WO2015130810A2 - Compositions and methods for the treatment of her2/neu over-expressing tumors - Google Patents
Compositions and methods for the treatment of her2/neu over-expressing tumors Download PDFInfo
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- WO2015130810A2 WO2015130810A2 PCT/US2015/017559 US2015017559W WO2015130810A2 WO 2015130810 A2 WO2015130810 A2 WO 2015130810A2 US 2015017559 W US2015017559 W US 2015017559W WO 2015130810 A2 WO2015130810 A2 WO 2015130810A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
- A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- a human subject is a child or adolescent.
- Listeria monocytogenes is an intracellular pathogen that primarily infects antigen presenting cells and has adapted for life in the cytoplasm of these cells.
- Host cells such as macrophages, actively phagocytose L. monocytogenes and the majority of the bacteria are degraded in the phagolysosome.
- Some of the bacteria escape into the host cytosol by perforating the phagosomal membrane through the action of a hemolysin, listeriolysin O (LLO).
- LLO listeriolysin O
- L. monocytogenes can polymerize the host actin and pass directly from cell to cell further evading the host immune system and resulting in a negligible antibody response to L. monocytogenes.
- HER2/neu also referred to herein as "Her-2" is a 185 kDa glycoprotein that is a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinases, and consists of an extracellular domain, a transmembrane domain, and an intracellular domain which is known to be involved in cellular signaling.
- EGFR epidermal growth factor receptor
- the Her2 antigen is overexpressed in 25 to 40% of all breast cancers and is also overexpressed in many cancers of the bone (osteosarcoma - OSA), ovaries, lung, pancreas, brain, and gastrointestinal tract.
- the overexpression of Her-2 is associated with uncontrolled cell growth and signaling, both of which contribute to the development of tumors. 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.
- OSA histologic heterogeneity, aggressive local disease and early metastases.
- OSA can occur in any bone but the limbs account for 75%-85% of all affected bones and where it is called 'appendicular osteosarcoma'.
- the remaining OSA affect the axial skeleton comprising maxilla, mandible, spine, cranium, ribs, nasal cavity, paranasal sinuses and pelvis.
- 95% of dogs have micrometastatic disease and despite amputation and chemotherapy, the median survival time is 10 months with most dogs euthanized due to progressive metastatic disease. Pulmonary metastatic disease is the principal cause of morbidity and mortality in both species.
- the present invention meets this need by providing a recombinant Listeria-HER2/ne vaccine strain that was generated using the LmddA vaccine vector which has a well-defined attenuation mechanism and is devoid of antibiotic selection markers and which has been found effective in treating canine osteosarcoma.
- the invention provided herein relates to an immunogenic composition
- an immunogenic composition comprising a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, and wherein administering the fusion protein to a subject having a HER2/neu-expressing tumor circumvents mutation avoidance by the tumor.
- circumventing mutation avoidance is due to epitope spreading.
- circumventing mutation avoidance is due to the chimeric nature of the antigen.
- the invention provided herein relates to a recombinant Listeria vaccine strain comprising a nucleic acid molecule, wherein and in another embodiment, the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a HER2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second 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.
- the invention provided herein relates to a method of treating a HER2/neu-expressing tumor growth or cancer in a subject, the method comprising the step of administering a recombinant attenuated Listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, wherein said nucleic acid molecule comprises a first open reading frame encoding said fusion polypeptide, wherein said 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 said recombinant Listeria vaccine strain.
- the subject is a human.
- a human subject may be an adult or a child.
- a subject is a canine.
- the chimeric HER2 is a canine chimeric HER2.
- the chimeric HER2 is a human chimeric HER2.
- administering said fusion polypeptide to said subject prevents escape mutations within said tumor.
- said human HER2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.
- the invention provided herein relates to a method of preventing a HER2/neu-expressing tumor growth or cancer.
- a method of treating a HER2/neu-expressing tumor growth or cancer results in increased overall survival of said subject.
- a method of treating a HER2/neu-expressing tumor growth or cancer results in a delay of metastatic disease in a subject.
- the treating results in an increased HER2/neu specific T cell response.
- this invention provides a method of eliciting an enhanced immune response against a HER2/neu-expressing tumor growth or cancer in a subject, the method comprising the step of administering a recombinant attenuated Listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, wherein said nucleic acid molecule comprises a first open reading frame encoding said fusion polypeptide, wherein said 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 lacking in the chromosome of said recombinant Listeria vaccine strain.
- said method of eliciting an enhanced immune response results in increased overall survival of said subject. In another embodiment, said method of eliciting an enhanced immune response results in a delay of metastatic disease in a subject. In another embodiment, said method of eliciting an enhanced immune response results in an increased HER2/neu specific T cell response.
- this invention provides a method of prolonging survival in a subject suffering a HER2/neu-expressing tumor growth or cancer, the method comprising the step of administering a recombinant attenuated Listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, wherein said nucleic acid molecule comprises a first open reading frame encoding said fusion polypeptide, wherein said 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 lacking in the chromosome of said recombinant Listeria vaccine strain.
- the subject is a human.
- a human subject may be an adult or a child.
- a subject is a canine.
- said method further comprises administering said recombinant attenuated Listeria following a relapse or metastasis in said subject.
- the invention provided herein relates to a method of delaying metastatic disease in a subject suffering from a HER2/neu-expressing tumor growth or cancer, the method comprising the step of administering a recombinant attenuated Listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, wherein said nucleic acid molecule comprises a first open reading frame encoding said fusion polypeptide, wherein said 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 lacking in the chromosome of said recombinant Listeria vaccine strain.
- a subject in a human may be an adult or a child.
- a subject is a canine.
- FIG. 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 ) 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).
- 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.
- 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-100 CCD camera.
- FIG. 6 Pixel intensity was graphed as number of photons per second per cm2 of surface area; this is shown as average radiance.
- Figure 7. Shows the first 18 patients vaccinated with ADXS31-164.
- Figure 8. Shows that ADXS31-164 administration does not cause early or late cardiac damage.
- 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
- Figure 10 Shows treatment schedule of combination ADXS31-164 and palliative radiation therapy (RT) in primary disease.
- Figure 11 Radiograph showing no evidence of metastatic disease in a dog following fracture of proximal humerus and also shows the presence of boney callus indicating fracture healing.
- 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.
- FIG. 13 Treatment-related adverse events and survival curves following ADXS-31- 164 administration.
- 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 marked proliferation and lysis in the bone metaphysis (arrows in A).
- OSA primary and metastatic osteosarcoma
- FIG. 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 and at 24 hours 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) OSA Specific Survival.
- 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 (G) and high (H) magnification showing focal accumulations of CD3+ T cells.
- K High magnification H&E stain of focal pneumonia showing large abnormal cells with mitotic figures surrounded by lymphocytes.
- L Vimentin stain of pneumonic region showing large cells, with mitotic figures surrounded by mononuclear 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 IC1 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), LVID (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.
- compositions and methods for preventing, treating and vaccinating against a Her2-neu antigen-expressing tumor and inducing an immune response against sub-dominant epitopes of the Her2-neu antigen, while circumventing mutation avoidance are provided herein.
- circumventing mutation avoidance is due to epitope spreading.
- circumventing mutation avoidance is due to the chimeric nature of the antigen.
- an immunogenic composition comprising a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, and wherein administering the fusion protein to a subject having an HER2/neu-expressing tumor prevents escape mutations within said tumor.
- a recombinant Listeria vaccine strain comprising the immunogenic composition.
- a subject is a human subject.
- the human subject is an adult or a child.
- the human subject is a child.
- a subject is a canine subject.
- the canine is a dog.
- a method of eliciting an enhanced immune response against a HER2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a recombinant Listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide.
- a method of preventing a HER2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a recombinant Listeria comprising nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide.
- a method of treating a HER2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a recombinant Listeria comprising nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide.
- a method of prolonging the survival of a subject having a HER2/neu-expressing tumor growth or cancer comprising the step of administering a recombinant Listeria comprising nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide.
- the subject is a human. In another embodiment, the subject is a canine.
- a method of delaying metastatic disease in a subject having a HER2/neu-expressing tumor growth or cancer comprising the step of administering a recombinant Listeria comprising nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide.
- the subject is a human. In another embodiment, the subject is a canine.
- a method of treating a HER2/neu-expressing tumor growth or cancer in a subject comprising the step of administering a recombinant attenuated Listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a HER2/neu chimeric antigen fused to an additional polypeptide, wherein said nucleic acid molecule comprises a first open reading frame encoding said fusion polypeptide, wherein said 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 said recombinant Listeria vaccine strain.
- the subject is a human.
- a human subject may be an adult or a child.
- a subject is a canine.
- the chimeric HER2 is a canine chimeric HER2.
- the chimeric HER2 is a human chimeric HER2.
- administering said fusion polypeptide to said subject prevents escape mutations within said tumor.
- said human HER2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.
- a recombinant Listeria vaccine 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 HER2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, and wherein the metabolic enzyme complements an endogenous gene that is lacking in the chromosome of the recombinant Listeria strain.
- the recombinant Listeria vaccine strain 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 lacking in the chromosome of the 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-based 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 chimeric HER2/neu or the ADXS-31-164 (expressing chimeric HER2/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.
- an attenuated auxotrophic Listeria vaccine strain provided herein 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 HER2/neu expression in vivo and in vitro by complementation of dal gene.
- ADXS31-164 expresses and secretes a chimeric HER2/neu protein fused to the first 441 amino acids of listeriolysin O (LLO), which in another embodiment, is a truncated and nonhemolytic LLO.
- LLO listeriolysin O
- ADXS31-164 exerts strong and antigen specific anti-tumor responses with ability to break tolerance toward HER2/neu in transgenic animals (see Examples, Figure 24).
- the ADXS31-164 strain is highly attenuated and has a better safety profile than previous Listeria vaccine generation, 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).
- ADXS31-164 strain is highly immunogenic, able to break tolerance toward the HER2/neu self-antigen and prevent tumor formation in HER2/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.
- 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).
- the Lm-LLO-ChHer2 strain is Lm-LLO-138, and comprises an antibiotic resistance gene and a prfA gene expressed from a plasmid.
- recombinant attenuated, antibiotic-free Zisien ' a-expressing chimeric antigens are useful for preventing, and treating a cancer or solid tumors, as exemplified herein.
- the tumor is a HER2/neu positive tumor.
- the cancer is a HER2/neu-expressing cancer.
- the cancer is breast cancer, a central nervous system (CNS) cancer, a head and neck cancer, an osteosarcoma (OS A), a canine osteosarcoma, Ewing's sarcoma (ES), or any HER2/neu-expressing cancer known in the art.
- 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.
- a cancer or solid tumor described herein is a result of relapse or metastatic disease.
- recombinant Listeria expressing a chimeric HER2/neu are useful as a therapeutic vaccine for the treatment of HER2/neu overexpressing solid tumors.
- a HER2/neu chimeric antigen provided herein is useful for treating HER2/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.
- provided herein is a nucleic acid molecule comprising a first open reading frame encoding a recombinant polypeptide provided herein, wherein the nucleic molecule resides within the recombinant Listeria vaccine strain.
- the nucleic acid molecule provided herein is used to transform the Listeria in order to arrive at a recombinant Listeria.
- the nucleic acid provided herein lacks a virulence gene.
- the 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 can be any gene known in the art to be associated with virulence in the recombinant Listeria.
- a metabolic gene, a virulence gene, etc., provided herein is lacking in a chromosome of the Listeria strain.
- the metabolic gene, virulence gene, etc., provided herein is lacking in the chromosome and in any episomal genetic element of the Listeria strain.
- 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.
- the term refers to plasmid vectors that integrate into the chromosome of the Listeria provided herein.
- the metabolic gene, virulence gene, etc. is lacking in the genome of the virulence strain.
- the virulence gene is mutated in the chromosome.
- the virulence gene is deleted from the chromosome.
- nucleic acids and plasmids provided herein do not confer antibiotic resistance upon a recombinant Listeria provided herein.
- a nucleic acid molecule provided herein comprises a plasmid.
- a nucleic acid molecule provided herein is a plasmid.
- a plasmid provided herein is an integration vector.
- a plasmid is a non-integration vector.
- a plasmid comprises an integration vector.
- an 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.
- the term "metabolic enzyme” may encompass an enzyme involved in synthesis of a nutrient required by the host bacteria.
- the term refers to an enzyme required for synthesis of a nutrient required by the host bacteria.
- the term refers to an enzyme involved in synthesis of a nutrient utilized by the host bacteria.
- the term refers to an enzyme involved in synthesis of a nutrient required for sustained growth of the host bacteria.
- the enzyme is required for synthesis of the nutrient.
- the term "Stably maintained” may encompass maintenance of a nucleic acid molecule or plasmid in a host cell or bacteria 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. In another embodiment, the period is more than 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.
- 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 HER2 chimeric protein or fragment thereof, vaccines and immunogenic compositions comprising same, and methods of inducing an anti-HER2 immune response and treating and vaccinating against a HER2- 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.
- a recombinant polypeptide provided herein comprises a fusion protein provided herein.
- the recombinant polypeptide is a fusion protein.
- a fusion protein provided herein comprises a chimeric HER2 antigen and 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 HER2/neu chimeric antigen.
- the additional polypeptide is functional.
- a fragment of the additional polypeptide is immunogenic.
- the additional polypeptide is immunogenic.
- a fusion protein provided herein comprises a non-hemolytic LLO protein or N-terminal fragment fused to a HER2/neu chimeric antigen provided herein.
- 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.
- a fusion protein of methods and compositions of the present invention comprises a truncated ActA- sequence from a Listeria organism.
- a truncated ActA consists of the first 390 amino acids of the wild type ActA protein as described in US Patent Serial No.
- the truncated ActA is an ActA-NlOO or a modified version thereof (referred to as ActA-NlOO*) in which a PEST motif has been deleted and containing the nonconservative QDNKR substitution as described in US Patent Publication Serial No. 2014/0186387.
- ActA proteins and fragments thereof augment antigen presentation and immunity in a similar fashion to LLO.
- a fusion protein provided herein comprises the HER2/neu antigen and an additional polypeptide.
- the additional polypeptide is a non-hemolytic 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. ActA proteins and fragments thereof augment antigen presentation and immunity in a similar fashion to LLO.
- 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.
- Fusion proteins comprising the HER2/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.
- DNA encoding the antigen provided herein can be produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately.
- the 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction.
- the amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence).
- the antigen is ligated into a plasmid. Each method represents a separate embodiment of the present invention.
- compositions of the present invention have 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 HER2 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 HER2 chimeric protein or fused to a fragment thereof.
- a HER2 chimeric protein of the methods and compositions of the present invention is a human HER2 chimeric protein.
- the HER2 chimeric protein is a mouse HER2 chimeric protein.
- the HER2 chimeric protein is a rat HER2 chimeric protein.
- the HER2 chimeric protein is a primate HER2 chimeric protein.
- the HER2 chimeric protein is a canine HER2 chimeric protein.
- the Her-2 protein is a HER2 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 "HER2/neu,” “Erbb2,” “v-erb-b2,” “c-erb-b2,” “neu,” or “cNeu.”
- HER2/neu is also referred to herein as "Her-2,” “Her-2 protein,” “HER2 protein,” or “HER2”).
- a Her2-neu chimeric protein provided herein harbors two of the extracellular and one intracellular fragments of HER2/neu antigen showing clusters of MHC- class I epitopes of the oncogene, where, in another embodiment, the chimeric protein, harbors 3 H2Dq and at least 17 of the mapped human MHC-class I epitopes of the HER2/neu antigen (fragments ECl, EC2, and IC1) (See Figure 1A). In another embodiment, the chimeric protein harbors at least 13 of the mapped human MHC-class I epitopes (fragments EC2 and IC1).
- the chimeric protein harbors at least 14 of the mapped human MHC-class I epitopes (fragments ECl and IC1). 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).
- 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 listeriolysin O
- the expression and secretion of the fusion protein tLLO-ChHer2 from the attenuated auxotrophic strain provided herein that expresses a chimeric HER2/neu antigen/LLO fusion protein is comparable to that of the Lm-LLO-ChHer2 in TCA precipitated cell culture supernatants after 8 hours of in vitro growth (See Figure IB).
- 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.
- a HER2/neu chimeric protein is encoded by the following nucleic acid sequence set forth in SEQ ID NO:l gagacccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacctgcccacca atgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcaggtcccactgcag aggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgctgaacaataccaccctgtcacaggggcctgtcacaggggcctgtcacaggggcctgtcacaggggcctgtg
- the HER2/neu chimeric protein comprises the sequence of SEQ ID NO: 2:
- 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 HER2/ ECI fragment is set forth in (SEQ ID NO:
- an amino acid sequence encoding a human her2/neu EC2 fragment is set forth in (SEQ ID NO: 71):
- an amino acid sequence encoding a canine her2/neu EC2 fragment is set forth in (SEQ ID NO: 72):
- an amino acid sequence encoding a human her2/neu IC1 fragment is set forth in (SEQ ID NO : 73) :
- an amino acid sequence encoding a canine her2/neu IC1 fragment is set forth in (SEQ ID NO : 74) :
- the human amino acid sequence of HER2 ECl fragment (SEQ ID NO: 69) has 89% identity with that of a canine HER2 ECl fragment (SEQ ID NO: 70).
- the human amino acid sequence of HER2 EC2 fragment (SEQ ID NO: 71) has 93% identity with that of a canine HER2 EC2 fragment (SEQ ID NO: 72).
- the human amino acid sequence of HER2 IC1 fragment (SEQ ID NO: 73) has 98% identity with that of a canine HER2 IC1 fragment (SEQ ID NO: 74).
- 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 HER2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the TM.
- the nucleic acid sequence encoding a rat-HER2/neu gene is comprises SEQ ID NO: 45:
- the nucleic acid sequence encoding a rat/HER2/neu ECl fragment comprises SEQ ID NO: 46:
- nucleic acid sequence encoding the rat HER2/neu EC2 fragment comprises SEQ ID NO: 47:
- nucleic acid sequence encoding the rat HER2/neu ICl fragment comprises SEQ ID NO: 48:
- the nucleic acid sequence of human-HER2/neu gene comprises SEQ ID NO: 49:
- the complete ECl human HER2/neu fragment spans from (58- 979 bp of the human HER2/neu gene) and is encoded by a nucleic acid sequence comprising SEQ ID NO: 54: GCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGC CAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTG GTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCC TGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGA GGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGA CAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCT GTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACA GAGATCTTGAAAGGAGGGAGGG
- the nucleic acid sequence encoding the human HER2/neu EC2 fragment implemented into the chimera spans from 1077-1554 bp of the human HER2/neu EC2 fragment and includes a 50 bp extension, and comprises SEQ ID NO: 51 :
- a complete EC2 human HER2/neu fragment spans from 907- 1504 bp of the human HER2/neu gene and is encoded by a nucleic acid sequence comprising SEQ ID NO: 55):
- CTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAG SEQ ID NO: 55 .
- nucleic acid sequence encoding the human HER2/neu IC1 fragment implemented into the chimera comprises SEQ ID NO: 52:
- nucleic acid sequence encoding the complete human HER2/neu IC1 fragment spans from 2034-3243 of the human HER2/neu gene and comprises 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 a nucleic acid sequence comprising SEQ ID NO: 3: atgaaaaaataatgctagttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaa taaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgtgacaaatgtgccgccaagaaaaggt tacaaagatggaaatgaaaatgaatatattgttgtggagaaaatccatcaatcaaaatgcagacatt
- the LLO protein comprises the sequence SEQ ID NO: 4:
- 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 a 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 non-hemolytic form of the LLO protein.
- a recombinant polypeptide encoded by a nucleic acid sequence of the methods and compositions of the present invention is a fusion protein comprising the chimeric HER2/neu antigen and an additional polypeptide, where in another embodiment, the fusion protein comprises, inter alia, an LLO fragment which in one embodiment is an LM non-hemolytic LLO protein or, in another embodiment, a truncated LLO (Examples herein).
- 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 (AA) sequence comprises, in another embodiment, a sequence selected from SEQ ID NO: 5-9.
- the PEST sequence is a PEST sequence from the Listeria monocytogenes ⁇ Lm) ActA protein.
- a PEST sequence comprises KTEEQPSEVNTGPR (SEQ ID NO: 5), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 6),
- 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 AA 35-51. In another embodiment, the PEST sequence is from Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK (SEQ ID NO: 10) at AA 38-54. In another embodiment, the PEST-like sequence is another PEST AA sequence derived from a prokaryotic organism. In another embodiment, the PEST sequence is any other PEST sequence known in the art. Each possibility represents a separate embodiment of the present invention.
- Fusion of an antigen to a PEST sequence of Lm enhanced cell mediated and antitumor 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 Lm.
- PEST AA sequences from other prokaryotic organisms can also be identified based on this method.
- Other prokaryotic organisms wherein PEST AA sequences would be expected to occur include, but are not limited to, other Listeria species.
- 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 composition comprising a recombinant polypeptide of the present invention.
- a vaccine consisting of a recombinant polypeptide of the present invention.
- a composition consisting of a recombinant polypeptide of the present invention is an immunogenic composition.
- nucleotide molecule encoding a recombinant polypeptide of the present invention.
- a vaccine comprising the nucleotide molecule.
- a composition comprising the nucleotide molecule.
- nucleotide molecule encoding a recombinant polypeptide of the present invention.
- nucleotide molecule of the present invention in another embodiment, provided herein is a recombinant polypeptide encoded by the nucleotide molecule of the present invention.
- 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.
- an immunogenic composition 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 or composition 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.
- the methods of the present invention comprise the step of administering a recombinant Listeria monocytogenes, in any form or embodiment as described herein. In one embodiment, the methods of the present invention consist of the step of administering a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein. In another embodiment, the methods of the present invention consist essentially of the step of administering a recombinant Listeria monocytogenes of the present invention, in any form or embodiment as described herein.
- 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-HER2/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 HER2 chimeric protein or a fragment thereof.
- the present invention provides a method of inducing an anti- HER2 immune response in a subject, comprising administering to the subject a recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to a HER2 chimeric protein or fused to a fragment thereof, thereby inducing an anti-HER2 immune response in a subject.
- 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 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.
- 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). In one embodiment, the signal peptide is native to the bacterium. In another embodiment, the signal peptide is foreign to the bacterium. In another embodiment, the signal peptide is a signal peptide from Listeria monocytogenes, such as a secAl signal peptide. In another embodiment, the signal peptide is a Usp45 signal peptide from Lactococcus lactis, or a Protective Antigen signal peptide from Bacillus anthracis. In another embodiment, the signal peptide is a secA2 signal peptide, such the p60 signal peptide from Listeria monocytogenes.
- 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-HER2 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 HER2 chimeric protein or fused to a fragment thereof, thereby inducing an anti-HER2 immune response in a subject.
- provided herein is a method of eliciting an enhanced immune response to a HER2/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 HER2-expressing tumor comprises an immune response to a subdominant epitope of the
- the immune response against the HER2-expressing tumor comprises an immune response to several subdominant epitopes of the HER2protein. In another embodiment, the immune response against the HER2-expressing tumor comprises an immune response to at least 1-5 subdominant epitopes of the HER2protein. In another embodiment, the immune response against the HER2-expressing tumor comprises an immune response to at least 1-10 subdominant epitopes of the HER2protein. In another embodiment, the immune response against the HER2-expressing tumor comprises an immune response to at least 1-17 subdominant epitopes of the HER2protein. In another embodiment, the immune response against the HER2-expressing tumor comprises an immune response to at least 17 subdominant epitopes of the HER2 protein.
- Point mutations or amino-acid deletions in the oncogenic protein HER2/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 HER2/neu antigen).
- trastuzumab a monoclonal antibody against an epitope located at the extracellular domain of the HER2/neu antigen.
- Described herein is a chimeric HER2/neu based composition which harbors two of the extracellular and one intracellular fragments of HER2/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 HER2/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.
- HER2/neu chimeric Listeria vaccines did not result in any escape mutations that could be associated with point mutations or amino acid deletions in the HER2/neu antigen (see Example 4 herein).
- a method of engineering a Listeria vaccine strain to express a HER2 chimeric protein or recombinant polypeptide expressing the chimeric protein comprising transforming a Listeria strain with a nucleic acid molecule.
- the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a HER2/neu chimeric antigen.
- the nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, and wherein said metabolic enzyme complements an endogenous gene that is lacking in the chromosome of the recombinant Listeria strain, thereby engineering a Listeria vaccine strain to express a HER2 chimeric protein.
- the methods and compositions provided herein further comprise an adjuvant, where in another embodiment, the adjuvant comprises a granulocyte/macrophage colony- stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21 , monophosphoryl lipid A, an unmethylated CpG-containing oligonucleotide or any adjuvant known in the art.
- GM-CSF granulocyte/macrophage colony- stimulating factor
- Attenuated Listeria strains such as LM 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 average skill in the art when equipped with the disclosure herein.
- 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).
- a nucleic acid molecule of the methods and compositions of the present invention is operably linked to a promoter/regulatory sequence.
- the first open reading frame of methods and compositions of the present invention is operably linked to a promoter/regulatory sequence.
- the nucleic acid molecule comprises a second open reading frame operably linked to a promoter/regulatory sequence.
- each of the open reading frames are operably linked to a promoter/regulatory sequence.
- 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
- a commercially available plasmid is used in the present invention.
- 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 to facilitate expression 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.
- 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.
- 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. 18:415-442).
- 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 Bacteriol, 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.
- Ligation of the non-hemolytic LLO and antigen sequences and insertion into a plasmid or vector produces a vector encoding non-hemolytic LLO joined to a terminus of the antigen.
- the two molecules are joined either directly or by a short spacer introduced by the restriction site.
- 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 AA 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.
- auxotroph strains and complementation systems are adopted for the use with this invention.
- the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain described herein.
- 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.
- a method of eliciting an enhanced immune response to a HER2/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 HER2/neu-expressing tumor comprises an immune response to at least one subdominant epitope of the HER2/neu protein.
- provided herein is a method of preventing an escape mutation in the treatment of HER2/neu 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.
- a method of preventing the onset of a HER2/neu antigen-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 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.
- 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 formation of a HER2/neu-expressing tumor in a subject comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
- the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain the provided herein.
- provided herein is a method of treating a Her2/neu- expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
- the method comprises the step of administering to the subject a composition comprising the recombinant Listeria vaccine strain provided herein.
- 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 administering a composition of the present invention.
- provided herein is a method of administering a vaccine of the present invention.
- a method of administering the 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.
- 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.
- 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 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.
- 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 HER2/ 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 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 recombinant polypeptide comprising an N-terminal fragment of a LLO protein fused to the HER2 chimeric protein or a fragment thereof or a recombinant nucleotide encoding the recombinant polypeptide, wherein the subject mounts an immune response against the HER2-expressing tumor, thereby delaying or inhibiting the metastatic disease emanating from a HER2- expressing tumor in a subject.
- the present invention provides a method of improving an antigenicity of a HER2 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 polypeptide, thereby improving an antigenicity of a HER2 chimeric protein.
- a method of improving an antigenicity of a HER2 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-HER2 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 for "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 treating, suppressing, or inhibiting 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 16% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 14% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 12% of the antigen-specific CD8 + T cells.
- the term refers to an epitope recognized by fewer than 10% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 8% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 6% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 5% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by over 4% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by fewer than 3% of the antigen-specific CD8 + T cells.
- 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 less than 1% of the antigen-specific CD8 + T cells. In another embodiment, the term refers to an epitope recognized by less than 0.5% of the antigen-specific CD8 + T cells.
- 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 LM 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 a growth of an HER2-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 HER2 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 a growth of an HER2-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 a growth on a HER2- 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.
- 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, and 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. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is pulmonary squamous adenocarcinoma. In another embodiment, the cancer is gastric adenocarcinoma. In another embodiment, the cancer is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof). In another embodiment, the cancer is an oral squamous cell carcinoma. In another embodiment, the cancer is non-small-cell lung carcinoma. In another embodiment, the cancer is a CNS carcinoma. In another embodiment, the cancer is an endometrial carcinoma. In another embodiment, the cancer is a bladder cancer.
- the cancer is mesothelioma. In another embodiment, the cancer is malignant mesothelioma (MM). In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is a prostate carcinoma. In another embodiment, the cancer is osteosarcoma. In another embodiment, the cancer is a HER2/neu expressing osteosarcoma. In another embodiment, the osteosarcoma is canine osteosarcoma. In another embodiment, the osteosarcoma is localized osteosarcoma. In another embodiment, the osteosarcoma is metastatic osteosarcoma. In another embodiment, the osteosarcoma is high grade osteosarcoma. In another embodiment, the osteosarcoma is canine appendicular osteosarcoma.
- 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 HER2 chimeric protein.
- the immunogenic composition further comprises a Listeria strain expressing the fusion.
- the present invention provides an immunogenic composition for treating cancer, the composition comprising a Listeria strain expressing a HER2 chimeric protein.
- a treatment protocol of the present invention is therapeutic.
- the protocol is prophylactic.
- the vaccines or compositions 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. Following such treatments, the vaccines of the present invention are administered so that the CTL response to a 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 or radiation treatment.
- 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 effect the growth of previously established tumors and to kill existing tumor cells.
- a "tumor antigen or fragment thereof,” “tumor-associated antigen or fragment thereof,” “heterologous antigen or fragment thereof,” or “antigen peptide or fragment thereof are used interchangeably herein and include any antigen known in the art including tumor antigens, angiogenic antigens, or infectious disease antigens.
- the antigen is a self-antigen.
- the antigen provided herein is derived is a tumor-associated antigen, which in one embodiment, is one of the following tumor antigens: a survivin, a MAGE (Melanoma- Associated Antigen E) protein, e.g.
- CEA carcinoembryonic antigen
- the antigen for the compositions and methods as provided herein are melanoma- associated antigens, which in one embodiment are TRP-2, MAGE-1 , MAGE-3, gp-100, tyrosinase, HSP-70, beta-HCG, or a combination thereof.
- Other tumor-associated antigens known in the art are also contemplated in the present invention.
- the antigen or fragment thereof is derived from an antigen selected from a HPV-E7 (from either an HPV16 or HPV18 strain), a HPV-E6 (from either an HPV16 or HPV18 strain), Her-2/neu, NY-ESO-1 , telomerase (TERT, SCCE, CEA, LMP-1, p53, carboxic anhydrase IX (CAIX), PSMA, a prostate stem cell antigen (PSCA), a HMW- MAA, WT-1, HIV-1 Gag, Proteinase 3, Tyrosinase related protein 2, PSA (prostate-specific antigen), EGFR-III, survivin, baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), LMP-1 , p53, PSMA, PSCA, Mucl, PSA (prostate-specific antigen), or a combination thereof.
- a HPV-E7 from either an HPV16 or HPV18 strain
- HPV-E6 from
- compositions and methods of this invention are used for vaccinating against a tumor or a cancer.
- a treatment protocol of the present invention is therapeutic.
- the protocol is prophylactic.
- the vaccines or compositions 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. 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, or conventional chemotherapy.
- 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 effect the growth of previously established tumors and to kill existing tumor cells.
- 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.
- Various embodiments of dosage ranges are contemplated by this invention.
- the dosage in the case of vaccine vectors, is in the range of 0.4 LDso/dose.
- the dosage is from about 0.4-4.9 LDso/dose.
- the dosage is from about 0.5-0.59 LDso/dose.
- the dosage is from about 0.6-0.69 LDso/dose.
- the dosage is from about 0.7-0.79 LDso/dose.
- the dosage is about 0.8 LDso/dose.
- 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.
- the dosage is 4 x 10 8 bacteria/dose. In another embodiment, 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.
- the dosage is 2 x 10 10 bacteria/dose. In another embodiment, 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 10 bacteria/dose. In another embodiment, the dosage is 8 x 10 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.
- the dosage is 8 x 10 11 bacteria/dose. In another embodiment, 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.
- Each possibility represents a separate embodiment of the present invention.
- the recombinant Listeria of methods and compositions of the present invention is, in one embodiment, stably transformed with a construct encoding a HER2 chimeric antigen or an LLO-HER2 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.
- Techniques for homologous recombination are well known in the art, and are described, for example, in Baloglu S, Boyle SM, et al. (Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein. Vet Microbiol 2005, 109(1-2): 11-7); and Jiang LL, Song HH, et al., (Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein.
- the construct or nucleic acid molecule is integrated into the Listerial chromosome using transposon insertion.
- Techniques for transposon insertion are well known in the art, and are described, inter alia, by Sun et al. (Infection and Immunity 1990, 58: 3770-3778) in the construction of DP-L967.
- Transposon mutagenesis has the advantage, in another embodiment, that a stable genomic insertion mutant can be formed but the disadvantage that the position in the genome where the foreign gene has been inserted is unknown.
- the construct or nucleic acid molecule is integrated into the Listeria! 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 Bacteriol 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.
- an Lm 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.
- Lm promoter 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 HER2 chimeric protein or LLO sequence of the present invention.
- the methods and compositions of the present invention utilize a HER2 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 65% 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 75% 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 85% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 90% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 95% homology with a nucleic acid encoding the signal peptide or the recombinant polypeptide or the fusion protein of the present invention.
- 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., 2001 , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y).
- methods of hybridization may be carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide.
- Hybridization conditions being, for example, overnight incubation at 42 °C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 ⁇ 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 monomeric 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 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 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.
- 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.
- the production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed.
- Each nucleic acid derivative represents a separate embodiment of the present invention.
- 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.
- the terms "contacting" or “administering,” in one embodiment, refer to directly contacting the cancer cell or tumor with a composition of the present invention. In another embodiment, the terms refer to indirectly contacting the cancer cell or tumor with a composition of the present invention.
- 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. Each possibility represents a separate embodiment of the present invention.
- the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention.
- Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
- Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals or organisms. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals or organisms. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
- 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 gelatin 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.
- the term "treating" refers to curing a disease.
- 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 pet dogs and cats, including dogs and cats 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. In another embodiment, the term “subject” also encompasses a human child.
- 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 antitumor 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.
- 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).
- HER2/neu HLA- A2 peptides were synthesized by EZbiolabs (Westfield, IN).
- C-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-HER2/neu antibody was purchased from Sigma.
- HER2/neu-pGEM7Z was kindly provided by Dr. Mark Greene at the University of Pennsylvania and contained the full-length human HER2/neu (hHer2) gene cloned into the pGEM7Z plasmid (Promega, Madison WI). This plasmid was used as a template to amplify three segments of hHER2/neu, namely, ECl, EC2, and IC1, by PCR using pfx DNA polymerase (Invitrogen) and the oligos indicated in Table 1. [00244] Table 1 : Primers for cloning of Human HER2-Chimera
- HER2/neu chimera construct was generated by direct fusion by the SOEing PCR method and each separate hHER2/neu segment as templates. Primers are shown in Table 2. [00246] Sequence of primers for amplification of different segments human Her2 regions
- 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).
- Interferon- ⁇ secretion by splenocytes from immunized mice Groups of 3-5 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 1 ⁇ of HLA- A2 specific peptides or 1 ⁇ g/ml of a recombinant His-tagged ChHer2 protein, produced in E. coli and purified by a nickel based affinity chromatography system. Samples from supernatants were obtained 24 or 72 hours later and tested for the presence of interferon- ⁇ (IFN- ⁇ ) using mouse IFN- ⁇ Enzyme- linked immunosorbent assay (ELIS A) kit according to manufacturer' s recommendations.
- IFN- ⁇ interferon- ⁇
- ELIS A mouse IFN- ⁇ Enzyme- linked immunosorbent assay
- 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 IC1 peptide pools plus IL-2 or IL-2 alone (to determine background counts). All assays were performed in duplicates. Plates were developed according to the manufacturer's instructions. Spots were counted using a CTL- Immunospot analyzer (C.T.L, Shaker Heights, OH). Number of spots were normalized by subtracting twice the number of spots counted in non-stimulated wells.
- CTL- Immunospot analyzer C.T.L, Shaker Heights, OH
- ADXS31-164 Effect of ADXS31-164 on regulatory T cells in spleens and tumors
- 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, LmddA-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 HER2/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 (dal) 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.
- EXAMPLE 2 ADXS31-164 IS AS IMMUNOGENIC AS LM-LLO-ChHER2.
- ADXS31-164 was also able to stimulate the secretion of IFN-y 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 HER2/neu antigen ( Figure 5C). [00257] Proper processing and presentation of the human MHC class I epitopes after immunizations with ADXS31-164 was tested in HLA-A2 mice.
- Splenocytes from immunized HLA-A2 transgenics were co-incubated for 72 hours with peptides corresponding to mapped HLA-A2 restricted epitopes located at the extracellular (HLYQGCQVV SEQ ID NO: 11 or KIFGSLAFL SEQ ID NO: 12) or intracellular (RLLQETELV SEQ ID NO: 13) domains of the HER2/neu molecule ( Figure 2C).
- HLYQGCQVV SEQ ID NO: 11 or KIFGSLAFL SEQ ID NO: 12 extracellular domains of the HER2/neu molecule
- RLLQETELV SEQ ID NO: 13 intracellular domains of the HER2/neu molecule
- EXAMPLE 3 ADXS31-164 WAS MORE EFFICACIOUS THAN LM-LLO-ChHER2 IN PREVENTING THE ONSET OF SPONTANEOUS MAMMARY TUMORS.
- ADXS31-164 Anti-tumor effects of ADXS31-164 were compared to those of Lm-LLO-ChHer2 in HER2/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, Zisen ' a-HER2/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.
- EXAMPLE 5 ADXS31-164 CAUSES A SIGNIFICANT DECREASE IN
- 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.
- 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 HER2/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, LmddA 164 and irrelevant vaccine Lm-LLO-NY were harvested.
- the DNA was purified from these samples and the DNA fragments corresponding to HER2/neu regions IC 1 , EC 1 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 detailed analysis of these sequences is shown below.
- Lm-LLO-NY-1 GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT
- Lm-LLO-NY-1 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT
- Lm-LLO-NY-2 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT
- Lm-LLO- 138-2 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGA ACGAGGACTT Lm-LLO-138-3 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT Lm-LLO- 138-4 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGA ACGAGGACTT Lm-ddA- 164- 1 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGA ACGAGGACTT Lm-ddA- 164-2 CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGA ACGAGGACTT
- Lm-LLO- 138-2 CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT Lm-ddA- 164-1
- EXAMPLE 7 PERIPHERAL IMMUNIZATION WITH ADXS31-164 CAN DELAY THE GROWTH OF A METASTATIC BREAST CANCER CELL LINE IN THE
- 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 luciferase and low levels of HER2/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 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.
- All dogs are to receive 4 weeks of carboplatin therapy. Four weeks after the last carboplatin dose, dogs are to receive ADXS-HER2 once every three weeks for a total of 3 doses. Group 1 (3 dogs) receive lxlO 8 CFU per dose, Group 2 (3 dogs) each receive 5xl0 8 CFU per dose and 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. Therefore 9-18 dogs may be treated in the initial study.
- EXAMPLE 9 PHASE 1 DOSE ESCALATION STUDY EVALUATING THE SAFETY OF ADXS-CHER2 IN COMPANION DOGS WITH HER2/NEU
- Group 1 (3 dogs) received the ADXS31-164 (Lm-human chimericHER2/neu) vaccine at 1x10 CFU per dose, Group 2 (3 dogs) each received 5x10 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 are administered as a slow intravenous infusion over 30 minutes.
- the dose chosen for Group 1 is the established safe dose for the chimeric ADXS31-164 recombinant in mice. In humans, the non-toxic dose for Lovaxin C 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 HER2/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). Results
- 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.
- One hour prior to vaccination patients receive 2 mg/kg diphenhydramine via intramuscular injection and 0.2mg/kg ondansetron as a slow intravenous push.
- the vaccine was kept at - 80°C and thawed patient-side. It was administered in 200mls of 0.9% NaCl over 30 mins.
- the infusion line is then flushed with 30 mis of Plasmalyte. Dogs are sent home with a three day course of amoxicillin (to start 72 hours post vaccination) and a 7 day course of liver supplement (S-adenosyl-methionine) that aids in cellular growth and repair.
- HER2/neu staining 5 micron thick serial sections of formalin fixed, decalcified, paraffin embedded tissues were mounted on negatively charged glass slides. Sections were heated at 80°C for 20 minutes, immersed in Pro Par (clearant) and rehydrated in ethanol. Antigen retrieval was performed by boiling sections in sodium citrate buffer (pH -9.0). Endogenous peroxidase was blocked using 3% hydrogen peroxide. Staining was performed with a rabbit anti-human HER2/neu antibody (Neu(c-18):sc-284, Santa Cruz Biotecnology) or a rabbit IgG isotype (Universal Negative Control serum, NC498, Biocare Medical).
- Bound antibody was detected using the Universal Streptavadin-Biotin2 System (DAKO/LSAB2, HRP). Tissues were stained with 3,3'-diaminobenzidine solution (DAKO) and counterstained with hematoxylin. Slides were viewed using a Nikon E600 infinity corrected upright microscope. Bright field images were acquired using a Nikon Digital Sight DS-Fil color camera and a NIS-Element BR3.0 for image analysis.
- CBC Complete Blood Count
- CS Chemistry Screen
- U Urinalysis
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Priority Applications (26)
Application Number | Priority Date | Filing Date | Title |
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EP15755609.3A EP3110942A4 (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors |
MX2016011114A MX2016011114A (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors. |
BR112016019534A BR112016019534A2 (en) | 2014-02-25 | 2015-02-25 | methods of treating tumor growth and eliciting an enhanced immune response against tumor growth |
NZ723750A NZ723750B2 (en) | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors | |
CA2940646A CA2940646A1 (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors |
US15/121,421 US20160361401A1 (en) | 2009-11-11 | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors |
CN201580010568.4A CN106661538A (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of HER2/NEU over-expressing tumors |
JP2016553897A JP2017507943A (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of HER2 / NEU overexpressing tumors |
RU2016137834A RU2016137834A (en) | 2014-02-25 | 2015-02-25 | COMPOSITIONS AND METHODS FOR TREATING TUMORS SUPERPRESSING HER2 / neu |
KR1020167025517A KR20160122829A (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors |
AU2015223136A AU2015223136A1 (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of HER2/Neu over-expressing tumors |
SG11201607036XA SG11201607036XA (en) | 2014-02-25 | 2015-02-25 | Compositions and methods for the treatment of her2/neu over-expressing tumors |
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Cited By (12)
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EP3113794A4 (en) * | 2014-03-05 | 2017-09-06 | Advaxis, Inc. | Methods and compositions for increasing a t-effector cell to regulatory t cell ratio |
US10055540B2 (en) | 2015-12-16 | 2018-08-21 | Gritstone Oncology, Inc. | Neoantigen identification, manufacture, and use |
US10058599B2 (en) | 2012-03-12 | 2018-08-28 | Advaxis, Inc. | Suppressor cell function inhibition following Listeria vaccine treatment |
US10064898B2 (en) | 2011-03-11 | 2018-09-04 | Advaxis, Inc. | Listeria-based adjuvants |
US10143734B2 (en) | 2014-02-18 | 2018-12-04 | Advaxis, Inc. | Biomarker directed multi-target immunotherapy |
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US11446369B2 (en) | 2007-05-10 | 2022-09-20 | Advaxis, Inc. | Compositions and methods comprising KLK3 or FOLH1 antigen |
US11885815B2 (en) | 2017-11-22 | 2024-01-30 | Gritstone Bio, Inc. | Reducing junction epitope presentation for neoantigens |
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Family Cites Families (2)
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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 |
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 |
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US11897927B2 (en) | 2016-11-30 | 2024-02-13 | Advaxis, Inc. | Immunogenic compositions targeting recurrent cancer mutations and methods of use thereof |
US11179339B2 (en) | 2017-09-19 | 2021-11-23 | Advaxis, Inc. | Compositions and methods for lyophilization of bacteria or listeria strains |
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Also Published As
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AU2015223136A1 (en) | 2016-09-22 |
CA2940646A1 (en) | 2015-09-03 |
IL247436A0 (en) | 2016-11-30 |
KR20240038103A (en) | 2024-03-22 |
WO2015130810A3 (en) | 2016-01-28 |
NZ723750A (en) | 2024-02-23 |
BR112016019534A2 (en) | 2017-10-24 |
SG11201607036XA (en) | 2016-09-29 |
MX2016011114A (en) | 2017-02-20 |
KR20160122829A (en) | 2016-10-24 |
EP3110942A4 (en) | 2017-08-30 |
RU2016137834A (en) | 2018-03-29 |
EP3110942A2 (en) | 2017-01-04 |
JP2017507943A (en) | 2017-03-23 |
CN106661538A (en) | 2017-05-10 |
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