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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
  • A61K39/02—Bacterial antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0208—Specific bacteria not otherwise provided for
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention provides methods of treating cervical cancers, comprising the step of administering to a subject a composition comprising a recombinant Listeria expressing a human papilloma virus antigen.
• the present invention provides a method of treating cervical cancers, comprising the step of administering a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain expressing a human papilloma virus antigen.
• Listeria monocytogenes ⁇ Lm is a food-borne gram-positive bacterium that can occasionally cause disease in humans, in particular elderly individuals, newborns, pregnant women and immunocompromised individuals.
• Lm has the ability to replicate in the cytosol of APCs after escaping from the phagolysosome, mainly through the action of the listeriolysin O (LLO) protein.
• LLO listeriolysin O
• Lm has been extensively investigated as a vector for cancer immunotherapy in pre-clinical models. Immunization of mice with JW-LLO-E7 induces regression of established tumors expressing E7 and confers long-term protection. The therapeutic efficacy of Lm-LLO- ⁇ correlates with its ability to induce E7-specific CTLs that infiltrate the tumor site, mature dendritic cells, reduce the number of intratumoral regulatory CD4 + CD25 + T cells and inhibit tumor angiogenesis.
• Lm has also a number of inherent advantages as an immunotherapy vector.
• the bacterium grows very efficiently in vitro without special requirements and it lacks LPS, which is a major toxicity factor in gram-negative bacteria, such as Salmonella.
• Genetically attenuated Lm vectors also offer additional safety as they can be readily eliminated with antibiotics, in case of serious adverse effects and unlike some viral vectors, no integration of genetic material into the host genome occurs.
• Persistent infection with high-oncogenic risk human papillomavirus (HR-HPV) types is recognized as a necessary, but not sufficient, cause of invasive carcinoma of the cervix (ICC).
• HPVs 16 and 18 are the most prevalent types in malignant lesions, accounting for over 70% of ICC and over 50% of high-grade precursor lesions.
• the HR- HPV E6 and E7 proteins are consistently expressed in dysplasias and carcinomas, disrupting the cell cycle regulatory proteins p53 and pRb, respectively.
• Cervical cancer is one of the most common cancers in women worldwide. But in the United States and other countries where cervical cancer screening is routine, this cancer is not so common. Most cervical cancer is caused by a virus called human papillomavirus, or HPV. You can get HPV by having sexual contact with someone who has it. There are many types of the HPV virus. Not all types of HPV cause cervical cancer. Some of them cause genital warts, but other types may not cause any symptoms. Most adults have been infected with HPV at some time. An infection may go away on its own. But sometimes it can cause genital warts or lead to cervical cancer.
• HPV human papillomavirus
• the present invention addresses this need by providing an attenuated live Listeria vaccine vector for treating cervical cancer.
• the present invention further provides a combination therapy comprising an attenuated live Listeria for treating cervical cancer.
• the present invention relates to a method of treating a persistent/ recurrent metastatic (squamous or non-squamous) cervical cancer (PRmCC) in a human subject, the method comprising the step of administering to said subject a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to an HPV-E7 antigen or a fragment thereof.
• PRmCC persistent/ recurrent metastatic (squamous or non-squamous) cervical cancer
• the present invention relates to a method of treating a cervical cancer in a human subject, the method comprising the step of administering to said subject a combination therapy comprising chemo-radiation and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to an HPV-E7 antigen or a fragment thereof, wherein said Listeria strain is administered at an initial dose of 5xl0 9 colony-forming units (CFU), and wherein a subsequent dose of Listeria strain is administered to said patient every 3 weeks, thereby treating said cervical cancer in said human subject.
• a combination therapy comprising chemo-radiation and a recombinant Listeria strain
• said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a re
• the present invention relates to a method of eliciting an anti- tumor cytotoxic T cell response against a PRmCC in a human subject comprising administering to said subject a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to an HPV-E7 antigen or a fragment thereof.
• the present invention relates to a method of eliciting an antitumor cytotoxic T cell response in a human subject comprising administering to said subject a combination therapy comprising a Listeria strain disclosed herein and chemo- radiation therapy.
• Lm-E7 and Lm-LLO-E7 use different expression systems to express and secrete E7.
• Lm-E7 was generated by introducing a gene cassette into the orfZ domain of the L. monocytogenes genome (A). The hly promoter drives expression of the hly signal sequence and the first five amino acids (AA) of LLO followed by FIPV-16 E7.
• A Lm- LLO-E7 was generated by transforming the prfA- strain XFL-7 with the plasmid pGG-55.
• pGG-55 has the hly promoter driving expression of a nonhemolytic fusion of LLO-E7.
• pGG-55 also contains the prfA gene to select for retention of the plasmid by XFL-7 in vivo.
• Lm-E7 and Lm-LLO-E7 secrete E7.
• Lm-Gag (lane 1), Lm-E7 (lane 2), Lm-LLO- P (lane 3), Lm-LLO-E7 (lane 4), XFL-7 (lane 5), and 10403S (lane 6) were grown overnight at 37°C in Luria-Bertoni broth. Equivalent numbers of bacteria, as determined by OD at 600 nm absorbance, were pelleted and 18 ml of each supernatant was TCA precipitated. E7 expression was analyzed by Western blot. The blot was probed with an anti-E7 mAb, followed by HRP -conjugated anti-mouse (Amersham), then developed using ECL detection reagents.
• FIG. 3 Tumor immunotherapeutic efficacy of LLO-E7 fusions. Tumor size in millimeters in mice is shown at 7, 14, 21, 28 and 56 days post tumor-inoculation. Naive mice: open-circles; Lm-LLO-E7: filled circles; Lm-E7: squares; Lm-Gag: open diamonds; and Lm-LLO-NP: filled triangles.
• FIG. 4 Splenocytes from Lm-LLO-E7-immunized mice proliferate when exposed to TC-1 cells.
• C57BL/6 mice were immunized and boosted with Lm-LLO-E7, Lm-E7, or control rLm strains.
• Splenocytes were harvested 6 days after the boost and plated with irradiated TC-1 cells at the ratios shown. The cells were pulsed with 3 ⁇ 4 thymidine and harvested.
• Cpm is defined as (experimental cpm) - (no-TC-1 control).
• FIG. 1 A. Induction of E7-specific IFN-gamma-secreting CD8 + T cells in the spleens and the numbers penetrating the tumors, in mice administered TC-1 tumor cells and subsequently administered Lm-E7, Lm-LLO-E7, Lm-ActA-E7, or no vaccine (naive).
• Figure 7 A Effect of passaging on bacterial load (virulence) of recombinant Listeria vaccine vectors. Top panel. Lm-Gag. Bottom panel. Lm-LLO-E7.
• Figure 7B Effect of passaging on bacterial load of recombinant Lm-E7 in the spleen. Average CFU of live bacteria per milliliter of spleen homogenate from four mice is depicted.
• Figure 8 shows induction of antigen-specific CD8 + T-cells for HIV-Gag and LLO after administration of passaged Lm-Gag versus unpassaged Lm-Gag.
• Mice were immunized with 10 3 (A, B, E, F) or 10 5 (C, D, G, H) CFU passaged Listeria vaccine vectors, and antigen-specific T-cells were analyzed.
• B, D, F, H unpassaged Listeria vaccine vectors.
• E-H immune response to an LLO peptide.
• FIG. 9A shows plasmid isolation throughout LB stability study.
• Figure 10B shows plasmid isolation throughout TB stability study.
• Figure IOC shows quantitation of TB stability study.
• Figure 10 shows numbers of viable bacteria chloramphenicol (CAP)-resistant and CAP-sensitive colony -forming units (CFU) from bacteria grown in LB. Dark bars: CAP + ; white bars: CAP " . The two dark bars and two white bars for each time point represent duplicate samples.
• CAP viable bacteria chloramphenicol
• CFU colony -forming units
• Figure 11 shows numbers of viable bacteria CAP -resistant and CAP-sensitive CFU from bacteria grown in TB. Dark bars: CAP + ; white bars: CAP. The two dark bars and two white bars for each time point represent duplicate samples. [0026] Figure 12. Actual chromatograms showing the region of the D133V mutation (arrows). The mixture ratio is shown in parentheses.
• Figure 13 Representation of the location of the ADV451, 452 and 453 primers and the segment of the prfA gene amplified in the reaction.
• Figure 14 Specificity of the PCR reaction using primers ADV451 and ADV453.
• Figure 15 Specificity of the PCR reaction using primers ADV452 and ADV453.
• Figure 16 Sensitivity of the PCR reaction to detect the wild-type prfA sequence using the primer ADV452 and 1 ng as the initial amount of DNA.
• Figure 17. Sensitivity of the PCR reaction to detect the wild-type prfA sequence using the primer ADV452 and 5 ng as the initial amount of DNA.
• Figure 18 Average density of the bands from the PCR depicted in figure 16.
• Figure 19 Average density of the bands from the PCR depicted in figure 17.
• Figure 20 Validation of the PCR reaction to detect the wild-type prfA sequence using the primer ADV452.
• Figure 21 Average density of the bands from the PCR depicted in figure 16.
• FIG 22 Analysis of the D133V prfA mutation in the JW-LLO-E7.
• A Original image used for densitometry;
• B Image was digitally enhanced to facilitate the visualization of the low density bands.
• Figure 24 The progression free survival (PFS) following initial treatment with ADXS11-001 was measured and demonstrates a median PFS of 3.1 months.
• Figure 25 Tumor shrinkage and response in 19 patients.
• FIG. 26 Exploratory analysis of overall survival (OS) in patients receiving per protocol treatment (3 doses of ADXS 11-001). Among the 26 patients treated, 18 completed the full per-protocol therapy (3 doses of axalimogene filolisbac over 3 months), and experienced a median overall survival exceeding one year (12.1 months) and a 12- month overall survival rate of 55.6 percent.
• OS overall survival
• Figure 27 Twelve-month survival was achieved irrespective of extent of prior therapy (1-3 lines).
• Figure 28 Depicts the clinical study schema, endpoints, and key eligibility criteria for the clinical trial described in Example 12.
• Figure 29 Depicts a CONSORT diagram for the clinical trial described in Example 12.
• Figure 30 Depicts 12-month survival for the clinical trial described in Example 12 compared to historical COG trial series in PRmCC.
• FIGS 31 A and 3 IB A) The 6-month survival rate is 42% (10/24) and median OS is 4.8 months (95% CI: 3.6-NR); median PFS is 2.6 months (95% CI: 2.0-3.2); B) Among the 50% of patients (12/24) who received 3 or more doses of ADXSl 1-001, median OS is NR (95% CI: 3.5-NR) with median follow-up of 9.2 months, and 6-month survival rate is 67%.
• Figure 32 Depicts timeline of clinical trial in a 66 year-old woman diagnosed with squamous cell cancer of the cervix, and subsequently surgically treated with radical hysterectomy. After pelvic recurrence 7 years post-hysterectomy, the patient received paclitaxel/carboplatin x 8 cycles (6 cycles with bevacizumab) ⁇ cisplatin (2 cycles) + pelvic radiation. Ten months later, there was systemic recurrence and the patient was enrolled in the clinical trial described in Example 12.
• Figure 33 Shows scans depicting durable complete response (CR).
• a method of treating a persistent/ recurrent metastatic (squamous or non-squamous) cervical cancer (PRmCC) in a human subject comprising the step of administering to said subject a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to an HPV-E7 antigen or a fragment thereof.
• a method of treating a cervical cancer in a human subject comprising the step of administering to said subject a combination therapy comprising chemo-radiation and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to an HPV-E7 antigen or a fragment thereof, wherein said Listeria strain is administered at an initial dose of 5xl0 9 colony-forming units (CFU), and wherein a subsequent dose of Listeria strain is administered to said patient every 3 weeks, thereby treating said cervical cancer in said human subject.
• a combination therapy comprising chemo-radiation and a recombinant Listeria strain
• said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an
• said method comprises administering to said subject at least one dose of chemo-radiation therapy prior to the administration of said recombinant Listeria strain. In another embodiment, said subject receives no more than 2 doses of chemo-radiation therapy prior to administering said recombinant Listeria strain.
• a method of eliciting an anti-tumor cytotoxic T cell response against a PRmCC in a human subject comprising administering to said subject a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to an HPV-E7 antigen or a fragment thereof.
• a method of eliciting an anti -tumor cytotoxic T cell response in a human subject comprising administering to said subject a recombinant Listeria strain at an initial dose of 5xl0 9 colony-forming units, and wherein a subsequent dose of Listeria strain is administered to said patient every 3 weeks, thereby eliciting an anti-tumor cytotoxic T cell response.
• said tumor is an HPV-E7- or HPV-E6- expressing tumor.
• a method of eliciting an anti-tumor cytotoxic T cell response in a human subject comprising administering to said subject a combination therapy comprising a Listeria strain disclosed herein and chemo-radiation therapy.
• kits for treating, protecting against, and inducing an immune response against a disease comprising the step of administering to a subject a recombinant Listeria strain, expressing a fusion peptide comprising a listeriolysin O (LLO) fragment and a heterologous antigen expressed by said disease or fragment thereof.
• LLO listeriolysin O
• the present invention also provides methods for inducing an anti- disease cytotoxic T-cell (CTL) response in a human subject and treating disorders, and symptoms associated with said disease comprising administration of the recombinant Listeria strain.
• CTL cytotoxic T-cell
• a recombinant Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising a first an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, and wherein said recombinant nucleic acid further comprises a second open reading frame encoding a mutant prfA gene.
• the mutant prfA gene is one that encodes a point mutation from amino acid D or Asp or Aspartate (or Aspartic acid) to amino acid V or Val or Valine at the 133 rd amino acid position.
• the recombinant Listeria is an attenuated Listeria.
• "Attenuation" and “attenuated” may encompass a bacterium, virus, parasite, infectious organism, prion, tumor cell, gene in the infectious organism, and the like, that is modified to reduce toxicity to a host.
• the host can be a human or animal host, or an organ, tissue, or cell.
• the bacterium to give a non-limiting example, can be attenuated to reduce binding to a host cell, to reduce spread from one host cell to another host cell, to reduce extracellular growth, or to reduce intracellular growth in a host cell.
• Attenuation can be assessed by measuring, e.g., an indicum or indicia of toxicity, the LD 50 , the rate of clearance from an organ, or the competitive index (see, e.g., Auerbuch, et al. (2001) Infect. Immunity 69:5953-5957).
• an attenuation results an increase in the LD 50 and/or an increase in the rate of clearance by at least 25%; more generally by at least 50%; most generally by at least 100% (2 -fold); normally by at least 5-fold; more normally by at least 10-fold; most normally by at least 50-fold; often by at least 100-fold; more often by at least 500- fold; and most often by at least 1000-fold; usually by at least 5000-fold; more usually by at least 10,000-fold; and most usually by at least 50,000-fold; and most often by at least 100,000-fold.
• Attenuated gene may encompass a gene that mediates toxicity, pathology, or virulence, to a host, growth within the host, or survival within the host, where the gene is mutated in a way that mitigates, reduces, or eliminates the toxicity, pathology, or virulence. The reduction or elimination can be assessed by comparing the virulence or toxicity mediated by the mutated gene with that mediated by the non -mutated (or parent) gene.
• “Mutated gene” encompasses deletions, point mutations, and frameshift mutations in regulatory regions of the gene, coding regions of the gene, non-coding regions of the gene, or any combination thereof.
• a method of treating an anal tumor or anal cancer in a human subject comprising the step of administering to said subject a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, thereby treating said anal tumor or anal cancer in said human subject.
• the recombinant Listeria expresses a fusion protein of N- terminal LLO and a heterologous antigen.
• the heterologous antigen is human papilloma virus E7 antigen (HPV-E7).
• HPV antigen is HPVE6.
• patients receive ADXS11-001 every 3 weeks during a 12- week treatment cycle.
• dose escalation is performed using the 3+3 design in 2 doses: 5xl0 9 colony-forming units (CFU; Dose Level 1) and lxlO 10 CFU (Dose Level 2).
• the recommended phase II dose are selected based on an observed dose-limiting toxicity (DLT) rate of ⁇ 33%.
• patients receive lxlO 9 colony-forming units (CFU), and a then receive subsequent dose every 1 month thereafter.
• patients receive lxlO 9 colony-forming units (CFU), and a then receive subsequent dose every 1 month thereafter for 3 months.
• efficacy is assessed using any method known in the art, which includes but is not limited to RECIST vl . l and immune-related RECIST.
• ADXS11-001 and “ADXS-HPV” are used interchangeably herein and refer to a Listeria monocytogenes comprising a nucleic acid encoding a truncated LLO fused to an HPV-E7 antigen.
• chemo-radiation regiment or chemo- radiation therapy for use in combination with the recombinant Listeria provided herein.
• the chemo-radiation therapy provided herein comprises mitomycin and fluorouracil (5-FU) and radiation therapy.
• the chemo- radiation therapy provided herein comprises cisplatin and radiation therapy.
• the chemo-radiation therapy comprises any other chemotherapeutic agents known in the art, including but not limited to, Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan, Nitrosoureas, Temozolomide, mitomycin, fluorouracil, Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, or Tioguanine (formerly Thioguanine).
• a chemo-radiation therapy provided herein comprises administering 2 courses of cisplatin with concurrent radiation (54 Gy in 30 fractions by intensity modulated radiation therapy).
• the regiment or therapy comprises administering 2-4 courses, 4-6 courses, 6-8 courses, 8-10 courses, 10-12 courses, 12-14 courses, or 14-16 courses of cisplatin or a suitable agent, with concurrent radiation.
• the radiation comprises 20-30 Gy, 30-40 Gy, 40-50 Gy, 50-60 Gy, 60-70 Gy, 70-80 Gy, 80-90 Gy, or 90-100 Gy.
• the radiation is provided in 10-20 fractions, 20-30 fractions, 30-40 fractions, 40-50 fractions, 50-60 fractions, 60-70 fractions, 70-80 fractions, 80-90 fractions, or 90-100 fractions. It will be understood by a skilled artisan that a clinician may adjust doses and schedules being administered to a subject as needed throughout a particular therapy disclosed herein.
• the subject receives a median of 1.5 (range 0-5) lines of systemic chemotherapy prior to the administration of said recombinant Listeria strain.
• a chemo-radiation therapy disclosed herein is administered prior to a first administration of said recombinant Listeria strain.
• said chemo-radiation therapy is administered following the administration of said recombinant Listeria strain.
• said chemo-radiation therapy is administered following a first administration of said recombinant Listeria strain and prior to one to three booster administrations of said recombinant Listeria strain.
• said chemo-radiation therapy is administered concurrently with said recombinant Listeria strain.
• the chemo-radiation regiment or chemo-radiation therapy disclosed herein comprises administering 2 courses of cisplatin with concurrent radiation (54 Gy in 30 fractions by intensity modulated radiation therapy).
• the radiation provided herein lasts about 6 weeks. In another embodiment, the radiation lasts 3 weeks. In another embodiment, the radiation lasts 4 weeks. In another embodiment, the radiation lasts, 5 weeks. In another embodiment, the radiation lasts 7 weeks. In another embodiment, the radiation lasts 8 weeks. In another embodiment, the radiation lasts 6-8 weeks. In another embodiment, the radiation lasts 4-6 weeks. In another embodiment, the radiation lasts 2-4 weeks. In another embodiment, the radiation lasts 8-10 weeks.
• a method of eliciting an anti-tumor cytotoxic T cell response in a human subject comprising administering to said subject a combination therapy disclosed herein.
• a method for inducing an immune response against a tumor or a cancer in a human subject comprising the step of administering to said subject a recombinant Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, wherein said recombinant nucleic acid further comprises a second open reading frame encoding a mutant prfA gene, thereby inducing an immune response against a tumor or a cancer.
• a method of treating a cancer in a human subject comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• the present invention provides a method of protecting a human subject against a cervical cancer, comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• the method further comprises the step of boosting the human subject with a recombinant Listeria strain of the present invention.
• the method further comprises the step of boosting the human subject with an immunogenic composition comprising a heterologous antigen or fragment thereof disclosed herein. In another embodiment, the method further comprises the step of boosting the human subject with an immunogenic composition that directs a cell of the subject to express the heterologous antigen. In another embodiment, the cell is a tumor cell. In another embodiment, the method further comprises the step of boosting the human subject with a vaccine or composition disclosed herein.
• the fragment thereof in the context of LLO proteins and ActA proteins disclosed herein refer to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues of the LLO or ActA proteins.
• the term refers to a peptide or polypeptide comprising an amino acid sequence of at least of at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues
• the fragment is a functional fragment that works as intended by the present invention (e.g. to elicit an immune response against a disease- associated antigen when in the form of an N-terminal LLO/heterologous antigen fusion protein or N-terminal ActA/heterologous antigen fusion protein).
• the fragment is functional in a non-fused form.
• the present invention provides codon optimization of a nucleic acid heterologous to Listeria, or of a nucleic acid endogenous to Listeria.
• the optimal codons utilized by L. monocytogenes for each amino acid are shown US Patent Publication 2007/0207170, which is hereby incorporated by reference herein.
• a nucleic acid is codon-optimized if at least one codon in the nucleic acid is replaced with a codon that is more frequently used by L. monocytogenes for that amino acid than the codon in the original sequence.
• the N-terminal LLO protein fragment and heterologous antigen are, in another embodiment, fused directly to one another.
• the genes encoding the N-terminal LLO protein fragment and the heterologous antigen are fused directly to one another.
• the N-terminal LLO protein fragment and the heterologous antigen are attached via a linker peptide.
• the N- terminal LLO protein fragment and the heterologous antigen are attached via a heterologous peptide.
• the N-terminal LLO protein fragment is N- terminal to the heterologous antigen.
• the N-terminal LLO protein fragment is the N-terminal-most portion of the fusion protein.
• vaccines of the present invention are efficacious at inducing immune responses against E7 and E6.
• the present invention provides a method of treating a cervical cancer in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, whereby the recombinant Listeria strain induces an immune response against the E7 antigen, thereby treating a cervical cancer in a human subject.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• a method of protecting a human subject against a cervical cancer comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, whereby the recombinant Listeria strain induces an immune response against the E7 antigen, thereby protecting a human subject against a cervical cancer.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• a method for inducing an immune response against a cervical cancer in a human subject comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, thereby inducing an immune response against a cervical cancer in a human subject.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• a method of treating a cervical cancer in a human subject comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an ActA protein and heterologous antigen, whereby the recombinant Listeria strain induces an immune response against the heterologous antigen, thereby treating a cervical cancer in a human subject.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• a method of protecting a human subject against a cervical cancer comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an ActA protein and a heterologous antigen, whereby the recombinant Listeria strain induces an immune response against the heterologous antigen, thereby protecting a human subject against a cervical cancer.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• a method for inducing an immune response against a cervical cancer in a human subject comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an heterologous protein and a heterologous antigen, thereby inducing an immune response against a cervical cancer in a human subject.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• the N-terminal ActA protein fragment and the heterologous antigen are, in another embodiment, fused directly to one another.
• the genes encoding the N-terminal ActA protein fragment and heterologous antigen are fused directly to one another.
• the N-terminal ActA protein fragment and heterologous antigen are attached via a linker peptide.
• the N- terminal ActA protein fragment and heterologous antigen are attached via a heterologous peptide.
• the N-terminal ActA protein fragment is N-terminal to the heterologous antigen.
• the N-terminal ActA protein fragment is the N-terminal-most portion of the fusion protein.
• a method of inducing an immune response against a cervical cancer in a human subject comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising a PEST amino acid sequence- containing peptide and a heterologous antigen, whereby the recombinant Listeria strain induces an immune response against the heterologous antigen, thereby treating a cervical cancer in a human subject.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• the method protects a human subject against a cervical. In another embodiment, the method treats a cervical cancer in said human subject.
• the PEST amino acid amino acid sequence-containing peptide and heterologous antigen are, in another embodiment, fused directly to one another.
• the genes encoding the PEST amino acid sequence-containing peptide and heterologous antigen are fused directly to one another.
• the PEST amino acid sequence-containing peptide and heterologous antigen are attached via a linker peptide.
• the PEST amino acid sequence-containing peptide and heterologous antigen are attached via a heterologous peptide.
• the PEST amino acid sequence-containing peptide is N-terminal to the heterologous antigen.
• the PEST amino acid sequence-containing peptide is the N-terminal-most portion of the fusion protein.
• a method for vaccinating a human subject against an HPV comprising the step of administering to the subject the recombinant Listeria strain provided herein, wherein the Listeria expresses an HPV E7 antigen and wherein the Listeria expresses a mutant prfA gene.
• the mutant prfA gene is a D133V prfA mutation.
• the mutant prfA gene is in a plasmid in said recombinant Listeria.
• the recombinant Listeria strain expresses the recombinant polypeptide.
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• the subject is at risk for developing an HPV-mediated carcinogenesis (e.g. a cervical cancer).
• the subject is HPV- positive.
• the subject exhibits cervical intraepithelial neoplasia.
• the subject exhibits a squamous intraepithelial lesion.
• the subject exhibits a dysplasia in the cervix.
• the HPV that is the target of methods of the present invention is, in another embodiment, an HPV 16.
• the HPV is an HPV-18.
• the HPV is selected from HPV-16 and HPV-18.
• the HPV is an HPV-31.
• the HPV is an HPV-35.
• the HPV is an HPV-39.
• the HPV is an HPV-45.
• the HPV is an HPV-51.
• the HPV is an HPV-52.
• the HPV is an HPV-58.
• the HPV is a high-risk HPV type.
• the HPV is a mucosal HPV type.
• a method of vaccinating a human subject against an antigen of interest comprising the step of administering intravenously to the human subject a recombinant Listeria strain comprising or expressing the antigen of interest, wherein the first peptide is selected from (a) an N-terminal fragment of an LLO protein; (b) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against an antigen of interest.
• a method of vaccinating a human subject against an antigen of interest comprising the step of administering intravenously to the human subject an immunogenic composition, comprising a fusion of a first peptide to the antigen of interest, wherein the first peptide is selected from (a) an N- terminal fragment of an LLO protein; and (b) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against an antigen of interest.
• a method of vaccinating a human subject against an antigen of interest comprising the step of administering intravenously to the human subject a recombinant Listeria strain comprising a recombinant polypeptide, the recombinant polypeptide comprising a first peptide fused to the antigen of interest, wherein the first peptide is selected from (a) an N-terminal fragment of an LLO protein; and (b) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against an antigen of interest.
• a method of inducing a CTL response in a human subject against an antigen of interest comprising the step of administering to the human subject a recombinant Listeria strain comprising or expressing the antigen of interest, thereby inducing a CTL response in a human subject against an antigen of interest.
• the step of administering is intravenous administration.
• a method for inducing a regression of a cancer in a subject comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• a method for suppressing a formation of a tumor in a subject comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• a method for inducing a remission of a cancer in a subject comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• a method for impeding a growth of a tumor in a human subject comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• a method for reducing a size of a tumor in a subject comprising the step of administering to the subject the recombinant Listeria strain disclosed herein.
• the disease is an infectious disease, an autoimmune disease, a respiratory disease, a pre-cancerous condition or a cancer.
• pre-cancerous condition may encompass dysplasias, preneoplastic nodules; macroregenerative nodules (MRN); low-grade dysplastic nodules (LG-DN); high-grade dysplastic nodules (HG-DN); biliary epithelial dysplasia; foci of altered hepatocytes (FAH); nodules of altered hepatocytes (NAH); chromosomal imbalances; aberrant activation of telomerase; re- expression of the catalytic subunit of telomerase; expression of endothelial cell markers such as CD31, CD34, and B H9 (see, e.g., Terracciano and Tomillo (2003) Pathologica 95:71-82; Su and Bannasch (2003) Toxicol.
• Pathol. 31 126-133; Rocken and Carl- McGrath (2001) Dig. Dis. 19:269-278; Kotoula, et al. (2002) Liver 22:57-69; Frachon, et al. (2001) J. Hepatol. 34:850-857; Shimonishi, et al. (2000) J. Hepatobiliary Pancreat. Surg. 7:542-550; Nakanuma, et al. (2003) J. Hepatobiliary Pancreat. Surg. 10:265-281).
• Methods for diagnosing cancer and dysplasia are disclosed (see, e.g., Riegler (1996) Semin. Gastrointest. Dis.
• an infectious disease is one caused by, but not limited to, any one of the following pathogens: BCG/Tuberculosis, Malaria, Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Rotavirus, Cholera, Diptheria-Tetanus, Pertussis, Haemophilus influenzae, Hepatitis B, Human papilloma virus, Influenza seasonal), Influenza A (H1N1) Pandemic, Measles and Rubella, Mumps, Meningococcus A+C, Oral Polio Vaccines, mono, bi and trivalent, Pneumococcal, Rabies, Tetanus Toxoid, Yellow Fever, Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox) and other related pox viruses, Francisella tularens
• the infectious disease is a livestock infectious disease.
• livestock diseases can be transmitted to man and are called "zoonotic diseases.”
• these diseases include, but are not limited to, Foot and mouth disease, West Nile Virus, rabies, canine parvovirus, feline leukemia virus, equine influenza virus, infectious bovine rhinotracheitis (IBR), pseudorabies, classical swine fever (CSF), IBR, caused by bovine herpesvirus type 1 (BHV-1) infection of cattle, and pseudorabies (Aujeszky's disease) in pigs, toxoplasmosis, anthrax, vesicular stomatitis virus, rhodococcus equi, Tularemia, Plague (Yersinia pestis), trichomonas.
• BHV-1 bovine herpesvirus type 1
• the disease provided herein is a cancer or a tumor.
• the tumor is cancerous.
• the cancer is breast cancer.
• the cancer is a cervical 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, it is a glioblastoma multiforme.
• 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 an endometrial carcinoma. In another embodiment, the cancer is a bladder cancer. In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is a prostate carcinoma.
• ovarian surface epithelial neoplasm e.g. a benign, proliferative or malignant variety thereof.
• the cancer is an oral squamous cell carcinoma.
• the cancer is non-small-cell lung carcinoma.
• the cancer is an endometrial carcinoma
• the cancer is oropharyngeal cancer. In another embodiment, the cancer is lung cancer. In another embodiment, the cancer is anal cancer. In another embodiment, the cancer is colorectal cancer. In another embodiment, the cancer is esophageal cancer.
• the cervical tumor targeted by methods of the present invention is, in another embodiment, a squamous cell carcinoma. In another embodiment, the cervical tumor is an adenocarcinoma. In another embodiment, the cervical tumor is an adenosquamous carcinoma. In another embodiment, the cervical tumor is a small cell carcinoma. In another embodiment, the cervical tumor is any other type of cervical tumor known in the art.
• the antigen provided herein is a heterologous tumor antigen, which is also referred to herein as "tumor antigen” "antigenic polypeptide,” or “foreign antigen.”
• the antigen is Human Papilloma Virus-E7 (HPV-E7) antigen, which in one embodiment, is from HPV16 (in one embodiment, GenBank Accession No. AAD33253) and in another embodiment, from HPV18 (in one embodiment, GenBank Accession No. P06788).
• the antigenic polypeptide is HPV-E6, which in one embodiment, is from HPV16 (in one embodiment, GenBank Accession No.
• the antigenic polypeptide is a Her/2-neu antigen.
• the antigenic polypeptide is Prostate Specific Antigen (PSA) (in one embodiment, GenBank Accession No. CAD30844, CAD54617, AAA58802, or NP_ 001639).
• PSA Prostate Specific Antigen
• the antigenic polypeptide is Stratum Corneum Chymotryptic Enzyme (SCCE) antigen (in one embodiment, GenBank Accession No. AAK69652, AAK69624, AAG33360, AAF01139, or AAC37551).
• the antigenic polypeptide is Wilms tumor antigen 1, which in another embodiment is WT-1 Telomerase (GenBank Accession. No. P49952, P22561, P_ 659032, CAC39220.2, or EAW68222.1).
• the antigenic polypeptide is hTERT or Telomerase (GenBank Accession. No. NM003219 (variant 1), NM198255 (variant 2), NM 198253 (variant 3), or NM 198254 (variant 4).
• the antigenic polypeptide is Proteinase 3 (in one embodiment, GenBank Accession No. M29142, M75154, M96839, X55668, NM 00277, M96628 or X56606).
• the antigenic polypeptide is Tyrosinase Related Protein 2 (TRP2) (in one embodiment, GenBank Accession No. NP-001913, AB 173976, AAP33051, or Q95119).
• TRP2 Tyrosinase Related Protein 2
• the antigenic polypeptide is High Molecular Weight Melanoma Associated Antigen (HMW-MAA) (in one embodiment, GenBank Accession No. NP-001888, AAI28111, or AAQ62842).
• the antigenic polypeptide is Testisin (in one embodiment, GenBank Accession No. AAF79020, AAF79019, AAG02255, AAK29360, AAD41588, or NP-659206).
• the antigenic polypeptide is NY-ESO-1 antigen (in one embodiment, GenBank Accession No. CAA05908, P78358, AAB49693, or NP-640343).
• the antigenic polypeptide is PSCA (in one embodiment, GenBank Accession No. AAH65183, NP-005663, NP-082492, 043653, or CAB97347).
• the antigenic polypeptide is Interleukin (IL) 13 Receptor alpha (in one embodiment, GenBank Accession No. NP-000631, NP-001551, NP-032382, NP_ 598751, NP_001003075, or NP-999506).
• the antigenic polypeptide is Carbonic anhydrase IX (CAIX) (in one embodiment, GenBank Accession No. CAI13455, CAI10985, EAW58359, NP-001207, NP-647466, or NP_001101426).
• the antigenic polypeptide is carcinoembryonic antigen (CEA) (in one embodiment, GenBank Accession No. AAA66186, CAA79884, CAA66955, AAA51966, AAD 15250, or AAA51970.).
• the antigenic polypeptide is MAGE-A (in one embodiment, GenBank Accession No.
• the antigenic polypeptide is survivin (in one embodiment, GenBank Accession No. AAC51660, AAY15202, ABF60110, NP-001003019, or NP_001082350).
• the antigenic polypeptide is GP100 (in one embodiment, GenBank Accession No. AAC60634, YP-655861, or AAB31176).
• the antigenic polypeptide is any other antigenic polypeptide known in the art.
• the antigenic peptide of the compositions and methods of the present invention comprise an immunogenic portion of the antigenic polypeptide.
• the antigen is HPV-E6. In another embodiment, the antigen is telomerase (TERT). In another embodiment, the antigen is LMP-1. In another embodiment, the antigen is p53. In another embodiment, the antigen is mesothelin. In another embodiment, the antigen is EGFRVIII. In another embodiment, the antigen is carboxic anhydrase IX (CAIX). In another embodiment, the antigen is PSMA. In another embodiment, the antigen is HMW-MAA. In another embodiment, the antigen is HIV-1 Gag. In another embodiment, the antigen is Tyrosinase related protein 2.
• the antigen is selected from HPV-E7, HPV-E6, Her-2, HIV-1 Gag, LMP-1, p53, PSMA, carcinoembryonic antigen (CEA), LMP-1, kallikrein-related peptidase 3 (KLK3), KLK9, Muc, Tyrosinase related protein 2, Mucl, FAP, IL-13R alpha 2, PSA (prostate-specific antigen), gp-100, heat-shock protein 70 (HSP-70), beta-HCG, EGFR- III, Granulocyte colony-stimulating factor (G-CSF), Angiogenin, Angiopoietin-1, Del-1, Fibroblast growth factors: acidic (aFGF) or basic (bFGF), Follistatin, Granulocyte colony - stimulating factor (G-CSF), Hepatocyte growth factor (HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine, Placental growth
• the antigen is a chimeric Her2/neu antigen as disclosed in US Patent Application Publication No. 2011/0142791, which is incorporated by reference herein in its entirety.
• the use of fragments of antigens disclosed herein is also encompassed by the present invention.
• the heterologous tumor antigen provided herein is a tumor-associated antigen, which in one embodiment, is one of the following tumor antigens: a MAGE (Melanoma- Associated Antigen E) protein, e.g.
• the antigen for the compositions and methods 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. It is to be understood that a skilled artisan would be able to use any heterologous antigen not mentioned herein but known in the art for use in the methods and compositions provided herein. It is also to be understood that the present invention provides, but is not limited by, an attenuated Listeria comprising a nucleic acid that encodes at least one of the antigens disclosed herein.
• the present invention encompasses nucleic acids encoding mutants, muteins, splice variants, fragments, truncated variants, soluble variants, extracellular domains, intracellular domains, mature sequences, and the like, of the disclosed antigens.
• nucleic acids encoding epitopes, oligo- and polypeptides of these antigens.
• codon optimized embodiments that is, optimized for expression in Listeria.
• the selected nucleic acid sequence can encode a full length or a truncated gene, a fusion or tagged gene, and can be a cDNA, a genomic DNA, or a DNA fragment, preferably, a cDNA. It can be mutated or otherwise modified as desired. These modifications include codon optimizations to optimize codon usage in the selected host cell or bacteria, i.e. Listeria.
• the selected sequence can also encode a secreted, cytoplasmic, nuclear, membrane bound or cell surface polypeptide.
• vascular endothelial growth factor is an important signaling protein involved in both vasculogenesis (the formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).
• VEGF activity is restricted mainly to cells of the vascular endothelium, although it does have effects on a limited number of other cell types (e.g. stimulation monocyte/macrophage migration).
• VEGF has been shown to stimulate endothelial cell mitogenesis and cell migration.
• VEGF also enhances microvascular permeability and is sometimes referred to as vascular permeability factor.
• all of the members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation.
• the VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region and an intracellular portion containing a split tyrosine-kinase domain.
• VEGF-A is a VEGFR-2 (KDR/Flk-1) ligand as well as a VEGFR-1 (Fit- 1 ) ligand.
• VEGFR- mediates almost all of the known cellular responses to VEGF.
• the function of VEGFR-l is less well defined, although it is thought to modulate VEGFR-2 signaling, in one embodiment, via sequestration of VEGF from VEGFR-2 binding, which in one embodiment, is particularly important during vasculogenesis in the embryo.
• VEGF-C and VEGF-D are ligands of the VEGFR-3 receptor, which in one embodiment, mediates lymphangiogenesis.
• compositions of the present invention comprise a VEGF receptor or a fragment thereof, which in one embodiment, is a VEGFR-2 and, in another embodiment, a VEGFR-l, and, in another embodiment, VEGFR-3.
• vascular Endothelial Growth Factor Receptor 2 is highly expressed on activated endothelial cells (ECs) and participates in the formation of new blood vessels.
• VEGFR2 binds all 5 isoforms of VEGF.
• signaling of VEGF through VEGFR2 on ECs induces proliferation, migration, and eventual differentiation.
• the mouse homologue of VEGFR2 is the fetal liver kinase gene-1 (Flk-1), which is a strong therapeutic target, and has important roles in tumor growth, invasion, and metastasis.
• VEGFR2 is also referred to as kinase insert domain receptor (a type III receptor tyrosine kinase) (KDR), cluster of differentiation 309 (CD309), FLK1, Ly73, Krd-1, VEGFR, VEGFR-2, or 6130401C07.
• KDR kinase insert domain receptor
• CD309 cluster of differentiation 309
• FLK1, Ly73 FLK1, Ly73
• Krd-1 kinase insert domain receptor
• VEGFR VEGFR-2
• 6130401C7 6130401C7
• the antigen is derived from a fungal pathogen, bacteria, parasite, helminth, or viruses.
• the antigen is selected from tetanus toxoid, hemagglutinin molecules from influenza virus, diphtheria toxoid, HTV gpl20, HIV gag protein, IgA protease, insulin peptide B, Spongospora subterranea antigen, vibriose antigens, Salmonella antigens, pneumococcus antigens, respiratory syncytial virus antigens, Haemophilus influenza outer membrane proteins, Helicobacter pylori urease, Neisseria meningitidis pilins, N.
• gonorrhoeae pilins the melanoma-associated antigens (TRP-2, MAGE-1, MAGE-3, gp-100, tyrosinase, MART-1, HSP-70, beta-HCG), human papilloma virus antigens El and E2 from type HPV-16, -18, -31, -33, -35 or -45 human papilloma viruses, the tumor antigens CEA, the ras protein, mutated or otherwise, the p53 protein, mutated or otherwise, Mucl, or pSA.
• the antigen is associated with one of the following diseases; cholera, diphtheria, Haemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid, Varicella-zoster, whooping cough3 yellow fever, the immunogens and antigens from Addison's disease, allergies, anaphylaxis, Bruton's syndrome, cancer, including solid and blood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, type 1 diabetes mellitus, acquired immune deficiency syndrome, transplant rejection, such as kidney, heart, pancreas, lung, bone, and liver transplants, Graves' disease, polyendoc
• an HPV E6 antigen is utilized instead of or in addition to an E7 antigen in a method of the present invention for treating, protecting against, or inducing an immune response against a cervical cancer.
• an ActA protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating, protecting against, or inducing an immune response against a cervical cancer.
• a PEST amino acid sequence-containing protein fragment is utilized instead of or in addition to an LLO fragment in a method of the present invention for treating, protecting against, or inducing an immune response against a cervical cancer.
• the present invention provides a method for inducing an anti-E7 cytotoxic T cell (CTL) response in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, thereby inducing an anti-E7 CTL response in a human subject.
• CTL cytotoxic T cell
• the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.
• the method further comprises the step of boosting the subject with a recombinant Listeria strain of the present invention.
• the method further comprises the step of boosting the subject with an immunogenic composition comprising an E7 antigen.
• the method further comprises the step of boosting the subject with an immunogenic composition that directs a cell of the subject to express an E7 antigen.
• the CTL response is capable of therapeutic efficacy against an HPV- mediated disease, disorder, or symptom.
• the CTL response is capable of prophylactic efficacy against an HPV-mediated disease, disorder, or symptom.
• the present invention provides a method of treating or ameliorating an HPV-mediated disease, disorder, or symptom in a subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, whereby the recombinant Listeria strain induces an immune response against the E7 antigen, thereby treating or ameliorating an HPV- mediated disease, disorder, or symptom in a subject.
• the subject is a human subject.
• the subject is a non-human mammal.
• the subject is any other type of subject known in the art.
• the HPV causing the disease, disorder, or symptom is, in another embodiment, an HPV 16.
• the HPV is an HPV-18.
• the HPV is an HPV-31.
• the HPV is an HPV-35.
• the HPV is an HPV-39.
• the HPV is an HPV-45.
• the HPV is an HPV-51.
• the HPV is an HPV-52.
• the HPV is an HPV-58.
• the HPV is a high-risk HPV type.
• the HPV is a mucosal HPV type.
• the HPV-mediated disease, disorder, or symptom is genital warts. In another embodiment, the HPV-mediated disease, disorder, or symptom is non-genital warts. In another embodiment, the HPV-mediated disease, disorder, or symptom is a respiratory papilloma. In another embodiment, the HPV-mediated disease, disorder, or symptom is any other HPV-mediated disease, disorder, or symptom known in the art.
• an HPV E6 antigen is utilized instead of or in addition to an E7 antigen in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.
• an ActA protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.
• a PEST amino acid sequence-containing protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.
• an HPV E6 antigen is utilized instead of or in addition to an E7 antigen in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.
• the antigen of methods and compositions disclosed herein is, in another embodiment, an HPV E7 protein.
• the antigen is an HPV E6 protein.
• the antigen is any other HPV protein known in the art.
• E7 antigen refers, in another embodiment, to an E7 protein. In another embodiment, the term refers to an E7 fragment. In another embodiment, the term refers to an E7 peptide. In another embodiment, the term refers to any other type of E7 antigen known in the art.
• the E7 protein of methods and compositions disclosed herein is, in another embodiment, an HPV 16 E7 protein.
• the E7 protein is an HPV-18 E7 protein.
• the E7 protein is an HPV-31 E7 protein.
• the E7 protein is an HPV-35 E7 protein.
• the E7 protein is an HPV-39 E7 protein.
• the E7 protein is an HPV-45 E7 protein.
• the E7 protein is an HPV-51 E7 protein.
• the E7 protein is an HPV-52 E7 protein.
• the E7 protein is an HPV-58 E7 protein.
• the E7 protein is an E7 protein of a high-risk HPV type.
• the E7 protein is an E7 protein of a mucosal HPV type.
• E6 antigen refers, in another embodiment, to an E6 protein. In another embodiment, the term refers to an E6 fragment. In another embodiment, the term refers to an E6 peptide. In another embodiment, the term refers to any other type of E6 antigen known in the art.
• the E6 protein of methods and compositions disclosed herein is, in another embodiment, an HPV 16 E6 protein.
• the E6 protein is an HPV-18 E6 protein.
• the E6 protein is an HPV-31 E6 protein.
• the E6 protein is an HPV-35 E6 protein.
• the E6 protein is an HPV-39 E6 protein.
• the E6 protein is an HPV-45 E6 protein.
• the E6 protein is an HPV-51 E6 protein.
• the E6 protein is an HPV-52 E6 protein.
• the E6 protein is an HPV-58 E6 protein.
• the E6 protein is an E6 protein of a high-risk HPV type.
• the E6 protein is an E6 protein of a mucosal HPV type.
• the immune response induced by methods and compositions of the present invention is, in another embodiment, a T cell response.
• the immune response comprises a T cell response.
• the response is a CD8 + T cell response.
• the response comprises a CD8 + T cell response.
• the N-terminal LLO protein fragment of methods and compositions of the present invention comprises, in another embodiment, SEQ ID No: 2.
• the fragment comprises an LLO signal peptide.
• the fragment comprises SEQ ID No: 2.
• the fragment consists approximately of SEQ ID No: 2.
• the fragment consists essentially of SEQ ID No: 2.
• the fragment corresponds to SEQ ID No: 2.
• the fragment is homologous to SEQ ID No: 2.
• the fragment is homologous to a fragment of SEQ ID No: 2.
• ALLO used in some of the Examples was 416 AA long (exclusive of the signal sequence), as 88 residues from the amino terminus which is inclusive of the activation domain containing cysteine 484 were truncated. It will be clear to those skilled in the art that any ALLO without the activation domain, and in particular without cysteine 484, are suitable for methods and compositions of the present invention.
• fusion of an E7 or E6 antigen to any ALLO including the PEST amino acid AA sequence, SEQ ID NO: 6, enhances cell mediated and anti-tumor immunity of the antigen.
• the LLO protein utilized to construct vaccines of the present invention has, in another embodiment, the sequence: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHA
• the full length active LLO protein is 504 residues long.
• the above LLO fragment is used as the source of the LLO fragment incorporated in a vaccine of the present invention.
• N-terminal fragment of an LLO protein utilized in compositions and methods of the present invention has the sequence:
• the LLO fragment corresponds to about AA 20-442 of an LLO protein utilized herein.
• the LLO fragment has the sequence:
• truncated LLO or "ALLO” refers to a fragment of LLO that comprises the PEST amino acid domain.
• the terms refer to an LLO fragment that comprises a PEST sequence.
• the terms refer to an LLO fragment that does not contain the activation domain at the amino terminus and does not include cysteine 484. In another embodiment, the terms refer to an LLO fragment that is not hemolytic. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of the activation domain. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of cysteine 484. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation at another location.
• the LLO fragment consists of about the first 441 AA of the LLO protein. In another embodiment, the LLO fragment consists of about the first 420 AA of LLO. In another embodiment, the LLO fragment is a non-hemolytic form of the LLO protein.
• the LLO fragment contains residues of a homologous LLO protein that correspond to one of the above AA ranges.
• the residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g. if the homologous LLO protein has an insertion or deletion, relative to an LLO protein utilized herein, then the residue numbers can be adjusted accordingly.
• the LLO fragment is any other LLO fragment known in the art.
• the dose is 5-500 x 10 8 CFU. In another embodiment, the dose is 7-500 x 10 8 CFU. In another embodiment, the dose is 10-500 x 10 8 CFU. In another embodiment, the dose is 20-500 x 10 8 CFU. In another embodiment, the dose is 30-500 x 10 8 CFU. In another embodiment, the dose is 50-500 x 10 8 CFU. In another embodiment, the dose is 70-500 x 10 8 CFU. In another embodiment, the dose is 100-500 x 10 8 CFU. In another embodiment, the dose is 150-500 x 10 8 CFU. In another embodiment, the dose is 5-300 x 10 8 CFU. In another embodiment, the dose is 5-200 x 10 8 CFU.
• the dose is 5-150 x 10 8 CFU. In another embodiment, the dose is 5- 100 x 10 8 CFU. In another embodiment, the dose is 5-70 x 10 8 CFU. In another embodiment, the dose is 5-50 x 10 8 CFU. In another embodiment, the dose is 5-30 x 10 8 CFU. In another embodiment, the dose is 5-20 x 10 8 CFU. In another embodiment, the dose is 1-30 x 10 9 CFU. In another embodiment, the dose is 1-20 x 10 9 CFU. In another embodiment, the dose is 2-30 x 10 9 CFU. In another embodiment, the dose is 1-10 x 10 9 CFU. In another embodiment, the dose is 2-10 x 10 9 CFU. In another embodiment, the dose is 3-10 x 10 9 CFU. In another embodiment, the dose is 2-7 x 10 9 CFU. In another embodiment, the dose is 2-5 x 10 9 CFU. In another embodiment, the dose is 3-5 x 10 9 CFU.
• the dose is 1 x 10 9 organisms. In another embodiment, the dose is 1.5 x 10 9 organisms. In another embodiment, the dose is 2 x 10 9 organisms. In another embodiment, the dose is 3 x 10 9 organisms. In another embodiment, the dose is 4 x 10 9 organisms. In another embodiment, the dose is 5 x 10 9 organisms. In another embodiment, the dose is 6 x 10 9 organisms. In another embodiment, the dose is 7 x 10 9 organisms. In another embodiment, the dose is 8 x 10 9 organisms. In another embodiment, the dose is 10 x 10 9 organisms. In another embodiment, the dose is 1.5 x 10 10 organisms. In another embodiment, the dose is 2 x 10 10 organisms.
• the dose is 2.5 x 10 10 organisms. In another embodiment, the dose is 3 x 10 10 organisms. In another embodiment, the dose is 3.3 x 10 10 organisms. In another embodiment, the dose is 4 x 10 10 organisms. In another embodiment, the dose is 5 x 10 10 organisms.
• the recombinant Listeria strain is administered to the human subject at a dose of 1 x 10 9 - 3.31 x 10 10 CFU.
• dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed, as determined by those skilled in the art.
• the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks (Q3W). In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks for a 12-week cycle. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 5 x 10 8 CFU - 1 x 10 10 CFU every 3 weeks for a 12-week cycle.
• the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks for a 24-week cycle. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks for a 48-week cycle. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks until at least 50% of tumor regression is achieved.
• the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 5 x 10 8 CFU - 1 x 10 10 CFU every 3 weeks until at least 50% of tumor regression is achieved. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks until at least 70%-80% of tumor regression is achieved. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks until at least 80-90% of tumor regression is achieved.
• the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks until complete tumor regression is achieved. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 5 x 10 8 CFU - 1 x 10 10 CFU every 3 weeks until complete tumor regression is achieved. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 5 x 10 8 CFU - 1 x 10 10 CFU every 3 weeks for three doses or repeated every three weeks until the patients experiences confirmed disease progression or complete response.
• the recombinant Listeria strain is administered at an initial dose of 5 x 10 9 CFU, followed by subsequent doses of 1 x 10 10 CFU every 3 weeks for three doses or repeated every 3 weeks until the patients experiences confirmed disease progression or complete response.
• the Listeria strain disclosed herein is administered to a subject as an 80 ml infusion over a 15 min period.
• the Listeria strain disclosed herein is administered as a 10-20 ml, 20-30 ml, 30-40 ml, 40-50 ml, 50-60 ml, 70-80 ml, 80-90 ml, 90-100 ml, 100-150 ml, 150-200 ml, 200-250 ml, or 250-300 ml infusion.
• the Listeria strain disclosed herein is administered as a 80 ml - 250 ml infusion.
• the Listeria strain disclosed herein is administered over a 5-10 min period, over a 10-20 min period, over a 20-30 min period, over a 30-40 min period, over a 40-50 min period, or over a 50-60 min period. It will be appreciated by a skilled artisan that the larger the infusion volume the longer the administration period will last.
• a treatment cycle begins with the first day of combination treatment and lasts for at least 12 weeks, 24 weeks or 48 weeks. In other embodiments, a treatment cycle begins with the first day of administration of combination treatment and lasts for at least 12 weeks, 24 weeks or 48 weeks. In other embodiments, a treatment cycle begins with the first day of administration of a chemo-radiation treatment and lasts for at least 12 weeks, 24 weeks or 48 weeks. In other embodiments, a treatment cycle begins with the first day of administration of a recombinant Listeria disclosed herein or a composition comprising the same and lasts for at least 12 weeks, 24 weeks or 48 weeks.
• a treatment cycle disclosed herein is followed by single maintenance or booster doses at intervals.
• a booster dose is administered Q2W, Q4W.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 2 months.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 3 months.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 2 months for a year period.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 3 months for year period.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 2 months for at least a year period.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 3 months for at least a year period.
• a booster dose is administered every 3 weeks for a total of 9 dosages (3 week x 3 cycle) followed by a single booster dose every 2 or 3 months for a 1 year to 3 year period.
• a booster dose is administered monthly for a total or 3 doses after an initial administration. In another embodiment, a booster dose is administered monthly for a total after an initial administration, for a total of 4-10 dosages. In another embodiment, a booster dose is administered monthly for a total after an initial administration, for a total of 10-20 dosages.
• a booster dose is administered every 3 or 4 weeks after an initial administration, for a total of 3 doses. In another embodiment, a booster dose is administered every 3 or 4 weeks for a total after an initial administration, for a total of 4- 10 dosages. In another embodiment, a booster dose is administered every 3 or 4 weeks for a total after an initial administration, for a total of 10-20 dosages.
• dosing is continued every 3-4 weeks until disease progression or complete response.
• the recombinant polypeptide of methods of the present invention is expressed by the recombinant Listeria strain.
• the expression is mediated by a nucleotide molecule carried by the recombinant Listeria strain.
• the recombinant Listeria strain expresses the recombinant polypeptide by means of a plasmid that encodes the recombinant polypeptide.
• the plasmid comprises a gene encoding a bacterial transcription factor.
• the plasmid encodes a Listeria transcription factor.
• the transcription factor is prfA.
• the prfA is a mutant prfA.
• the prfA in said plasmid within said Listeria encodes a D133V amino acid mutation.
• the transcription factor is any other transcription factor known in the art.
• the plasmid comprises a gene encoding a metabolic enzyme.
• the metabolic enzyme is a bacterial metabolic enzyme.
• the metabolic enzyme is a Listeria! metabolic enzyme.
• the metabolic enzyme is an amino acid metabolism enzyme.
• the amino acid metabolism gene is involved in a cell wall synthesis pathway.
• the metabolic enzyme is the product of a D-amino acid aminotransferase gene (dat).
• the metabolic enzyme is the product of an alanine racemase gene (dal).
• the metabolic enzyme is any other metabolic enzyme known in the art.
• a method of present invention further comprises the step of boosting the human subject with a recombinant Listeria strain of the present invention.
• the recombinant Listeria strain used in the booster inoculation is the same as the strain used in the initial "priming" inoculation.
• the booster strain is different from the priming strain.
• the same doses are used in the priming and boosting inoculations.
• a larger dose is used in the booster.
• a smaller dose is used in the booster.
• a method of present invention further comprises the step of inoculating the human subject with an immunogenic composition comprising the E7 antigen.
• the immunogenic composition comprises a recombinant E7 protein or fragment thereof.
• the immunogenic composition comprises a nucleotide molecule expressing a recombinant E7 protein or fragment thereof.
• the non-Listerial inoculation is administered after the Listerial inoculation. In another embodiment, the non-Listerial inoculation is administered before the Listerial inoculation.
• Boosting refers, in another embodiment, to administration of an additional vaccine dose to a subject.
• 2 boosts or a total of 3 inoculations
• 3 boosts are administered.
• 4 boosts are administered.
• 5 boosts are administered.
• 6 boosts are administered.
• more than 6 boosts are administered.
• the recombinant Listeria strain of methods and compositions of the present invention is, in another embodiment, a recombinant Listeria monocytogenes strain.
• the Listeria strain is a recombinant Listeria seeligeri strain.
• the Listeria strain is a recombinant Listeria grayi strain.
• the Listeria strain is a recombinant Listeria ivanovii strain.
• the Listeria strain is a recombinant Listeria murrayi strain.
• the Listeria strain is a recombinant Listeria welshimeri strain.
• the Listeria strain is a recombinant strain of any other Listeria species known in the art.
• the present invention provides a number of listerial species and strains for making or engineering an attenuated Listeria of the present invention.
• the Listeria strain is L. monocytogenes 10403S wild type (see Bishop and Hinrichs (1987) J. Immunol. 139: 2005-2009; Lauer, et al. (2002) J. Bact. 184: 4177-4186.)
• the Listeria strain is L. monocytogenes DP-L4056 (phage cured) (see Lauer, et al. (2002) J. Bact. 184: 4177-4186).
• the Listeria strain is L.
• the Listeria strain is L. monocytogenes DP-L4029, which is phage cured, deleted in ActA (see Lauer, et al. (2002) J. Bact. 184: 4177-4186; Skoble, et al. (2000) J. Cell Biol. 150: 527-538).
• the Listeria strain is L.
• the Listeria strain is L. monocytogenes DP -L4097 (LLO-S44A) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101 : 13832-13837; supporting information).
• the Listeria strain is L. monocytogenes DP -L4364 (delta lplA; lipoate protein ligase) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci.
• the Listeria strain is L. monocytogenes DP -L4405 (delta inlA) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101 : 13832-13837; supporting information).
• the Listeria strain is L. monocytogenes DP-L4406 (delta inlB) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101 : 13832-13837; supporting information).
• the Listeria strain is L.
• the Listeria strain is L. monocytogenes CS-L0002 (delta ActA-delta lplA) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101 : 13832-13837; supporting information).
• the Listeria strain is L. monocytogenes CS-L0003 (L461T-delta lplA) (see Brockstedt, et al. (2004) Proc. Natl.
• the Listeria strain is L. monocytogenes DP-L4038 (delta ActA-LLO L461T) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101 : 13832-13837; supporting information).
• the Listeria strain is L. monocytogenes DP-L4384 (S44A-LLO L461T) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101 : 13832-13837; supporting information).
• the Listeria strain is L. monocytogenes.
• the Listeria strain is L. monocytogenes DP-L4017 (10403S hly (L461T), having a point mutation in hemolysin gene (see U.S. Provisional Pat. Appl. Ser. No. 60/490,089 filed Jul. 24, 2003).
• the Listeria strain is L. monocytogenes EGD (see GenBank Acc. No. AL591824).
• the Listeria strain is L. monocytogenes EGD-e (see GenBank Acc. No. NC_003210. ATCC Acc. No. BAA-679).
• the Listeria strain is L. monocytogenes DP -L4029 deleted in uvrAB (see U.S. Provisional Pat. Appl. Ser. No. 60/541,515 filed Feb. 2, 2004; US Provisional Pat. Appl. Ser. No. 60/490,080 filed Jul. 24, 2003).
• the Listeria strain is L. monocytogenes ActA-/inlB - double mutant (see ATCC Acc. No. PTA-5562).
• the Listeria strain is L. monocytogenes lplA mutant or hly mutant (see U.S. Pat. Applic. No. 20040013690 of Portnoy, et. al).
• the Listeria strain is L.
• the present invention encompasses reagents and methods that comprise the above listerial strains, as well as these strains that are modified, e.g., by a plasmid and/or by genomic integration, to contain a nucleic acid encoding one of, or any combination of, the following genes: hly (LLO; listeriolysin); iap (p60); inlA; inlB; inlC; dal (alanine racemase); dat (D-amino acid aminotransferase); plcA; plcB; actA; or any nucleic acid that mediates growth, spread, breakdown of a single walled vesicle, breakdown of a double walled vesicle, binding to a host cell, uptake by a host cell.
• the present invention is not to be limited by the particular strains disclosed above
• 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 sub-strains 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 as described herein (e.g. in Example 12). In another embodiment, the passaging is performed by any other method known in the art.
• the recombinant Listeria strain utilized in methods of the present invention has been stored in a frozen cell bank. In another embodiment, the recombinant Listeria strain has been stored in a lyophilized cell bank.
• the cell bank of methods and compositions of the present invention is a master cell bank.
• the cell bank is a working cell bank.
• the cell bank is Good Manufacturing Practice (GMP) cell bank.
• the cell bank is intended for production of clinical-grade material.
• the cell bank conforms to regulatory practices for human use.
• the cell bank is any other type of cell bank known in the art.
• Good Manufacturing Practices are defined, in another embodiment, by (21 CFR 210-211) of the United States Code of Federal Regulations. In another embodiment, “Good Manufacturing Practices” are defined by other standards for production of clinical - grade material or for human consumption; e.g. standards of a country other than the United States.
• a recombinant Listeria strain utilized in methods of the present invention is from a batch of vaccine doses.
• a recombinant Listeria strain utilized in methods of the present invention is from a frozen or lyophilized stock produced by methods provided in US Patent Ser. No. 8,114,414, which is incorporated by reference herein.
• a peptide of the present invention is a fusion peptide.
• fusion peptide refers to a peptide or polypeptide comprising 2 or more proteins linked together by peptide bonds or other chemical bonds.
• the proteins are linked together directly by a peptide or other chemical bond.
• the proteins are linked together with 1 or more AA (e.g. a "spacer") between the 2 or more proteins.
• a vaccine of the present invention further comprises an adjuvant.
• the adjuvant utilized in methods and compositions of the present invention is, in another embodiment, a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein.
• the adjuvant comprises a GM-CSF protein.
• the adjuvant is a nucleotide molecule encoding GM-CSF.
• the adjuvant comprises a nucleotide molecule encoding GM-CSF.
• the adjuvant is saponin QS21.
• the adjuvant comprises saponin QS21.
• the adjuvant is monophosphoryl lipid A.
• the adjuvant comprises monophosphoryl lipid A. In another embodiment, the adjuvant is SBAS2. In another embodiment, the adjuvant comprises SBAS2. In another embodiment, the adjuvant is an unmethylated CpG-containing oligonucleotide. In another embodiment, the adjuvant comprises an unmethylated CpG- containing oligonucleotide. In another embodiment, the adjuvant is an immune- stimulating cytokine. In another embodiment, the adjuvant comprises an immune- stimulating cytokine. In another embodiment, the adjuvant is a nucleotide molecule encoding an immune-stimulating cytokine.
• the adjuvant comprises a nucleotide molecule encoding an immune-stimulating cytokine.
• the adjuvant is or comprises a quill glycoside.
• the adjuvant is or comprises a bacterial mitogen.
• the adjuvant is or comprises a bacterial toxin.
• the adjuvant is or comprises any other adjuvant known in the art.
• a nucleotide of the present invention is operably linked to a promoter/regulatory sequence that drives expression of the encoded peptide in the Listeria strain.
• Promoter/regulatory sequences useful for driving constitutive expression of a gene are well known in the art and include, but are not limited to, for example, the PhiyA, PActA, and p60 promoters of Listeria, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ promoter.
• inducible and tissue specific expression of the nucleic acid encoding a peptide of the present invention is accomplished by placing the nucleic acid encoding the peptide under the control of an inducible or tissue specific promoter/regulatory sequence.
• tissue specific or inducible promoter/regulatory sequences which are useful for his purpose include, but are not limited to the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter.
• a promoter that is induced in response to inducing agents such as metals, glucocorticoids, and the like, is utilized.
• the invention includes the use of any promoter/regulatory sequence, which is either known or unknown, and which is capable of driving expression of the desired protein operably linked thereto.
• N-terminal fragment of an ActA protein utilized in methods and compositions of the present invention has, in another embodiment, the sequence set forth in SEQ ID NO: 4:
• the ActA fragment comprises the sequence set forth in SEQ ID NO: 4.
• the ActA fragment is any other ActA fragment known in the art.
• the recombinant nucleotide encoding a fragment of an ActA protein comprises the sequence set forth in SEQ ID NO: 5:
• a PEST amino acid AA sequence is fused to the E7 or E6 antigen.
• recombinant Listeria strains expressing PEST amino acid sequence-antigen fusions induce anti-tumor immunity (Example 3) and generate antigen-specific, tumor-infiltrating T cells (Example 4).
• enhanced cell mediated immunity was demonstrated for fusion proteins comprising an antigen and LLO containing the PEST amino acid AA sequence KENS IS SM APP A SPP ASPKTPIEKKH ADEIDK (SEQ ID NO: 6).
• the PEST amino acid AA sequence has, in another embodiment, a sequence selected from SEQ ID NO: 7-12.
• the PEST amino acid sequence is a PEST amino acid sequence from the LM ActA protein.
• the PEST amino acid sequence is KTEEQP SEVNTGPR (SEQ ID NO: 7), K AS VTDT SEGDLD S SMQ S ADESTPQPLK (SEQ ID NO: 8), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 9), or
• the PEST amino acid sequence is from Streptolysin O protein of Streptococcus sp. In another embodiment, the PEST amino acid sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 11) at AA 35-51. In another embodiment, the PEST amino acid sequence is from Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK (SEQ ID NO: 12) at AA 38-54. In another embodiment, the PEST amino acid sequence is another PEST amino acid AA sequence derived from a prokaryotic organism. In another embodiment, the PEST amino acid sequence is any other PEST amino acid sequence known in the art.
• PEST amino acid sequences 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. Alternatively, PEST amino acid AA sequences from other prokaryotic organisms can also be identified based by this method. Other prokaryotic organisms wherein PEST amino acid AA sequences would be expected to include, but are not limited to, other Listeria species.
• the PEST amino acid sequence is embedded within the antigenic protein.
• "fusion" refers to an antigenic protein comprising both the antigen and the PEST amino acid amino acid sequence either linked at one end of the antigen or embedded within the antigen.
• the PEST amino acid sequence is identified using any other method or algorithm known in the art, e.g the CaSPredictor (Garay-Malpartida HM, Occhiucci JM, Alves J, Belizario JE. Bioinformatics. 2005 Jun;21 Suppl 1 :i 169-76). In another embodiment, the following method is used:
• a PEST index is calculated for each 30-35 AA stretch by assigning a value of 1 to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gin.
• the coefficient value (CV) for each of the PEST residue is 1 and for each of the other AA (non-PEST) is 0.
• the LLO protein, ActA protein, or fragment thereof of the present invention need not be that which is set forth exactly in the sequences set forth herein, but rather other alterations, modifications, or changes can be made that retain the functional characteristics of an LLO or ActA protein fused to an antigen as set forth elsewhere herein.
• the present invention utilizes an analog of an LLO protein, ActA protein, or fragment thereof. Analogs differ, in another embodiment, from naturally occurring proteins or peptides by conservative AA sequence differences or by modifications which do not affect sequence, or by both.
• E7 protein or a fragment thereof is fused to a LLO protein, ActA protein, or PEST amino acid sequence-containing peptide to generate a recombinant peptide of methods of the present invention.
• the E7 protein that is utilized (either whole or as the source of the fragments) has, in another embodiment, the sequence:
• the E7 protein is a homologue of SEQ ID No: 13. In another embodiment, the E7 protein is a variant of SEQ ID No: 13. In another embodiment, the E7 protein is an isomer of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of a homologue of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of a variant of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of an isomer of SEQ ID No: 13.
• the sequence of the E7 protein is: MHGPK ATLQDIVLHLEPQNEIP VDLLCHEQL SD SEEENDEIDGVNHQHLP ARRAE PQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (SEQ ID No: 14).
• the E6 protein is a homologue of SEQ ID No: 14.
• the E6 protein is a variant of SEQ ID No: 14.
• the E6 protein is an isomer of SEQ ID No: 14.
• the E6 protein is a fragment of SEQ ID No: 14.
• the E6 protein is a fragment of a homologue of SEQ ID No: 14.
• the E6 protein is a fragment of a variant of SEQ ID No: 14.
• the E6 protein is a fragment of an isomer of SEQ ID No: 14.
• the E7 protein has a sequence set forth in one of the following GenBank entries: M24215, NC_004500, V01116, X62843, or M14119.
• the E7 protein is a homologue of a sequence from one of the above GenBank entries.
• the E7 protein is a variant of a sequence from one of the above GenBank entries.
• the E7 protein is an isomer of a sequence from one of the above GenBank entries.
• the E7 protein is a fragment of a sequence from one of the above GenBank entries.
• the E7 protein is a fragment of a homologue of a sequence from one of the above GenBank entries.
• the E7 protein is a fragment of a variant of a sequence from one of the above GenBank entries.
• the E7 protein is a fragment of an isomer of a sequence from one of the above GenBank entries.
• E6 protein or a fragment thereof is fused to a LLO protein, ActA protein, or PEST amino acid sequence-containing peptide to generate a recombinant peptide of methods of the present invention.
• the E6 protein that is utilized (either whole or as the source of the fragments) has, in another embodiment, the sequence:
• the E6 protein is a homologue of SEQ ID No: 15.
• the E6 protein is a variant of SEQ ID No: 15.
• the E6 protein is an isomer of SEQ ID No: 15.
• the E6 protein is a fragment of SEQ ID No: 15.
• the E6 protein is a fragment of a homologue of SEQ ID No: 15. In another embodiment, the E6 protein is a fragment of a variant of SEQ ID No: 15. In another embodiment, the E6 protein is a fragment of an isomer of SEQ ID No: 15.
• the sequence of the E6 protein is: MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVY RDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPL NPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (SEQ ID No: 16).
• the E6 protein is a homologue of SEQ ID No: 16.
• the E6 protein is a variant of SEQ ID No: 16.
• the E6 protein is an isomer of SEQ ID No: 16.
• the E6 protein is a fragment of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of a homologue of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of a variant of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of an isomer of SEQ ID No: 16. [00203] In another embodiment, the E6 protein has a sequence set forth in one of the following GenBank entries: M24215, M14119, NC_004500, V01116, X62843, or M14119. In another embodiment, the E6 protein is a homologue of a sequence from one of the above GenBank entries.
• the E6 protein is a variant of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is an isomer of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of a homologue of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of a variant of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of an isomer of a sequence from one of the above GenBank entries.
• homology refers to identity to an LLO sequence (e.g. to one of SEQ ID No: 1-3) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 78%.
• “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 88%.
• “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 97%.
• homology refers to identity to one of SEQ ID No: 1-3 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-3 of 100%.
• “homology” refers to identity to an E7 sequence (e.g. to one of SEQ ID No: 13-14) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 62%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 75%.
• “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 87%.
• “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 96%.
• homology refers to identity to one of SEQ ID No: 13-14 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of 100%.
• “homology” refers to identity to an E6 sequence (e.g. to one of SEQ ID No: 15-16) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 78%.
• “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 88%.
• “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 97%.
• homology refers to identity to one of SEQ ID No: 15-16 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of 100%.
• homology refers to identity to a PEST amino acid sequence (e.g. to one of SEQ ID No: 6-12) or to an ActA sequence (e.g. to one of SEQ ID No: 4-5) of greater than 70%.
• “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 60%.
• “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 64%.
• homoology refers to identity to one of SEQ ID No: 6- 12 or SEQ ID No: 4-5 of greater than 68%.
• “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 82%.
• “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6- 12 or SEQ ID No: 4-5 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 90%.
• “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 97%.
• homology refers to identity to one of SEQ ID No: 6- 12 or SEQ ID No: 4-5 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6-12 or SEQ ID No: 4-5 of 100%.
• Protein and/or peptide homology for any AA sequence listed herein is determined, in one embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of AA sequences, utilizing any of a number of software packages available, via established methods. Some of these packages include the FASTA, BLAST, MPsrch or Scanps packages, and employ, in other embodiments, 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 LLO protein, ActA protein, or fragment thereof is attached to the antigen by chemical conjugation.
• glutaraldehyde is used for the conjugation.
• the conjugation is performed using any suitable method known in the art.
• fusion proteins of the present invention are prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods discussed below.
• subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated, in another embodiment, to produce the desired DNA sequence.
• DNA encoding the fusion protein is produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately.
• the 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction.
• the amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence).
• the insert is then ligated into a plasmid.
• the LLO protein, ActA protein, or fragment thereof and the antigen, or fragment thereof are conjugated by a means known to those of skill in the art.
• the antigen, or fragment thereof is conjugated, either directly or through a linker (spacer), to the ActA protein or LLO protein.
• the chimeric molecule is recombinantly expressed as a single-chain fusion protein.
• a fusion peptide of the present invention is synthesized using standard chemical peptide synthesis techniques.
• the chimeric molecule is synthesized as a single contiguous polypeptide.
• the LLO protein, ActA protein, or fragment thereof; and the antigen, or fragment thereof are synthesized separately, then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule, thereby forming a peptide bond.
• the ActA protein or LLO protein and antigen are each condensed with one end of a peptide spacer molecule, thereby forming a contiguous fusion protein.
• the peptides and proteins of the present invention are prepared by solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, 111.; or as described by Bodanszky and Bodanszky (The Practice of Peptide Synthesis, 1984, Springer- Verlag, New York).
• SPPS solid-phase peptide synthesis
• a suitably protected AA residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin.
• “Suitably protected” refers to the presence of protecting groups on both the alpha-amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial AA, and couple thereto of the carboxyl end of the next AA in the sequence of the desired peptide. This AA is also suitably protected.
• the carboxyl of the incoming AA can be activated to react with the N-terminus of the support-bound AA by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an "active ester” group such as hydroxybenzotriazole or pentafluorophenly esters.
• the present invention provides a kit comprising vaccine of the present invention, an applicator, and instructional material that describes use of the methods of the invention.
• kit kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure.
• the term "subject” may enccompass a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae.
• the subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans.
• the term "subject” does not exclude an individual that is normal in all respects.
• TC-1 The C57BL/6 syngeneic TC-1 tumor was immortalized with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene.
• TC-1 provided by T. C. Wu (Johns Hopkins University School of Medicine, Baltimore, MD) is a highly tumorigenic lung epithelial cell expressing low levels of with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene.
• TC-1 was grown in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 100 ⁇ nonessential amino acids, 1 mM sodium pyruvate, 50 micromolar (mcM) 2-ME, 400 microgram (mcg)/ml G418, and 10% National Collection Type Culture-109 medium at 37° with 10% CO2.
• C3 is a mouse embryo cell from C57BL/6 mice immortalized with the complete genome of HPV 16 and transformed with pEJ-ras.
• EL-4/E7 is the thymoma EL-4 retrovirally transduced with E7. L. monocytogenes strains and propagation
• Listeria strains used were Lm-LLO-E7 (hly-E7 fusion gene in an episomal expression system; Figure 1A), Lm-E7 (single-copy E7 gene cassette integrated into Listeria genome), Lm-LLO-NP ("DP-L2028”; hly-NP fusion gene in an episomal expression system), and Lm-Gag ("ZY-18"; single-copy HIV-1 Gag gene cassette integrated into the chromosome).
• E7 was amplified by PCR using the primers 5'- GGCTCGAGC ATGGAGAT AC ACC-3 ' (SEQ ID No: 17; Xhol site is underlined) and 5'- GGGGACT AGTTT ATGGTTTCTGAGAAC A-3 1 (SEQ ID No: 18; Spel site is underlined) and ligated into pCR2.1 (Invitrogen, San Diego, CA). E7 was excised from pCR2.1 by Xhol/ Spel digestion and ligated into pGG-55.
• the hly-E7 fusion gene and the pluripotential transcription factor prfA were cloned into pAM401, a multicopy shuttle plasmid (Wirth R et al, J Bacterid, 165: 831, 1986), generating pGG-55.
• the hly promoter drives the expression of the first 441 AA of the hly gene product, (lacking the hemolytic C-terminus, referred to below as " ⁇ ,” and having the sequence set forth in SEQ ID No: 25), which is joined by the Xhol site to the E7 gene, yielding a hly-E7 fusion gene that is transcribed and secreted as LLO-E7.
• GGGGGCTAGCCCTCCTTTGATTAGTATATTC-3' (SEQ ID No: 19; Nhel site is underlined) and 5'-CTCCCTCGAGATCATAATTTACTTCATC-3' (SEQ ID No: 20; Xhol site is underlined).
• the prfA gene was PCR amplified using primers 5'- GACT AC AAGGACGATGACCGAC AAGTGATAACCCGGGATCT AAAT AAATCCG TTT-3' (SEQ ID No: 27; Xbal site is underlined) and 5'- CCCGTCGACC AGCTCTTCTTGGTGAAG-3 1 (SEQ ID No: 21; Sail site is underlined).
• Lm-E7 was generated by introducing an expression cassette containing the hly promoter and signal sequence driving the expression and secretion of E7 into the orfZ domain of the LM genome.
• E7 was amplified by PCR using the primers 5'- GCGGATCCCATGGAGATACACCTAC-3' (SEQ ID No: 22; BamHI site is underlined) and 5'-GCTCTAGATTATGGTTTCTGAG-3' (SEQ ID No: 23; Xbal site is underlined). E7 was then ligated into the pZY-21 shuttle vector.
• LM strain 10403S was transformed with the resulting plasmid, pZY-21-E7, which includes an expression cassette inserted in the middle of a 1.6-kb sequence that corresponds to the orfX, Y, Z domain of the LM genome.
• the homology domain allows for insertion of the E7 gene cassette into the orfZ domain by homologous recombination.
• Clones were screened for integration of the E7 gene cassette into the orfZ domain.
• Bacteria were grown in brain heart infusion medium with (Lm-LLO-E7 and Lm-LLO-NP) or without (Lm-E7 and ZY-18) chloramphenicol (20 ⁇ g/ml). Bacteria were frozen in aliquots at -80°C. Expression was verified by Western blotting ( Figure 2).
• Listeria strains were grown in Luria-Bertoni medium at 37°C and were harvested at the same optical density measured at 600 nm. The supernatants were TCA precipitated and resuspended in lx sample buffer supplemented with 0.1 N NaOH. Identical amounts of each cell pellet or each TCA-precipitated supernatant were loaded on 4-20% Tris- glycine SDS-PAGE gels (NOVEX, San Diego, CA).
• the gels were transferred to polyvinylidene difluoride and probed with an anti-E7 monoclonal antibody (mAb) (Zymed Laboratories, South San Francisco, CA), then incubated with HRP -conjugated anti-mouse secondary Ab (Amersham Pharmacia Biotech, Little Chalfont, U.K.), developed with Amersham ECL detection reagents, and exposed to Hyperfilm (Amersham Pharmacia Biotech).
• mAb monoclonal antibody
• Tumors were measured every other day with calipers spanning the shortest and longest surface diameters. The mean of these two measurements was plotted as the mean tumor diameter in millimeters against various time points. Mice were sacrificed when the tumor diameter reached 20 mm. Tumor measurements for each time point are shown only for surviving mice.
• spleens were harvested.
• Splenocytes were established in culture with irradiated TC-1 cells (100: 1, splenocytes:TC- 1) as feeder cells; stimulated in vitro for 5 days, then used in a standard 51 Cr release assay, using the following targets: EL-4, EL-4/E7, or EL-4 pulsed with E7 H-2b peptide (RAHYNIVTF, SEQ ID NO: 24).
• E:T cell ratios were 80: 1, 40: 1, 20: 1, 10: 1, 5 : 1, and 2.5 : 1. Following a 4-h incubation at 37°C, cells were pelleted, and 50 ⁇ supernatant was removed from each well. Samples were assayed with a Wallac 1450 scintillation counter (Gaithersburg, MD). The percent specific lysis was determined as [(experimental counts per minute (cpm)- spontaneous cpm)/(total cpm - spontaneous cpm)] x 100.
• C57BL/6 mice were immunized with 0.1 LD50 and boosted by i.p. injection 20 days later with 1 LD50 Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag.
• spleens were harvested from immunized and naive mice.
• Splenocytes were established in culture at 5 x 10 5 /well in flat-bottom 96-well plates with 2.5 x 10 4 , 1.25 x 10 4 , 6 x 10 3 , or 3 x 10 3 irradiated TC-1 cells/well as a source of E7 Ag, or without TC-1 cells or with 10 ⁇ g/ml Con A.
• C57BL/6 mice were immunized intravenously (i.v.) with 0.1 LD50 Lm-LLO-E7 or Lm-E7 and boosted 30 days later.
• Three-color flow cytometry for CD8 (53-6.7, PE conjugated), CD62 ligand (CD62L; MEL- 14, APC conjugated), and E7 H-2Db tetramer was performed using a FACSCalibur® flow cytometer with CellQuest® software (Becton Dickinson, Mountain View, CA).
• Splenocytes harvested 5 days after the boost were stained at room temperature (rt) with H-2Db tetramers loaded with the E7 peptide (RAHYNIVTF) or a control (HIV-Gag) peptide.
• Tetramers were used at a 1/200 dilution and were provided by Dr. Larry R. Pease (Mayo Clinic, Rochester, MN) and by the NIAID Tetramer Core Facility and the NIH AIDS Research and Reference Reagent Program. Tetramer + , CD8 + , CD62L low cells were analyzed.
• mice 24 C57BL/6 mice were inoculated with 5 x 10 5 B16F0-Ova cells. On days 3, 10 and 17, groups of 8 mice were immunized with 0.1 LD50 Lm-OVA (10 6 cfu), Lm-LLO- OVA (10 8 cfu) and eight animals were left untreated.
• Lm-E7 and Lm-LLO-E7 were compared for their abilities to impact on TC-1 growth.
• Subcutaneous tumors were established on the left flank of C57BL/6 mice. Seven days later tumors had reached a palpable size (4-5 mm).
• Mice were vaccinated on days 7 and 14 with 0.1 LD50 Lm-E7, Lm-LLO-E7, or, as controls, Lm-Gag and Lm-LLO-NP.
• Lm-LLO-E7 induced complete regression of 75% of established TC-1 tumors, while tumor growth was controlled in the other 2 mice in the group ( Figure 3). By contrast, immunization with Lm-E7 and Lm-Gag did not induce tumor regression.
• EXAMPLE 2 LM-LLO-E7 TREATMENT ELICITS TC-1 SPECIFIC SPLENOCYTE PROLIFERATION
• EXAMPLE 3 FUSION OF E7 TO LLP, ActA, OR A PEST AMINO ACID SEQUENCE ENHANCES E7-SPECIFIC IMMUNITY AND GENERATES TUMOR-INFILTRATING E7-SPECIFIC CD8 + CELLS
• [00233] 500 mcl (microliter) of MATRIGEL®, comprising 100 mcl of 2 x 10 5 TC-1 tumor cells in phosphate buffered saline (PBS) plus 400 mcl of MATRIGEL® (BD Biosciences, Franklin Lakes, N.J.) were implanted subcutaneously on the left flank of 12 C57BL/6 mice (n 3). Mice were immunized intraperitoneally on day 7, 14 and 21, and spleens and tumors were harvested on day 28. Tumor MATRIGELs were removed from the mice and incubated at 4 °C overnight in tubes containing 2 milliliters (ml) of RP 10 medium on ice.
• PBS phosphate buffered saline
• MATRIGEL® BD Biosciences, Franklin Lakes, N.J.
• Tumors were minced with forceps, cut into 2 mm blocks, and incubated at 37 °C for 1 hour with 3 ml of enzyme mixture (0.2 mg/ml collagenase-P, 1 mg/ml DNAse-1 in PBS). The tissue suspension was filtered through nylon mesh and washed with 5% fetal bovine serum + 0.05% of NaN 3 in PBS for tetramer and IFN-gamma staining.
• Splenocytes and tumor cells were incubated with 1 micromole (mem) E7 peptide for 5 hours in the presence of brefeldin A at 10 7 cells/ml.
• Cells were washed twice and incubated in 50 mcl of anti-mouse Fc receptor supernatant (2.4 G2) for 1 hour or overnight at 4 °C.
• Cells were stained for surface molecules CD8 and CD62L, permeabilized, fixed using the permeabilization kit Golgi-stop® or Golgi-Plug® (Pharmingen, San Diego, Calif), and stained for IFN-gamma.
• H-2D tetramer was loaded with phycoerythrin (PE)- conjugated E7 peptide (RAHYNIVTF, SEQ ID NO: 24), stained at rt for 1 hour, and stained with anti-allophycocyanin (APC) conjugated MEL-14 (CD62L) and FITC- conjugated CD8 D at 4 °C for 30 min. Cells were analyzed comparing tetramer + CD8 + CD62L low cells in the spleen and in the tumor.
• PE phycoerythrin
• APC anti-allophycocyanin conjugated MEL-14
• CD8 D FITC- conjugated CD8 D
• mice were implanted with TC-1 tumor cells and immunized with either Lm-LLO-E7 (1 x 10 7 CFU), Lm-E7 (1 x 10 6 CFU), or Lm-ActA-E7 (2 x 10 8 CFU), or were untreated (naive).
• Tumors of mice from the Lm-LLO-E7 and Lm-ActA-E7 groups contained a higher percentage of IFN-gamma-secreting CD8 + T cells ( Figure 5 A) and tetramer-specific CD8 + cells ( Figure 5B) than in Lm-E7 or naive mice.
• mice were administered Lm-LLO-E7, Lm- PEST-E7, Lm-APEST-E7, or Lm-E7epi, and levels of E7-specific lymphocytes within the tumor were measured.
• Mice were treated on days 7 and 14 with 0.1 LD50 of the 4 vaccines. Tumors were harvested on day 21 and stained with antibodies to CD62L, CD8, and with the E7/Db tetramer. An increased percentage of tetramer-positive lymphocytes within the tumor were seen in mice vaccinated with Lm-LLO-E7 and Lm-PEST-E7 (Figure 6A). This result was reproducible over three experiments ( Figure 6B).
• Lm-LLO-E7, Lm-ActA-E7, and Lm-PEST-E7 are each efficacious at induction of tumor-infiltrating CD8 + T cells and tumor regression.
• EXAMPLE 4 PASSAGING OF LISTERIA VACCINE VECTORS THROUGH MICE ELICITS INCREASED IMMUNE RESPONSES TO HETEROLOGOUS AND
• L. monocytogenes strain 10403S, serotype 1 was the wild type organism used in these studies and the parental strain of the constructs described below.
• Strain 10403S has an LD50 of approximately 5 x 10 4 CFU when injected intraperitoneally into BALB/c mice.
• "Lm-Gag” is a recombinant LM strain containing a copy of the HIV-1 strain HXB (subtype B laboratory strain with a syncytia-forming phenotype) gag gene stably integrated into the listerial chromosome using a modified shuttle vector pKSV7. Gag protein was expressed and secreted by the strain, as determined by Western blot. All strains were grown in brain-heart infusion (BHI) broth or agar plates (Difco Labs, Detroit, Mich).
• Bacteria from a single clone expressing the passenger antigen and/or fusion protein were selected and cultured in BHI broth overnight. Aliquots of this culture were frozen at " 70°C with no additives. From this stock, cultures were grown to 0.1-0.2 O.D. at 600 nm, and aliquots were again frozen at -70°C with no additives. To prepare cloned bacterial pools, the above procedure was used, but after each passage a number of bacterial clones were selected and checked for expression of the target antigen, as described herein. Clones in which expression of the foreign antigen was confirmed were used for the next passage.
• mice 6-8 week old female BALB/c (H-2d) mice were purchased from Jackson Laboratories (Bar Harbor, Me) and were maintained in a pathogen-free microisolator environment. The titer of viable bacteria in an aliquot of stock culture, stored frozen at -70 °C, was determined by plating on BHI agar plates on thawing and prior to use. In all, 5 x 10 5 bacteria were injected intravenously into BALB/c mice. After 3 days, spleens were harvested, homogenized, and serial dilutions of the spleen homogenate were incubated in BHI broth overnight and plated on BHI agar plates.
• Lymphocytes were cultured for 5 hours in complete RPMI-10 medium supplemented with 50 U/ml human recombinant IL-2 and 1 microliter/ml Brefeldin A (GolgistopTM; PharMingen, San Diego, CA) in the presence or absence of either the cytotoxic T-cell (CTL) epitope for HIV-GAG (AMQMLKETI; SEQ ID No: 25), Listeria LLO (GYKDGNEYI; SEQ ID No: 26) or the HPV virus gene E7 (RAHYNIVTF (SEQ ID
• CTL cytotoxic T-cell epitope for HIV-GAG
• AQMLKETI cytotoxic T-cell epitope for HIV-GAG
• AQMLKETI cytotoxic T-cell epitope for HIV-GAG
• AQMLKETI cytotoxic T-cell epitope for HIV-GAG
• AQMLKETI cytotoxic T-cell epitope for HIV-GAG
• GYKDGNEYI
• This plasmid was used to complement a prfA negative mutant so that in a live host, selection pressures would favor conservation of the plasmid, because without it the bacterium is avirulent. All 3 constructs had been propagated extensively in vitro for many bacterial generations.
• EXAMPLE 5 A PrfA-CONTAINING PLASMID IS STABLE IN AN LM STRAIN WITH A PrfA DELETION IN THE ABSENCE OF ANTIBIOTICS
• L. monocytogenes strain XFL7 contains a 300 base pair deletion in the prfA gene XFL7 carries pGG55 which partially restores virulence and confers CAP resistance, and is described in United States Patent Application Publication No. 200500118184.
• the lysis solution was then incubated at 37°C for 15 minutes before neutralization.
• the plasmid DNA was resuspended in 30 ⁇ ⁇ rather than 50 ⁇ ⁇ to increase the concentration.
• the cells were incubated for 15min in PI buffer + Lysozyme, then incubated with P2 (lysis buffer) and P3 (neutraliztion buffer) at room temperature.
• plasmid DNA was subjected to agarose gel electrophoresis, followed by ethidium bromide staining. While the amount of plasmid in the preps varied slightly between samples, the overall trend was a constant amount of plasmid with respect to the generational number of the bacteria ( Figures 9A-B). Thus, pGG55 exhibited stability in strain XFL7, even in the absence of antibiotic.
• Plasmid stability was also monitored during the stability study by replica plating on agar plates at each stage of the subculture. Consistent with the results from the agarose gel electrophoresis, there was no overall change in the number of plasmid-containing cells throughout the study in either LB or TB liquid culture ( Figures 10 and 11, respectively).
• the primers used for amplification of the prfA gene and discrimination of the D133V mutation are shown in Table 1.
• Stock solutions of the primers ADV451, 452 and 453 were prepared by diluting the primers in TE buffer to 400 ⁇ . An aliquot of the stock solution was further diluted to 20 ⁇ in water (PCR grade) to prepare a working solution. Primers were stored at -20°C. The reagents used in the PCR are shown in Table 2.
• pGG55 plasmids with (pGG55 D133V) and without (pGG55 WT) the prfA mutation were extracted and purified by midipreparations either from E. coli or Listeria monocytogenes using the PureLinkTM HiPure Plasmid Midiprep Kit (Invitrogen, K2100- 05), according to the manufacturer's instructions.
• plasmid purification from Listeria bacterial strains carrying the pGG55 D133V or WT plasmids were streak plated from frozen stocks in BHI agar plates supplemented with chloramphenicol (25 ⁇ g/ml).
• a single colony from each strain was grown in 5 ml of selective medium (BHI broth with 25 ⁇ g/ml of chloramphenicol) for 6 hours with vigorous shaking at 37°C and subinoculated 1 :500 in 100 ml of selective medium for overnight growth under similar conditions.
• Bacteria from the overnight culture were harvested by centrifugation at 4,000 x g for 10 minutes and resuspended buffer R3 (resuspension buffer) containing 2 mg/ml of lysozyme (Sigma, L7001). The bacteria suspension was incubated for at least 1 hour at 37°C before proceeding to the regular protocol.
• Concentration and purity of the eluted plasmids were measured in a spectrophotometer at 260nm and 280nm.
• the pGG55 D133V and WT plasmids were resuspended in water to a final concentration of 1 ng/ ⁇ from the midiprep stock solution.
• serial 10-fold dilutions from the 1 ng/ ⁇ solution were prepared, corresponding to dilutions from 10 "1 to lo- 7 .
• the reaction mixture contained lx PCR buffer, 1.5 mM MgCb, 0.8 mM dNTPs, 0.4 ⁇ of each primer, 0.05 U/ ⁇ of Taq DNA polymerase and 0.04 ng/ ⁇ of the pGG55 D133V template plasmid.
• 10 tubes were required and the key components in each tube in a 25 ⁇ reaction are shown in the Table 3.
• a master mix was prepared with enough reagents for 11 reactions as shown in Table 4, and 24 ⁇ of this PCR mix was added to each tube.
• Table 3 Set of individual PCR reactions to validate the method to detect the presence of wild-type prfA sequence in Lm-LL - ⁇ samples.
• dNTPs Deoxynucleotides
• Deoxynucleotides mix (dATP, dCTP, dGTP and dTTP) 0.5 ⁇
• Primer ADV452 (20 ⁇ ) 0.5 ⁇
• Primer ADV453 (20 ⁇ ) 0.5 ⁇
• a pGG55 WT (1 ng in tube 3; 100 pg in tube 4; 10 pg in tube 5; 1 pg in tube 6; 100 fg in tube 7; 10 fg in tube 8; 1 fg in tube 9; 0.1 fg in tube 10).
• Sequencing of the plasmids was done using the dideoxy sequencing method.
• the plasmids pGG55 D133V and pGG55 WT were mixed at different ratios (1 : 1, 1 : 10, 1; 100, 1 : 1,000 and 1 : 10,000).
• the total amount of plasmid in the mixture was kept constant (500 ⁇ g) and the plasmid containing the wild-type sequence was 10-fold serially diluted in relation to the D133V plasmid to determine the sensitivity of the method.
• EXAMPLE 6 SEQUENCING IS NOT A SENSITIVE METHOD TO DETECT THE REVERSION OF THE D133V MUTATION.
• EXAMPLE 7 DEVELOPMENT OF A HIGHLY SPECIFIC AND SENSITIVE PCR METHOD TO DETECT REVERSION OF THE D133V MUTATION.
• the ADV453 primer is the reverse primer located approximately 300 bp downstream the annealing site of the ADV451 and ADV452 primers ( Figure 13).
• the expected PCR band obtained with the primers ADV451 or ADV452 and ADV453 is 326 bp.
• the ADV451 primer should only amplify the pGG55 D133V plasmid, whereas the ADV452 would be specific to the wild-type prfA sequence.
• EXAMPLE 8 SPECIFICITY OF THE PCR METHOD.
• the sensitivity of the reaction was tested using 1 ng of template DNA.
• decreasing amounts of DNA corresponding to 10-fold dilutions from 10 "1 to 10 "7 ), were also included in the reaction to estimate the sensitivity.
• the primers ADV452 and ADV453 were used.
• the sensitivity of the method was 1 in 100,000 (data not shown). As shown in figure 5, increasing the number of PCR cycles to 37 improved the visual sensitivity of the method to 10 "6 for the detection of D133V revertants, without significantly compromising the specificity.
• This strain is approx. 4 -5 logs more attenuated than the wild-type parent strain 10403S and secretes the fusion protein tLLO-E7.
• This immunotherapy is based on the backbone XFL7, which is derived from 10403S by the irreversible deletion in the virulence gene transcription activator prfA.
• PrfA regulates the transcription of several virulence genes such as Listeriolysin O (LLO), ActA, PlcA (phospholipase A), PlcB (phospholipase B) etc that are required for in vivo intracellular growth and survival of L. monocytogenes.
• the plasmid pGG55 is retained by the Lm-LLO-E7 in vitro by means of selection with 'chloramphenicol' .
• Lm- LLO-E7 carries a copy of mutated prfA (D133V), which has been demonstrated to be less active than wild-type PrfA in DNA binding and activating the transcription of virulence genes.
• mutated prfA D133V
• complementation with mutated prfA resulted in approx. 40 fold reduction in the amount of secreted LLO from Lm-LLO-E7 when compared to wild-type strain 10403S.
• Lm-LLO-E7 exhibits a reduced expression of the virulence genes that are regulated by PrfA such as actA, inlA, inlB, inlC, plcB etc.
• PrfA such as actA, inlA, inlB, inlC, plcB etc.
• the complementation with mutated copy of prfA possibly causes a reduction in the expression of different virulence genes that are regulated by PrfA resulting in overall attenuation of approx. 4-5 logs.
• EXAMPLE 11 HIGH-DOSE TREATMENT WITH ADXS11-001, A LISTERIA MONOCYTOGENES (LM)-LISTERIOLYSIN O (LLO) IMMUNOTHERAPY, IN WOMEN WITH CERVICAL CANCER
• NCT02164461 Phase I, dose-escalation, open-label study (NCT02164461) enrolling women aged >18 years with persistent, metastatic, or recurrent squamous/adenocarcinoma of the cervix and documented disease progression (not amenable to surgery/standard radiotherapy).
• Additional eligibility criteria include: measurable and/or evaluable disease per Response Evaluation Criteria in Solid Tumors (RECIST vl . l); Eastern Cooperative Oncology Group (ECOG) performance status of 0-1; and ⁇ 2 prior treatments for metastatic disease. Patients had measurable disease (RECIST vl . l) with documented progression on/intolerance to prior therapy, and ECOG PS 0-1. The primary endpoint was the safety and tolerability of ADXS11-001; secondary endpoints included evaluating tumor response and progression-free survival, and assessing correlative immunologic studies. Patients receive ADXS11-001 every 3 weeks during a 12-week treatment cycle.
• Dose escalation was performed using the 3+3 design in 2 doses: 5xl0 9 colony-forming units (CFU; Dose Level 1 (DL1)) and lxlO 10 CFU (Dose Level 2 (DL2)).
• the recommended phase II dose are selected based on an observed dose-limiting toxicity (DLT) rate of ⁇ 33%.
• Efficacy is assessed using RECIST vl . l and immune-related RECIST. Blood samples are collected in cycle 1 only and used for immune monitoring and cytokine/chemokine analysis.
• DL1 Dose Level 1
• EXAMPLE 12 ADXSl 1-001 IMMUNOTHERAPY IN THE TREATMENT OF PERSISTENT/RECURRENT METASTATIC SQUAMOUS OR NON- SQUAMOUS CELL CARCINOMA OF THE CERVIX: RESULTS FROM STAGE I OF A PHASE II STUDY
• N -67 Simon 2 Stage design; single-arm, 2-stage, phase II multicenter study (NCT01266460)
• PRmCC Persistent/recurrent metastatic
• stage 1 Following preliminary analysis of stage 1, the study was amended to allow for continuous (>3 doses) treatment with ADXSl 1-001 at 28-day intervals, until clinical progression, confirmed radiologic disease progression, intolerable toxicity, or patient refusal of treatment
• FIGO Federation Internationale de Gynecologie et d'Obstetrique
• GOG Gynecologic Oncology Group
• PS performance status.
• This Phase II study represents the second Simon 2-stage trial of persistent or recurrent metastatic (squamous or non-squamous cell) carcinoma of the cervix (PRmCC) in Group history to meet the protocol-specific required efficacy and safety criteria to progress to stage 2.
• the study further presents data from Stage 1 of this ongoing two-stage Phase 2 study of Advaxis's lead Lm TechnologyTM immunotherapy, axalimogene filolisbac (ADXS-HPV, also known as ADXS11-001), in patients with PRmCC who have progressed on at least one prior line of systemic therapy.
• ADXS-HPV also known as ADXS11-001
• ADXS11-001 was well tolerated, with Grade 1-2 fatigue, chills and fever the most commonly reported AEs.
• the CONSORT diagram depicts the total number of patients enrolled and subsequently treated in stages 1 and 2, as well as distribution of ADXS11-001 doses received.
• 10 patients were actively receiving ADXS11-001.
• the baseline demographics and clinical characteristics of patients enrolled in stage 1 and in stage 2 are presented in Table 7 above.
• the primary endpoint 12-month survival rate cannot be calculated due to limited median follow-up of 8.7 months.

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