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WO2016011320A1 - Système de largage d'antigènes hétérologues bivalent dérivé de listéria - Google Patents

Système de largage d'antigènes hétérologues bivalent dérivé de listéria Download PDF

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WO2016011320A1
WO2016011320A1 PCT/US2015/040855 US2015040855W WO2016011320A1 WO 2016011320 A1 WO2016011320 A1 WO 2016011320A1 US 2015040855 W US2015040855 W US 2015040855W WO 2016011320 A1 WO2016011320 A1 WO 2016011320A1
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another embodiment
protein
seq
vector
tumor
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Anu Wallecha
Poonam MOLLI
Robert Petit
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Ayala Pharmaceuticals Inc
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Advaxis Inc
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Priority claimed from US14/341,215 external-priority patent/US9650639B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

Definitions

  • ANTIGENS FIELD OF INVENTION Disclosed herein are recombinant Listeria strains comprising nucleotides encoding two or more heterologous antigens each fused to a truncated LLO, an N-terminal ActA or a PEST-sequence, methods of preparing same, and methods of inducing an immune response, and treating, inhibiting, or suppressing cancer or tumors comprising administering same.
  • BACKGROUND OF THE INVENTION [0002] A great deal of pre-clinical evidence and early clinical trial data suggests that the anti- tumor capabilities of the immune system can be harnessed to treat patients with established cancers.
  • the vaccine strategy takes advantage of tumor antigens associated with various types of cancers.
  • LLO listeriolysin-O
  • Lm-derived peptides also have access to MHC class II presentation via the phagolysosome.
  • Cancer is a complex disease and combined therapeutic approaches are more likely to succeed. Not only tumor cells, but also the microenvironment that supports tumor growth, must be targeted to maximize the therapeutic efficacy. Most immunotherapies focus on single antigens to target tumor cells and therefore they have shown limited success against human cancers. A single therapeutic agent capable of targeting multiple targets, such as tumor cells and tumor microenvironment simultaneously would have an advantage over other immunotherapeutic approaches, especially if it results in a synergistic anti-tumor effect.
  • the invention provided herein relates to a recombinant Listeria comprising a nucleotide molecule comprising a first and a second open reading frame each encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen or a functional fragment thereof fused to an endogenous PEST-containing amino acid sequence.
  • said nucleotide molecule further comprises a third open reading frame encoding a third polypeptide, wherein said third polypeptide comprises a heterologous antigen or a functional fragment thereof fused to an endogenous PEST-containing amino acid sequence.
  • the invention relates to a recombinant Listeria comprising a first nucleic acid molecule comprising an open reading frame encoding a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) or a fragment thereof fused to a truncated Listeriolysin O protein, and a second nucleic acid molecule comprising an open reading frame encoding a recombinant polypeptide comprising a chimeric HER2 antigen or a fragment thereof fused to an endogenous an LLO protein.
  • the invention relates to a method of inducing an anti-tumor immune response in a subject comprising administering to said subject the recombinant Listeria provided herein.
  • the immune response allows treating, suppressing, or inhibiting a cancer in a subject.
  • the invention relates to a recombinant Listeria strain comprising a first nucleotide molecule and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence, wherein said first nucleotide molecule is in an extrachromosomal episome or plasmid, and wherein said second nucleotide molecule is integrated into the Listeria genome.
  • the invention relates to a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence.
  • the invention relates to a recombinant Listeria strain comprising a first and a second nucleic acid molecule, said first nucleic acid molecule encoding a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) antigen or a functional fragment thereof fused to a truncated Listeriolysin O (LLO), and wherein said second nucleic acid molecule encodes a chimeric HER (cHER2) protein fused to an endogenous LLO.
  • the invention relates to a method of increasing the efficacy of a Listeria-based immunotherapy, said method comprising sequentially or concomitantly administering two or more recombinant Listeria strains to a subject having a tumor.
  • the Listeria strains that are administered sequentially or concomitantaly each comprise a nucleic acid molecule, said nucleic acid molecule encoding a recombinant polypeptide comprising a heterologous antigen fused to a PEST-containing polypeptide.
  • the heterologous antigen is chimeric HER (cHER2), CA9, PSA or HMW-MAA-C.
  • the invention relates to a method of producing a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence, said method comprising the steps of: a) Obtaining a plasmid
  • the invention relates to a method of producing the recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST- containing amino acid sequence, said method comprising the steps of: a) Obtaining a plasmid
  • FIG. 1 Map of the pADV134 plasmid.
  • B Proteins from LmddA-134 culture supernatant were precipitated, separated in a SDS-PAGE, and the LLO-E7 protein detected by Western-blot using an anti-E7 monoclonal antibody.
  • the antigen expression cassette consists of hly promoter, ORF for truncated LLO and human PSA gene (klk3).
  • C Map of the pADV142 plasmid.
  • FIG. 1 Schematic representation of monovalent and bivalent plasmids. Restriction sites that were used for cloning of antigen 1 (XhoI and SpeI) and antigen 2 (XbaI and SacI or BglII) are indicated. The black arrow represents the direction of transcription.
  • p15 ori and RepR refers to E.coli and Listeria origin of replication.
  • tLLO is truncated Listeriolysin O protein. Bacillus-dal gene codes for D-alanine racemase which complements for the synthesis of D-alanine in Lm ⁇ dal dat strain.
  • FIG. 1 Plasmid stability in vitro of LmddA-LLO-PSA if cultured with and without selection pressure (D-alanine). Strain and culture conditions are listed first and plates used for CFU determination are listed after.
  • B Clearance of LmddA-LLO-PSA in vivo and assessment of potential plasmid loss during this time. Bacteria were injected i.v. and isolated from spleen at the time point indicated. CFUs were determined on BHI and BHI + D-alanine plates.
  • Figure 5. In vivo clearance of the strain LmddA-LLO-PSA after administration of 10 8 CFU in C57BL/6 mice.
  • FIG. 1 (A) Schematic representation of the chromosomal region of the Lmdd-143 and LmddA-143 after klk3 integration and actA deletion; (B) The klk3 gene is integrated into the Lmdd and LmddA chromosome.
  • the Lm-tLLO-HMW-MMA 2160-2258 (also referred as Lm-LLO-HMW-MAA-C) strain secretes a ⁇ 62 kDa band corresponding to the tLLO-HMW-MAA 2160-2258 fusion protein (B).
  • Mice immunized with the Lm-LLO-HMW-MAA-C impeded the growth of established tumors (C).
  • Sequential tissues were either stained with the pan-vessel marker anti-CD31 or the anti-NG2 antibody for the HMW-MAA mouse homolog AN2, in conjunction with anti-CD8 ⁇ for possible TILs.
  • Group 3 shows isotype controls for the above antibodies and DAPI staining used as a nuclear marker. A total of 5 tumors were analyzed and a single representative image from each group is shown. CD8 + cells around blood vessels are indicated by arrows.
  • LmddA244G was transformed with the plasmid containing expression cassette for the fusion protein tLLO-HMC (pAdv168) (B) resulting in strain LmddA244G/168.
  • LmddA strain was transformed with plasmid pAdv164, which contains expression cassette for tLLO-cHer2 fusion protein to create LmddA164 vaccine.
  • LmddA backbone was transformed with plasmid pAv168, which contains expression cassette for tLLO-HMC (2160-2258 amino acid residues at the C-terminus of HMW-MAA or CSPG4) fusion protein to create LmddA168 vaccine.
  • FIG. 16 A. Established NT2 tumors were implanted with treated with mono therapies and bivalent therapy on days 6, 13 and 20. The na ⁇ ve group is untreated mice. B. The percent tumor free mice in different treatment and untreated na ⁇ ve group. C. The volume of established NT2 tumors after of LmddA244G-168 treatment. [00031] Figure 17. A. Generation of Her2 specific immune responses in mice after administration of monovalent (LmddA164) as well as bivalent immunotherapy (LmddA244G-168) expressing chimera Her2.
  • the Her2 specific immune responses were evaluated in an ELIspot based assay using FvB IC1 peptide epitope -RLLQETELV (Seavey et al 2009, Clin Cancer Res. 2009 Feb 1;15(3):924-32.
  • the Her2 specific immune responses were evaluated in an ELISA based assay using affinity purified HMA- MAA-C protein fragment.
  • Figure 18 Immunohistochemical (IHC) staining of tumors anti-CD3 antibody on day 27 of the tumor regression study.
  • NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated na ⁇ ve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168).
  • A untreated na ⁇ ve group
  • B mono immunotherapy
  • C mono immunotherapy
  • LmddA168 mono immunotherapy
  • D bivalent immunotherapy
  • NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated na ⁇ ve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168).
  • A untreated na ⁇ ve group
  • B mono immunotherapy
  • C mono immunotherapy
  • LmddA168 mono immunotherapy
  • D bivalent immunotherapy
  • NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated na ⁇ ve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168).
  • A untreated na ⁇ ve group
  • B mono immunotherapy
  • C mono immunotherapy
  • LmddA168 mono immunotherapy
  • D bivalent immunotherapy
  • NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated na ⁇ ve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168).
  • A untreated na ⁇ ve group
  • B mono immunotherapy
  • C mono immunotherapy
  • LmddA168 mono immunotherapy
  • LmddA244G-168 bivalent immunotherapy
  • Established 4T1 tumors were treated with mono therapies and bivalent therapy on days 1, 8, and 15.
  • the na ⁇ ve group is untreated mice.
  • Figure 25 Established NT2 tumors were treated with mono therapies, bivalent therapy, or sequential mono therapies.
  • the na ⁇ ve group is untreated mice.
  • a recombinant Listeria comprising a recombinant nucleic acid molecule comprising a first and a second open reading frame each encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen or a functional fragment thereof fused to a PEST-containing amino acid sequence.
  • said nucleotide molecule further comprises a third open reading frame encoding a third polypeptide, wherein said third polypeptide comprises a heterologous antigen or a functional fragment thereof fused to an endogenous PEST-containing amino acid sequence.
  • a method of inducing an anti-tumor immune response in a subject comprising the step of administering to said subject the recombinant Listeria provided herein.
  • the immune response allows treating, suppressing, or inhibiting a cancer in a subject.
  • a recombinant Listeria strain comprising a first nucleotide molecule and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence, wherein said first nucleotide molecule is in an extrachromosomal episome or plasmid, and wherein said second nucleotide molecule is integrated into the Listeria genome.
  • the invention discloses, in another embodiment, a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence.
  • the invention discloses, in another embodiment, a recombinant Listeria strain comprising a first and a second nucleic acid molecule, wherein said first nucleic acid molecule encodes a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) antigen or a functional fragment thereof fused to a truncated Listeriolysin O (LLO), and wherein said second nucleic acid molecule encodes a chimeric HER (cHER2) protein fused to an endogenous LLO.
  • a recombinant Listeria strain comprising a first and a second nucleic acid molecule, wherein said first nucleic acid molecule encodes a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) antigen or a functional fragment thereof fused to a truncated Listeriolysin O (LLO), and wherein said second nucleic acid molecule encodes a chimeric HER (cHER2) protein fuse
  • the invention discloses, in another embodiment, a method of increasing the efficacy of a Listeria-based immunotherapy, the method comprising sequentially or concomitantly administering two or more recombinant Listeria strains to a subject having a tumor.
  • the Listeria strains that are administered sequentially or concomitantaly each comprise a nucleic acid molecule, said nucleic acid molecule encoding a recombinant polypeptide comprising a heterologous antigen fused to a PEST-containing polypeptide.
  • the heterologous antigen is chimeric HER (cHER2), CA9, PSA or HMW-MAA-C.
  • the method of increasing the efficacy of a Listeria- based immunotherapy enhances an antigen-specific immune response as a result of administering the same.
  • a method of producing a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST- containing amino acid sequence, said method comprising the steps of: a) Obtaining a plasmid
  • a method of producing the recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST- containing polypeptide, said method comprising the steps of: a) Obtaining a plasmid
  • a recombinant Listeria strain comprising a bivalent episomal expression vector, the vector comprising a first, and at least a second nucleic acid molecule encoding a heterologous antigenic polypeptide or a functional fragment thereof, wherein the first and the second nucleic acid molecules each encode the heterologous antigenic polypeptide or functional fragment thereof in an open reading frame with an endogenous PEST-containing polypeptide.
  • the term“bivalent” or“multivalent,” when in reference to a nucleotide molecule, plasmid, or vector may encompass a nucleotide molecule, nucleic acid, DNA sequence, plasmid, and the like, that expresses two (bivalent), three (multivalent), or more (multivalent) heterologous antigens each individually fused to a PEST-containing sequence (such as an N-terminal or truncated LLO, N-terminal ActA, or a PEST sequence or PEST peptide).
  • a bivalent plasmid encodes two heterologous antigens.
  • a bivalent plasmid encodes two different heterologous antigens.
  • the term“bivalent” is used interchangeably herein with “dual”.
  • a multivalent plasmid encodes three different heterologous antigens.
  • the term“bivalent” or“multivalent,” when in reference to a recombinant Listeria strain may encompass a Listeria strain that is capable of expressing two (bivalent) or more (multivalent) heterologous antigens.
  • the term “bivalent” or “multivalent,” when in reference to a recombinant Listeria strain may encompass a Listeria strain that expresses two (bivalent) or more (multivalent) heterologous antigens.
  • a bivalent Listeria strain comprises a bivalent plasmid that expresses two heterologous antigens from a plasmid.
  • a bivalent Listeria strain expresses one heterologous antigen from the genome, (following integration of the heterologous antigen into the Listeria genome), and another heterologous antigen from a plasmid present in the cytoplasm of said Listeria strain.
  • a multivalent Listeria strain expresses three heterologous antigens from a plasmid.
  • a multivalent Listeria strain expresses three heterologous antigens, one from the genome (following integration of the heterologous antigen into the Listeria genome) and two from a plasmid present in the cytoplasm of the Listeria strain.
  • a multivalent Listeria strain expresses three heterologous antigens, two from the genome (following integration of the heterologous antigen into the Listeria genome) and one from a plasmid present in the cytoplasm of the Listeria strain.
  • a multivalent Listeria strain comprises the ability to express three or more heterologous antigens in total, where at least one is expressed from either the genome or from a plasmid (in any desired combination, i.e.- 3 from the plasmid and 1 from the genome, 2 from the plasmid and 2 from the genome, 1 from the plasmid and 3 from the genome, etc.).
  • a bivalent Listeria strain expresses one heterologous antigen from the genome in the context of a fusion protein with a PEST-containing polypeptide, (following integration of the heterologous antigen into the frame of the gene encoding the PEST-containing polypeptide in the Listeria genome), and another heterologous antigen from a plasmid present in the cytoplasm of said Listeria strain, also in the context of a fusion protein with a PEST-containing polypeptide.
  • a multivalent Listeria strain expresses three heterologous antigens from a plasmid, all in the context of a fusion protein with a PEST-containing polypeptide.
  • a multivalent Listeria strain expresses three heterologous antigens, one from the genome (following integration of the heterologous antigen into the Listeria genome) and two heterologous antigens from a plasmid present in the cytoplasm of the Listeria strain, all in the context of a PEST-containing polypeptide.
  • a multivalent Listeria strain expresses three heterologous antigens, two from the genome (following integration of the heterologous antigen into the Listeria genome) and one from a plasmid present in the cytoplasm of the Listeria strain, all in the context of a fusion protein with a PEST-containing polypeptide.
  • a multivalent Listeria strain comprises the ability to express three or more heterologous antigens in total, where at least one is expressed from either the genome or from a plasmid (in any desired combination, i.e.- 3 from the plasmid and 1 from the genome, 2 from the plasmid and 2 from the genome, 1 from the plasmid and 3 from the genome, etc.,) all in the context of a fusion protein with a PEST- containing polypeptide.
  • the PEST-containing polypeptide is a truncated LLO, an N- terminal ActA protein, or a PEST sequence.
  • antigen refers to any polypeptide that are loaded onto and presented on MHC class I and/or class II molecules on a host’s cell’s surface and can be recognized or detected by an immune cell of the host, thereby leading to the mounting of an immune response against the polypeptide, peptide or cell presenting the same.
  • the immune response may also extend to other cells within the host, including diseased cells such as tumor or cancer cells that express the same polypeptides or peptides.
  • an antigen may be foreign, that is, heterologous to the host and is referred to as a“heterologous antigen” herein.
  • a heterologous antigen is heterologous to a Listeria strain provided herein that recombinantly expresses said antigen.
  • a heterologous antigen is heterologous to the host and a Listeria strain provided herein that recombinantly expresses said antigen.
  • the antigen is a self-antigen, which is an antigen that is present in the host but the host does not elicit an immune response against it because of immunologic tolerance. It will be appreciated by a skilled artisan that a heterologous antigen as well as a self-antigen may encompass a tumor antigen, a tumor-associated antigen or an angiogenic antigen.
  • a heterologous antigen may encompass an infectious disease antigen.
  • the terms“recombinant Listeria” and“live-attenuated Listeria” are used interchangeably here and refer to a Listeria comprising at least one attenuating mutation, deletion or inactivation that expresses a fusion protein of an antigen fused to a truncated LLO, truncated ActA or PEST sequence embodied herein.
  • a recombinant Listeria provided herein is a recombinant Listeria monocytogenes.
  • nucleic acid “nucleotide,”“nucleic acid molecule,”“oligonucleotide,” or“nucleotide molecule” are used interchangeably herein and may encompass a string of at least two base-sugar-phosphate combinations, as will be appreciated by a skilled artisan.
  • the terms include, in one embodiment, DNA and RNA. It will also be appreciated by a skilled artisan that the terms may encompass the monomeric units of nucleic acid polymers.
  • RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes.
  • siRNA and miRNA has been described (Caudy AA et al, Genes & Devel 16: 2491-96 and references cited therein).
  • DNA may be in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups.
  • these forms of DNA and RNA may be single, double, triple, or quadruple stranded.
  • the term may also encompass artificial nucleic acids that may contain other types of backbones but the same bases.
  • phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353-57; and Raz NK et al Biochem Biophys Res Commun. 297:1075- 84.
  • the production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed.
  • amino acid or “amino acids” are understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid may include both D- and L-amino acids.
  • a nucleic acid molecule operably integrated into a genome as an open reading frame with an endogenous polypeptide is a nucleic acid molecule that has integrated into a genome in the same open reading frame as an endogenous polypeptide.
  • endogenous may encompass an item that has developed or originated within the reference organism or arisen from causes within the reference organism.
  • endogenous refers to native.
  • fragment may encompass a protein or polypeptide that is shorter or comprises fewer amino acids than the full length protein or polypeptide.
  • a fragment is an N-terminal fragment.
  • a fragment is a C-terminal fragment.
  • a fragment is an intrasequential section of the protein or peptide.
  • a fragment as provided herein is a functional fragment, which may encompass an immunogenic fragment.
  • a fragment has more than 5 amino acids.
  • a fragment has 10-20 amino acids, 20-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-350 amino acids, or 350-500 amino acids.
  • the term“fragment” refers to a nucleic acid that is shorter or comprises fewer nucleotides than the full length nucleic acid.
  • a fragment is a 5’-terminal fragment.
  • a fragment is a 3’-terminal fragment.
  • a fragment encodes an intrasequential section of the protein.
  • a fragment has more than 5 nucleotides.
  • a fragment has 10-20 nucleotides, 20-50 nucleotides, 50-100 nucleotides, 100-200 nucleotides, 200-350 nucleotides, 350-500 or 500-1000 nucleotides.
  • the term“functional” within the meaning of the invention may encompass the innate ability of a protein, peptide, nucleic acid, fragment or a variant thereof to exhibit a biological activity.
  • a biological activity may encompass having the potential to elicit an immune response when used as provided herein, an illustration of which may be to be used as part of a fusion protein).
  • Such a biological function may encompass its binding property to an interaction partner, e.g., a membrane-associated receptor, or its trimerization property.
  • these biological functions may in fact be changed, e.g., with respect to their specificity or selectivity, but with retention of the basic biological function.
  • the term“functional fragment” may encompass an immunogenic fragment that is capable of eliciting an immune response when administered to a subject alone or as part of a pharmaceutical composition comprising a recombinant Listeria strain expressing said immunogenic fragment.
  • a functional fragment has biological activity as will be understood by a skilled artisan and as further provided herein.
  • a multivalent plasmid that delivers at least two antigens.
  • the plasmid is a dual or bivalent plasmid.
  • the dual, bivalent or multivalent plasmid is episomal in nature in that it remains extrachromosomal.
  • the dual, or multivalent plasmid comprises sequences for integration into the Listeria chromosome.
  • the recombinant nucleic acid backbone of a plasmid disclosed herein comprises SEQ ID NO: 1.
  • a bivalent plasmid backbone comprises at least two nucleic acid sequences encoding at least two antigens.
  • the bivalent plasmid backbone comprises a nucleic acid sequences having at least two open reading frames encoding at least two antigens.
  • the bivalent plasmid backbone comprises a nucleic acid sequences having two open reading frames encoding two antigens.
  • the multivalent plasmid backbone comprises a nucleic acid sequences having at least three open reading frames encoding at least three antigens.
  • the multivalent plasmid backbone comprises at least three nucleic acid sequences having at least three open reading frames encoding at least three antigens. In another embodiment, the multivalent plasmid backbone comprises a nucleic acid sequences having three open reading frames encoding three antigens. In another embodiment, the multivalent plasmid backbone comprises three nucleic acid sequences having three open reading frames encoding three antigens.
  • the antigens encoded by the bivalent Listeria strains disclosed herein include CA9, chimeric HER2 (cHER2), and HMW-MAA or a fragment thereof (see Examples 11-16 herein).
  • HMW-MAA fragment is HMW-MAA- C (HMC).
  • a Listeria strain LmddA244G disclosed herein comprises a nucleic acid sequence comprising an open reading frame encoding a cHER2 fused to an endogenous nucleic acid comprising an open reading frame encoding an LLO protein (see SEQ ID NO: 2).
  • the endogenous LLO- CHER2 fusion is a homolog of SEQ ID NO: 2. In another embodiment, the endogenous LLO- CHER2 fusion is a variant of SEQ ID NO: 2. In another embodiment, the endogenous LLO- CHER2 fusion is an isomer of SEQ ID NO: 2. [00072] In one embodiment, the amino acid sequence of the fusion between a cHER2 and an endogenous LLO comprises SEQ ID NO: 3
  • the amino acid sequence of endogenous LLO protein comprises SEQ ID NO: 4, M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S V A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N N A V N T L V E R W N E K Y A Q A Y S N V S A K I D Y D D E M A Y S E S Q L I A
  • LmddA164 comprises a nucleic acid sequence comprising an open reading frame encoding tLLO fused to cHER2, wherein said nucleic acid sequence comprises SEQ ID NO: 5: atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaata aagaaaattcaattttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgtgacaaatgtgccgccaagaaaagtaagtaccaagaaaagta
  • plasmid pAdv168 comprises SEQ ID NO: 5.
  • the truncated LLO-CHER2 fusion is a homolog of SEQ ID NO: 5.
  • the truncated LLO-CHER2 fusion is a variant of SEQ ID NO: 5.
  • the truncated LLO-CHER2 fusion is an isomer of SEQ ID NO: 5.
  • an amino acid sequence of a tLLO fused to a cHER2 comprises SEQ ID NO: 6:
  • the truncated LLO-cHER2 fusion is a homolog of SEQ ID NO: 6. In another embodiment, the truncated LLO-cHER2 fusion is a variant of SEQ ID NO: 6. In another embodiment, the truncated LLO-cHER2 fusion is an isomer of SEQ ID NO: 6.
  • an amino acid sequence of a truncated LLO comprises SEQ ID NO: 7: M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S M A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N A V N T L V E R W N E K Y A Q A Y P N V S A K I D Y D D E M A
  • LmddA168 comprises a nucleic acid sequence comprising an open reading frame encoding tLLO fused to HMW-MAA-C (HMC) comprises SEQ ID NO: 8: atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaata aagaaaattcaattttcatccatggcaccaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgtgacaaatgtgccgccaagaaaagtaagtatataccacggagatgca
  • plasmid pAdv168 comprises SEQ ID NO: 8.
  • the truncated LLO-HMC fusion is a homolog of SEQ ID NO: 8.
  • the truncated LLO-HMC fusion is a variant of SEQ ID NO: 8.
  • the truncated LLO-HMC fusion is an isomer of SEQ ID NO: 8.
  • an amino acid sequence of a tLLO fused to an HMC antigen comprises SEQ ID NO: 9: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEI DKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAIS SLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVN TLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKM QEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLK LSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDL RDILKKGATFNRETPG
  • the truncated LLO-HMC fusion is a homolog of SEQ ID NO: 9. In another embodiment, the truncated LLO-HMC fusion is a variant of SEQ ID NO: 9. In another embodiment, the truncated LLO-HMC fusion is an isomer of SEQ ID NO: 9.
  • the sequence of HMC comprises SEQ ID NO: 10: A T E P Y N A A R P Y S V A L L S V P E A A R T E A G K P E S S T P T G E P G P M A S S P E P A V A K G G F L S F L E A N M F S V I I P M C L V L L L L A L I L P L L F Y L R K R N K T G K H D V Q D Y K D D D D D K (SEQ ID NO: 10).
  • the antigens are heterologous antigens to the bacteria host carrying the plasmid.
  • the antigens are heterologous antigens to the Listeria host carrying the plasmid.
  • the recombinant episomal nucleic acid sequence encoding the plasmid backbone and at least two heterologous antigens comprises SEQ ID NO: 11.
  • the recombinant episomal nucleic acid sequence encoding the plasmid backbone and at least two heterologous antigens consists of SEQ ID NO: 11.
  • a vaccine comprising a recombinant Listeria strain, wherein said Listeria further comprises a bivalent or multivalent plasmid disclosed herein and an adjuvant, cytokine, chemokine, or a combination thereof.
  • an immunotherapy comprising a recombinant Listeria strain, wherein said Listeria further comprises a bivalent or multivalent plasmid disclosed herein and an adjuvant, cytokine, chemokine, or a combination thereof.
  • a pharmaceutical formulation comprising a recombinant Listeria strain, wherein said Listeria further comprises the bivalent or multivalent plasmid disclosed herein and an adjuvant, cytokine, chemokine, or a combination thereof.
  • a recombinant Listeria strain comprising a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide or fragment thereof, wherein the first nucleic acid molecule is integrated into the Listeria genome in an open reading frame with an endogenous LLO gene and wherein the second nucleic acid molecule is present in an episomal expression vector or plasmid within the recombinant Listeria strain.
  • the first nucleic acid molecule encodes a chimeric HER2 (cHER2) protein fused to an endogenous LLO protein and the second nucleic acid molecule encodes an HMW-MAA peptide fused to a truncated LLO, truncated ActA protein, or PEST sequence.
  • this invention provides a recombinant Listeria strain comprising a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide fused to a truncated LLO, a truncated or N-terminal ActA protein or a PEST sequence.
  • the first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with an endogenous nucleic acid sequence encoding a an LLO protein, an ActA protein or a PEST sequence.
  • the first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid sequence encoding LLO.
  • the first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid sequence encoding ActA.
  • the integration does not eliminate the functionality of LLO.
  • the integration does not eliminate the functionality of ActA.
  • the functionality of LLO or ActA is its native functionality.
  • the LLO functionality is allowing the organism to escape from the phagolysosome, while in another embodiment, the LLO functionality is enhancing the immunogenicity of a polypeptide to which it is fused.
  • a recombinant Listeria of the present invention retains LLO function, which in one embodiment, is hemolytic function and in another embodiment, is antigenic function. Other functions of LLO are known in the art, as are methods and assays for evaluating LLO functionality.
  • a recombinant Listeria of the present invention has wild-type virulence, while in another embodiment, a recombinant Listeria of the present invention has attenuated virulence.
  • a recombinant Listeria of the present invention is avirulent. In one embodiment, a recombinant Listeria of the present invention is sufficiently virulent to escape the phagolysosome and enter the cytosol. In one embodiment, a recombinant Listeria of the present invention expresses a fused antigen-LLO protein.
  • the integration of the first nucleic acid molecule into the Listeria genome does not disrupt the structure nor, in another embodiment, the function of the endogenous LLO gene, ActA gene, or PEST-containing gene. In one embodiment, the integration of the first nucleic acid molecule into the Listeria genome does not disrupt the ability of said Listeria to escape the phagolysosome.
  • either the second nucleic acid is integrated into the genome while the first is expressed from a plasmid.
  • the second nucleic acid molecule is operably integrated into the Listeria genome with said first nucleic acid molecule in an open reading frame with an endogenous polypeptide comprising a PEST sequence.
  • the first and second nucleic acid molecules are integrated in frame with a nucleic acid sequence encoding an LLO protein, while in another embodiment, they are integrated in frame with a nucleic acid sequence encoding an ActA protein.
  • the second nucleic acid molecule is operably integrated into the Listeria genome in an open reading frame with a nucleic acid sequence encoding a polypeptide comprising a PEST sequence in a site that is distinct from the integration site of the first nucleic acid molecule.
  • the first nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding an LLO protein, while the second nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding an ActA protein, while in another embodiment, the first nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding an ActA protein, while the second nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding a LLO protein.
  • this invention provides a recombinant Listeria strain comprising a first nucleic acid molecule encoding a first heterologous antigenic polypeptide or fragment thereof and a second nucleic acid molecule encoding a second heterologous antigenic polypeptide or fragment thereof, wherein said first nucleic acid molecule is integrated into the Listeria genome such that the first heterologous antigenic polypeptide and an LLO, ActA or PEST sequence are expressed as a fusion protein.
  • the first heterologous antigenic polypeptide and the LLO, ActA or PEST sequence are translated in a single open reading frame, while in another embodiment, the first heterologous antigenic polypeptide and the LLO, ActA or PEST sequence are fused after being translated separately.
  • the Listeria genome comprises a deletion of the endogenous ActA gene, which in one embodiment is a virulence factor. In one embodiment, such a deletion provides a more attenuated and thus safer Listeria strain for human use.
  • the antigenic polypeptide is integrated in frame with LLO in the Listeria chromosome.
  • the integrated nucleic acid molecule is integrated into the ActA locus.
  • the chromosomal nucleic acid encoding ActA is replaced by a nucleic acid molecule encoding an antigen.
  • the Listeria strain comprises an inactivation of the endogenous actA gene.
  • the Listeria strain comprises an truncation of the endogenous actA gene.
  • the Listeria strain comprises a non-functional replacement of the endogenous actA gene.
  • the Listeria strain comprises a substitution of the endogenous actA gene. All of the above-mentioned modifications fall within the scope of what is considered to be a“mutation” of the endogenous actA gene.
  • the Listeria strain provided herein comprises a mutation, deletion or an inactivation of the endogenous dal/dat and actA genes and such a Listeria strain is referred to herein as an“LmddA” strain.
  • the Listeria strain provided herein comprises a mutation, deletion or an inactivation of the endogenous dal/dat/actA and prfA genes.
  • the bivalent or multivalent plasmids provided herein comprise a replication control region.
  • a recombinant nucleic acid molecule encoding the bivalent or multivalent plasmid provided herein comprises a replication control region.
  • the plasmid control region regulates replication of the recombinant nucleic acid molecule.
  • the plasmid control region comprises an open reading frame encoding a transcription repressor that represses heterologous antigen expression from the first or at least the second nucleic acid molecule.
  • the plasmid control region comprises an open reading frame encoding transcription inducer that induces heterologous antigen expression from the first or at least the second nucleic acid molecule.
  • the plasmid control region comprises an open reading frame encoding a transcription repressor that represses heterologous antigen expression from a first, second or third nucleic acid molecule.
  • the plasmid control region comprises an open reading frame encoding a transcription inducer that induces heterologous antigen expression from the first, second or third nucleic acid molecule.
  • the plasmid replication regulation region enables the regulation of expression of exogenous heterologous antigen from each of the first or at least the second open reading frame of a recombinant nucleic acid molecule comprised by the Listeria or the plasmid disclosed herein.
  • the plasmid replication regulation region enables the regulation of expression of exogenous heterologous antigen from each of the first, second or third open reading frames.
  • measuring metabolic burden in a bacteria such as a Listeria is accomplished by any means know in the art at the time of the invention which include but are not limited to, measuring growth rates of the vaccine strain, optical density readings, colony forming unit (CFU) plating, and the like.
  • the metabolic burden on the bacterial cell is determined by measuring the viability of the bacterial cell. Methods of measuring bacteria viability are readily known and available in the art, some of which include but are not limited to, bacteria plating for viability count, measuring ATP, and flow cytometry. In ATP staining, detection is based on using the luciferase reaction to measure the amount of ATP from viable cells, wherein the amount of ATP in cells correlates with cell viability.
  • this method can be used in various ways, also known in the art, for example after employing the use of viability dyes which are excluded by live bacterial cells and are absorbed or adsorbed by a dead bacterial cells.
  • viability dyes which are excluded by live bacterial cells and are absorbed or adsorbed by a dead bacterial cells.
  • a skilled artisan would readily understand that these and any other methods known in the art for measuring bacterial viability can be used in the present invention. It is to be understood that a skilled artisan would be able to implement the knowledge available in the art at the time of the invention for measuring growth rates of the vaccine strain or expression of marker genes by the vaccine strain that enable determining the metabolic burden of the vaccine strain expressing multiple heterologous antigens or functional fragments thereof.
  • the integrated nucleic acid molecule is integrated into the Listeria chromosome.
  • said first nucleic acid molecule is a vector designed for site- specific homologous recombination into the Listeria genome.
  • the construct or heterologous gene is integrated into the Listerial chromosome using homologous recombination.
  • Techniques for homologous recombination are well known in the art, and are described, for example, in Frankel, FR, Hegde, S, Lieberman, J, and Y Paterson. Induction of a cell-mediated immune response to HIV gag using Listeria monocytogenes as a live vaccine vector. J. Immunol. 155: 4766 - 4774.
  • the construct or heterologous gene is integrated into the Listerial chromosome using transposon insertion.
  • Techniques for transposon insertion are well known in the art, and are described, inter alia, by Sun et al. (Infection and Immunity 1990, 58: 3770-3778) in the construction of DP-L967.
  • Transposon mutagenesis has the advantage, in one embodiment, that a stable genomic insertion mutant can be formed.
  • the position in the genome where the foreign gene has been inserted by transposon mutagenesis is unknown.
  • the construct or heterologous gene is integrated into the Listerial chromosome using phage integration sites (Lauer P, Chow MY et al, Construction, characterization, and use of two LM site-specific phage integration vectors. J Bacteriol 2002;184(15): 4177-86).
  • an integrase gene and attachment site of a bacteriophage e.g. U153 or PSA listeriophage
  • is used to insert the heterologous gene into the corresponding attachment site which can be any appropriate site in the genome (e.g.
  • the first nucleic acid sequence of methods and compositions as provided herein is operably linked to a promoter/regulatory sequence.
  • the second nucleic acid sequence is operably linked to a promoter/regulatory sequence.
  • each of the nucleic acid sequences disclosed herein are operably linked to a promoter/regulatory sequence.
  • the promoter/regulatory sequence is present on an episomal plasmid cmprising said nucleic acid sequence.
  • an endogenous Listeria promoter/regulatory sequence controls the expression of a nucleic acid sequence of the methods and compositions of the present invention.
  • a nucleic acid sequence disclosed herein is operably linked to a promoter, regulatory sequence, or a combination thereof that drives expression of the encoded peptide in the Listeria strain.
  • Promoter, regulatory sequences, and combinations thereof useful for driving constitutive expression of a gene are well known in the art and include, but are not limited to, for example, the P hlyA , P actA , hly, and p60 promoters of Listeria, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter, and the B.
  • inducible and tissue specific expression of the nucleic acid encoding a peptide as provided herein is accomplished by placing the nucleic acid encoding the peptide under the control of an inducible or tissue-specific promoter/regulatory sequence.
  • tissue-specific or inducible regulatory sequences, promoters, and combinations thereof which are useful for his purpose include, but are not limited to the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter.
  • a promoter that is induced in response to inducing agents such as metals, glucocorticoids, and the like, is utilized.
  • a regulatory sequence is a promoter, while in another embodiment, a regulatory sequence is an enhancer, while in another embodiment, a regulatory sequence is a suppressor, while in another embodiment, a regulatory sequence is a repressor, while in another embodiment, a regulatory sequence is a silencer.
  • the nucleic acid construct used for integration to the Listeria genome contains an integration site.
  • the site is a PhSA (phage from Scott A) attPP' integration site. PhSA is, in another embodiment, the prophage of L.
  • the site is any another integration site known in the art.
  • the nucleic acid construct contains an integrase gene.
  • the integrase gene is a PhSA integrase gene.
  • the integrase gene is any other integrase gene known in the art.
  • the nucleic acid construct is a plasmid. In another embodiment, the nucleic acid construct is a shuttle plasmid. In another embodiment, the nucleic acid construct is an integration vector. In another embodiment, the nucleic acid construct is a site-specific integration vector. In another embodiment, the nucleic acid construct is any other type of nucleic acid construct known in the art. [000107]
  • the integration vector of methods and compositions disclosed herein is, in another embodiment, a phage vector. In another embodiment, the integration vector is a site-specific integration vector. In another embodiment, the vector further comprises an attPP’ site. [000108] In another embodiment, the integration vector is a U153 vector. In another embodiment, the integration vector is an A118 vector.
  • the integration vector is a PhSA vector.
  • the vector is an A511 vector (e.g. GenBank Accession No: X91069).
  • the vector is an A006 vector.
  • the vector is a B545 vector.
  • the vector is a B053 vector.
  • the vector is an A020 vector.
  • the vector is an A500 vector (e.g. GenBank Accession No: X85009).
  • the vector is a B051 vector.
  • the vector is a B052 vector.
  • the vector is a B054 vector.
  • the vector is a B055 vector.
  • the vector is a B056 vector. In another embodiment, the vector is a B101 vector. In another embodiment, the vector is a B110 vector. In another embodiment, the vector is a B1 ll vector. In another embodiment, the vector is an A153 vector. In another embodiment, the vector is a D441 vector. In another embodiment, the vector is an A538 vector. In another embodiment, the vector is a B653 vector. In another embodiment, the vector is an A513 vector. In another embodiment, the vector is an A507 vector. In another embodiment, the vector is an A502 vector. In another embodiment, the vector is an A505 vector. In another embodiment, the vector is an A519 vector. In another embodiment, the vector is a B604 vector.
  • the vector is a C703 vector. In another embodiment, the vector is a B025 vector. In another embodiment, the vector is an A528 vector. In another embodiment, the vector is a B024 vector. In another embodiment, the vector is a B012 vector. In another embodiment, the vector is a B035 vector. In another embodiment, the vector is a C707 vector. [000110] In another embodiment, the vector is an A005 vector. In another embodiment, the vector is an A620 vector. In another embodiment, the vector is an A640 vector. In another embodiment, the vector is a B021 vector. In another embodiment, the vector is an HSO47 vector. In another embodiment, the vector is an H10G vector. In another embodiment, the vector is an H8/73 vector.
  • the vector is an H19 vector. In another embodiment, the vector is an H21 vector. In another embodiment, the vector is an H43 vector. In another embodiment, the vector is an H46 vector. In another embodiment, the vector is an H107 vector. In another embodiment, the vector is an H108 vector. In another embodiment, the vector is an H110 vector. In another embodiment, the vector is an H163/84 vector. In another embodiment, the vector is an H312 vector. In another embodiment, the vector is an H340 vector. In another embodiment, the vector is an H387 vector. In another embodiment, the vector is an H391/73 vector. In another embodiment, the vector is an H684/74 vector. In another embodiment, the vector is an H924A vector.
  • the vector is a 184 vector. In another embodiment, the vector is a 575 vector. In another embodiment, the vector is a 633 vector. In another embodiment, the vector is a 699/694 vector. In another embodiment, the vector is a 744 vector. In another embodiment, the vector is a 900 vector. In another embodiment, the vector is a 1090 vector. In another embodiment, the vector is a 1317 vector. In another embodiment, the vector is a 1444 vector. In another embodiment, the vector is a 1652 vector. In another embodiment, the vector is a 1806 vector. In another embodiment, the vector is a 1807 vector. In another embodiment, the vector is a 1921 /959 vector. In another embodiment, the vector is a 1921/11367 vector.
  • the vector is a 1921/11500 vector. In another embodiment, the vector is a 1921/11566 vector. In another embodiment, the vector is a 1921/12460 vector. In another embodiment, the vector is a 1921/12582 vector. In another embodiment, the vector is a 1967 vector. In another embodiment, the vector is a 2389 vector. In another embodiment, the vector is a 2425 vector. In another embodiment, the vector is a 2671 vector. In another embodiment, the vector is a 2685 vector. In another embodiment, the vector is a 3274 vector. In another embodiment, the vector is a 3550 vector. In another embodiment, the vector is a 3551 vector. In another embodiment, the vector is a 3552 vector. In another embodiment, the vector is a 4276 vector.
  • the vector is a 4277 vector. In another embodiment, the vector is a 4292 vector. In another embodiment, the vector is a 4477 vector. In another embodiment, the vector is a 5337 vector. In another embodiment, the vector is a 5348/11363 vector. In another embodiment, the vector is a 5348/11646 vector. In another embodiment, the vector is a 5348/12430 vector. In another embodiment, the vector is a 5348/12434 vector. In another embodiment, the vector is a 10072 vector. In another embodiment, the vector is a 11355C vector. In another embodiment, the vector is a 11711A vector. In another embodiment, the vector is a 12029 vector. In another embodiment, the vector is a 12981 vector.
  • the vector is a 13441 vector. In another embodiment, the vector is a 90666 vector. In another embodiment, the vector is a 90816 vector. In another embodiment, the vector is a 93253 vector. In another embodiment, the vector is a 907515 vector. In another embodiment, the vector is a 910716 vector. In another embodiment, the vector is a NN-Listeria vector. In another embodiment, the vector is a O1761 vector. In another embodiment, the vector is a 4211 vector. In another embodiment, the vector is a 4286 vector. In another embodiment, the integration vector is any other site- specific integration vector known in the art that is capable of infecting Listeria. [000111] .
  • the integration vector or plasmid of methods and compositions as provided herein does not confer antibiotic resistance to the Listeria vaccine strain. In another embodiment, the integration vector or plasmid does not contain an antibiotic resistance gene..
  • the present invention provides an recombinant nucleic acid encoding a recombinant polypeptide. In one embodiment, the nucleic acid comprises a sequence sharing at least 80% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 85% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein.
  • the nucleic acid comprises a sequence sharing at least 90% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 95% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 97% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 99% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein.
  • a method of producing a recombinant Listeria strain comprising a bivalent plasmid encoding two distinct heterologous antigens.
  • the plasmid is a multivalent plasmid that encodes 3 or more distinct heterologous antigens.
  • the plasmid is a multivalent plasmid that encodes 4 or more distinct heterologous antigens.
  • the plasmid is a multivalent plasmid that encodes 5 or more distinct heterologous antigens.
  • the recombinant Listeria provided herein expresses at leas at one antigen encoded by the plasmids disclosed herein.
  • a method of producing a recombinant Listeria strain expressing two distinct heterologous antigens in another embodiment, expresses at least 3 or more distinct heterologous antigens. In another embodiment, the recombinant Listeria expresses 4 or more distinct heterologous antigens. In another embodiment, the recombinant Listeria expresses 5 or more distinct heterologous antigens.
  • the method of producing a recombinant Listeria comprises transforming said recombinant Listeria with nucleic acid comprising a bivalent or multivalent plasmid.
  • the plasmid is an episomal plasmid that remains extrachromosomal.
  • the plasmid is an integrative plasmid.
  • the method provided herein comprises expressing the antigens and fusion proteins disclosed herein under conditions conducive to protein expression..
  • the nucleic acids described herein comprise DNA vectors, RNA vectors, plasmids (extrachromosomal and/or integrative), etc., that may be used in the methods provided herein for generating any of the compositions disclosed herein.
  • the recombinant Listeria strain may express more than two antigens, some of which are expressed from one or more nucleic acid molecules integrated into the Listeria chromosome and some of which are expressed via one or more episomal expression plasmids or vectors present in the recombinant Listeria strain.
  • a recombinant Listeria strain as provided herein comprises two or more episomal expression plasmids, each of which expresses at least one distinct antigenic polypeptide.
  • one or more of the antigens are expressed as a fusion protein with LLO, which in one embodiment, is non-hemolytic LLO or truncated LLO.
  • a recombinant Listeria strain as provided herein targets tumors by eliciting immune responses to two separate antigens, which are expressed by two different cell types, which in one embodiment are a cell surface antigen and an anti-angiogenic polypeptide, while in another embodiment, a recombinant Listeria strain as provided herein targets tumors by eliciting an immune response to two different antigens expressed by the same cell type. In another embodiment, a recombinant Listeria strain as provided herein targets tumors by eliciting an immune response to two different antigens as described hereinbelow or as are known in the art.
  • a heterologous antigen disclosed herein is associated with the local tissue environment that is further associated with the development of or metastasis of cancer.
  • the heterologous antigen provided herein is associated with tumor immune evasion or resistance to cancer.
  • the heterologous antigen provided herein is an angiogenic antigen.
  • a first antigen of the compositions and methods of the present invention is directed against a specific cell surface antigen or tumor target, and a second antigen is directed against an angiogenic antigen or tumor microenvironment.
  • the first and second antigens of the compositions and methods of the present invention are polypeptides expressed by tumor cells, or in another embodiment, polypeptides expressed in a tumor microenvironment.
  • the first antigen of the compositions and methods of the present invention is a polypeptide expressed by a tumor and the second antigen of the compositions and methods of the present invention is a receptor target, NO Synthetase, Arg-1, or other enzyme known in the art.
  • a method of producing a recombinant Listeria strain expressing two antigens comprising, in one embodiment, genetically fusing a first nucleic acid encoding a first antigen and a second nucleic acid encoding a second antigen into the Listeria genome in an open reading frame with a native polypeptide comprising a PEST sequence.
  • the expressing said first and second antigens are produced under conditions conducive to antigenic expression in said recombinant Listeria strain.
  • the recombinant Listeria strain of the composition and methods as provided herein comprises an episomal expression vector comprising the second nucleic acid molecule encoding a heterologous antigen.
  • the second nucleic acid molecule encoding a heterologous antigen is present in said episomal expression vector in an open reading frame with a polypeptide comprising a PEST sequence.
  • the terms “heterologous antigen,” “antigen,” are used interchangeably herein.
  • an episomal expression vector of the methods and compositions as provided herein comprises an antigen fused in frame to a nucleic acid sequence encoding a PEST-like AA sequence.
  • the antigen is HMW- MAA, and in another embodiment, a HMW-MAA fragment.
  • the PEST-like AA sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 12).
  • the PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID No: 13).
  • fusion of an antigen to any LLO sequence that includes one of the PEST-like AA sequences enumerated herein can enhance cell mediated immunity against HMW-MAA.
  • the PEST-like AA sequence is a PEST-like sequence from a Listeria ActA protein.
  • the PEST-like sequence is KTEEQPSEVNTGPR (SEQ ID NO: 14), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 15), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 16), or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 17).
  • the PEST-like sequence is from Listeria seeligeri cytolysin, encoded by the lso gene.
  • the PEST-like sequence is RSEVTISPAETPESPPATP (SEQ ID NO: 18).
  • the PEST-like sequence is from Streptolysin O protein of Streptococcus sp.
  • the PEST-like sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 19) at AA 35-51.
  • the PEST-like sequence is from Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK (SEQ ID NO: 20) at AA 38-54.
  • the PEST-like sequence has a sequence selected from SEQ ID NO: 14-20.
  • the PEST-like sequence has a sequence selected from SEQ ID NO: 12-20.
  • the PEST-like sequence is another PEST-like AA sequence derived from a prokaryotic organism.
  • the PEST sequence is any other PEST sequence known in the art, including, but not limited to, those disclosed in United States Patent Publication No. 2014/0186387, which is incorporated by reference herein in its entirety [000126] Identification of PEST-like sequences is well known in the art, and is described, for example in Rogers S et al (Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 1986; 234(4774):364-8, incorporated herein by reference) and Rechsteiner M et al (PEST sequences and regulation by proteolysis.
  • PEST-like sequence refers, in another embodiment, to a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues.
  • P proline
  • E glutamic acid
  • S serine
  • T threonine residues
  • the PEST-like sequence is flanked by one or more clusters containing several positively charged amino acids.
  • the PEST-like sequence mediates rapid intracellular degradation of proteins containing it.
  • the PEST-like sequence fits an algorithm disclosed in Rogers et al.
  • the PEST-like sequence fits an algorithm disclosed in Rechsteiner et al.
  • the PEST-like sequence contains one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein degradation.
  • a sequence referred to herein as a PEST-like sequence is a PEST sequence.
  • PEST-like sequences of prokaryotic organisms are identified in accordance with methods such as described by, for example Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21:267-271) for LM and in Rogers S et al (Science 1986; 234(4774):364-8).
  • PEST-like AA sequences from other prokaryotic organisms can also be identified based on this method.
  • PEST-like AA sequences would be expected to include, but are not limited to, other Listeria species.
  • the PEST-like sequence fits an algorithm disclosed in Rogers et al.
  • the PEST-like sequence fits an algorithm disclosed in Rechsteiner et al.
  • the PEST-like sequence is identified using the PEST-find program. [000128]
  • identification of PEST motifs is achieved by an initial scan for positively charged amino acids R, H, and K within the specified protein sequence. All amino acids between the positively charged flanks are counted and only those motifs are considered further, which contain a number of amino acids equal to or higher than the window-size parameter.
  • a PEST-like sequence must contain at least 1 P, 1 D or E, and at least 1 S or T.
  • the quality of a PEST motif is refined by means of a scoring parameter based on the local enrichment of critical amino acids as well as the motif's hydrophobicity. Enrichment of D, E, P, S and T is expressed in mass percent (w/w) and corrected for 1 equivalent of D or E, 1 of P and 1 of S or T.
  • calculation of hydrophobicity follows in principle the method of J. Kyte and R.F. Doolittle (Kyte, J and Dootlittle, RF. J. Mol. Biol. 157, 105 (1982), incorporated herein by reference.
  • Kyte-Doolittle hydropathy indices which originally ranged from - 4.5 for arginine to +4.5 for isoleucine, are converted to positive integers, using the following linear transformation, which yielded values from 0 for arginine to 90 for isoleucine.
  • Hydropathy index 10 * Kyte-Doolittle hydropathy index + 45
  • a potential PEST motif ’s hydrophobicity is calculated as the sum over the products of mole percent and hydrophobicity index for each amino acid species.
  • PEST score 0.55 * DEPST - 0.5 * hydrophobicity index.
  • PEST score 0.55 * DEPST - 0.5 * hydrophobicity index.
  • “PEST sequence,”“PEST-like sequence” or“PEST-like sequence peptide” are used interchangeably here and refer to a peptide having a score of at least +5, using the above algorithm.
  • the term refers to a peptide having a score of at least 6.
  • the peptide has a score of at least 7.
  • the score is at least 8.
  • the score is at least 9.
  • the score is at least 10.
  • the score is at least 11.
  • the score is at least 12. In another embodiment, the score is at least 13. In another embodiment, the score is at least 14. In another embodiment, the score is at least 15. In another embodiment, the score is at least 16. In another embodiment, the score is at least 17. In another embodiment, the score is at least 18. In another embodiment, the score is at least 19. In another embodiment, the score is at least 20. In another embodiment, the score is at least 21. In another embodiment, the score is at least 22. In another embodiment, the score is at least 22. In another embodiment, the score is at least 24. In another embodiment, the score is at least 24. In another embodiment, the score is at least 25. In another embodiment, the score is at least 26. In another embodiment, the score is at least 27. In another embodiment, the score is at least 28.
  • the score is at least 29. In another embodiment, the score is at least 30. In another embodiment, the score is at least 32. In another embodiment, the score is at least 35. In another embodiment, the score is at least 38. In another embodiment, the score is at least 40. In another embodiment, the score is at least 45.
  • the PEST sequence is identified using any other method or algorithm known in the art, e.g the CaSPredictor (Garay-Malpartida HM, Occhiucci JM, Alves J, Belizario JE. Bioinformatics. 2005 Jun;21 Suppl 1:i169-76).
  • a PEST index is calculated for each stretch of appropriate length (e.g. a 30-35 amino acid 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 residue is 1 and for each of the other amino acids (non-PEST) is 0.
  • Each method for identifying a PEST sequence represents a separate embodiment as provided herein.
  • the PEST sequence is any other PEST sequence known in the art.
  • the present invention provides fusion proteins, which in one embodiment, are expressed by Listeria.
  • such fusion proteins comprise fusions to a tLLO, a truncated ActA or a PEST sequence.
  • PEST sequence may encompass cases wherein a protein fragment comprises a PEST sequence having surrounding sequences other than the PEST sequence.
  • the protein fragment consists of the PEST sequence.
  • “fusion” refers to two peptides or protein fragments either linked together at their respective ends or embedded one within the other.
  • fused may also encompass an operable linkage by covalent bonding.
  • the term encompasses recombinant fusion (of nucleic acid sequences or open reading frames thereof).
  • a recombinant Listeria strain of the compositions and methods as provided herein comprises a full length LLO polypeptide, which in one embodiment, is hemolytic.
  • the recombinant Listeria strain comprises a non-hemolytic LLO polypeptide.
  • the polypeptide is an LLO fragment.
  • the oligopeptide is a complete LLO protein.
  • the polypeptide is any LLO protein or fragment thereof known in the art.
  • an LLO protein fragment is utilized in compositions and methods as provided herein.
  • a truncated LLO protein is encoded by the episomal expression vector as provided herein that expresses a polypeptide, that is, in one embodiment, an antigen, in another embodiment, an angiogenic factor, or, in another embodiment, both an antigen and angiogenic factor.
  • the LLO fragment is an N-terminal fragment.
  • the N-terminal LLO fragment has the sequence: [00054] MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIE KKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQ NNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKI VVKNATKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGT AFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQ ALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNI IKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNE LAVIKNN
  • an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 21.
  • the LLO AA sequence is a homologue of SEQ ID No: 21.
  • the LLO AA sequence is a variant of SEQ ID No: 21.
  • the LLO AA sequence is a fragment of SEQ ID No: 21.
  • the LLO AA sequence is an isoform of SEQ ID No: 21.
  • the LLO fragment has the sequence: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEI DKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQV VNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNAT KSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVN NSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNA ENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFK AVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKN NSEYIETTSKAYTD
  • an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 22.
  • the LLO AA sequence is a homologue of SEQ ID No: 22.
  • the LLO AA sequence is a variant of SEQ ID No: 22.
  • the LLO AA sequence is a fragment of SEQ ID No: 22.
  • the LLO AA sequence is an isoform of SEQ ID No: 22.
  • the LLO protein used in the compositions and methods as provided herein comprises the following sequence: [000145] MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIE KKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSIN QNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDN KIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKF GTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQ LQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVEL TNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLK
  • an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID NO: 23.
  • the LLO AA sequence is a homologue of SEQ ID NO: 23.
  • the LLO AA sequence is a variant of SEQ ID NO: 23. In another embodiment, the LLO AA sequence is a fragment of SEQ ID NO: 23. In another embodiment, the LLO AA sequence is an isoform of SEQ ID NO: 23.
  • Each possibility represents a separate embodiment as provided herein.
  • the LLO protein used in the compositions and methods as provided herein has, in another embodiment, the sequence: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADE IDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQ VVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNA TKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAV NNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVN AENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSF KAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNEL
  • an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID NO: 24.
  • the LLO AA sequence is a homologue of SEQ ID NO: 24.
  • the LLO AA sequence is a variant of SEQ ID NO: 24.
  • the LLO AA sequence is a fragment of SEQ ID NO: 24.
  • the LLO AA sequence is an isoform of SEQ ID NO: 24.
  • the amino acid sequence of the LLO polypeptide of the compositions and methods as provided herein is from the Listeria monocytogenes 10403S strain, as set forth in Genbank Accession No.: ZP_01942330, EBA21833, or is encoded by the nucleic acid sequence as set forth in Genbank Accession No.: NZ_AARZ01000015 or AARZ01000015.1.
  • the LLO sequence for use in the compositions and methods as provided herein is from Listeria monocytogenes, which in one embodiment, is the 4b F2365 strain (in one embodiment, Genbank accession number: YP_012823), the EGD- e strain (in one embodiment, Genbank accession number: NP_463733), or any other strain of Listeria monocytogenes known in the art.
  • the LLO sequence for use in the compositions and methods as provided herein is from Flavobacteriales bacterium HTCC2170 (in one embodiment, Genbank accession number: ZP_01106747 or EAR01433; in one embodiment, encoded by Genbank accession number: NZ_AAOC01000003).
  • proteins that are homologous to LLO in other species such as alveolysin, which in one embodiment, is found in Paenibacillus alvei (in one embodiment, Genbank accession number: P23564 or AAA22224; in one embodiment, encoded by Genbank accession number: M62709) may be used in the compositions and methods as provided herein.
  • Other such homologous proteins are known in the art.
  • Each LLO protein and LLO fragment represents a separate embodiment of the methods and compositions as provided herein.
  • homologues of LLO from other species may be used to create a fusion protein of LLO with an antigen of the compositions and methods as provided herein, which in one embodiment, is HMW-MAA, and in another embodiment is a fragment of HMW-MAA.
  • the LLO fragment of methods and compositions as provided herein is a PEST-like domain.
  • an LLO fragment that comprises a PEST sequence is utilized as part of a composition or in the methods as provided herein.
  • the LLO fragment does not contain the activation domain at the carboxy terminus.
  • the LLO fragment does not include cysteine 484.
  • the LLO fragment is a non-hemolytic fragment. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of the activation domain. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of cysteine 484. In another embodiment, an LLO sequence is rendered non-hemolytic by deletion or mutation at another location. [000152] In another embodiment, the LLO fragment consists of about the first 441 AA of the LLO protein. In another embodiment, the LLO fragment comprises about the first 400-441 AA of the 529 AA full length LLO protein. In another embodiment, the LLO fragment corresponds to AA 1-441 of an LLO protein disclosed herein.
  • the LLO fragment consists of about the first 420 AA of LLO. In another embodiment, the LLO fragment corresponds to AA 1-420 of an LLO protein disclosed herein. In another embodiment, the LLO fragment consists of about AA 20-442 of LLO. In another embodiment, the LLO fragment corresponds to AA 20-442 of an LLO protein disclosed herein. In another embodiment, any ⁇ LLO without the activation domain comprising cysteine 484, and in particular without cysteine 484, are suitable for methods and compositions as provided herein. [000153] In another embodiment, the LLO fragment corresponds to the first 400 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 300 AA of an LLO protein.
  • the LLO fragment corresponds to the first 200 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 100 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 50 AA of an LLO protein, which in one embodiment, comprises one or more PEST-like sequences. [000154] In another embodiment, the LLO fragment is a non-hemolytic LLO. In another embodiment, the non-hemolytic LLO comprises one or more PEST-like sequences. [000155] In another embodiment, the LLO fragment contains residues of a homologous LLO protein that correspond to one of the above AA ranges. The residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g.
  • a recombinant Listeria strain of the methods and compositions as provided herein comprise a nucleic acid molecule operably integrated into the Listeria genome as an open reading frame with an endogenous ActA sequence.
  • a recombinant Listeria strain of the methods and compositions as provided herein comprise an episomal expression vector comprising a nucleic acid molecule encoding fusion protein comprising an antigen fused to an ActA or a truncated ActA.
  • the expression and secretion of the antigen is under the control of an actA promoter and ActA signal sequence and it is expressed as fusion to 1-233 amino acids of ActA (truncated ActA or tActA).
  • the truncated ActA consists of the first 390 amino acids of the wild type ActA protein as described in US Patent Serial No. 7,655,238, which is incorporated by reference herein in its entirety.
  • the truncated ActA is an ActA-N100 or a modified version thereof (referred to as ActA- N100*) in which a PEST motif has been deleted and containing the nonconservative QDNKR substitution as described in US Patent Publication Serial No. 2014/0186387.
  • the antigen is HMW-MAA, while in another embodiment, it’s an immunogenic fragement of HMW-MAA.
  • the present invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof.
  • the present invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof.
  • the Her-2 chimeric protein of the methods and compositions of the present invention is a human Her-2 chimeric protein.
  • the Her-2 protein is a mouse Her-2 chimeric protein. In another embodiment, the Her-2 protein is a rat Her-2 chimeric protein. In another embodiment, the Her-2 protein is a primate Her-2 chimeric protein. In another embodiment, the Her-2 protein is a Her-2 chimeric protein of any other animal species or combinations thereof known in the art. Each possibility represents a separate embodiment of the present invention. [000159] In another embodiment, a Her-2 protein is a protein referred to as“HER- 2/neu,”“Erbb2,”“v-erb-b2,”“c-erb-b2,”“neu,” or“cNeu.” Each possibility represents a separate embodiment of the present invention.
  • the Her2-neu chimeric protein harbors two of the extracellular and one intracellular fragments of Her2/neu antigen showing clusters of MHC-class I epitopes of the oncogene, where, in another embodiment, the chimeric protein, harbors 3 H2Dq and at least 17 of the mapped human MHC-class I epitopes of the Her2/neu antigen (fragments EC1, EC2, and IC1) as described in US Patent Application Serial No. 12/945,386, which is incorporated by reference herein in its entirety.
  • the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and expressed and secreted by the Listeria monocytogenes attenuated auxotrophic strain LmddA.
  • the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and is expressed from the chromosome of a recombinant Listeria provided herein, while an additional antigen is expressed from a plasmid present within the recombinant Listeria disclosed herein.
  • the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and is expressed from a plasmid of a recombinant Listeria provided herein, while an additional antigen is expressed from the chromosome of the recombinant Listeria provided herein.
  • a recombinant Listeria provided herein is a Listeria monocytogenes attenuated auxotrophic strain LmddA.
  • the HER-2 chimeric protein is encoded by a nucleic acid sequence comprising SEQ ID NO:25 [000162] acccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacctgccc accaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcaaccaagtgaggcaggtcccac tgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgctgaaca ataccaccctgtcacaggggcctccccaggaggcctgcaggaggctgca
  • the Her-2 chimeric protein comprises SEQ ID NO: 26: T H L D M L R H L Y Q G C Q V V Q G N L E L T Y L P T N A S L S F L Q D I Q E V Q G Y V L I A H N Q V R Q V P L Q R L R I V R G T Q L F E D N Y A L A V L D N G D P L N N T T P V T G A S P G G L R E L Q L R S L T E I L K G G G V L I Q R N P Q L C Y Q D T I L W K N I Q E F A G C K K I F G S L A F L P E S F D G D P A S N T A P L Q P E Q L Q V F E T L E E I T G Y L Y I S A W P D S L P D L S V F Q N L Q V I R G R I L H N G A Y S L T L Q G L
  • the HER2 chimeric protein or fragment thereof of the methods and compositions provided herein does not include a signal sequence thereof. In another embodiment, omission of the signal sequence enables the HER2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the signal sequence.
  • the fragment of a HER2 chimeric protein of methods and compositions of the present invention does not include a transmembrane domain (TM) thereof. In one embodiment, omission of the TM enables the HER2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the TM.
  • the nucleic acid sequence of rat-Her2/neu gene is CCGGAATCGCGGGCACCCAAGTGTGTACCGGCACAGACATGAAGTTGCGGCTCC CTGCCAGTCCTGAGACCCACCTGGACATGCTCCGCCACCTGTACCAGGGCTGTCA GGTAGTGCAGGGCAACTTGGAGCTTACCTACGTGCCTGCCAATGCCAGCCTCTCA TTCCTGCAGGACATCCAGGAAGTTCAGGGTTACATGCTCATCGCTCACAACCAGG TGAAGCGCGTCCCACTGCAAAGGCTGCGCATCGTGAGAGGGACCCAGCTCTTTGA GGACAAGTATGCCCTGGCTGTGCTAGACAACCGAGATCCTCAGGACAATGTCGCC GCCTCCACCCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGT CTCACAGAGATCCTGAAGGGAGGGAGGGGTTTTGATCCGTGGGAACCCTCAGGACAATGTCGCC GCCTCCAGGCAGAACCCCAGAGGGGCTGCGGGAG
  • the nucleic acid sequence encoding the rat/her2/neu EC1 fragment is: CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGA GATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGAC ATGGTTTTGTGGAAGGACGTCTTCCGCAAGAATAACCAACTGGCTCCTGTCGATA TAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAA TCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACCATCTGT ACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGCCATGAGC AGTGTGCCGCAGGCTGCACGGGCCCCAAGCA (SEQ ID NO: 28).
  • the nucleic acid sequence encoding the rat her2/neu EC2 fragment is:
  • the nucleic acid sequence encoding the rat her2/neu IC1 fragment is: CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGG AGCTAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGG GCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTT GAGAGAAAACACATCTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGT GATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACAT CCACAGTACAGCTGGTGACACAGCTTATGCCCTACGGCTGCCTTCTGGACCATGT CCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCAACTGGTGTGTTCAG ATTGCCAAGGGAATGCTACCATGT CCGAAATGAGCTACCATGT CCGAAATGAGCTACCATGT CCGAAATGAGCTACCATGT
  • the nucleic acid sequence of human-Her2/neu gene is: ATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGCCCCCCG GAGCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTG CCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGT GGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTC CTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGA GGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGA CAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCT GTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCACA GAGATCTTGAAAGG
  • the nucleic acid sequence encoding the human her2/neu EC1 fragment implemented into the chimera spans from 120-510 bp of the human EC1 region and is set forth in (SEQ ID NO: 32).
  • the complete EC1 human her2/neu fragment spans from (58- 979 bp of the human her2/neu gene and is set forth in (SEQ ID NO: 33).
  • the nucleic acid sequence encoding the human her2/neu EC2 fragment implemented into the chimera spans from 1077-1554 bp of the human her2/neu EC2 fragment and includes a 50 bp extension, and is set forth in (SEQ ID NO: 34).
  • complete EC2 human her2/neu fragment spans from 907-1504 bp of the human her2/neu gene and is set forth in (SEQ ID NO: 35).
  • nucleic acid sequence encoding the human her2/neu IC1 fragment implemented into the chimera is set forth in (SEQ ID NO: 36).
  • CAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAA TCTTAGACGAAGCATACGTGATGGCTGGTGGGCTCCCCATATGTCTCCCGCCTT CTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATG GCTGCCTCTTAGACT (SEQ ID NO: 36).
  • the nucleic acid sequence encoding the complete human her2/neu IC1 fragment spans from 2034-3243 of the human her2/neu gene and is set forth in (SEQ ID NO: 37).
  • the present invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a carbonic anhydrase 9 (or carbonic anhydrase IX) protein or fused to a fragment thereof.
  • the present invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a carbonic anhydrase 9 or fused to a fragment thereof.
  • the carbonic anhydrase 9 protein of the methods and compositions of the present invention is a human carbonic anhydrase 9 protein.
  • the carbonic anhydrase 9 protein is a mouse carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a rat carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a primate carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a carbonic anhydrase 9 protein of any other animal species or combinations thereof known in the art. [000180] In one embodiment, the terms “carbonic anhydrase 9”, “carbonic anhydrase IX”, and“CA9”, are used interchangeably herein.
  • the nucleic acid sequence of the human-CA9 gene is: cagaggttgccccggatgcaggaggattccccttgggaggaggctcttggggaagatgacccactgggcgaggaggatctgcc cagtgaagaggattcacccagagaggaggatccacccggagaggaggatctacctggagaggaggatctacctggagaggaggatctacctggagaggaggatctacctggagaggaggat ctacctggagaggaggat ctacctgaggctccctgaagttagaggatctacctactgttgaggctcctggagatcctcaaga accccagaataatgcccacagggacaaagaaggggatgaccagagtcattggcgctatggaggcgacccgcctggccccggg
  • the CA9 nucleic acid sequence is a homolog of SEQ ID NO: 38. In another embodiment, the CA9 nucleic acid sequence is a variant of SEQ ID NO: 38. In another embodiment, the CA9 nucleic acid sequence is a fragment of SEQ ID NO: 38. In another embodiment the CA9 nucleic acid sequence is any sequence known in the art including, but not limited to, those set forth in GenBank Accession nos. NM_001216.2, XM_006716867.1, XM_006716868.1, and X66839.1.
  • the amino acid sequence encoded by the human CA9 gene provided herein is: Q R L P R M Q E D S P L G G S S G E D D D P L G E E D L P S E E D S P R E E D P P G E E D L P G E E D L P G E E D L P E V K P K S E E E G S L K L E D L P T V E A P G D P Q E P Q N N A H R D K E G D D D Q S H W R Y G G D P P P W P R V S P A C A G R F Q S P V D I R P Q L A A F C P A L R P L E L L G F Q L P P L P E L R L R N N G H S V Q L T L P P G L E M A L G P G R E Y R A L Q L H L H W G A A G R P G S E H T V E G H R F P A E I H V V H L S T A F A R V D E
  • the CA9 amino acid sequence is a homolog of SEQ ID NO: 39. In another embodiment, the CA9 amino acid sequence is a variant of SEQ ID NO: 39. In another embodiment, the CA9 amino acid sequence is an isomer of SEQ ID NO: 39. In another embodiment, the CA9 amino acid sequence is a fragment of SEQ ID NO: 39. In another embodiment the CA9 amino acid sequence is any sequence known in the art including, but not limited to, those set forth in GenBank Accession nos. NP_001207.2, XP_006716930.1, XP_006716931.1, and CAA47315.1.
  • the nucleic acid sequence encoding a truncated LLO-CA9 fusion comprises SEQ ID NO: 40: atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaata aagaaaattcaatttcatccatggcaccaccagcatctccgctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaag gttacaagatggaaaatgtgccgccaagaaaag gttacaagatggaaatgtgccgccaagaaa
  • the truncated LLO-CA9 fusion is a homolog of SEQ ID NO: 40. In another embodiment, the truncated LLO-CA9 fusion is a variant of SEQ ID NO: 40. In another embodiment, the truncated LLO-CA9 fusion is an isomer of SEQ ID NO: 40.
  • an amino acid sequence comprising a tLLO fused to CA9 comprises SEQ ID NO: 41 MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHAD EIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADI QVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKN ATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKA VNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGV NAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNS SFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTT
  • the truncated LLO-CA9 fusion is a homolog of SEQ ID NO: 41. In another embodiment, the truncated LLO-CA9 fusion is a variant of SEQ ID NO: 41. In another embodiment, the truncated LLO-CA9 fusion is an isomer of SEQ ID NO: 41.
  • the LmddA strain provided herein comprises a mutation
  • an antigen of the methods and compositions as provided herein is fused to an ActA protein, which in one embodiment, is an N-terminal fragment of an ActA protein, which in one embodiment, comprises or consists of the first 390 AA of ActA, in another embodiment, the first 418 AA of ActA, in another embodiment, the first 50 AA of ActA, in another embodiment, the first 100 AA of ActA, which in one embodiment, comprise a PEST sequence such as that provided in SEQ ID NO: 2.
  • an N- terminal fragment of an ActA protein utilized in methods and compositions as provided herein comprises or consists of the first 150 AA of ActA, in another embodiment, the first approximately 200 AA of ActA, which in one embodiment comprises 2 PEST sequences as described herein.
  • an N-terminal fragment of an ActA protein utilized in methods and compositions as provided herein comprises or consists of the first 250 AA of ActA, in another embodiment, the first 300 AA of ActA.
  • the ActA fragment contains residues of a homologous ActA protein that correspond to one of the above AA ranges. The residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g.
  • the N-terminal portion of the ActA protein comprises 1, 2, 3, or 4 PEST sequences, which in one embodiment are the PEST sequences specifically mentioned herein, or their homologs, as described herein or other PEST sequences as can be determined using the methods and algorithms described herein or by using alternative methods known in the art.
  • An N-terminal fragment of an ActA protein utilized in methods and compositions as provided herein has, in another embodiment, the sequence set forth in SEQ ID NO: 42: MRAMMVVFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPRYETAR EVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAAINEEASG ADRPAIQVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVNKKKVAKES VADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWVRDKIDENPEVK KAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPATSEPS SFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIIRETASSLDSSF TRGDLASLRNAINRHSQNFSDFPPIPTEEELNGRGGRP (SEQ ID NO:
  • the ActA fragment comprises the sequence set forth in SEQ ID NO: 42. In another embodiment, the ActA fragment is any other ActA fragment known in the art. In another embodiment, the ActA protein is a homologue of SEQ ID NO: 42. In another embodiment, the ActA protein is a variant of SEQ ID NO: 42. In another embodiment, the ActA protein is an isoform of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of a homologue of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of a variant of SEQ ID NO: 42.
  • the ActA protein is a fragment of an isoform of SEQ ID NO: 42.
  • the recombinant nucleotide encoding a fragment of an ActA protein comprises the sequence set forth in SEQ ID NO: 43: atgcgtgcgatgatggtggttttcattactgccaattgcattacgattaaccccgacataatatttgcagcgacagatagcgaagattcta gtctaaacacagatgaatgggaagaagaaaaaacagaagagcaaccaagcgaggtaaatacgggaccaagatacgaaactgcac gtgaagttcacgtgatattaaagaactagaaaaatcgaataaagtgagaaatacgaacaa
  • the recombinant nucleotide has the sequence set forth in SEQ ID NO: 43.
  • the recombinant nucleotide comprises any other sequence that encodes a fragment of an ActA protein.
  • An N-terminal fragment of an ActA protein utilized in methods and compositions as provided herein has, in another embodiment, the sequence set forth in SEQ ID NO: 44: MRAMMVVFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPRYETA REVSSRDIEELEKSNKVKNTNKADLIAMLKAKAEKGPNNNNNNGEQTGNVAINEEA SGVDRPTLQVERRHPGLSSDSAAEIKKRRKAIASSDSELESLTYPDKPTKANKRKVA KESVVDASESDLDSSMQSADESTPQPLKANQKPFFPKVFKKIKDAGKWVRDKIDENP EVKKAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPTP SEPSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIMRETAPS LDSSFTSGDLASLRSAINRHSENFSDFPLIPTEEELNGRGGRP (SEQ ID NO:
  • the ActA fragment comprises the sequence set forth in SEQ ID NO: 44. In another embodiment, the ActA fragment is any other ActA fragment known in the art. In another embodiment, the ActA protein is a homologue of SEQ ID NO: 44. In another embodiment, the ActA protein is a variant of SEQ ID NO: 44. In another embodiment, the ActA protein is an isoform of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of a homologue of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of a variant of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of an isoform of SEQ ID NO: 44. [000191] In another embodiment, a truncated ActA protein comprises the sequence set forth in SEQ ID NO: 45:
  • a truncated ActA sequence provided herein is further fused to an hly signal peptide at the N-terminus.
  • the truncated ActA fused to hly signal peptide comprises SEQ ID NO: 46:
  • the recombinant nucleotide encoding a fragment of an ActA protein comprises the sequence set forth in SEQ ID NO: 47: atgcgtgcgatgatggtagtttcattactgccaactgcattacgattaaccccgacataatatttgcagcgacagatagcgaagattcca gtctaaacacagatgaatgggaagaagaaaaaacagaagagcagccaagcgaggtaaatacgggaccaagatacgaaactgcacg tgaagtaagttcacgtgatattgaggaactagaaaaatcgaataaaaaaatacgaacaagcagacctaaaaaactagaaaaatcgaataaaaaaatacgaacaagcagacctaaaa
  • the recombinant nucleotide has the sequence set forth in SEQ ID NO: 47.
  • the recombinant nucleotide comprises any other sequence that encodes a fragment of an ActA protein.
  • the ActA fragment is another ActA fragment known in the art, which in one embodiment, is any fragment comprising a PEST sequence.
  • the ActA fragment is amino acids 1-100 of the ActA sequence.
  • the ActA fragment is amino acids 1-200 of the ActA sequence.
  • the ActA fragment is amino acids 200-300 of the ActA sequence.
  • the ActA fragment is amino acids 300-400 of the ActA sequence. In another embodiment, the ActA fragment is amino acids 1-300 of the ActA sequence.
  • a recombinant nucleotide as provided herein comprises any other sequence that encodes a fragment of an ActA protein. In another embodiment, the recombinant nucleotide comprises any other sequence that encodes an entire ActA protein. Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • the ActA sequence for use in the compositions and methods as provided herein is from Listeria monocytogenes, which in one embodiment, is the EGD strain, the 10403S strain (Genbank accession number: DQ054585) the NICPBP 54002 strain (Genbank accession number: EU394959), the S3 strain (Genbank accession number: EU394960), the NCTC 5348 strain (Genbank accession number: EU394961), the NICPBP 54006 strain (Genbank accession number: EU394962), the M7 strain (Genbank accession number: EU394963), the S19 strain (Genbank accession number: EU394964), or any other strain of Listeria monocytogenes which is known in the art.
  • the sequence of the deleted actA region in the strain, Lmdd ⁇ actA is as follows: [000197] gcgccaaatcattggttgattggtgaggatgtctgtgtgtgcgtgggtcgcgagatgggcgaataagaagcattaaagatcct gacaaatataatcaagcggctcatatgaaagattacgaatcgcttccactcacagaggaaggcgactggggcggagttcattataatag tggtatcccgaataaagcagcctataatactatcactaaacttggaaagaaaaaacagaacagctttattttcgcgctttaaagtactatttt aacgtaccgatgcgaaaaaaagcgcttcaaca
  • the underlined region contains actA sequence element that is present in the Lmdd ⁇ actA strain.
  • the bold sequence gtcgac represent the site of junction of the N-T and C-T sequence.
  • the recombinant Listeria strain of the compositions and methods as provided herein comprise a first or second nucleic acid molecule that encodes a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA), or, in another embodiment, a fragment of HMW-MAA.
  • HMW-MAA High Molecular Weight-Melanoma Associated Antigen
  • HMW-MAA is also known as the melanoma chondroitin sulfate proteoglycan (MCSP), and in another embodiment, is a membrane-bound protein of 2322 residues.
  • MCSP melanoma chondroitin sulfate proteoglycan
  • HMW-MAA is expressed on over 90% of surgically removed benign nevi and melanoma lesions, and is also expressed in basal cell carcinoma, tumors of neural crest origin (e.g. astrocytomas, gliomas, neuroblastomas and sarcomas), childhood leukemias, and lobular breast carcinoma lesions.
  • HMW-MAA is highly expressed on both activated pericytes and pericytes in tumor angiogeneic vasculature which, in another embodiment is associated with neovascularization in vivo.
  • immunization of mice with the recombinant Listeria expressing a fragment of HMW-MAA decreases the number of pericytes in the tumor vasculature.
  • HMW-MAA a murine homolog of HMW-MAA, known as NG2 or AN2, has 80% homology to HMW-MAA, as well as similar expression pattern and function.
  • HMW-MAA is highly expressed on both activated pericytes and pericytes in tumor angiogenic vasculature.
  • activated pericytes are associated with neovascularization in vivo.
  • activated pericytes are involved in angiogenesis.
  • angiogenesis is important for survival of tumors.
  • pericytes in tumor angiogenic vasculature are associated with neovascularization in vivo.
  • activated pericytes are important cells in vascular development, stabilization, maturation and remodeling. Therefore, in one embodiment, besides its role as a tumor-associated antigen, HMW-MAA is also a potential universal target for anti-angiogenesis using an immunotherapeutic approach. As described herein (Example 8), results obtained using an Lm-based vaccine against this antigen has supported this possibility. [000201]
  • one of the antigens of the methods and compositions provided herein is expressed in activated pericytes.
  • the HMW-MAA protein from which HMW-MAA fragments as provided herein are derived is, in another embodiment, a human HMW-MAA protein.
  • the HMW-MAA protein is a mouse protein.
  • the HMW-MAA protein is a rat protein.
  • the HMW-MAA protein is a primate protein.
  • the HMW-MAA protein is from any other species known in the art.
  • the HMW-MAA protein is melanoma chondroitin sulfate proteoglycan (MCSP).
  • an AN2 protein is used in methods and compositions as provided herein.
  • an NG2 protein is used in methods and compositions as provided herein.
  • the HMW-MAA protein of methods and compositions as provided herein has the sequence: [000204] MQSGRGPPLPAPGLALALTLTMLARLASAASFFGENHLEVPVATALTDIDLQ LQFSTSQPEALLLLAAGPADHLLLQLYSGRLQVRLVLGQEELRLQTPAETLLSDSIPHT VVLTVVEGWATLSVDGFLNASSAVPGAPLEVPYGLFVGGTGTLGLPYLRGTSRPLRG CLHAATLNGRSLLRPLTPDVHEGCAEEFSASDDVALGFSGPHSLAAFPAWGTQDEGT LEFTLTTQSRQAPLAFQAGGRRGDFIYVDIFEGHLRAVVEKGQGTVLLHNSVPVADG QPHEVSVHINAHRLEISVDQYPTHTSNRGVLSYLEPRGSLLLGGLDA
  • an HMW-MAA AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 49.
  • the HMW-MAA AA sequence is a homologue of SEQ ID No: 49.
  • the HMW-MAA AA sequence is a variant of SEQ ID No: 49.
  • the HMW-MAA AA sequence is a fragment of SEQ ID No: 49.
  • the HMW-MAA AA sequence is an isoform of SEQ ID No: 49.
  • the HMW-MAA protein of methods and compositions as provided herein is encoded by the sequence: [000206] atgcagtcccggccgcggcccccacttccagcccccggcctggccttggctttgaccctgactatgttggccagacttgc atccgcggcttccttcttcggtgagaaccacctggaggtgcctgtggccacggctctgaccgacatagacctgcagctgcagttctcca cgtcccagccccgaagccctccttctctggcagcaggcccagctgaccacctcctgctgcagctctactctggacgctgcaggtcag acttgttctgggccaggaggagctgaggctgcaggtcagactccag
  • the recombinant nucleotide has the sequence set forth in SEQ ID NO: 50.
  • an HMW-MAA-encoding nucleotide of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 50.
  • the HMW-MAA-encoding nucleotide is a homologue of SEQ ID No: 50.
  • the HMW-MAA-encoding nucleotide is a variant of SEQ ID No: 50.
  • the HMW-MAA-encoding nucleotide is a fragment of SEQ ID No: 50.
  • the HMW-MAA-encoding nucleotide is an isoform of SEQ ID No: 50.
  • the HMW-MAA protein of methods and compositions as provided herein has an AA sequence set forth in a GenBank entry having an Accession Numbers selected from NM_001897 and X96753.
  • the HMW-MAA protein is encoded by a nucleotide sequence set forth in one of the above GenBank entries.
  • the HMW-MAA protein comprises a sequence set forth in one of the above GenBank entries.
  • the HMW-MAA protein is a homologue of a sequence set forth in one of the above GenBank entries.
  • the HMW- MAA protein is a variant of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is a fragment of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is an isoform of a sequence set forth in one of the above GenBank entries.
  • the HMW-MAA fragment utilized in the present invention comprises, in another embodiment, AA 360-554. In another embodiment, the fragment consists essentially of AA 360-554. In another embodiment, the fragment consists of AA 360-554. In another embodiment, the fragment comprises AA 701-1130.
  • the fragment consists essentially of AA 701-1130. In another embodiment, the fragment consists of AA 701-1130. In another embodiment, the fragment comprises AA 2160-2258. In another embodiment, the fragment consists essentially of 2160-2258. In another embodiment, the fragment consists of 2160-2258.
  • the recombinant Listeria of the compositions and methods as provided herein comprise a plasmid that encodes a recombinant polypeptide that is, in one embodiment, angiogenic, and in another embodiment, antigenic.
  • the polypeptide is HMW-MAA, and in another embodiment, the polypeptide is a HMW-MAA fragment.
  • the plasmid further encodes a non-HMW-MAA peptide.
  • the non-HMW-MAA peptide enhances the immunogenicity of the polypeptide.
  • the HMW-MAA fragment of methods and compositions as provided herein is fused to the non-HMW-MAA AA sequence.
  • the HMW-MAA fragment is embedded within the non-HMW-MAA AA sequence.
  • an HMW-MAA-derived peptide is incorporated into an LLO fragment, ActA protein or fragment, or PEST-like sequence.
  • the non-HMW-MAA peptide is, in one embodiment, a listeriolysin (LLO) oligopeptide.
  • the non-HMW-MAA peptide is an ActA oligopeptide.
  • the non-HMW-MAA peptide is a PEST-like oligopeptide.
  • fusion to LLO, ActA, PEST-like sequences and fragments thereof enhances the cell-mediated immunogenicity of antigens.
  • fusion to LLO, ActA, PEST- like sequences and fragments thereof enhances the cell-mediated immunogenicity of antigens in a variety of expression systems.
  • the non-HMW-MAA peptide is any other immunogenic non-HMW-MAA peptide known in the art.
  • Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • the recombinant Listeria strain of the compositions and methods as provided herein express a heterologous antigen that is expressed by a tumor cell.
  • the recombinant Listeria strain of the compositions and methods as provided herein comprise a first or second nucleic acid molecule that encodes a Prostate Specific Antigen (PSA), which in one embodiment, is a marker for prostate cancer that is highly expressed by prostate tumors, which in one embodiment is the most frequent type of cancer in American men and, in another embodiment, is the second cause of cancer related death in American men.
  • PSA is a kallikrein serine protease (KLK3) secreted by prostatic epithelial cells, which in one embodiment, is widely used as a marker for prostate cancer.
  • the recombinant Listeria strain as provided herein comprises a nucleic acid molecule encoding KLK3 protein.
  • the KLK3 protein has the sequence: [000214] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK NRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEE FLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCN GVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 51; GenBank Accession No. CAA32915).
  • the KLK3 protein is a homologue of SEQ ID No: 51. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 51. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 51. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 51.
  • the KLK3 protein has the sequence: [000216] IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVIL LGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAEL TDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHP QKVTKFMLCAGRWTGGKSTCSGDSGGPLVCYGVLQGITSWGSEPCALPERPSLYTK VVHYRKWIKDTIVANP (SEQ ID No: 52).
  • the KLK3 protein is a homologue of SEQ ID No: 52.
  • the KLK3 protein is a variant of SEQ ID No: 52. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 52. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 52.
  • the KLK3 protein has the sequence: IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSL FHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVK VMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTK FMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR KWIKDTIVANP (SEQ ID No: 53; GenBank Accession No. AAA59995.1).
  • the KLK3 protein is a homologue of SEQ ID No: 53. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 53. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 53. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 53.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: ggtgtcttaggcacactggtcttggagtgcaaaggatctaggcacgtgaggctttgtatgaagaatcggggatcgtacccaccccctgtt tctgtttcatcctgggcatgtctcctctgcctttgtccctagatgaagtctccatgagctacaagggcctggtgcatccagggtgatctagt aattgcagaacagcaagtgctagctctcccccttccacagctctgggtgtgggagggggttgtccagcctccagcagcatgggga gggccttggtggtgccagcagcatgggga gggccttggtgccagca
  • the KLK3 protein is encoded by residues 401..446, 1688..1847, 3477..3763, 3907..4043, and 5413..5568 of SEQ ID No: 54.
  • the KLK3 protein is encoded by a homologue of SEQ ID No: 54.
  • the KLK3 protein is encoded by a variant of SEQ ID No: 54.
  • the KLK3 protein is encoded by an isomer of SEQ ID No: 54.
  • the KLK3 protein is encoded by a fragment of SEQ ID No: 54.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSWVILITELTMPALPMVL HGSLVPWRGGV (SEQ ID No: 55; GenBank Accession No.
  • the KLK3 protein is a homologue of SEQ ID No: 55. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 55. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 55. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 55.
  • Each possibility represents a separate embodiment as provided herein.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggtgtggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgg gctggtgg gcacccccagtgggtcctcacagctgcccactg
  • the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 56.
  • the KLK3 protein is encoded by a homologue of SEQ ID No: 56.
  • the KLK3 protein is encoded by a variant of SEQ ID No: 56.
  • the KLK3 protein is encoded by an isomer of SEQ ID No: 56.
  • the KLK3 protein is encoded by a fragment of SEQ ID No: 56.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRK (SEQ ID No: 57; GenBank Accession No. NP_001025221).
  • the KLK3 protein is a homologue of SEQ ID No: 57.
  • the KLK3 protein is a variant of SEQ ID No: 57.
  • the sequence of the KLK3 protein comprises SEQ ID No: 57.
  • the KLK3 protein is an isomer of SEQ ID No: 57.
  • the KLK3 protein is a fragment of SEQ ID No: 57.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttccacccttccgtgacgtggattggtgc tgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggca gggcagtctgcggtgtctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaagtgagtaggggcctggggtc
  • the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 58.
  • the KLK3 protein that is the source of the KLK3 peptide has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD SGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 59; GenBank Accession No. NP_001025220).
  • the KLK3 protein is a homologue of SEQ ID No: 29. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 59. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 59. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 59.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggtgctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgg gcacccccagtgggtcctcacagctgcccactgcatcaggaaca
  • the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 60.
  • the KLK3 protein is encoded by a homologue of SEQ ID No: 60.
  • the KLK3 protein is encoded by a variant of SEQ ID No: 60.
  • the KLK3 protein is encoded by an isomer of SEQ ID No: 60.
  • the KLK3 protein is encoded by a fragment of SEQ ID No: 60.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRKPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYAS GWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSG DSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 61; GenBank Accession No. NP_001025219).
  • the KLK3 protein is a homologue of SEQ ID No: 61.
  • the KLK3 protein is a variant of SEQ ID No: 61. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 61. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 61.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggtgctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaagccaggtgatgactccagc cacgacctcatgctgctcccgctgctgctgcatcaggaagccaggtgatgact
  • the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 62.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 63; GenBank Accession No. NP_001639).
  • the KLK3 protein is a homologue of SEQ ID No: 63. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 63. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 63. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 63.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggtgctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgg gctggtgg gcacccccagtgggtcctcacagctgcccact
  • the KLK3 protein is encoded by residues 42-827 of SEQ ID No: 64.
  • the KLK3 protein is encoded by a homologue of SEQ ID No: 64.
  • the KLK3 protein is encoded by a variant of SEQ ID No: 64.
  • the KLK3 protein is encoded by an isomer of SEQ ID No: 64.
  • the KLK3 protein is encoded by a fragment of SEQ ID No: 64.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 65 GenBank Accession No. AAX29407.1).
  • the KLK3 protein is a homologue of SEQ ID No: 65. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 65. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 65. In another embodiment, the sequence of the KLK3 protein comprises SEQ ID No: 65. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 65.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: gggggagccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtgg attggtgctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctct ct cgtggcagtggcagtctgcggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatc tgctgggttcatcctgcatcaggaacaaaagcgtgatc tgctgtg
  • the KLK3 protein is encoded by residues 47-832 of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 66.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD SGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVA (SEQ ID No: 67; GenBank Accession No. AJ459782).
  • the KLK3 protein is a homologue of SEQ ID No: 67.
  • the KLK3 protein is a variant of SEQ ID No: 67. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 67. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 67.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSVSHPYSQDLEGKGEWG
  • the KLK3 protein is a homologue of SEQ ID No: 68.
  • the KLK3 protein is a variant of SEQ ID No: 68.
  • the KLK3 protein is an isomer of SEQ ID No: 68.
  • the sequence of the KLK3 protein comprises SEQ ID No: 68.
  • the KLK3 protein is a fragment of SEQ ID No: 68.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGERGHGWGDAGEGASPDCQAEALSPPTQHPSPDRELGSFLSL PAPLQAHTPSPSILQQSSLPHQVPAPSHLPQNFLPIAQPAPCSQLLY (SEQ ID No: 69 GenBank Accession No. AJ459784).
  • the KLK3 protein is a homologue of SEQ ID No: 69.
  • the KLK3 protein is a variant of SEQ ID No: 69.
  • the sequence of the KLK3 protein comprises SEQ ID No: 69.
  • the KLK3 protein is an isomer of SEQ ID No: 69.
  • the KLK3 protein is a fragment of SEQ ID No: 69.
  • the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID NO: 70 GenBank Accession No.
  • the KLK3 protein is a homologue of SEQ ID No: 70. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 70. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 70. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 70.
  • the KLK3 protein is encoded by a nucleotide molecule having the sequence: aagtttcccttctcccagtccaagaccccaaatcaccacaaaggacccaatccccagactcaagatatggtctgggcgctgtcttgtgtc tcctaccctgatccctgggttcaactctgctcccagagcatgaagcctctccaccagcaccaacctgcaaacctagggaag attgacagaattcccagcctttcccagctccccctgcccatgtcccaggactcccagccttggttctctctgcccatgtcccaggactcccagccttggttctctctgccccccgtgtcttttcaaaccca catcctaaatccatctcttt
  • the KLK3 protein is encoded by residues 67-1088 of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 71.
  • the KLK3 protein is encoded by a sequence set forth in one of the following GenBank Accession Numbers: BC005307, AJ310938, AJ310937, AF335478, AF335477, M27274, and M26663.
  • the KLK3 protein is encoded by a sequence set forth in one of the above GenBank Accession Numbers. Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • the KLK3 protein is encoded by a sequence set forth in one of the following GenBank Accession Numbers: NM_001030050, NM_001030049, NM_001030048, NM_001030047, NM_001648, AJ459782, AJ512346, or AJ459784.
  • GenBank Accession Numbers NM_001030050, NM_001030049, NM_001030048, NM_001030047, NM_001648, AJ459782, AJ512346, or AJ459784.
  • Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • the KLK3 protein is encoded by a variation of any of the sequences described herein wherein the sequence lacks MWVPVVFLTLSVTWIGAAPLILSR (SEQ ID NO: 72).
  • the KLK3 protein has the sequence that comprises a sequence set forth in one of the following GenBank Accession Numbers: X13943, X13942, X13940, X13941, and X13944. [000239] In another embodiment, the KLK3 protein is any other KLK3 protein known in the art. [000240] In another embodiment, the KLK3 peptide is any other KLK3 peptide known in the art. In another embodiment, the KLK3 peptide is a fragment of any other KLK3 peptide known in the art. Each type of KLK3 peptide represents a separate embodiment of the methods and compositions as provided herein.
  • KLK3 peptide refers, in another embodiment, to a full-length KLK3 protein. In another embodiment, the term refers to a fragment of a KLK3 protein. In another embodiment, the term refers to a fragment of a KLK3 protein that is lacking the KLK3 signal peptide. In another embodiment, the term refers to a KLK3 protein that contains the entire KLK3 sequence except the KLK3 signal peptide.“KLK3 signal sequence” refers, in another embodiment, to any signal sequence found in nature on a KLK3 protein. In another embodiment, a KLK3 protein of methods and compositions as provided herein does not contain any signal sequence.
  • the kallikrein-related peptidase 3 that is the source of a KLK3 peptide for use in the methods and compositions as provided herein is a PSA protein.
  • the KLK3 protein is a P-30 antigen protein.
  • the KLK3 protein is a gamma-seminoprotein protein.
  • the KLK3 protein is a kallikrein 3 protein.
  • the KLK3 protein is a semenogelase protein.
  • the KLK3 protein is a seminin protein.
  • the KLK3 protein is any other type of KLK3 protein that is known in the art.
  • the KLK3 protein is a splice variant 1 KLK3 protein. In another embodiment, the KLK3 protein is a splice variant 2 KLK3 protein. In another embodiment, the KLK3 protein is a splice variant 3 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 1 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 2 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 3 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 4 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 5 KLK3 protein.
  • the KLK3 protein is a transcript variant 6 KLK3 protein. In another embodiment, the KLK3 protein is a splice variant RP5 KLK3 protein. In another embodiment, the KLK3 protein is any other splice variant KLK3 protein known in the art. In another embodiment, the KLK3 protein is any other transcript variant KLK3 protein known in the art. [000244] In another embodiment, the KLK3 protein is a mature KLK3 protein. In another embodiment, the KLK3 protein is a pro-KLK3 protein. In another embodiment, the leader sequence has been removed from a mature KLK3 protein of methods and compositions as provided herein.
  • the KLK3 protein that is the source of a KLK3 peptide of methods and compositions as provided herein is a human KLK3 protein.
  • the KLK3 protein is a primate KLK3 protein.
  • the KLK3 protein is a KLK3 protein of any other species known in the art.
  • one of the above KLK3 proteins is referred to in the art as a“KLK3 protein.”
  • KLK3-LLO fusions are provided in US Patent Ser. No. 9,012,141 which is incorporated by reference herein in its entirety.
  • the antigen of interest is HPV-E7.
  • the antigen is HPV-E6.
  • the antigen is Her-2/neu. In another embodiment, the antigen is NY-ESO-1. In another embodiment, the antigen is telomerase (TERT). In another embodiment, the antigen is stratum corneum chymotryptic enzyme (SCCE) and variants thereof. In another embodiment, the antigen is CEA. In another embodiment, the antigen is LMP-1. In another embodiment, the antigen is p53. In another embodiment, the antigen is carboxic anhydrase IX (CAIX). In another embodiment, the antigen is PSMA. In another embodiment, the antigen is prostate stem cell antigen (PSCA). In another embodiment, the antigen is HMW-MAA. In another embodiment, the antigen is WT-1.
  • the antigen is HIV-1 Gag. In another embodiment, the antigen is Proteinase 3. In another embodiment, the antigen is Tyrosinase related protein 2. In another embodiment, the antigen is PSA (prostate-specific antigen). In another embodiment, the antigen is selected from HPV-E7, HPV-E6, Her-2, NY- ESO-1, telomerase (TERT), SCCE, HMW-MAA, WT-1, HIV-1 Gag, CEA, LMP-1, p53, PSMA, PSCA, Proteinase 3, Tyrosinase related protein 2, Muc1, PSA (prostate-specific antigen), or a combination thereof.
  • the antigen is a tumor-associated antigen, which in one embodiment, is one of the following tumor antigens: a MAGE (Melanoma-Associated Antigen E) protein, e.g. MAGE 1, MAGE 2, MAGE 3, MAGE 4, a tyrosinase; carbonic anhydrase 9 (CA9), a mutant ras protein; a mutant p53 protein; p97 melanoma antigen, a ras peptide or p53 peptide associated with advanced cancers; the HPV 16/18 antigens associated with cervical cancers, KLH antigen associated with breast carcinoma, CEA (carcinoembryonic antigen) associated with colorectal cancer, gp100, mesothelin, EGFRvIII, a MART1 antigen associated with melanoma, or the PSA antigen associated with prostate cancer.
  • a MAGE Melnoma-Associated Antigen E
  • MAGE 1 MAGE 1
  • MAGE 3 MAGE 4
  • the antigen for the compositions and methods as provided herein are melanoma-associated antigens, which in one embodiment are TRP-2, MAGE-1, MAGE-3, gp-100, tyrosinase, HSP-70, beta-HCG, or a combination thereof.
  • the recombinant nucleic acid provided herein may encode two separate antigens that serve as tumor targets, which in one embodiment are Prostate Specific Antigen (PSA) and Prostate Cancer Stem Cell (PSCA) antigen.
  • the recombinant nucleic acid molecule provided herein encodes two separate antigens that serve as tumor targets, which in one embodime are cHer2 and HMW-MAA-C.
  • the recombinant nucleic acid molecule provided herein encodes two separate antigens that serve as tumor targets, which in one embodime are cHer2 and CA9.
  • the each invidiual antigen of the two or more antigens expressed by a Listeria as provided herein complement or synergize the immune response.
  • the heterologousn antigen provided herein is an angiogenic antigen that affects vascular growth.
  • the recombinant nucleic acid provided herein may encode two polypeptides each comprising an angiogenic antigen that affect vascular growth fused to a PEST-containing peptide provided herein.
  • the angiogenic antigen is any angiogenic antigen known in the art, including but not limited toEGFR-III and its related family members, VEGFR and its related family members, HMW-MAA.
  • the heterologous antigen provided herein may serve as both a tumor antigen an angiogenic factor.
  • the heterologous antigen is a tumor antigen.
  • the heterologous antigen is an inhibitor of the function or expression of ARG-1 or NOS or combination.
  • an inhibitor of NOS is N G -mono-methyl-L-arginine (L-NMMA), N G -nitro-L-arginine methyl ester (L- NAME), 7-NI, L-NIL, or L-NIO.
  • N-omega-nitro-L-arginine a nitric oxide synthase inhibitor and L-arginine competitive inhibitor may be encoded by the nucleic acid.
  • the second nucleic acid may encode an mRNA that inhibits function or expression of ARG-1 or NOS.
  • a heterologous antigen expressed by the Listeria of the present invention may be a neuropeptide growth factor antagonist, which in one embodiment is [D- Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, [Arg6, D-Trp7,9, NmePhe8]substance P(6-11).
  • the antigen is derived from a fungal pathogen, bacteria, parasite, helminth, or viruses.
  • the antigen is selected from tetanus toxoid, hemagglutinin molecules from influenza virus, diphtheria toxoid, HIV gp120, HIV gag protein, IgA protease, insulin peptide B, Spongospora subterranea antigen, vibriose antigens, Salmonella antigens, pneumococcus antigens, respiratory syncytial virus antigens, Haemophilus influenza outer membrane proteins, Helicobacter pylori urease, Neisseria meningitidis pilins, N.
  • gonorrhoeae pilins human papilloma virus antigens E1 and E2 from type HPV-16, -18, -31, -33, -35 or -45 human papilloma viruses, or a combination thereof.
  • the antigen is associated with one of the following diseases; cholera, diphtheria, Haemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid, Varicella-zoster, whooping cough3 yellow fever, the immunogens and antigens from Addison's disease, allergies, anaphylaxis, Bruton's syndrome, cancer, including solid and blood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, type 1 diabetes mellitus, acquired immune deficiency syndrome, transplant rejection, such as kidney, heart, pancreas, lung, bone, and liver transplants, Graves' disease, polyen
  • the immune response induced by methods and compositions as provided herein is, in another embodiment, a T cell response.
  • the immune response comprises a T cell response.
  • the response is a CD8+ T cell response.
  • the response comprises a CD8 + T cell response.
  • a recombinant Listeria of the compositions and methods as provided herein comprise an angiogenic polypeptide.
  • anti-angiogenic approaches to cancer therapy are very promising, and in one embodiment, one type of such anti-angiogenic therapy targets pericytes.
  • molecular targets on vascular endothelial cells and pericytes are important targets for antitumor therapies.
  • the platelet-derived growth factor receptor (PDGF-B/PDGFR- ⁇ ) signaling is important to recruit pericytes to newly formed blood vessels.
  • angiogenic polypeptides as provided herein inhibit molecules involved in pericyte signaling, which in one embodiment, is PDGFR- ⁇ .
  • the compositions of the present invention comprise an angiogenic factor, or an immunogenic fragment thereof, where in one embodiment, the immunogenic fragment comprises one or more epitopes recognized by the host immune system.
  • an angiogenic factor is a molecule involved in the formation of new blood vessels.
  • the angiogenic factor is VEGFR2.
  • an angiogenic factor of the present invention is Angiogenin; Angiopoietin-1; Del-1; Fibroblast growth factors: acidic (aFGF) and basic (bFGF); Follistatin; Granulocyte colony-stimulating factor (G-CSF); Hepatocyte growth factor (HGF) /scatter factor (SF); Interleukin-8 (IL-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; Transforming growth factor-alpha (TGF-alpha); Transforming growth factor-beta (TGF-beta); Tumor necrosis factor-alpha (TNF-alpha); Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF).
  • G-CSF Granulocyte colony-stimulating factor
  • HGF Hepatocyte
  • an angiogenic factor is an angiogenic protein.
  • a growth factor is an angiogenic protein.
  • an angiogenic protein for use in the compositions and methods of the present invention is Fibroblast growth factors (FGF); VEGF; VEGFR and Neuropilin 1 (NRP-1); Angiopoietin 1 (Ang1) and Tie2; Platelet-derived growth factor (PDGF; BB-homodimer) and PDGFR; Transforming growth factor-beta (TGF- ⁇ ), endoglin and TGF- ⁇ receptors; monocyte chemotactic protein-1 (MCP-1); Integrins ⁇ V ⁇ 3, ⁇ V ⁇ 5 and ⁇ 5 ⁇ 1; VE-cadherin and CD31; ephrin; plasminogen activators; plasminogen activator inhibitor-1; Nitric oxide synthase (NOS) and COX-2; AC133; or Id1/Id3.
  • FGF Fibroblast growth factors
  • VEGF VE
  • an angiogenic protein for use in the compositions and methods of the present invention is an angiopoietin, which in one embodiment, is Angiopoietin 1, Angiopoietin 3, Angiopoietin 4 or Angiopoietin 6.
  • endoglin is also known as CD105; EDG; HHT1; ORW; or ORW1.
  • endoglin is a TGFbeta co-receptor.
  • target antigens include, but is not limited to: Wilm’s tumor-1 associated protein (Wt-1 ), including Isoforms A, B, C, and D; MHC class I chain-related protein A (MICA); MHC class I chain-related protein B (MICB); gastrin and peptides thereof; gastrin/CCK-2 receptor (CCK-B); Glypican-3; Coactosin-like protein; Prostate acid phosphatase (PAP); Six-transmembrane epithelial antigen of prostate (STEAP); Prostate carcinoma antigen-1 (PCTA-1); Prostate tumor-inducing gene-1 (PTI-1); Prostate-specific gene with homology to G protein-coupled receptor; Prostase; Cancer-testis antigens; SCP-1; SSX-1, SSX-2, SSX-4; GAGE; CT7; CT8; CT10; LAGE-1; GAGE-3/6, GAGE-1, GAGE-2, GAGE
  • cancer vaccines as provided herein generate effector T cells that are able to infiltrate the tumor, destroy tumor cells and eradicate the disease.
  • naturally occurring tumor infiltrating lymphocytes are associated with better prognosis in several tumors, such as colon, ovarian and melanoma.
  • tumors without signs of micrometastasis have an increased infiltration of immune cells and a Th1 expression profile, which correlate with an improved survival of patients.
  • the infiltration of the tumor by T cells has been associated with success of immunotherapeutic approaches in both pre-clinical and human trials.
  • the infiltration of lymphocytes into the tumor site is dependent on the up-regulation of adhesion molecules in the endothelial cells of the tumor vasculature, generally by proinflammatory cytokines, such as IFN- ⁇ , TNF- ⁇ and IL-1.
  • proinflammatory cytokines such as IFN- ⁇ , TNF- ⁇ and IL-1.
  • adhesion molecules have been implicated in the process of lymphocyte infiltration into tumors, including intercellular adhesion molecule 1 (ICAM-1), vascular endothelial cell adhesion molecule 1 (V-CAM-1), vascular adhesion protein 1 (VAP- 1) and E-selectin.
  • IAM-1 intercellular adhesion molecule 1
  • V-CAM-1 vascular endothelial cell adhesion molecule 1
  • VAP- 1 vascular adhesion protein 1
  • E-selectin E-selectin
  • cancer vaccines as provided herein increase TILs, up-regulate adhesion molecules (in one embodiment, ICAM-1, V-CAM-1, VAP-1, E- selectin, or a combination thereof), up-regulate proinflammatory cytokines (in one embodiment, IFN- ⁇ , TNF- ⁇ , IL-1, or a combination thereof), or a combination thereof.
  • adhesion molecules in one embodiment, ICAM-1, V-CAM-1, VAP-1, E- selectin, or a combination thereof
  • proinflammatory cytokines in one embodiment, IFN- ⁇ , TNF- ⁇ , IL-1, or a combination thereof
  • the compositions and methods as provided herein provide anti- angiogenesis therapy, which in one embodiment, may improve immunotherapy strategies.
  • compositions and methods as provided herein circumvent endothelial cell anergy in vivo by up-regulating adhesion molecules in tumor vessels and enhancing leukocyte-vessel interactions, which increases the number of tumor infiltrating leukocytes, such as CD8 + T cells.
  • tumor infiltrating leukocytes such as CD8 + T cells.
  • enhanced anti-tumor protection correlates with an increased number of activated CD4 + and CD8 + tumor-infiltrating T cells and a pronounced decrease in the number of regulatory T cells in the tumor upon VEGF blockade.
  • a tumor-associated antigen delivered to a host afflicted by a tumor as described herein, will have a synergistic effect in impacting tumor growth and a more potent therapeutic efficacy.
  • targeting pericytes through vaccination will lead to cytotoxic T lymphocyte (CTL) infiltration, destruction of pericytes, blood vessel destabilization and vascular inflammation, which in another embodiment is associated with up-regulation of adhesion molecules in the endothelial cells that are important for lymphocyte adherence and transmigration, ultimately improving the ability of lymphocytes to infiltrate the tumor tissue.
  • CTL cytotoxic T lymphocyte
  • concomitant delivery of a tumor-specific antigen generate lymphocytes able to invade the tumor site and kill tumor cells.
  • the platelet-derived growth factor receptor (PDGF-B/PDGFR- ⁇ ) signaling is important to recruit pericytes to newly formed blood vessels.
  • inhibition of VEGFR-2 and PDGFR- ⁇ concomitantly induces endothelial cell apoptosis and regression of tumor blood vessels, in one embodiment, approximately 40% of tumor blood vessels.
  • said recombinant Listeria strain is an auxotrophic Listeria strain.
  • said auxotrophic Listeria strain is a dal/dat mutant.
  • the nucleic acid molecule is stably maintained in the recombinant bacterial strain in the absence of antibiotic selection.
  • auxotrophic mutants useful as vaccine vectors may be generated in a number of ways.
  • D-alanine auxotrophic mutants can be generated, in one embodiment, via the disruption of both the dal gene and the dat gene to generate an attenuated auxotrophic strain of Listeria which requires exogenously added D- alanine for growth.
  • the generation of AA strains of Listeria deficient in D-alanine may be accomplished in a number of ways that are well known to those of skill in the art, including deletion mutagenesis, insertion mutagenesis, and mutagenesis which results in the generation of frameshift mutations, mutations which cause premature termination of a protein, or mutation of regulatory sequences which affect gene expression.
  • mutagenesis can be accomplished using recombinant DNA techniques or using traditional mutagenesis technology using mutagenic chemicals or radiation and subsequent selection of mutants.
  • deletion mutants are preferred because of the accompanying low probability of reversion of the auxotrophic phenotype.
  • mutants of D-alanine which are generated according to the protocols presented herein may be tested for the ability to grow in the absence of D-alanine in a simple laboratory culture assay. In another embodiment, those mutants which are unable to grow in the absence of this compound are selected for further study.
  • other genes involved in synthesis of a metabolic enzyme, as provided herein may be used as targets for mutagenesis of Listeria.
  • said auxotrophic Listeria strain comprises an episomal expression vector comprising a metabolic enzyme that complements the auxotrophy of said auxotrophic Listeria strain.
  • the construct is contained in the Listeria strain in an episomal fashion.
  • the foreign antigen is expressed from a vector harbored by the recombinant Listeria strain.
  • said episomal expression vector lacks an antibiotic resistance marker.
  • an antigen of the methods and compositions as provided herein is genetically fused to an oligopeptide comprising a PEST sequence.
  • said endogenous polypeptide comprising a PEST sequence is LLO.
  • said endogenous polypeptide comprising a PEST sequence is ActA.
  • the metabolic enzyme complements an endogenous metabolic gene that is lacking in the remainder of the chromosome of the recombinant bacterial strain.
  • the endogenous metabolic gene is mutated in the chromosome. In another embodiment, the endogenous metabolic gene is deleted from the chromosome. In another embodiment, said metabolic enzyme is an amino acid metabolism enzyme. In another embodiment, said metabolic enzyme catalyzes a formation of an amino acid used for a cell wall synthesis in said recombinant Listeria strain. In another embodiment, said metabolic enzyme is an alanine racemase enzyme. In another embodiment, said metabolic enzyme is a D-amino acid transferase enzyme. [000268] In another embodiment, the metabolic enzyme catalyzes the formation of an amino acid (AA) used in cell wall synthesis. In another embodiment, the metabolic enzyme catalyzes synthesis of an AA used in cell wall synthesis.
  • AA amino acid
  • the metabolic enzyme catalyzes synthesis of an AA used in cell wall synthesis.
  • the metabolic enzyme is involved in synthesis of an AA used in cell wall synthesis.
  • the AA is used in cell wall biogenesis.
  • the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component.
  • the metabolic enzyme is encoded by an alanine racemase gene (dal) gene.
  • the dal gene encodes alanine racemase, which catalyzes the reaction L-alanine ⁇ D-alanine.
  • the dal gene of methods and compositions of the methods and compositions as provided herein is encoded, in another embodiment, by the sequence: [000272] atggtgacaggctggcatcgtccaacatggattgaaatagaccgcgcagcaattcgcgaaatataaaaaatgaacaaa ataaactcccggaaagtgtcgacttatgggcagtagtcaaagctaatgcatatggtcacggaattatcgaagttgctaggacggcgaaa gaagctggagcaaaggtttctgcgtagccattttagatgaggcactggctcttagagaagctggatttcaagatgactttattcttgtgctt ggtgcaaccagaaaagaagatgctaatctggcagccaaaccacat
  • nucleotide encoding dal is homologous to SEQ ID No: 73. In another embodiment, the nucleotide encoding dal is a variant of SEQ ID No: 73. In another embodiment, the nucleotide encoding dal is a fragment of SEQ ID No: 73. In another embodiment, the dal protein is encoded by any other dal gene known in the art.
  • the dal protein has the sequence: [000274] MVTGWHRPTWIEIDRAAIRENIKNEQNKLPESVDLWAVVKANAYGHGIIEV ARTAKEAGAKGFCVAILDEALALREAGFQDDFILVLGATRKEDANLAAKNHISLTVF REDWLENLTLEATLRIHLKVDSGMGRLGIRTTEEARRIEATSTNDHQLQLEGIYTHFA TADQLETSYFEQQLAKFQTILTSLKKRPTYVHTANSAASLLQPQIGFDAIRFGISMYGL TPSTEIKTSLPFELKPALALYTEMVHVKELAPGDSVSYGATYTATEREWVATLPIGYA DGLIRHYSGFHVLVDGEPAPIIGRVCMDQTIIKLPREFQTGSKVTIIGKDHGNTVTADD AAQYLDTINYEVTCLLNERIPRKYIH (SEQ ID No: 74; GenBank Accession No: AF038428).
  • the dal protein is homologous to SEQ ID No: 74. In another embodiment, the dal protein is a variant of SEQ ID No: 74. In another embodiment, the dal protein is an isomer of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of a homologue of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of a variant of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of an isomer of SEQ ID No: 74.
  • the dal protein is any other Listeria dal protein known in the art. In another embodiment, the dal protein is any other gram-positive dal protein known in the art. In another embodiment, the dal protein is any other dal protein known in the art. [000276] In another embodiment, the dal protein of methods and compositions as provided herein retains its enzymatic activity. In another embodiment, the dal protein retains 90% of wild-type activity. In another embodiment, the dal protein retains 80% of wild-type activity. In another embodiment, the dal protein retains 70% of wild-type activity. In another embodiment, the dal protein retains 60% of wild-type activity.
  • the dal protein retains 50% of wild-type activity. In another embodiment, the dal protein retains 40% of wild-type activity. In another embodiment, the dal protein retains 30% of wild-type activity. In another embodiment, the dal protein retains 20% of wild-type activity. In another embodiment, the dal protein retains 10% of wild-type activity. In another embodiment, the dal protein retains 5% of wild-type activity.
  • the metabolic enzyme is encoded by a D-amino acid aminotransferase gene (dat).
  • D-glutamic acid synthesis is controlled in part by the dat gene, which is involved in the conversion of D-glu + pyr to alpha-ketoglutarate + D-ala, and the reverse reaction.
  • a dat gene utilized in the present invention has the sequence set forth in GenBank Accession Number AF038439.
  • the dat gene is any another dat gene known in the art.
  • nucleotide encoding dat is homologous to SEQ ID No: 75. In another embodiment, the nucleotide encoding dat is a variant of SEQ ID No: 75. In another embodiment, the nucleotide encoding dat is a fragment of SEQ ID No: 75. In another embodiment, the dat protein is encoded by any other dat gene known in the art.
  • the dat protein has the sequence: [000282] MKVLVNNHLVEREDATVDIEDRGYQFGDGVYEVVRLYNGKFFTYNEHIDR LYASAAKIDLVIPYSKEELRELLEKLVAENNINTGNVYLQVTRGVQNPRNHVIPDDFP LEGVLTAAAREVPRNERQFVEGGTAITEEDVRWLRCDIKSLNLLGNILAKNKAHQQN ALEAILHRGEQVTECSASNVSIIKDGVLWTHAADNLILNGITRQVIIDVAKKNGIPVKE ADFTLTDLREADEVFISSTTIEITPITHIDGVQVADGKRGPITAQLHQYFVEEITRACGE LEFAK (SEQ ID No: 76; GenBank Accession No: AF038439).
  • the dat protein is homologous to SEQ ID No: 76. In another embodiment, the dat protein is a variant of SEQ ID No: 76. In another embodiment, the dat protein is an isomer of SEQ ID No: 45. In another embodiment, the dat protein is a fragment of SEQ ID No: 76. In another embodiment, the dat protein is a fragment of a homologue of SEQ ID No: 76. In another embodiment, the dat protein is a fragment of a variant of SEQ ID No: 76. In another embodiment, the dat protein is a fragment of an isomer of SEQ ID No: 76. [000283] In another embodiment, the dat protein is any other Listeria dat protein known in the art.
  • the dat protein is any other gram-positive dat protein known in the art. In another embodiment, the dat protein is any other dat protein known in the art. [000284] In another embodiment, the dat protein of methods and compositions of the methods and compositions as provided herein retains its enzymatic activity. In another embodiment, the dat protein retains 90% of wild-type activity. In another embodiment, the dat protein retains 80% of wild-type activity. In another embodiment, the dat protein retains 70% of wild- type activity. In another embodiment, the dat protein retains 60% of wild-type activity. In another embodiment, the dat protein retains 50% of wild-type activity. In another embodiment, the dat protein retains 40% of wild-type activity. In another embodiment, the dat protein retains 30% of wild-type activity.
  • the dat protein retains 20% of wild-type activity. In another embodiment, the dat protein retains 10% of wild-type activity. In another embodiment, the dat protein retains 5% of wild-type activity.
  • the metabolic enzyme is encoded by dga. D-glutamic acid synthesis is also controlled in part by the dga gene, and an auxotrophic mutant for D- glutamic acid synthesis will not grow in the absence of D-glutamic acid (Pucci et al, 1995, J Bacteriol. 177: 336-342). In another rembodiment, the recombinant Listeria is auxotrophic for D-glutamic acid.
  • a further example includes a gene involved in the synthesis of diaminopimelic acid.
  • Such synthesis genes encode beta-semialdehyde dehydrogenase, and when inactivated, renders a mutant auxotrophic for this synthesis pathway (Sizemore et al, 1995, Science 270: 299-302).
  • the dga protein is any other Listeria dga protein known in the art.
  • the dga protein is any other gram- positive dga protein known in the art.
  • the metabolic enzyme is encoded by an alr (alanine racemase) gene.
  • the metabolic enzyme is any other enzyme known in the art that is involved in alanine synthesis.
  • the metabolic enzyme is any other enzyme known in the art that is involved in L-alanine synthesis. In another embodiment, the metabolic enzyme is any other enzyme known in the art that is involved in D-alanine synthesis.
  • the recombinant Listeria is auxotrophic for D- alanine. Bacteria auxotrophic for alanine synthesis are well known in the art, and are described in, for example, E. coli (Strych et al, 2002, J. Bacteriol. 184:4321-4325), Corynebacterium glutamicum (Tauch et al, 2002, J.
  • the metabolic enzyme is an amino acid aminotransferase.
  • the metabolic enzyme is encoded by serC, a phosphoserine aminotransferase.
  • the metabolic enzyme is encoded by asd (aspartate beta-semialdehyde dehydrogenase), involved in synthesis of the cell wall constituent diaminopimelic acid.
  • the metabolic enzyme is encoded by gsaB- glutamate-1-semialdehyde aminotransferase, which catalyzes the formation of 5- aminolevulinate from (S)-4-amino-5-oxopentanoate.
  • the metabolic enzyme is encoded by HemL, which catalyzes the formation of 5-aminolevulinate from (S)-4- amino-5-oxopentanoate.
  • the metabolic enzyme is encoded by aspB, an aspartate aminotransferase that catalyzes the formation of oxalozcetate and L-glutamate from L-aspartate and 2-oxoglutarate.
  • the metabolic enzyme is encoded by argF-1, involved in arginine biosynthesis.
  • the metabolic enzyme is encoded by aroE, involved in amino acid biosynthesis.
  • the metabolic enzyme is encoded by aroB, involved in 3-dehydroquinate biosynthesis.
  • the metabolic enzyme is encoded by aroD, involved in amino acid biosynthesis.
  • the metabolic enzyme is encoded by aroC, involved in amino acid biosynthesis.
  • the metabolic enzyme is encoded by hisB, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisD, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisG, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by metX, involved in methionine biosynthesis. In another embodiment, the metabolic enzyme is encoded by proB, involved in proline biosynthesis. In another embodiment, the metabolic enzyme is encoded by argR, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by argJ, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by thiI, involved in thiamine biosynthesis.
  • the metabolic enzyme is encoded by LMOf2365_1652, involved in tryptophan biosynthesis.
  • the metabolic enzyme is encoded by aroA, involved in tryptophan biosynthesis.
  • the metabolic enzyme is encoded by ilvD, involved in valine and isoleucine biosynthesis.
  • the metabolic enzyme is encoded by ilvC, involved in valine and isoleucine biosynthesis.
  • the metabolic enzyme is encoded by leuA, involved in leucine biosynthesis.
  • the metabolic enzyme is encoded by dapF, involved in lysine biosynthesis.
  • the metabolic enzyme is encoded by thrB, involved in threonine biosynthesis (all GenBank Accession No.
  • the metabolic enzyme is a tRNA synthetase.
  • the metabolic enzyme is encoded by the trpS gene, encoding tryptophanyltRNA synthetase.
  • the metabolic enzyme is any other tRNA synthetase known in the art.
  • the LmddA strain provided herein comprises a mutation, deletion or an inactivation of the dal/dat and actA chromosomal genes.
  • a recombinant Listeria strain as provided herein has been passaged through an animal host.
  • the passaging maximizes efficacy of the strain as a vaccine vector.
  • the passaging stabilizes the immunogenicity of the Listeria strain.
  • the passaging stabilizes the virulence of the Listeria strain.
  • the passaging increases the immunogenicity of the Listeria strain.
  • the passaging increases the virulence of the Listeria strain.
  • the passaging removes unstable sub- strains of the Listeria strain.
  • the passaging reduces the prevalence of unstable sub-strains of the Listeria strain.
  • the passaging attenuates the strain, or in another embodiment, makes the strain less virulent.
  • the recombinant Listeria strain of the methods and compositions as provided herein is, in another embodiment, a recombinant Listeria monocytogenes strain.
  • the Listeria strain is a recombinant Listeria seeligeri strain.
  • the Listeria strain is a recombinant Listeria grayi strain.
  • the Listeria strain is a recombinant Listeria ivanovii strain.
  • the Listeria strain is a recombinant Listeria murrayi strain. In another embodiment, the Listeria strain is a recombinant Listeria welshimeri strain. In another embodiment, the Listeria strain is a recombinant strain of any other Listeria species known in the art. In another embodiment, the sequences of Listeria proteins for use in the methods and compositions as provided herein are from any of the above-described strains.
  • a Listeria monocytogenes strain as provided herein is the EGD strain, the 10403S strain, the NICPBP 54002 strain, the S3 strain, the NCTC 5348 strain, the NICPBP 54006 strain, the M7 strain, the S19 strain, or another strain of Listeria monocytogenes which is known in the art.
  • the recombinant Listeria strain is a vaccine strain, which in one embodiment, is a bacterial vaccine strain.
  • the recombinant Listeria strain utilized in methods of the present invention has been stored in a frozen cell bank.
  • the recombinant Listeria strain has been stored in a lyophilized cell bank.
  • the cell bank of methods and compositions of the present invention is a master cell bank.
  • the cell bank is a working cell bank.
  • the cell bank is Good Manufacturing Practice (GMP) cell bank.
  • the cell bank is intended for production of clinical-grade material.
  • the cell bank conforms to regulatory practices for human use.
  • the cell bank is any other type of cell bank known in the art. Each possibility represents a separate embodiment of the present invention.
  • “Good Manufacturing Practices” are defined, in another embodiment, by (21 CFR 210–211) of the United States Code of Federal Regulations. In another embodiment,“Good Manufacturing Practices” are defined by other standards for production of clinical-grade material or for human consumption; e.g. standards of a country other than the United States. Each possibility represents a separate embodiment of the present invention.
  • a recombinant Listeria strain utilized in methods of the present invention is from a batch of vaccine doses.
  • a recombinant Listeria strain utilized in methods of the present invention is from a frozen stock produced by a method disclosed herein.
  • a recombinant Listeria strain utilized in methods of the present invention is from a lyophilized stock produced by a method disclosed herein.
  • a cell bank, frozen stock, or batch of vaccine doses of the present invention exhibits viability upon thawing of greater than 90%.
  • the thawing follows storage for cryopreservation or frozen storage for 24 hours.
  • the storage is for 2 days.
  • the storage is for 3 days.
  • the storage is for 4 days.
  • the storage is for 1 week.
  • the storage is for 2 weeks.
  • the storage is for 3 weeks.
  • the storage is for 1 month.
  • a cell bank, frozen stock, or batch of vaccine doses of the present invention is cryopreserved by a method that comprises growing a culture of the Listeria strain in a nutrient media, freezing the culture in a solution comprising glycerol, and storing the Listeria strain at below -20 degrees Celsius. In another embodiment, the temperature is about -70 degrees Celsius.
  • the temperature is about -70 - -80 degrees Celsius.
  • the culture e.g. the culture of a Listeria vaccine strain that is used to produce a batch of Listeria vaccine doses
  • the culture is inoculated from a cell bank.
  • the culture is inoculated from a frozen stock.
  • the culture is inoculated from a starter culture.
  • the culture is inoculated from a colony.
  • the culture is inoculated at mid-log growth phase.
  • the culture is inoculated at approximately mid-log growth phase.
  • the culture is inoculated at another growth phase.
  • the solution used for freezing contains glycerol in an amount of 2-20%. In another embodiment, the amount is 2%. In another embodiment, the amount is 20%. In another embodiment, the amount is 1%. In another embodiment, the amount is 1.5%. In another embodiment, the amount is 3%. In another embodiment, the amount is 4%. In another embodiment, the amount is 5%. In another embodiment, the amount is 2%. In another embodiment, the amount is 2%. In another embodiment, the amount is 7%. In another embodiment, the amount is 9%. In another embodiment, the amount is 10%. In another embodiment, the amount is 12%. In another embodiment, the amount is 14%.
  • the amount is 16%. In another embodiment, the amount is 18%. In another embodiment, the amount is 222%. In another embodiment, the amount is 25%. In another embodiment, the amount is 30%. In another embodiment, the amount is 35%. In another embodiment, the amount is 40%.
  • the solution used for freezing contains another colligative additive or additive with anti-freeze properties, in place of glycerol. In another embodiment, the solution used for freezing contains another colligative additive or additive with anti-freeze properties, in addition to glycerol. In another embodiment, the additive is mannitol. In another embodiment, the additive is DMSO. In another embodiment, the additive is sucrose.
  • a vaccine is a composition which elicits an immune response to an antigen or polypeptide in the composition as a result of exposure to the composition.
  • the vaccine additionally comprises an adjuvant, cytokine, chemokine, or combination thereof.
  • the vaccine or composition additionally comprises antigen presenting cells (APCs), which in one embodiment are autologous, while in another embodiment, they are allogeneic to the subject.
  • APCs antigen presenting cells
  • a“vaccine” is a composition which elicits an immune response in a host to an antigen or polypeptide in the composition as a result of exposure to the composition.
  • the immune response is to a particular antigen or to a particular epitope on the antigen.
  • the vaccine may be a peptide vaccine, in another embodiment, a DNA vaccine.
  • the vaccine may be contained within and, in another embodiment, delivered by, a cell, which in one embodiment is a bacterial cell, which in one embodiment, is a Listeria.
  • a vaccine may prevent a subject from contracting or developing a disease or condition, wherein in another embodiment, a vaccine may be therapeutic to a subject having a disease or condition.
  • a vaccine of the present invention comprises a composition of the present invention and an adjuvant, cytokine, chemokine, or combination thereof.
  • the present invention provides an immunogenic composition comprising a recombinant Listeria of the present invention.
  • the immunogenic composition of methods and compositions of the present invention comprises a recombinant vaccine vector of the present invention.
  • the immunogenic composition comprises a plasmid of the present invention.
  • the immunogenic composition comprises an adjuvant.
  • a vector of the present invention may be administered as part of a vaccine composition.
  • a vaccine of the present invention is delivered with an adjuvant.
  • the adjuvant favors a predominantly Th1-mediated immune response.
  • the adjuvant favors a Th1-type immune response.
  • the adjuvant favors a Th1-mediated immune response.
  • the adjuvant favors a cell-mediated immune response over an antibody- mediated response.
  • the adjuvant is any other type of adjuvant known in the art.
  • the immunogenic composition induces the formation of a T cell immune response against the target protein.
  • the adjuvant is MPL. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is a TLR agonist. In another embodiment, the adjuvant is a TLR4 agonist. In another embodiment, the adjuvant is a TLR9 agonist. In another embodiment, the adjuvant is Resiquimod®. In another embodiment, the adjuvant is imiquimod. In another embodiment, the adjuvant is a CpG oligonucleotide. In another embodiment, the adjuvant is a cytokine or a nucleic acid encoding same. In another embodiment, the adjuvant is a chemokine or a nucleic acid encoding same.
  • the adjuvant is IL-12 or a nucleic acid encoding same. In another embodiment, the adjuvant is IL-6 or a nucleic acid encoding same. In another embodiment, the adjuvant is a lipopolysaccharide. In another embodiment, the adjuvant is as described in Fundamental Immunology, 5th ed (March 2003): William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; Chapter 43: Vaccines, GJV Nossal, which is hereby incorporated by reference. In another embodiment, the adjuvant is any other adjuvant known in the art. Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • provided herein is a method of inducing an immune response to an antigen in a subject comprising administering a recombinant Listeria strain to said subject.
  • a method of inducing an anti-angiogenic immune response to an antigen in a subject comprising administering a recombinant Listeria strain to said subject.
  • said recombinant Listeria strain comprises a first and second nucleic acid molecule.
  • each said nucleic acid molecule encodes a heterologous antigen.
  • said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with an endogenous polypeptide comprising a PEST sequence.
  • a method of treating, suppressing, or inhibiting at least one cancer in a subject comprising administering a recombinant Listeria strain to said subject.
  • said recombinant Listeria strain comprises a first and second nucleic acid molecule.
  • each said nucleic acid molecule encoding a heterologous antigen.
  • said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid sequence encoding an endogenous polypeptide comprising a PEST sequence.
  • at least one of said antigens is expressed by at least one cell of said cancer cells.
  • provided herein is a method of delaying the onset to a cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of delaying the progression to a cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of extending the remission to a cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of decreasing the size of an existing tumor in a subject comprising administering a recombinant Listeria strain to said subject.
  • provided herein is a method of preventing the growth of an existing tumor in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of preventing the growth of new or additional tumors in a subject comprising administering a recombinant Listeria strain to said subject. [000314] In one embodiment, cancer or tumors may be prevented in specific populations known to be susceptible to a particular cancer or tumor.
  • such susceptibilty may be due to environmental factors, such as smoking, which in one embodiment, may cause a population to be subject to lung cancer, while in another embodiment, such susceptbility may be due to genetic factors, for example a population with BRCA1/2 mutations may be susceptible, in one embodiment, to breast cancer, and in another embodiment, to ovarian cancer.
  • one or more mutations on chromosome 8q24, chromosome 17q12, and chromosome 17q24.3 may increase susceptibility to prostate cancer, as is known in the art.
  • Other genetic and environmental factors contributing to cancer susceptibility are known in the art.
  • the recombinant Listeria strain is administered to the subject at a dose of 1 x 10 6 - 1 x 10 7 CFU. In another embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10 7 - 1 x 10 8 CFU. In another embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10 8 - 3.31 x 10 10 CFU. In another embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10 9 - 3.31 x 10 10 CFU. In another embodiment, the dose is 5-500 x 10 8 CFU. In another embodiment, the dose is 7-500 x 10 8 CFU.
  • the dose is 10-500 x 10 8 CFU. In another embodiment, the dose is 20-500 x 10 8 CFU. In another embodiment, the dose is 30-500 x 10 8 CFU. In another embodiment, the dose is 50-500 x 10 8 CFU. In another embodiment, the dose is 70-500 x 10 8 CFU. In another embodiment, the dose is 100- 500 x 10 8 CFU. In another embodiment, the dose is 150-500 x 10 8 CFU. In another embodiment, the dose is 5-300 x 10 8 CFU. In another embodiment, the dose is 5-200 x 10 8 CFU. In another embodiment, the dose is 5-15 x 10 8 CFU. In another embodiment, the dose is 5-100 x 10 8 CFU.
  • the dose is 5-70 x 10 8 CFU. In another embodiment, the dose is 5-50 x 10 8 CFU. In another embodiment, the dose is 5-30 x 10 8 CFU. In another embodiment, the dose is 5-20 x 10 8 CFU. In another embodiment, the dose is 1-30 x 10 9 CFU. In another embodiment, the dose is 1-20 x 10 9 CFU. In another embodiment, the dose is 2-30 x 10 9 CFU. In another embodiment, the dose is 1-10 x 10 9 CFU. In another embodiment, the dose is 2-10 x 10 9 CFU. In another embodiment, the dose is 3-10 x 10 9 CFU. In another embodiment, the dose is 2-7 x 10 9 CFU. In another embodiment, the dose is 2-5 x 10 9 CFU.
  • the dose is 3-5 x 10 9 CFU. [000316] In another embodiment, the dose is 1 x 10 7 organisms. In another embodiment, the dose is 1.5 x 10 7 organisms. In another embodiment, the dose is 2 x 10 8 organisms. In another embodiment, the dose is 3 x 10 7 organisms. In another embodiment, the dose is 4 x 10 7 organisms. In another embodiment, the dose is 5 x 10 7 organisms. In another embodiment, the dose is 6 x 10 7 organisms. In another embodiment, the dose is 7 x 10 7 organisms. In another embodiment, the dose is 8 x 10 7 organisms. In another embodiment, the dose is 10 x 10 7 organisms. In another embodiment, the dose is 1.5 x 10 8 organisms.
  • the dose is 2 x 10 8 organisms. In another embodiment, the dose is 2.5 x 10 8 organisms. In another embodiment, the dose is 3 x 10 8 organisms. In another embodiment, the dose is 3.3 x 10 8 organisms. In another embodiment, the dose is 4 x 10 8 organisms. In another embodiment, the dose is 5 x 10 8 organisms.
  • Each dose and range of doses represents a separate embodiment of the present invention [000317] In another embodiment, the dose is 1 x 10 9 organisms. In another embodiment, the dose is 1.5 x 10 9 organisms. In another embodiment, the dose is 2 x 10 9 organisms. In another embodiment, the dose is 3 x 10 9 organisms. In another embodiment, the dose is 4 x 10 9 organisms.
  • the dose is 5 x 10 9 organisms. In another embodiment, the dose is 6 x 10 9 organisms. In another embodiment, the dose is 7 x 10 9 organisms. In another embodiment, the dose is 8 x 10 9 organisms. In another embodiment, the dose is 10 x 10 9 organisms. In another embodiment, the dose is 1.5 x 10 10 organisms. In another embodiment, the dose is 2 x 10 10 organisms. In another embodiment, the dose is 2.5 x 10 10 organisms. In another embodiment, the dose is 3 x 10 10 organisms. In another embodiment, the dose is 3.3 x 10 10 organisms. In another embodiment, the dose is 4 x 10 10 organisms. In another embodiment, the dose is 5 x 10 10 organisms.
  • Boosting may encompass administering an additional vaccine or immunogenic composition or recombinant Listeria strain dose to a subject.
  • 2 boosts or a total of 3 inoculations
  • 3 boosts are administered.
  • 4 boosts are administered.
  • 5 boosts are administered.
  • 6 boosts are administered.
  • more than 6 boosts are administered.
  • a method of present invention further comprises the step of boosting the human subject with a recombinant Listeria strain as provided herein.
  • the recombinant Listeria strain used in the booster inoculation is the same as the strain used in the initial“priming” inoculation.
  • the booster strain is different from the priming strain.
  • the same doses are used in the priming and boosting inoculations.
  • a larger dose is used in the booster.
  • a smaller dose is used in the booster.
  • the methods of the present invention further comprise the step of administering to the subject a booster vaccination.
  • the booster vaccination follows a single priming vaccination.
  • a single booster vaccination is administered after the priming vaccinations.
  • two booster vaccinations are administered after the priming vaccinations.
  • three booster vaccinations are administered after the priming vaccinations.
  • the period between a prime and a boost vaccine is experimentally determined by the skilled artisan.
  • the period between a prime and a boost vaccine is 1 week, in another embodiment it is 2 weeks, in another embodiment, it is 3 weeks, in another embodiment, it is 4 weeks, in another embodiment, it is 5 weeks, in another embodiment it is 6-8 weeks, in yet another embodiment, the boost vaccine is administered 8-10 weeks after the prime vaccine.
  • a method of the present invention further comprises boosting the human subject with a recombinant Listeria strain provided herein.
  • a method of the present invention comprises the step of administering a booster dose of an immunogenic composition comprising the recombinant Listeria strain provided herein.
  • the booster dose is an alternate form of said immunogenic composition.
  • the methods of the present invention further comprise the step of administering to the subject a booster immunogenic composition.
  • the booster dose follows a single priming dose of said immunogenic composition .
  • a single booster dose is administered after the priming dose.
  • two booster doses are administered after the priming dose.
  • three booster doses are administered after the priming dose.
  • the period between a prime and a boost dose of an immunogenic composition comprising the recombinant Listeria provided herein is experimentally determined by the skilled artisan.
  • the dose is experimentally determined by a skilled artisan.
  • the period between a prime and a boost dose is 1 week, in another embodiment it is 2 weeks, in another embodiment, it is 3 weeks, in another embodiment, it is 4 weeks, in another embodiment, it is 5 weeks, in another embodiment it is 6-8 weeks, in yet another embodiment, the boost dose is administered 8-10 weeks after the prime dose of the immunogenic composition.
  • Heterologous "prime boost" strategies have been effective for enhancing immune responses and protection against numerous pathogens. Schneider et al., Immunol. Rev.
  • 6,500,432 is directed to methods of enhancing an immune response of nucleic acid vaccination by simultaneous administration of a polynucleotide and polypeptide of interest.
  • simultaneous administration means administration of the polynucleotide and the polypeptide during the same immune response, preferably within 0-10 or 3-7 days of each other.
  • the antigens contemplated by the patent include, among others, those of Hepatitis (all forms), HSV, HIV, CMV, EBV, RSV, VZV, HPV, polio, influenza, parasites (e.g., from the genus Plasmodium), and pathogenic bacteria (including but not limited to M. tuberculosis, M.
  • the first or second nucleic acid molecule encodes a prostate specific antigen (PSA) and the method is for treating, inhibiting or suppressing prostate cancer.
  • PSA prostate specific antigen
  • the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing ovarian cancer.
  • the first or second nucleic acid molecule encodes PSA and the method is treating, inhibiting, or suppressing metastasis of prostate cancer, which in one embodiment, comprises metastasis to bone, and in another embodiment, comprises metastasis to other organs.
  • the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing metastasis of prostate cancer to bones.
  • the method is for treating, inhibiting, or suppressing metastatis of prostate cancer to other organs.
  • the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing breast cancer.
  • the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing both ovarian and breast cancer.
  • the first or second nucleic acid molecule encodes a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) and the method is for treating, inhibiting or suppressing melanoma.
  • HMW-MAA High Molecular Weight-Melanoma Associated Antigen
  • the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing breast cancer.
  • the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing ovarian cancer.
  • the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing benign nevi lesions. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing basal cell carcinoma. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing a tumor of neural crest origin, which in one embodiment, is an astrocytoma, glioma, neuroblastoma, sarcoma, or combination thereof.
  • the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing a childhood leukemia, which in one embodiment, is Childhood Acute Lymphoblastic Leukemia, and in another embodiment, is Childhood Acute Myeloid Leukemia (which in one embodiment, is acute myelogenous leukemia, acute myeloid leukemia, acute myelocytic leukemia, or acute non-lymphocytic leukemia) and in another embodiment, is acute lymphocytic leukemia (which in one embodiment, is called acute lymphoblastic leukemia, and in another embodiment, is acute myelogenous leukemia (also called acute myeloid leukemia, acute myelocytic leukemia, or acute non-lymphocytic leukemia) and in another embodiment, is Hybrid or mixed lineage leukemia.
  • Childhood Acute Lymphoblastic Leukemia which in one embodiment, is acute myelogenous leukemia, acute myeloid leukemia, acute my
  • the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing Chronic myelogenous leukemia or Juvenile Myelomonocytic Leukemia (JMML).
  • the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing lobular breast carcinoma lesions.
  • the cancer that is the target of methods and compositions as provided herein is, in another embodiment, a melanoma.
  • the cancer is a sarcoma.
  • the cancer is a carcinoma.
  • the cancer is a mesothelioma (e.g.
  • the cancer is a glioma. In another embodiment, the cancer is a germ cell tumor. In another embodiment, the cancer is a choriocarcinoma. [000325] In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is a carcinomatous lesion of the pancreas. In another embodiment, the cancer is pulmonary adenocarcinoma. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is pulmonary squamous adenocarcinoma.
  • the cancer is gastric adenocarcinoma.
  • the cancer is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof).
  • the cancer is an oral squamous cell carcinoma.
  • the cancer is non small-cell lung carcinoma.
  • the cancer is an endometrial carcinoma.
  • the cancer is a bladder cancer.
  • the cancer is a head and neck cancer.
  • the cancer is a prostate carcinoma.
  • the cancer is a non-small cell lung cancer (NSCLC).
  • the cancer is a colon cancer.
  • the cancer is a lung cancer.
  • the cancer is an ovarian cancer. In another embodiment, the cancer is a uterine cancer. In another embodiment, the cancer is a thyroid cancer. In another embodiment, the cancer is a hepatocellular carcinoma. In another embodiment, the cancer is a thyroid cancer. In another embodiment, the cancer is a liver cancer. In another embodiment, the cancer is a renal cancer. In another embodiment, the cancer is a kaposis. In another embodiment, the cancer is a sarcoma. In another embodiment, the cancer is another carcinoma or sarcoma. Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • the compositions and methods as provided herein can be used to treat solid tumors related to or resulting from any of the cancers as described hereinabove.
  • the tumor is a Wilms’ tumor.
  • the tumor is a desmoplastic small round cell tumor.
  • the present invention provides a method of impeding angiogenesis of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria encoding a heterologous antigen.
  • the antigen is HMW-MAA.
  • the antigen is fibroblast growth factor (FGF).
  • the antigen is vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the antigen is any other antigen known in the art to be involved in angiogenesis.
  • the methods and compositions of impeding angiogenesis of a solid tumor in a subject comprise administering to the subject a composition comprising a recombinant Listeria encoding two heterologous antigens.
  • the methods and compositions of impeding angiogenesis of a solid tumor in a subject comprise administering to the subject a composition comprising a mixture of two recombinant Listeria strains wherein each strain encodes a different heterologous antigens.
  • the methods and compositions of impeding angiogenesis of a solid tumor in a subject comprise administering to the subject a composition comprising a recombinant Listeria strains encioding first heterologous antigen, followed by administering to the subject a composition comprising a recombinant Listeria strains encioding second heterologous antigen.
  • one of the two heterologous antigens is HMW-MAA.
  • the antigen is any other antigen known in the art to be involved in angiogenesis.
  • the prostate cancer model used to test methods and compositions as provided herein is the TPSA23 (derived from TRAMP-C1 cell line stably expressing PSA) mouse model.
  • the prostate cancer model is a 178-2 BMA cell model.
  • the prostate cancer model is a PAIII adenocarcinoma cells model.
  • the prostate cancer model is a PC-3M model.
  • the prostate cancer model is any other prostate cancer model known in the art. Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • the vaccine is tested in human subjects, and efficacy is monitored using methods well known in the art, e.g. directly measuring CD4 + and CD8 + T cell responses, or measuring disease progression, e.g. by determining the number or size of tumor metastases, or monitoring disease symptoms (cough, chest pain, weight loss, etc).
  • the present invention provides a method of treating benign prostate hyperplasia (BPH) in a subject.
  • the present invention provides a method of treating Prostatic Intraepithelial Neoplasia (PIN) in a subject
  • BPH benign prostate hyperplasia
  • PIN Prostatic Intraepithelial Neoplasia
  • a recombinant Listeria strain comprising a nucleic acid molecule operably integrated into the Listeria genome.
  • said nucleic acid molecule encodes (a) an endogenous polypeptide comprising a PEST sequence and (b) a polypeptide comprising an antigen in an open reading frame.
  • a method of treating, suppressing, or inhibiting at least one tumor in a subject comprising administering a recombinant Listeria strain to said subject.
  • said recombinant Listeria strain comprises a first and second nucleic acid molecule.
  • each said nucleic acid molecule encodes a heterologous antigen.
  • said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a native polypeptide comprising a PEST sequence and wherein said antigen is expressed by at least one cell of said tumor.
  • antigen is used herein to refer to a substance that when placed in contact with an organism, results in a detectable immune response from the organism.
  • An antigen may be a lipid, peptide, protein, carbohydrate, nucleic acid, or combinations and variations thereof.
  • “variant” refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them, for example splice variants.
  • “isoform” refers to a version of a molecule, for example, a protein, with only slight differences compared to another isoform, or version, of the same protein.
  • isoforms may be produced from different but related genes, or in another embodiment, may arise from the same gene by alternative splicing.
  • isoforms are caused by single nucleotide polymorphisms.
  • “fragment” refers to a protein or polypeptide that is shorter or comprises fewer amino acids than the full length protein or polypeptide.
  • fragment refers to a nucleic acid that is shorter or comprises fewer nucleotides than the full length nucleic acid.
  • the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment is an intrasequential section of the protein, peptide, or nucleic acid. In one embodiment, the fragment is a functional fragment. In another embodiment, the fragment is an immunogenic fragment. In one embodiment, a fragment has 10-20 nucleic or amino acids, while in another embodiment, a fragment has more than 5 nucleic or amino acids, while in another embodiment, a fragment has 100-200 nucleic or amino acids, while in another embodiment, a fragment has 100-500 nucleic or amino acids, while in another embodiment, a fragment has 50-200 nucleic or amino acids, while in another embodiment, a fragment has 10- 250 nucleic or amino acids.
  • immunogenicity or“immunogenic” is used herein to refer to the innate ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response in an animal when the protein, peptide, nucleic acid, antigen or organism is administered to the animal.
  • enhancing the immunogenicity in one embodiment, refers to increasing the ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response in an animal when the protein, peptide, nucleic acid, antigen or organism is administered to an animal.
  • the increased ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response can be measured by, in one embodiment, a greater number of antibodies to a protein, peptide, nucleic acid, antigen or organism, a greater diversity of antibodies to an antigen or organism, a greater number of T-cells specific for a protein, peptide, nucleic acid, antigen or organism, a greater cytotoxic or helper T-cell response to a protein, peptide, nucleic acid, antigen or organism, and the like.
  • a“homologue” refers to a nucleic acid or amino acid sequence which shares a certain percentage of sequence identity with a particular nucleic acid or amino acid sequence.
  • a sequence useful in the composition and methods as provided herein may be a homologue of a particular LLO sequence or N-terminal fragment thereof, ActA sequence or N-terminal fragment thereof, or PEST-like sequence described herein or known in the art. In one embodiment, such a homolog maintains
  • a sequence useful in the composition and methods as provided herein may be a homologue of an antigenic polypeptide, which in one embodiment, is CA9, cHER2 or HMW- MAA or a functional fragment thereof.
  • a homolog of a polypeptide and, in one embodiment, the nucleic acid encoding such a homolog, of the present invention maintains the functional characteristics of the parent polypeptide.
  • a homolog of an antigenic polypeptide of the present invention maintains the antigenic characteristic of the parent polypeptide.
  • a sequence useful in the composition and methods as provided herein may be a homologue of any sequence described herein.
  • a homologue shares at least 70% identity with a particular sequence.
  • a homologue shares at least 72% identity with a particular sequence.
  • a homologue shares at least 75% identity with a particular sequence.
  • a homologue shares at least 78% identity with a particular sequence.
  • a homologue shares at least 80% identity with a particular sequence.
  • a homologue shares at least 82% identity with a particular sequence.
  • a homologue shares at least 83% identity with a particular sequence. In another embodiment, a homologue shares at least 85% identity with a particular sequence. In another embodiment, a homologue shares at least 87% identity with a particular sequence. In another embodiment, a homologue shares at least 88% identity with a particular sequence. In another embodiment, a homologue shares at least 90% identity with a particular sequence. In another embodiment, a homologue shares at least 92% identity with a particular sequence. In another embodiment, a homologue shares at least 93% identity with a particular sequence. In another embodiment, a homologue shares at least 95% identity with a particular sequence. In another embodiment, a homologue shares at least 96% identity with a particular sequence.
  • a homologue shares at least 97% identity with a particular sequence. In another embodiment, a homologue shares at least 98% identity with a particular sequence. In another embodiment, a homologue shares at least 99% identity with a particular sequence. In another embodiment, a homologue shares 100% identity with a particular sequence.
  • “functional” within the meaning of the invention is used herein to refer to the innate ability of a protein, peptide, nucleic acid, fragment or a variant thereof to exhibit a biological activity or function.
  • a biological function is its binding property to an interaction partner, e.g., a membrane-associated receptor, and in another embodiment, its trimerization property.
  • these biological functions may in fact be changed, e.g., with respect to their specificity or selectivity, but with retention of the basic biological function.
  • “treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein.
  • treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof.
  • “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • “preventing” or“impeding” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof.
  • “suppressing” or“inhibiting” refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.
  • symptoms are primary, while in another embodiment, symptoms are secondary.
  • “primary” refers to a symptom that is a direct result of a particular disease or disorder
  • “secondary” refers to a symptom that is derived from or consequent to a primary cause.
  • the compounds for use in the present invention treat primary or secondary symptoms or secondary complications.
  • “symptoms” may be any manifestation of a disease or pathological condition.
  • the term “comprising” refers to the inclusion of other recombinant polypeptides, amino acid sequences, or nucleic acid sequences, as well as inclusion of other polypeptides, amino acid sequences, or nucleic acid sequences, that may be known in the art, which in one embodiment may comprise antigens or Listeria polypeptides, amino acid sequences, or nucleic acid sequences.
  • the term“consisting essentially of” refers to a composition for use in the methods as provided herein, which has the specific recombinant polypeptide, amino acid sequence, or nucleic acid sequence, or fragment thereof.
  • compositions for use in the methods as provided herein are administered intravenously.
  • the vaccine is administered orally, whereas in another embodiment, the vaccine is administered parenterally (e.g., subcutaneously, intramuscularly, and the like).
  • the compositions or vaccines are administered as a suppository, for example a rectal suppository or a urethral suppository.
  • the pharmaceutical compositions are administered by subcutaneous implantation of a pellet. In a further embodiment, the pellet provides for controlled release of an agent over a period of time.
  • the pharmaceutical compositions are administered in the form of a capsule.
  • the route of administration may be parenteral.
  • the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation (aerosol), nasal aspiration (spray), intranasal (drops), sublingual, oral, aerosol or suppository or a combination thereof.
  • Such an aerosol may comprise any agent described herein.
  • compositions as set forth herein may be in a form suitable for intracranial administration, which in one embodiment, is intrathecal and intracerebroventricular administration.
  • the regimen of administration will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, body weight, and response of the individual patient, etc.
  • parenteral application particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories and enemas. Ampoules are convenient unit dosages. Such a suppository may comprise any agent described herein.
  • sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the new compounds and use the lyophilisates obtained, for example, for the preparation of products for injection.
  • pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • compositions of this invention are pharmaceutically acceptable.
  • pharmaceutically acceptable refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound for use in the present invention.
  • compositions of or used in the methods of this invention may be administered alone or within a composition.
  • compositions of this invention admixture with conventional excipients i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds may be used.
  • suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • they can also be combined where desired with other active
  • Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • the compositions of the methods and compositions as provided herein may comprise the composition of this invention and one or more additional compounds effective in preventing or treating cancer.
  • the additional compound may comprise a compound useful in chemotherapy, which in one embodiment, is Cisplatin.
  • Ifosfamide, Fluorouracilor5-FU, Irinotecan, Paclitaxel (Taxol), Docetaxel, Gemcitabine, Topotecan or a combination thereof may be administered with a composition as provided herein for use in the methods as provided herein.
  • fusion proteins as provided herein are prepared by a process comprising subcloning of appropriate sequences, followed by expression of the resulting nucleotide.
  • subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated, in another embodiment, to produce the desired DNA sequence.
  • DNA encoding the fusion protein is produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately.
  • the 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction.
  • the amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence).
  • the insert is then ligated into a plasmid.
  • the present invention also provides a recombinant Listeria comprising a nucleic acid molecule encoding a polypeptide comprising a heterologous antigen or fragment thereof fused to a PEST-containing sequence, wherein said nucleic acid molecule is remains episomaly in said Listeria.
  • a recombinant Listeria capable of expressing and secreting two distinct heterologous antigens.
  • the first and second antigen are distinct.
  • said first and second antigens are concomitantly expressed.
  • said first or second antigen are expressed at the same level. In another embodiment, said first or second antigen are differentially expressed. In another embodiment, gene or protein expression is determined by methods that are well known in the art which in another embodiment comprise real-time PCR, northern blotting, immunoblotting, etc. In another embodiment, said first or second antigen’s expression is controlled by an inducible system, while in another embodiment, said first or second antigen’s expression is controlled by a constitutive promoter. In another embodiment, inducible expression systems are well known in the art. [000358] In one embodiment, provided herein is a method of preparing a recombinant Listeria capable of expressing and secreting two distinct heterologous antigens that target tumor cells and angiogenesis concomitantly.
  • said method of preparing said recombinant Listeria comprises the steps of genetically fusing a first antigen into the genome that is operably linked to an open reading frame encoding a first polypeptide or fragment thereof comprising a PEST sequence and transforming said recombinant Listeria with an episomal expression vector encoding a second antigen that is operably linked to an open reading frame encoding a second polypeptide or fragment thereof comprising a PEST sequence.
  • said method of preparing said recombinant Listeria comprises the steps of genetically fusing a first antigen into the genome that is operably linked to an open reading frame encoding a first polypeptide or fragment thereof comprising a PEST sequence and genetically fusing a second antigen that is operably linked to an open reading frame encoding a second polypeptide or fragment thereof comprising a PEST sequence.
  • Methods for transforming bacteria are well known in the art, and include calcium- chloride competent cell-based methods, electroporation methods, bacteriophage-mediated transduction, chemical, and physical transformation techniques (de Boer et al, 1989, Cell 56:641-649; Miller et al, 1995, FASEB J., 9:190-199; Sambrook et al.
  • the Listeria vaccine strain as provided herein is transformed by electroporation.
  • Each method represents a separate embodiment of the methods and compositions as provided herein.
  • a method of inducing an immune response to an antigen in a subject comprising administering a recombinant Listeria strain to said subject, wherein said recombinant Listeria strain comprises a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide or fragment thereof, wherein said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid encoding an endogenous polypeptide comprising a PEST sequence.
  • provided herein is a method of inhibiting the onset of cancer, said method comprising the step of administering a recombinant Listeria composition that expresses two distinct heterologous antigens specifically expressed in said cancer.
  • a method of treating a subject having a tumor or cancer said method comprising the step of administering a pharmaceutical composition or formulation comprising a recombinant Listeria provided hereinthat expresses two or more distinct heterologous antigens specifically expressed on said tumor.
  • the recombinant Listeria expressing two or more heterologous antigens fused to a PEST-containing sequence targets two or more different tumors or cancers, or metastases in a subject having said tumors or cancers or metastases.
  • a PEST-containing sequence such as N-terminal LLO, N- terminal ActA, or a PEST sequence or peptide
  • a method of ameliorating symptoms that are associated with a cancer in a subject comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer.
  • provided herein is a method of protecting a subject from cancer, said method comprising the step of administering a recombinant Listeria composition that expresses two distinct heterologous antigens specifically expressed in said cancer
  • a method of delaying onset of cancer said method comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer.
  • a method of treating metastatic cancer said method comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer.
  • a method of preventing metastatic canceror micrometastatis comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer.
  • the recombinant Listeria composition is administered orally or parenterally.
  • a pharmaceutical composition comprising the recombinant Listeria provided herein is administered intravenously, subcutaneuosly, intranasally, intramuscularly, or injected into a tumor site or into a tumor.
  • nucleic acids or“nucleotide” refers to a string of at least two base-sugar-phosphate combinations.
  • the term includes, in one embodiment, DNA and RNA.“Nucleotides” refers, in one embodiment, to the monomeric units of nucleic acid polymers.
  • RNA may be, in one embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes.
  • DNA may be in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups.
  • these forms of DNA and RNA may be single, double, triple, or quadruple stranded.
  • the term also includes, in another embodiment, artificial nucleic acids that may contain other types of backbones but the same bases.
  • the artificial nucleic acid is a PNA (peptide nucleic acid).
  • PNA contain peptide backbones and nucleotide bases and are able to bind, in one embodiment, to both DNA and RNA molecules.
  • the nucleotide is oxetane modified. In another embodiment, the nucleotide is modified by replacement of one or more phosphodiester bonds with a phosphorothioate bond.
  • the artificial nucleic acid contains any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353-57; and Raz NK et al Biochem Biophys Res Commun. 297:1075-84.
  • nucleic acids The production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed. Each nucleic acid derivative represents a separate embodiment as provided herein.
  • the terms“polypeptide,”“peptide” and“recombinant peptide” refer, in another embodiment, to a peptide or polypeptide of any length.
  • a peptide or recombinant peptide as provided herein has one of the lengths enumerated above for an HMW-MAA fragment.
  • the term“peptide” refers to native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and/or peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • antigenic polypeptide is used herein to refer to a polypeptide, peptide or recombinant peptide as described hereinabove that is foreign to a host and leads to the mounting of an immune response when present in, or, in another embodiment, detected by, the host.
  • Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • the peptides as provided herein may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • oligonucleotide is interchangeable with the term "nucleic acid”, and may refer to a molecule, which may include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also refers to sequences that include any of the known base analogs of DNA and RNA.
  • “Stably maintained” refers, in another embodiment, to maintenance of a nucleic acid molecule or plasmid in the absence of selection (e.g. antibiotic selection) for 10 generations, without detectable loss.
  • the period is 15 generations. In another embodiment, the period is 20 generations. In another embodiment, the period is 25 generations. In another embodiment, the period is 30 generations. In another embodiment, the period is 40 generations. In another embodiment, the period is 50 generations. In another embodiment, the period is 60 generations. In another embodiment, the period is 80 generations. In another embodiment, the period is 100 generations. In another embodiment, the period is 150 generations. In another embodiment, the period is 200 generations. In another embodiment, the period is 300 generations. In another embodiment, the period is 500 generations. In another embodiment, the period is more than 500 generations. In another embodiment, the nucleic acid molecule or plasmid is maintained stably in vitro (e.g. in culture).
  • nucleic acid molecule or plasmid is maintained stably in vivo. In another embodiment, the nucleic acid molecule or plasmid is maintained stably both in vitro and in vitro.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid may include both D- and L-amino acids.
  • nucleic acid or “nucleic acid sequence” refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form.
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid.
  • the term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved thereover for the purposes desired.
  • nucleic-acid-like structures with synthetic backbones are examples of synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'- N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see, e.g., Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Mulligan (1993) J.
  • PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appi. Pharmacol. 144:189-197.
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
  • the term "recombination site” or “site-specific recombination site” refers to a sequence of bases in a nucleic acid molecule that is recognized by a recombinase (along with associated proteins, in some cases) that mediates exchange or excision of the nucleic acid segments flanking the recombination sites.
  • the recombinases and associated proteins are collectively referred to as “recombination proteins” see, e.g., Landy, A., (Current Opinion in Genetics & Development) 3:699-707; 1993).
  • a "phage expression vector” or “phagemid” refers to any phage-based recombinant expression system for the purpose of expressing a nucleic acid sequence of the methods and compositions as provided herein in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell.
  • a phage expression vector typically can both reproduce in a bacterial cell and, under proper conditions, produce phage particles.
  • the term includes linear or circular expression systems and encompasses both phage-based expression vectors that remain episomal or integrate into the host cell genome.
  • the term“operably linked” may mean that the transcriptional and translational regulatory nucleic acid, is positioned relative to any coding sequences in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5' to the coding region.
  • the term“open reading frame” or “ORF” may encompass a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. In another embodiment, the start and stop ends of the ORF are not equivalent to the ends of the mRNA, but they are usually contained within the mRNA.
  • ORFs are located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene.
  • a nucleic acid molecule operably integrated into a genome as an open reading frame with an endogenous polypeptide is a nucleic acid molecule that has integrated into a genome in the same open reading frame as an endogenous polypeptide.
  • the present invention provides a fusion polypeptide comprising a linker sequence.
  • a "linker sequence" refers to an amino acid sequence that joins two heterologous polypeptides, or fragments or domains thereof.
  • a linker is an amino acid sequence that covalently links the polypeptides to form a fusion polypeptide.
  • a linker typically includes the amino acids translated from the remaining recombination signal after removal of a reporter gene from a display vector to create a fusion protein comprising an amino acid sequence encoded by an open reading frame and the display protein.
  • the linker can comprise additional amino acids, such as glycine and other small neutral amino acids.
  • heterologous as used herein describes a nucleic acid, amino acid, peptide, polypeptide, or protein derived from a different species than the reference species.
  • a Listeria strain expressing a heterologous polypeptide in one embodiment, would express a polypeptide that is not native or endogenous to the Listeria strain, or in another embodiment, a polypeptide that is not normally expressed by the Listeria strain, or in another embodiment, a polypeptide from a source other than the Listeria strain.
  • heterologous may be used to describe something derived from a different organism within the same species.
  • the heterologous antigen is expressed by a recombinant strain of Listeria, and is processed and presented to cytotoxic T-cells upon infection of mammalian cells by the recombinant strain.
  • the heterologous antigen expressed by Listeria species need not precisely match the corresponding unmodified antigen or protein in the tumor cell or infectious agent so long as it results in a T-cell response that recognizes the unmodified antigen or protein which is naturally expressed in the mammal.
  • a method of the present invention further comprises boosting the subject with a recombinant Listeria strain provided herein.
  • a method of the present invention comprises the step of administering a booster dose of vaccine comprising the recombinant Listeria strain provided herein.
  • "fused” refers to operable linkage by covalent bonding.
  • the term includes recombinant fusion (of nucleic acid sequences or open reading frames thereof).
  • the term includes chemical conjugation.
  • “Transforming,” in one embodiment, refers to engineering a bacterial cell to take up a plasmid or other heterologous DNA molecule.
  • “transforming” refers to engineering a bacterial cell to express a gene of a plasmid or other heterologous DNA molecule.
  • conjugation is used to introduce genetic material and/or plasmids into bacteria.
  • Methods for conjugation are well known in the art, and are described, for example, in Nikodinovic J et al (A second generation snp-derived Escherichia coli- Streptomyces shuttle expression vector that is generally transferable by conjugation. Plasmid. 2006 Nov;56(3):223-7) and Auchtung JM et al (Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12554-9).
  • Methods represent a separate embodiment of the methods and compositions as provided herein.
  • “Metabolic enzyme” refers, in another embodiment, to an enzyme involved in synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme required for synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient utilized by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient required for sustained growth of the host bacteria. In another embodiment, the enzyme is required for synthesis of the nutrient.
  • Each possibility represents a separate embodiment of the methods and compositions as provided herein.
  • Attenuation may encompass a diminution in the ability of the bacterium to cause disease in an animal.
  • pathogenic characteristics of the attenuated Listeria strain have been lessened compared with wild-type Listeria, although the attenuated Listeria is capable of growth and maintenance in culture.
  • the lethal dose at which 50% of inoculated animals survive is preferably increased above the LD.sub.50 of wild-type Listeria by at least about 10-fold, more preferably by at least about 100-fold, more preferably at least about 1,000 fold, even more preferably at least about 10,000 fold, and most preferably at least about 100,000-fold.
  • An attenuated strain of Listeria is thus one which does not kill an animal to which it is administered, or is one which kills the animal only when the number of bacteria administered is vastly greater than the number of wild type non-attenuated bacteria which would be required to kill the same animal.
  • an attenuated bacterium should also be construed to mean one which is incapable of replication in the general environment because the nutrient required for its growth is not present therein. Thus, the bacterium is limited to replication in a controlled environment wherein the required nutrient is provided.
  • the attenuated strains of the present invention are therefore environmentally safe in that they are incapable of uncontrolled replication.
  • the Listeria as provided herein expresses a heterologous polypeptide, as described herein, in another embodiment, the Listeria as provided herein secretes a heterologous polypeptide, as described herein, and in another embodiment, the Listeria as provided herein expresses and secretes a heterologous polypeptide, as described herein.
  • the Listeria as provided herein comprises a heterologous polypeptide, and in another embodiment, comprises a nucleic acid that encodes a heterologous polypeptide.
  • Listeria strains as provided herein may be used in the preparation of vaccines or immunotherapies described herein.
  • Listeria strains as provided herein may be used in the preparation of peptide vaccines. Methods for preparing peptide vaccines are well known in the art and are described, for example, in EP1408048, United States Patent Application Number 20070154953, and OGASAWARA et al (Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8995-8999, October 1992).
  • peptide evolution techniques are used to create an antigen with higher immunogenicity. Techniques for peptide evolution are well known in the art and are described, for example in United States Patent 6773900.
  • the vaccines of the methods and compositions as provided herein may be administered to a host vertebrate animal, preferably a mammal, and more preferably a human, either alone or in combination with a pharmaceutically acceptable carrier.
  • the vaccine is administered in an amount effective to induce an immune response to the Listeria strain itself or to a heterologous antigen which the Listeria species has been modified to express.
  • the amount of vaccine to be administered may be routinely determined by one of skill in the art when in possession of the present disclosure.
  • a pharmaceutically acceptable carrier may include, but is not limited to, sterile distilled water, saline, phosphate buffered solutions or bicarbonate buffered solutions.
  • the pharmaceutically acceptable carrier selected and the amount of carrier to be used will depend upon several factors including the mode of administration, the strain of Listeria and the age and disease state of the vaccinee.
  • administration of the vaccine may be by an oral route, or it may be parenteral, intranasal, intramuscular, intravascular, intrarectal, intraperitoneal, or any one of a variety of well-known routes of administration.
  • the route of administration may be selected in accordance with the type of infectious agent or tumor to be treated.
  • the present invention provides a recombinant Listeria strain comprising a nucleic acid molecule encoding a heterologous antigenic polypeptide or fragment thereof, wherein said nucleic acid molecule is operably integrated into the Listeria genome in an open reading frame with an endogenous PEST-containing gene.
  • the term "about” as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.
  • subject refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • kits comprising the pharmaceutical compositions or formulations comprising the recombinant Listeria provided herein.
  • Plasmids and strains [000400] The sequence of the plasmid pAdv142 (6523 bp) was as follows: [000401] cggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaggctgc accggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagc ggaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagc cgttttccataggctccgccccctgacaagcat
  • EXAMPLE 1 Construction of attenuated Listeria strain-Lmdd ⁇ actA and insertion of the human klk3 gene in frame to the hly gene in the Lmdd and Lmdda strains.
  • the strain Lm dal dat (Lmdd) was attenuated by the irreversible deletion of the virulence factor, ActA.
  • An in-frame deletion of actA in the Lmdaldat (Lmdd) background was constructed to avoid any polar effects on the expression of downstream genes.
  • the Lm dal dat ⁇ actA contains the first 19 amino acids at the N-terminal and 28 amino acid residues of the C-terminal with a deletion of 591 amino acids of ActA.
  • the actA deletion mutant was produced by amplifying the chromosomal region corresponding to the upstream (657 bp-oligo’s Adv 271/272) and downstream (625 bp- oligo’s Adv 273/274) portions of actA and joining by PCR.
  • the sequence of the primers used for this amplification is given in the Table 2.
  • the upstream and downstream DNA regions of actA were cloned in the pNEB193 at the EcoRI/PstI restriction site and from this plasmid, the EcoRI/PstI was further cloned in the temperature sensitive plasmid pKSV7, resulting in ⁇ actA/pKSV7 (pAdv120).
  • the plasmid pAdv134 contained the antigen expression cassette tLLO-E7.
  • the LmddA strain was transformed with the pADV134 plasmid and expression of the LLO-E7 protein from selected clones confirmed by Western blot ( Figure 2B).
  • the Lmdd system derived from the 10403S wild-type strain lacks antibiotic resistance markers, except for the Lmdd streptomycin resistance.
  • pAdv134 was restricted with XhoI/XmaI to clone human PSA, klk3 resulting in the plasmid, pAdv142.
  • the new plasmid, pAdv142 ( Figure 2C, Table 1) contains Bacillus dal (B-Dal) under the control of Listeria p60 promoter.
  • the shuttle plasmid, pAdv142 complemented the growth of both E. coli ala drx MB2159 as well as Listeria monocytogenes strain Lmdd in the absence of exogenous D-alanine.
  • the antigen expression cassette in the plasmid pAdv142 consists of hly promoter and LLO-PSA fusion protein ( Figure 2C). [000407]
  • the plasmid pAdv142 was transformed to the Listeria background strains, LmddactA strain resulting in Lm-ddA-LLO-PSA.
  • the in vitro stability of the plasmid was examined by culturing the LmddA-LLO- PSA Listeria strain in the presence or absence of selective pressure for eight days.
  • the selective pressure for the strain LmddA-LLO-PSA is D-alanine. Therefore, the strain LmddA- LLO-PSA was passaged in Brain-Heart Infusion (BHI) and BHI+ 100 ⁇ g/ml D-alanine.
  • CFUs were determined for each day after plating on selective (BHI) and non-selective (BHI+D- alanine) medium.
  • Plasmid maintenance in vivo was determined by intravenous injection of 5 x 10 7 CFU LmddA-LLO-PSA, in C57BL/6 mice. Viable bacteria were isolated from spleens homogenized in PBS at 24 h and 48 h.
  • LmddA-LLO-PSA is a recombinant Listeria strain that secretes the episomally expressed tLLO-PSA fusion protein.
  • mice were immunized with LmddA- LLO-PSA at various doses and toxic effects were determined.
  • LmddA-LLO-PSA caused minimum toxic effects (data not shown).
  • the results suggested that a dose of 10 8 CFU of LmddA-LLO-PSA was well tolerated by mice. Virulence studies indicate that the strain LmddA-LLO-PSA was highly attenuated.
  • Mouse macrophage-like cell line such as J774A.1 were infected in vitro with Listeria constructs and intracellular growth was quantified.
  • the positive control strain, wild type Listeria strain 10403S grows intracellularly, and the negative control XFL7, a prfA mutant, cannot escape the phagolysosome and thus does not grow in J774 cells.
  • the intracytoplasmic growth of LmddA-LLO-PSA was slower than 10403S due to the loss of the ability of this strain to spread from cell to cell ( Figure 5B). The results indicate that LmddA- LLO-PSA has the ability to infect macrophages and grow intracytoplasmically.
  • EXAMPLE 5 Immunogenicity of the strain-LmddA-LLO-PSA in C57BL/6 mice [000413]
  • the PSA-specific immune responses elicited by the construct LmddA-LLO-PSA in C57BL/6 mice were determined using PSA tetramer staining. Mice were immunized twice with LmddA-LLO-PSA at one week intervals and the splenocytes were stained for PSA tetramer on day 6 after the boost.
  • LmddA-142 LmddA-LLO-PSA
  • TPSA Tramp-C1- PSA
  • mice were subcutaneously implanted with 2 x 10 6 TPSA cells. When tumors reached the palpable size of 4-6 mm, on day 6 after tumor inoculation, mice were immunized three times at one week intervals with 10 8 CFU LmddA-142, 10 7 CFU Lm-LLO-PSA (positive control) or left untreated. The na ⁇ ve mice developed tumors gradually (Figure 7A).
  • mice immunized with LmddA-142 were all tumor-free until day 35 and gradually 3 out of 8 mice developed tumors, which grew at a much slower rate as compared to the na ⁇ ve mice ( Figure 7B).
  • Five out of eight mice remained tumor free through day 70.
  • Lm-LLO-PSA-vaccinated mice had fewer tumors than na ⁇ ve controls and tumors developed more slowly than in controls ( Figure 7C).
  • the construct LmddA-LLO-PSA could regress 60 % of the tumors established by TPSA cell line and slow the growth of tumors in other mice. Cured mice that remained tumor free were rechallenged with TPSA tumors on day 68.
  • mice were subcutaneously implanted with a mixture of tumors and matrigel followed by two immunizations at seven day intervals with naiive or control (Lm-LLO-E7) Listeria, or with LmddA-LLO-PSA.
  • Tumors were excised on day 21 and were analyzed for the population of CD8 + CD62L low PSA tetramer+ and CD4 + CD25 + FoxP3 + regulatory T cells infiltrating in the tumors.
  • TILs tumor infiltrating lymphocytes
  • the construct LmddA- LLO-PSA had a stronger impact in decreasing the frequency of CD4 + CD25 + FoxP3 + T-regs in tumors when compared to the na ⁇ ve and Lm-LLO-E7 immunized group ( Figure 7B).
  • the LmddA-142 vaccine can induce PSA-specific CD8 + T cells that are able to infiltrate the tumor site ( Figure 8A).
  • Lmdd-143 and LmddA-143 secretes a functional LLO despite the PSA fusion.
  • the Lmdd-143 and LmddA-143 contain the full-length human klk3 gene, which encodes the PSA protein, inserted by homologous recombination downstream and in frame with the hly gene in the chromosome. These constructs were made by homologous recombination using the pKSV7 plasmid (Smith and Youngman, Biochimie.
  • EXAMPLE 9 A recombinant Lm strain secreting a LLO-HMW-MAA fusion protein results in a broad antitumor response.
  • the Lm-tLLO-HMW-MMA 2160-2258 (also referred as Lm-LLO-HMW-MAA-C) is based on the avirulent Lm XFL-7 strain and a pGG55-based plasmid.
  • EXAMPLE 10 Immunization of mice with Lm-LLO-HMW-MAA-C induces infiltration of the tumor stroma by CD8 + T cells and a significant reduction in the pericyte coverage in the tumor vasculature.
  • NT-2 cells do not express the HMW-MAA homolog NG2
  • immunization of FVB/N mice with Lm-LLO-HMW-MAA-C significantly impaired the growth of NT-2 tumors and eventually led to tumor regression (Figure 12D).
  • This tumor model was used to evaluate CD8 + T cells and pericytes in the tumor site by immunofluorescence.
  • Lm-LLO-HMW-MAA-C vaccination impacts blood vessel formation in the tumor site by targeting pericytes.
  • EXAMPLE 11 Development of a recombinant L. monocytogenes vector with enhanced anti-tumor activity by concomitant expression and secretion of LLO-PSA and tLLO- HMW-MAA 2160-2258 fusion proteins, eliciting immune responses to both heterologous antigens. Materials and Methods: [000428] Construction of the pADV168 plasmid.
  • the HMW-MAA-C fragment is excised from a pCR2.1 -HMW-MAA 2160-2258 plasmid by double digestion with XhoI and XmaI restriction endonucleases. This fragment is cloned in the pADV134 plasmid already digested with XhoI and XmaI to excise the E7 gene.
  • the pADV168 plasmid (Fig. 14B) is electroporated into electrocompetent the dal (-) dat (-) E. coli strain MB2159 and positive clones screened for RFLP and sequence analysis.
  • Lmdd-143/168, LmddA-143/168 and the control strains LmddA- 168, Lmdd-143/134 and LmddA-143/134 Lmdd, Lmdd-143 and LmddA-143 is transformed with either pADV168 (Fig. 14B) or pADV134 plasmid. Transformants are selected on Brain- Heart Infusion-agar plates supplemented with streptomycin (250 @g/ml) and without D- alanine (BHIs medium).
  • Individual clones are screened for LLO-PSA, tLLO-HMW-MAA 2160- 2258 and tLLO-E7 secretion in bacterial culture supernatants by Western-blot using an anti- LLO, anti-PSA or anti-E7 antibody.
  • a selected clone from each strain will be evaluated for in vitro and in vivo virulence. Each strain is passaged twice in vivo to select the most stable recombinant clones. Briefly, a selected clone from each construct is grown and injected i.p to a group of 4 mice at 1x10 8 CFU/mouse. Spleens are harvested on days 1 and 3, homogenized and plated on BHIs-agar plates.
  • fusion proteins LLO-ChHer2 and tLLO-HMC were detected by western blot using anti-LLO and anti-FLAG antibodies respectively (see Fig. 14C).
  • Hemolytic assay To determine the ability of genomic LLO to cause phagolysosomal escape a hemolytic assay was performed using secreted supernatant of control wild type 10403S and LmddA244G-168 and sheep red blood cells as target cells.
  • In vitro intracellular replication in J774 cells An in vitro intracellular growth assay was perfromed using a murine macrophage-like J774 cell line.
  • J774 cells were infected for 1 hour in medium without antibiotics at MOI of 1:1 with either one of the mono vaccines (LmddA164 and LmddA168–each generated by transforming an individual Listeria strain with pADV164 and another with pADV168, to arrive at LmddA164 and LmddA168, respectively– see Fig. 14B) or bivalent immunotherapy.
  • LmddA164 and LmddA168 each generated by transforming an individual Listeria strain with pADV164 and another with pADV168, to arrive at LmddA164 and LmddA168, respectively– see Fig. 14B
  • bivalent immunotherapy At 1 h post-nfection, cells were treated with 10 ⁇ g/ml of gentamicin to kill extracellular bacteria. .. Samples were harvested at regular time intervals and cells lysed with water to quantify the number of intracellular CFU.
  • An LD 50 of >1 x 10 8 constitutes an acceptable value based on previous experience with other Lm-based vaccines.
  • RESULTS [000434] Once the pADV168 plasmid is successfully constructed, it is sequenced for the presence of the correct HMW-MAA sequence. This plasmid in these new strains express and secrete the LLO fusion proteins specific for each construct. These strains are highly attenuated, with an LD50 of at least 1x10 8 CFU and likely higher than 1x10 9 CFU for the actA-deficient (LmddA) strains, which lack the actA gene and consequently the ability of cell- to-cell spread.
  • LmddA actA-deficient
  • LmddA-cHer2/HMC A recombinant Lm (LmddA-cHer2/HMC) was generated.
  • This Lm strain expresses and secretes a chimeric Her2 (cHer2) protein chromosomally as fusion to genomic Listeriolysin O (LLO) and a fragment of HMW-MAA 2160-2258 (also named HMW-MAA C or HMC) using a plasmid as fusion to truncated LLO (tLLO), to target tumor cells and tumor vasculature concomitantly referred as LmddA244G-168.
  • cHer2 chimeric Her2
  • LLO genomic Listeriolysin O
  • HMW-MAA C also named HMW-MAA C or HMC
  • the expression and secretion of both the fusion proteins tLLO-HMC and LLO-cHer2 from LmddA244G-168 was detected by western blot using anti-FLAG and anti-LLO specific antibodies (Fig. 14B). Furthermore, the vaccine LmddA244G-168 was passaged twice in vivo in mice to stabilize the virulence of LmddA-244G and to confirm that it retained the expression of recombinant fusion proteins (Fig. 14B). The vaccine LmddA244G-168 retained its ability to express and secrete both the fusion proteins, tLLO-HMC and LLO-cHer2 after two in vivo mice passages.
  • the strain LmddA244G-168 expresses chromosomal LLO as fusion protein LLO- cHer2 which may impact the functional ability of LLO to cause phagolysosomal escape.
  • LLO- cHer2 fusion protein LLO- cHer2
  • EXAMPLE 12 Detection of immune responses and anti-tumor effects elicited upon immunization with Lmdd-244G/168.
  • Immune responses to cHer2 and HMW-MAA are studied in mice upon immunization with Lmdd-244G-168 strain using standard methods, such as detection of IFN- ⁇ production against these antigens. The therapeutic efficacy of dual-expression vectors are tested in the NT2 breast tumor model.
  • IFN- ⁇ ELISpot We evaluated the ability of bivalent immunotherapy to generate immune responses specific for the two antigens Her2 and HMW-MAA in FvB mice.
  • mice were immunized with different immunotherapies such as LmddA134 (Lm-control), LmddA164 and LmddA244G/168 on day 0 and boosted on day 14.
  • Her2/neu specific immune responses were detected in the spleens harvested on day 21.
  • the IFN- ⁇ ELispot assay was done according to the kit instructions and spleen cells were stimulated with peptide epitope specific for the intracellular region (RLLQETELV) (SEQ ID NO: 84). [000440] IFN- ⁇ ELISA.
  • the generation of HMW-MAA-C specific immune responses in the splenocytes of immunized mice was determined by stimulating cells with HMA-MAA-C protein for 2 days.
  • the IFN- ⁇ release was detected by ELISA performed using mouse interferon-gamma ELISA kit.
  • Anti-tumor efficacy The antitumor efficacy was examined using mouse NT2 breast tumor model. FvB mice were implanted with 1 x 10 6 NT2 cells on day 0 and established tumors on right flank were treated starting day 6 with three immunizations at weekly intervals with different immunotherapies. Tumors were monitored twice a week until the end of the study. Mice were euthanized if the tumor diameter was greater than 1.5 cm. RESULTS [000442] Next, the anti-tumor therapeutic efficacy of LmddA244G was examined using mouse NT2 breast tumor model.
  • the FvB mice bearing established NT2 tumors on right flank were treated with three immunizations at one week interval with different immunotherapies expressing either mono antigens LmddA164 (ChHer2), LmddA168 (HMC) or bivalent immunotherapy LmddA244G-168.
  • Treatment with both mono- and bivalent-immunotherapy caused a reduction of NT2 tumor as indicated in Figure 16A and 16C.
  • a stronger impact on the control of NT2 tumor growth was observed after treatment with bivalent- immunotherapy.
  • Additional analysis on the percent tumor free mice in each group confirmed that treatment with bivalent immunotherapy generated maximum tumor-free mice (70 %) when compared to mono-immunotherapy (less than 40 %) treated groups.
  • TILs tumor infiltrating lymphocytes and endothelial cell- adhesion molecules induced upon immunization with Lmdd-143/168 or LmddA-143/168.
  • the tumors from mice immunized with either Lmdd-143/168 or LmddA-143/168 are analyzed by immunofluorescence to study expression of adhesion molecules by endothelial cells, blood vessel density and pericyte coverage in the tumor vasculature, as well as infiltration of the tumor by immune cells, including CD8 and CD4 T cells.
  • TILs specific for the PSA antigen are characterized by tetramer analysis and functional tests.
  • mice are immunized with LmddA-142, LmddA-168, a control Lm vaccine or left untreated.
  • the tumors are surgically excised, washed in ice-cold PBS and minced with a scalpel. The tumors are treated with dispase to solubilize the Matrigel and release single cells for analysis.
  • PSA-specific CD8 + T cells are stained with a PSA65-74 H-2Db tetramer-PE and anti-mouse CD8-FITC, CD3-PerCP-Cy5.5 and CD62L-APC antibodies.
  • TILs are stained with CD4-FITC, CD3-PerCP-Cy5.5 and CD25-APC and subsequently permeabilized for FoxP3 staining (anti-FoxP3-PE, Milteny Biotec). Cells are analyzed by a FACS Calibur cytometer and CellQuestPro software (BD Biosciences). [000447] Immunofluorescence.
  • the TPSA23 tumors embedded in matrigel are surgically excised and a fragment immediately cryopreserved in OCT freezing medium.
  • the tumor fragments are cryosectioned for 8-10 ⁇ m thick sections.
  • samples are thawed and fixed using 4% formalin.
  • sections are stained with antibodies in blocking solution in a humidified chamber at 37°C for 1 hour.
  • DAPI Intravid stains
  • ⁇ SMA intracellular stains
  • This Lm strain expresses and secretes a chimeric Her2 (cHer2) protein chromosomally as fusion to genomic Listeriolysin O (LLO) and a fragment of human Carbonic Anhydrase 9 (CA9) using a plasmid as fusion to truncated LLO (tLLO), to multiply target tumor cells.
  • cHer2 chimeric Her2 protein chromosomally as fusion to genomic Listeriolysin O (LLO) and a fragment of human Carbonic Anhydrase 9 (CA9) using a plasmid as fusion to truncated LLO (tLLO), to multiply target tumor cells.
  • mice were given the boost of each vaccine on day 10. On day 11, 4 mice in each group were euthanized and tumors were measured. On day 18, 4-5 mice in each group were euthanized and tumors were measured. On day 21, the remaining mice in each group were euthanized and the tumors were measured. Results [000459] On day 4, the tumors are barely palpable, so no measurements were made. [000460] Table 4
  • the mice in the dual vaccine group had the smallest tumors ( Figure 21). This may be due to the level of control on tumor growth that was seen early on. [000462]
  • the dual vaccine shows an initial level of tumor control in the 4T1 model that is higher than levels achieved with the mono-vaccines or the control mice as the dual vaccine groups have the smallest tumor burden at the end of the experiment (see Figure 22 and 23).
  • EXAMPLE 15 Anti-tumor efficacy of a dual cHER2- HMW-MAA Listeria vaccine on the growth of 4T1 tumors implanted in the mammary glands of Balb/c mice.
  • LmddA-cHer2/HMW-MAA A recombinant Lm (LmddA-cHer2/HMW-MAA) was generated. This Lm strain expresses and secretes a chimeric Her2 (cHer2) protein chromosomally as fusion to genomic Listeriolysin O (LLO) and high molecular weight melanoma associated antigen (HMW- MAA) using a plasmid as fusion to truncated LLO (tLLO), to multiply target tumor cells.
  • LLO genomic Listeriolysin O
  • HMW- MAA high molecular weight melanoma associated antigen
  • mice were given the boost of each vaccine on day 8. On day 15, 4 mice in each group were euthanized and tumors were measured. . Mice were given another boost of each vaccine on day 15. On day 15, 21, 28 and 35, 4-5 mice in each group were euthanized and tumors were measured. On days 42, the remaining mice in each group were euthanized and the tumors were measured. Results [000470] The results are summarized in Figure 24. The graphs show that the dual vaccine (recombinant Listeria expressing two heterologous antigens) has a large impact on the tumor volume (Figure 24). The volumes of tumors in mice receiving bivalent therapy were smaller than both the control and the mono- HMW-MMA and cHER2 vaccinated mice.
  • the PBS and PSA control mice have tumors that are comparable in volume to the mono- HMW-MMA and cHER2 groups.
  • the dual vaccine shows an initial level of tumor control in the 4T1 model that is higher than levels achieved with the mono-vaccines or the control mice as the dual vaccine groups have the smallest tumor burden at the end of the experiment (see Figure 24).
  • EXAMPLE 16 Comparative study of anti-tumor efficacy of a dual and sequential cHER2- HMW-MAA Listeria vaccine on the growth of NT2 breast tumor model.
  • Experimental Details [000473] The antitumor efficacy was examined using mouse NT2 breast tumor model.
  • FvB mice were implanted with 1 x 10 6 NT2 cells on day 0 and established tumors on right flank were treated starting day 6 with three immunizations at weekly intervals with different immunotherapies. Tumors were monitored twice a week until the end of the study. Mice were euthanized if the tumor diameter was greater than 1.5 cm. [000474] Table 6
  • mice bearing established NT2 tumors on right flank were treated with five immunizations of 1x10 8 at one week intervals with different immunotherapies expressing either mono antigens LmddA164 (ChHer2), LmddA168 (HMC), or combination of therapies expressing both antigens administered simultaneously (bivalent therapy) (see Table 1).

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Abstract

L'invention concerne des souches de Listéria recombinées comprenant des nucléotides codant pour deux ou davantage d'antigènes hétérologues qui sont chacun fusionnés avec un LLO tronqué, un ActA N-terminal ou une séquence PEST, des procédés de préparation de celles-ci, et des méthodes d'induction d'une réponse immunitaire, et de traitement, d'inhibition ou de suppression d'un cancer ou de tumeurs, comprenant l'administration de celles-ci.
PCT/US2015/040855 2014-07-18 2015-07-17 Système de largage d'antigènes hétérologues bivalent dérivé de listéria Ceased WO2016011320A1 (fr)

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US201462026085P 2014-07-18 2014-07-18
US62/026,085 2014-07-18
US14/341,215 US9650639B2 (en) 2008-05-19 2014-07-25 Dual delivery system for heterologous antigens
US14/341,215 2014-07-25
US201462076406P 2014-11-06 2014-11-06
US62/076,406 2014-11-06

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US11179339B2 (en) 2017-09-19 2021-11-23 Advaxis, Inc. Compositions and methods for lyophilization of bacteria or listeria strains
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US11446369B2 (en) 2007-05-10 2022-09-20 Advaxis, Inc. Compositions and methods comprising KLK3 or FOLH1 antigen
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US10143734B2 (en) 2014-02-18 2018-12-04 Advaxis, Inc. Biomarker directed multi-target immunotherapy
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US10847253B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
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US11897927B2 (en) 2016-11-30 2024-02-13 Advaxis, Inc. Immunogenic compositions targeting recurrent cancer mutations and methods of use thereof
US11179339B2 (en) 2017-09-19 2021-11-23 Advaxis, Inc. Compositions and methods for lyophilization of bacteria or listeria strains
US12239738B2 (en) 2017-09-19 2025-03-04 Advaxis, Inc. Compositions and methods for lyophilization of bacteria or listeria strains
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens

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