Patent for “Compositions containing angiogenic factors, and their methods of use”

Animal Health – Matthew Seavey, Yvonne Paterson, Paulo Maciag, Duane Sewell, University of Pennsylvania Penn

Search Patent for “Compositions containing angiogenic factors, and their methods of use”

Abstract for “Compositions containing angiogenic factors, and their methods of use”

“The invention provides recombinant Listeria strains that contain an angiogenic element, recombinant peptides comprising an angiogenic factor operatively linked with a polypeptide comprising PEST-like sequences, recombinant nuclear molecules encoding the same, and related vaccines and immunogenic and therapeutic techniques utilizing such.”

Background for “Compositions containing angiogenic factors, and their methods of use”

“Targeting tumor cells involved in angiogenesis can slow down rapidly growing tumors by restricting oxygen and nutrients supply, or increasing susceptibility to chemotherapy depending on the strategy employed. This is done by increasing the efficacy of a drug delivered via vascular network restructuring. In mice, tumor cells have been shown to resist anti-angiogenesis treatments. Human studies of several treatments have also been conducted. The tumor microenvironment (TME), which recruits myeloid derived suppressor cells, is responsible for the necessary angiogenesis switch that allows for tumor growth and eventual dissemination.

Multiple investigators have shown repeatedly the importance of targeting tumorangiogenesis due to its central role in invasion and growth. Targeting endothelial and pericytes would be beneficial, as they are crucial for tumor survival. Tumor cells can also mutate or downregulate MHC Class I molecules, which is required for T-cell-mediated responses.

“However, 30 years after angiogenesis was demonstrated to play an enabling function in cancer, modern medicine continues to seek out new compounds and therapeutics that target the tumor vasculature. Most therapeutics are administered in multiple doses and can cause side effects.

“In one embodiment, this invention provides a recombinant Listeria Strain that expresses an angiogenic factor.”

“In another embodiment, the invention provides a vaccine that contains a recombinant Listeria strain with an angiogenic factor.”

“In one embodiment, this invention provides a strain of recombinant Listeria that expresses an angiogenic element. The angiogenic factors are a vascular growth factor receptor-2 polypeptide (VEGFR2), an endoglin or an immunogenic fragment thereof.

“In another embodiment, this invention provides a method for inducing an anti?VEGFR2 immune reaction in a subject. This involves administering to said subject an immunogenic fragment or a recombinant Listeria-expressing a vascular growth factor receptor-2(VEGFR2) polypeptide.

“In another embodiment, this invention provides a method for treating cancer in a subject. It involves administering to said subject a composition containing a recombinant Listeria Strain expressing an angiogenic element.”

“In another embodiment, this invention provides a method for inhibiting or suppressing cancer in a subject. It includes administering to said subject a composition consisting of a recombinant Listeria Strain expressing an angiogenic element.”

“In another embodiment, this invention provides a method to prevent the recurrence or a type of tumor in a subject. It involves administering to said subject a composition containing a recombinant Listeria strain that expresses an angiogenic factor.”

“In another embodiment, this invention provides a method for inhibiting metastasis in a subject. It involves administering to said subject a composition consisting of a recombinant Listeria Strain expressing an angiogenic element.”

“In another embodiment, this invention provides a recombinant peptide that contains an angiogenic element operatively linked with a polypeptide consisting of a PEST-like sequence.”

“Another embodiment of the invention provides a vaccine that contains a recombinant protein containing an angiogenic element operatively linked with a polypeptide consisting of a PEST-like sequence as well as an adjuvant.”

“In another embodiment, this invention provides a vector for a recombinant vaccine that encodes an angiogenic protein and is operatively linked with a polypeptide that contains a PEST-like sequence.”

“In another embodiment, this invention provides a method for inducing an Anti-VEGFR2 immune reaction in a subject. This involves administering an immunogenic composition consisting of a recombinant peptide comprising either a vascular growth factor receptor-2(VEGFR2) polypeptide, or an immunogenic fragment thereof. The immunogenic composition is operatively linked with a polypeptide comprising an PEST-like sequence.

“In another embodiment, this invention provides a method for treating cancer in a subject. It involves administering to said subject an composition consisting of a recombinant protein containing an angiogenic element operatively linked with a polypeptide consisting of a PEST-like sequence.

“In another embodiment, this invention provides a method for inhibiting or suppressing cancer in a subject. It involves administering to said subject an composition consisting of an angiogenic element operatively linked with a polypeptide comprising PEST-like sequence.

“In another embodiment, this invention provides a method to prevent the recurrence or a type of tumor in a subject. It involves administering to said subject a composition that contains an angiogenic agent operatively linked with a polypeptide consisting of a PEST-like sequence.”

“Another embodiment of the invention is a method to inhibit metastasis in a tumor in an individual. This involves administering to the subject a composition that contains an angiogenic agent operatively linked with a polypeptide consisting of a PEST-like sequence.”

“In another embodiment, this invention provides a nucleotide molecular molecule that encodes a recombinant peptide consisting of an angiogenic element operatively linked with a polypeptide consisting of a PEST-like sequence.”

“In another embodiment, this invention provides a vaccine consisting of a nucleotide molecular encoding an angiogenic factor and operatively linked with a polypeptide comprising an adjuvant and a PEST-like sequence.

“In another embodiment, this invention provides a vector for recombinant vaccines that contains a nucleotide molecular encoding an angiogenic element and a polypeptide consisting of a PEST-like sequence.

“Another embodiment of the invention provides a method for inducing an anti?VEGFR2 immune reaction in a subject. This involves administering an immunogenic composition consisting of a nucleotide mole molecule encoding an immunogenic polypeptide that contains a vascular growth factor receptor-2 (VEGFR2) protein or an immunogenic fragment thereof. The immunogenic composition is operatively linked with a polypeptide comprising an PEST-like sequence.

“In another embodiment, this invention provides a method for treating cancer in a subject. It involves administering to said subject an composition that contains a nucleotide molecular code encoding a protein recombinant to an angiogenic factor and a polypeptide consisting of a PEST-like sequence.

“In another embodiment, this invention provides a method for inhibiting or suppressing cancer in a subject. It involves administering to said subject an composition consisting of a nucleotide molecular molecule that encodes an angiogenic element operatively linked with a polypeptide comprising PEST-like sequence.

“Another embodiment of the invention provides a method for preventing the return of a cancer in a subject. It involves administering to said subject a composition that contains a nucleotide molecular encoding an angiogenic element operatively linked with a polypeptide consisting of a PEST-like sequence.

“In another embodiment, this invention provides a method for inhibiting metastasis in a tumor in an individual. It involves administering to the subject a composition that contains a nucleotide molecular code encoding a synthetic recombinant polypeptide containing an angiogenic and a PEST-like sequence.

“In one embodiment, this invention provides recombinant Listeria species comprising an angiogenic factor, recombinant peptides comprising an angiogenic factor operatively linked with a polypeptide comprising an AEST-like sequence, and recombinant nucleotide molecular molecules encoding the same, as well as related vaccines and immunogenic and therapeutic techniques utilizing such.”

“In another embodiment, this invention provides a recombinant Listeria species that expresses an angiogenic or immunogenic fragment.”

“Another embodiment of the invention provides a recombinant Listeria species that contains a nucleic Acid encoding an immunogenic or angiogenic fragment.”

“In one embodiment, this invention provides recombinant Listeria strains consisting of vascular endothelial Growth Factor-2 (VEGFR2)-peptides, as well as recombinant Polypeptides comprising VEGFR2 linked to a polypeptide comprising PEST-like sequences, recombinant Nucleotide molecules encoding the same, related vaccines and related immunogenic or therapeutic methods.”

“In another embodiment, this invention provides a recombinant Listeria variety that expresses a vascular growth factor receptor-2(VEGFR2) polypeptide (or an immunogenic fragment thereof).

“In another embodiment, this invention provides a recombinant Listeria species that expresses an endolin polypeptide (or an immunogenic fragment thereof) in accordance with the present invention.”

“In another embodiment, this invention provides a strain of recombinant Listeria that contains a nucleic acids encoding either a vascular growth factor receptor-2(VEGFR2) polypeptide (or an immunogenic fragment thereof).

“In another embodiment, this invention provides a recombinant Listeria species that contains a nucleic Acid encoding an endoglin monopeptide or an immunogenic segment thereof.”

“In another embodiment, a recombinant Listeria species is provided by the present invention that contains a recombinant nucleotide molecular of the invention.” The present invention also provides a recombinant Listeria species that contains a recombinant mononucleotide from the invention.

“The recombinant Listeria strain is a method and composition of the present invention. In another embodiment, it is a recombinant Listeria monocytogenes. Another embodiment of the Listeria strain is a Recombinant Listeria Seeligeri strain. Another embodiment of the Listeria strain includes a recombinant Listeria greyi strain. Another embodiment of the Listeria strain includes a recombinant Listeria Ivanovii strain. Another embodiment of the Listeria strain includes a recombinant Listeria ivanovii strain. Another embodiment of the Listeria strain includes a recombinant Listeria shelshimeri strain. Another embodiment of the Listeria strain includes a recombinant strain from any other Listeria species. One embodiment of the Listeria Strain is a Listeria Strain with LLO. In another embodiment, it is a Listeria Strain with ActA. In another embodiment, it is a Listeria Strain with PEST-like sequences.

“In one embodiment, Listeria moncytogenes can be described as a gram positive facultative intracellular bacteria that is capable of infecting phagocytic cell lines and which makes it an attractive delivery vehicle for foreign proteins. Lm is released from the phagosome by the hemolytic virus factor LLO. This gene encodes Lm and allows it to reproduce in the cytoplasm. Lm could be engineered to express an interest gene fused to LLO’s first 441 amino acid. This will exclude the hemolytic domain LLO. The PEST domain, which is essential for adjuvant activity in the fused protein, also allows Lm to be engineered.

“In another embodiment, the Listeria-strain is reduced by the deletion of a gene. Another embodiment of the Listeria strain is affected by the deletion of more than one gene. Another embodiment of the Listeria strain is affected by the deletion or inactivation a gene. Another embodiment of the Listeria strain is affected by deletion or inactivation more than one gene.

“In another embodiment, hly is the gene that has been mutated. Another embodiment has the gene that has been mutated as actA. Plc A is another example of a gene that has been mutated. PlcB is another example of a gene that has been mutated. Another embodiment of the gene that has been mutated is mpl. In another embodiment, inl A is the gene that has been mutated. In another embodiment, inlA is the gene that has been mutated. Another embodiment has the gene that is mutated as bsh.

“Another embodiment of the Listeria strain is an autotrophic mutant. Another embodiment of the Listeria strain has a deficiency in a gene that encodes a vitamin synthesis gene. Another embodiment shows that the Listeria strain lacks a gene that encodes pantothenic acid synase.

“In another embodiment, the Listeria variety is deficient for an AA metabolism enzyme. Another embodiment shows that the Listeria strain lacks a D-glutamic Acid synthase gene. Another embodiment of the problem is that the Listeria strain lacks the dat gene. Another embodiment shows that the Listeria strain has a deficiency in the dal genes. Another embodiment shows that the Listeria strain has a deficiency in the dga genes. Another embodiment shows that the Listeria strain lacks a gene responsible for the synthesis diaminopimelic acids. CysK. Another embodiment of the gene is vitamin B12 independent methionine syntheticase. Another embodiment of the gene is trpA. Another embodiment of the gene is called trpB. Another embodiment of the gene is trpE. Another embodiment of the gene is asnB. Another embodiment of the gene is gltD. Another embodiment of the gene is gltB. Another embodiment has the gene leuA. Another embodiment has the gene as argG. Another embodiment of the gene is called thrC. Another embodiment of this invention is that the Listeria strain has a deficiency in any one or more genes.

“Another embodiment shows that the Listeria strain lacks a synthase gene. Another embodiment of the gene is an AAS synthesis gene. The gene can also be called folP. Another embodiment of the gene is dihydrouridine synase family protein. Another embodiment of the gene is ispD. Another embodiment of the gene is ispF. Another embodiment of the gene is phosphoenolpyruvate synase. In another embodiment, it is hisF. In another embodiment, it is hisH. Another embodiment of the gene is fliI. Another embodiment of the gene is ribosomal large-subunit pseudouridine synthase. Another embodiment of the gene is ispD. Another embodiment of the gene is bifunctional GMP synase/glutamine amidotransferase. The gene is also known as cobS in another embodiment. The gene is also cobB in another embodiment. Another embodiment of the gene is cobB. In another embodiment, the gene is uroporphyrin-III C-methyltransferase/uroporphyrinogen-III synthase. Another embodiment of the gene is cobQ. Another embodiment of the gene is uppS. Another embodiment of the gene is truB. Another embodiment of the gene is dxs. Another embodiment of the gene is mvaS. Another embodiment of the gene is dapA. Another embodiment of the gene is ispG. Another embodiment of the gene is folC. Citrate synthase is another example. Another embodiment of the gene is argJ. In another embodiment, the gene is 3-deoxy-7-phosphoheptulonate synthase. In another embodiment, the gene is indole-3-glycerol-phosphate synthase. Another embodiment of the gene is anthranilate synase/glutamine amidotransferase. Another embodiment of the gene is menB. Another embodiment of the gene is menaquinone specific isochorismate synase. In another embodiment, the gene is phosphoribosylformylglycinamidine synthase I or II. In another embodiment, the gene is phosphoribosylaminoimidazole-succinocarboxamide synthase. Another embodiment of the gene is carB. In another embodiment, it is carA. Another embodiment of the gene is thyA. In another embodiment, it is mgsA. Another embodiment of the gene is aroB. Another embodiment of the gene is hepB. Another embodiment of the gene is rluB. Another embodiment of the gene is ilvB. In another embodiment, it is ilvN. In another embodiment, it is also called alsS. Another embodiment of the gene is fabF. Another embodiment of the gene is called fabH. Another embodiment of the gene is pseudouridine synase. Another embodiment of the gene is called pyrG. Another embodiment of the gene is truA. Another embodiment of the gene is pabB. Another embodiment of the gene is an atp synase gene (e.g. atpC, atpD-2, aptG, atpA-2, etc).”

“In another embodiment, it is called phoP. The gene can also be called aroA in another embodiment. Another embodiment of the gene is aroC. Another embodiment has the gene aroD. Another embodiment of the gene is plcB.

“In another embodiment, the Listeria strain lacks a peptide transportationer. In another embodiment, the gene is ABC transporter/ATP-binding/permease protein. In another embodiment, the gene is oligopeptide ABC transporter/oligopeptide-binding protein. Another embodiment of the gene is the oligopeptide ABCtransporter/permease proteins. In another embodiment, the gene is zinc ABC transporter/zinc-binding protein. Another embodiment of the gene is sugar ABCtransporter. Another embodiment of the gene is phosphate transportationer. Another embodiment of the gene is ZIP Zinc Transporter. Another embodiment of the gene is drug resistance carrier of the EmrB/QacA families. Another embodiment of the gene is a sulfate transportationer. Another embodiment of the gene is proton dependent oligopeptide carrier. Another embodiment of the gene is magnesium transporter. Another embodiment of the gene is formate/nitrite transportationer. In another embodiment, the gene is spermidine/putrescine ABC transporter. Another embodiment of the gene is Na/Pi cotransporter. Another embodiment of the gene is sugarphosphate transporter. Another embodiment of the gene is glutamine ABCtransporter. Another embodiment of the gene is major facilitator family carrier. Another embodiment of the gene is L-proline ABC transporter/glycine betaine. Another embodiment of the gene is molybdenum-ABC transporter. Another embodiment of the gene is techoic acids ABC transporter. Another embodiment of the gene is cobalt ABCtransporter. Another embodiment of the gene is ammonium transportationer. Another embodiment of the gene is the amino acid ABC transporter. Another embodiment of the gene is cell division ABCtransporter. Another embodiment of the gene is manganeseAB transporter. Another embodiment of the gene is iron compound ABCtransporter. Another embodiment of the gene is maltose/maltodextrin ABS transporter. Another embodiment of the gene is drug resistance carrier of the family Bcr/CflA.

“In another embodiment, a gene is a subunit one of the above proteins.”

“In one embodiment compositions of this invention induce strong innate stimulation interferon-gamma. This in one embodiment has anti-angiogenic qualities. A Listeria of the invention inducing strong innate stimulation interferon-gamma (Dominiecki and al., Cancer Immunol Imother.) 2005 May; 54(5):477-88. Epub 2004 Oct. 6. incorporated by reference in its entirety by Beatty and Paterson J Immunol 2001 February 15;166(4):2276-82, incorporated by reference in its entirety One embodiment of anti-angiogenic Listeria properties is mediated by CD4+T cells (Beatty & Paterson 2001). Another embodiment of anti-angiogenic properties in Listeria is mediated by CD8+T cells. Another embodiment of IFN-gamma secretion in response to Listeria vaccination is mediated either by NK cells or NKT cells, Th1CD4+ T cell, TC1CD8+ T cell, or a combination thereof.

“In another embodiment, compositions according to the invention incite production of one or more antiangiogenic factors or proteins. IFN-gamma is the anti-angiogenic factor in one embodiment. Another embodiment of the anti-angiogenic proteins is pigment epithelium derived factor (PEDF), angiostatin, endostatin, fms-like Tyrosine Kinase (1 sFlt)-1, or soluble endoglin (2 sEng). One embodiment of the invention involves Listeria in the release anti-angiogenic factor. In this embodiment, the Listeria also plays a therapeutic role and acts as a vector to introduce an antigen to a subject.

“Each Listeria strain, type and combination of them represents an individual embodiment of the present invention.”

“In another embodiment, the recombinant Listeria is transformed with a construct that encodes an antigen or LLO-antigen fuse. To facilitate subcloning, one embodiment of the construct includes a polylinker. There are many methods for creating recombinant Listeria; each technique is an individual embodiment of the present invention.

“In one embodiment, a construct that is useful in the compositions or methods of the present invention can be expressed from the Listeria genome.”

“In another embodiment, the construct (or heterologous gene) is integrated into the Listerial Genome by homologous Recombination. Homologous recombination techniques are well-known in the art and are described in Frankel F R. Hegde, S. Lieberman, J and Y Paterson. Listeria monocytogenes are used as a vaccine vector to induce a cell-mediated immune reaction to HIV gag. J. Immunol. 155: 4766-4774. 1995; Mata M. Yao, Z. Zubair. A, Syres. K and Y Paterson. Evaluation of a recombinant Listeria moncytogenes expressing an HIV-protein that protects mice from viral challenge. Vaccine 19;1435-45, 2001. Boyer, J D. Robinson, T M. Maciag. Peng, X. Johnson, R S. Pavlakis. G. Lewis, M G. Shen, A. Siliciano. R. Brown, C R. Weiner, D. and Y Paterson. DNA prime Listeria boost inducing a cellular immune reaction to SIV antigens within the Rhesus Macaque model. This is capable of limited suppression SIV239 virus replication. Virology. 333: 88-101, 2005. Homologous recombination can also be performed in another embodiment as described in U.S. Pat. No. 6,855,320. A temperature sensitive plasmid can be used in another embodiment to select the recombinants. Each technique is an individual embodiment of the present invention.

“In another embodiment, the construct (or heterologous gene) is integrated into the Listerial Genome using transposon insert. Transposon insertion techniques are well-known in the art and have been described by Sun et. al. Infection and Immunity 1990, 58.3770-3778 describes the construction of DP?L967. In another embodiment, transposon mutagenesis offers the advantage of forming a stable genetic insertion mutant. Another embodiment does not specify the location of the transposon-mutagenesis insertable foreign gene in the genome.

“In another embodiment, the construct (or heterologous gene) is integrated into the Listerial chromosome by using phage Integration sites (Lauer, P., Chow M Y et all, Construction, characterization and use of two LM-site-specific phage vectors. J Bacteriol 2002, 184(15), 4177-86. Another embodiment uses an integrase and attachment sites of a bacteria (e.g. U153 or PSA listeriophage is used to insert heterologous genes into the appropriate attachment site. This can be any site in the genome (e.g. comK or the 3. End of the arg-tRNA gene. Endogenous prophages can be cured by removing the attachment site used prior to integrating the construct or heterologous genes. This method can also result in single-copy integratants. Each possibility is a different embodiment of the present invention.

“In one embodiment, the composition of this invention is expressed using an episomal vector from a Listeria strain. The construct can be carried by the Listeria strain using an episomal vector. Another embodiment of the construct is carried on a Listeria strain with a plasmid. This technique has been used to create LM vectors that contain antigen fusion protein. Lm-GG/E7 is a combination of a prfA deletion mutant and a plasmid that contains a copy the prfA gene, and an E7 copy fused to a LLO (hly), gene. This was done to remove the enzyme’s hemolytic activity. The organism maintained functional LLO via an endogenous copy of hly. Another embodiment of the plasmid includes an antibiotic resistance gene. Another embodiment contains a gene that encodes a virulence protein, which is not present in the genome of the modified Listeria strain. Another embodiment of the virulence element is prfA. Another embodiment of the virulence element is LLO. Another embodiment of the virulence factors is ActA. Another embodiment of the virulence factors is any one of the genes listed above as targets for attenuation. Another embodiment uses the virulence factors to refer to any other virulence factors known in the art. Each possibility is a different embodiment of the present invention.

“In another embodiment, the recombinant protein of the present invention is fused with a Listerial Protein, such as PI?PLC or a construct encoding it. A signal sequence from a secreted Listerial protein, such as ActA, hemolysin or phospholipases, is fused with the antigen-encoding genes in another embodiment. Another embodiment uses a signal sequence from the recombinant vaccination vector. A signal sequence that is functional in the recombinant vaccination vector can be used in another embodiment. Each possibility is a distinct embodiment of the present invention.

“In an alternative embodiment, the construct is contained within the Listeria strain as an episomal form.” Another embodiment of the foreign antigen is expressed using a vector contained in the recombinant Listeria species.

“Each Listeria method represents a distinct embodiment of the present invention.”

“In another embodiment, the recombinant Listeria strain has been passed through an animal host. Another embodiment maximizes the effectiveness of the strain as an immunogenic vector. Another embodiment of the passaging is to stabilize the immunogenicity and virulence of the Listeria strain. Another embodiment of the passaging is to stabilize the virulence in the Listeria strain. Another embodiment increases the immunogenicity for the Listeria strain. Another embodiment of the passaging is to increase the virulence and virility of the Listeria strain. Another embodiment of the passaging is to remove unstable sub-strains from the Listeria strain. Another embodiment reduces the number of unstable sub-strains in the Listeria strain by passaging. Passaging can be used to reduce the strain’s virulence or attenuate it. One embodiment of the invention states that the Listeria is passed through a mammal. In this case, it is a mouse. This invention allows for the use of mammals like mice, rabbits and guinea-pigs as well as gerbils, gerbils and rats. These mammals are well-known in the art. They are readily available to skilled artisans through a variety wholesalers, distributors and laboratories such as Jackson Laboratories (Bar Harbor). The art includes methods for passing a recombinant Listeria strain via an animal host. These are described in U.S. Patent Application Ser. No. 10/541,614. Each possibility is a different embodiment of the present invention.”

“In addition, the invention also includes an isolated nucleic acids encoding a truncated ActA or LLO or PEST protein, and an isolated nucleic acids encoding an immunogenic or VEGFR2 fragment or endoglin, respectively, operably linked with a nucleic Acid comprising a promoter/regulatory sequencing such that the nucleic is preferably capable to direct expression of the protein encoded in the nucleic. The invention includes expression vectors and methods to introduce exogenous genetic material into cells. This is in addition to concomitant expression in cells of the exogenous genetic material, such as the ones described in Sambrook et. al. (1989), Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. New York. Ausubel et al. (1997, Current Protocols in Molecular Biology. John Wiley & Sons. New York).

“Another embodiment of the invention is?nucleic acid? or ?nucleotide? A string of at most two base-sugar phosphate combinations. In one embodiment, the term encompasses DNA and RNA. ?Nucleotides? In one embodiment, the monomeric units in nucleic acid polymers are referred to. In one embodiment, RNA can be expressed in the following forms: tRNA(transfer RNA), small nuclear RNA, snRNA [small nuclear RNA], rRNA[ribosomal] RNA), messenger RNA], anti-senseRNA, small inhibitoryRNA (siRNA), microRNA (miRNA), and ribozymes. It has been explained how siRNA and microRNA are used (Caudy, A. A. et al. Genes & Devel 16 : 2491-96; and the references cited therein). In other embodiments, DNA can also be in the form of plasmid, viral, linear, chromosomal, or derivatives thereof. These forms of DNA or RNA can also be single, double-, triple-, or quadruple stranded. In another embodiment, artificial nucleic acid can also be included. These artificial nucleic acid may contain different types of backbones, but the same bases. One embodiment of the artificial nucleic acids is a PNA (peptide nuclear acid). PNA are composed of peptide backbones as well as nucleotide base and can bind to DNA and RNA in one embodiment. Another embodiment modifies the nucleotide with oxetane. Another embodiment modifies the nucleotide by replacing one or more phosphodiester bonds in the nucleotide with a phosphorothioate link. Another embodiment of artificial nucleic acid includes any variant of the native phosphate backbone known to the art. PNA and phosphothiorate nucleic acid use are well-known to those who are skilled in the art. They are described in Raz N K et al Biochem Biophys Res Commun 279.1075-84. Those skilled in the art know how to produce and use nucleic acid. This is described in Molecular Cloning (2001), Sambrook, and Russell. Methods in Enzymology: Methods to molecular cloning of eukaryotic cell (2003) Purchio, G. C. Fareed. Each nucleic acid derivative is an embodiment of the present invention.

“In one embodiment, an isolated nucleic acid?” A nucleic acid fragment or segment that has been isolated from its adjacent sequences in a naturally occurring state. This term can also be used to describe nucleic acids that have been substantially purified of other components that naturally accompany them, such as DNA, RNA, or proteins. This term includes, for instance, a recombinant genome that is integrated into a vector or autonomously replicating virus or into the genomic DNA a prokaryote and eukaryote. It also includes a cDNA, a genomic or fragment of cDNA produced by restriction enzyme digestion or PCR. A recombinant gene that encodes additional polypeptide sequences is also included.

“In one embodiment, a vector is provided by the present invention that contains an oligonucleotide encoded with a polypeptide. The term “oligonucleotide” is used in one embodiment. A short nucleic acid polymer containing twenty or fewer bases. The present invention is a vector that contains a polynucleotide encoded with a polypeptide. The term “polynucleotide” is used in one embodiment. A chain of nucleotides that is longer than 5 in one embodiment and more than 10 in another embodiment. In another embodiment, it’s more than 20 in another embodiment. In another embodiment, it’s more than 50. An oligonucleotide, polynucleotide, or nucleic acid can refer to prokaryotic sequences or eukaryotic DNA sequences. This term can also be used to refer to sequences that contain any of the known base analogues of DNA or RNA.

“In one embodiment, the polynucleotides described in the present invention can be linked. In one embodiment, for example, the polynucleotides that encode LLO, Flk1?E1, Flk1?E2, or Flk1?I1 are operably linked. One embodiment of the term “operably linked” is used. The?operably linked? signifies that the single-stranded or dual-stranded nucleic acids moiety contains the two polynucleotides that are arranged in the nucleic acids moiety in such an arrangement that they are expressed together. A promoter that is operably linked with the coding area of a gene can promote transcription.

“In one embodiment, a mononucleotide according to the present invention includes a promoter/regulatory sequence. In one embodiment, this promoter/regulatory is located at the 5? The desired protein coding sequence should be at the end so that it causes the cell to express the desired protein. The nucleic acid that encodes the desired protein and its promoter/regulatory sequence together form a “transgene.”

“In one embodiment, the isolated nucleic acids of the present invention are expressed under the control a promoter. This promoter could be a hly promoter or prfA promoter or ActA promoter. A promoter can also be used to express a polylpeptide from the invention, as described in another embodiment. Another embodiment uses a CMV promoter or CAG promoter. The art also recognizes other possible promoters.

“In one embodiment, the expression?promoter/regulatory sequencing? A nucleic acid sequence that is necessary for the expression of a gene product which is operably linked with the promoter/regulatory sequencing. This sequence could be the core promoter sequence in some cases, but it may also include an enhancer and other regulatory elements that are necessary for the expression of the gene products. For example, the promoter/regulatory sequence could be one that expresses the gene in a tissue-specific manner.

“In one embodiment, a “constitutive” is a nucleotide sequence that encodes or specifies a gene product. A promoter is a sequence of nucleotides that, when linked to a polynucleotide that encodes or specifies the gene product, causes the product to be produced within a human cell.

“Inducible” is a term that refers to a ‘inducible’. A promoter is a sequence of nucleotides that is operably linked to a polynucleotide that encodes or specifies the product of a gene. The promoter causes the product to be produced in living cells substantially only when the inducer corresponds to the promoter.

“A?tissue-specific’ is one embodiment. A promoter is a sequence of nucleotides that is operably linked to a polynucleotide that encodes or specifies the product. The gene product is produced in living cells substantially only if it is a cell from the tissue type indicated by the promoter.

“In another embodiment, a nucleic acid is provided that encodes a recombinant protein of the present invention. One embodiment of the isolated nucleic acids contains a sequence that shares at least 85% homology to a nucleic Acid encoding a polypeptide recombinant according to the present invention. Another embodiment of the isolated nucleic acids includes a sequence that shares at least 90% homology to a nucleic Acid encoding the recombinant protein of the invention. Another embodiment of the isolated nucleic acids includes a sequence that shares at least 95% homology to a nucleic Acid encoding the recombinant protein of the invention. Another embodiment of the isolated nucleic acids includes a sequence that shares at least 97% homology to a nucleic Acid encoding the recombinant protein of the invention. Another embodiment of the isolated nucleic acids includes a sequence that shares at least 97% homology with a nucleic Acid encoding a polypeptide recombinant to the present invention.

“The invention therefore includes a vector containing an isolated nucleic acids of the present invention. It is well-known that a desired nucleic acids can be incorporated into a vector. The choice of vectors is also well-known, as explained in Sambrook et. al. (1989), Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. New York. Ausubel et al. (1997, Current Protocols in Molecular Biology. John Wiley & Sons. New York.

“The invention also contains cells, viruses, proviruses and the like that contain such vectors. The art is well-versed in methods for creating cells that contain vectors and/or exogenous DNA. See, for example, Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. New York. Ausubel et al. (1997, Current Protocols in Molecular Biology. John Wiley & Sons. New York).

“In another embodiment, an expression vector is a virus. Sambrook et. al. describe methods for creating and using recombinant genes. (2001, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory. New York), and Brent et al. (2003, Current Protocols in Molecular Biology John Wiley & Sons New York). Each possibility is a different embodiment of the present invention.

“In an alternative embodiment, the vector may be intracellular pathogen. Another embodiment of the vector comes from a cytosolic disease. Another embodiment of the vector comes from an intracellular pathogen. An intracellular pathogen can induce a predominant cell-mediated immune response in another embodiment. Another embodiment of the vector is a Salmonella strain. Another embodiment of the vector is a BCG-strain. Another embodiment of the vector is a bacteria vector. A second embodiment of the invention allows dendritic cells to be transduced using a vector to increase the immune response. In one embodiment, this is done by increasing CTL activity.

“Another embodiment induces a predominant Th1-type immune reaction with the recombinant vaccination vector.”

“In another embodiment, the vector can be selected from Salmonella sp. Shigella sp. BCG, L. monocytogenes E. coli and S. gordonii. Another embodiment delivers the fusion proteins via recombinant bacteria vectors that have been modified to escape phagolysosomal Fusion and live in cells’ cytoplasm. Another embodiment of the vector is a virus vector. Other embodiments select the vector from Vaccinia and Avipox. Adenovirus, Adenovirus AAV, Vacciniavirus NYVAC, Modified vaccinia Strain Ankara (MVA), Semliki forest virus, Venezuelan equine encephalitis viruses, retroviruses, and herpes virus. Another embodiment of the vector is a naked-DNA vector. Another embodiment of the vector includes any other vector that is known to the art. Each possibility is a different embodiment of the present invention.

“In one embodiment, a Listeria is provided by the invention. In one embodiment, a Listeria strain contains an isolated nucleic acids or vector of this invention. In one embodiment, a ?Listeria vaccine strain? This term is used to describe a recombinant Listeria organism expressing a VEGFR2 protein or a part thereof or an endoglin protein or a portion thereof.

“Another embodiment of the invention includes a vaccine that contains a recombinant Listeria strain and optionally an adjuvant. The present invention also provides a vaccine that contains a recombinant polypeptide and optionally an adjuvant. The present invention also provides a vaccine that includes a recombinant Oligonucleotide and optionally an adjuvant.

“A vaccine according to the invention may also include an adjuvant in one embodiment. One embodiment of the vaccine includes an adjuvant, cytokine or combination thereof. A vaccine is a composition that triggers an immune response in response to antigens or polypeptides contained in the composition. APCs can be added to a vaccine or composition in another embodiment. In one embodiment they are autologous while in another they are allogeneic.

“In one embodiment, a ?vaccine? A composition that elicits an immune reaction in a host to an antigen/polypeptide contained in the composition. One embodiment of the immune response to the composition is to a specific antigen or an epitope. One embodiment may contain a peptide vaccine while another might include a DNA vaccine. Another embodiment may contain the vaccine within and deliver it by a cell. In one embodiment, this cell is a Listeria cell. A vaccine in one embodiment may prevent a subject developing or contracting a disease. In another embodiment, the vaccine may be used to treat a patient with a condition or disease. The compositions of this invention have both therapeutic and preventive effects. A vaccine according to the invention may include a mixture of the composition and an adjuvant, chemotherapy, cytokine or other combination.

“The present invention also provides an immunogenic composition that contains a recombinant protein of the invention. Another embodiment of the immunogenic composition of methods or compositions of this invention includes a vector that encodes a recombinant protein of the invention. Another embodiment of the immunogenic composition includes a plasmid that encodes a recombinant protein from the present invention. An adjuvant may also be included in the immunogenic composition. A vector of the invention can be administered in one embodiment as part of a vaccine composition. Each option represents an individual embodiment of the present invention.

“In another embodiment, the immunogenic composition used in methods and compositions according to the present invention includes a recombinant vaccination vector. Another embodiment of the recombinant vaccination vector includes a plasmid that encodes a recombinant protein from the present invention. Another embodiment of the recombinant vaccination vector includes an isolated nucleic acids of the present invention. Another embodiment of the recombinant vaccination vector includes an isolated nucleic acids that encode a recombinant protein. Each possibility is a different embodiment of the present invention.

“An immunogenic composition consisting of methods and compositions according to the present invention includes, in another embodiment, an adjuvant which favors a Th1-type immune reaction. Another embodiment favors a Th1-mediated immune reaction. Another embodiment favors a Th1-type immune reaction. Another embodiment favors a Th1-mediated immune reaction. Another embodiment favors a cell-mediated immune reaction over an antibody-mediated one. Another embodiment of the adjuvant includes any other adjuvant that is known to the art. Another embodiment induces a T cell immune reaction against the target protein. Each possible embodiment is a different embodiment of the present invention.

“In one embodiment, a predominantly Th1-type immune reaction? It refers to an immune reaction in which IFNgamma is secreted. It can also refer to an immune response that secretes tumor necrosis factor 0. It can also refer to an immune response that secretes IL-2. Each possibility is a different embodiment of the present invention.

“The present invention also provides a recombinant peptide that comprises a VEGFR2 or fragment of a VEGFR2 peptide and is operatively linked with a nonVEGFR2 peptide from a nonhemolytic listriolysin polypeptide, ActA polypeptide, a PEST-like or fragment thereof. One embodiment of the invention provides a fragment with the same or similar properties and functions as the full-length peptide or protein. This can be demonstrated using tools and assays that are well-known in the art. Below are detailed descriptions of the properties and functions of full-length peptides or proteins according to the invention.

“Another embodiment of the invention provides a polypeptide that is recombinant and includes an endoglin, or a fraction thereof, operatively linked with a nonendoglin-like polypeptide. This polypeptide can be either a nonhemolytic listeriolysin(LLO) polypeptide (ActA polypeptide), a ActA polypeptide, a PEST-like or fragment thereof. The fragment may have the same or similar properties and functions as the full-length peptide or protein in one embodiment. This can be proven using tools and assays that are well-known in the art. Below are detailed descriptions of the properties and functions of full-length peptides or proteins according to the invention.

“In one embodiment, this invention provides a recombinant Listeria species that contains a nucleic Acid encoding an immunogenic or angiogenic fragment.”

“In one embodiment, this invention provides a recombinant peptide that contains an angiogenic or immunogenic fragment, which is operatively linked with a polypeptide consisting of a PEST-like sequence.”

“In one embodiment, compositions of this invention include an angiogenic element or an immunogenic fraction thereof. In one embodiment, an immunogenic fragment includes one or more epitopes that are recognized by the host immune. An angiogenic factor can be defined as a molecule that is involved in the formation new blood vessels. VEGFR2 is the angiogenic element in one embodiment. Endoglin is another embodiment of the angiogenic factors. Another embodiment of the invention’s angiogenic factors is Angiogenin, Angiopoietin-1 and Del-1; Fibroblast growth proteins: acidic (aFGF-BB) and basic (bFGF-BB); Follistatin. An angiogenic factor can also be an angiogenic protein. A growth factor can be described as an angiogenic proteins in one embodiment. One embodiment of the invention provides an angiogenic protein that can be used in compositions and methods. It is Fibroblast growth proteins (FGF); VEGF, VEGFR and Neuropilin 1(NRP-1); Angiopoietin 1 and Tie2 respectively; Platelet-derived Growth Factor (PDGF; BBhomodimer and PDGFR); Transforming growth factor beta (TGF-beta). Endoglin, TGF-? TGF-?, endoglin and monocyte chemotactic proteins-1 (MCP-1); Integrins?V.3,?V.5 and?5?1?; VE?cadherin (VE-cadherin); ephrin (plasminogen activaters; plasminogen inhibitor-1; Nitric oxygen synthese (NOS), COX-2; AC133; and Id1/Id3. Angiopoietin is an angiogenic protein that can be used in the compositions or methods of the present invention. In one embodiment it is Angiopoietin 1. Angiopoietin 3. Angiopoietin 4. Endoglin can also be known as CD105, EDG, HHT1 and ORW in one embodiment. Endoglin can be a TGFbeta receptor in one embodiment.

HMW-MAA is not considered an angiogenic factor in one embodiment. HMW-MAA is another angiogenic factor that can be used in the compositions or methods of the invention. HMWMAA, in one embodiment, is also known by the following: HMWMAA (High molecular weight melanomas associated antigen; HMWMAA; NG2); melanoma chondroitin-sulfate prooglycan; MSK16; HMWMAA; HMW-MAA (MEL-CSPG); or chondroitin-sulfate prosoglycan 4 (4 (CSPG4)). Other angiogenic factors may also be known and can be used in compositions and methods according to the invention.

The compositions and methods described in the present invention target the tumor vasculature. The compositions and methods described in the present invention show profound epitope spread. This is in one embodiment a process in which epitopes that are not cross-reactive with inducing epitopes become major targets of an ongoing immune reaction. One embodiment of the Examples below shows that an immuno response against tumor antigens may be induced by a vaccine targeting tumor vasculature. This allows skilled artisans to treat, suppress or inhibit tumors without having to target a specific antigen. The compositions of this invention can be used as a universal vaccine against cancer or tumors. In one embodiment, it is not dependent upon a specific tumor antigen for effectiveness. One embodiment confers tumor specificity because tumors have poor pericyte and vasculature coverage.

“In one embodiment, the compositions of the present invention are extremely effective in the methods of the present invention, in one embodiment, for protracted periods of time, because antigen loss variants are unlikely to occur, because angiogenic-related polypeptides such as VEGFR2/HMW-MAA/Endoglin are crucial for general functioning, and therefore will not be lost or replaced. The compositions and methods described in one embodiment will not cause harm to normal tissue with well-developed pericyte and vasculature coverage. However, they will target tumor vasculature which is less well-developed and has poor pericyte and vasculature coverage.

“In another embodiment, the compositions and methods described in the present invention use a specific fragment of an angiogenic protein. In one embodiment, the fragment used in the compositions and methods of the present invention are based on analyzing the angiogenic factor amino acid sequence for regions that contain T cell epitopes, which in one embodiment, are determined by running the angiogenic factor sequence through an epitope predictor program, several of which are known in the art (for example, SYFPEITHI: http://www.syfpeithi.de/ or RANKpep: http://bio.dfci.harvard.edu/RANKPEP/), and in another embodiment, are determined by predictive epitope mapping. In another embodiment, a hydrophobicity map, which in one embodiment, is Expasy: http://ca.expasy.org/, is used either alone or in conjunction with epitope prediction programs to identify a fragment of an angiogenic factor for use in the compositions and methods of the present invention. Another embodiment uses human sequences homologous with angiogenic factors sequences in other animals, such as mice and rats. These species are known to contain T cell epitopes. Another embodiment uses angiogenic factors fragments that are known to contain T cell epitopes.

VEGFR2 is an angiogenic factor that can be used in compositions and methods according to the present invention.

“Vasculogenesis, the formation of embryonic circulatory systems and angiogenesis are two important functions of vascular endothelial Growth Factor (VEGF). VEGF activity in one embodiment is limited to the vascular endothelium cells, but it can have an effect on a small number of other types of cells (e.g. stimulation of monocyte/macrophage movement In vitro, VEGF was shown to stimulate cell migration and endothelial cell proliferation. VEGF can also increase microvascular permeability, which is sometimes called the vascular permeability factor.

“In one embodiment, all of the members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation. The VEGF receptors contain an extracellular portion that is composed of 7 immunoglobulin like domains, a single transmembrane spanning area and an intracellular portion that contains a split tyrosine kinase domain.

“In one embodiment, VEGF-1 (Flt-1 ligand) is a VEGFR-2 ligand (KDR/Flk-1). VEGFR-1 mediates nearly all known cellular responses of VEGF in one embodiment. Although VEGFR-1’s function is not well understood, it is believed to modulate VEGFR-2 signals in one embodiment via sequestration VEGF from VEGFR-2-binding. This is especially important in embryo vasculogenesis. One embodiment of VEGFR-3 receptor ligands VEGF C and VEGF D mediates lymphangiogenesis.

“In one embodiment the compositions according to the present invention include a VEGF receptor, or a fragment thereof. In one embodiment it is a VEGFR-2, while in another it is a VEGFR-1 and in yet another an VEGFR-3.

Summary for “Compositions containing angiogenic factors, and their methods of use”