the cells of the subject ex vivo; in another embodiment, the composition is administered to the cells of a donor ex vivo; in another embodiment, the composition is administered to the cells of a donor in vivo, then is transferred to the subject. Each possibility represents a separate embodiment of the present invention.
[00265] In one embodiment, the cancer treated by a method of the present invention is breast cancer. In another embodiment, the cancer is a Her2 containing cancer. In another embodiment, the cancer is a melanoma. In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is a carcinomatous lesion of the pancreas. In another embodiment, the cancer is pulmonary adenocarcinoma. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is pulmonary squamous adenocarcinoma. In another embodiment, the cancer is gastric adenocarcinoma. In another embodiment, the cancer is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof). In another embodiment, the cancer is an oral squamous cell carcinoma. In another embodiment, the cancer is non small-cell lung carcinoma. In another embodiment, the cancer is a CNS carcinoma. In another embodiment, the cancer is an endometrial carcinoma. In another embodiment, the cancer is a bladder cancer. In another embodiment, the cancer is mesothelioma. In another embodiment, the cancer is malignant mesothelioma (MM). In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is a prostate carcinoma.
[00266] In one embodiment, the cancer is an osteosarcoma, which in one embodiment is a cancerous bone tumor. In one embodiment, the osteosarcoma is any one of the following subtypes: osteoblastic, chondroblastic, fibroblastic OSA, telangiectatic OSA, small cell OSA, low-grade central OSA, periosteal OSA, paraosteal OSA, secondary OSA, high-grade periosteal OSA, or extraskeletal OSA.
[00267] In another embodiment, the cancer is a Her-2/neu expressing osteosarcoma. In one embodiment, the osteosarcoma is canine osteosarcoma. In another embodiment, the osteosarcoma is localized osteosarcoma. In another embodiment, the osteosarcoma is metastatic osteosarcoma. In another embodiment, the osteosarcoma is high grade osteosarcoma. In another embodiment, the osteosarcoma is canine appendicular osteosarcoma. In another embodiment, the cancer is pulmonary metastatic disease. Each possibility represents a separate embodiment of the present invention.
[00268] In another embodiment of the methods of the present invention, the subject mounts an immune response against the antigen-expressing tumor or target antigen, thereby mediating the anti-tumor effects.
[00269] In another embodiment, the present invention provides an immunogenic composition for treating cancer, the composition comprising a fusion of a truncated LLO to a Her-2 chimeric protein. In another embodiment, the immunogenic composition further comprises a Listeria strain expressing the fusion. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides an immunogenic composition for treating cancer, the composition comprising a Listeria strain expressing a Her-2 chimeric protein.
[00270] In another embodiment, provided herein is an immunogenic composition comprising a recombinant form of Listeria of the present invention.
[00271] In one embodiment, a treatment protocol of the present invention is therapeutic. In another embodiment, the protocol is prophylactic. In another embodiment, the vaccines of the present invention are used to protect people at risk for cancer such as breast cancer or other types of Her2-containing tumors because of familial genetics or other circumstances that predispose them to these types of ailments as will be understood by a skilled artisan. In another embodiment, the vaccines are used as a cancer immunotherapy after debulking of tumor growth by surgery, conventional chemotherapy or radiation treatment. In another embodiment, the vaccines are combined with radiation treatment and either surgery, conventional chemotherapy or both. Following such treatments, the vaccines of the present invention are administered so that the CTL response to the tumor antigen of the vaccine destroys remaining metastases and prolongs remission from the cancer. In another embodiment, vaccines are used as a cancer immunotherapy in combination with surgery, conventional chemotherapy, radiation treatment, or any combination thereof. In another embodiment, such combination treatment is used in subjects that cannot undergo amputation. In another embodiment, such combination treatment is used in subjects with primary osteosarcoma that cannot undergo amputation. In another embodiment, vaccines of the present invention are used to affect the growth of previously established tumors and to kill existing tumor cells. Each possibility represents a separate embodiment of the present invention.
[00272] In another embodiment, the vaccines and immunogenic compositions utilized in any of the methods described above have any of the characteristics of vaccines and immunogenic compositions of the present invention. Each characteristic represents a separate embodiment of the present invention. It is to be understood that compositions described in the context of the compositions and uses of the present invention may be referred to as immunogenic compositions and vice versa.
[00273] Various embodiments of dosage ranges are contemplated by this invention. In one embodiment, in the case of vaccine vectors, the dosage is in the range of 0.4 LDso/dose. In another embodiment, the dosage is from about 0.4-4.9 LDso/dose. In another embodiment the dosage is from about 0.5-0.59 LDso/dose. In another embodiment the dosage is from about 0.6-0.69 LDso/dose. In another embodiment the dosage is from about 0.7-0.79 LDso/dose. In another embodiment the dosage is about 0.8 LDso/dose. In another embodiment, the dosage is 0.4 LD50/dose to 0.8 of the LD50/dose.
[00274] In another embodiment, the dosage is 107 bacteria/dose. In another embodiment, the dosage is 1.5 x 107 bacteria/dose. In another embodiment, the dosage is 2 x 107 bacteria/dose. In another embodiment, the dosage is 3 x 107 bacteria/dose. In another embodiment, the dosage is 4 x 107 bacteria/dose. In another embodiment, the dosage is 6 x 107 bacteria/dose. In another embodiment, the dosage is 8 x 107 bacteria/dose. In another embodiment, the dosage is 1 x 108 bacteria/dose. In another embodiment, the dosage is 1.5 x 108 bacteria/dose. In another embodiment, the dosage is 2 x 108 bacteria/dose. In another embodiment, the dosage is 3 x 108 bacteria/dose. In another embodiment, the dosage is 4 x 108 bacteria/dose.
In another embodiment, the dosage is 6 x 108 bacteria/dose. In another embodiment, the dosage is 8 x 108 bacteria/dose. In another embodiment, the dosage is 1 x 109 bacteria/dose. In another embodiment, the dosage is 1.5 x 109 bacteria/dose. In another embodiment, the dosage is 2 x 109 bacteria/dose. In another embodiment, the dosage is 3 x 109 bacteria/dose. In another embodiment, the dosage is 5 x 109 bacteria/dose. In another embodiment, the dosage is 6 x 109 bacteria/dose. In another embodiment, the dosage is 8 x 109 bacteria/dose. In another embodiment, the dosage is 1 x 1010 bacteria/dose. In another embodiment, the dosage is 1.5 x 1010 bacteria/dose. In another embodiment, the dosage is 2 x 1010 bacteria/dose. In another embodiment, the dosage is 3 x 1010 bacteria/dose. In another embodiment, the dosage is 5 x 1010 bacteria/dose. In another embodiment, the dosage is 6 x 10 bacteria/dose. In another embodiment, the dosage is 8 x 10 bacteria/dose. In another embodiment, the dosage is 8 x 109 bacteria/dose. In another embodiment, the dosage is 1 x 1011 bacteria/dose. In another embodiment, the dosage is 1.5 x 1011 bacteria/dose. In another embodiment, the dosage is 2 x 1011 bacteria/dose. In another embodiment, the dosage is 3 x 1011 bacteria/dose. In another embodiment, the dosage is 5 x 1011 bacteria/dose. In another embodiment, the dosage is 6 x 1011 bacteria/dose. In another embodiment, the dosage is 8 x 1011 bacteria/dose. In another embodiment, the dosage is 5.0 x 108 bacteria/dose. In another embodiment, the dosage is 3.3 x 109 bacteria/dose. In another embodiment, a composition for the use in the methods provided herein comprises 3.3 x 109 Listeria/dose. Each possibility represents a separate embodiment of the present invention.
[00275] In one embodiment, a vaccine or immunogenic composition of the present invention is administered alone to a subject. In another embodiment, the vaccine or immunogenic composition is administered together with another cancer therapy, which in one embodiment is radiation therapy. Each possibility represents a separate embodiment of the present invention.
[00276] The recombinant Listeria of methods and compositions of the present invention is, in one embodiment, stably transformed with a construct encoding a Her-2 chimeric antigen or an LLO-Her-2 chimeric antigen fusion. In one embodiment, the construct contains a polylinker to facilitate further subcloning. Several techniques for producing recombinant Listeria are known.
[00277] In one embodiment, the construct or nucleic acid molecule is integrated into the Listerial chromosome using homologous recombination. Techniques for homologous recombination are well known in the art, and are described, for example, in Baloglu S, Boyle SM, et al. (Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein.
Vet Microbiol 2005, 109(1-2): 11-7); and Jiang LL, Song HH, et al., (Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein. Acta Biochim Biophys Sin (Shanghai) 2005, 37(1): 19-24). In another embodiment, homologous recombination is performed as described in United States Patent No. 6,855,320. In this case, a recombinant Listeria Monocytogenes strain that expresses E7 was made by chromosomal integration of the E7 gene under the control of the hly promoter and with the inclusion of the hly signal sequence to ensure secretion of the gene product, yielding the recombinant referred to as Lm-AZ/E7. In another embodiment, a temperature sensitive plasmid is used to select the recombinants. Each technique represents a separate embodiment of the present invention. [00278] In another embodiment, the construct or nucleic acid molecule is integrated into the Listerial chromosome using transposon insertion. Techniques for transposon insertion are well known in the art, and are described, inter alia, by Sun et al. (Infection and Immunity 1990, 58: 3770-3778) in the construction of DP-L967. Transposon mutagenesis has the advantage, in another embodiment, that a stable genomic insertion mutant can be formed but the disadvantage that the position in the genome where the foreign gene has been inserted is unknown.
[00279] In another embodiment, the construct or nucleic acid molecule is integrated into the Listerial chromosome using phage integration sites (Lauer P, Chow MY et al, Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J Bacterid 2002;184(15): 4177-86). In certain embodiments of this method, an integrase gene and attachment site of a bacteriophage (e.g. U153 or PSA listeriophage) is used to insert the heterologous gene into the corresponding attachment site, which may be any appropriate site in the genome (e.g. comK or the 3' end of the arg tRNA gene). In another embodiment, endogenous prophages are cured from the attachment site utilized prior to integration of the construct or heterologous gene. In another embodiment, this method results in single-copy integrants. Each possibility represents a separate embodiment of the present invention.
[00280] In another embodiment, one of various promoters is used to express the antigen or fusion protein containing same. In one embodiment, a Listeria monocytogenes promoter is used, e.g. promoters for the genes hly, actA, pica, plcB and mpl, which encode the Listerial proteins hemolysin, actA, phosphotidylinositol-specific phospholipase, phospholipase C, and metalloprotease, respectively. Each possibility represents a separate embodiment of the present invention.
[00281] In another embodiment, methods and compositions of the present invention utilize a homologue of a Her-2 chimeric protein or LLO sequence of the present invention. In another embodiment, the methods and compositions of the present invention utilize a Her-2 chimeric protein from a non-human mammal. The terms "homology," "homologous," etc, when in reference to any protein or peptide, refer in one embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
[00282] In another embodiment, the term "homology," when in reference to any nucleic acid sequence similarly indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence.
[00283] In another embodiment, the present invention provides an isolated nucleic acid encoding a signal peptide or a recombinant polypeptide or fusion protein of the present invention. In one embodiment, the isolated nucleic acid comprises a sequence sharing at least
65% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 75% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 85% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 90% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 95% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 97% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 99% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention.
[00284] Homology is, in one embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid sequence homology may include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
[00285] In another embodiment, "homology" refers to identity to a sequence selected from a sequence (nucleic acid or amino acid sequence) provided herein of greater than 65%. In another embodiment, "homology" refers to identity to a sequence selected from a sequence provided herein of greater than 70%. In another embodiment, the identity is greater than 75%. In another embodiment, the identity is greater than 78%. In another embodiment, the identity is greater than 80%. In another embodiment, the identity is greater than 82%. In another embodiment, the identity is greater than 83%. In another embodiment, the identity is greater than 85%. In another embodiment, the identity is greater than 87%. In another embodiment, the identity is greater than 88%. In another embodiment, the identity is greater than 90%. In another embodiment, the identity is greater than 92%. In another embodiment, the identity is greater than 93%. In another embodiment, the identity is greater than 95%. In another embodiment, the identity is greater than 96%. In another embodiment, the identity is greater than 97%. In another embodiment, the identity is greater than 98%. In another embodiment, the identity is greater than 99%. In another embodiment, the identity is 100%. Each possibility represents a separate embodiment of the present invention.
[00286] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al.,
2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). For example, methods of hybridization may be carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42 °C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.
[00287] In one embodiment of the present invention, "nucleic acids" refers to a string of at least two base-sugar-phosphate combinations. The term includes, in one embodiment, DNA and RNA. "Nucleotides" refers, in one embodiment, to the monomelic units of nucleic acid polymers. RNA may be, in one embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy AA et al, Genes & Devel 16: 2491-96 and references cited therein). DNA may be in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA may be single, double, triple, or quadruple stranded. The term also includes, in another embodiment, artificial nucleic acids that may contain other types of backbones but the same bases. In one embodiment, the artificial nucleic acid is a PNA (peptide nucleic acid). PNA contain peptide backbones and nucleotide bases and are able to bind, in one embodiment, to both DNA and RNA molecules. In another embodiment, the nucleotide is oxetane modified. In another embodiment, the nucleotide is modified by replacement of one or more phosphodiester bonds with a phosphorothioate bond. In another embodiment, the artificial nucleic acid contains any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353- 57; and Raz NK et al Biochem Biophys Res Commun. 297:1075-84. The production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed. Each nucleic acid derivative represents a separate embodiment of the present invention.
[00288] Protein and/or peptide homology for any amino acid sequence listed herein is determined, in one embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via established methods. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example. Each method of determining homology represents a separate embodiment of the present invention.
[00289] In another embodiment, the present invention provides a kit comprising a reagent utilized in performing a method of the present invention. In another embodiment, the present invention provides a kit comprising a composition, tool, or instrument of the present invention.
[00290] The terms "contacting" or "administering," in one embodiment, refer to directly contacting the cancer cell or tumor with a composition of the present invention. In another embodiment, the terms refer to indirectly contacting the cancer cell or tumor with a composition of the present invention. In another embodiment, methods of the present invention include methods in which the subject is contacted with a composition of the present invention after which the composition is brought in contact with the cancer cell or tumor by diffusion or any other active transport or passive transport process known in the art by which compounds circulate within the body.
[00291] In another embodiment, methods of this invention may include at least a single administration of a composition of this invention, wherein in another embodiment, methods of this invention may include multiple administrations of a composition of this invention.
Each possibility represents a separate embodiment of the present invention.
[00292] In one embodiment, the present invention provides methods in which recombinant Listeria is administered only once. In another embodiment, Listeria is administered twice. In another embodiment, Listeria is administered three times. In another embodiment, Listeria is administered four times. In another embodiment, Listeria is administered more than four times. In another embodiment, Listeria is administered multiple times. In another embodiment, Listeria is administered at regular intervals, which in one embodiment, may be daily, weekly, every two weeks, every three weeks, or every month. Each possibility represents a separate embodiment of the present invention.
[00293] In one embodiment, the present invention provides methods in which radiation therapy is administered only once. In another embodiment, radiation therapy is administered twice. In another embodiment, radiation therapy is administered three times. In another embodiment, radiation therapy is administered four times. In another embodiment, radiation therapy is administered more than four times. In another embodiment, radiation therapy is administered multiple times. In another embodiment, radiation therapy is administered at regular intervals, which in one embodiment, may be daily, weekly, every two weeks, every three weeks, or every month. Each possibility represents a separate embodiment of the present invention.
[00294] In one embodiment, the radiation therapy is administered prior to the administration of the recombinant attenuated Listeria. In another embodiment, the radiation therapy is administered twice prior to the first administration of the recombinant attenuated Listeria. In another embodiment, the radiation therapy is administered three times prior to the first administration of the recombinant attenuated Listeria.
[00295] In another embodiment, the recombinant attenuated Listeria is administered prior to the administration of the radiation therapy. In another embodiment, the recombinant attenuated Listeria is administered twice prior to the first administration of the radiation therapy. In another embodiment, the recombinant attenuated Listeria is administered three times prior to the first administration of the radiation therapy.
[00296] In another embodiment, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention.
Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals or organisms. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals or organisms. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
Pharmaceutical Compositions
[00297] It will be appreciated by a skilled artisan that the terms "immunogenic composition", "composition" and "pharmaceutical composition" may be used interchangeably. It is also to be understood that administration of such compositions enhances an immune response, or increase a T effector cell to regulatory T cell ratio or elicit an anti-tumor immune response, as further provided herein.
[00298] In one embodiment, the immunogenic composition provided herein comprises a recombinant Listeria provided herein.
[00299] In one embodiment, a "combination therapy" refers to the combination of radiation therapy described herein administered in conjunction with, or prior to administration of a composition comprising the recombinant Listeria provided herein.
[00300] The pharmaceutical compositions containing vaccines and compositions of the present invention are, in another embodiment, administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra- peritonealy, intra- ventricularly, intra-cranially, intra-vaginally or intra-tumorally.
[00301] In another embodiment of the methods and compositions provided herein, the vaccines or compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment of the present invention, the active ingredient is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelating capsule.
[00302] In another embodiment, the vaccines or compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
[00303] In one embodiment, the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.
[00304] In one embodiment, repeat administrations (booster doses) of compositions of this invention may be undertaken immediately following the first course of treatment or after an interval of days, weeks or months to achieve tumor regression. In another embodiment, repeat doses may be undertaken immediately following the first course of treatment or after an interval of days, weeks or months to achieve suppression of tumor growth. Assessment may be determined by any of the techniques known in the art, including diagnostic methods such as imaging techniques, analysis of serum tumor markers, biopsy, or the presence, absence or amelioration of tumor associated symptoms.
[00305] In one embodiment, a subject is administered a booster dose every 1-2 weeks, every 2-3 weeks, every 3-4 weeks, every 4-5 weeks, every 6-7 weeks, every 7-8 weeks, or every 9-
10 weeks in order to achieve the intended anti-tumor response. In one embodiment, a subject is administered a booster dose every 1-2 months, every 2-3 months, every 3-4 months, every 4-5 months, every 6-7 months, every 7-8 months, or every 9-10 months in order to achieve the intended anti-tumor response.
[00306] In one embodiment, the term "treating" refers to curing a disease. In another embodiment, "treating" refers to preventing a disease. In another embodiment, "treating" refers to reducing the incidence of a disease. In another embodiment, "treating" refers to ameliorating symptoms of a disease. In another embodiment, "treating" refers to increasing performance free survival or overall survival of a patient. In another embodiment, "treating" refers to stabilizing the progression of a disease. In another embodiment, "treating" refers to inducing remission. In another embodiment, "treating" refers to slowing the progression of a disease. The terms "reducing", "suppressing" and "inhibiting" refer in another embodiment to lessening or decreasing. Each possibility represents a separate embodiment of the present invention.
[00307] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.
[00308] It is to be understood by the skilled artisan that the term "subject" can encompass a mammal including an adult human or a human child, teenager or adolescent in need of therapy for, or susceptible to, a condition or its sequelae, and also may include non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. It will also be appreciated that the term may encompass livestock. The term "subject" does not exclude an individual that is normal in all respects.
[00309] In one embodiment, the term "subject" also encompasses dogs that cannot undergo amputation. In another embodiment, the term "subject" also encompasses humans that cannot undergo surgery. In another embodiment, the term "subject" also encompasses humans that cannot undergo amputation.
[00310] It will be appreciated by the skilled artisan that the term "mammal" for purposes of treatment refers to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals, such as canines, including dogs, and horses, cats, cattle, pigs, sheep, etc.
[00311] A "therapeutically effective amount", in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder. A "therapeutically effective amount" of a vaccine provided herein for purposes of treatment of tumor may be determined empirically and in a routine manner.
[00312] In one embodiment, compositions for use in the methods of the present invention comprise a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain. In another embodiment, the metabolic enzyme complements an endogenous gene that is lacking in the chromosome of said recombinant attenuated Listeria strain.
[00313] In one embodiment, "mutated" or "mutant" describes a deletion. In another embodiment, "mutated" or "mutant" describes an inactivation. In another embodiment,
"mutated" or "mutant" describes a truncation. In another embodiment, "mutated" or "mutant" describes an addition. In another embodiment, "mutated" or "mutant" describes a substitution. In another embodiment, "mutated" or "mutant" describes insertion of a premature stop codon. In another embodiment, "mutated" or "mutant" describes a change to one or more nucleic acids within a gene which disrupts expression of the gene.
[00314] In one embodiment, "radiation therapy" or "radiotherapy" refers to the medical use of ionizing radiation as part of cancer treatment to control or eradicate malignant cells. Radiotherapy may be used for curative, adjuvant, or palliative treatment. Suitable types of radiotherapy include conventional external beam radiotherapy, stereotactic radiation therapy (e.g., Axesse, Cyberknife, Gamma Knife, Novalis, Primatom, Synergy, X-Knife,
TomoTherapy or Trilogy), Intensity-Modulated Radiation Therapy, particle therapy (e.g., proton therapy), brachytherapy, delivery of radioisotopes, intraoperative radiotherapy, Auger therapy, Volumetric modulated arc therapy (VMAT), Virtual simulation, 3-dimensional conformal radiation therapy, and intensity-modulated radiation therapy, etc. It is to be understood that this list is not meant to be limiting. [00315] In one embodiment, radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. In one embodiment, X-rays, gamma rays, and charged particles are types of radiation that may be used for cancer treatment. In one embodiment, radiation therapy kills cancer cells by damaging their DNA either directly or by creating free radicals within the cells that can in turn damage the DNA.
[00316] In one embodiment, the radiation may be delivered by a machine outside the body (external-beam radiation therapy), or in another embodiment, it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachy therapy).
[00317] In one embodiment, systemic radiation therapy uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells.
[00318] In one embodiment, the present invention provides a method for concomitantly treating radiation insensitive cancers such as osteosarcomas with standard radiation in combination with immunotherapy, such as administration of recombinant Listeria in a regimen which requires shorter radiation treatment times, thus ameliorating side effects ordinarily associated with radiation treatment.
[00319] In one embodiment, the radiation is administered according to this invention by standard techniques with standard megavoltage equipment, such as AECL Theratron 80, Varian Clinac 4 or Varian Clinac. In one embodiment, the maximum size of the radiation portal should be no greater than 300 cm2. In one embodiment, a suitable does is between about 15 Gy and 35 Gy, with the specific dose dependent on the area of the body treated. Thus, a dose to the spinal cord would be about 35 Gy, whereas a dose to the bilateral kidneys would be about 15 Gy and to the whole liver 20 Gy. Breaks in the therapy are at the discretion of the clinician taking into consideration the patients tolerance for radiation therapy.
[00320] In another embodiment, radiation dosages in the combination therapy are administered in sequence. In another embodiment, radiation dosages in the combination therapy are administered in on consecutive days as exemplified herein (see Example 11). In another embodiment, radiation doses of 8Gy in the combination therapy are on consecutive days for a total dose of 16 Gy prior to administration of Multiple doses (up to 8) of ADXS31-
164 were given once every 3 weeks as demonstrated in Examples herein (see Example 11). In another embodiment, radiation doses of 8Gy in the combination therapy are on consecutive days for a total dose of 16 Gy prior to administration of Multiple doses (up to 8) of up to 3.3xl09 CFU ADXS31-164 were given once every 3 weeks as demonstrated in Examples herein (see Example 11). In another embodiment, radiation doses of 8Gy in the combination therapy are on consecutive days for a total dose of 16 Gy prior to administration of Multiple doses (up to 8) of 3.3xl09 CFU ADXS31-164 were given once every 3 weeks, as demonstrated in Examples herein (see Example 11). In one embodiment, the multiple doses range of ADXS31-164 provided herein is from 1-8, 8-15, 15-25, or as many as required to achieve an intended therapeutic goal.
[00321] In one embodiment, total radiation doses administered for a combination therapy provide herein range from 70-80 Gy. In another embodiment, total radiation doses administered for a combination therapy provided herein range from 10-26 GY. In another embodiment, radiation doses ranging from 10-26 GY are administered in sequence. In one embodiment, the sequence may be hourly, daily, weekly or bi-weekly. In one embodiment, radiation doses ranging from 70-80 Gy are administered in sequence. In another embodiment, radiation doses ranging from 10-26 GY are administered. In another embodiment, radiation doses are approximately α/β=5.4 Gy and μ=1.73 Gy-1 for an adult male.
[00322] In one embodiment, the radiation therapy described in the present invention as part of a combination therapy is palliative radiation therapy. In one embodiment, radiation therapy may be given with palliative intent. In one embodiment, palliative treatments are intended to relieve symptoms and reduce the suffering caused by cancer or a tumor. In another embodiment, the radiation therapy described herein as part of a combination therapy is meant to cure the cancer or tumor.
[00323] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES
[00324] Materials and Methods
[00325] Oligonucleotides were synthesized by Invitrogen (Carlsbad, CA) and DNA sequencing was done by Genewiz Inc, South Plainfield, NJ. Flow cytometry reagents were purchased from Becton Dickinson Biosciences (BD, San Diego, CA). Cell culture media, supplements and all other reagents, unless indicated, were from Sigma (St. Louise, MO). Her-2/neu HLA-A2 peptides were synthesized by EZbiolabs (Westfield, IN). Complete
RPMI 1640 (C-RPMI) medium contained 2mM glutamine, 0.1 mM non-essential amino acids, and ImM sodium pyruvate, 10% fetal bovine serum, penicillin/streptomycin, Hepes (25mM). The polyclonal anti-LLO antibody was described previously and anti-Her-2/neu antibody was purchased from Sigma.
[00326] Mice and Cell Lines [00327] All animal experiments were performed according to approved protocols by IACUC at the University of Pennsylvania or Rutgers University. FVB/N mice were purchased from Jackson laboratories (Bar Harbor, ME). The FVB/N Her-2/neu transgenic mice, which overexpress the rat Her-2/neu onco-protein were housed and bred at the animal core facility at the University of Pennsylvania. The NT-2 tumor cell line expresses high levels of rat Her-2/neu protein, was derived from a spontaneous mammary tumor in these mice and grown as described previously. DHFR-G8 (3T3/neu) cells were obtained from ATCC and were grown according to the ATCC recommendations. The EMT6-Luc cell line was a generous gift from Dr. John Ohlfest (University of Minnesota, MN) and was grown in complete C-RPMI medium. Bioluminescent work was conducted under guidance by the Small Animal Imaging Facility (SAIF) at the University of Pennsylvania (Philadelphia, PA).
[00328] Listeria constructs and antigen expression
[00329] Her-2/neu-pGEM7Z was kindly provided by Dr. Mark Greene at the University of Pennsylvania and contained the full-length human Her-2/neu (hHer2) gene cloned into the pGEM7Z plasmid (Promega, Madison WI). This plasmid was used as a template to amplify three segments of hHer-2/neu, namely, ECl, EC2, and IC1, by PCR using pfx DNA polymerase (Invitrogen) and the oligos indicated in Table 1.
[00330] Table 2: Primers for cloning of Human her-2-Chimera
Table 2
Figure imgf000075_0001
Her-2- GTGGCCCGGGTCTAGATTAGTCTAAGAGGCAGCCATAGG 2034-2424 679-808 Chimera ( ) (SEQ ID NO: 62)
[00331] The Her-2/neu chimera construct was generated by direct fusion by the SOEing PCR method and each separate hHer-2/neu segment as templates. Primers are shown in Table 3.
[00332] Sequence of primers for amplification of different segments human Her2 regions.
Table 3
Figure imgf000076_0001
[00333] ChHer2 gene was excised from pAdvl38 using Xhol and Spel restriction enzymes, and cloned in frame with a truncated, non-hemolytic fragment of LLO in the Lmdd shuttle vector, pAdvl34. The sequences of the insert, LLO and hly promoter were confirmed by DNA sequencing analysis. This plasmid was electroporated into electro-competent act A, dal, dat mutant Listeria monocytogenes strain, LmddA and positive clones were selected on Brain Heart infusion (BHI) agar plates containing streptomycin (250μg/ml). In some experiments similar Listeria strains expressing hHer-2/neu (Lm-hHer2) fragments were used for comparative purposes. These have been previously described. In all studies, an irrelevant Listeria construct (Lm-control) was included to account for the antigen independent effects of Listeria on the immune system. Lm-controls were based on the same Listeria platform as ADXS31-164, but expressed a different antigen such as HPV16-E7 or NY-ESO-1. Expression and secretion of fusion proteins from Listeria were tested. Each construct was passaged twice in vivo.
[00334] Cytotoxicity assay
[00335] Groups of 3-5 FVB/N mice were immunized three times with one week intervals with 1 x 108 colony forming units (CFU) of Lm-LLO-ChHer2, ADXS31-164, Lm-hHer2 ICI or Lm-control (expressing an irrelevant antigen) or were left naive. NT-2 cells were grown in vitro, detached by trypsin and treated with mitomycin C (250 μg/ml in serum free C-RPMI medium) at 37°C for 45 minutes. After 5 washes, they were co-incubated with splenocytes harvested from immunized or naive animals at a ratio of 1 :5 (Stimulator: Responder) for 5 days at 37°C and 5% C02. A standard cytotoxicity assay was performed using europium labeled 3T3/neu (DHFR-G8) cells as targets according to the method previously described. Released europium from killed target cells was measured after 4 hour incubation using a spectrophotometer (Perkin Elmer, Victor2) at 590 nm. Percent specific lysis was defined as (lysis in experimental group-spontaneous lysis)/(Maximum lysis-spontaneous lysis).
[00336] Interferon-γ secretion by splenocytes from immunized mice
[00337] Groups of 3-5 FVB/N or HLA-A2 transgenic mice were immunized three times with one week intervals with 1 x 108 CFU of ADXS31-164, a negative Listeria control (expressing an irrelevant antigen) or were left naive. Splenocytes from FVB/N mice were isolated one week after the last immunization and co-cultured in 24 well plates at 5 x 106 cells/well in the presence of mitomycin C treated NT-2 cells in C-RPMI medium.
Splenocytes from the HLA-A2 transgenic mice were incubated in the presence of ΙμΜ of HLA-A2 specific peptides or ^g/ml of a recombinant His-tagged ChHer2 protein, produced in E. coli and purified by a nickel based affinity chromatography system. Samples from supematants were obtained 24 or 72 hours later and tested for the presence of interferon-γ (IFN-γ) using mouse IFN-γ Enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer's recommendations.
[00338] INF-γ ELISpot Assay
[00339] Cryopreserved PBMC from each indicated time point were thawed, rested overnight at 37°C and then counted. Cells were stimulated with 2.5 uM pools of overlapping human Her-2/neu peptides (l lmers overlapping by 5 amino acids) that represent the ECl,
EC2 and ICI domains of Her-2/neu present in the chimeric vaccine, and recombinant human IL-2 (Invitrogen, Fredrick, MD) for 5 days. Cells were harvested, washed twice in 1 x PBS and counted. IFN-γ ELISpot assays were performed according to the manufacturer's protocol using a commercial canine IFN-γ ELISpot assay kit (R&D Systems, Minneapolis, MN). Briefly, 0.8 - 2 x 105 stimulated cells were incubated with 2.5 uM of ECl, EC2 or ICI peptide pools plus IL-2 or IL-2 alone (to determine background counts). All assays were performed in duplicates. Plates were developed according to the manufacturer's instructions. Spots were counted using a CTL-Immunospot analyzer (C.T.L, Shaker Heights, OH). Number of spots were normalized by subtracting twice the number of spots counted in non- stimulated wells.
[00340] Tumor studies in Her2 transgenic animals
[00341] Six weeks old FVB/N rat Her-2/neu transgenic mice (9-14/group) were immunized 6 times with 5 x 108 CFU of Im-LLO-ChHer2, ADXS31-164 or Lm-control. They were observed twice a week for the emergence of spontaneous mammary tumors, which were measured using an electronic caliper, for up to 52 weeks. Escaped tumors were excised when they reached a size 1cm2 in average diameter and preserved in RNAlater at -20°C. In order to determine the effect of mutations in the Her-2/neu protein on the escape of these tumors, genomic DNA was extracted using a genomic DNA isolation kit, and sequenced.
[00342] Effect of ADXS31-164 on regulatory T cells in spleens and tumors
[00343] Mice were implanted subcutaneously (s.c.) with 1 x 106 NT-2 cells. On days 7, 14 and 21, they were immunized with 1 x 108 CFUs of ADXS31-164, Lm<M -control or left naive. Tumors and spleens were extracted on day 28 and tested for the presence of CD3+/CD4+/FoxP3+ Tregs by FACS analysis. Briefly, splenocytes were isolated by homogenizing the spleens between two glass slides in C-RPMI medium. Tumors were minced using a sterile razor blade and digested with a buffer containing DNase (12U/ml), and collagenase (2mg/ml) in PBS. After 60 min incubation at RT with agitation, cells were separated by vigorous pipetting. Red blood cells were lysed by RBC lysis buffer followed by several washes with complete RPMI-1640 medium containing 10% FBS. After filtration through a nylon mesh, tumor cells and splenocytes were resuspended in FACS buffer (2% FBS/PBS) and stained with anti-CD3-PerCP-Cy5.5, CD4-FITC, CD25-APC antibodies followed by permeabilization and staining with anti-Foxp3-PE. Flow cytometry analysis was performed using 4-color FACS calibur (BD) and data were analyzed using cell quest software (BD).
[00344] Statistical analysis
[00345] The log-rank Chi-Squared test was used for survival data and student's i-test for the
CTL and ELISA assays, which were done in triplicates. A p-value of less than 0.05 (marked as *) was considered statistically significant in these analyzes. All statistical analysis was done with either Prism software, V.4.0a (2006) or SPSS software, V.15.0 (2006). For all FVB/N rat Her-2/neu transgenic studies we used 8-14 mice per group, for all wild-type FVB/N studies we used at least 8 mice per group unless otherwise stated. All studies were repeated at least once except for the long term tumor study in Her-2/neu transgenic mouse model.
EXAMPLE 1:
GENERATION OF L. MONOCYTOGENES STRAINS THAT SECRETE LLO
FRAGMENTS FUSED TO Her-2 FRAGMENTS : CONSTRUCTION OF
ADXS31-164.
[00346] Construction of the chimeric Her-2/neu gene (ChHer2) was described previously. Briefly, ChHer2 gene was generated by direct fusion of two extracellular (aa 40-170 and aa 359-433) and one intracellular fragment (aa 678-808) of the Her-2/neu protein by SOEing PCR method. The chimeric protein harbors most of the known human MHC class I epitopes of the protein. ChHer2 gene was excised from the plasmid, pAdvl38 (which was used to construct Lm-LLO-ChHer2) and cloned into LmddA shuttle plasmid, resulting in the plasmid pAdvl64 (Figure 1A). There are two major differences between these two plasmid backbones. 1) Whereas pAdvl38 uses the chloramphenicol resistance marker (cat) for in vitro selection of recombinant bacteria, pAdvl64 harbors the D-alanine racemase gene (dot) from bacillus subtilis, which uses a metabolic complementation pathway for in vitro selection and in vivo plasmid retention in LmddA strain which lacks the dal-dat genes. This vaccine platform was designed and developed to address FDA concerns about the antibiotic resistance of the engineered Listeria vaccine strains. 2) Unlike pAdvl38, pAdvl64 does not harbor a copy of the prfA gene in the plasmid (see sequence below and Figure 1A), as this is not necessary for in vivo complementation of the Lmdd strain. The LmddA vaccine strain also lacks the actA gene (responsible for the intracellular movement and cell-to-cell spread of Listeria) so the recombinant vaccine strains derived from this backbone are 100 times less virulent than those derived from the Lmdd, its parent strain. LmddA -based vaccines are also cleared much faster (in less than 48 hours) than the Lmdd-b&sed vaccines from the spleens of the immunized mice. The expression and secretion of the fusion protein tLLO-ChHer2 from this strain was comparable to that of the Lm-LLO-ChHer2 in TCA precipitated cell culture supernatants after 8 hours of in vitro growth (Figure IB) as a band of -104 KD was detected by an anti-LLO antibody using Western Blot analysis. The Listeria backbone strain expressing only tLLO was used as negative control.
[00347] pAdvl64 sequence (7075 base pairs) (see Figure 1):
[00348] cggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctg caccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcga gcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaa agccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactata aagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccg cgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccg accgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattg atttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttac ctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgc agaccaaaacgatctcaagaagatcatcttattaatcagataaaatatttctagccctcctttgattagtatattcctatcttaaagttactttta tgtggaggcattaacatttgttaatgacgtcaaaaggatagcaagactagaataaagctataaagcaagcatataatattgcgtttcatctt tagaagcgaatttcgccaatattataattatcaaaagagaggggtggcaaacggtatttggcattattaggttaaaaaatgtagaaggag agtgaaacccatgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcat ctgcattcaataaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacac gcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgcc gccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaa gttgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctg taaaacgtgattcattaacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaa cgttaacaacgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgat gacgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcg caatcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttcca gatttttcggcaaagctgttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgt atggccgtcaagtttatttgaaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtct caggtgatgtagaactaacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcat cgacggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaa caaacttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaatt aacatcgatcactctggaggatacgttgctcaattcaacatttcttgggatgaagtaaattatgatctcgagacccacctggacatgctcc gccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacctgcccaccaatgccagcctgtccttcctgcagg atatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcaggtcccactgcagaggctgcggattgtgcgaggc acccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgctgaacaataccacccctgtcacaggggcctcc ccaggaggcctgcgggagctgcagcttcgaagcctcacagagatcttgaaaggaggggtcttgatccagcggaacccccagctct gctaccaggacacgattttgtggaagaatatccaggagtttgctggctgcaagaagatctttgggagcctggcatttctgccggagag ctttgatggggacccagcctccaacactgccccgctccagccagagcagctccaagtgtttgagactctggaagagatcacaggtta cctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctgcaagtaatccggggacgaattctgcacaat ggcgcctactcgctgaccctgcaagggctgggcatcagctggctggggctgcgctcactgagggaactgggcagtggactggccc tcatccaccataacacccacctctgcttcgtgcacacggtgccctgggaccagctctttcggaacccgcaccaagctctgctccacact gccaaccggccagaggacgagtgtgtgggcgagggcctggcctgccaccagctgtgcgcccgagggcagcagaagatccgga agtacacgatgcggagactgctgcaggaaacggagctggtggagccgctgacacctagcggagcgatgcccaaccaggcgcag atgcggatcctgaaagagacggagctgaggaaggtgaaggtgcttggatctggcgcttttggcacagtctacaagggcatctggatc cctgatggggagaatgtgaaaattccagtggccatcaaagtgttgagggaaaacacatcccccaaagccaacaaagaaatcttagac gaagcatacgtgatggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacggtgcagctggtgacac agcttatgccctatggctgcctcttagactaatctagacccgggccactaactcaacgctagtagtggatttaatcccaaatgagccaac agaaccagaaccagaaacagaacaagtaacattggagttagaaatggaagaagaaaaaagcaatgatttcgtgtgaataatgcacg aaatcattgcttatttttttaaaaagcgatatactagatataacgaaacaacgaactgaataaagaatacaaaaaaagagccacgaccag ttaaagcctgagaaactttaactgcgagccttaattgattaccaccaatcaattaaagaagtcgagacccaaaatttggtaaagtatttaat tactttattaatcagatacttaaatatctgtaaacccattatatcgggtttttgaggggatttcaagtctttaagaagataccaggcaatcaatt aagaaaaacttagttgattgccttttttgttgtgattcaactttgatcgtagcttctaactaattaattttcgtaagaaaggagaacagctgaat gaatatcccttttgttgtagaaactgtgcttcatgacggcttgttaaagtacaaatttaaaaatagtaaaattcgctcaatcactaccaagcc aggtaaaagtaaaggggctatttttgcgtatcgctcaaaaaaaagcatgattggcggacgtggcgttgttctgacttccgaagaagcga ttcacgaaaatcaagatacatttacgcattggacaccaaacgtttatcgttatggtacgtatgcagacgaaaaccgttcatacactaaag gacattctgaaaacaatttaagacaaatcaataccttctttattgattttgatattcacacggaaaaagaaactatttcagcaagcgatatttt aacaacagctattgatttaggttttatgcctacgttaattatcaaatctgataaaggttatcaagcatattttgttttagaaacgccagtctatgt gacttcaaaatcagaatttaaatctgtcaaagcagccaaaataatctcgcaaaatatccgagaatattttggaaagtctttgccagttgatc taacgtgcaatcattttgggattgctcgtataccaagaacggacaatgtagaattttttgatcccaattaccgttattctttcaaagaatggc aagattggtctttcaaacaaacagataataagggctttactcgttcaagtctaacggttttaagcggtacagaaggcaaaaaacaagtag atgaaccctggtttaatctcttattgcacgaaacgaaattttcaggagaaaagggtttagtagggcgcaatagcgttatgtttaccctctctt tagcctactttagttcaggctattcaatcgaaacgtgcgaatataatatgtttgagtttaataatcgattagatcaacccttagaagaaaaag aagtaatcaaaattgttagaagtgcctattcagaaaactatcaaggggctaatagggaatacattaccattctttgcaaagcttgggtatc aagtgatttaaccagtaaagatttatttgtccgtcaagggtggtttaaattcaagaaaaaaagaagcgaacgtcaacgtgttcatttgtca gaatggaaagaagatttaatggcttatattagcgaaaaaagcgatgtatacaagccttatttagcgacgaccaaaaaagagattagaga agtgctaggcattcctgaacggacattagataaattgctgaaggtactgaaggcgaatcaggaaattttctttaagattaaaccaggaag aaatggtggcattcaacttgctagtgttaaatcattgttgctatcgatcattaaattaaaaaaagaagaacgagaaagctatataaaggcg ctgacagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaaattggcagaacgccccaaaacggacccacaactcgat ttgtttagctacgatacaggctgaaaataaaacccgcactatgccattacatttatatctatgatacgtgtttgtttttctttgctggctagctta attgcttatatttacctgcaataaaggatttcttacttccattatactcccattttccaaaaacatacggggaacacgggaacttattgtacag gccacctcatagttaatggtttcgagccttcctgcaatctcatccatggaaatatattcatccccctgccggcctattaatgtgacttttgtg cccggcggatattcctgatccagctccaccataaattggtccatgcaaattcggccggcaattttcaggcgttttcccttcacaaggatgt cggtccctttcaattttcggagccagccgtccgcatagcctacaggcaccgtcccgatccatgtgtctttttccgctgtgtactcggctcc gtagctgacgctctcgccttttctgatcagtttgacatgtgacagtgtcgaatgcagggtaaatgccggacgcagctgaaacggtatctc gtccgacatgtcagcagacgggcgaaggccatacatgccgatgccgaatctgactgcattaaaaaagccttttttcagccggagtcca gcggcgctgttcgcgcagtggaccattagattctttaacggcagcggagcaatcagctctttaaagcgctcaaactgcattaagaaata gcctctttctttttcatccgctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttgcggtcaagaattgccatcacgttctg aacttcttcctctgtttttacaccaagtctgttcatccccgtatcgaccttcagatgaaaatgaagagaaccttttttcgtgtggcgggctgc ctcctgaagccattcaacagaataacctgttaaggtcacgtcatactcagcagcgattgccacatactccgggggaaccgcgccaag caccaatataggcgccttcaatccctttttgcgcagtgaaatcgcttcatccaaaatggccacggccaagcatgaagcacctgcgtcaa gagcagcctttgctgtttctgcatcaccatgcccgtaggcgtttgctttcacaactgccatcaagtggacatgttcaccgatatgttttttca tattgctgacattttcctttatcgcggacaagtcaatttccgcccacgtatctctgtaaaaaggttttgtgctcatggaaaactcctctctttttt cagaaaatcccagtacgtaattaagtatttgagaattaattttatattgattaatactaagtttacccagttttcacctaaaaaacaaatgatga gataatagctccaaaggctaaagaggactataccaactatttgttaattaa (SED ID NO: 53)
EXAMPLE 2:
ADXS31-164 IS AS IMMUNOGENIC AS LM-LLO-ChHER2.
[00349] Immunogenic properties of ADXS31-164 in generating anti-Her-2/neu specific cytotoxic T cells were compared to those of the Zm-LLO-ChHer2 vaccine in a standard CTL assay. Both vaccines elicited strong but comparable cytotoxic T cell responses toward Her-
2/neu antigen expressed by 3T3/neu target cells. Accordingly, mice immunized with a Listeria expressing only an intracellular fragment of Her2-fused to LLO showed lower lytic activity than the chimeras which contain more MHC class I epitopes. No CTL activity was detected in naive animals or mice injected with the irrelevant Listeria vaccine (Figure 2A). ADXS31-164 was also able to stimulate the secretion of IFN-γ by the splenocytes from wild type FVB/N mice (Figure 2B). This was detected in the culture supernatants of these cells that were co-cultured with mitomycin C treated NT-2 cells, which express high levels of Her-2/neu antigen (Figure 5C).
[00350] Proper processing and presentation of the human MHC class I epitopes after immunizations with ADXS31-164 was tested in HLA-A2 mice. Splenocytes from immunized HLA-A2 transgenics were co-incubated for 72 hours with peptides corresponding to mapped HLA-A2 restricted epitopes located at the extracellular (HLYQGCQVV SEQ ID NO: 11 or KIFGSLAFL SEQ ID NO: 12) or intracellular (RLLQETELV SEQ ID NO: 13) domains of the Her-2/neu molecule (Figure 2C). A recombinant ChHer2 protein was used as positive control and an irrelevant peptide or no peptide as negative controls. The data from this experiment show that ADXS31-164 is able to elicit anti-Her-2/neu specific immune responses to human epitopes that are located at different domains of the targeted antigen.
EXAMPLE 3:
ADXS31-164 WAS MORE EFFICACIOUS THAN LM-LLO-ChHER2 IN
PREVENTING THE ONSET OF SPONTANEOUS MAMMARY TUMORS.
[00351] Anti-tumor effects of ADXS31-164 were compared to those of Lm-LLO-ChHer2 in Her-2/neu transgenic animals which develop slow growing, spontaneous mammary tumors at 20-25 weeks of age. All animals immunized with the irrelevant Listeria-control vaccine developed breast tumors within weeks 21-25 and were sacrificed before week 33. In contrast, Listeria-Her-2/neu recombinant vaccines caused a significant delay in the formation of the mammary tumors. On week 45, more than 50% of ADXS31-164 vaccinated mice (5 out of 9) were still tumor free, as compared to 25% of mice immunized with Lm-LLO-ChHer2. At week 52, 2 out of 8 mice immunized with ADXS31-164 still remained tumor free, whereas all mice from other experimental groups had already succumbed to their disease (Figure 3).
These results indicate that despite being more attenuated, ADXS31-164 is more efficacious than Lm-LLO-ChHer2 in preventing the onset of spontaneous mammary tumors in Her-2/neu transgenic animals.
EXAMPLE 4:
MUTATIONS IN HER-2/NEU GENE UPON IMMUNIZATION WITH ADXS31-164.
[00352] Mutations in the MHC class I epitopes of Her-2/neu have been considered responsible for tumor escape upon immunization with small fragment vaccines or trastuzumab (Herceptin), a monoclonal antibody that targets an epitope in the extracellular domain of Her-2/neu. To assess this, genomic material was extracted from the escaped tumors in the transgenic animals and sequenced the corresponding fragments of the neu gene in tumors immunized with the chimeric or control vaccines. Mutations were not observed within the Her-2/neu gene of any vaccinated tumor samples suggesting alternative escape mechanisms (data not shown).
EXAMPLE 5:
ADXS31-164 CAUSES A SIGNIFICANT DECREASE IN INTRA- IT MORAL T
REGULATORY CELLS.
[00353] To elucidate the effect of ADXS31-164 on the frequency of regulatory T cells in spleens and tumors, mice were implanted with NT-2 tumor cells. Splenocytes and intra- tumoral lymphocytes were isolated after three immunizations and stained for Tregs, which were defined as CD3+/CD4+/CD25+/FoxP3+ cells, although comparable results were obtained with either FoxP3 or CD25 markers when analyzed separately. The results indicated that immunization with ADXS31-164 had no effect on the frequency of Tregs in the spleens, as compared to an irrelevant Listeria vaccine or the naive animals (See Figure 4). In contrast, immunization with the Listeria vaccines caused a considerable impact on the presence of Tregs in the tumors (Figure 5A). Whereas in average 19.0% of all CD3+ T cells in untreated tumors were Tregs, this frequency was reduced to 4.2% for the irrelevant vaccine and 3.4% for ADXS31-164, a 5-fold reduction in the frequency of intra-tumoral Tregs (Figure 5B). The decrease in the frequency of intra-tumoral Tregs in mice treated with either of the LmddA vaccines could not be attributed to differences in the sizes of the tumors. In a representative experiment, the tumors from mice immunized with ADXS31-164 were significantly smaller [mean diameter (mm) +SD, 6.71+0.43, n=5] than the tumors from untreated mice (8.69+0.98, n=5, p<0.01) or treated with the irrelevant vaccine (8.41+1.47, n=5, p=0.04), whereas comparison of these last two groups showed no statistically significant difference in tumor size (p=0.73). The lower frequency of Tregs in tumors treated with LmddA vaccines resulted in an increased intratumoral CD8/Tregs ratio, suggesting that a more favorable tumor microenvironment can be obtained after immunization with LmddA vaccines. However, only the vaccine expressing the target antigen Her-2/neu (ADXS31-164) was able to reduce tumor growth, indicating that the decrease in Tregs has an effect only in the presence on antigen-specific responses in the tumor.
EXAMPLE 6:
NO ESCAPE MUTATIONS WERE INTRODUCED BY LISTERIA VACCINE EXPRESSING HER-2 CHIMERA.
[00354] Tumor samples of the mice immunized with different vaccines such as Lm-LLO- 138, LmddA164 and irrelevant vaccine Lm-LLO-NY were harvested. The DNA was purified from these samples and the DNA fragments corresponding to Her-2/neu regions
ICl, ECl and EC2 were amplified and were sequenced to determine if there were any immune escape mutations. The alignment of sequence from each DNA was performed using CLUSTALW. The results of the analysis indicated that there were no mutations in the DNA sequences harvested from tumors. The reference sequences are listed below:
[00355] Alignment of EC2 (975 -1029 bp of Her-2-neu)
[00356] GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGC CCTGTGCT (SEQ ID NO: 14)
[00357] CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCC ATCACCAGTGAC (SEQ ID NO: 15)
[00358] AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCA
TTTTTGCCGGAG (SEQ ID No: 16)
[00359] AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGC AGCTCCAAGTG (SEQ ID No: 17)
[00360] TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGG CCAGACAGTCTC (SEQ ID NO: 18)
[00361] CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTC TCCACGATGGC (SEQ ID NO: 19)
[00362] GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTG CGCTCACTGCGG (SEQ ID NO: 20)
[00363] GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCT TTGTACACACT (SEQ ID NO: 21)
[00364] GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCAC AGTGGGAACCGG (SEQ ID NO: 22)
[00365] CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGT GCCCACGGGCAC (SEQ ID NO: 23)
[00366] TGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTC
GGGGCCAGGAG (SEQ ID NO: 24)
[00367] Alignment of IC1 (2114-3042 bp of Her-2-neu)
[00368] CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAG AGACGGAGC (SEQ ID NO: 25)
[00369] TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACA AGGGCATCTGGA (SEQ ID NO: 26)
[00370] TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGA GAGAAAACACAT (SEQ ID NO: 27)
[00371] CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTG
GTGTGGGTTCTC (SEQ ID NO: 28)
[00372] CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCT
GGTGACACAGC (SEQ ID NO: 29)
[00373]
TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAG GCTCCC (SEQ ID NO: 30)
[00374] AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACC TGGAGGACGTGC(SEQ ID NO: 31)
[00375] GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTC CCAACCACGTCA(SEQ ID NO: 32)
[00376] AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAG AGTACCATGCAG (SEQ ID NO: 33)
[00377] ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCA GACGCCGGTTCA(SEQ ID NO: 34)
[00378] CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGA
TGACTTTTGGGG (SEQ ID NO: 35)
[00379] CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGG AGAAGGGAGAA (SEQ ID NO: 36)
[00380] CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGG TCAAATGTT (SEQ ID NO: 37) [00381 ] GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAG AATTTT (SEQ ID NO: 38)
ACTT (SEQ ID NO: 39)
[00383] Alignment of ECl (399-758 bp of Her-2-neu)
[00384] CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCT CACAGAGATCCT (SEQ ID NO: 40)
CATGGTTTTGTG (SEQ ID NO: 41)
[00386] CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGG GGTGAGAGTCC (SEQ ID NO: 42)
[00387] GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGC CCGGTGCAAGGG (SEQ ID NO: 43)
[00388] CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCAC GGGCCCCAAGCA (SEQ ID NO: 44)
EXAMPLE 7:
PERIPHERAL IMMUNIZATION WITH ADXS31-164 CAN DELAY THE
GROWTH OF A METASTATIC BREAST CANCER CELL LINE IN THE
BRAIN.
[00389] Mice were immunized IP with ADXS31-164 or irrelevant Lm-control vaccines and then implanted intra-cranially with 5,000 EMT6-Luc tumor cells, expressing lucif erase and low levels of Her-2/neu (Figure 6C). Tumors were monitored at different times post- inoculation by ex vivo imaging of anesthetized mice. On day 8 post-tumor inoculation, tumors were detected in all control animals, but none of the mice in ADXS31-164 group showed any detectable tumors (Figure 6A and B). ADXS31-164 could clearly delay the onset of these tumors, as on day 11 post- tumor inoculation, all mice in the negative control group had already succumbed to their tumors, but all mice in ADXS31-164 group were still alive and only showed small signs of tumor growth. These results strongly suggest that the immune responses obtained with the peripheral administration of ADXS31-164 could possibly reach the central nervous system and that LmddA-b&sed vaccines might have a potential use for treatment of CNS tumors.
EXAMPLE 8:
TREATMENT OF CANINE OSTEASARCOMA BY IMMUNIZATION WITH ADXS31-
164. [00390] Canine Osteosarcoma is a cancer of long (leg) bones that is a leading killer of large dogs over the age of 10 years. Standard treatment is amputation immediately after diagnosis, followed by chemotherapy. Invariably, however, the cancer metastasizes to the lungs. With chemotherapy, dogs survive about 18 months compared to 6-12 months, without treatment. The HER2 antigen is believed to be present in up to 50% of osteosarcoma.
ADXS31-164 creates an immune attack on cells expressing this antigen and has been developed to treat human breast cancer.
[00391] Dogs with a histological diagnosis of osteosarcoma and evidence of expression of HER2/neu by malignant cells are eligible for enrollment.
Canine Osteosarcoma Trial
[00392] In the first regiment the limbs are amputated, followed by round of chemotherapy treatment. 3 doses of Her-2 vaccine are subsequently administered with or without a 6 month interval booster.
[00393] All dogs are to receive 4 weeks of carboplatin therapy. Four weeks after the last carboplatin dose, dogs are to receive ADXS-HER2 once every three weeks for a total of
3 doses. Group 1 (3 dogs) receive lxlO8 CFU per dose, Group 2 (3 dogs) each receive 5xl08
CFU per dose and Group 3 (3 dogs) receives lxlO9 CFU per dose. Additional dogs are added to a Group to gather more data should if a potentially dose limiting toxicities, be observed.
Therefore 9-18 dogs may be treated in the initial study.
[00394] In the second regiment, the same as the first regiment is repeated with the exception that only a single dose of vaccine is administered before chemotherapy (1 month before)for a total of 4 doses.
[00395] Further, in both regiments a single dose is administered a month after chemotherapy.
EXAMPLE 9:
PHASE 1 DOSE ESCALATION STUDY EVALUATING THE SAFETY OF ADXS- cHER2 IN COMPANION DOGS WITH HER-2/NEU OVEREXPRESSING CANINE
OSTEOSARCOMA.
[00396] A pilot phase I dose escalation study was performed to determine the dose of a L. monocytogenes expressing human Her-2/neu recombinant vaccine that can safely and effectively stimulate tumor-specific immunity in dogs with osteosarcoma. The tumors of all dogs presenting to PennVet for limb amputation due to suspected or confirmed OSA were routinely harvested and evaluated histopathologically to confirm the diagnosis of OSA. In addition, tumor sections from all dogs were evaluated by IHC and Western blot analysis to determine whether the tumor expresses Her-2/neu. Only dogs with a histological diagnosis of OSA and evidence of expression of Her-2/neu by malignant cells were eligible for enrollment. Single cell suspensions of tumor tissue taken at surgery were cryopreserved and used as autologous tumor targets in chromium release assays to determine anti-tumor immunity.
[00397] Up to 18 privately owned dogs with appendicular OSA and confirmed expression of Her2-neu were enrolled (Figure 7). At enrollment (3 weeks post last carboplatin treatment), all dogs received basic clinical laboratory tests including a Complete Blood Count (CBC), Chemistry Screen (CS) and urinalysis (UA) and a baseline evaluation of cardiac function by echocardiography and measurement of cardiac-specific Troponin I (cTnl) levels. Thoracic radiographs were taken to determine whether pulmonary metastases are present. Only dogs with no evidence of pulmonary metastases were eligible for inclusion in the study. At the time of enrollment, peripheral blood mononuclear cells (PBMCs) are collected to assess baseline levels of anti-tumor immunity (see Assessment of anti-tumor immunity). Furthermore, blood was taken to evaluate baseline immune function to ensure they were no longer immune suppressed by carboplatin. Only dogs with functionally intact immune systems were eligible to receive the Listeria vaccine.
[00398] Lm recombinant dosing and data capture
[00399] All dogs were vaccinated using a single Lm-huHer-2/neu recombinant vaccine. The first Lm-huHer2-neu vaccine were given three weeks after the last carboplatin dose and were given once every three weeks after this for a total of 3 doses (Figure 7).
[00400] Group 1 (3 dogs) received the ADXS31-164 (Lm-hucHer-2/neu) vaccine at lxl 08 CFU per dose, Group 2 (3 dogs) each received 5xl08 CFU per dose, Group 3 (3 dogs) receive lxlO9 CFU per dose, and 3.3 x 109 CFU per dose (1 dog). Recombinant Lm was administered as a slow intravenous infusion over 30 minutes. The dose chosen for Group 1 is the established safe dose for the chimeric huHer-2/neu recombinant in mice. In humans, the non-toxic dose for Lm-LLO-E7 is only one log higher than that established in mice, and this dose is the dose evaluated in Group 3 in this pilot trial.
[00401] At the time of Lm administration, dogs were monitored for evidence of systemic adverse effects. During infusion, heart rate and rhythm was monitored by ECG and respiratory rate were recorded. Further, heart damage was monitored using ultrasound and by measuring Troponin I levels (Figure 8). Following infusion, dogs were monitored closely for 48 hours. Core body temperature was monitored continuously for <12 hours post infusion using the Vital Sense continuous body temperature monitoring system by MiniMitter Respironics (routinely used in our Veterinary Clinical Trials Center, VCIC). Pulse rate, rhythm and quality, respiratory rate and effort, were monitored and recorded every hour for the first 6 hours then every 4 hours thereafter, as well as blood pressure and temperature (Figure 9). All symptoms consistent with immune stimulation are noted and fluids, analgesics, anti-emetics and anti-histamines are used as necessary to control severe reactions. All dogs were observed six times a day and any signs of toxicological effects of the recombinants including discomfort, lethargy, nausea, vomiting and diarrhea were recorded. Blood samples were taken at 24, 48 and 72 hours after the first ADXS31-164 vaccine for cultures to assess the clearance of Lm after systemic administration.
[00402] Assessment of anti-tumor immunity
[00403] Three weeks following the last carboplatin dose, dogs receive a routine clinical examination and baseline blood work including CBC, CS, UA and cTnl levels. PBMCs are taken at this time for baseline evaluation of anti-tumor immunity. Repeat immune assessment is performed at the time of each vaccination and three weeks after the last vaccination. PBMCs are analyzed for Her-2/neu specific T cell responses by CFSE proliferation, cytokine production (ELISpot and qRT-PCR) and CTL assay against autologous tumor targets as outlined below (Figure 12).
[00404] To date, we have performed a total of 41 infusions of ADXS31-164 in 16 dogs.
Figure imgf000089_0001
[00405] ADXS31-164 dose has ranged from 1 x 108, 5 x 108, 1 x 109 and 3.3 x 109 CFU.
Figure imgf000089_0002
enzymes
5 x 10s 9 3 Fever, nausea, vomiting, elevated liver enzymes
l x lOy 17 10 Fever, nausea, vomiting, elevated liver enzymes, thrombocytopenia.3 x 10y 1 1 Nausea, vomiting,
[00406] Standard operating procedure for vaccine administration
[00407] A standard operating procedure was developed for the administration of ADXS31- 164. One hour prior to vaccination, patients receive 2 mg/kg diphenhydramine via intramuscular injection and 0.2mg/kg ondansetron as a slow intravenous push. The vaccine was kept at -80°C and thawed patient-side. It was administered in 200mls of 0.9% NaCl over 30 mins. The infusion line is then flushed with 30 mis of Plasmalyte. Dogs are sent home with a three day course of amoxicillin (to start 72 hours post vaccination) and a 7 day course of liver supplement (S-adenosyl-methionine) that aids in cellular growth and repair.
[00408] The primary endpoint of the study was to determine the maximum tolerated dose of
ADXS31-164.
[00409] Doses up to 3.3 x 109 were well tolerated in dogs ranging in body weight from 25kg to 67kg. All side effects reported were grade I toxicities and the maximum tolerated dose has yet to be reached. Side effects routinely occurred within 2-4 hours of vaccine administration. High fevers usually resolved with intravenous isotonic fluids delivered at maintenance rate
(4mls/kg/hour) for 2-4 hours. In two cases where fevers reached 104.7 and above, a single subcutaneous injection of carprofen induced normothermia within 1-2 hours. Nausea and vomiting was usually self-limiting but in cases where several episodes are noted, 1 mg/kg cerenia is administered and this was very effective at preventing further nausea and vomiting. A total of 5 dogs developed mild, grade I elevations in liver enzymes within 48 hours of vaccine administration - these resolved by one week post vaccination.
[00410] Clearance of Listeria
[00411] After performing blood cultures on all 16 dogs vaccinated to date there was no detectable Listeria in the peripheral circulation of any of the dogs at 24 hours post vaccination. Shedding of Listeria in the urine and feces of vaccinated dogs was not assessed.
[00412] Secondary endpoints for the study are progression-free survival and overall survival. A statistically significant overall survival advantage in dogs with osteosarcoma has been observed when ADXS31-164 is administered after limb amputation and 4 doses of carboplatin. Early results from the first two dose groups (6 dogs) show a significant survival advantage in dogs that received ADXS31-164 compared to 6 dogs whose owners elected not to participate in the trial but who were followed for survival (p=0.003) (Figure 13). The mean survival time for unvaccinated dogs is 239.5 days. The mean survival time for vaccinated dogs has not yet been reached. This remains true when all dogs within the intent to treat group are included in analysis.
[00413] In conclusion, there was no evidence of significant short or long-term side effects on the cardiovascular, hematopoietic, hepatic, or renal systems. Moreover, administration of ADXS31-164 in the presence of minimal residual disease can delay/prevent metastatic disease and prolong overall survival of dogs with Her-2/neu positive osteosarcoma.
EXAMPLE 10:
PHASE 1 CLINICAL TRIAL EVALUATING ADXS31-164 IN THE SPONTANEOUS
CANINE MODEL OF OSTEROSARCOMA (OSA).
[00414] Vaccine manufacture
[00415] Design and generation of ADXS31-164. Briefly, the dal dat actA mutant strain of Listeria monocytogenes {Lm) was transfected with the pADV plasmid carrying a chimeric human HER2/neu construct. The construct contains 2 extracellular domains (ECl and EC2) and one intracellular domain (IC1) of the human HER2/neu molecule that contain the majority of HLA-A2 restricted immunodominant epitopes, fused to a truncated listeriolysin O construct. The transfer plasmid also contains the bacillus p60 dal gene and is maintained within the mutant Lm via auxotrophic complementation. There is no bacterial resistance cassette. Vaccines were manufactured by Vibalogics GmbH (Cuxhaven, Germany) and stored at -80°C prior to use.
[00416] Histopathology, Staging and Immunohistochemistry
[00417] Histopathological assessment of all primary appendicular osteosarcoma tumors was performed by a board certified veterinary pathologist (J.E.). Tumors were described as osteoblastic, chondroblastic, fibroblastic and telangiectatic based on histological features.
Primary tumors were scored based on mitotic index, nuclear pleomorphism and the amount of matrix and necrosis present. Histological scores were converted into a grade (I, II or III).
[00418] For HER2/neu staining, 5 micron thick serial sections of formalin fixed, decalcified, paraffin embedded tissues were mounted on negatively charged glass slides. Sections were heated at 80°C for 20 minutes, immersed in Pro Par (clearant) and rehydrated in ethanol.
Antigen retrieval was performed by boiling sections in sodium citrate buffer (pH -9.0).
Endogenous peroxidase was blocked using 3% hydrogen peroxide. Staining was performed with a rabbit anti-human HER2/neu antibody (Neu(c-18):sc-284, Santa Cruz Biotecnology) or a rabbit IgG isotype (Universal Negative Control serum, NC498, Biocare Medical). Bound antibody was detected using the Universal Streptavadin-Biotin2 System (DAKO/LSAB2, HRP). Tissues were stained with 3,3'-diaminobenzidine solution (DAKO) and counterstained with hematoxylin. Slides were viewed using a Nikon E600 infinity corrected upright microscope. Bright field images were acquired using a Nikon Digital Sight DS-Fil color camera and a NIS-Element BR3.0 for image analysis. Tissue sections were evaluated and scored for HER2/neu positivity by a board certified pathologist (J.E.) based on the percentage of neoplastic cells staining for HER2/neu (<10% = 1, 10%-50% = 2, >50% = 3) and the intensity of HER2/neu staining (weak = 1, moderate = 2, strong = 3). Scores were based on cells analyzed within 10 hpf for each tissue section. A combined HER2/neu score was obtained by multiplying the two separate scores given for percentage of tumor cells positive for HER2/neu staining and HER2/neu staining intensity. Only dogs with greater than 10% of their tumor cells staining positive for HER2/neu were eligible for trial enrollment.
[00419] Eligibility Criteria and Clinical Trial Design
[00420] Dogs with a histopathological and immunohistochemical diagnosis of HER2/neu positive OSA that had undergone primary tumor removal either by limb amputation or limb- sparing surgery and had received 4 doses of 300mg/m2 carboplatin given once every 3 weeks (or once every 4 weeks if myelosuppression occurred) as adjuvant chemotherapy were eligible for screening. Dogs were screened three weeks after their last carboplatin treatment. A thorough physical examination, Complete Blood Count (CBC), Chemistry Screen (CS) and Urinalysis (UA) were performed to determine general health status. Basic innate and adaptive immune function was tested using a flow cytometric neutrophil oxidative burst assay and mitogen-induced lymphocyte proliferation assay respectively. Baseline cardiac status was evaluated by electrocardiography, echocardiography and serum cardiac troponin I levels. Thoracic radiographs were performed to determine the presence of pulmonary metastatic disease (see Figure 14B). Only those dogs found to be systemically healthy with intact innate and adaptive immune function, no evidence of underlying cardiac disease and no evidence of pulmonary metastatic disease were eligible for enrollment. Dogs that died during the course of the study underwent necropsy. The presence and location of metastatic disease was recorded and histopathology and immunohistochemistry to evaluate HER2/neu expression in metastatic lesions were performed.
[00421] Immune analysis
[00422] Neutrophil oxidative burst assay. Red blood cells in sodium heparin anti-coagulated blood were lysed using 0.83% NH4C1 and the remaining white blood cells were washed twice in 1 x PBS. Cells were labeled with 15ug/ml of dihydrorhodamine 123 (DHR-123; Molecular Probes, Grand Island, NY) and activated with 3 nM phorbol 12-myristate 13- acetate (PMA, Sigma, St. Louis, MO) for 30 minutes at 37 C. Cells were placed on ice for 15 minutes prior to flow cytometric analysis. Cells were acquired on a FACS Canto cytometer (BD Biosciences, San Jose, CA) and analyzed using FloJo software (Treestar, San Carlos, CA).
[00423] Lymphocyte proliferation assay. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from sodium heparin anti-coagulated whole blood by density centrifugation. PBMCs were washed twice in 1 x PBS and counted. Cells were labeled with 5uM CFSE and stimulated with 1.25uM Concanavalin A at 37°C for 5 days. Cells were harvested, washed twice in FACS buffer, labeled with APC-conjugated rat anti-canine CD4 and PE conjugated rat anti-canine CD8 antibodies (Serotec, Raleigh, NC) and analyzed by flow cytometry. For immune function analysis, peripheral blood taken from healthy colony dogs (IACUC #804197) was used as a positive control.
[00424] T cell subset analysis. PBMCs taken at baseline, prior to each vaccination, at re- stage and at every 2 months thereafter were analyzed for CD4 and CD8 T cell subsets. Briefly, cryopreserved cells were thawed and washed twice in FACS buffer (lx PBS, 0.2%
BSA fraction V, and 4 mM sodium azide) prior to surface staining with mouse anti-canine CD3, PE-labeled rat anti-dog CD8 or Alexa-labeled rat anti-dog CD4 (Serotec, Raleigh, NC). Cells were incubated with the vital dye 7-ADD immediately prior to flow cytometric acquisition. Total CD4+ and CD8+ T cell numbers were calculated from the flow cytometric percentages and total lymphocyte counts determined using a Cell Dyn 3700CS Hematology analyzer.
[00425] Vaccine Administration
[00426] Prior to vaccination, dogs received the 5HT3 antagonist ondansetron (0.2mg/kg) intravenously and the HI receptor blocker, diphenhydramine (2mg/kg) intramuscularly to prevent nausea and anaphylaxis respectively. A standard 3+3 clinical trial design was employed. ADXS31-164 was administered at the following doses; Group 1 (2 x 108 CFU) , Group 2 (5 x 108 CFU), Group 3 (1 x 109 CFU) and Group 4 (3.3 x 109 CFU). ADXS31-164 was diluted in lOOmls 0.9% NaCl (Groups 1 and 2) and 200mls 0.9% NaCl (Groups 3 and 4) and administered intravenously over 30 minutes. Temperature, pulse, respiratory rate, heart rate and rhythm (by EKG) and blood pressure were monitored every hour following infusion.
In cases where body temperature exceeded 103°F, dogs were placed on intravenous Plasmalyte at 4mls/kg/hr until their temperature fell below 103°F. Dogs were monitored every hour for signs of lethargy, nausea or vomiting. Blood samples were drawn 24 hours and one week post vaccination to assess for any changes in hematological or biochemical parameters and blood cultures were performed at 24 hours post vaccination to determine persistance of live bacteria in the blood stream. All dogs received a short course of amoxycillin and S-Adenosylmethionine (SAMe) 72 hours after vaccination to kill any remaining listeria and provide anti-oxidant support to the liver.
[00427] Owners with dogs that were free of metastatic disease at least 5 months after receiving the last vaccine in the initial series were offered the option to receive a booster vaccine at a standard dose of 1 x 109 CFU. Booster vaccines were administered as described and dogs were monitored after infusion as described above.
[00428] Toxicity
[00429] Toxicity was graded according to the Veterinary Co-operative Oncology Group- Common Terminology Criteria for Adverse Events (VCOG-CTCAE). Assessment of cardiac toxicity was performed through serial electrocardiograms, echocardiograms and serum cardiac troponin I levels at baseline, at the time of each vaccination, 3 weeks after the last vaccination and every 2 months thereafter until death. Parameters assessed included Left Ventricular Fractional Shortening (LVFS) and Left Ventricular Internal Dimension in diastole (LVIDd) and Left Ventricular Internal Dimension in systole (LVIDs). LVIDd and
LVIDs were normalized to body weight to account for the wide range of body size amongst dogs.
[00430] ELISpot analysis
[00431] Cryopreserved PBMC from each indicated time point were thawed, rested overnight at 37 °C and then counted. Cells were stimulated with 2.5 uM pools of overlapping human HER2/Neu peptides (l lmers overlapping by 5 amino acids) that represent the EC 1, EC2 and IC1 domains of HER2/Neu present in the chimeric vaccine, and recombinant human IL-2 (Invitrogen, Fredrick, MD) for 5 days. Cells were harvested, washed twice in 1 x PBS and counted. IFN-γ ELISpot assays were performed according to the manufacturer's protocol using a commercial canine IFN-γ ELISpot assay kit (R&D Systems, Minneapolis,
MN). Briefly, 0.8 - 2 x 105 stimulated cells were incubated with 2.5 uM of ECl, EC2 or IC1 peptide pools plus IL-2 or IL-2 alone (to determine background counts). All assays were performed in duplicates. Plates were developed according to the manufacturer's instructions. Spots were counted using a CTL-Immunospot analyzer (C.T.L, Shaker Heights, OH).
[00432] Primary and Secondary Outcome Measures
[00433] Time To Metastasis (TTM) was calculated as the time between amputation and development of metastatic disease. OSA Specific Survival was calculated as the time between amputation and death. Patients that died of unrelated causes were censored at the time of their death.
RESULTS [00434] Eighteen dogs that fulfilled the eligibility criteria were enrolled in this phase I clinical trial. The age, breed, sex, tumor location, subtype, grade and HER2/neu status were recorded (Table 4). A standard 3+3 clinical trial design was employed. ADXS31-164 was administered at the following doses; Group 1: 2 x 108 CFU (n=3), Group 2: 5 x 108 CFU (n=3), Group 3: 1 x 109 CFU (n=9), and Group 4: 3 x 109 CFU (n=3). Five additional dogs with pre-existing pulmonary metastatic disease, identified at the time of screening also received ADXS31-164 on a compassionate care basis (Table 4). Four of these dogs had strong HER2/neu staining in >50 of neoplastic cells from their primary tumor. Three of these dogs had multiple pulmonary metastatic nodules and two dogs had a single metastatic nodule at screening. Dogs with multiple pulmonary nodules received one vaccine each before disease progression and withdrawal from the study for alternative treatments. The two dogs with single nodules received the full course of three vaccines each. Dogs with preexisting metastatic disease received either 1 x 109 CFU (n=3) or 3 x 109 CFU (n=2) ADXS31-164 (Table 5).
[00435] Figure 15 shows a schematic of the time-line of the phase 1 clinical trial, wherein three vaccinations were administered following amputation and follow-up chemotherapy.
[00436] Table 4: Signalment and tumor characteristics of enrolled dogs
Figure imgf000095_0001
[00437] Table 5: Signalment and tumor characteristics of dogs with pre-existing metastatic disease treated on a compassionate care basis.
Figure imgf000096_0001
RESULTS
[00438] Safety and Toxicity=S&iety was evaluated for all 23 vaccinated dogs. All dogs tolerated ADXS31-164 administration well with only transient, low grade toxicities observed on the day of vaccination (Table 6). A statistically significant increase in body temperature occurred 4 hours after ADXS31-164 administration in all groups irrespective of dose (Fig 9A). Hypotension was not observed at any time point or at any dose (Fig. 9B). 8/18 dogs (without pre-existing metastatic disease) and 3/5 dogs (with pre-existing metastatic disease) developed fevers of >103°F within 4 hours of vaccination and were given intravenous fluids at that time. Three dogs received a single dose of a non-steroid anti-inflammatory drug to reduce body temperature. In all cases, fevers resolved without further intervention. Transient lethargy, nausea and vomiting that did not require therapeutic intervention occurred within 4 hours of vaccination regardless of dose. In two dogs transient single or bigeminal ventricular premature contractions were identified shortly after vaccination. One dog with pre-existing metastatic disease developed ventricular tachycardia within 2 hours of vaccination. Treatment with lidocaine, procainamide, sotalol and corticosteroids had little effect however, the arrhythmia resolved within 72 hours. Transient, but statistically significant increases in white blood cell and neutrophil counts occurred 24 hours after ADXS31-164 and were accompanied by a transient decrease in platelets and lymphocytes (Fig. 17). Although there was no correlation between ADXS31-164 dose and magnitude of hematological change, there was a significant difference in the magnitude of white blood cell, neutrophil and monocyte responses between dogs that survived and those that died (Fig. 18A-F). Mild, transient increases in the serum concentrations of liver enzymes occurred in approximately half of the dogs, consistent with mild inflammation caused by the hepatotropic Listeria (Table 6). All changes identified in the peripheral blood were asymptomatic and resolved within one week of ADXS31-164 administration. No significant changes in renal function were documented in any dog. 19/23 dogs had blood cultures performed 24 hours after ADXS31-164 administration and all were negative, consistent with rapid clearance of the highly attenuated LmddA strain.
[00439] Given that HER2/neu targeted monoclonal antibodies cause cardio toxicity we evaluated biomarkers of cardiac damage and echocardiographic measures of dysfunction including cardiac troponin I, fractional shortening ( ), LVIDd and LVIDs at baseline, prior to each vaccination and every 2 months thereafter. No significant, sustained changes in cardiac troponin I, fractional shortening, LVIDd or LVIDs were identified in any of the vaccinated dogs (Fig. 26 A-D). One dog in Group 3 showed a stepwise increase in serum cardiac troponin I at the time of each vaccination however, this was not accompanied by echocardiographic signs of dysfunction. Values returned to baseline following the last vaccination and were not elevated on repeat assessments.
[00440] Throughout the clinical trial cardiac troponin I levels were measured along with fractional shortening, Left Ventricular Internal Diameter in systole (LVIDs) and LVID in diastole (LVIDd) as shown in Figure 25 (A-D), there was no evidence of long or short-term cardio toxicity following administration of ADXS31 - 164.
[00441] Table 6 below presents data showing minimal treatment related adverse events were reported during the clinical trial.
[00442] Table 6: Treatment Related Adverse Events occurring at or within 48 hours of ADXS31-164 vaccination.
Number of Dogs with Treatment Related Adverse Events
Figure imgf000098_0001
[00443] Conclusion: ADSX31-164 toxicities were low grade and transient
[00444] Immune Response to ADXS31-164
[00445] The results presented in Figure 18 demonstrate that an early immune response to ADXS31-164 in dogs receiving the vaccines predicted survival of the dogs. Figure 18 shows that ADXS31-164 induced increases in WBC, neutrophil and monocytes counts, which correlated with survival and were accompanied by a transient decrease in platelets and lymphocytes (Figure 17).
[00446] The ability of ADXS31-164 to induce and maintain an immune response, and in particular to induce HER2/Neu specific T cell immunity was assessed during the clinical trial. In order to evaluate the immune response and to determine if a HER2/Neu specific T cell response was induced by ADXS31-164, HER2/Neu specific T cell numbers were assessed by IFN-γ ELISpot. Samples were taken at baseline (3 weeks post carboplatin), at every vaccination and every 2 months thereafter. Figure 19 shows the results of the ELISpot assay.
[00447] HER2/neu Specific Immune Responses. Immunological responses against the human ECl, EC2 and IC1 domains of HER2/neu (sharing 89%, 93% and 98% identity with canine HER2/neu respectively) were detected at baseline in 4/18, 6/18 and 1/18 dogs respectively. Induced IFN-γ responses against one or more of the HER2/neu domains were detected in 7 dogs 3 weeks after the third ADXS31-164 vaccination (Table 7). Five of these dogs developed immune responses against the highly conserved IC1 domain. Five additional dogs developed IFN-γ responses against the IC1 domain 2 months later. Three additional dogs developed IFN-γ responses against either EC2 alone, EC2 and IC1 or ECl, EC2 and EC3 at the time of relapse (dogs 001, 002 and 017). 3 dogs that developed immunological responses against HER2/neu during their initial vaccination series were evaluated by IFN-γ ELISpot over 15 to 17 months. HER2/Neu specific IFN-γ responses were not maintained however, the dogs remained free of metastatic disease during this time. 10 dogs received additional booster vaccinations, of the 6 evaluable, 2 dogs had detectable increases in HER2/neu specific IFN-γ responses 2 months after booster vaccination. Of the 8 dogs that relapsed, 5 had no increase in HER2/neu specific IFN-γ responses 3 weeks after ADXS31- 164.
[00448] Table 7
Figure imgf000100_0001
[00449] Booster vaccinations. Ten of the 18 do gs without metastatic disease at enrollment were administered a single booster vaccine between 5 and 10 months after the initial vaccine series. Four of these dogs received additional booster vaccines given between 4 and 15 months after the first booster vaccine. Similar low grade, transient side effects were noted at the time of booster vaccination as with the initial vaccination series.
[00450] Figure 20 (A and B) show that repeat booster vaccinations also stimulated HER2 specific immunity. Repeat booster vaccinations were administered at 6 and 10 months for animal 289-003, and at 8 months for animal 289-004.
[00451] Clinical Outcomes. 8/18 dogs in the vaccinated group relapsed, 4 with pulmonary metastatic disease and 4 with bone metastases. Two dogs with bone metastases progressed to pulmonary metastases. One dog with a bone lesion in her sacrum died from aspiration pneumonia and one dog with a solitary pulmonary nodule died of nephroblastoma however, necropsy specimens from bone and lung lesions respectively were not available for histopathological confirmation of metastatic osteosarcoma. These two dogs were censored from OSA specific survival analysis. Dogs that relapsed received different rescue chemo- and radiation therapies at the discretion of the primary clinician. The 4 dogs with bone metastases were treated with analgesics only (1 dog), palliative radiation alone (1 dog) or in combination with chemotherapy (2 dogs). Two dogs received Adriamycin and 1 dog received palladia for the treatment of pulmonary metastatic disease. Median OSA specific survival for vaccinated dogs has not yet been reached. Kaplan-Meier survival curves for TTM and OS A Specific Survival are shown in Fig. 21. Overall survival rates at 1 and 2 years for vaccinated dogs are 71.4% and 57% respectively. Of the 12 dogs that developed HER2/neu specific IFN-γ responses within 2 months of vaccination, 9 are still alive (3 dogs > 900 days, 1 dog>700 days, 3 dogs > 400 days and 2 dogs > 300 days and 7 remain tumor free to date (Table 7)). The results presented in Figure 24 demonstrate that ADXS31-164 breaks the tolerance to HER2/Neu. This may be significant for the treatment of OSA as well as other HER2/Neu tumors and/or cancers.
[00452] Necropsy findings. 6/18 dogs died during the study period and necropsies were performed on 4 of these dogs. Three dogs were found to have multifocal grade II and III metastatic osteosarcoma involving the lungs (3 dogs), bone (2 dogs), mediastinum (1 dog) and kidney (1 dog). One dog, euthanized on account of a large progressive renal mass was found to have nephroblastoma. This dog also had a single pulmonary nodule but this was unfortunately not evaluated by histopathology.
[00453] Survival, Prolonged Survival, Tumor Progression following Administration of ADXS31-164
[00454] Three dogs with multiple metastatic pulmonary nodules at screening and treated on a compassionate care basis received one vaccine each before disease progression and removal from the study. The two dogs presenting with solitary metastatic pulmonary nodules at the time of screening received all three vaccines (see Table 5 for signalment and tumor characteristics). Progressive pulmonary metastatic disease occurred in one of these dogs despite vaccination. No additional pulmonary lesions developed in the second dog despite the pre-exisiting pulmonary nodule doubling in size every 3 weeks (Fig. 22A and B). CT scan one week after the last vaccination, confirmed the absence of additional metastatic lesions and the dog underwent metastatectomy. Prior to surgery, the dye indocyanine green (ICG), used to detect tumor margins and areas of inflammation, was administered intravenously and at surgery, fluorescence under near infra-red light was seen in the pulmonary nodule and several other areas of healthy appearing pulmonary parenchyma near the solitary nodule (Fig. 22C and D). Histopathology of the pulmonary nodule revealed metastatic OSA with large areas of hemorrhage and necrosis, surrounded by a thick fibrous capsule (Fig. 22E). IHC showed an accumulation of CD3+ T cells around the fibrous capsule with very few T cells within the nodule itself (Fig. 22G and H). Other areas identified by near infra-red fluorescence showed focal areas of T cell infiltrates (Fig. 22F, 221 and 22J). T cells were seen surrounding abnormally large, vimentin positive cells with prominent mitotic figures (Fig. 22K and 22L). These findings suggest that single metastatic sarcoma cells may be effectively targeted by tumor specific T cells within the lung and provide a possible mechanism by which ADXS31-164 prevents metastatic pulmonary disease. The dog recovered well from surgery and remained free of pulmonary metastatic disease for 5 months before developing widespread aggressive, HER2/neu+ metastatic disease in the subcutaneous tissue (osteoblastic, grade II and chondroblastic, grade III), mediastinum (osteoblastic, grade II) and diaphragm (osteoblastic, grade III).
Results show that despite induction of HER2/neu specific T cell responses, off-tumor side effects were not identified, hence induction of HER2/neu specific T cells is responsible for elimination of HER2/neu positive metastatic cells and long term protection from disease recurrence. This is supported by the timing of HER2/Neu-specific T cell expansion which in 5 dogs occurred approximately 8 months post diagnosis, when many dogs will develop metastatic disease and by the histopathological findings of focal T cell responses within the pulmonary parenchyma of one dog following vaccination and metastatectomy.
[00455] The results presented in Figure 22 and Figure 23 demonstrate that administration of ADXS31-164 delays and/or prevents metastatic disease and prolongs the overall survival in dogs with spontaneous HER2+ osteosarcoma. As can be seen in both figures, dogs receiving vaccine had significantly extended survival time, while the median survival for those dogs receiving vaccine has not yet been reached.
[00456] While our study demonstrates the effectiveness of this approach in preventing metastatic disease, vaccination with ADXS31-164 was unable to induce regression of pre-existing gross, pulmonary metastatic disease in 5 dogs treated on a compassionate care basis. In one dog this appeared to be associated with a failure of T cells to penetrate the fibrous capsule surrounding the metastatic lesion or for those cells to survive within the established tumor microenvironment (Fig. 22C). However, in the same dog, focal areas of T cell infiltrates surrounding large, actively dividing mesenchymal cells, purported to be metastatic OSA cells were identified in grossly normal lung parenchyma and unexpectedly, following metastatectomy this dog did not develop further pulmonary metastatic disease. Taken together, these data suggest that ADXS31-164 prevents pulmonary metastatic disease through its ability to induce potent innate immune responses that may sensitize metastatic OSA cells to FAS/FASL mediated apoptosis and adaptive immune responses in the form of HER2/Neu specific T cells that eliminate micrometastatic pulmonary disease.
[00457] Conclusions:
[00458] At the time of filing this application 12/18 dogs have not developed pulmonary metastatic disease, demonstrating that ADXS31-164 prevents metastatic disease in a subject suffering from spontaneous HER2+ osteosarcoma when administered in the setting of minimal residual disease. Vaccinated dogs showed a statistically significant increase in overall survival compared to a historical HER2/Neu+ control group. Median survival in the HER2/Neu+ control dogs (n=l l) was 316 days (p=0.032) wherein the median survival in ADSX31-164 treated dogs has not been reached. Further, the results indicate that ADXS31-164 breaks peripheral tolerance to the highly conserved
IC1 domain of HER2/Neu (Figure 26). The magnitude of the increase in leucocytes within 24 hours of ADXS31-164 administration (Figure 18) correlates with survival, suggesting that outcome depends in part upon the ability of the dog's immune system to respond to the vaccine. Importantly, this study showed that administration of up to 3 x 109 CFU of ADXS31-164 to dogs with spontaneous OS A is safe and causes only transient, low grade side effects at the time of administration. Moreover, prevention of pulmonary metastatic disease maybe in part associated with CD3+ T cell mediated elimination of microscopic metastatic disease in the lung. This work has important implications for pediatric OSA and other human cancers that express HER2/Neu.
[00459] Moreover, here we show that administration of ADXS31-164 in doses up to 3.3 x 10Λ9 CFU are safe in the dog and despite inducing HER2/neu specific immunity, do not lead to short or long term cardio toxicity. On target, off tumor side effects including cardio toxicity has been associated with the administration of large numbers of HER2/neu specific T cells or when trastuzumab has been used concurrently with anthracyclines. We employed a standard chemotherapy protocol without doxorubicin to reduce any potential risk of cardio toxicity.
[00460] Our study demonstrates that ADXS31-164 can prevent pulmonary metastatic disease in dogs with OSA. These results demonstrate safety and unprecedented survival times in dogs with OSA and pave the way to investigate the ability of ADXS31-164 to prevent metastatic disease in patients with HER2/neu expressing tumors including pediatric osteosarcoma and mammary carcinoma.
EXAMPLE 11:
COMBINATION ADXS31-164 AND RADIATION THERAPY FOR THE
TREATMENT OF CANINE AND HUMAN OSTEOSARCOMA (OSA) and
PULMONARY METASTATIC DISEASE.
[00461] A recombinant Listeria monocytogenes expressing a human chimeric Her-2/neu construct (ADXS31-164) used in combination with palliative radiation to prevent pulmonary metastatic disease and prolong overall survival in dogs with spontaneous appendicular osteosarcoma is described. Given the similarities between canine and human osteosarcoma, we believe that this combination will be effective therapy for human disease. [00462] Materials and Methods
[00463] Vaccine preparation
[00464] The details of the construction of ADXS31-164 vaccine have been described above.
The ADXS31-164 vaccine stocks were prepared and stored as 1 ml aliquots in freezer at -70 °C. Before injection, vaccine stocks were thawed at 37 °C for 2-3 min and then washed twice with phosphate-buffer saline (PBS) and resuspended in PBS at a final concentration of
5xl08 colony forming units (CFU)/ml. Each dog was immunized intraperitoneally with 200 μΐ of this suspension.
[00465] RT and immunotherapy
[00466] Ten systemically healthy dogs with histopathologically confirmed, treatment naive,
HER2+ appendicular OSA, and no evidence of cardiac or metastatic disease were enrolled.
All dogs received 16Gy of RT in two fractions on consecutive days, followed by the first of 8 intravenous doses of ADXS31-164 (3.3 x 10Λ9 CFU per dose) given once every 3 weeks.
Immunization with the Listeria- based vaccine was performed every 3 weeks (e.g., on days 7, 28, 49, 70, 91, 112, 133 and 154) (Figure 10). Immune analysis was also performed on the day of immunization.
[00467] On days 4 and 5, external beam RT of 8 Gy was delivered using a Siemens 6 MV linear accelerator. The RT was given under general anesthesia. Tumors were evaluated clinically every three weeks and radiographically at baseline, at the fourth vaccine administration (day 70) and at the eighth vaccine administration (day 154). At these time points, thoracic radiographs were performed to determine the presence of pulmonary metastatic disease.
[00468] A bone biopsy to confirm the diagnosis of osteosarcoma was performed at the time of enrollment. Complete Blood Count (CBC), Chemistry Screen (CS), Urinary Analysis (UA), electrocardiogram (EKG)/Echocardiogram/ serum concentration of cardiac troponin I
(cTnl) and radiographs of the affected limb and the thorax were performed on Days 0, 70, and 133 and every 2 months thereafter until euthanasia. On the day of euthanasia Complete Blood Count (CBC), Chemistry Screen (CS), Urinary Analysis (UA), Immune analysis, electrocardiogram (EKG)/Echocardiogram/ serum concentration of cardiac troponin I (cTnl); and necropsy were performed.
[00469] ELISpot assayCryopreserved PBMC from each indicated time point were thawed, rested overnight at 37 °C and then counted. Cells were stimulated with 2.0 uM pools of overlapping human HER2/Neu peptides (l lmers overlapping by 5 amino acids) that represent the ECl, EC2 and ICl domains of HER2/Neu present in the chimeric vaccine, and recombinant human IL-2 (Invitrogen, Fredrick, MD) for 5 days. Cells were harvested, washed twice in 1 x PBS and counted. IFN-γ ELISpot assays were performed according to the manufacturer's protocol using a commercial canine IFN-γ ELISpot assay kit (R&D Systems, Minneapolis, MN). Briefly, 0.1 - 3 x 105 stimulated cells were incubated with 2.5 uM of ECl, EC2 or IC1 peptide pools or none (to determine background counts). All assays were performed in duplicates. Plates were developed according to the manufacturer's instructions. Spots were counted using a CTL-Immunospot analyzer (C.T.L, Shaker Heights, OH).
Results
[00470] We evaluated the use of ADXS31-164 as adjuvant therapy in dogs with spontaneous osteosarcoma as described herein above. ADXS31-164 was administered to dogs with spontaneous appendicular OS A following 16 Gy RT administered on two consecutive days. Up to 8 doses of ADXS31-164 were administered. This work showed repeat administrations of 3.3 x 109 CFU of ADXS31-164 to be safe.
[00471] The potential synergy between radiation therapy and ADXS31-164 to promote antitumor immunity (in particular the generation of Her-2/neu specific T cells), retard the progression of the primary tumor and prevent/delay pulmonary metastatic disease was then explored.
[00472] Figure 10 describes the treatment protocol. Dogs were screened on day 0 for enrollment in the trial. Screening included evaluation of baseline blood tests, urinalysis, cardiac evaluation, thoracic and affected limb radiographs and bone biopsy to confirm the diagnosis of osteosarcoma. Palliative radiation was given on 2 consecutive days following enrollment. Multiple doses (up to 8) of ADXS31-164 were given once every 3 weeks following palliative radiation therapy with only transient, low-grade, side effects (data not shown). Thoracic and limb radiographs were repeated at day 70 and 154. Lameness scores were assigned by two board certified veterinary orthopedic surgeons to each dog at each time point based on their evaluation of videos taken of each dog at each time point. Owners also filled in a validated pain questionnaire that documented the owners perception of quality of life (Figure 28). 5/10 dogs are still alive, three without evidence of tumor progression or pulmonary metastatic disease. At the time of writing, median survival is 285 days which compares favorably with a historical median survival of 136 days achieved with RT alone. In one patient, presenting for trial enrollment with a pathological fracture of the proximal humerus that was stabilized by 2 bone plates and an intramedullary pin, radiographs show evidence of the fracture healing and no evidence of pulmonary metastatic disease three months after radiation therapy and ADXS31-164 administration, (Figure 11). [00473] Moreover, results show that palliative radiation therapy in conjunction with ADXS31-164 therapy reduces lysis, promotes tumor consolidation, and prolongs survival of subjects (Figure 29 A-Dand Figure 30-A-B). Lateral (Figure 29A) and AP (Figure 29C) radiographic views of a distal tibial osteosarcoma lesion demonstrating marked cortical bone remodeling, and reduction in lysis, following 16Gy radiation (given as 8Gy on 2 consecutive days starting on 9.13.2014) and 3 doses of ADXS31-164 (11.25.2014). Lateral (Figure 29B) and AP (Figure 29D) radiographic views of a distal radial osteosarcoma lesion treated with 16Gy radiation (given as 8Gy on 2 consecutive days starting on 7.16.2014) and 3 doses of ADXS31-164 (10.13.2014). Note the significant reduction in swelling and bony lysis within the distal portion of the radius (compare radiographs dated 10.13.2014 with 7.16.2014 in Figure 29B). There is increased bone density on the medial aspect of the distal tibia (compare radiographs dated 10.13.2014 with 7.16.2014 in Figure 29D). There is a small minimally displaced bone fracture of the medial aspect of the distal radius seen on the 10.13.2014 radiographs in Figure 29D. Therefore, ADXS31-164 may be used without chemotherapy; in combination with radiation and potentially in the neo-adjuvant setting, prior to amputation and chemotherapy to prevent metastatic disease.
[00474] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

What is claimed is:
1. A method of treating a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, said fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, said nucleic acid further comprising a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
2. The method of claim 1, wherein said Her-2/neu chimeric antigen is encoded by the sequence set forth in SEQ ID NO: 2.
3. The method of claims 1-2, wherein said subject is a human adult or child.
4. The method of claims 1-2, wherein said subject is a canine.
5. The method of any one of claims 1-4, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.
6. The method of claim 1, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the canine MHC-class I epitopes.
7. The method of any one of claims 1-6, wherein said nucleic acid molecule is integrated into the Listeria genome.
8. The method of any one of claims 1-6, wherein said nucleic acid molecule is in a plasmid in said recombinant attenuated Listeria strain and wherein said plasmid is stably maintained in said recombinant attenuated Listeria strain in the absence of antibiotic selection.
9. The method of any one of claims 1-8, wherein said recombinant Listeria comprises a deletion in the actA virulence gene.
10. The method of any one of claims 1-9, wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment.
11. The method of any one of claims 1-10, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.
12. The method of any one of claims 1-11, wherein said radiation therapy is administered prior to administration of said recombinant attenuated Listeria strain.
13. The method of any one of claims 1-12, wherein said radiation therapy is administered on 2 consecutive days in 8Gy doses each for a total of 16 Gy.
14. The method of any one of claims 1-13, wherein said recombinant attenuated Listeria strain is ADXS31-164.
15. The method of any one of claims 1-14, wherein said recombinant attenuated Listeria is administered multiple times at a dose of 3.3x109 CFU per administration.
16. The method of claim 15, wherein said recombinant attenuated Listeria is administered from one and up to 8 times every 3 weeks.
17. The method of claims 15-16, further comprising administering a booster treatment.
18. The method of claim 17, wherein said booster treatment is administered every two months.
19. The method of any one of claims 1-18, wherein said recombinant attenuated Listeria strain is administered with an independent adjuvant, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
20. The method of any one of claims 1-19, wherein said tumor growth or cancer is a relapse or metastasis.
21. The method of any one claims 1-20, wherein said cancer is osteosarcoma.
22. The method of claim 21, wherein said metastasis is pulmonary metastatic disease.
23. A method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, said fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, said nucleic acid further comprising a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
24. The method of claim 23, wherein said Her-2/neu chimeric antigen is encoded by the sequence set forth in SEQ ID NO: 2.
25. The method of claims 23-24, wherein said subject is a human adult or child.
26. The method of claims 23, wherein said subject is a canine.
27. The method of any one of claims 23-26, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.
28. The method of any one of claims 23 or 26, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the canine MHC-class I epitopes.
29. The method of any one of claims 23-28, wherein said nucleic acid molecule is integrated into the Listeria genome.
30. The method of any one of claims 23-29, wherein said nucleic acid molecule is in a plasmid in said recombinant attenuated Listeria strain and wherein said plasmid is stably maintained in said recombinant attenuated Listeria strain in the absence of antibiotic selection.
31. The method of any one of claims 23-30, wherein said recombinant Listeria comprises a deletion in the actA virulence gene.
32. The method of any one of claims 23-31 , wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment.
33. The method of any one of claims 23-32, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.
34. The method of any one of claims 23-33, wherein said radiation therapy is administered prior to administration of said recombinant attenuated Listeria strain.
35. The method of any one of claims 23-34, wherein said radiation therapy is administered on 2 consecutive days in 8Gy doses each for a total of 16 Gy.
36. The method of any one of claims 23-35, wherein said recombinant attenuated Listeria strain is ADXS31-164.
37. The method of any one of claims 23-36, wherein said recombinant attenuated Listeria is administered multiple times at a dose of 3.3x109 CFU per administration.
38. The method of claim 37, wherein said recombinant attenuated Listeria is administered from one and up to 8 times every 3 weeks.
39. The method of claims 37-38, further comprising administering a booster treatment.
40. The method of claim 39, wherein said booster treatment is administered every two months.
41. The method of any one of claims 23-40, wherein said recombinant attenuated Listeria strain is administered with an independent adjuvant, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
42. The method of any one of claims 23-41, wherein said tumor growth or cancer is a relapse or metastasis.
43. The method of any one claims 23-42, wherein said cancer is osteosarcoma.
44. The method of claim 43, wherein said metastasis is pulmonary metastatic disease.
45. The method of claim 23-44, wherein said eliciting an enhanced immune response results in an increased Her-2/neu specific T cell response.
46. A method of prolonging survival in a subject suffering from a Her-2/neu-expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide, and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
47. The method of claim 46, wherein said Her-2/neu chimeric antigen is encoded
by the sequence set forth in SEQ ID NO: 2.
48. The method of claims 46-47, wherein said subject is a human adult or child.
49. The method of claim 46-47, wherein said subject is a canine.
50. The method of any one of claims 46-48, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.
51. The method of claim 46, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the canine MHC-class I epitopes.
52. The method of any one of claims 46-51, wherein said nucleic acid molecule is integrated into the Listeria genome.
53. The method of any one of claims 46-51, wherein said nucleic acid molecule is in a plasmid in said recombinant attenuated Listeria strain and wherein said plasmid is stably maintained in said recombinant attenuated Listeria strain in the absence of antibiotic selection.
54. The method of any one of claims 46-53, wherein said recombinant Listeria comprises a deletion in the actA virulence gene.
55. The method of any one of claims 46-54, wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment.
56. The method of any one of claims 46-55, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.
57. The method of any one of claims 46-56, wherein said radiation therapy is administered prior to administration of said recombinant attenuated Listeria strain.
58. The method of any one of claims 46-57, wherein said radiation therapy is administered on 2 consecutive days in 8Gy doses each for a total of 16 Gy.
59. The method of any one of claims 46-58, wherein said recombinant attenuated Listeria strain is ADXS31-164.
60. The method of any one of claims 46-59, wherein said recombinant attenuated Listeria is administered multiple times at a dose of 3.3x109 CFU per administration.
61. The method of claim 60, wherein said recombinant attenuated Listeria is administered from one and up to 8 times every 3 weeks.
62. The method of claims 60-61, further comprising administering a booster treatment.
63. The method of claim 62, wherein said booster treatment is administered every two months.
64. The method of any one of claims 46-63, wherein said recombinant attenuated Listeria strain is administered with an independent adjuvant, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
65. The method of any one of claims 46-64, wherein said tumor growth or cancer is a relapse or metastasis.
66. The method of any one claims 46-65, wherein said cancer is osteosarcoma.
67. The method of claim 65, wherein said metastasis is pulmonary metastatic disease.
68. A method of delaying metastatic disease in a subject suffering from a Her-2/neu- expressing tumor growth or cancer comprising the step of administering a combination of radiation therapy and a recombinant attenuated Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a Her-2/neu chimeric antigen fused to an additional polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is mutated in the chromosome of said recombinant attenuated Listeria strain, and wherein the administration of said radiation therapy comprises at least two administrations of said radiation therapy.
69. The method of claim 68, wherein said Her-2/neu chimeric antigen is encoded
by the sequence set forth in SEQ ID NO: 2.
70. The method of claims 68-69, wherein said subject is a human adult or child.
71. The method of claim 68-69, wherein said subject is a canine.
72. The method of any one of claims 68-71, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.
73. The method of claim 68, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the canine MHC-class I epitopes.
74. The method of any one of claims 68-73, wherein said nucleic acid molecule is integrated into the Listeria genome.
75. The method of any one of claims 68-73, wherein said nucleic acid molecule is in a plasmid in said recombinant attenuated Listeria strain and wherein said plasmid is stably maintained in said recombinant attenuated Listeria strain in the absence of antibiotic selection.
76. The method of any one of claims 68-75, wherein said recombinant Listeria comprises a deletion in the actA virulence gene.
77. The method of any one of claims 68-76, wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment.
78. The method of any one of claims 68-77, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.
79. The method of any one of claims 68-78, wherein said radiation therapy is administered prior to administration of said recombinant attenuated Listeria strain.
I l l
80. The method of any one of claims 68-79, wherein said radiation therapy is administered on 2 consecutive days in 8Gy doses each for a total of 16 Gy.
81. The method of any one of claims 68-80, wherein said recombinant attenuated Listeria strain is ADXS31-164.
82. The method of any one of claims 68-81, wherein said recombinant attenuated Listeria is administered multiple times at a dose of 3.3x109 CFU per administration.
83. The method of claim 82, wherein said recombinant attenuated Listeria is administered from one and up to 8 times every 3 weeks.
84. The method of claims 82-83, further comprising administering a booster treatment.
85. The method of claim 84, wherein said booster treatment is administered every two months.
86. The method of any one of claims 68-85, wherein said recombinant attenuated Listeria strain is administered with an independent adjuvant, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
87. The method of any one of claims 68-86, wherein said tumor growth or cancer is a relapse or metastasis.
88. The method of any one claims 68-87, wherein said cancer is osteosarcoma.
89. The method of claim 88, wherein said metastasis is pulmonary metastatic disease.
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AU2015253737A AU2015253737A1 (en) 2014-05-02 2015-04-02 Combination immuno therapy and radiotherapy for the treatment of Her-2-positive cancers
SG11201609135VA SG11201609135VA (en) 2014-05-02 2015-04-02 Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers
KR1020167033949A KR20170002552A (en) 2014-05-02 2015-04-02 Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers
EP15786740.9A EP3137107A4 (en) 2014-05-02 2015-04-02 Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers
CA2947677A CA2947677A1 (en) 2014-05-02 2015-04-02 Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers
CN201580036168.0A CN106794234A (en) 2014-05-02 2015-04-02 Combined immunization therapy and radiotherapy for treating the positive cancers of HER 2
TW105109639A TW201707715A (en) 2015-03-26 2016-03-25 Combination immuno therapy and radiotherapy for the treatment of Her-2-positive cancers
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US201462076411P 2014-11-06 2014-11-06
US62/076,411 2014-11-06
USPCT/US2015/017559 2015-02-25
PCT/US2015/017559 WO2015130810A2 (en) 2014-02-25 2015-02-25 Compositions and methods for the treatment of her2/neu over-expressing tumors
US14/669,629 2015-03-26
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