JP2018527382A - Recombinant Listeria vaccine strain and method of use in cancer immunotherapy - Google Patents

Abstract

The present invention provides a method of treating cervical cancer comprising administering to a subject a composition comprising a recombinant Listeria expressing a human papillomavirus antigen. In another embodiment, the present invention provides a method of treating cervical cancer comprising administering a combination therapy comprising chemoradiotherapy and a recombinant Listeria strain expressing a human papillomavirus antigen. [Selection figure] None

Description

  The present invention provides a method of treating cervical cancer comprising the step of administering to a subject a composition comprising a recombinant Listeria expressing a human papillomavirus antigen. In another embodiment, the present invention provides a method of treating cervical cancer comprising administering a combination therapy comprising chemoradiotherapy and a recombinant Listeria strain expressing a human papillomavirus antibody.

Listeria monocytogenes (Lm) is a food-borne Gram-positive bacterium that can occasionally cause disease in humans, especially older individuals, newborns, pregnant women, and individuals with immune disorders. In addition to strong activation of innate immunity and the induction of cytokine responses that enhance antigen-presenting cell (APC) function, Lm escapes phagolysosomes primarily through the activity of listeriocin O (LLO) protein before the APC cytosol. Have the ability to replicate within. This unique intracellular life cycle allows antigens secreted by Lm to be processed and presented in the context of both MHC class I and MHC class II molecules, resulting in potent cytotoxic CD8 ⁺ and Th1. Produces a CD4 ⁺ T cell mediated immune response. Lm has been extensively studied in preclinical models as a vector for cancer immunotherapy. Immunization of mice with Lm-LLO-E7 induces regression of established tumors expressing E7 and provides long-term protection. The therapeutic efficacy of Lm-LLO-E7 correlates with its ability to induce E7-specific CTLs, which infiltrate the tumor site, mature dendritic cells, and regulate CD4 ⁺ CD25 ⁺ in the tumor Reduces the number of T cells and inhibits tumor angiogenesis.

  Lm also has a number of inherent advantages as an immunotherapy vector. Bacteria grow very efficiently in vitro without special requirements, and this lacks LPS, a major virulence factor in Gram-negative bacteria (such as Salmonella). Genetically attenuated Lm vectors can be easily removed using antibiotics in the event of serious adverse effects and, unlike some viral vectors, in the genetic material host genome. It also provides additional safety because no integration into it occurs.

  Persistent infection with human papillomavirus (HR-HPV) type at high carcinogenic risk is recognized as a cause of invasive cervical cancer (ICC) that is necessary but not sufficient. HPV 16 and 18 are the most frequent types of malignant lesions, accounting for more than 70% of ICC and more than 50% of high grade precursor lesions. HR-HPVE6 and E7 proteins are consistently expressed in dysplasia and carcinoma and interfere with the cell cycle regulatory proteins p53 and pRb, respectively. Both dysplastic and invasive malignancies, as well as the essential expression of E6 and E7 by viral origin of these proteins, make them excellent targets for HPV therapeutic vaccines.

  Cervical cancer is one of the most common cancers for women around the world. However, in the United States and other countries where cervical cancer screening is routinely performed, this cancer is not so common. Most cervical cancers are caused by a virus called human papillomavirus, or HPV. By having sexual contact with others who carry this virus, HPV may be transmitted. There are many types of HPV viruses. Not all types of HPV cause cervical cancer. Some HPVs cause genital warts, while other types may not cause any symptoms. Many adults are infected with HPV at some point. Infection may disappear spontaneously. However, occasionally it can cause genital warts or develop cervical cancer.

  According to estimates by the American Cancer Society, about 12,900 new cases of cervical cancer in the United States will be diagnosed with invasive cervical cancer in 2015. About 4,100 women die from cervical cancer. In addition, it is estimated that 528,000 people are newly diagnosed with cervical cancer each year and 266,000 people are associated death. Survival rate is less than 30% in persistent / recurrent metastatic squamous cell cervical cancer (PRmCC) and historical 12-month survival rate never exceeds this rate It was. With the availability of first-class platinum-based doublet chemotherapy ± bevacizumab, median survival is 13-17 months. However, patients who have progressed after at least one (line) systemic treatment can only live for 4-7 months.

  Furthermore, there is no approved treatment after primary treatment failure. Therefore, there is a need for new treatment methods in cervical cancer including PRmCC.

  The present invention addresses this need by providing a live attenuated Listeria vaccine vector for treating cervical cancer. The invention further provides a combination therapy comprising live attenuated Listeria for the treatment of cervical cancer.

  In one aspect, the invention relates to a method of treating persistent / recurrent metastatic (squamous or non-squamous epithelial) cervical cancer (PRmCC) in a human subject, the method comprising a recombinant Listeria strain Wherein said Listeria strain comprises a recombinant nucleic acid, said nucleic acid comprising a recombinant polypeptide comprising an N-terminal fragment of LLO protein fused to HPV-E7 antigen or fragment thereof. Contains the first open reading frame to encode.

In another aspect, the invention relates to a method of treating cervical cancer in a human subject, said method comprising administering to said subject a combination therapy comprising chemo-radiotherapy and a recombinant Listeria strain. Wherein said Listeria strain comprises a recombinant nucleic acid, said nucleic acid comprising a first polypeptide encoding a recombinant polypeptide comprising an N-terminal fragment of LLO protein fused to HPV-E7 antigen or fragment thereof. Including an open reading frame, in which case the aforementioned Listeria strain is administered at an initial dose of 5 × 10 ⁹ colony forming units (CFU), and in this case, the dose of the Listeria strain from the second onwards is every 3 weeks. Administered to the aforementioned patient, thereby treating said cervical cancer in said human subject.

  In a related aspect, the invention relates to a method for inducing an anti-tumor cytotoxic T cell response against PRmCC in a human subject, the method comprising administering a recombinant Listeria strain to said subject, The Listeria strain comprises a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of the LLO protein fused to the HPV-E7 antigen or fragment thereof.

  In a related aspect, the invention relates to a method of eliciting an anti-tumor cytotoxic T cell response in a human subject, the method comprising a combination therapy comprising a Listeria strain disclosed herein and chemoradiotherapy. Administration.

  Other features and advantages of the present invention will become apparent from the following detailed description, examples, and drawings. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the mode for carrying out the present invention, the mode for carrying out the present invention and specific examples thereof are not While preferred embodiments of the invention are shown, it should be understood that they have been presented by way of example only.

  The subject matter relating to the invention is specifically pointed out and distinctly claimed in the concluding portion of the specification. However, both the structure and method of operation of the present invention, together with its objects, features and advantages, can best be understood by referring to the following detailed description when read in conjunction with the accompanying drawings. .

Figure 1: Lm-E7 and Lm-LLO-E7 express and secrete E7 using different expression systems. Lm-E7, It was generated by introducing a gene cassette into the orfZ domain of the monocytogenes genome (A). The hly promoter causes expression of the hly signal sequence and the first five amino acids (AA) of LLO followed by HPV-16 E7. B), Lm-LLO-E7 was generated by transforming prfA-strain XFL-7 with plasmid pGG-55. pGG-55 has an hly promoter that drives expression of a non-hemolytic fusion of LLO-E7. pGG-55 also contains the prfA gene and selects for plasmid retention by XFL-7 in vivo.

Figure 2: Lm-E7 and Lm-LLO-E7 secrete E7. Lm-Gag (lane 1), Lm-E7 (lane 2), Lm-LLO-NP (lane 3), Lm-LLO-E7 (lane 4), XFL-7 (lane 5), and 10403S (lane 6) Were grown overnight at 37 ° C. in Luria-Bertani medium. Equivalent numbers of bacteria determined by OD absorbance at 600 nm were pelleted and 18 mL of each supernatant was TCA precipitated. E7 expression was analyzed by Western blot. Blots were probed with anti-E7 mAb followed by HRP-conjugated anti-mouse (Amersham) and then developed using ECL detection reagent.

Figure 3: Tumor immunotherapy efficacy of LLO-E7 fusion Tumor size in mice is shown in millimeters at 7, 14, 21, 28 and 56 days after tumor inoculation. Naive mouse: white circle, Lm-LLO-E7: black circle, Lm-E7: square, Lm-Gag: white diamond, and Lm-LLO-NP: black triangle.

FIG. 4: Spleen cells from Lm-LLO-E7 immunized mice proliferate when exposed to TC-1 cells. C57BL / 6 mice were immunized and boosted with Lm-LLO-E7 strain, Lm-E7 strain, or control rLm strain. Splenocytes were harvested 6 days after boost and plated in culture dishes at the indicated ratio with irradiated TC-1 cells. Cells were pulse labeled with ³ H thymidine and harvested. cpm is defined as (experimental cpm)-(non-TC-1 control).

FIG. Intraspleen in mice administered TC-1 tumor cells followed by Lm-E7, Lm-LLO-E7, Lm-ActA-E7, or no vaccine (untreated) Of E7-specific IFN-gamma-secreting CD8 ⁺ T cells and the number of tumors penetrating. B. Induction and penetration of E7-specific CD8 ⁺ cells in the spleen and tumor of the mice described in (A).

FIG. 6: Listeria construct containing a PEST region that elicits a higher percentage of E7-specific lymphocytes in the tumor. A. Representative data from one experiment. B. Mean and SE of data from all three experiments.

FIG. 7A: Effect of passage on bacterial load (disease toxicity) of recombinant Listeria vaccine vector. Upper panel: Lm-Gag. Lower panel: Lm-LLO-E7. FIG. 7B: Effect of passage on bacterial load of recombinant Lm-E7 in the spleen. Shown is the average CFU of bacteria per milliliter of spleen homogenate from 4 mice.

FIG. 8: Induction of antigen-specific CD8 ⁺ T cells against HIV-Gag and LLO after administration of passaged Lm-Gag and non-passaged 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 analyzed for antigen-specific T cells. B, D, F, H: Listeria vaccine vector not passaged. AD: immune response to MHC class I HIV-Gag peptides. EH: immune response to LLO peptide. I: Splenocytes from mice immunized with 10 ⁵ CFU passaged Lm-Gag stimulated with a control peptide from HPV E7.

FIG. 9A shows plasmid separation throughout the LB stability study. FIG. 10B: shows plasmid separation throughout the TB stability study. FIG. 10C shows the quantification of the TB stability test.

FIG. 10: Number of bacterial chloramphenicol (CAP) resistant and CAP sensitive colony forming units (CFU) from bacteria grown in LB. Colored bar: CAP ⁺ ; Open bar: CAP ⁻ . Two colored bars and two open bars for each time point represent duplicate samples.

FIG. 11: Number of bacterial CAP resistant and CAP sensitive CFUs from bacteria grown in TB. Colored bar: CAP ⁺ ; Open bar: CAP. Two colored bars and two open bars for each time point represent duplicate samples.

FIG. 12: Actual chromatogram showing the region of D133V mutation (arrow). The mixing ratio is shown in parentheses.

FIG. 13: Presentation of ADV451, 452, and 453 primers and the location of the segment of the prfA gene amplified in the reaction.

FIG. 14: Specificity of PCR reaction using primers ADV451 and ADV453.

FIG. 15: Specificity of PCR reaction using primers ADV452 and ADV453.

FIG. 16: Sensitivity of PCR reaction to detect wild type prfA sequence using primer ADV452 and initial DNA amount of 1 ng.

FIG. 17: Sensitivity of PCR reaction to detect wild type prfA sequence using primer ADV452 and initial DNA amount of 5 ng.

FIG. 18: Average density of bands from the PCR illustrated in FIG.

FIG. 19: Average density of bands from the PCR illustrated in FIG.

FIG. 20: Validation of PCR reaction to detect wild type prfA sequence using primer ADV452.

FIG. 21 is the average density of bands from the PCR illustrated in FIG.

FIG. 22: Analysis of D133V prfA mutation in Lm-LLO-E7. A: Original image used for density measurement, B: Image was digitally enhanced to facilitate visualization of low density bands.

Figure 23: have PRmCC,> patients also progressed after receiving systemic treatment of ADXS11-001 once (lines), indicating the 12-month survival rate of 38.5% (n = 10/26 ).

FIG. 24: Progression-free survival (PFS) after initial treatment with ADXS11-001 was measured, indicating 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 Par Protocol treatment (3 doses of ADXS11-001). Of the 26 patients who received treatment, 18 completed all par-protocol treatment (3 doses of axarimogene filolisbac over 3 months) and median overall survival was Over one year (12.1 months), the 12-month overall survival was 55.6 percent.

FIG. 27: 12-month survival was achieved regardless of the extent of previous treatment (1-3 times (line)).

FIG. 28: Describes clinical trial summary, endpoints and key eligibility criteria for the clinical trial described in Example 12.

FIG. 29: Describes the CONSORT diagram for the clinical trial described in Example 12.

FIG. 30 describes the 12-month survival rate of the clinical trial described in Example 12 compared to the PRmCC historical COG test system.

31A and 31B: A) 6-month survival rate is 42% (10/24), median OS is 4.8 months (95% CI: 3.6-NR); median PFS is 2.6 Months (95% CI: 2.0-3.2); B) 50% (12/24) of patients who received 3 or more doses of ADXS11-001, median OS was NR (95% CI: 3. 5-NR), the median follow-up period is 9.2 months, and the 6-month survival rate is 67%.

Figure 32: Describes the clinical trial timeline in a 66 year old woman diagnosed with squamous cell cervical cancer and subsequently treated with radical hysterectomy. After 7 years of total hysterectomy, this patient received 8 cycles of paclitaxel / carboplatin (6 cycles combined with bevacizumab) → cisplatin (2 cycles) + pelvic radiation therapy. Ten months later, there was a generalized recurrence and this patient was enrolled in the clinical trial described in Example 12.

FIG. 33: shows a scanned image representing long-term complete disappearance (CR).

  In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

  As used herein, including the appended claims, the singular forms of the words “a,” “an,” and “the”, unless the context clearly dictates otherwise. Contains multiple corresponding references. All references listed herein include each published document, sequences obtained by GenBank accession number, patent applications, patents, sequence listings, nucleotide or oligopeptide or polypeptide sequences of the sequence listing, and The foregoing publications and patent document drawings are incorporated by reference to the same extent as if they were specifically and individually indicated to be incorporated by reference. The term “invention” refers to an embodiment of the invention or to some embodiments of the invention. Unless otherwise stated, the term “invention” does not necessarily refer to all embodiments of the invention.

Abbreviations The following abbreviations are used throughout the detailed description and examples of the present invention.
APC antigen-presenting cell BID twice daily CFU colony forming unit Lm Listeria monocytogenes NCBI National Biotechnology Information Center NCI National Cancer Institute PFS progression-free survival OS overall survival ORR open reading frame PRmCC persistence / Recurrent Metastatic Squamous Cell Cervical Cancer PCR Polymerase Chain Reaction Q2W Administered once every 2 weeks Q3W Administered once every 3 weeks Q4W Administered once every 4 weeks QD Administered once a day RECIST Response Evaluation Criteria in Solid Tumors (Evaluation criteria for solid tumors)
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TIL Tumor infiltrating lymphocyte UTR Untranslated region

  In one embodiment, disclosed herein is a method of treating persistent / recurrent metastatic (squamous or non-squamous cells) cervical cancer (PRmCC) in a human subject, said method comprising recombinant Administering a Listeria strain to said subject, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising an N-terminal fragment of LLO protein fused to HPV-E7 antigen or fragment thereof A first open reading frame encoding a recombinant polypeptide

In another embodiment, disclosed herein is a method of treating cervical cancer in a human subject, said method comprising administering to said subject a combination therapy comprising chemo-radiation therapy and a recombinant Listeria strain. A first open reading encoding a recombinant polypeptide comprising an N-terminal fragment of the LLO protein fused to an HPV-E7 antigen or fragment thereof. Including the frame, in which the aforementioned Listeria strain is administered at an initial dose of 5 × 10 ⁹ colony forming units (CFU), and in this case the second and subsequent administrations of the Listeria strain are administered every 3 weeks in the aforementioned patient To treat the aforementioned cervical cancer in said human subject. In another embodiment, the method includes administering to the subject at least one dose of chemoradiotherapy prior to administration of the recombinant Listeria strain. In another embodiment, the subject has received no more than two doses of chemo-radiotherapy prior to administration of the recombinant Listeria strain.

In one embodiment, a method of eliciting an anti-tumor cytotoxic T cell response against PRmCC in a human subject is disclosed herein, the method comprising administering a recombinant Listeria strain to the subject. Wherein said Listeria strain comprises a recombinant nucleic acid, said nucleic acid comprising a first open reading encoding a recombinant polypeptide comprising an N-terminal fragment of LLO protein fused to HPV-E7 antigen or fragment thereof. Contains a frame. In one embodiment, a method of eliciting an anti-tumor cytotoxic T cell response in a human subject is disclosed herein, wherein the method is as described above with an initial dose of 5 × 10 ⁹ colony forming units of a recombinant Listeria strain. And subsequent administration of the Listeria strain is administered to the aforementioned patient every 3 weeks, thereby eliciting an anti-tumor cytotoxic T cell response . In another embodiment, the aforementioned tumor is an HPV-E7 expressing tumor or an HPV-E6 expressing tumor.

  In another embodiment, a method for eliciting an anti-tumor cytotoxic T cell response in a human subject is disclosed herein, the method comprising a combination therapy comprising a Listeria strain disclosed herein and chemoradiotherapy as described above. Including administering to a subject.

In some embodiments, the present invention discloses a method for treating, preventing, and inducing an immune response thereto, wherein the method is expressed by a listeriolysin O (LLO) fragment and the aforementioned disease. Administering to the subject a recombinant Listeria strain that expresses a fusion peptide comprising the heterologous antigen or fragment thereof. The present invention relates to a method for inducing an anti-disease cytotoxic T cell (CTL) response in a human subject comprising the administration of a recombinant Listeria strain, and to treat disorders and symptoms associated with the aforementioned diseases. A method is also provided. In one embodiment, a recombinant Listeria strain, wherein said recombinant Listeria strain comprises a recombinant nucleic acid and said nucleic acid comprises a first N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof. Provided herein is a recombinant Listeria strain comprising a first open reading frame encoding a type polypeptide, and wherein the recombinant nucleic acid further comprises a second open reading frame encoding a mutant PrfA gene. ing. In one embodiment, the mutated prfA gene encodes a point mutation from amino acid D at position 133, or Asp, or aspartate (or aspartic acid) to amino acid V, or Val, or valine. It is. In another embodiment, the recombinant Listeria is an attenuated Listeria. “Attenuated” and “Attenuated” are genes in bacteria, viruses, parasites, infectious organisms, prions, tumor cells, infectious organisms that have been modified to reduce toxicity to the host, And the like. The host can be a human or animal host, or an organ, tissue, or cell. Non-limiting examples include: reducing binding to a host cell, reducing diffusion from one host cell to another, reducing extracellular growth, or host Bacteria can be attenuated to reduce intracellular growth within the cell. For example, attenuation can be assessed by measuring toxic sign (s), LD ₅₀ , removal rate from organs, or competitive index (eg, Auerbuch, et al. (2001) Infect. Immunity 69). : 5953-5957). Generally, attenuation results in at least 25%, more typically at least 50%, most typically at least 100% (2 times), typically at least 5 times more Typically at least 10 times, most typically at least 50 times, often at least 100 times, more often at least 500 times, and most often at least 1000 times, usually at least 5000 times, more usual cases It results in an increase in LD ₅₀ and / or an increased removal rate of at least 10,000 times, and most usually at least 50,000 times, and most often at least 100,000 times.

  The term “attenuated gene” may encompass a gene that mediates toxicity, pathology, or virulence to the host, grows in the host, or survives in the host, where the gene is toxic, Those of skill in the art will appreciate that mutations are made to reduce, reduce, or eliminate pathology or virulence. Reduction or elimination can be assessed by comparing the virulence or toxicity mediated by the mutated gene with that mediated by the unmutated (or parent) gene. A “mutated gene” includes deletions, point mutations, and frameshift mutations in the regulatory region of the gene, the coding region of the gene, the non-coding region of the gene, or any combination thereof. In one embodiment, provided herein is a method of treating an anal tumor or anal cancer in a human subject, the method comprising administering a combination therapy comprising chemoradiotherapy and a recombinant Listeria strain to said subject. Wherein said Listeria strain comprises a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of LLO protein fused to a heterologous antigen or fragment thereof. Thereby treating the aforementioned anal tumor or anal cancer of said human subject.

  In one embodiment, the recombinant Listeria expresses an N-terminal LLO and a heterologous antigen fusion protein. In another embodiment, the heterologous antigen is human papillomavirus E7 antigen (HPV-E7). In another embodiment, the HPV antigen is HPVE6.

In one embodiment, the patient is administered ADXS11-001 every 3 weeks for a 12 week treatment cycle. In another embodiment, dose escalation is performed using the 3 + 3 design in the following two doses: 5 × 10 ⁹ colony forming units (CFU; dose level 1) and 1 × ^{10 10} CFU (dose level 2).

  In another embodiment, the recommended phase II dose is selected based on the observed <33% dose limiting toxicity (DLT) rate.

In one embodiment, the patient is administered 1 × 10 ⁹ colony forming units (CFU) and then receives a subsequent dose every month thereafter. In another embodiment, the patient is administered 1 × 10 ⁹ colony forming units (CFUs) and then receives subsequent doses every month for the subsequent 3 months. In one embodiment, efficacy is assessed using any method known in the art, including but not limited to RECIST v1.1 and immune-related RECIST.

  In one embodiment, the terms “ADXS11-001” and “ADXS-HPV” are used interchangeably herein and contain a nucleic acid encoding a truncated LLO fused to an HPV-E7 antigen. Refers to Listeria monocytogenes.

  In one embodiment, provided herein is a chemoradiotherapy or chemoradiotherapy for use with a recombinant Listeria provided herein. In another embodiment, the chemoradiotherapy provided herein includes mitomycin and fluorouracil (5-FU) and radiation therapy. In another embodiment, the chemoradiotherapy provided herein comprises cisplatin and radiation therapy. In another embodiment, chemoradiotherapy comprises cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosourea, temozolomide, mitomycin, fluorouracil, azacitidine, azathioprine, capecitabine, cytarabine, doxyfluridine, gemcitabine, hydroxyurea, mercaptopurine, Including methotrexate, or any other chemotherapeutic agent known in the art, including but not limited to thioguanine (formerly Thioguanine).

  In one embodiment, the chemoradiotherapy provided herein comprises administering two courses of cisplatin with concurrent combination radiation (54 Gy in 30 fractions by intensity modulated radiation therapy). In another embodiment, the regimen or therapy is 2-4 cool, 4-6 cool, 6-8 cool, 8-10 cool, 10-12 cool, 12-14 cool, or 14-16 cool cisplatin or Including administering an appropriate agent simultaneously with radiation therapy. In another embodiment, radiation therapy 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. In another embodiment, radiation therapy is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, Or provided in 90-100 splits. One skilled in the art will appreciate that the clinician can adjust the dose and schedule administered to a subject as needed throughout the particular treatment regimen disclosed herein.

  In one embodiment, the subject has received median 1.5 (range 0-5) systemic chemotherapy prior to administration of the aforementioned recombinant Listeria strain.

  In one embodiment, the chemoradiotherapy disclosed herein is performed prior to the first administration of the aforementioned recombinant Listeria strain. In another embodiment, chemoradiotherapy is performed after administration of the aforementioned recombinant Listeria strain. In another embodiment, said chemoradiotherapy is performed after the first administration of said recombinant Listeria strain and before 1-3 boosters of said recombinant Listeria strain. In another embodiment, said chemoradiotherapy is performed simultaneously with said recombinant Listeria strain.

  In one embodiment, the chemoradiotherapy or chemoradiotherapy disclosed herein comprises administering two courses of cisplatin with concurrent combination radiation (54 Gy in 30 fractions by intensity modulated radiation therapy).

  In one embodiment, the radiation provided herein lasts for about 6 weeks. In another embodiment, the radiation is continued for 3 weeks. In another embodiment, the radiation is continued for 4 weeks. In another embodiment, the radiation is continued for 5 weeks. In another embodiment, the radiation is continued for 7 weeks. In another embodiment, the radiation is continued for 8 weeks. In another embodiment, the radiation is continued for 6-8 weeks. In another embodiment, the radiation is continued for 4-6 weeks. In another embodiment, the radiation lasts 2-4 weeks. In another embodiment, the radiation lasts 8-10 weeks.

  In another embodiment, disclosed herein is a method of eliciting an anti-tumor cytotoxic T cell response in a human subject comprising administering to the aforementioned subject a combination therapy disclosed herein.

  In one embodiment, disclosed herein is a method of inducing an immune response against a tumor or cancer in a human subject, said method administering a recombinant Listeria strain comprising a recombinant nucleic acid to said subject. Wherein said nucleic acid comprises 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, said recombinant nucleic acid comprising a mutated prfA gene A second open reading frame encoding, thereby eliciting an immune response against the tumor or cancer.

  In one embodiment, a method of treating cancer in a subject is disclosed herein, comprising administering to the subject a recombinant Listeria strain disclosed herein. In another embodiment, the present invention provides a method of protecting a human subject from cervical cancer, comprising administering to the subject a recombinant Listeria strain disclosed herein. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide. In another embodiment, the method further comprises boosting the human subject with a recombinant Listeria strain of the invention. In another embodiment, the method further comprises boosting the human subject with an immunogenic composition comprising a heterologous antigen or fragment thereof disclosed herein. In another embodiment, the method further comprises boosting the human subject with an immunogenic composition that directs the subject's cells to express the heterologous antigen. In another embodiment, the cell is a tumor cell. In another embodiment, the method further comprises boosting the human subject with an immunogenic composition comprising a heterologous antigen or fragment thereof disclosed herein.

  In one embodiment, the LLO protein disclosed herein and fragments thereof in the context of an ActA protein refer to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues of the LLO or ActA protein. In another embodiment, the term includes 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 acids of an LLO or ActA protein or polypeptide. Residue, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues Group, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250 contiguous amino acid residues of the amino acid sequence, at least 300 contiguous amino acid residues, at least 350 Continued amino acid residue refers to a peptide or polypeptide comprising an amino acid sequence of at least 400 contiguous amino acid residues, or at least 450 contiguous amino acid residues.

  In another embodiment, a fragment is used in the present invention (eg, 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). A functional fragment that works as intended. In another embodiment, the fragment is functional in an unfused form.

  In certain embodiments, the invention provides for codon optimization of nucleic acids that are heterologous to, or endogenous to, Listeria. L. The optimal codons utilized for each amino acid by monocytogenes are shown in US Patent Publication No. 2007/0207170, which is incorporated herein by reference. At least one codon in the nucleic acid is A nucleic acid is codon optimized if it is replaced by a codon that is used more frequently than the codon in the original sequence for that amino acid by monocytogenes.

  In another embodiment, the N-terminal LLO protein fragment and the heterologous antigen are fused directly to each other. In another embodiment, the N-terminal LLO protein fragment and the gene encoding the heterologous antigen are fused directly to each other. In another embodiment, the N-terminal LLO protein fragment and the heterologous antigen are attached via a linker peptide. In another embodiment, the N-terminal LLO protein fragment and the heterologous antigen are attached via a heterologous peptide. In another embodiment, the N-terminal LLO protein fragment is N-terminal to the heterologous antigen. In another embodiment, the N-terminal LLO protein fragment is the N most terminal portion of the fusion protein.

  As disclosed herein, recombinant Listeria strains expressing LLO-antigen fusion induce anti-tumor immunity (Example 1), elicit antigen-specific T cell proliferation (Example 2), and antigen-specificity. T cells that are targeted and infiltrate the tumor are generated (Example 3). Thus, the vaccine of the present invention is effective in eliciting an immune response against E7 and E6.

  In another embodiment, the present invention provides a method of treating cervical cancer in a human subject, comprising administering to the subject a recombinant Listeria strain, wherein the recombinant Listeria strain comprises LLO protein and A recombinant polypeptide comprising an N-terminal fragment of the HPV E7 antigen, whereby the recombinant Listeria strain elicits an immune response against the E7 antigen, thereby treating cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the present invention discloses a method for protecting a human subject from cervical cancer, comprising administering to the subject a recombinant Listeria strain, wherein the recombinant Listeria strain comprises LLO protein and HPV E7 antigen. A recombinant polypeptide comprising an N-terminal fragment of the recombinant Listeria strain, thereby eliciting an immune response against the E7 antigen, thereby protecting human subjects from cervical cancer. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the present invention discloses a method for eliciting an immune response against cervical cancer in a human subject, comprising administering to the subject a recombinant Listeria strain, Listeria strains comprise a recombinant polypeptide comprising an LLO protein and an N-terminal fragment of HPV E7 antigen, thereby eliciting an immune response against cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the present invention discloses a method of treating cervical cancer in a human subject, comprising administering to the subject a recombinant Listeria strain, wherein the recombinant Listeria strain is ActA protein and heterologous. A recombinant polypeptide comprising an N-terminal fragment of the antigen, whereby the recombinant Listeria strain elicits an immune response against the heterologous antigen, thereby treating cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the present invention discloses a method for protecting a human subject from cervical cancer, comprising administering to the subject a recombinant Listeria strain, wherein the recombinant Listeria strain comprises ActA protein and a heterologous antigen. A recombinant polypeptide comprising an N-terminal fragment, whereby the recombinant Listeria strain elicits an immune response against the heterologous antigen, thereby protecting the human subject from cervical cancer. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the present invention discloses a method for eliciting an immune response against cervical cancer in a human subject, comprising administering to the subject a recombinant Listeria strain, Listeria strains comprise a recombinant polypeptide comprising a heterologous protein and an N-terminal fragment of a heterologous antigen, thereby eliciting an immune response against cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the N-terminal ActA protein fragment and the heterologous antigen are fused directly to each other. In another embodiment, the gene encoding the N-terminal ActA protein fragment and the gene encoding the heterologous antigen are directly fused to each other. In another embodiment, the N-terminal ActA protein fragment and the heterologous antigen are attached via a linker peptide. In another embodiment, the N-terminal ActA protein fragment and the heterologous antigen are connected via a heterologous peptide. In another embodiment, the N-terminal ActA protein fragment is N-terminal to the heterologous antigen. In another embodiment, the N-terminal ActA protein fragment is the most N-terminal portion of the fusion protein.

  In another embodiment, the invention discloses a method of eliciting an immune response against cervical cancer in a human subject, comprising administering to the subject a recombinant Listeria strain, wherein the recombinant Listeria strain is a PEST amino acid. A recombinant polypeptide comprising a sequence-containing peptide and a heterologous antigen, whereby the recombinant Listeria strain elicits an immune response against the heterologous antigen, thereby treating cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide. In another embodiment, the method protects a human subject from cervical cancer. In another embodiment, the method treats cervical cancer in the aforementioned human subject.

  The PEST amino acid sequence-containing peptide and the heterologous antigen are, in another embodiment, fused directly to each other. In another embodiment, the PEST amino acid sequence-containing peptide and the gene encoding the heterologous antigen are fused directly to each other. In another embodiment, the PEST amino acid sequence-containing peptide and the heterologous antigen are attached via a linker peptide. In another embodiment, the PEST amino acid sequence-containing peptide and the heterologous antigen are attached via the heterologous peptide. In another embodiment, the PEST amino acid sequence-containing peptide is N-terminal to a heterologous antigen. In another embodiment, the PEST amino acid sequence-containing peptide is the N-terminal portion of the fusion protein.

  In another embodiment, the present invention discloses a method for vaccinating a human subject against HPV, comprising administering to the subject a recombinant Listeria strain provided herein, It expresses the HPV E7 antigen and Listeria expresses the mutated prfA gene. In another embodiment, the mutated prfA gene is a D133V prfA mutation. In another embodiment, the mutated prfA gene is in a plasmid in the aforementioned recombinant Listeria strain. In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide.

  In another embodiment, the subject is at risk of increasing HPV-mediated carcinogenicity (eg, cervical cancer). In another embodiment, the subject is HPV positive. In another embodiment, the subject exhibits a cervical intraepithelial neoplasia. In another embodiment, the subject exhibits a squamous intraepithelial lesion. In another embodiment, the subject exhibits dysplasia of the cervix.

  The HPV that is the target of the method of the invention is HPV 16 in another embodiment. In another embodiment, the HPV is HPV-18. In another embodiment, the HPV is selected from HPV-16 and HPV-18. In another embodiment, the HPV is HPV-31. In another embodiment, the HPV is HPV-35. In another embodiment, the HPV is HPV-39. In another embodiment, the HPV is HPV-45. In another embodiment, the HPV is HPV-51. In another embodiment, the HPV is HPV-52. In another embodiment, the HPV is HPV-58. In another embodiment, the HPV is a high risk HPV type. In another embodiment, the HPV is a mucosal HPV type.

  In another embodiment, the present invention discloses a method of vaccinating a human subject against an antigen of interest, wherein the method intravenously administers a recombinant Listeria strain comprising or expressing the antigen of interest to a human subject. The first peptide is selected from (a) an N-terminal fragment of the LLO protein, (b) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against the antigen of interest.

  In another embodiment, the present invention discloses a method of vaccinating a human subject against a subject antigen, the method comprising an immunogenic composition comprising a fusion of a first peptide to the subject antigen. V. Administering to a human subject, wherein the immunogenic peptide is selected from (a) an N-terminal fragment of an LLO protein, (b) a peptide containing a PEST amino acid sequence, thereby vaccine against an antigen of interest to a human subject Inoculate.

  In another embodiment, the present invention discloses a method of inoculating a human subject with a vaccine against an antigen of interest, the method comprising intravenously administering to the human subject a recombinant Listeria strain comprising the recombinant polypeptide; The recombinant polypeptide comprises a first peptide fused to an antigen of interest, wherein the first peptide is selected from (a) an N-terminal fragment of an LLO protein, (b) a peptide containing a PEST amino acid sequence, thereby human Vaccinate the subject against the antigen of interest.

  In another embodiment, the present invention discloses a method of inducing a CTL response to a subject antigen in a human subject, the method comprising administering to the human subject a recombinant Listeria strain comprising or expressing the subject antigen. Thereby inducing a CTL response to the antigen of interest within the human subject. In another embodiment, the administering step is intravenous administration.

  In another embodiment, the present invention discloses a method for inducing cancer regression in a subject, comprising administering to the subject a recombinant Listeria strain disclosed herein.

  In another embodiment, the present invention discloses a method of reducing the incidence of cancer recurrence in a subject, comprising administering to the subject a recombinant Listeria strain disclosed herein.

  In another embodiment, the present invention discloses a method of inhibiting tumor formation in a subject, comprising administering to the subject a recombinant Listeria strain disclosed herein.

  In another embodiment, the present invention discloses a method of inducing cancer remission in a subject, comprising administering to the subject a recombinant Listeria strain disclosed herein.

  In another embodiment, the present invention discloses a method of preventing tumor growth in a human subject, comprising administering to the subject a recombinant Listeria strain disclosed herein.

  In another embodiment, the present invention discloses a method for reducing the size of a tumor in a subject, comprising administering to the subject a recombinant Listeria strain disclosed herein.

  In one embodiment, the disease is an infectious disease, autoimmune disease, respiratory disease, precancerous condition, or cancer.

  The term “pre-cancerous condition” refers to dysplasia, pre-neoplastic nodule; macroregenerative nodules (MRN); mild dysplastic nodule (LG-DN); highly dysplastic nodule (HG-DN); Degenerative hepatocyte lesions (FAH); Degenerated hepatocyte nodules (NAH); Chromosome imbalance; Abnormal activation of telomerase; Reexpression of the catalytic subunit of telomerase; Endothelial cells such as CD31, CD34, and BNH9 Those skilled in the art will appreciate that it may involve the expression of markers (eg, Terracciano and Tomilo (2003) Pathology 95: 71-82; Su and Bannash (2003) Toxicol. Pathol. 31: 126). -133; Roc en and Carl-McGrath (2001) Dig.Dis.19: 269-278; Kotoula, et al. (2002) River 22: 57-69; Frachoon, 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 have been disclosed (see, eg, Riegler (1996) Semin. Gastrointest. Dis. 7: 74-87; Benvegnu, et al. (1992) Liver 12: 80-83; Giannini, et al. (1987) Hepatogastrenterol. 34: 95-97; Anthony (1976) Cancer Res. 36: 2579-2583).

  In one embodiment, the infectious disease is caused by one of (but not limited to) the following pathogens. BCG / tuberculosis, malaria, Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, rotavirus, cholera, diphtheria-tetanus, pertussis, H. influenzae, hepatitis B, human papillomavirus, seasonal influenza), pan Onset influenza A (H1N1), measles and rubella, mumps, meningococcal A + C, oral polio vaccine, monovalent, bivalent and trivalent, pneumococci, rabies, tetanus toxoid, yellow fever, Bacillus anthracis ( Anthrax), Clostridium botulinum toxin (Botulinum toxicosis), Yersinia pestis (Pest), Great pressure ulcer (Minorpoxia), and other related poxviruses, Francisella larensis (varicosis), viral hemorrhagic fever, arena Virus (LCM, Junin virus, Machupo virus Guanaritovirus, Lassa fever, Bunyavirus (Hantavirus, Rift Valley fever), Flavivirus (Dengue), Filovirus (Ebola, Marburg), Burkholderia Shade male, Koksiella Berneti (Q fever), Brucella species ( Brucellosis), Burkholderia male (nasal fin), Chlamydia sitash (parrot disease), ricin toxin (from Ricinus communis), cepstridium perfringen epsilon toxin, Staphylococcus enterotoxin B, rash typhoid (rickettsia Prowazeki), other rickettsia food and water-borne pathogens, bacteria (diarrheagenic Escherichia coli, pathogenic Vibrio, Shigella species, Salmonella BCG /, Campylobacter jejuni, Yersinia enterocolitica) Sivirus, Hepatitis A, West Nile virus, Lacrosse, California encephalitis, VEE, EEE, WEE, Japanese encephalitis virus, Kasanur forest virus, Nipah virus, Hanta virus, Tick-borne hemorrhagic fever virus, Chikungunya virus, Crimea-Congo hemorrhagic fever Virus, tick-borne encephalitis virus, hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), human papilloma virus (HPV), protozoa (cryptospodium parvum, cyclospora caietanen) Cis, rumble flagellates, dysentery amoeba, toxoplasma), fungi (microsporidia), yellow fever, tuberculosis including drug-resistant TB, rabies, prions, coronavirus severe acute respiratory symptoms (SARS-CoV) , Coccidioides posadasi, coccidioides imimitis, bacterial mania, trachoma chlamydia, cytomegalovirus, inguinal granulomas, flexible gonococci, gonorrhea, syphilis treponema, trichomonas, or in the art not listed here Any other infectious disease known.

  In another embodiment, the aforementioned infection is a livestock infection. In another embodiment, livestock diseases can be transmitted to humans and are referred to as “zoozoic infections”. In another embodiment, these diseases include foot-and-mouth disease, West Nile virus, rabies, canine parvovirus, feline leukemia virus, equine influenza virus, bovine infectious rhinotracheitis (IBR), pseudorabies, swine fever (CSF), bovine 1 IBR resulting from infection with bovine herpesvirus (BHV-1), and pseudorabies in pigs (Aujeszky's disease), toxoplasmosis, anthrax, vesicular stomatitis virus, Rhodococcus equi, savage disease, plague (Yersinia pestis), Examples include, but are not limited to, Trichomonas.

  In one embodiment, the disease provided herein is a cancer or tumor. In one embodiment, the tumor is cancerous. In another embodiment, the cancer is breast cancer. In another embodiment, the cancer is cervical cancer. In another embodiment, the cancer is a Her2 containing cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is a pancreatic cancer lesion. In another embodiment, the cancer is lung adenocarcinoma. In another embodiment, this is glioblastoma multiforme. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is lung squamous adenocarcinoma. In another embodiment, the cancer is gastric adenocarcinoma. In another embodiment, the cancer is an ovarian surface epithelial neoplasm (eg, its benign, proliferative, or malignant variant). In another embodiment, the cancer is oral squamous cell carcinoma. In another embodiment, the cancer is non-small cell lung cancer. In another embodiment, the cancer is endometrial cancer. In another embodiment, the cancer is bladder cancer. In another embodiment, the cancer is head and neck cancer. In another embodiment, the cancer is prostate cancer. In another embodiment, 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 the method of the present invention is a squamous cell carcinoma in another embodiment. In another embodiment, the cervical tumor is an adenocarcinoma. In another embodiment, the cervical tumor is adenosquamous cell 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.

  In one embodiment, the antigen provided herein is a heterologous tumor antigen, which is also referred to herein as a “tumor antigen”, “antigenic polypeptide”, or “foreign antigen”. In another embodiment, the antigen is a human papillomavirus-E7 (HPV-E7) antigen, which in one embodiment is derived from HPV16 (in one embodiment, GenBank accession number AAD33253), and another embodiment. Then, it is derived from HPV18 (in one embodiment, GenBank accession number P06788). In another embodiment, the antigenic polypeptide is HPV-E6, which in one embodiment is derived from HPV16 (in one embodiment, GenBank accession number AAD33252, AAM51854, AAM51853, or AAB67615), and another In an embodiment is derived from HPV18 (in one embodiment, GenBank accession number P06463). In another embodiment, the antigenic polypeptide is a Her / 2-neu antigen. In another embodiment, the antigenic polypeptide is prostate specific antigen (PSA) (in one embodiment, GenBank accession number CAD30844, CAD54617, AAA58802, or NP_001639). In another embodiment, the antigenic polypeptide is stratum corneum chymotrypsin-like enzyme (SCCE) antigen (in one embodiment, GenBank accession number AAK69665, AAK69624, AAG33360, AAF01139, or AAC37551). In another embodiment, the antigenic polypeptide is Wilms tumor antigen 1, which in another embodiment is WT-1 telomerase (GenBank accession number P49952, P22561, NP — 659032, CAC39220.2, or EAW68222.1). is there. In another embodiment, the antigenic polypeptide is hTERT or telomerase (GenBank accession number NM003219 (variant 1), NM198255 (variant 2), NM198253 (variant 3), or NM198254 (variant 4)). In one embodiment, the antigenic polypeptide is proteinase 3 (in one embodiment, GenBank accession number M29142, M75154, M96839, X55668, NM00277, M96628, or X56606) In another embodiment, the antigenic polypeptide. Tyrosinase-related protein 2 (TRP2) (in one embodiment, GenBank accession number NP — 001913, ABI 73976, AAP 33051, or Q95119) In another embodiment, an antigenic polypeptide Is a high molecular weight melanoma associated antigen (HMW-MAA) (in one embodiment, GenBank accession number NP_001888, AAI28111, or AAQ62842) In another embodiment, the antigenic polypeptide is testisin (in one embodiment, GenBank). Accession number AAF79020, AAF79019, AAG02255, AAK29360, AAD41588, or NP_659206. In another embodiment, the antigenic polypeptide is a NY-ESO-1 antigen (in one embodiment, GenBank accession numbers CAA05908, P78358, AAB49963, Or NP — 640343. In another embodiment, the antigenic polypeptide is PSCA (in one embodiment, GenBank accession number AAH65183, NP — 0). In another embodiment, the antigenic polypeptide is an interleukin (IL) 13 receptor alpha (in one embodiment, GenBank accession numbers NP_000631, NP_001551, NP_033871, NP_5983075, NP_001003075). In another embodiment, the antigenic polypeptide is carbonic anhydrase IX (CAIX) (in one embodiment, GenBank accession numbers CAI13455, CAI10985, EAW58359, NP_001207, NP_647466, or NP_001101426). In another embodiment, the antigenic polypeptide is a carcinoembryonic antigen (CEA) (in one embodiment, a GenBank recipient). Number AAA66186, CAA79884, CAA66955, AAA51966, AAD15250 or AAA51970,. ). In another embodiment, the antigenic polypeptide is MAGE-A (in one embodiment, GenBank accession number NP_7868585, NP_78684, NP_005352, NP_004979, NP_005358, or NP_005353). In another embodiment, the antigenic polypeptide is survivin (in one embodiment, GenBank accession number AAC51660, AAY15202, ABF60110, NP_001003019, or NP_001082350). In another embodiment, the antigenic polypeptide is GP100 (in one embodiment, GenBank accession number AAC60634, YP — 655861, or AAB31176). In another embodiment, the antigenic polypeptide is any other antigenic polypeptide known in the art. In another embodiment, the antigenic peptide of the composition and method of the invention comprises an immunogenic portion of the antigenic polypeptide.

  In another embodiment, 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 carbonic 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. In another embodiment, the antigen is 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, Muc1, FAP, IL-13Rα2, PSA (prostate specific antigen), gp-100, heat shock protein 70 (HSP-70), β-HCG, EGFR- III, granulocyte colony stimulating factor (G-CSF), angiogenin, angiopoietin-1, Del-1, fibroblast growth factor: acidic (aFGF) or basic (bFGF), follistatin, granulocyte colony stimulating factor (G -CSF), hepatocyte growth factor (HGF) / dispersion factor (SF), interleukin-8 ( L-8), leptin, midkine, placental growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), progranulin, proliferin, trait Transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF) / vascular permeability factor (VPF), VEGFR, VEGFR2 (KDR / FLK-1) or fragment thereof, FLK-1 or epitope thereof, FLK-E1, FLK-E2, FLK-I1, endoglin or fragment thereof, neuropilin 1 (NRP-1), angiopoietin 1 (Ang1), Tie2, platelet derived growth factor (PDGF), platelet derived growth factor receptor (PDG) R), transforming growth factor-β (TGF-β), endoglin, TGF-β receptor, monocyte chemotactic protein-1 (MCP-1), VE-cadherin, CD31, ephrin, ICAM-1, V-CAM-1, VAP-1, E-selectin, plasminogen activator, plasminogen activator inhibitor-1, nitric oxide synthase (NOS), COX-2, AC133, or Id1 / Id3 , Angiopoietin 3, Angiopoietin 4, Angiopoietin 6, CD105, EDG, HHT1, ORW, ORW1 or TGFβ co-receptor, or combinations thereof. In one embodiment, the antigen is a chimeric Her2 / neu antigen described in US Patent Application Publication No. 2011/0142791, which is hereby incorporated by reference in its entirety. The use of fragments of the antigens disclosed herein is also encompassed by the present invention.

  In another embodiment, the heterologous tumor antigen provided herein is a tumor associated antigen, which in one embodiment is one of the following tumor antigens: MAGE (melanoma associated antigen E) protein, eg, MAGE 1, MAGE 2, MAGE 3, MAGE 4, tyrosinase; mutant ras protein; mutant p53 protein; p97 melanoma antigen, ras peptide or p53 peptide associated with advanced cancer HPV16 / 18 antigen associated with cervical cancer, KLH antigen associated with breast cancer, CEA (carcinoembryonic antigen) associated with colon cancer, MART1 antigen associated with melanoma, or PSA associated with prostate cancer antigen. In another embodiment, the antigen for the compositions and methods provided herein is a melanoma-associated antigen, which in one embodiment is TRP-2, MAGE-1, MAGE-3, gp. -100, tyrosinase, HSP-70, β-HCG, or combinations thereof. Those skilled in the art will appreciate that any heterologous antigen not described herein but known in the art could be used in the methods and compositions provided herein. It should also be understood that the present invention provides, but is not limited to, attenuated Listeria comprising a nucleic acid encoding at least one of the antigens disclosed herein. The present invention encodes mutants, mutant proteins, splice variants, fragments, truncation variants, soluble variants, extracellular domains, intracellular domains, mature sequences, and the like of the disclosed antigens Nucleic acids to be included. Nucleic acids encoding the epitopes, oligos, and polypeptides of these antigens are provided. Also provided are codon optimized embodiments that optimize expression in Listeria. All cited references, GenBank accession numbers, and nucleic acids, peptides, and polypeptides disclosed herein are hereby incorporated by reference in their entirety. In another embodiment, the selected nucleic acid sequence can encode a full-length gene or truncation gene, fusion gene or tagged gene, which can be cDNA, genomic DNA, or a DNA fragment, preferably It can be cDNA. This can be mutated or otherwise modified as desired. These modifications include codon optimization to optimize codon usage within the selected host cell or bacterium, ie, Listeria. The selected sequence can also encode a secreted polypeptide, cytoplasmic polypeptide, nuclear polypeptide, membrane-bound polypeptide, or cell surface polypeptide.

  In one embodiment, vascular endothelial growth factor (VEGF) is an important signaling protein involved in both angiogenesis (embryonic circulation formation) and angiogenesis (blood vessel growth from existing vasculature). In one embodiment, VEGF activity is primarily restricted to cells of the vascular endothelium, which also has an effect on a limited number of other cell types (eg, stimulated monocyte / macrophage migration). In vitro, VEGF has been shown to stimulate endothelial cell mitogenesis and cell migration. This is often referred to as vascular permeability factor because VEGF also enhances microvascular permeability.

  In one embodiment, all members of the VEGF family stimulate cellular responses by binding to the tyrosine kinase receptor (VEGFR) on the cell surface, causing them to dimerize and activate through transphosphorylation. Let The VEGF receptor has an extracellular portion that is composed of seven immunoglobulin-like domains, a single transmembrane region, and an intracellular portion that contains a split tyrosine kinase domain.

  In one embodiment, VEGF-A is a VEGFR-2 (KDR / Flk-1) ligand, as well as a VEGFR-1 (Flt-1) ligand. In one embodiment, VEGFR- mediates most known cellular responses to VEGF. Although the function of VEGFR-1 is not well defined, it is believed that in one embodiment it regulates VEGFR-2 signaling through sequestration of VEGF from VEGFR-2 binding, and in one embodiment this is within the embryo. It is particularly important during angiogenesis. In one embodiment, VEGF-C and VEGF-D are ligands for the VEGFR-3 receptor, which in one embodiment mediates lymphangiogenesis.

  In one embodiment, the composition of the invention comprises a VEGF receptor or fragment thereof, which in one embodiment is VEGFR-2, in another embodiment, VEGFR-1, In an embodiment, it is VEGFR-3.

  In one embodiment, vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed on activated endothelial cells (EC) and is involved in the formation of new blood vessels. In one embodiment, VEGFR2 binds all five isoforms of VEGF. In one embodiment, VEGF signaling through VEGFR2 on EC induces proliferation, migration, and terminal differentiation. In one embodiment, the mouse homologue of VEGFR2 is fetal liver kinase gene-1 (Flk-1), which is a potent therapeutic target and has an important role in tumor growth, invasion, and metastasis. In one embodiment, VEGFR2 is also a kinase insert domain receptor (type III receptor tyrosine kinase) (KDR), surface antigen class 309 (CD309), FLK1, Ly73, Krd-1, VEGFR, VEGFR-2, or 6130401C07 Called.

  In another embodiment, the antigen is derived from a fungal pathogen, bacteria, parasite, helminth, or virus. In another embodiment, the antigen is a tetanus toxoid, hemagglutinin molecule from influenza virus, diphtheria toxoid, HIV gp120, HIV gag protein, IgA protease, insulin peptide B, powdery scab fungus antigen, vibriobacterium antigen, Salmonella antigen, pneumococcal antigen, respiratory syncytial virus antigen, H. influenzae outer membrane protein, Helicobacter pylori urease, Neisseria meningitidis pilin, Neisseria gonorrhoeae pilin, melanoma-related antigen (TRP-2, MAGE-1, MAGE- 3, gp-100, tyrosinase, MART-1, HSP-70, β-HCG), type HPV-16, -18, -31, -33, -35, or -45, human papillomavirus antigens E1 and E2, Tumor antigen CEA, mutated or Not ras protein, mutated or non p53 protein is selected from Muc1 or pSA,.

  In other embodiments, the antigen is associated with one of the following diseases. Cholera, diphtheria, hemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, smallpox, pneumococcal pneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid, varicella zoster Pertussis 3, yellow fever, immunogen and antigen from Addison's disease, allergy, anaphylaxis, Bruton syndrome, cancer including solid and blood tumors, eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, type 1 diabetes, Acquired immune deficiency syndrome, transplant rejection such as kidney, heart, pancreas, lung, bone, and liver, Graves' disease, polyendocrine autoimmune disease, hepatitis, microscopic polyarteritis, nodular polyarteritis, pemphigus , Primary biliary cirrhosis, pernicious anemia, celiac disease, antibody-mediated nephritis, glomerulonephritis, rheumatic disease, systemic lupus erythematosus, arthritis Machi, seronegative spondyloarthropathy, rhinitis, Sjogren's syndrome, systemic sclerosis, sclerosing cholangitis, Wegener's granulomatosis, herpes zosteritis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain Valley Syndrome, myasthenia gravis, Lambert Eaton myasthenia syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, hives, infant transient hypogammaglobulinemia, myeloma, X-linked high Igm Syndrome, Wiscott-Aldrich syndrome, telangiectasia ataxia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia, Waldenstrom macroglobulinemia, amyloidosis , Chronic lymphocytic leukemia, non-Hodgkin lymphoma, malaria perisporozoite protein, microbial antigen, viral antigen, autoantigen, and lister A disease.

  In another embodiment, in the method of the invention for treating cervical cancer, protecting from cervical cancer, or eliciting an immune response against cervical cancer, in place of or in addition to E7 antigen HPV E6 antigen is utilized.

  In another embodiment, in the methods disclosed herein for treating cervical cancer, protecting against cervical cancer, or eliciting an immune response against cervical cancer, instead of LLO fragment or LLO fragment In addition, ActA protein fragments are utilized.

  In another embodiment, in the method of the invention for treating cervical cancer, protecting from cervical cancer, or eliciting an immune response against cervical cancer, instead of or in addition to LLO fragment PEST amino acid sequence-containing protein fragments are utilized.

  In another embodiment, the present invention provides a method for inducing an anti-E7 cytotoxic T cell (CTL) response in a human subject, comprising administering a recombinant Listeria strain to the subject, Type Listeria strains comprise a recombinant polypeptide comprising an LLO protein and an N-terminal fragment of HPV E7 antigen, thereby inducing an anti-E7 CTL response in a human subject. In another embodiment, the recombinant Listeria strain comprises a plasmid encoding the recombinant polypeptide. In another embodiment, the method further comprises boosting the subject with a recombinant Listeria strain of the invention. In another embodiment, the method further comprises boosting the subject with an immunogenic composition comprising the E7 antigen. In another embodiment, the method further comprises boosting the subject with an immunogenic composition that directs the subject's cells to express the E7 antigen. In another embodiment, the CTL response has the ability to be therapeutically effective against HPV mediated diseases, HPV mediated disorders, or HPV mediated symptoms. In another embodiment, the CTL response has the ability of prophylactic efficacy against HPV mediated diseases, HPV mediated disorders, or HPV mediated symptoms.

  In another embodiment, the invention provides a method of treating or ameliorating a subject's HPV-mediated disease, HPV-mediated disorder, or HPV-mediated condition, comprising administering a recombinant Listeria strain to the subject. Wherein the recombinant Listeria strain comprises a recombinant polypeptide comprising an LLO protein and an N-terminal fragment of HPV E7 antigen, whereby the recombinant Listeria strain elicits an immune response against the E7 antigen, whereby To treat or ameliorate an HPV-mediated disease, HPV-mediated disorder, or HPV-mediated condition. In another embodiment, the subject is a human subject. In another embodiment, the subject is a non-human mammal. In another embodiment, the subject is any other type of subject known in the art.

  In another embodiment, the HPV causing the disease, disorder, or symptom is HPV 16. In another embodiment, the HPV is HPV-18. In another embodiment, the HPV is HPV-31. In another embodiment, the HPV is HPV-35. In another embodiment, the HPV is HPV-39. In another embodiment, the HPV is HPV-45. In another embodiment, the HPV is HPV-51. In another embodiment, the HPV is HPV-52. In another embodiment, the HPV is HPV-58. In another embodiment, the HPV is a high risk HPV type. In another embodiment, the HPV is a mucosal HPV type.

  In another embodiment, the HPV-mediated disease, HPV-mediated disorder, or HPV-mediated condition is genital wart. In another embodiment, the HPV-mediated disease, HPV-mediated disorder, or HPV-mediated condition is non-genital warts. In another embodiment, the HPV-mediated disease, HPV-mediated disorder, or HPV-mediated condition is respiratory papilloma. In another embodiment, the HPV-mediated disease, HPV-mediated disorder, or HPV-mediated condition is any other HPV-mediated disorder, HPV-mediated disorder, or HPV-mediated condition known in the art. .

  In another embodiment, HPV E6 antigen is utilized to treat or ameliorate an HPV mediated disease, disorder, or symptom in place of or in addition to the E7 antigen of the methods disclosed herein. The

  In another embodiment, ActA protein fragments are utilized to treat or ameliorate HPV-mediated diseases, disorders, or symptoms in place of or in addition to LLO fragments of the methods disclosed herein. The

  In another embodiment, the PEST amino acid sequence-containing protein fragment is used to treat or ameliorate an HPV-mediated disease, disorder, or symptom in place of or in addition to the LLO fragment of the methods disclosed herein. Used for

  In another embodiment, HPV E6 antigen is utilized to treat or ameliorate an HPV mediated disease, disorder, or symptom in place of or in addition to the E7 antigen of the methods disclosed herein. The

  The antigen of the methods and compositions disclosed herein is, in another embodiment, HPV E7 protein. In another embodiment, the antigen is HPV E6 protein. In another embodiment, 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 the methods and compositions disclosed herein is, in another embodiment, an HPV 16 E7 protein. In another embodiment, the E7 protein is an HPV-18 E7 protein. In another embodiment, the E7 protein is an HPV-31 E7 protein. In another embodiment, the E7 protein is an HPV-35 E7 protein. In another embodiment, the E7 protein is an HPV-39 E7 protein. In another embodiment, the E7 protein is an HPV-45 E7 protein. In another embodiment, the E7 protein is an HPV-51 E7 protein. In another embodiment, the E7 protein is an HPV-52 E7 protein. In another embodiment, the E7 protein is an HPV-58 E7 protein. In another embodiment, the E7 protein is a high risk HPV type E7 protein. In another embodiment, the E7 protein is a mucosal HPV type E7 protein.

  “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 the methods and compositions disclosed herein is, in another embodiment, an HPV 16 E6 protein. In another embodiment, the E6 protein is an HPV-18 E6 protein. In another embodiment, the E6 protein is an HPV-31 E6 protein. In another embodiment, the E6 protein is an HPV-35 E6 protein. In another embodiment, the E6 protein is an HPV-39 E6 protein. In another embodiment, the E6 protein is an HPV-45 E6 protein. In another embodiment, the E6 protein is an HPV-51 E6 protein. In another embodiment, the E6 protein is an HPV-52 E6 protein. In another embodiment, the E6 protein is an HPV-58 E6 protein. In another embodiment, the E6 protein is a high risk HPV type E6 protein. In another embodiment, the E6 protein is a mucosal HPV type E6 protein.

In another embodiment, the immune response elicited by the methods and compositions of the present invention is a T cell response. In another embodiment, the immune response comprises a T cell response. In another embodiment, the response is a CD8 ⁺ T cell response. In another embodiment, the response comprises a CD8 ⁺ T cell response.

  In another embodiment, the N-terminal LLO protein fragment of the methods and compositions of the invention comprises SEQ ID NO: 2. In another embodiment, the fragment comprises an LLO signal peptide. In another embodiment, the fragment comprises SEQ ID NO: 2. In another embodiment, the fragment consists essentially of SEQ ID NO: 2. In another embodiment, the fragment consists essentially of SEQ ID NO: 2. In another embodiment, the fragment corresponds to SEQ ID NO: 2. In another embodiment, the fragment is homologous to SEQ ID NO: 2. In another embodiment, the fragment is homologous to the fragment of SEQ ID NO: 2. The ΔLLO used in some examples was 416 AA long (not including signal sequence) since 88 residues from the amino terminus including the activation domain containing cysteine 484 were cleaved. It was. It will be apparent to those skilled in the art that any ΔLLO that does not have an activation domain and in particular does not have cysteine 484 is suitable for the methods and compositions of the present invention. In another embodiment, fusion of the E7 or E6 antigen to any ΔLLO comprising the PEST amino acid AA sequence, SEQ ID NO: 6, enhances the mediated cell and antigen anti-tumor immunity.

The LLO protein utilized to construct the vaccine of the present invention, in another embodiment, has the following sequence:
(GenBank accession number P13128; SEQ ID NO: 1; the nucleic acid sequence is represented by GenBank accession number X15127 (SEQ ID NO: 33)). The first 25 AA of the proprotein corresponding to this sequence is a signal sequence, which is cleaved from it when LLO is secreted by the bacteria. Thus, in this embodiment, the full length active LLO protein is 504 residues long. In another embodiment, the above LLO fragments are used as a source of LLO fragments that are incorporated into the vaccines of the invention.

In another embodiment, the N-terminal fragment of the LLO protein utilized in the compositions and methods of the invention has the following sequence:
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD (SEQ ID NO: 2).

  In another embodiment, the LLO fragment corresponds to approximately AA 20-442 of the LLO protein utilized herein.

In another embodiment, the LLO fragment has the following sequence:
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTD (SEQ ID NO: 3).

  In another embodiment, “truncated LLO” or “ΔLLO” refers to a fragment of LLO comprising a PEST amino acid domain. In another embodiment, these terms refer to an LLO fragment that includes a PEST sequence.

  In another embodiment, the term refers to an LLO fragment that does not contain an activation domain at the amino terminus and does not contain cysteine 484. In another embodiment, these terms refer to LLO fragments that are not hemolytic. In another embodiment, the LLO fragment is rendered non-hemolytic by an activation domain deletion or mutation. 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 elsewhere.

  In another embodiment, the LLO fragment consists of approximately the first 441 AA of the LLO protein. In another embodiment, the LLO fragment consists of approximately the first 420 AA of the LLO. In another embodiment, the LLO fragment is a non-hemolytic form of the LLO protein.

  In another embodiment, the LLO fragment contains residues of a homologous LLO protein corresponding to one of the above AA ranges. In another embodiment, the residue numbers do not necessarily correspond exactly to the residue numbers listed above, eg, homologous LLO proteins are inserted or deleted compared to the LLO proteins utilized herein. The residue number can be adjusted as appropriate.

  In another embodiment, the LLO fragment is any other LLO fragment known in the art.

In another embodiment, the dose is 5-500 × 10 ⁸ CFU. In another embodiment, the dose is 7-500 × 10 ⁸ CFU. In another embodiment, the dose is 10-500 × 10 ⁸ CFU. In another embodiment, the dose is 20-500 × 10 ⁸ CFU. In another embodiment, the dose is 30-500 × 10 ⁸ CFU. In another embodiment, the dose is 50-500 × 10 ⁸ CFU. In another embodiment, the dose is 70-500 × 10 ⁸ CFU. In another embodiment, the dose is 100-500 × 10 ⁸ CFU. In another embodiment, the dose is 150-500 × 10 ⁸ CFU. In another embodiment, the dose is 5-300 × 10 ⁸ CFU. In another embodiment, the dose is 5-200 × 10 ⁸ CFU. In another embodiment, the dose is 5-150 × 10 ⁸ CFU. In another embodiment, the dose is 5-100 × 10 ⁸ CFU. In another embodiment, the dose is 5-70 × 10 ⁸ CFU. In another embodiment, the dose is 5-50 × 10 ⁸ CFU. In another embodiment, the dose is 5-30 × 10 ⁸ CFU. In another embodiment, the dose is 5-20 × 10 ⁸ CFU. In another embodiment, the dose is 1-30 × 10 ⁹ CFU. In another embodiment, the dose is 1-20 × 10 ⁹ CFU. In another embodiment, the dose is 2-30 × 10 ⁹ CFU. In another embodiment, the dose is 1-10 × 10 ⁹ CFU. In another embodiment, the dose is 2-10 × 10 ⁹ CFU. In another embodiment, the dose is 3-10 × 10 ⁹ CFU. In another embodiment, the dose is 2-7 × 10 ⁹ CFU. In another embodiment, the dose is 2-5 × 10 ⁹ CFU. In another embodiment, the dose is 3-5 × 10 ⁹ CFU.

In another embodiment, the dose is 1 × 10 ⁹ organism. In another embodiment, the dose is 1.5 × 10 ⁹ organisms. In another embodiment, the dose is 2 × 10 ⁹ organisms. In another embodiment, the dose is 3 × 10 ⁹ organisms. In another embodiment, the dose is 4 × 10 ⁹ organisms. In another embodiment, the dose is 5 × 10 ⁹ organisms. In another embodiment, the dose is 6 × 10 ⁹ organisms. In another embodiment, the dose is 7 × 10 ⁹ organisms. In another embodiment, the dose is 8 × 10 ⁹ organisms. In another embodiment, the dose is 10 × 10 ⁹ organisms. In another embodiment, the dose is 1.5 × 10 ¹⁰ organisms. In another embodiment, the dose is 2 × 10 ¹⁰ organisms. In another embodiment, the dose is 2.5 × 10 ¹⁰ organisms. In another embodiment, the dose is 3 × 10 ¹⁰ organisms. In another embodiment, the dose is 3.3 × 10 ¹⁰ organisms. In another embodiment, the dose is 4 × 10 ¹⁰ organisms. In another embodiment, the dose is 5 × 10 ¹⁰ organisms.

In another embodiment, the recombinant Listeria strain is administered to the human subject at a dose of 1 × 10 ^{9 to} 3.31 × 10 ¹⁰ CFU. In some embodiments, dosage levels below the lower limit of the aforementioned range may be sufficient, while in other cases, larger doses may still be used as determined by one skilled in the art.

In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then every third week (Q3W) at a dose of 1 × ^{10 10} CFU for subsequent doses. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU for a 12 week cycle, and then every 3 weeks at doses of 1 × ^{10 10} CFU for subsequent doses. In another embodiment, the recombinant Listeria strain is administered at a first dose of 5 × 10 ⁹ CFU for a 12-week cycle, and then every 3 weeks at a dose of 5 × ^{10 8} CFU-1 × ^{10 10} CFU for the second and subsequent cycles. The In another embodiment, the recombinant Listeria strain is administered at a first dose of 5 × 10 ⁹ CFU for a 24 week cycle, and then every third week at a dose of 1 × ^{10 10} CFU for subsequent doses. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU for a 48-week cycle, and then every 3 weeks for subsequent doses at a dose of 1 × ^{10 10} CFU. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at doses of 1 × ^{10 10} CFU for subsequent doses every 3 weeks until at least 50% tumor regression is obtained. The In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at subsequent doses of 5 × ^{10 8} CFU-1 × ^{10 10} CFU for at least 50% tumor regression for 3 weeks. Every dose. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at subsequent doses of 1 × ^{10 10} CFU every 3 weeks until at least 70-80% tumor regression is obtained. Be administered. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at subsequent doses of 1 × ^{10 10} CFU every 3 weeks until at least 80-90% tumor regression is obtained. Be administered. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at subsequent doses of 1 × ^{10 10} CFU every 3 weeks until complete tumor regression is obtained. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at subsequent doses of 5 × ^{10 8} CFU-1 × ^{10 10} CFU every 3 weeks until complete tumor regression is obtained. Be administered. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at subsequent doses of 5 × ^{10 8} CFU-1 × ^{10 10} CFU every 3 weeks for 3 doses Or is administered repeatedly every 3 weeks until the patient is confirmed for disease progression or until complete disappearance. In another embodiment, the recombinant Listeria strain is administered at an initial dose of 5 × 10 ⁹ CFU and then at a dose of 1 × ^{10 10} CFU for subsequent doses administered every 3 weeks for 3 doses, or patient Is administered repeatedly every 3 weeks until confirmation of disease progression or until complete disappearance is experienced.

  In one embodiment, the Listeria strain disclosed herein is administered to a subject as an 80 ml infusion over 15 minutes. In another embodiment, the Listeria strain disclosed herein is 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 It is administered as an infusion of ˜150 ml, 150-200 ml, 200-250 ml, or 250-300 ml. In another embodiment, the Listeria strain disclosed herein is administered as an 80 ml to 250 ml infusion. In another embodiment, the Listeria strain disclosed herein is for 5-10 minutes, 10-20 minutes, 20-30 minutes, 30-40 minutes, 40-50 minutes, or 50- Administered over 60 minutes. One skilled in the art will recognize that the higher the infusion volume, the longer the administration time.

  In some embodiments, the treatment cycle begins on the first day of the combination therapy and lasts at least 12 weeks, 24 weeks or 48 weeks. In another embodiment, the treatment cycle begins on the first day of the combination therapy administration and lasts at least 12 weeks, 24 weeks or 48 weeks. In another embodiment, the treatment cycle begins on the first day of chemoradiotherapy administration and lasts at least 12 weeks, 24 weeks or 48 weeks. In other embodiments, the treatment cycle is initiated on the first day of administration of the recombinant Listeria disclosed herein or a composition comprising the Listeria and continues for at least 12 weeks, 24 weeks, or 48 weeks.

  In another embodiment, a single maintenance dose or booster dose is administered at intervals after the treatment cycle disclosed herein.

  In another embodiment, the term “second and subsequent” and “booster” doses of recombinant Listeria are used herein in that they are administered after the initial administration of the recombinant Listeria disclosed herein. Used interchangeably in the book.

  In another embodiment, the booster dose is administered at Q2W, Q4W. In another embodiment, booster doses are administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, followed by a single booster dose every 2 months. In another embodiment, booster doses are administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, followed by a single booster dose every 3 months. In another embodiment, booster doses are administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, followed by a single booster dose every 2 months for 1 year. In another embodiment, booster doses are administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, followed by a single booster dose every 3 months for 1 year. In another embodiment, booster doses are administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, and then a single booster dose is administered every 2 months for at least 1 year . In another embodiment, the booster dose is administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, then a single booster dose is administered every 3 months for at least 1 year . In another embodiment, the booster dose is administered every 3 weeks (3 weeks x 3 cycles) for a total of 9 doses, then a single booster every 2 or 3 months for 1-3 years A dose is administered.

  In one embodiment, the booster dose is administered monthly for a total or 3 doses after the initial administration. In another embodiment, the booster dose is administered monthly relative to the total number after the initial administration, with a total of 4-10 doses administered. In another embodiment, the booster dose is administered monthly relative to the total number after the initial administration, with a total of 10-20 doses administered

  In one embodiment, the booster dose is administered every 3 or 4 weeks after the initial administration, for a total of 3 doses administered. In another embodiment, the booster dose is administered every 3 or 4 weeks for a total after the initial administration, with a total of 4-10 doses administered. In another embodiment, the booster dose is administered every 3 or 4 weeks for a total after the initial administration, with a total of 10-20 doses administered.

  In one embodiment, dosing is continued every 3-4 weeks until disease progresses or disappears completely.

  Other dosing changes are the same q4 weeks, especially when concomitantly better with the combination regimen, and hybrid to adjuvant treatment, in which case patients receive q3 weeks x3 “cycle” dosing Then receive a single infusion of the same dose as a “booster” or “maintenance” dose every 2 or 3 months until a certain time point (1-2 years). In another embodiment, the method disclosed herein is followed by a single maintenance dose or booster dose at a predetermined interval, including but not limited to the interval disclosed herein. Is called.

  In another embodiment, the recombinant polypeptide of the method of the invention is expressed by a recombinant Listeria strain. In another embodiment, expression is mediated by nucleotide molecules carried by the recombinant Listeria strain.

  In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide with a plasmid encoding the recombinant polypeptide. In another embodiment, the plasmid contains a gene encoding a bacterial transcription factor. In another embodiment, the plasmid encodes a Listeria transcription factor. In another embodiment, the transcription factor is prfA. In another embodiment, prfA is a mutated prfA. In another embodiment, prfA in said plasmid within said Listeria encodes a D133V amino acid mutation. In another embodiment, the transcription factor is any other transcription factor known in the art.

  In another embodiment, the plasmid contains a gene encoding a metabolic enzyme. In another embodiment, the metabolic enzyme is a bacterial metabolic enzyme. In another embodiment, the metabolic enzyme is a Listeria metabolic enzyme. In another embodiment, the metabolic enzyme is an amino acid metabolic enzyme. In another embodiment, the amino acid metabolism gene is involved in the cell wall synthesis pathway. In another embodiment, the metabolic enzyme is the product of a D-amino acid aminotransferase gene (dat). In another embodiment, the metabolic enzyme is the product of an alanine racemase gene (dal). In another embodiment, the metabolic enzyme is any other metabolic enzyme known in the art.

  In another embodiment, the method of the invention further comprises boosting a human subject with the recombinant Listeria strain of the invention. In another embodiment, the recombinant Listeria strain used in the booster inoculation is the same as the strain used in the initial “preparation” inoculation. In another embodiment, the booster strain is different from the primed strain. In another embodiment, the same dose is used in prime and booster inoculations. In another embodiment, a larger dose is used for the booster. In another embodiment, a smaller dose is used for the booster.

  In another embodiment, the methods of the invention further comprise inoculating a human subject with an immunogenic composition comprising an E7 antigen. In another embodiment, the immunogenic composition comprises a recombinant E7 protein or fragment thereof. In another embodiment, the immunogenic composition comprises a nucleotide molecule that expresses a recombinant E7 protein or fragment thereof. In another embodiment, non-Listeria inoculation is performed after Listeria inoculation. In another embodiment, non-Listeria inoculation is performed prior to Listeria inoculation.

  In another embodiment, “boosting” refers to administration of an additional vaccine dose to a subject. In another embodiment of the method of the invention, two boosts (ie, a total of three inoculations) are administered. In another embodiment, 3 boosts are administered. In another embodiment, 4 boosts are administered. In another embodiment, 5 boosts are administered. In another embodiment, 6 boosts are administered. In another embodiment, 7 or more boosts are administered.

  In another embodiment, the recombinant Listeria strain of the methods and compositions of the present invention is a recombinant Listeria monocytogenes strain. In another embodiment, the Listeria strain is a recombinant Listeria shiligeri strain. In another embodiment, the Listeria strain is a recombinant Listeria gray strain. In another embodiment, the Listeria strain is a recombinant Listeria Ivanobi strain. In another embodiment, the Listeria strain is a recombinant Listeria mula strain. In another embodiment, the Listeria strain is a recombinant Listeria Welsimelli strain. In another embodiment, the Listeria strain is a recombinant strain of any other Listeria species known in the art.

  The present invention provides several Listeria species and strains for making or modifying the attenuated Listeria of the present invention. In one embodiment, 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). Then, the Listeria strain is L. Monocytogenes DP-L4056 (phage curing) (see Lauer, et al. (2002) J. Bact. 184: 4177-4186). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4027, which is phage-cured and deleted within the hly gene (Lauer, et al. (2002) J. Bact. 184: 4177-4186; Jones and Portnoy (1994) Infect.Immunity 65: 5608-5613). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4029, which is phage-cured and deleted in ActA (Lauer, et al. (2002) J. Bact. 184: 4177-4186; Skobble, et al. (2000). J. Cell Biol. 150: 527-538). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4042 (Delta PEST) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4097 (LLO-S44A) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4364 (Delta lplA, lipote protein ligase) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4405 (Delta inlA) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4406 (Delta inlB) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes CS-L0001 (Delta ActA-Delta inlB) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes CS-L0002 (Delta ActA-Delta lplA) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes CS-L0003 (L461T-Delta lplA) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4038 (Delta ActA-LLO L461T) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4384 (S44A-LLO L461T) (see Blockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. Monocytogenes. Mutations in lipoic acid protein (see O'Riordan, et al. (2003) Science 302: 462-464). In another embodiment, the Listeria strain is L. Monocytogenes DP-L4017 (10403S hly (L461T) with a point mutation in the hemolysin gene (see US Provisional Application No. 60 / 490,089, filed July 24, 2003). In another embodiment, the Listeria strain is L. monocytogenes EGD (see GenBank accession number AL591824) In another embodiment, the Listeria strain is L. monocytogenes EGD-e (GenBank accession number NC — 003210). ATCC Accession No. BAA-679) In another embodiment, the Listeria strain is a L. monocytogenes uvrAB lacking DP-L4029 (US Patent Provisional Application No. 60/60, filed Feb. 2, 2004). No. 541,515, U.S. Provisional Application No. 60/4, filed July 24, 2003 In another embodiment, the Listeria strain is an L. monocytogenes ActA− / inlB− double mutant (see ATCC accession number PTA-5562) In another embodiment, Listeria. The strain is an L. monocytogenes lplA mutant or an hly mutant (see Portnoy et al. US Patent Application No. 20040136690) In another embodiment, the Listeria strain is an L. monocytogenes DAL / DAT (See US Patent Application No. 2005048081 (Frankel and Portnoy applications).) The present invention includes the Listeria strains described above, as well as hly (LLO; Listeriolysin); iap (p60); inlA; inlB; inlC; dal (alanine racemase); dat (D -Amino acid aminotransferase); plcA; plcB; those strains modified to contain nucleic acids encoding one or any combination of the genes of actA, for example by plasmid and / or genomic integration, or growth, spread And reagents and methods comprising any nucleic acid that mediates the degradation of single walled vesicles, the degradation of double walled vesicles, binding to host cells, and uptake by host cells. Is not limited by the particular strain disclosed above.

  In another embodiment, the recombinant Listeria strain of the present invention has been passaged through an animal host. In another embodiment, passaging maximizes the effectiveness of the strain as a vaccine vector. In another embodiment, the passaging stabilizes the immunogenicity of the Listeria strain. In another embodiment, the passaging stabilizes the pathogenic toxicity of the Listeria strain. In another embodiment, the passaging increases the immunogenicity of the Listeria strain. In another embodiment, the passaging increases the virulence of the Listeria strain. In another embodiment, the passaging removes an unstable subline of the Listeria strain. In another embodiment, the passaging reduces the prevalence of unstable sublines of the Listeria strain. In another embodiment, the Listeria strain comprises a genomic insert of a gene encoding an antigen-containing recombinant peptide. In another embodiment, the Listeria strain carries a plasmid containing a gene encoding an antigen-containing recombinant peptide. In another embodiment, passaging is performed as described herein (eg, in Example 12). In another embodiment, the passaging is performed by any other method known in the art.

  In another embodiment, the recombinant Listeria strain utilized in the methods of the invention is stored in a frozen cell bank. In another embodiment, the recombinant Listeria strain is stored in a frozen cell bank.

  In another embodiment, the cell bank of the methods and compositions of the invention is a master cell bank. In another embodiment, the cell bank is a working cell bank. In another embodiment, the cell bank is a manufacturing and quality control standard (GMP) cell bank. In another embodiment, the cell bank is intended for the production of clinical grade material. In another embodiment, the cell bank is compatible with regulatory enforcement for human use. In another embodiment, the cell bank is any other type of cell bank known in the art.

  In another embodiment, “standards for manufacturing control and quality control” are defined by (21 CFR 210-211) of the US Federal Regulations. In another embodiment, “standards for manufacturing control and quality control” are defined by other standards for clinical grade material production or for human consumption, such as standards from countries other than the United States.

  In another embodiment, the recombinant Listeria strain utilized in the methods of the invention is derived from a batch of vaccine doses.

  In another embodiment, the recombinant Listeria strain utilized in the methods of the invention is a frozen or produced by the method provided in US Pat. No. 8,114,414 (incorporated herein by reference). From lyophilized stock.

  In another embodiment, the peptide of the invention is a fusion peptide. In another embodiment, “fusion peptide” refers to a peptide or polypeptide comprising two or more proteins linked together by peptide bonds or other chemical bonds. In another embodiment, the aforementioned proteins are directly linked to each other by peptide bonds or other chemical bonds. In another embodiment, the proteins are linked to each other by one or more AA (eg, “spacers”) between the two or more proteins.

  In another embodiment, the vaccine of the present invention further comprises an adjuvant. In another embodiment, the adjuvant used in the methods and compositions of the invention is granulocyte / macrophage colony stimulating factor (GM-CSF) protein. In another embodiment, the adjuvant comprises GM-CSF protein. In another embodiment, the adjuvant is a nucleotide molecule encoding GM-CSF. In another embodiment, the adjuvant comprises a nucleotide molecule encoding GM-CSF. In another embodiment, the adjuvant is saponin QS21. In another embodiment, the adjuvant comprises saponin QS21. In another embodiment, the adjuvant is monophosphoryl lipid A. In another embodiment, 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 immunostimulatory cytokine. In another embodiment, the adjuvant comprises an immunostimulatory cytokine. In another embodiment, the adjuvant is a nucleotide molecule encoding an immunostimulatory cytokine. In another embodiment, the adjuvant comprises a nucleotide molecule that encodes an immunostimulatory cytokine. In another embodiment, the adjuvant is or includes a quill glycoside. In another embodiment, the adjuvant is or includes a bacterial mitogen. In another embodiment, the adjuvant is or includes a bacterial toxin. In another embodiment, the adjuvant is or includes any other adjuvant known in the art.

In another embodiment, the nucleotides of the invention are operably linked to a promoter / regulatory sequence that drives expression of the encoded peptide in a Listeria strain. Promoter / regulatory sequences useful for causing constitutive expression of genes are well known in the art and include, for example, the Listeria P _hlyA promoter, the P _ActA promoter, and the p60 promoter, Streptococcus bac promoter, Streptococcus Examples include, but are not limited to, the Myces griseus sgiA promoter and the Bacillus thuringiensis phaZ promoter. In another embodiment, inducible and tissue specific expression of a nucleic acid encoding a peptide of the invention places the nucleic acid encoding the peptide under the control of an inducible or tissue specific promoter / regulatory sequence. Carried out by. Examples of tissue specific or inducible promoter / regulatory sequences useful for this purpose include, but are not limited to, the MMTV LTR inducible promoter, and the SV40 late enhancer / promoter. Another embodiment utilizes a promoter that is induced in response to an inducing agent such as a metal, glucocorticoid, and the like. Thus, the present invention provides any promoter / regulatory sequence that is either a known or unknown promoter / regulatory sequence that can cause expression of a desired protein that is operably linked to that sequence. It is understood to include the use of sex sequences.

The N-terminal fragment of the ActA protein utilized in the methods and compositions of the present invention, in another embodiment, has the sequence shown in SEQ ID NO: 4.
In EmuarueiemuemubuibuiefuaitieienushiaitiaienuPidiaiaiefueieitidiesuidiesuesueruenutidiidaburyuiiikeitiiikyuPiesuibuienutiJipiaruwaiitieiaruibuiesuesuarudiaikeiieruikeiesuenukeibuiaruenutienukeieidieruaieiemuerukeiikeieiikeiJipienuaienuenuenuenuesuikyutiienueieiaienuiieiesujieidiaruPieiaikyubuiiaruarueichiPijieruPiesudiesueieiiaikeikeiaruarukeieiaieiesuesudiesuieruiesuerutiwaiPidikeiPitikeibuienukeikeikeibuieikeiiesubuieidieiesuiesudierudiesuesuemukyuesueidiiesuesuPikyuPierukeieienukyukyuPiefuefuPikeibuiefukeikeiaikeidieijikeidaburyubuiarudikeiaidiienuPiibuikeikeieiaibuidikeiesueijieruaidikyueruerutikeikeikeiesuiibuienueiesudiefupipipipitidiiieruaruerueierupiitiPiemueruerujiefuenueiPieitiesuiPiesuesuefuiefupipipipitidiiieruaruerueierupiitiPiemueruerujiefuenueiPieitiesuiPiesuesuefuiefupipipipitiidiieruiaiaiaruitieiesuesuerudiesuesuefutiarujidierueiesueruaruenueiaienuRHSQNFSDFPPIPTEEELNGRGGRP. In another embodiment, ActA fragment comprises the sequence shown in SEQ ID NO: 4. In another embodiment, the ActA fragment is any other ActA fragment known in the art. In another embodiment, the recombinant nucleotide encoding a fragment of the ActA protein comprises the sequence shown in SEQ ID NO: 5.
In another embodiment, the recombinant nucleotide has the sequence shown in SEQ ID NO: 5. In another embodiment, the recombinant nucleotide comprises any other sequence encoding a fragment of the ActA protein.

  In another embodiment of the methods and compositions of the invention, the PEST amino acid AA sequence is fused to the E7 or E6 antigen. As disclosed herein, recombinant Listeria strains expressing PEST amino acid sequence-antigen fusions elicit anti-tumor immunity (Example 3) and generate antigen-specific, tumor-infiltrating T cells ( Example 4). In addition, cell-mediated enhancement of immunity was demonstrated against a fusion protein comprising an antigen and LLO containing the PEST amino acid AA sequence KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 6).

  Thus, fusion of an antigen to other LM PEST amino acid sequences and PEST amino acid sequences derived from other prokaryotes also enhances the immunogenicity of the antigen. In another embodiment, the PEST amino acid AA sequence has a sequence selected from SEQ ID NOs: 7-12. In another embodiment, the PEST amino acid sequence is a PEST amino acid sequence derived from an LM ActA protein. In another embodiment, the PEST amino acid sequence is KTEEQPSEVNTGPR (SEQ ID NO: 7), KASVTDTSEGDLDSSMQSADESTTPQPLK (SEQ ID NO: 8), KNEEVNASDFPPPPTDDEELR (SEQ ID NO: 9), or RGGIPTSEEFSSSLNSGDFDTDIDSETIDE IDSETSETID In another embodiment, the PEST amino acid sequence is derived from a Streptococcus sp. In another embodiment, the PEST amino acid sequence is derived from Streptococcus pyogenes streptolysin O, eg, KQNASTETTTNEQPK (SEQ ID NO: 11) at AA 35-51. In another embodiment, the PEST amino acid sequence is from Streptococcus equisimilis streptolidine O, eg, KQNTANTETTTTTNEQPK (SEQ ID NO: 12) in 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.

  Other prokaryotic PEST amino acid sequences can be identified, for example, according to methods such as those described by Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21: 267-271) for LM. Alternatively, PEST amino acid AA sequences from other prokaryotes can also be identified based on this method. Other prokaryotes for which the PEST amino acid AA sequence is expected include, but are not limited to, other Listeria species. In another embodiment, the PEST amino acid sequence is embedded in the antigenic protein. Thus, in another embodiment, a “fusion” comprises an antigenic protein comprising both an antigen and a PEST amino acid sequence, either linked to one end of the antigen or embedded within the antigen. Point to.

  In another embodiment, the PEST amino acid sequence is determined by any other method or algorithm known in the art, for example, CaSPredictor (Garay-Malpartida HM, Octichicc JM, Alves J, Belizario JE. Bioinformatics. 2005 Jun; 21 Sup. 1: i169-76). In another embodiment, the following method is used.

  The 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 Gln. The coefficient value (CV) for each of the PEST residues is 1 and the coefficient value for each of the other AA (non-PEST) is 0.

  Each method for identifying a PEST amino acid sequence represents a separate embodiment of the present invention.

  In another embodiment, the LLO protein, ActA protein, or fragment thereof of the invention need not be shown exactly as shown herein, but is fused to the antigen shown elsewhere herein. Other alterations, modifications, or changes can be made that retain the functional characteristics of the LLO or ActA protein. In another embodiment, the present invention utilizes analogs of LLO protein, ActA protein, or fragments thereof. In another embodiment, the analog differs from the naturally-occurring protein or peptide by differences in conserved AA sequences, by modifications that do not affect the sequence, or both.

  In another embodiment, either the entire E7 protein or a fragment thereof is fused to an LLO protein, ActA protein, or PEST amino acid sequence-containing peptide to produce a recombinant peptide of the methods of the invention. In another embodiment, the E7 protein utilized (either in whole or as a fragment source) is the following sequence: MHGDPTLHEYMLDLQPETTDLYCYEQLDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTRLLCVQSTHVDIRTCIDLMGTGQQ In another embodiment, 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 an isomeric fragment of SEQ ID NO: 13.

  In another embodiment, the sequence of the E7 protein is MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEDIGVNHQHLPARRAEPQRHTMLCMCCKCEAIRELVVESSADDDLRAFQQLFNLTLSFVCCPWCASQQ (SEQ ID NO: 14). In another embodiment, the E6 protein is a homologue of SEQ ID NO: 14. In another embodiment, the E6 protein is a variant of SEQ ID NO: 14. In another embodiment, the E6 protein is an isomer of SEQ ID NO: 14. In another embodiment, the E6 protein is a fragment of SEQ ID NO: 14. In another embodiment, the E6 protein is a fragment of a homologue of SEQ ID NO: 14. In another embodiment, the E6 protein is a fragment of a variant of SEQ ID NO: 14. In another embodiment, the E6 protein is an isomeric fragment of SEQ ID NO: 14.

  In another embodiment, the E7 protein has a sequence set forth in one of the following GenBank entries: M24215, NC_004500, V01116, X62843, or M14119. In another embodiment, the E7 protein is a homologue of a sequence of one of the above GenBank entries. In another embodiment, the E7 protein is a variant of one of the above GenBank entries. In another embodiment, the E7 protein is an isomer of a sequence of one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of a sequence of one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of a homologue of a sequence of one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of a variant of one of the above GenBank entries. In another embodiment, the E7 protein is an isomeric fragment of one of the above GenBank entries.

  In another embodiment, either the entire E6 protein or a fragment thereof is fused to an LLO protein, ActA protein, or a PEST amino acid sequence-containing peptide to produce a recombinant peptide of the methods of the invention. In another embodiment, with a (either in the as a source of whole or fragments) utilized by the E6 protein, sequence EmueichikyukeiarutieiemuefukyudiPikyuiaruPiarukeieruPikyuerushitiierukyutitiaieichidiaiaieruishibuiwaishikeikyukyuerueruaruaruibuiwaidiefueiefuarudierushiaibuiwaiarudijienuPiwaieibuishidikeishierukeiefuwaiesukeiaiesuiwaiarueichiwaishiwaiesueruwaijititieruikyukyuwaienukeiPierushidierueruaiarushiaienushikyukeiPierushiPiiikeikyuarueichierudikeikeikyuaruefueichiNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO: 15). In another embodiment, the E6 protein is a homologue of SEQ ID NO: 15. In another embodiment, the E6 protein is a variant of SEQ ID NO: 15. In another embodiment, the E6 protein is an isomer of SEQ ID NO: 15. In another embodiment, the E6 protein is a fragment of SEQ ID NO: 15. In another embodiment, 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 an isomeric fragment of SEQ ID NO: 15.

  In another embodiment, the sequence of the E6 protein is MARFEDPTRRPYKLPDLCTELNTSLQDIITCVYCKTVLELTEVFEFAKDLFVVYRDSIPHAACHKCIDFYSRIRQRQRQRQRQRQRQRQRQRQRQRQRQRQRQRQRQRQR In another embodiment, the E6 protein is a homologue of SEQ ID NO: 16. In another embodiment, the E6 protein is a variant of SEQ ID NO: 16. In another embodiment, the E6 protein is an isomer of SEQ ID NO: 16. In another embodiment, 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 an isomeric fragment of SEQ ID NO: 16.

  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. In another embodiment, 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 an isomeric fragment of a sequence from one of the above GenBank entries.

  In another embodiment, “homology” refers to greater than 70% identity to an LLO sequence (eg, one of SEQ ID NOs: 1-3). In another embodiment, “homology” refers to greater than 64% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 68% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 72% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 75% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 78% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 80% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 82% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 83% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 85% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 87% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 88% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 90% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 92% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 93% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 95% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 96% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 97% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 98% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to greater than 99% identity to one of SEQ ID NOs: 1-3. In another embodiment, “homology” refers to 100% identity to one of SEQ ID NOs: 1-3.

  In another embodiment, “homology” refers to greater than 70% identity to the E7 sequence (eg, one of SEQ ID NOs: 13-14). In another embodiment, “homology” refers to greater than 62% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 64% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 68% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 72% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 75% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 78% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 80% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 82% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 83% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 85% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 87% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 88% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 90% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 92% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 93% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 95% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 96% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 97% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 98% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to greater than 99% identity to one of SEQ ID NOs: 13-14. In another embodiment, “homology” refers to 100% identity to one of SEQ ID NOs: 13-14.

  In another embodiment, “homology” refers to greater than 70% identity to the E6 sequence (eg, one of SEQ ID NOs: 15-16). In another embodiment, “homology” refers to greater than 64% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 68% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 72% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 75% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 78% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 80% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 82% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 83% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 85% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 87% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 88% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 90% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 92% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 93% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 95% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 96% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 97% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 98% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to greater than 99% identity to one of SEQ ID NOs: 15-16. In another embodiment, “homology” refers to 100% identity to one of SEQ ID NOs: 15-16.

  In another embodiment, “homology” refers to identity to a PEST amino acid sequence (eg, one of SEQ ID NOs: 6-12) or ActA sequence (eg, one of SEQ ID NOs: 4-5). It means higher than 70%. In another embodiment, “homology” refers to greater than 60% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 64% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 68% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 72% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 75% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 78% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 80% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 82% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 83% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 85% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 87% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 88% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 90% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 92% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 93% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 95% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 96% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 97% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 98% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to greater than 99% identity to SEQ ID NO: 6-12, or one of SEQ ID NOs: 4-5. In another embodiment, “homology” refers to 100% identity to SEQ ID NOs: 6-12, or one of SEQ ID NOs: 4-5.

  Protein and / or peptide homology to any AA sequence listed herein, in one embodiment, by methods well described in the art, including immunoblot analysis, or through established methods Determined through computer algorithm analysis of AA sequences using any number of software packages available. Some of these packages include the FASTA, BLAST, MPsrch, or Scans packages, and in other embodiments, these include, for example, the use of Smith-Waterman algorithms and / or extensive analysis / Use local or BLOCKS alignment. The method for determining each homology represents a separate embodiment of the present invention.

  In another embodiment, the LLO protein, ActA protein, or fragment thereof is attached to the antigen by chemical linkage. In another embodiment, glutaraldehyde is used for conjugation. In another embodiment, the coupling is accomplished using any suitable method known in the art.

  In another embodiment, the fusion proteins of the invention are prepared by any suitable method including, for example, cloning and restriction of appropriate sequences, or direct chemical synthesis by the methods discussed below. In another embodiment, the partial sequence is cloned and the appropriate partial sequence is split using appropriate restriction enzymes. In another embodiment, the fragments are then ligated to produce the desired DNA sequence. In another embodiment, the DNA encoding the fusion protein is produced using a DNA amplification method such as, for example, polymerase chain reaction (PCR). First, the new end sections on either side of the native DNA are amplified separately. The 5 'end of one amplified sequence encodes a peptide linker, while the 3' end of another amplified sequence also encodes a peptide linker. Since the 5 'end of the first fragment is the complement of the 3' end of the second fragment, use the two fragments as overlapping templates in a third PCR reaction (eg after partial purification on LMP agarose) can do. The amplified sequence consists of a codon, a section on the carboxy side of the open site (here forming the amino sequence), a linker, and a sequence on the amino side of the open site (here forming the carboxyl sequence). It will contain. The insert is then ligated to the plasmid.

  In another embodiment, the LLO protein, ActA protein, or fragment thereof, and the antigen, or fragment thereof, are combined by means known to those skilled in the art. In another embodiment, the antigen or fragment thereof is bound to ActA protein or LLO protein either directly or through a linker (spacer). In another embodiment, the chimeric molecule is recombinantly expressed as a single chain fusion protein.

  In another embodiment, the fusion peptides of the invention are synthesized using standard chemical peptide synthesis techniques. In another embodiment, the chimeric molecule is synthesized as a single continuous polypeptide. In another embodiment, the LLO protein, ActA protein, or fragment thereof, and the antigen or fragment thereof are synthesized separately and then fused by condensation between the amino terminus of one molecule and the carboxyl terminus of the other molecule; This forms a peptide bond. In another embodiment, the ActA protein or LLO protein, and the antigen are each condensed with one end of the peptide spacer molecule, thereby forming a continuous fusion protein.

  In another embodiment, the peptides and proteins of the present invention are prepared by Stewart et al. In Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill. Or as described by Bodanzky and Bodanzky (The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York). In another embodiment, a suitably protected AA residue is attached through its carboxyl group to a derivatized, insoluble polymer support (such as a 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 any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents, and reaction conditions used throughout the synthesis, and can be removed under conditions that do not affect the final peptide product. Stepwise synthesis of oligopeptides is performed by removing the N-protecting group from the initial AA and ligating it to the next AA carboxyl end of the desired peptide sequence. This AA is also suitably protected. Carboxyimide, symmetrical anhydride, or formation of a “active ester” group, such as hydroxybenzotriazole or pentafluorophenyl ester, to form a reactive group that supports the carboxyl of incoming AA N It can be activated to react with the termini.

  In another embodiment, the present invention provides a kit comprising a vaccine, applicator of the invention, and instructional material illustrating the use of the method of the invention. Although model kits are described below, other useful kit contents will be apparent to those of skill in the art in view of the present disclosure.

  The term “about” in quantitative terms is plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment. Those skilled in the art will appreciate that this may include plus or minus 20%.

  One of skill in the art will appreciate that the term “subject” may encompass mammals, including humans, who require therapy for, or are sensitive to symptoms or sequelae. Subjects may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice, and humans. The term “subject” does not exclude individuals that are normal in any respect.

  All references cited herein are indicated as each individual publication, database entry (eg, Genbank sequence or GeneID entry), patent application, or patent is specifically and individually incorporated by reference. Are incorporated by reference to the same extent. This statement of incorporation by reference is rule 37C. F. R. Based on §1.57 (b) (1), applicants intend to relate to any and all individual publications, database entries (eg, Genbank sequences or GeneID entries), patent applications, or patents, each of which Even if such a citation is not immediately adjacent to a built-in statement with a dedicated reference, rule 37 C.I. F. R. Explicitly specified according to §1.57 (b) (2). If the inclusion of a dedicated statement of incorporation by reference is in the description, this general statement of incorporation by reference will never weaken. Citation of references herein is not intended as an admission that such references are pertinent existing technology, nor constitutes an endorsement of the content or date of these publications or documents.

  The following examples are presented in order to more fully illustrate the embodiments of the invention. However, these should never be understood as limiting the broad scope of the present invention.

Experimental details section Example 1: LLO-antigen fusion induces anti-tumor immunity
Materials and Experimental Methods (Examples 1-2)
Cell line C57BL / 6 syngeneic TC-1 tumors were immortalized with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene. T.A. C. TC-1 provided by Wu (Johns Hopkins University School of Medicine, Baltimore, MD) expressed low levels of HPV-16 E6 and E7 and was transformed with the c-Ha-ras oncogene. It is a highly epithelial lung epithelial cell. TC-1 with RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 100 μM non-essential amino acid, 1 mM sodium pyruvate, 50 micromolar (mcM) Cultured in 2-ME, 400 microgram (mcg) / mL G418, and 10% National Collection Type Culture-109 medium at 37 ° with 10% CO ₂ . C3 is a mouse embryo cell from a C57BL / 6 mouse that has been immortalized with the complete genome of HPV 16 and transformed with pEJ-ras. EL-4 / E7 is a thymoma EL-4 transduced with E7 by a retrovirus.
L. Monocytogenes strains and growth

The Listeria strains used were Lm-LLO-E7 (hly-E7 fusion gene in the episomal expression system, FIG. 1A), Lm-E7 (single copy E7 gene cassette integrated into the Listeria genome), Lm-LLO. -NP ("DP-L2028", hly-NP fusion gene in episomal expression system), and Lm-Gag ("ZY-18", single copy HIV-1 Gag gene cassette integrated into the chromosome). The E7, primer 5'-GG CTCGAG CATGGAGATACACC-3 ' ( with SEQ ID NO: 17, XhoI site is underlined) and 5'-GGGG ACTAGT TTATGGTTTCTGAGAACA-3' ( SEQ ID NO: 18, SpeI site underlined) using PCR And ligated into pCR2.1 (Invitrogen San Diego, Calif., USA). E7 was excised from pCR2.1 by XhoI / SpeI digestion and ligated to pGG-55. The hly-E7 fusion gene and the pluripotent transcription factor prfA were cloned into pAM401, which is a multicopy shuttle plasmid (Wirth R et al, J Bacteriol, 165: 831, 1986) to prepare pGG-55. The hly promoter drives the expression of the first 441 AA of the hly gene product (lacking the hemolytic C-terminus, hereinafter referred to as “ΔLLO” and having the sequence shown in SEQ ID NO: 25), which is driven by the XhoI site. It is linked to the E7 gene and transcribed as LLO-E7 to obtain a secreted hly-E7 fusion gene. Transformation with pGG-55 of a Listeria prfA negative strain, XFL-7 (provided by Dr. Hao Shen, University of Pennsylvania) was selected for plasmid retention in vivo (FIGS. 1A-FIG. 1B). The hly promoter and gene fragment are primers 5′-GGGG GCTAGC CCTCCTTTGATTTAGTAATTC-3 ′ (SEQ ID NO: 19, NheI site is underlined), and 5′-CTCC CTCGAG ATCATATATTTACTTCATTC-3 ′ (SEQ ID NO: 20, XhoI site is underlined) It was created using. The prfA gene has primers 5′-GACTACAGAGCGGATGGACCGACAAGTGATAA CCCGGG ATCTAAATAAATCCCGTTT-3 ′ (SEQ ID NO: 27, XbaI site is underlined), and 5′-CCC GTCGAC CAGCCTCTTTTGGTGAAG-3 ′ (site number is 21) PCR amplified. Lm-E7 was generated by introducing an expression cassette containing the hly promoter and a signal sequence that causes expression and secretion of E7 into the orfZ domain of the LM genome. PCR amplification of E7 using primers 5′-GC GGATCC CATGGAGATACACCTACC-3 ′ (SEQ ID NO: 22, BamHI site underlined) and 5′-GC TCTAGA TTATGGTTTCTGAG-3 ′ (SEQ ID NO: 23, XbaI site underlined) did. E7 was then ligated into the pZY-21 shuttle vector. The resulting plasmid pZY-21-E7 was transformed into LM strain 10403S, which contains an expression cassette inserted in the middle of the 1.6 kb sequence corresponding to the orfX, Y, Z domains of the LM genome. Its homology domain allows 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 cultured in brain heart infusion medium containing chloramphenicol (20 μg / mL) (Lm-LLO-E7 and Lm-LLO-NP), or the bacteria were cultured in the same medium without chloramphenicol. Cultured (Lm-E7 and ZY-18). Bacteria were frozen in aliquots at -80 ° C. Expression was verified by Western blot (Figure 2).
Western blot

Listeria strains were grown at 37 ° C. in Luria-Bertani medium and harvested at the same optical density measured at 600 nm. The supernatant was TCA precipitated and resuspended in 1 × sample buffer supplemented with 0.1 N NaOH. The same amount of each cell pellet or each TCA precipitation supernatant was loaded onto a 4-20% Trisglycine SDS-PAGE gel (NOVEX, San Diego, CA). The gels were transferred to polyvinylidene difluoride and examined with anti-E7 monoclonal antibody (mAb) (Zymed Laboratories, South San Francisco, Calif.) And then HRP-conjugated anti-mouse secondary Ab (Amersham Pharmacia Biotech, Little UK). -Chalfont), developed with Amersham ECL detection reagent, and exposed to Hyperfilm (Amersham Pharmacia Biotech).
Measuring tumor growth

Tumors were measured every other day with calipers on the shortest and longest diameters of the surface. The average of these two measurements was plotted against various time points in millimeters as the average tumor diameter. Mice were sacrificed when the tumor diameter reached 20 mm. Tumor measurements for each time point are shown only for live mice.
Effect of recombinant Listeria on established tumor growth

6-8 week old C57BL / 6 mice (Charles River) received 2 × 10 ⁵ TC-1 cells subcutaneously in the left flank. One week after tumor inoculation, the tumor reached a palpable size of 4-5 mm in diameter. Groups of 8 mice were then grouped on days 7 and 14 with 0.1 LD ₅₀ of Lm-LLO-E7 (10 ⁷ CFU), Lm-E7 (10 ⁶ CFU), Lm-LLO-NP (10 ⁷ CFU), or Lm-Gag (5 × 10 ⁵ CFU).
⁵¹ Cr release assay

Six to eight week old C57BL / 6 mice were immunized intraperitoneally with 0.1 LD ₅₀ of Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag. The spleen was collected 10 days after immunization. Splenocytes are established by culturing with irradiated TC-1 cells as feeder cells (100: 1, splenocytes: TC-1), stimulated in vitro for 5 days, and then standard ⁵¹ using the following targets: Used for Cr release assay. E-7 H-2b peptide pulsed with EL-4, EL-4 / E7, or EL-4 (RAHYNIVTF, SEQ ID NO: 24). The E: T cell ratios were repeated three times: 80: 1, 40: 1, 20: 1, 10: 1, 5: 1, and 2.5: 1. After 4 hours incubation at 37 ° C., the cells were pelleted and 50 μl of supernatant was removed from each well. Samples were assayed using a Wallac 1450 scintillation counter (Gaithersburg, MD). The specific lysis rate was determined as [(count per minute by experiment (cpm) −spontaneous cpm) / (total cpm−spontaneous cpm)] × 100
TC-1 specific growth

With Lm-LLO-E7, Lm- E7, Lm-LLO-NP or Lm-Gag, immunized C57BL / 6 mice 0.1LD _50, it was boosted by intraperitoneal injection with _{1LD 50} after 20 days. Six days after the boost, spleens were collected from immunized and naive mice. With 2.5 × 10 ⁴ cells, 1.25 × 10 ⁴ cells, 6 × 10 ³ cells, or 3 × 10 ³ cells irradiated TC-1 cells per well as E7 Ag source, or TC-1 cells Splenocytes were established by culturing at a density of 5 × 10 ⁵ cells / well in flat-bottom 96-well plates with or without 10 μg / ml Con A. After 45 hours, cells were pulsed with 0.5 μCi [ ³ H] thymidine / well. Plates were harvested 18 hours later using a Tomtec harvester 96 (Orange, Connecticut, USA) and growth was assessed using a Wallac 1450 scintillation counter. The change in cpm was calculated as cpm without experimental cpm-Ag.
Flow cytometry analysis

C57BL / 6 mice were immunized intravenously (iv) with 0.1 LD ₅₀ of Lm-LLO-E7 or Lm-E7 and boosted 30 days later. Three-color flow cytometry for CD8 (53-6.7, PE binding), CD62 re-cancerous (CD62L, MEL-14, APC binding), and E7 H-2Db tetramer, FACSCalibur® flow site Performed using a meter and CellQuest® software (Becton Dickinson, Mountain View, Calif.). Splenocytes harvested 5 days after boost were stained at room temperature (rt) with H-2Db tetramer loaded with E7 peptide (RAHYNIVTF) or control (HIV-Gag) peptide. The tetramer was diluted to 1/200 and used. These tetramers are known as Dr. Larry R. Provided by Pease (May Clinic, Rochester, MN) and NIAID Tetramer Core Facility and NIH AIDS Research and standard reagent programs. Tetramer ⁺ , CD8 ⁺ , CD62L ^low cells were analyzed.
B16F0-Ova experiment

Twenty-four C57BL / 6 mice were inoculated with 5 × 10 ⁵ B16F0-Ova cells. Immunizing groups of 8 mice with 0.1 LD ₅₀ Lm-OVA (10 ⁶ cfu), Lm-LLO-OVA (10 ⁸ cfu) on days 3, 10 and 17; Eight mice were left untreated.
statistics

For tumor diameter comparison, mean tumor size and SD for each group were determined and statistical significance was determined by Student's t-test. p ≦ 0.05 was considered significant.
result

Lm-E7 and Lm-LLO-E7 were compared for their ability to affect TC-1 proliferation. A subcutaneous tumor was established on the left flank of C57BL / 6 mice. After 7 days, the tumor reached palpable size (4-5 mm). On days 7 and 14, mice were vaccinated with 0.1 LD ₅₀ of Lm-E7, Lm-LLO-E7, or Lm-Gag and Lm-LLO-NP as controls. Lm-LLO-E7 induced 75% complete regression of established TC-1 tumors while tumor growth was controlled in the other 2 mice in this group (FIG. 3). In contrast, immunization with Lm-E7 and Lm-Gag did not induce tumor regression. This experiment was repeated multiple times and was always very similar. In addition, similar results were achieved for Lm-LLO-E7 under different immunization protocols. In another experiment, mice with established 5 mm TC-1 tumors could be cured with a single immunization.

  In other experiments, similar results were obtained using two other E7 expressing tumor cell lines, C3 and EL-4 / E7. To confirm the effectiveness of vaccination with Lm-LLO-E7, tumor-removed animals were reloaded with TC-1 tumor cells or EL-4 / E7 tumor cells on day 60 or 40, respectively. . Animals immunized with Lm-LLO-E7 remained tumor free until the end of the experiment (124 days for TC-1 and 54 days for EL-4 / E7).

Thus, expression of the antigen as a fusion protein with ΔLLO enhances the immunogenicity of the antigen.
Example 2: LM-LLO-E7 treatment elicits TC-1-specific splenocyte proliferation

To measure the induction of T cells by Lm-E7 with Lm-LLO-E7, a TC-1-specific proliferative response, a measure of antigen-specific immunity, was measured in immunized mice. Splenocytes from mice immunized with Lm-LLO-E7 were expanded when exposed to irradiated TC-1 cells as a source of E7 in splenocytes. The TC-1 ratio is 20: 1, 40: 1, 80: 1, and 160: 1 (FIG. 4). In contrast, splenocytes from mice immunized with Lm-E7 and rLm controls only showed background levels of proliferation.

Example 3: Fusion of E7 to LLO, ActA, or PEST amino acid sequences enhances E7-specific immunity and generates tumor infiltrating E7-specific CD8 ⁺ cells
Materials and experimental methods

Contains 100 mcl (microliters) of 2 × 10 ⁵ TC-1 tumor cells in phosphate buffered saline (PBS) plus 400 mcl MATRIGEL® (BD Biosciences, Franklin Lakes, NJ) 500 mcl MATRIGEL® was implanted subcutaneously into the left flank of 12 C57BL / 6 mice (n = 3). Mice were immunized intraperitoneally on days 7, 14, and 21, and spleens and tumors were harvested on day 28. Tumor MATRIGEL was removed from the mice and incubated overnight at 4 ° C. in a tube containing 2 milliliters (mL) of RP 10 medium on ice. The tumor was minced using forceps and cut into 2 mm blocks and incubated with 3 ml of enzyme mixture (0.2 mg / ml collagenase P, 1 mg / ml DNAse-1 in PBS) for 1 hour at 37 ° C. For tetramer and IFN-γ staining, the tissue suspension was filtered through a nylon mesh and washed with 5% fetal calf serum + 0.05% NaN ₃ in PBS.

10 ⁷ cells / mL splenocytes and tumor cells were incubated with 1 micromole (mcm) of E7 peptide for 5 hours in the presence of brefeldin A. Cells were washed twice and incubated in 50 mcl anti-mouse Fc receptor supernatant (2.4 G2) at 4 ° C. for 1 hour or overnight. Cells were stained for surface molecules CD8 and CD62L, permeabilized, fixed using permeabilization kits Golgi-stop® or Golgi-Plug® (Pharmingen, San Diego, Calif.), And IFN-γ Was stained for. 500,000 events were acquired using a 2 laser flow cytometer, FACSCalibur and analyzed using Cellquest Software (Becton Dickinson, Franklin Lakes, NJ). The percentage of IFN-γ secreting cells in activated (CD62L ^low ) CD8 ⁺ T cells was calculated.

For tetramer staining, the H-2D ^b tetramer was loaded with phycoerythrin (PE) -conjugated E7 peptide (RAHYNIVTF, SEQ ID NO: 24), stained for 1 hour at room temperature, and anti-allophycocyanin (APC) bound Stained with MEL-14 (CD62L) and FITC-conjugated CD8 • for 30 minutes at 4 ° C. Cells were analyzed by comparing tetramer ⁺ CD8 ⁺ CD62L ^low cells in spleen and tumor.
result

To analyze the ability of Lm-ActA-E7 to enhance antigen-specific immunity, mice were transplanted with TC-1 tumor cells and Lm-LLO-E7 (1 × 10 ⁷ CFU), Lm-E7 (1 × 10 6). ⁶ CFU) or Lm-ActA-E7 (2 × 10 ⁸ CFU) or untreated (naïve). Tumors of mice from the Lm-LLO-E7 and Lm-ActA-E7 groups have a higher percentage of IFN-γ secreting CD8 ⁺ T cells (FIG. 5A) and tetramer specific than Lm-E7 or untreated mice. CD8 ⁺ cells (FIG. 5B).

In another experiment, tumor-bearing mice were administered Lm-LLO-E7, Lm-PEST-E7, Lm-ΔPEST-E7, or Lm-E7epi, and the level of E7-specific lymphocytes in the tumor was measured. Mice were treated on days 7 and 14 with four vaccines of 0.1 LD ₅₀ . Tumors were harvested on day 21 and stained with antibodies against CD62L, CD8, and with E7 / Db tetramer. An increased percentage of tetramer positive lymphocytes within the tumor was seen in mice vaccinated with Lm-LLO-E7 and Lm-PEST-E7 (FIG. 6A). This result was reproducible over three experiments (FIG. 6B).

Thus, Lm-LLO-E7, Lm-ActA-E7, and Lm-PEST-E7 are each effective in inducing tumor-infiltrating CD8 ⁺ T cells and tumor regression.
Example 4: Passage of a Listeria vaccine vector through mice elicits an increased immune response against heterologous and endogenous antigens
Materials and experimental methods <br/> Bacterial strains

L. Monocytogenes strain 10403S, serotype 1 (ATCC, Manassas, VA, USA) is the wild-type organism used in these studies and the parent strain of the construct described below. Strain 10403S has an LD ₅₀ of approximately 5 × 10 ⁴ CFU when injected intraperitoneally into BALB / c mice. “Lm-Gag” is a copy of the HIV-1 strain HXB (subtype B laboratory strain containing syncytium-forming phenotype) gag gene that is stably integrated into the Listeria chromosome using the modified shuttle vector pKSV7. Recombinant LM strain containing. Gag protein was expressed and secreted by the strain as determined by Western blot. All strains were grown in Brain Heart Infusion (BHI) medium or agar plates (Difco Labs, Detroit, Michigan, USA).
Bacterial culture

Bacteria from a single clone expressing passenger antigen and / or fusion protein were selected and cultured overnight in BHI medium. An aliquot of this culture without additives ^- and frozen at 70 ° C.. From this stock, cultures were 0.1-0.2 O.D. D. And aliquots were frozen again at -70 ° C. without additives. The above procedure was used to prepare a cloned bacterial pool, but after each passage, a number of bacterial clones were selected and examined for target antigen expression as described herein. did. Clones with confirmed foreign antigen expression were used for the next passage.
Bacterial passage in mice

Female BALB / c (H-2d) mice 6-8 weeks old were purchased from Jackson Laboratories (Bar Harbor, Maine, USA) and maintained in a microisolator environment free of pathogens. Bacterial titers in aliquots of stock cultures cryopreserved at -70 ° C were determined by plating on BHI agar plates during thawing and prior to use. A total of 5 × 10 ⁵ bacteria were injected intravenously into BALB / c mice. Three days later, spleens were harvested and homogenized and serial dilutions of spleen homogenates were incubated overnight in BHI medium and plated on BHI agar plates. Aliquots are 0.1-0.2 O.D. for further passage. D. And then frozen at -70 ° C., and the bacterial titer was determined again by serial dilution. After the first passage (passage 0), this series of procedures was repeated a total of 4 times.
Intracellular cytokine staining for IFN-γ

Lymphocytes were placed in complete RPMI-10 medium supplemented with 50 U / mL human recombinant IL-2 and 1 microliter / mL Brefeldin A (Golgistop ™; PharMingen, San Diego, Calif.). 1 micromolar concentration of cytotoxic T cell (CTL) epitope against HIV-GAG (AMQMLKETI; SEQ ID NO: 25), Listeria LLO (GYKDGNEYI; SEQ ID NO: 26) or HPV virus gene E7 (RAHYNIVTF; SEQ ID NO: 24) In the presence or absence of. Cells were first surface stained, then washed and subjected to intracellular cytokine staining using the Cytofix / Cytoperm kit according to the manufacturer's recommendations (PharMingen, San Diego, Calif.). For intracellular IFN-γ staining, FITC-conjugated rat anti-mouse IFN-γ monoclonal antibody (clone XMG 1.2) and its isotype control Ab (rat IgG1; both obtained from PharMingen) were used. A total of 10 ⁶ cells in PBS containing 1% bovine serum albumin and 0.02% sodium azide (FACS buffer), was stained for 30 min at 4 ° C., followed by FACS buffer 3 Washed twice. Sample data was acquired on either a FACScan ™ flow cytometer or a FACSCalibur ™ instrument (Becton Dickinson, San Jose, Calif.). Three-color flow for CD8 (PERCP binding, rat anti-mouse, clone 53-6.7, Pharmingen, San Diego, Calif., USA), CD62L (APC binding, rat anti-mouse, clone MEL-14), and intracellular IFN-γ Cytometry was performed using a FACSCalibur ™ flow cytometer and data was further analyzed using CELLQuest software (Becton Dickinson, Mountain View, Calif., USA). Cells were gated on CD8 high and CD62L ^low prior to analysis for CD8 ⁺ and intracellular IFN-γ staining.
result
Passaging in mice increases the virulence of recombinant Listeria monocytogenes

  Three different constructs were used to determine the effect of passage on recombinant Listeria vaccine vectors. Two of these constructs carry the genomic insertion of the passenger antigen. The first construct contains the HIV gag gene (Lm-Gag) and the second construct contains the HPV E7 gene (Lm-E7). The third construct (Lm-LLO-E7) has a fusion gene for the passenger antigen (HPV E7) fused to a truncated version of LLO and a gene encoding prfA (a positive regulator controlling Listeria virulence factor) Contains a plasmid. Since this plasmid was used to complement the prfA negative mutation, the selection pressure would support the preservation of the plasmid in the living host, since without it the bacteria would be non-virulent. All three constructs have been extensively propagated in vitro for many bacterial generations.

Bacterial passage, as measured by the number of bacteria surviving in the spleen, results in increased bacterial virulence at each of the first two passages. For Lm-Gag and Lm-LLO-E7, disease toxicity increased at each passage up to passage 2 (FIG. 7A). The plasmid-containing construct, Lm-LLO-E7, showed the most dramatic increase in disease toxicity. Prior to passage, the initial immunization dose of Lm-LLO-E7 needed to increase the bacteria to 10 ⁷ and the spleen had to be taken on day 2 to recover the bacteria (while An initial dose of 10 ⁵ bacteria for Lm-Gag was taken on day 3). After the initial passage, the standard dose of Lm-LLO-E7 was sufficient to allow the third day collection. For Lm-E7, the degree of virulence increased by a factor of 1.5 over bacteria that were not passaged (FIG. 7B). Thus, passage through mice increases the virulence of the Listeria vaccine strain.
The passage is L. Increases the ability of monocytogenes to induce CD8 ⁺ T cells

Next, the effect of passage on the induction of antigen-specific CD8 ⁺ T cells was determined using MHC-class I specific using the HIV-Gag peptides AMQMLKETI (SEQ ID NO: 25) and LLO91-99 (GYKDGNEYI; SEQ ID NO: 26). As determined by intracellular cytokine staining using a dominant immunodominant peptide. Injection of 10 ³ CFU of passaged bacteria (Lm-Gag) into mice induced a significant number of HIV-Gag-specific CD8 ⁺ T cells, but the same dose of non-passaged Lm-Gag was: No detectable Gag-specific CD8 ⁺ T cells were induced. Increasing the dose of non-passaged bacteria 100-fold did not compensate for these relative non-disease toxicities. In fact, no detectable Gag-specific CD8 ⁺ T cells were induced at higher doses. The same dose increase with passage bacteria increased the induction of Gag-specific T cells by 50% (FIG. 8). Since the same pattern of induction of antigen-specific CD8 ⁺ cells was observed with LLO-specific CD8 ⁺ T cells, which were observed with LLO, an endogenous Listeria antigen, these results were generated by the characteristics of the passenger antigen It shows that it is not. Thus, passage through mice increases the immunogenicity of the Listeria vaccine strain.
Example 5: PrfA-containing plasmid is stable in LM strains with PrfA deletion in the absence of antibiotics
Materials and experimental methods <br/> Bacteria

L. Monocytogenes strain XFL7 contains a 300 base pair deletion in the prfA gene, and XFL7 carries pGG55 that partially restores disease toxicity and confers CAP resistance, which is a US patent application. It is described in the publication No. 20050118184.
Development of a protocol for plasmid extraction from Listeria

  1 mL of Listeria monocytogenes Lm-LLO-E7 research working cell bank vial was inoculated into 27 mL of BH1 medium containing 34 μg / mL CAP and grown at 37 ° C. and 200 rpm for 24 hours.

  Seven 2.5 mL samples of the culture were pelleted (5 minutes at 15000 rpm) and the pellets were incubated at 37 ° C. for various lengths of time from 0-60 minutes with 50 μl of lysozyme solution.

Lysozyme solution:
-29 [mu] l 1 M dipotassium hydrogen phosphate-21 [mu] l 1 M potassium dihydrogen phosphate-500 [mu] l 40% sucrose (filter sterilized through a 0.45 / [mu] m filter)
-450 [mu] l water-60 [mu] l lysozyme (50 mg / mL)

After incubation with lysozyme, the suspension was centrifuged as before and the supernatant was discarded. Each pellet was then subjected to plasmid extraction by a modified version of the QIAprep Spin Miniprep Kit® (Qiagen, Germany, Maryland) protocol. Changes to the protocol are as follows:
1. The volumes of Buffers PI, P2, and N3 were all increased by a factor of 3 to allow complete lysis of the increased biomass.
2. Prior to the addition of P2, 2 mg / mL lysozyme was added to the resuspended cells. The lysis solution was then incubated at 37 ° C. for 15 minutes before neutralization.
3. To increase the concentration, plasmid DNA was resuspended in 30 μl instead of 50 μl.

  In other experiments, cells were incubated for 15 minutes in P1 buffer + lysozyme and then incubated at room temperature with P2 (lysis buffer) and P3 (neutralization buffer).

  An equal volume of isolated plasmid DNA from each secondary culture was run on a 0.8% agarose gel stained with ethidium bromide and visualized to examine any signs of structural or separation instability.

  The results show that as the incubation time with lysozyme increases (to the optimum level of incubation for approximately 50 minutes), the L. It was shown that the efficiency of plasmid extraction from Monocytogenes Lm-LLO-E7 is increased.

These results provide an effective method for plasmid extraction from Listeria vaccine strains.
Replica plating

Dilutions of the original culture were plated with or without 34 μg / mL CAP on plates containing LB or TB agar. The difference between the count on selective agar and the count on non-selective agar was used to determine if there was any overall separation instability of the plasmid.
result

L. in the absence of antibiotics. Genetic stability of the pGG55 plasmid of monocytogenes strain XFL7 (ie, the extent to which the plasmid is maintained by bacteria or remains stably associated with bacteria in the absence of selection pressure (eg, antibiotic selection pressure) ) Luria-Bertani medium (LB: 5 g / L NaCl, 10 g / ml soy peptone, 5 g / L yeast extract) and Terrific Broth medium (TB: 10 g / L glucose, 11.8 g / L soy peptone, 23. 6 g / L yeast extract, 2.2 g / L KH ₂ PO ₄ , 9.4 g / L K ₂ HPO ₄ ) in both overlapping media, evaluated by continuous subculture. 50 mL of fresh medium in a 250 mL baffled shake flask was inoculated with a constant number of cells (1 ODmL), which was then subcultured at 24 hour intervals. The culture was incubated at 37 ° C. and 200 rpm in an orbital shaker. In each subculture, the OD ₆₀₀ was measured and used to calculate the elapsed cell doubling time (or generation) until reaching 30 generations in LB and reaching 42 generations in TB. A known number of cells (15 ODmL) at each subculture stage (approximately every 4 generations) were pelleted by centrifugation and plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep® protocol described above. After purification, the plasmid DNA was subjected to agarose gel electrophoresis and subsequently stained with ethidium bromide. Although the amount of plasmid in the preparation varied slightly from sample to sample, the overall trend was that the amount of plasmid was constant with respect to the number of bacterial generations (FIGS. 9A-9B). Thus, pGG55 exhibited stability in strain XFL7 even in the absence of antibiotics.

  Plasmid stability was also monitored by replica plating on agar plates at each stage of the secondary culture during the stability test. Consistent with the results from agarose gel electrophoresis, there was no overall change in the number of plasmid-containing cells in either LB or TB liquid cultures throughout the study (FIGS. 10 and 11, respectively).

These findings demonstrate that the plasmid encoding prfA exhibits stability without antibiotics in Listeria strains that contain mutations in prfA.

Materials and Methods (Examples 6-10)

  PCR reagents:

  The primers used to amplify the prfA gene and differentiate the D133V mutation are shown in Table 1. Stock solutions of primers ADV451, 452, and 453 were prepared by diluting the primers to 400 μM in TE buffer. An aliquot of the stock solution was further diluted to 20 μM in water (PCR grade) to prepare a diluted standard solution. Primers were stored at -20 ° C. Table 2 shows the reagents used in PCR.

Table 1. Primers ADV451, 452, and 453.

Table 2 PCR reagents
Plasmid DNA preparation

The pGG55 plasmid with (pGG55 D133V) and without (pGG55 WT), with or without the prfA mutation, was transformed into E. coli using the PureLink ™ HiPure Plasmid Midiprep Kit (Invitrogen, K2100-05) according to the manufacturer's instructions. Extracted with Midiprep from either Listeria monocytogenes and purified. For plasmid purification from Listeria, bacterial strains carrying pGG55 D133V or WT plasmids were streaked from frozen stocks into BHI agar plates supplemented with chloramphenicol (25 μg / mL). Single colonies from each strain were grown at 37 ° C. with vigorous shaking for 6 hours in 5 mL selective medium (BHI medium containing 25 μg / mL chloramphenicol) and overnight under similar conditions. Subcultured 100 mL selective medium at 1: 500 for growth. Bacteria from overnight cultures were collected by centrifugation at 4,000 × g for 10 minutes and resuspended in buffer R3 (resuspension buffer) containing 2 mg / mL lysozyme (Sigma, L7001). . The bacterial suspension was incubated at 37 ° C. for at least 1 hour before proceeding to the standard protocol. The concentration and purity of the eluted plasmid were measured with a spectrophotometer at 260 nm and 280 nm. To prepare the template DNA, pGG55 D133V and WT plasmids were resuspended in water to a final concentration of 1 ng / μl from the midiprep stock solution. For the pGG55 WT plasmid, 10-fold serial dilutions from 1 ng / μl solutions corresponding to dilutions from 10 ⁻¹ to 10 ⁻⁷ were prepared.
PrfA-specific PCR protocol for testing clinical grade materials

The reaction mixture was 1 × PCR buffer, 1.5 mM MgCl ₂ , 0.8 mM dNTP, 0.4 μM of each primer, 0.05 U / μl Taq DNA polymerase, and 0.04 ng / μl pGG55 D133V template. Contained the plasmid. Ten tubes are required for each test and the major components in each tube in a 25 μl reaction are shown in Table 3. For PCR reactions, a master mix containing enough reagents for 11 reactions was prepared as shown in Table 4, and 24 μl of this PCR mix was added to each tube. Subsequently, a total of 1 μl of serially diluted pGG55 WT plasmid was added to the corresponding tube: 1 ng in tube 3, 100 pg in tube 4, 10 pg in tube 5, 1 pg in tube 6, tube 100 fg in 7, 10 fg in tube 8, 1 fg in tube 9, and 0.1 fg in tube 10. This serial dilution was used to calibrate the standard curve for the purpose of determining method sensitivity. In addition, 0.5 μl water and 0.5 μl primer ADV451 (20 μM stock) were added to tube 1 and 1 μl water was added to tube 2 to complete a final volume of 25 μl. The amount of each reagent per tube for a 25 μl reaction is shown in Table 5. Table 6 shows the PCR cycle conditions used in the reaction.

  After completion of the PCR reaction, 5 μl of gel loading buffer (6 × with bromophenol blue) is added to each sample and 10 μl is analyzed by electrophoresis in a 1.2% agarose gel in TBE buffer. did. The dimensions of the gel are 7 cm x 7 cm x 1 cm and have 15 sample well (1 mm x 2 mm) combs. The gel was run at 100V for about 30 minutes until the bromophenol blue dye reached the center of the gel. The gel was stained in ethidium bromide (0.5 μg / mL) for 20 minutes and destained in water for 10 minutes. The gel was visualized by illumination with UV light and photographed. Images were analyzed using band density measurement software (Quantity One version 4.5.1, BioRad).

Table 3. A set of individual PCR reactions to validate the method of detecting the presence of wild type prfA sequences in Lm-LLO-E7 samples.

Table 4 Preparation of master PCR mix.

Table 5 PCR protocol for validation of methods to detect the presence of wild type prfA sequence using primers ADV451, 452 and 453.

Table 6 PCR cycle conditions for detecting the presence of wild type prfA sequence using primers ADV451, 452 and 453
Sequencing:

Plasmid sequencing was performed using the dideoxy sequencing method. Plasmids pGG55 D133V and pGG55 WT were mixed in 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 μg) and the plasmid containing the wild type sequence was serially diluted 10-fold with respect to the D133V plasmid to determine the sensitivity of the method.

Results Example 6: Sequencing is not a sensitive method for detecting reversion of the D133V mutation.

In order to estimate the sensitivity of sequencing in detecting the wild type prfA sequence, pGG55 D133V and WT plasmids were mixed in different ratios and sequenced. The results are shown in FIG. 12 and revealed that sequencing has high specificity in distinguishing prfAD133V mutations (FIG. 12). On the other hand, the sensitivity is low and the maximum dilution of wild type prfA pGG55 plasmid with a detectable peak in the sequence is 10 to 1 (FIG. 12). In conclusion, sequencing is very specific but the sensitivity of this method is low and not suitable for screening for the presence of rare events such as revertants of the prfA D133V mutation within the Lm-LLO-E7 sample .

Example 7: Development of a highly specific and sensitive PCR method for detecting reversion of the D133V mutation.

Given the low sensitivity of sequencing to detect rare events, it is necessary to develop more sensitive methods with similar specificity to detect the reversion of D133V mutation to wild type Indispensable. To achieve this goal, we designed a PCR-based method that specifically amplifies the wild-type sequence, which includes at least one prfA for 10,000,000 copies of the D133V mutated sequence. Sufficient sensitivity to detect wild type copies. Three primers were designed for this method. That is, ADV451, ADV452, and ADV453 (Table 1). Both ADV451 and ADV452 are forward primers and differ in the last nucleotide at the 3 ′ position so as to distinguish the A → T (D133V) mutation at position 398 of the prfA gene. The ADV453 primer is a reverse primer located approximately 300 bp downstream of the annealing sites of ADV451 and ADV452 primers (FIG. 13). The expected PCR band obtained using primers ADV451, or ADV452, and ADV453 is 326 bp. Under severe conditions, the ADV451 primer should only amplify the pGG55 D133V plasmid, while ADV452 will be specific for the wild type prfA sequence.

Example 8: Specificity of PCR method.

  The reaction using primer ADV451 was very specific, amplifying the mutant D133V prfA sequence (lanes 1 to 3) but not the wild type sequence (lanes 4 to 6). However, when 5 ng of template DNA was used, a very faint band could be detected in lane 4, but not 1 ng (FIG. 14).

As shown in FIG. 15, the reaction with the ADV452 primer only amplifies the wild type prfA sequence (lanes 4, 5, and 6) and when pGG55 carrying the D133V prfA mutation was used as a template (lane 1, 2 and 3), no band was detected even when 5 ng of plasmid was used in the reaction (FIG. 16). In conclusion, the PCR reaction with primers ADV451 and ADV452 is very specific and can distinguish the A ← → T (D133V) mutation at position 398 of the prfA gene in the pGG55 plasmid. Based on these results, an amount of 1 ng was selected as the standard amount of template DNA used in the reaction.

Example 9: Sensitivity of PCR method

The sensitivity of the reaction was tested using 1 ng template DNA. Against plasmid carrying the wild-type prfA sequence ^{(corresponding} to 10-fold dilution of 10 -1 ^{to 10 -7)} DNA in an amount gradually decreases to estimate the sensitivity was also included in the reaction. In these reactions, only primers ADV452 and ADV453 were used. In a PCR reaction of 30 cycles (10 cycles at an annealing temperature of 53 ° C. and an additional 20 cycles at an annealing temperature of 50 ° C.), the sensitivity of the method was 1: 100,000 (data not shown). As shown in FIG. 5, increasing the number of PCR cycles to 37 improved the visibility of the method to 10 ⁻⁶ without significant loss of specificity for detecting D133V revertants. . When 1 ng of plasmid was used as the initial amount of DNA, a clear band was seen at 10 ⁻⁶ dilution, corresponding to the detection level of 1 copy of wild type sequence for 1 million D133V mutations. After longer exposure, only a very weak band was visible in lane 1 and lane 9, reconfirming the robust specificity of the method. On the other hand, when starting with 5 ng of DNA, the band could be easily detected at 10 ⁻⁷ dilution, increasing the sensitivity of the PCR. However, a band of similar intensity can also be detected using the pGG55 D133V plasmid, which shows the limit of the specificity of the method (FIG. 17). This band observed with the pGG55 D133V plasmid is an increase in cycle number. This may be due to non-specific amplification of the D133V mutation with primer ADV452, which can accumulate significantly. These results show that the sensitivity limit for this method is between 1: 1 and 1,000,000 to 1: 10,000,000 without significant loss of specificity.

Example 10: Recombinant Listeria expressing LLO fusion protein in E7 (LM-LLO-E7)

This strain is attenuated approximately 4-5 logs from the wild-type parent strain 10403S and secretes the fusion protein tLLO-E7. This immunotherapy is based on backbone XFL7, which is derived from 10403S by an irreversible deletion in the virulence gene transcription activator prfA. PrfA is an in vivo intracellular growth such as Listeriocin O (LLO), ActA, PlcA (phospholipase A), PlcB (phospholipase B) and L. It regulates the transcription of several pathogenic genes required for the survival of monocytogenes. Plasmid pGG55 is retained by Lm-LLO-E7 in vitro by selection means using “chloramphenicol”. However, because of the in vivo retention of the plasmid by Lm-LLO-E7, it carries a copy of the mutant prfA (D133V), which is more active than wild-type PrfA in activating DNA binding and transcription of the virulence gene. Low activity has been demonstrated. It was observed that complementation with mutant prfA resulted in an approximately 40-fold reduction in the amount of LLO secreted from Lm-LLO-E7 compared to wild type strain 10403S. This implies that the strain Lm-LLO-E7 may exhibit reduced expression of pathogenic genes regulated by PrfA such as actA, inlA, inlB, inlC, plcB. In Lm-LLO-E7, complementation with a mutated copy of prfA may result in decreased expression of different virulence genes regulated by PrfA, resulting in an overall attenuation of approximately 4-5 logs. Bring about

Example 11: High dose treatment method using ADXS11-001, Listeria monocytogenes (LM) -Listeriolysin O (LLO) immunotherapy in women with cervical cancer

  This is a phase I dose escalation open-label study (NCT02164461), 18 years of age or older with persistent, metastatic, or recurrent cervical squamous / adenocarcinoma and documented disease progression Of women (not eligible for surgery / standard radiotherapy) are registered.

Additional eligibility criteria include: Diseases that can be measured and / or evaluated according to guidelines for determining the therapeutic effect of solid cancer (RECIST v1.1: Response Evaluation Criteria in Solid Tumors); US East Coast Cancer Clinical The general status of the test group (ECOG: Eastern Cooperative Oncology Group) is 0-1; and for metastatic disease, 2 or less before treatment. The patient had measurable disease (RECIST v1.1), progress was recorded / tolerated for previous treatment, ECOG PS was 0-1. The primary endpoint was the safety and tolerability of ADXS11-001; secondary endpoints included assessment of tumor response, assessment of progression-free survival, and analysis of correlated immunity tests. Patients receive ADXS11-001 every 3 weeks for a 12-week treatment cycle. Dose escalation was performed using a 3 + 3 design with the following two doses: 5 × 10 ⁹ colony forming units (CFU; dose level 1 (DL1)) and 1 × ^{10 10} CFU (dose level 2 (DL2)). The recommended phase II dose is selected based on the observed <33% dose limiting toxicity (DLT) rate. Efficacy is analyzed using RECIST v1.1 and immune related RECIST. Blood samples are taken only in the first cycle and used for immune monitoring and cytokine / chemokine analysis.
result

  Registration to dose level 1 (DL1) is complete (n = 6). Initially three patients were enrolled in the first dose cohort; one patient experienced grade 3 hypotension as a DLT, for which an additional 3 patients were enrolled. The average age is 51.3 years, 66.7% (n = 4) has ECOG 0 as reference value, and 83.3% (n = 5) patients have squamous histology Yes. All patients had previously received median 1.5 (range 0-5) systemic chemotherapy in addition to concurrent cisplatin chemoradiotherapy. A total of 16 ADXS11-001 doses were safely administered; dose level 2 (DL2) was initiated on an developmental basis. Revised data for the determination of the maximum tolerated dose and efficacy of ADXS11-001 is presented.

Nine of 10 enrolled patients were treated (DL1, n = 6; DL2, n = 3). The median age was 53 years old. 78% had ECOG PS 0 and 33% had less than 3 (line) systemic therapies. Overall, 36 ADXS11-001 doses were administered, of which DL2 was 8. All patients experienced one or more adverse events (AE), of which treatment-related AE (TRAE) was reported in 8 out of 9 patients. TRAEs that occurred in more than two patients were chills, vomiting, hypotension, tachycardia, fever, and nausea. These were 99% grade 1-2, and one patient (DL1) experienced a single grade 3 DLT (hypotension) and no grade 4-5 TRAEs were reported. One DL2 patient has been on treatment for over 9 months. Treatment continues and a partial response is recorded.

Example 12 Results from Phase I of the ADXS11-001 Immunotherapy Phase II Trial in the Treatment of Persistent / Recurrent Metastatic Squamous Cell or Nonsquamous Cell Cervical Cancer Study Design and Qualification And therapeutic intervention

Subjects: N = about 67, Simon two-stage design; single group, 2-stage, phase II multicenter study (NCT01266460)
- Age:> 18 years of age - Eligibility: Except for those who received as a first-line treatment of components, received a> 1 times prior systemic administration chemotherapy of for PRmCC, persistent / recurrent metastatic (PRmCC) squamous epithelium Cell cervical cancer or non-squamous cell cervical cancer. Previous bevacizumab was allowed but not required.
-Measurable disease > 1 target lesion (RECIST 1.1)

1 × 10 ⁹ colony forming units of ADXS11-001 were administered as a 250 mL infusion over 60 minutes on day 1 every 28 days. Patients initially received up to three doses, followed by clinical progression, confirmation of disease progression with radiation, confirmation of intolerable toxicity, or confirmation of patient treatment refusal.

  After stage 1 preliminary analysis, the study was revised and continued with ADXS11-001 at 28-day intervals until clinical progression, confirmation of disease progression with radiation, intolerable toxicity, or patient treatment refusal Treatment was allowed (> 3 doses).

  All patients received antihistamine, anti-inflammatory, and antiemetic premedication, and a 7-day course of oral antibiotic treatment began approximately 72 hours after each ADXS11-001 infusion.

  The study summary, endpoints and key eligibility criteria are shown in FIG. Co-primary endpoint: 12-month survival rate and tolerability / safety of ADXS11-001. Secondary endpoints: progression-free survival (PFS), overall survival (OS), and objective response rate (ORR)

Statistical method

Sample size calculation is based on the expected null ratio of patients who survived 12 months in a historical study = 20%-detected a 15% increase in survival at 12 months (up to 35%) with a unilateral significance level of 0.10 90% power to do.
-Stage 1 target sample size = 27, and Stage 2 = 36.
-The test proceeded to stage 2 registration after the conditional power at the end of stage 1 was determined to be ≧ 20%.
-N = 29 patients were enrolled in stage 1 and 26 patients were treated with at least one ADXS11-001 medication.

Table 7 Standard demographics

Table 8 Study treatment exposure

Table 9 Safety (1)

Table 10 Safety (2)
result

  This Phase II trial is a persistent or recurrent metastatic (squamous or non-squamous cell) in group history that meets the protocol-specific essential efficacy and safety criteria to advance to Stage 2. 2 represents a second Simon 2-stage test for cervical cancer (PRmCC). The study further clarified Advaxis-led Lm Technology ™ immunotherapeutic agent Axarimogene philolisback (ADXS-HPV) in patients with ongoing PRmCC who had undergone at least one systemic treatment (line). , Also known as ADXS11-001), represents data from stage 1 of this ongoing two-stage phase 2 trial.

In patients who have PRmCC and who are still undergoing systemic therapy > 1 (line), ADXS11-001 shows a 38.5% 12-month survival rate (n = 10/26) (FIG. 23). ). Furthermore, the median duration of progression-free survival (PFS) was 3.1 months (FIG. 24).

  ADXS11-001 was well tolerated and was the most commonly reported AE with grade 1-2 fatigue, chills, and fever.

  Only 5 patients experienced treatment-related grade 3 or grade 4 AEs (n = 4 grade 3, n = 1 grade 4).

  Of the 26 patients treated, 18 completed all per-protocol treatment (3 doses of Axarimogene phyllolisback over 3 months) Median overall survival over 1 year (12. 1 month), and 55.6 percent 12-month overall survival (Figure 26). A 12-month survival was obtained regardless of the extent of previous treatment (1-3 times (line)) (Figure 27), Axarimogene phyllolisback was well tolerated, grades 1-2 Fatigue, chills, and fever were the most commonly reported AEs.

The CONSORT diagram (FIG. 29) shows the total number of patients enrolled and subsequently treated in stages 1 and 2 and the distribution of administered ADXS11-001 doses. Of the 26 patients treated at stage 1, the median number of dosing was 3 (range 1-3), and 69% (n = 18) received the maximum 3 doses. Of the 24 patients treated at stage 2, the median number of doses was 2.5 (range 1-6) and 50% (n = 12) received 3 or more doses. At the time of clinical trial injunction, it was subsequently lifted, but 10 patients voluntarily received ADXS11-001. Of these, 4 (40%) patients received 3 or more doses, while 6 (60%) patients received less than 3 doses. Baseline demographics and clinical characteristics of patients enrolled in stage 1 and stage 2 are shown in Table 7 above.
Safety Tolerability

  All patients (26; 100%) treated at stage 1 of the study had at least one adverse event (AE). In 24 (92%) patients, these AEs were treatment related (TRAE). Nineteen (73%) had only grade 1-2 TRAEs, 4 (15%) had grade 3 TRAEs, and 1 (4%) patients probably had related grade 4 TRAEs. It was. The most common TRAEs> 30% were fatigue, chills, fever, nausea and headache (Table 10)

The safety findings of patients enrolled in stage 2 were similar to the findings reported in detail in stage 1.
Effectiveness

For patients treated in Stage 1 of this study (n = 26):
-The 12-month survival rate is 38.5% (n = 10/26).
-Patients who survived at 12 months (n = 10/26) were once (3 of 8 patients), 2 (6 of 14 patients), or 3 (4) Experience with systemic medication (line) of 1 patient) suggests that this 12-month survival rate was obtained regardless of the extent of prior treatment.
-Median OS is 7.7 months (95% confidence interval ([CI]: 3.9-12.4) and median progression-free survival (PFS) is 3.1 months (95% CI: 2.8) -3.7, Figures 23 and 24).
-The investigator's assessment of the best tumor effect was reported in 20 of 26 treated patients.
-Seven patients (27%) had stable disease (SD) and 10 patients (38%) had progressive disease (PD).
-The remaining 6 patients could not be assessed for efficacy by investigator.
-A post-hoc analysis of 18 (69%) patients out of 26 who received all three per protocol administrations of ADXS11-001 was> 1 year median OS (12.1 months [95% CI: 6.8—not reached (NR)]) and a 12-month survival rate of 55.6% (FIG. 26).

The 12-month survival rate in GOG / NRG-0265 is ^{indistinguishable from} the historical GOG clinical trial system of PRmCC ^3-15,18 (FIG. 30).

For patients treated in stage 2 of the study (n = 24):
-The 12-month survival rate for primary endpoints cannot be calculated because of a limited median follow-up of 8.7 months.
-The 6-month survival rate is 42% (10/24) and the median OS is 4.8 months (95% CI: 3.6-NR) (Figure 31A).
-Due to clinical trial injunction, 10 out of 24 (42%) patients were discontinued or died, and ADXS11-001 was discontinued. The median PFS is 2.6 months (95% CI: 2.0-3.2) (FIG. 31B) and is similar to the value observed in stage 1.
-50% (12/24) of patients receiving ADXS11-001 at least 3 times had a median follow-up of 9.2 months and median OS was NR (95% CI: 3.5-NR ), The 6-month survival rate is 67% (FIG. 31B).
-The investigator assessment of the best tumor effect was reported in 20 of 24 patients treated.
-1 patient (4%) experienced complete disappearance (CR), 8 (33%) experienced SD and 11 (46%) had PD.

  Representative images of medical history and long-term CR are shown in FIGS.

  In patients with PRmCC who have progressed more than once (line) after receiving systemic treatment, ADXS11-001 is well tolerated and the 12-month survival rate is 38.5% (n = 10/26). Stage 2 findings reinforce the rationale for the more controlled ADXS11-001 study in PRmCC and were pre-treated with large amounts of bevacizumab (31% and 83% in stage 1 and stage 2 respectively), especially A consistent survival benefit is proposed in patients who received more than two doses of immunotherapy.

  While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. Accordingly, it is to be understood that all such modifications and changes that fall within the true spirit of the invention are covered by the appended claims.

Claims (39)

  A method of treating persistent / recurrent metastatic (squamous or non-squamous cells) cervical cancer (PRmCC) in a human subject comprising administering to said subject a recombinant Listeria strain The Listeria strain comprises a recombinant nucleic acid, the nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of the LLO protein fused to an HPV-E7 antigen or fragment thereof; Method. The Listeria strain is administered at an initial dose of 5 × 10 ⁹ colony forming units (CFU), and subsequent doses of the Listeria strain are administered to the subject three times every three weeks, or when the patient has disease progression. The method of claim 1, wherein the cervical cancer in the human subject is treated by repeated administration every three weeks until confirmation is received or complete disappearance is achieved. 3. The method of any one of claims 1-2, wherein the second and subsequent doses comprise 5 x ¹⁰ < ⁸ > CFU up to 1.0 x ¹⁰ < ¹⁰ > CFU of the Listeria strain.   4. The method of any one of claims 1-3, wherein the method is performed over a 12 week treatment cycle.   5. The method of claim 4, wherein a single maintenance dose or booster dose is administered after the method at intervals. The Listeria strain is administered at an initial dose of 1 × 10 ⁹ colony forming units (CFU), and in this case, subsequent doses of the Listeria strain are administered to the patient every 3 weeks or every 4 weeks. The method of claim 1, wherein:   7. The method of claim 6, wherein the Listeria strain is administered as an 80-250 ml infusion over 15 minutes per dose.   7. The method of any one of claims 5-6, wherein the Listeria strain is administered for a total of 3 months or about 12 weeks.   9. The method of any one of claims 1-8, wherein dosing is continued every 3-4 weeks until the disease has progressed or has completely disappeared.   10. The method according to any one of claims 1 to 9, wherein the recombinant nucleic acid further comprises a second open reading frame encoding a mutated prfA gene.   11. The method of claim 10, wherein the mutated prfA gene encodes a D133V mutation.   12. The method of any one of claims 10-11, wherein the mutated prfA gene complements a prfA genomic mutation or deletion.   12. The method according to any one of claims 1 to 11, wherein the administering step is intravenous or oral administration.   14. The method of any one of claims 1 to 13, wherein the N-terminal fragment of LLO protein comprises SEQ ID NO: 2.   The method according to any one of claims 1 to 14, wherein the recombinant Listeria strain is a recombinant Listeria monocytogenes strain.   16. The method of any one of claims 1-15, wherein the recombinant Listeria strain has been passaged through an animal host prior to the administering step.   17. The method of any one of claims 1-16, further comprising inoculating the human subject with an immunogenic composition that leads to contain or express the E7 antigen.   18. The method according to any one of claims 1 to 17, wherein the recombinant Listeria strain is stored in a frozen or lyophilized cell bank. A method of treating cervical cancer in a human subject comprising administering to said subject a combination therapy comprising chemo-radiation therapy and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, The nucleic acid comprises a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of LLO protein fused to HPV-E7 antigen or fragment thereof, wherein the Listeria strain comprises 5 × 10 ⁹ colony forming units A method wherein the first dose of (CFU) is administered, and subsequent doses of Listeria strains are administered to the patient every three weeks, thereby treating the cervical cancer in the human subject.   3 doses of the second and subsequent doses are administered, or the second and subsequent doses are repeated every 3 weeks until the patient is confirmed for disease progression or experiences complete disappearance, The method of claim 19.   21. The method of any one of claims 1 to 20, wherein the cervical cancer is a persistent / recurrent metastatic (squamous or non-squamous cell) cervical cancer. The method according to any one of claims 19 to 21, wherein the second and subsequent doses comprise 5.0 x ^{10 8 to} 1.0 x ^{10 10} CFU of the Listeria strain.   22. The method of claims 19-21, wherein the method is performed over a 12 week treatment cycle.   24. The method of claim 23, wherein after the method, a single maintenance dose or booster dose is administered at intervals.   25. The method of any one of claims 19-24, wherein the method further comprises administering at least one dose of chemoradiation therapy to the subject prior to administration of the recombinant Listeria strain.   26. The method of claim 25, wherein the subject has never received more than one dose of chemoradiotherapy.   27. The method of claims 25-26, wherein the chemoradiotherapy is cisplatin.   28. The method of claim 27, wherein the chemoradiotherapy comprises administering two courses of cisplatin with simultaneous combined radiation (54 Gy in 30 fractions by intensity modulated radiation therapy).   30. The method of claim 28, wherein the radiation therapy is continued for about 6 weeks.   30. The method of claims 19-29, wherein the subject receives median 1.5 (range 0-5) systemic chemotherapy prior to administration of the recombinant Listeria strain.   31. The method of claims 19-30, wherein the recombinant nucleic acid further comprises a second open reading frame encoding a mutated prfA gene.   32. The method of claim 31, wherein the mutated prfA gene encodes a D133V mutation.   33. The method of any one of claims 31 to 32, wherein the mutated prfA gene complements a prfA genomic mutation or deletion.   34. The method of claims 19-33, wherein the administering step is intravenous or oral administration.   35. The method of any one of claims 19 to 34, wherein the N-terminal fragment of LLO protein comprises SEQ ID NO: 2.   36. The method according to any one of claims 19 to 35, wherein the recombinant Listeria strain is a recombinant Listeria monocytogenes strain.   37. A method according to any one of claims 19 to 36, wherein the recombinant Listeria strain has been passaged through an animal host prior to the administering step.   38. The method of any one of claims 19 to 37, further comprising inoculating the human subject with an immunogenic composition that leads to contain or express the E7 antigen.   39. The method of any one of claims 19 to 38, wherein the recombinant Listeria strain is stored in a frozen or lyophilized cell bank.