EP3137107A1 - 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 cancersInfo
- Publication number
- EP3137107A1 EP3137107A1 EP15786740.9A EP15786740A EP3137107A1 EP 3137107 A1 EP3137107 A1 EP 3137107A1 EP 15786740 A EP15786740 A EP 15786740A EP 3137107 A1 EP3137107 A1 EP 3137107A1
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- EP
- European Patent Office
- Prior art keywords
- another embodiment
- neu
- recombinant
- recombinant attenuated
- radiation therapy
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- 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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US14/268,436 US20140234370A1 (en) | 2009-11-11 | 2014-05-02 | Compositions and methods for prevention of escape mutation in the treatment of her2/neu over-expressing tumors |
US201462076411P | 2014-11-06 | 2014-11-06 | |
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 US10016617B2 (en) | 2009-11-11 | 2015-03-26 | Combination immuno therapy and radiotherapy for the treatment of Her-2-positive cancers |
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MA (1) | MA39942A (en) |
MX (1) | MX2016014367A (en) |
SG (1) | SG11201609135VA (en) |
WO (1) | WO2015167748A1 (en) |
Families Citing this family (9)
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US9012141B2 (en) | 2000-03-27 | 2015-04-21 | Advaxis, Inc. | Compositions and methods comprising KLK3 of FOLH1 antigen |
CA2829960A1 (en) | 2011-03-11 | 2012-09-20 | John Rothman | Listeria-based adjuvants |
BR112014022662A2 (en) | 2012-03-12 | 2017-10-03 | Advaxis Inc | INHIBITION OF SUPPRESSOR CELL FUNCTION FOLLOWING LISTERIA VACCINE TREATMENT |
RU2016136855A (en) | 2014-02-18 | 2018-03-22 | Адваксис, Инк. | BIOMARKER-DIRECTED MULTI-PURPOSE IMMUNOTHERAPY |
CN106459887A (en) | 2014-04-24 | 2017-02-22 | 阿德瓦希斯公司 | Recombinant listeria vaccine strains and methods of producing the same |
MA41644A (en) | 2015-03-03 | 2018-01-09 | Advaxis Inc | LISTERIA-BASED COMPOSITIONS INCLUDING A MINIGEN EXPRESSION SYSTEM CODING PEPTIDES, AND METHODS OF USE THEREOF |
TW201805013A (en) * | 2016-06-03 | 2018-02-16 | 美商依圖比克斯公司 | Compositions and methods for tumor vaccination and immunotherapy involving HER2/NEU |
CN110506107A (en) | 2016-11-30 | 2019-11-26 | 阿德瓦希斯公司 | Target the immunogenic composition and its application method of relapsed cancer mutation |
CA3075849A1 (en) | 2017-09-19 | 2019-03-28 | Advaxis, Inc. | Compositions and methods for lyophilization of bacteria or listeria strains |
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US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US6855320B2 (en) | 2000-03-29 | 2005-02-15 | The Trustees Of The University Of Pennsylvania | Fusion of non-hemolytic, truncated form of listeriolysin O to antigens to enhance immunogenicity |
US7432304B2 (en) * | 2001-05-30 | 2008-10-07 | The Regents Of The University Of Michigan | Small molecule antagonists of Bcl-2 family proteins |
WO2005037233A2 (en) * | 2003-10-15 | 2005-04-28 | Medimmune, Inc. | Listeria-based epha2 vaccines |
US9017660B2 (en) * | 2009-11-11 | 2015-04-28 | Advaxis, Inc. | Compositions and methods for prevention of escape mutation in the treatment of Her2/neu over-expressing tumors |
JP5985397B2 (en) * | 2009-11-11 | 2016-09-06 | アドバクシス インコーポレイテッド | Recombinant Listeria strain and immunogenic composition containing the same |
WO2011100754A1 (en) * | 2010-02-15 | 2011-08-18 | The Trustees Of The University Of Pennsylvania | Live listeria-based vaccines for central nervous system therapy |
ES2613630T3 (en) * | 2010-05-23 | 2017-05-25 | Aduro Biotech, Inc. | Methods and compositions that use Listeria for an auxiliary cancer treatment |
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SG11201609135VA (en) | 2016-11-29 |
CN106794234A (en) | 2017-05-31 |
KR20170002552A (en) | 2017-01-06 |
JP2017514904A (en) | 2017-06-08 |
IL248704A0 (en) | 2017-01-31 |
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