JP2012518417A - Compositions and methods for preventing and / or treating cancer using PA-CARD - Google Patents

Abstract

The present invention relates to methods and materials for killing cancer cells using bacterially derived proteins. The present invention specifically relates to azurin, Laz, Pa-CARD, and the fusion protein Azu-H. 8 and H.I. 8-Azu and their use in killing leukemia cells and / or ovarian cancer cells.
[Selection] Figure 1

Description

Related Applications This application was filed on US Provisional Application No. 61 / 154,236, filed on February 20, 2009, and May 19, 2009, based on US Patent Acts 119 and 120. Claims priority to US Provisional Application No. 61 / 179,435 and is a continuation-in-part of US Patent Application No. 12 / 314,703 filed on December 15, 2008. A continuation-in-part of U.S. Patent Application No. 11 / 508,173, filed on August 23, and a continuation of U.S. Patent Application No. 11 / 244,105, filed on October 6, 2005 This is a continuation-in-part of US patent application Ser. No. 11 / 488,695 filed on Jul. 19, 2006. The entire contents of these applications are hereby incorporated by reference in their entirety.

  Leukemia is a malignant cancer of the bone marrow and blood. Leukemia is characterized by the accumulation of abnormal blood cells in an uncontrolled manner, leading to inhibition of normal blood cell function and often death. In 2008, an estimated 44,270 people in the United States were newly affected with leukemia. Leukemia is the fifth most common cause of cancer death in men and the sixth most common cause of cancer death in women. Leukemia causes more deaths in children under the age of 20 than any other cancer. ("Cancer facts and figures 2008", Atlanta: American Cancer Society (2008); Xie Y et al., Cancer 97: 2229-35 (2003)). Certain types of leukemia are very difficult to treat, for example, acute promyelocytic leukemia (APL) is a rare but fatal disease, with 30% of patients responding to standard chemotherapy Not too much.

  Many current therapies are effective to some extent, but exhibit significant adverse side effects due to the lack of specificity for cancer cells. The success of a highly effective drug known as Gleevec®, called imatinib, has greatly accelerated efforts to develop target-specific therapeutics. Gleevec® specifically inhibits Bcr-Abl tyrosine kinase. In chronic myelogenous leukemia (CML), chromosomal translocation results in constitutive activation of Bcr-Abl tyrosine kinase, which in turn leads to activation of cell signaling that promotes cancer growth. Retinoic acid is a hormone involved in cell differentiation and, together with arsenic trioxide (As2O3), is a suitable therapeutic agent for APL.

  However, the development of resistance is a major problem with drugs that target a single attack point, such as a kinase, and the conformation specific binding properties of the drug may change. When tolerance increases in leukemia patients, clinicians are forced to rely on traditional combination drug therapies, which combine tumor-specific drugs to create a synergistic effect of drug activity and create non-specific drugs. Allows for use at lower doses and explores other effective treatments.

  The present invention relates to compositions and methods of use of cytotoxic peptides that kill cancer, particularly leukemia and / or ovarian cancer. One aspect of the invention is an isolated peptide capable of killing cancer cells comprising a caspase mobilization (CARD) -like domain. Isolated peptides may be derived from bacteria, in particular Pseudomonas aeruginosa. In some embodiments, the isolated peptide is Pa-CARD. In other embodiments, the isolated peptide comprises or consists of SEQ ID NO: 27. In some embodiments, the isolated peptide can kill leukemia cells, fibrosarcoma cells, ovarian cancer cells, and / or breast cancer cells. In other embodiments, the isolated peptide is chemically modified to increase or optimize its blood half-life.

  One or more proteins selected from the group consisting of isolated peptides capable of killing cancer cells, comprising a caspase mobilization (CARD) -like domain, Laz, H8-Azu, and Azu-H8 It also relates to a method of killing cancer cells by contacting cells with cells. In some embodiments, the cancer cells are selected from the group consisting of leukemia cells, fibrosarcoma cells, ovarian cancer cells, and breast cancer cells. The method may further comprise contacting the cell with one or more cytotoxic agents capable of killing cancer cells. These cytotoxic agents may be, but are not limited to, cisplatin, Gleevec®, retinoic acid, 5'-aza-2'-deoxycytidine, and arsenic trioxide. In certain embodiments, the method comprises contacting a cancer cell with cisplatin. In another embodiment, the cancer cell is contacted with the one or more cytotoxic agents at about the same time as the one or more proteins.

  The present invention comprises a group of isolated peptides capable of killing cancer cells comprising a caspase mobilization (CARD) -like domain, Laz, H8-Azu, and Azu-H8 in a mammalian patient suffering from cancer. Also relates to a method comprising administering one or more proteins selected from. In some embodiments, the cancer is selected from the group consisting of leukemia, fibrosarcoma, ovarian cancer, and breast cancer. In further embodiments, one or more cytotoxic agents capable of killing cancer cells can be administered to the patient. Cytotoxic agents may include, but are not limited to, cisplatin, Gleevec®, retinoic acid, 5'-aza-2'-deoxycytidine, and arsenic trioxide. In a further embodiment, the additional cytotoxic agent administered to the patient is cisplatin. In another embodiment, the one or more cytotoxic agents are administered at about the same time as the one or more proteins.

  The present invention relates to azurin and Laz H. et al. It also relates to a method comprising killing leukemia cells by contacting the cells with a peptide comprising 8 regions. In a further embodiment, the leukemic cells are azurin and Laz H. Contact with the peptide containing the 8 regions simultaneously or nearly simultaneously.

  The present invention provides a method for treating azurin and Laz H. It also relates to a method comprising administering a peptide comprising 8 regions. In further embodiments, azurin and Laz H. et al. The peptide comprising the 8 regions is administered to the patient simultaneously or nearly simultaneously.

  The present invention relates to Laz, azurin, H. et al. 8-Azu, Azu-H. By contacting the leukemic cells with one or more proteins selected from the group consisting of isolated peptides capable of killing cancer cells, comprising 8, and a caspase mobilization (CARD) -like domain It also relates to a method comprising inducing cell differentiation.

  The present invention relates to Laz, azurin, H. et al. 8-Azu, Azu-H. By contacting the cancer cell with one or more proteins selected from the group consisting of 8 and an isolated peptide capable of killing the cancer cell comprising a caspase mobilization (CARD) -like domain It also relates to a method comprising selectively entering, wherein the cancer cells are selected from the group consisting of leukemia cells and ovarian cancer cells.

  The present invention relates to Laz, azurin, H. et al. 8-Azu, Azu-H. And contacting the cancer cell with one or more proteins selected from the group consisting of isolated peptides capable of killing the cancer cell, comprising a caspase mobilization (CARD) -like domain. It also relates to a method comprising inducing cell cycle arrest. The cancer cell may be selected from the group consisting of leukemia cells, fibrosarcoma cells, breast cancer cells, and ovarian cancer cells. In further embodiments, the protein increases cellular Weel protein levels. In another embodiment, the protein causes depletion of phosphorylated AKT-Ser-473. In yet another embodiment, the protein increases cellular Weel protein levels and causes depletion of phosphorylated AKT-Ser-473.

  The present invention comprises inducing apoptosis of cancer cells by caspase 3 activation by contacting the cancer cells with an isolated peptide capable of killing the cancer cells comprising a caspase mobilization (CARD) -like domain. Also related to the method. In a further embodiment, the cancer cell is an ovarian cancer cell.

  The present invention regulates the expression of NF-kB signaling pathway genes in cancer cells by contacting the cancer cells with an isolated peptide containing a caspase mobilization (CARD) -like domain that can kill the cancer cells. It also relates to a method involving the above. In a further embodiment, the cancer cell is an ovarian cancer cell.

  The present invention also relates to an expression vector encoding an isolated peptide capable of killing cancer cells comprising a caspase mobilization (CARD) -like domain. In certain embodiments, the expression vector encodes Pa-CARD.

  The invention also relates to a pharmaceutical composition comprising an isolated peptide capable of killing cancer cells comprising a caspase mobilization (CARD) -like domain. In a further embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition is azurin, Laz, H. et al. 8-Azu, and Azu-H. A protein selected from the group consisting of 8; In another embodiment, the pharmaceutical composition further comprises one or more cytotoxic agents capable of killing cancer cells. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier suitable for intravenous injection.

  The present invention also relates to a method comprising administering to a patient suffering from leukemia one or more of the pharmaceutical compositions disclosed in the present invention in a therapeutically effective amount. In further embodiments, the pharmaceutical composition is administered to the patient in a manner selected from the group consisting of intravenous, topical, subcutaneous, intramuscular, oral, and intratumoral.

  Another aspect of the invention is a nucleic acid molecule that encodes one or more of the isolated peptides disclosed herein.

  Another aspect of the present invention is a kit comprising one or more of the pharmaceutical compositions described herein.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 Neisseria gonorrhoeae (Neisseria gonorrhoeae) laz gene in the genome DNA coding sequence, a Genbank accession number Y00530 (ctggcaggct tgacgcttcg atacgctctg tttcggtcag gctggtcccg aaaccggaaa aaccgccgaa aaccaatacc ctgcatttga gtaaggctgc gctggagagt ttcggttcgg cggcggcaaa gttggaaaaa cggcatcccg aattggcgga ggcattggca aacttggtta gaaggcatgg ggcataaaaat gttaacgggga atttgtgtaa acatccgtta atttatag aa gtaaaggata atgggtctaa tactaaagaa ataggttcgg ggtaaaattg ccccttttaa agtaaacgat tgtaaacttg cagacaggct ttgatttcaa atgaaatttg tagcaaaatg ccgccccgaa acatctgttt gtgcaacgcg gcggaatctt tttcaaggtt ttgttaatgg cggttgcact ttgatttctg taaaaccgaa tattatttta tcgattggag atttaccatg aaagcttatc tggctctgat ttctgccgcc gttatcggtt tggctgcctg ctctcaagaa cctgccgcgc ctgctgccga gcaactcct gctgctgaag cacccgcttc cgaagcgcct gccgccgaag ctgctcctgc agatgctgcc gaagcccctg ctgccggcaa ttgtgcggca actgtcgaat ccaacgacaa tatgcagttc aacaccaaag acatccaagt cagcaaagca tgtaaagagt ttaccatcac tctgaaacat accggtacgc aacccaaagc cagcatgggt cacaaccttg tgattgccaa agctgaagac atggacggcg tatttaaaga cggcgtaggt gctgccgata ccgactatgt caaacctgac gatgcgcgcg ttg ttgccca caccaaactg atcggcggcg gcgaagagtc ttccctgact ctggatcctg ccaaattggc tgacggcgac tacaaatttg cctgcacttt cccgggtcac ggtgctttga tgaacggcaa agtgactttg gtcgattaat ccgcttaaag tctcaaaaga cggacagcct gctttgtgca ggctgtttta ttataaaatg actgcttgaa aagtgccccg ttgagaacga aaacatgaat ccgtttgaaa).

  SEQ ID NO: 2 is a genomic DNA coding sequence of Pseudomonas aeruginosa azurin gene (ctttttcatg cagcggatcg ctcgcgcatc acttcagggt cagggtgccc ttcatcagcg cggagtggcc cgggaaggtg cagaagaaca tgtactgctc gccttccttc agcttggaga cgtcgaaggt caccgagtcc ttctcgcccg agccgatcag cttggtgtgg gcgatgacac ggctgtcgtc gggcttcagg taatccttgt ccaggccgga agccatgccg tcggtgacca cgccctgcat gtcggcggcg gtgctcagta c cagttgtg gcccatgacg ttcttcggca ggttgccggg gtgggacagg ttgacggtga actgcttgca gctcttgtcg acggtgatgg cattggtgtt gaactgcatc tggtcgttac cctggatgtc caccgagcac tcggcagcca gcagtggcgc actgagcagg gacagcaggg ataccgcagc gagtttacgt agcatggagc agcctcctag gcaggttggg cgatgaatcc tgaaagagca gactgcccga tcgggcaccg).

  SEQ ID NO: 3 is the H. coli of Neisseria gonorrhoe laz gene. 8 region genomic DNA coding sequence (tgctctcaag aacctgccgc gcctgctgcc gagcaactc ctgccggtga agaccccgct

  SEQ ID NO: 4 is a forward primer for PCR amplification of the Neisseria gonorrhoeae Laz coding gene (laz) (ccggatattcc ggcagggagt ttgtataatat ccg).

  SEQ ID NO: 5 is a reverse primer for PCR amplification of Neisseria gonorrhoeae Laz coding gene (laz) (ggggtaccgc cgtggcaggc atacagcatt tcaatcgg).

  SEQ ID NO: 6 is a forward primer for PCR amplification of the 3.1 kb fragment of pUC18-laz (ggcaggcagg gctttcggcag catctgc).

  SEQ ID NO: 7 is a reverse primer for PCR amplification of the 3.1 kb fragment of pUC18-laz (ctgcaggctg actcttagagg atccccg).

  SEQ ID NO: 8 is a forward primer for PCR amplification of a 0.4 kb fragment of pUC19-paz (gccgagtgct cgggtggacacat ccgg).

  SEQ ID NO: 9 is a reverse primer for PCR amplification of a 0.4 kb fragment of pUC19-paz (tactcgagtc actcagggt cagggtg).

  SEQ ID NO: 10 is a forward primer for PCR amplification of the 3.3 kb fragment of pUC19-paz (cttcagggtc agggtgccct tcatc).

  SEQ ID NO: 11 is a reverse primer for PCR amplification of the 3.3 kb fragment of pUC19-paz (ctgcagggtg actcttagag atccccg).

  SEQ ID NO: 12 is a forward primer for PCR amplification of the 0.13 kb fragment of pUC18-laz (tgctctcaag aacctgccgc gcctgc).

  SEQ ID NO: 13 is a reverse primer for PCR amplification of a 0.13 kb fragment of pUC18-laz (taggacccctt aggcaggcagg ggctttggggca gcatctgc).

  SEQ ID NO: 14 is a forward primer for PCR amplification of the GST-encoding gene from pGEX-5X-3 (cgagctcatg tcccctatac tagttattgg g).

  SEQ ID NO: 15 is a reverse primer for PCR amplification of the GST-encoding gene from pGEX-5X-3 (cccagagtttt cagggcccc cacgaccttc gatagagacc).

  SEQ ID NO: 16 is a signal peptide of pUC18-laz to laz and H.264. It is a forward primer for PCR amplification of the 8 coding region (ggaattcat tgaaagcttta tctggc).

  SEQ ID NO: 17 is a signal peptide from pUC18-laz to laz and H.264. Reverse primer for PCR amplification of the 8 coding region (ccggatattcg gcaggcagggg cttcggc).

  SEQ ID NO: 18 is pUC18-laz to H.P. Forward primer for PCR amplification of the 8 coding region (cgggatcccc tgctctcaag aacctgccgc gee).

  SEQ ID NO: 19 is from pUC18-laz to H.P. Reverse primer for PCR amplification of the 8 coding region (cgaattttt aggcaggcagg ggctttcggca gcatctgcagg).

  SEQ ID NO: 20 is pGEX-5X-3-H. 8 to GST-H. It is a forward primer for PCR amplification of the 8 fusion region (cgagctcatg tcccctatac taggtattgg g).

  SEQ ID NO: 21 is pGEX-5X-3-H. 8 to GST-H. Reverse primer for PCR amplification of the 8 fusion region (ccgctcgagt caggcagcagg gggctttgggg ag).

  SEQ ID NO: 22 is the amino acid sequence of the Neisseria gonorrhoeae strain F62Laz protein, Genbank accession number Y00530 (Cys Ser Gln Glu Pro Ala Ala Ala A Ala Pro Ala Asp Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys Ala Ser Met Gly His Asn Leu Val Ile Ala Lys Ala Glu Asp Met Asp Gly Val Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Asp Tyr Lys Phe Ala Cys Thr Phe Pro Gly ).

  SEQ ID NO: 23 is the amino acid sequence of Pseudomonas aeruginosaazurin (Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp Gln Met Gln Phe Asn Thr AsL Asl Ser His Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val Thr Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr Met Phe Phe Cys Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr Leu Thr Leu Lys).

  SEQ ID NO: 24 is a H. coli derived from Neisseria gonorrhoeae strain F62Laz protein. It is the amino acid sequence of 8 regions (Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Gly Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala

  SEQ ID NO: 25 is the amino acid sequence of a peptapeptide motif (Ala Ala Glu Ala Pro).

  SEQ ID NO: 26 is P.I. It is the amino acid sequence of Elginosa arginine deiminase (ADI). Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val Asn Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu Arg Gly Ile Asp Val Leu Glu Met His Asn Leu Leu Thr Glu Thr Ile Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Ala Asp Ser Val Gly Leu Gly Leu Thr Ser Glu Leu Arg Ser Trp Leu Glu Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu Ile Gly Gly Val Ala Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn Ile Leu Lys Met Tyr Arg Glu Tyr Leu Gly His Ser Phe Leu Leu Pro Pro Leu Pro Thn Thr Thr Thr Thr Gly Val Thr Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu Ile Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu Gly Gly Asp Val Met Pro Ile Gly Asn Gly Val Val Leu Ile Gly Met Gly Glu Arg Ser Ser Ser Arg Gln Ala Ile Gly Gly Gly Val Ile Val Ala Gly Leu Pro Lys Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg Asp Leu Ile Val Pro Phe Ser Leu Arg Pro Asp Pro Ser Ser Pro Tyr Gly Met Asn Ile Arg Arg Glu Glu Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu Lys Lys Leu Arg Val Val Glu Thr Gly Asn Ser Phe Ala Glu Arg Glu Arg Pro Gly Val Val Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala Gly Val Glu Val Ile Thr Ile Ser Ala Ser Glu Leu Gly Arg Gly Arg Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asp Tyr).

  SEQ ID NO: 27 is P.I. Elginosa ADI CARD region, amino acid sequence of residues 75 to 225 (Leu Leu Thr Glu Thr Ile Gln As Pro Arg Ser Trp Leu Glu Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu Ile Gly Gly Val Ala Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn Ile Leu Lys Met Tyr Arg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu ro Pro Leu Pro Asn Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu Ile Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu Gly).

  SEQ ID NO: 28 is P.I. It is a nucleotide sequence of a forward primer for PCR amplification of the CARD motif of the gene of Aeruginosa ADI (ATGCAACATC TGCTGACCGA GACCATCCAG).

  SEQ ID NO: 29 is a P.I. It is the nucleotide sequence of the reverse primer for PCR amplification of the CARD motif of the gene of Elginosa ADI (CAGGTCGAGG AGCCGTGGTC CTTGTC).

FIG. 1 is a schematic diagram of laz (A) derived from Neisseria gonorrhoeae and paz (B) derived from Pseudomonas aeruginosa. P. for cloning and high expression in E. coli. The Elginosa azurin gene was composed of the azurin gene itself (paz) and a signal peptide (psp) sequence that determines its periplasmic location (B). laz H. 8 region is the 5 ′ end (pUC18-H.8-paz) (C) of the paz gene containing the Neisserial signal sequence nsp, or the 3 ′ end of the paz gene (pUC19-paz-H.8) (D ) In-frame cloning. The detailed procedure for preparing the construct is shown in Example 1. azurin-like sequence of Neisseria gonorrhoeae present in naz, laz gene; nsp, Neisseria signal peptide sequence. In either case, the signal peptide sequence is cleaved to produce mature Paz (periplasm) and Laz (surface exposed) proteins. (E) SDS-PAGE of Laz, Paz, and fusion proteins. Laz, H.M. 8-Paz, or Paz-H. H. 8 etc. The anomalous migration of the 8 fusion proteins (all about 17 kDa) was performed by rapid H. 8 containing proteins have been previously described (Cannon, Clin. Microbiol. Rev. 2: S1-S4 (1989); Fisete et al., J. Biol. Chem. 278: 46252-46260 (2003) )). FIG. FIG. 6 is a graph illustrating the degree to which 8-Paz fusion protein is cytotoxic to various cancer cells. (A) Synthetic H. pylori for glioblastoma LN-229 cells. 8 peptides, Paz, Laz, and H. at the carboxy terminus of Paz (Paz-H.8) and the amino terminus of Paz (H.8-Paz). Cytotoxicity of 8 fusions. Cells were treated with three different concentrations (10, 20, and 40 μM) of protein for 6, 12, and 24 hours. An MTT assay was performed to determine the extent of viable cells that was the basis for cytotoxicity (percent cell death). To calculate the percent cytotoxicity, the value of untreated viable cells was taken as 100% and Paz, Laz, and H.P. The number of viable cells in samples treated with 8-fusion protein was determined. The degree of cytotoxicity (%) was then determined from the number of dead cells. (B) H. human breast cancer MCF-7 cells. 8 peptides, Paz, Paz-H. 8, H. 8-Paz and Laz cytotoxicity. All the processing conditions are the same as the above (A). FIG. 3 shows the entry of various fluorescently labeled azurin-related proteins into glioblastoma LN-229 and breast cancer MCF-7 cells. (A) H. aurea to which Alexa fluor (registered trademark) 568 is bound. 8 peptides, Paz, Paz-H. 8, H. 8-Paz and Laz (20 μM each) were incubated with LN-229 cells on a cover glass at 37 ° C. for 30 minutes, after which images were taken. (B) Internalization of various Alexa fluor® 568 binding proteins described in (A) into MCF-7 cells, visualized with a confocal microscope. (C) Laz internalization was visualized with a confocal microscope. Various concentrations (2, 4, 8, and 16 μM) of fluorescently labeled Laz were incubated with LN-229 cells at 37 ° C. for 30 minutes. The nucleus is blue labeled with DAPI (4,6-diamidino-2-phenylindole). (D) Alexa fluor® 568 conjugated Laz (10 μM) was incubated with LN-229 cells for various periods (5, 10, 20, and 30 minutes) at 37 ° C. Internalization was visualized with a confocal microscope. (E) Alexa fluor® 568-conjugated Paz (10 μM) was incubated with LN-229 cells on a cover glass at 37 ° C. for various times, after which images were taken. Little measurable fluorescence was detected (E). FIG. 4 is a bar graph showing the quantification of fluorescence found in the confocal microscope images of FIGS. (A) Quantification of fluorescence in the image of FIG. 3A. Quantification of azurin protein fluorescence was performed by using Adobe (R) Photoshop (R). Error bars represent the standard deviation of the fluorescence of three different cells in a single sample. (B) Quantification of fluorescence in the image of FIG. 3B. Quantification was performed as in FIG. 4A. (C) Quantification of fluorescence in the image of FIG. 3C. Quantification was performed as in FIG. 4A. (D) Quantification of fluorescence in the image of FIG. 3D. Quantification was performed as in FIG. 4A. FIG. 5 is a diagram showing an image of the labeled fusion protein in the cell, and a graph of uptake and cytotoxicity, respectively. H. The combined treatment with 8-GST fusion protein facilitates the uptake of Alexa fluor® 568-labeled Paz into glioblastoma LN-229 cells. Unlabeled 20 μM (A) H. 8, (B) GST, (C) GST-H. 8, (D) H. 8-GST, (E) PBS buffer, and 20 μM Alexa fluor® 568 conjugated Paz were incubated with LN-229 cells at 37 ° C. for 30 minutes. Internalization was visualized with a confocal microscope. (F) Synthesis H. in the presence or absence of Paz. 8 peptides, GST, and GST-H. 8 / H. Cytotoxicity of 8-GST fusion derivatives. Approximately 5 × 10 ³ LN-229 cells were seeded in 96-well culture plates and each with 20 μM H.P. with (+ Paz) or without (−Paz) 20 μM Paz. 8 peptide, GST, GST-H. 8, H. Treated with 8-GST or the same volume of PBS buffer for 24 hours. FIG. 6 shows Paz, H. et al. With IRdye® 800CW (LI-COR Biotechnology, Lincoln, Nebra) bound. It is a brain image of the mouse | mouth which injected 8-Paz and Laz. (A) Brain image of a living mouse. 500 μg Paz bound to IRdye® 800 CW, H.P. 8-Paz and Laz were injected intraperitoneally into living nude mice. After 24 hours, the mice were sacrificed, the brains were removed, and fluorescence was detected and measured with a LI-COR Odyssey® infrared imaging system. (B) An image of the head midbrain region of the nude mouse brain processed as in (A). The mouse brain was cut horizontally and images were taken. FIG. 7 shows H. coli in E. coli. SDS-PAGE, Western blot and confocal microscopy images for localization of 8-Gst fusion protein. (A) Cloned gst, H. 8-gst, or gst-H. E. coli BL21 (DE3) cells with 8 genes were cultured at 37 ° C. with 0.1 mM IPTG. The cell pellet was washed twice with PBS and the whole cell lysate was run on SDS-PAGE. Coomassie blue staining was used for protein detection. (B) The above procedure was repeated, but this time, the whole cell lysate and the contents of the periplasmic space were both isolated separately, electrophoresed by SDS-PAGE (20 μg protein), and GST or GST-H. Eight fusion proteins were detected by Western blot using a monoclonal anti-GST antibody to determine total protein and periplasmic concentrations. (C) Cloned gst, H. 8-gst, or gst-H. E. coli strain BL21 (DE) cells carrying 8 genes were cultured at 37 ° C. with 0.4 mM IPTG (Table 2). 1 ml of each of these bacterial cultures was centrifuged and the resulting bacterial pellet was collected. After washing twice with PBS, 1 ml of 1% FBS-PBS containing anti-GST antibody (1: 2000) was added. The cell suspension was incubated for 1 hour and then washed twice with PBS. Bacterial cells were incubated with FITC-conjugated anti-rabbit IgG for 30 minutes in 1% FBS-PBS. Cells were washed again to remove unbound antibody and fixed with ethanol on ice. E. coli samples treated with DAPI (providing blue coloration) were observed with a confocal microscope (× 100 objective lens), and single cells were also photographed. (D) pUC19-paz (P. aeruginosaazurin), pUC19-laz (Nicelia), pUC18-H. 8-paz, or pUC18-paz-H. E. coli cells harboring 8 were cultured overnight in the presence of 0.1 mM IPTG at 37 ° C. 0.5 ml of such culture was centrifuged and the resulting bacterial pellet was washed twice with cold PBS. Anti-azurin antibody (1: 500) in 1 ml 1% FBS-PBS was added and incubated for 1 hour on ice. After washing twice with PBS, FITC-conjugated anti-rabbit antibody was added, incubated on ice for 30 minutes, washed twice with PBS, and fixed with cold ethanol. Bacterial samples were observed with a confocal microscope (x100 objective). FIG. 8 is a graph showing cytotoxicity determined by MTT assay. (A and B) Cytotoxicity of HL60 cells (A) and K562 cells (B) treated with 10 μM Laz, azurin (Azu), H8-Azu, and Azu-H8, or 5 μM DAC as a positive control. Determined after 24, 48, and 72 hours in the MTT assay. (C) Low concentration: K562 cells treated with 3.75 nM, 37.5 nM, 75 nM, 2.5 μM, 5 μM, and 10 μM Laz, Azu, H8-Azu, Azu-H8, or 5 μM DAC as a positive control. . (D) Cytotoxicity determined by MTT assay of HL60 cells treated with various concentrations, 1 μM, 2.5 μM, 5 μM, and 10 μM Laz, Azu, H8-Azu, Azu-H8, or 5 μM DAC. Data are expressed as the mean (± standard error) of three separate experiments performed in triplicate. All numbers are significantly different from controls (p <0.05). FIG. 9 is a fluorescence microscopic image of cell shape changes induced by Laz and azurin in HL60 and K562 cells. Laz induces morphological changes in HL60 and K562 cells, as shown by fluorescence microscopy. (A) and (B) represent untreated HL60 cells and K562 cells. HL60 cells (C) or K562 cells (D) treated with 10 μM Laz for 48 hours undergo differentiation, which is a process that subsequently leads to cell apoptosis. Arrows point to differentiated granulomatous cells. Cytotoxicity was high, but fewer morphological changes were seen in HL60 cells (E) or K562 cells (F) treated with 10 μM azurin for 48 hours. FIG. 10 is a confocal microscope image showing the selective entry of Laz and azurin into HL60 and K562 cells. Panel 1: Both azurin and Laz do not enter normal peripheral blood mononuclear cells (PBMC) after 1 hour incubation at 10 μM concentration (A and B). Azurin, like Laz (C), enters K562 cells after 1 hour incubation (D). Azurin, like Laz (E), enters HL60 cells after 1 hour incubation (F). Panel 2: Compared to control K562 cells, Azu-H8, H8-Azu enters such K562 cells, similar to Laz. FIG. 11 is a graph that charts cell cycle progression in the absence and presence of azurin, Laz, and DAC. K562 cells were treated with 10 μM Laz, azurin, and 5 μM DAC for 48 hours as previously described, fixed, stained, and analyzed for DNA content. FIG. 12 is an image of an immunoblot. This figure shows the phosphorylation status of AKT (AKT-P-Ser473) and Weel protein by immunoblotting of proteins in Laz and Pa-CARD treated K562 and HL-60 cells in cytoplasmic and nuclear extracts Provide level analysis. Cytoplasmic proteins and nuclei comparing levels of Weel and phospho-AKT serine 473 from HL60 and K562 cells after 48 hours treatment with 10 μM Laz or 10 μM Pa-CARD compared to untreated cell controls Western blot analysis of the protein is shown. FIG. 13 shows P. cerevisiae that exhibits cytotoxic activity against leukemia cells. FIG. 2 is a schematic diagram of the CARD domain of ADI from Aeruginosa. (A) Structure-based sequence alignments indicate that CARD domains are present in Pa-ADI and Ma-ADI but not in other members of the superfamily enzymes that modify guanidino groups such as DDAH and AGAT . The CARD domain is a mammalian caspase-9. DDAH, dimethylarginine dimethylaminohydrolase; AGAT, arginine: glycine amidinotransferase CARD domain. The β-strand and α-helix are shown as arrows and elongated bands, respectively. The Roman numerals I to V are five ββαβ subunits. The clip portion (α-helix module) retains structural homology to Pa-ADI and the 6α-helix module that forms the CARD domain of caspase-9 protein molecules. Found in M. arginiini ADI (Ma-ADI). N, N-terminal; C, C-terminal. (B) P.I. SDS-PAGE gel run of purified 46 kDa ADI and 17 kDa CARD protein from Aeruginosa. (C) Cytotoxic activity of Pa-ADI and Pa-CARD against fibrosarcoma (HT-1080), breast cancer (MCF-7), and leukemia (HL60) cells. Cells were incubated with 10 μM Pa-ADI or Pa-CARD for 48 hours, after which cell viability was determined by MTT assay. FIG. 14 is an image showing structural features of arginine deiminase (ADI) enzymes derived from Mycoplasma arginini and Pseudomonas aeruginosa. (A) P.I. Ribbon diagram of Elginosa ADI (1RXX_A) with two domains highlighted (MolMol program). (B) Here is a copy of the diagram shown in FIG. 13A. (C) The described P.I. It is a schematic diagram which shows the whole folding structure and topology of an Elginosa ADI (Pa-ADI). FIG. 15 is a gel image and graph showing purification of Pa-ADI and Pa-CARD and arginine deiminase enzyme activity. (A) P.I. SDS-PAGE gel migration of purified ADI and CARD proteins from Aeruginosa. (B) Kinetics of ADI activity of purified Pa-ADI and Pa-CARD at 37 ° C. and pH 7.2. Citrulline, the product of the enzyme reaction, was measured at 490 nm. The arginine deiminase enzyme activity of Pa-CARD was not detected. FIG. 16 is a graph showing that Pa-CARD has higher cytotoxicity than Pa-ADI for a range of cancer cells. (A) Fibrosarcoma (HT-1080), breast cancer (MCF-7), and ovarian cancer (SKOV-3) cells were incubated with 20 μM Pa-ADI or Pa-CARD for 48 hours, after which cell viability was measured by MTT. Determined by assay. The average number of live cells per well (± standard error) was determined in triplicate cultures from three experiments. All numbers are significantly different from controls (p <0.05). (B) Dose-dependent cytotoxicity against ovarian cancer SKOV-3 cells as shown by Pa-CARD. Expressed as a function of time (24, 48, and 72 hours). (C) The same experiment was performed using Pa-ADI. The average number of live cells per well (± standard error) was determined in triplicate cultures from 3 experiments as described in A (p <0.05). FIG. 17 is a graph showing the comparative cytotoxic effect of Pa-CARD, azurin, and cisplatin in ovarian cancer SKOV-3 cells. (A) All 20 μM Pa-ADI, Pa-CARD, azurin, and cisplatin were incubated with either SKOV-3 cells or normal ovarian HOSE6-3 cells for 48 hours, after which cell viability was determined by MTT assay. . (B) Pa-CARD shows some additive effect with cisplatin compared to individual treatments with SKOV-3 cells. FIG. 18 is an image showing that Pa-CARD induces apoptosis in SKOV-3 cells by caspase activation but not in HOSE6-3 cells. A TUNEL assay was used to detect apoptosis-induced DNA strand breaks by measuring the incorporation of fluorescein-tagged dUTP using an in situ cell death detection fluorescein kit (Roche). SKOV-3 and HOSE6-3 cells were grown on 8-well Lab Tek chamber slides and incubated with 10 μM Pa-CARD, Pa-ADI, and azurin for 48 hours. An untreated negative control or a BSA treated negative control was also maintained in parallel (bottom row). Cell nuclei were stained blue with DAPI. The cells observed under the green (A) and blue (B) channels, as well as the overlay image (C) (cyan) are shown. FIG. 19 is a graph showing caspase activation by Pa-CARD. Caspase activity was measured with a caspase-Glo ™ 3/7 assay kit purchased from Promega. Pa-ADI and Pa-CARD were used at two different concentrations to assess dose response effects. The increase in caspase activity is expressed as a fold increase compared to controls without drug treatment.

Definitions As used herein, the term “cell” includes both the singular or plural of the term unless specifically stated as “single cell”.

  As used herein, the term “cytotoxic peptide” means a peptide of the invention that is selectively cytotoxic to cancer cells, particularly leukemia cells, but not normal cells.

  As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably and refer to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of the corresponding natural amino acid. This term also applies to natural amino acid polymers. The terms “polypeptide”, “peptide”, and “protein” include, but are not limited to, glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. Modifications are also included. It will be appreciated that polypeptides are not necessarily completely linear. For example, a polypeptide may be branched as a result of ubiquitination, generally circular (as a result of post-translational events, including those resulting from natural processing events and non-naturally occurring artificial manipulations. There are cases where there is a branch or no branch. Cyclic polypeptides, branched polypeptides, and branched cyclic polypeptides may be synthesized by non-translatable natural processes, as well as by fully synthetic methods. A synthetic peptide is a peptide produced without the assistance of cellular components. Synthetic methods for producing peptides are well known in the art and are commercially available. Furthermore, the present invention contemplates the use of both methionine-containing amino terminal variants and methionine-free amino terminal variants of the proteins of the invention.

  As used herein, the term “symptoms” refers to the anatomical and physiological from normality that constitutes a normal state disorder in a living animal or part thereof and disrupts or alters the performance of physical functions. Including technical deviations.

  As used herein, the term “inhibits cell proliferation” means delay or stop cell division and / or cell proliferation. The term also includes inhibition of cell development or increase in cell death.

  As used herein, the term “affected by” includes presently showing signs of symptoms, and there are no significant signs of symptoms during and upon recovery from symptoms Including even cases.

  As used herein, the term “treatment” includes preventing, reducing, stopping, or reversing the progression or severity of symptoms or signs associated with the condition being treated. Thus, the term “treatment” includes medical, therapeutic and / or prophylactic administration, as appropriate. Treatment can also include preventing or reducing the onset of precancerous lesions or symptoms such as cancer.

  A “therapeutically effective amount” prevents, reduces, halts, or reverses the onset of a particular symptom of a subject being treated, or partially describes an existing indication of a particular symptom of a subject being treated An amount effective to alleviate the whole or all. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

  The term “substantially pure” as used herein, when used to modify a protein or other cellular product of the invention, eg, other protein and / or activity inhibition. Refers to proteins isolated from growth media or cell contents in a form that is substantially free or contaminated with compounds. The term “substantially pure” refers to a numerical value of at least about 75% of the isolated fraction or at least “75% substantially pure” in dry weight, more specifically, the term “ “Substantially pure” refers to a compound of the active compound that is at least about 85% or at least “85% substantially pure” by dry weight. Most specifically, the term “substantially pure” refers to a compound of the active compound that is at least about 95% or at least “95% substantially pure” by dry weight. The term “substantially pure” is used, for example, to modify a synthetically produced protein or compound of the invention when the synthetic protein is isolated from reagents and byproducts of the synthesis reaction (pluralization). You can also

  The term “pharmaceutical grade” as used herein, when referring to a peptide or compound of the invention, substantially consists of components normally associated with the substance found in its natural state, including synthetic reagents and by-products. A peptide or compound that has been isolated or essentially isolated and is substantially or essentially isolated from a component that may impair its use as a medicament. For example, “pharmaceutical grade” peptides may be isolated from any carcinogen. In some cases, “pharmaceutical grade” designates a peptide or compound that is substantially or essentially isolated from any substance that may render the composition unsuitable for intravenous administration to a patient. Therefore, it may be modified by the intended method of administration, such as “venous pharmaceutical grade”. For example, “intravenous pharmaceutical grade” peptides may be isolated from surfactants such as SDS, and antibacterial agents such as azides.

  The phrases “isolated”, “purified” or “biologically pure” are substantially or essentially free from components normally associated with materials found in their native state. Refers to a substance. Thus, isolated peptides according to the present invention preferably do not contain substances normally associated with peptides in their in situ environment. An “isolated” region refers to a region that does not include the entire sequence of the polypeptide from which the region is derived. An “isolated” nucleic acid, protein, or corresponding fragment thereof has been substantially removed from its in vivo environment, and thus is not limited to, but includes, nucleotide sequencing, restriction digests, designated site mutations Induction and subcloning into nucleic acid fragment expression vectors, and obtaining substantially pure amounts of protein or protein fragment may be manipulated by those skilled in the art.

  As used herein with respect to peptides, the term “variant” refers to an amino acid sequence variant that may have amino acid substitutions, deletions, or insertions compared to a wild-type polypeptide. The mutant may be a truncated form of the wild type peptide. “Deletion” is the removal of one or more amino acids from within the wild-type protein, and “cleavage” is the removal of one or more amino acids from one or more ends of the wild-type protein It is. Therefore, a mutant peptide can be produced by manipulating a gene encoding a polypeptide. Variants can be engineered to alter the basic composition or characteristics of a polypeptide but not at least some of its fundamental activities. For example, a “variant” of a Neisseria peptide or Pa-CARD peptide may be a mutant Neisseria peptide or Pa-CARD peptide that retains the ability to kill leukemia cells. In some cases, variant peptides are synthesized with unnatural amino acids such as ε- (3,5-dinitrobenzoyl) -Lys residues. (Ghadiri and Fernholz, J. Am. Chem. Soc, 112: 9633-9635 (1990)). In some embodiments, the variant has no more than 20, 19, 18, 17, or 16 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 15, 14, 13, 12, or 11 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 10, 9, 8, or 7 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has 6 substitutions, deletions, or insertions compared to the wild-type peptide. In some embodiments, the variant has no more than 5 or 4 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 3, 2, or 1 amino acid substitutions, deletions, or insertions compared to the wild-type peptide. In some embodiments, variants are generated using the techniques and methods described in US patent application Ser. No. 12 / 389,120, the entire disclosure of which is incorporated herein by reference.

  The term “amino acid” as used herein refers to an amino acid moiety comprising any natural or non-natural or synthetic amino acid residue, ie 1, 2, or 3 or more carbon atoms, typically By any moiety comprising at least one carboxyl residue and at least one amino residue linked directly by one (α) carbon atom. The term “residue” is synonymous with “amino acid”.

  The term “derivative” as used herein with respect to a peptide refers to a peptide derived from the subject peptide. Induction includes chemically modifying the peptide such that the peptide still retains some of its fundamental activity. For example, a “derivative” of a Neisseria peptide or Pa-CARD peptide may be a chemically modified Neisseria peptide or Pa-CARD peptide that retains its ability to kill leukemia cells and / or ovarian cancer cells. Good. Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, or glycosylation of peptides. In addition, a derivative peptide is a fusion of a polypeptide or fragment thereof with, but is not limited to, another peptide, drug molecule, or other therapeutic agent, or pharmaceutical, or a chemical compound such as a detectable probe. There may be. In some embodiments, derivatives are produced using the techniques and methods described in US patent application Ser. No. 12 / 389,120, the entire disclosure of which is incorporated herein by reference.

  The term “percent amino acid sequence identity (%)” is defined as the percentage of amino acid residues in a polypeptide that are identical to the amino acid residues of a candidate sequence when the two sequences are aligned. To determine% amino acid identity, sequences are aligned and, if necessary, gaps are introduced to achieve maximum% sequence identity, conservative substitutions are not considered part of sequence identity . Amino acid sequence alignment procedures for determining percent identity are well known to those of skill in the art. In many cases, peptide sequences are aligned using publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software. In a specific embodiment, Blastp (National Center for Biotechnology Information, Bethesda, MD) is used using the default parameters for long complexity filters, expect 10, word size 3, extent 11, and extension 1. The

When aligning amino acid sequences, the% amino acid sequence identity of a given amino acid sequence A relative to or relative to a given amino acid sequence B is (or alternatively to a given amino acid sequence B Can be rephrased as a given amino acid sequence A having or containing a certain% amino acid sequence identity compared to or with B), can be calculated as follows:
Amino acid sequence identity% = X / Y * 100
Where X is the number of amino acid residues scored as identity matches by alignment of the A and B sequence alignment programs or algorithms;
Y is the total number of amino acid residues of B.

  If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% A amino acid sequence identity to B will not be equal to the% B amino acid sequence identity to A. When comparing a longer sequence to a shorter sequence, the shorter sequence would be the “B” sequence. For example, if a truncated peptide is compared to the corresponding wild-type polypeptide, the truncated peptide will be the “B” sequence.

General The present invention relates to bacterial peptides that exhibit cytotoxic effects on cancer cells but not to normal cells, as well as compositions comprising variants, derivatives and structural equivalents of such peptides, and mammals. A method for preventing the onset of cancer, treating mammalian cancer, and killing mammalian cancer cells is provided. In particular, the compositions and methods disclosed herein can be used to prevent, treat, and / or kill leukemia and / or ovarian cancer.

  Many cupredoxin proteins, particularly Pseudomonas aeruginosaazurin, and their truncated forms have the ability to preferentially enter and kill many types of solid mammalian cancer cells, both in vivo and in vitro It has been shown. (Yamada et al., Cell. Biol. 7: 1418-1431 (2005); Hiraoka et al., PNAS 101: 6427-6432 (2004); Hiraoka et al., Biochem. Biophys. Res. Comm. 338: 1284-1290 (2005)). Also, U.S. Patent Nos. 7,491,394, 7,381,701, and 7,084,105, and U.S. Patent Application Nos. 11 / 488,693, 11 / 950,165, See 11 / 854,654 and 12 / 338,480. These disclosures are incorporated herein by reference in their entirety. Azurin-like genes include Neisseria gonorrhoeae and N. cerevisiae. It is present in many gonococci and meningococci such as N. meningitidis. (Gotschlich and Seiff, FEMS Microbiol. Lett. 43: 253-255 (1987); Kawula et al., Mol. Microbiol. 1: 179-185 (1987)). Azurin is produced by a number of pathogenic bacteria, and there is significant sequence homology among such genes (Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)).

  The protein epitope named “H.8” is conserved in pathogenic Neisseria species and Detected by the binding of a monoclonal antibody designated 8. Another Neisseria gonorrhoeae gene laz is Encodes a protein that cross-reacts with 8 monoclonal antibodies. (Hayashi and Wu, J. Bioenerg. Biomembr. 22: 451-471 (1990)).

  Laz is a gonococcal cell surface protein that contains the signal peptide lipoprotein consensus sequence recognized by the bacterial enzyme signal peptidase II, which processes this sequence to convert cysteine residues by fatty acids and glycerol. N-terminal acylation results. (Hayashi and Wu, ibid .; Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)). Laz lipoprotein is about 17 kDa and is an N-terminal 39 amino acid region containing an incomplete pentapeptide repeat of the motif Ala-Ala-Glu-Ala-Pro (AAEAP (SEQ ID NO: 25)). Includes 8 regions. (Gotschlich and Seiff, ibid .; Kawula et al., Ibid .; Woods et al., Mol. Microbiol. 3: 43-48 (1989)). The 39 amino acid N-terminal region of Laz is preceded by P.I. There is a 127 amino acid region that is highly homologous to Erginosaazurin. (Cannon, Clin. Microbiol. Rev. 2: S1-S4 (1989)). Laz is involved in protection against oxidative stress and copper toxicity and increases survival in an ex vivo primary human uterine vaginal epithelial assay. (Wu et al., Infect. Immun. 73: 8444-8448 (2005)).

  It is now possible that azurin-like proteins derived from Laz proteins, Neisseria gonorrhoeae and other Neisseria species can specifically enter and kill brain cancer cells such as glioblastoma cells and other tumors. It turns out. See Examples 2 and 7. In addition, H. of Laz protein. The 8 region, when fused to either its N-terminus or C-terminus, It has now been found that Elginosaazurin can be given the ability to enter and kill glioblastoma cells. See Examples 2 and 3.

  H. It has now also been found that the 8 region does not need to be physically associated with a co-administered protein such as azurin in order to confer to the protein the ability to enter glioblastoma cells. See Example 5. H. 8 and H. fused to the N-terminus of GST. Both 8 increased the entry of physically unbound azurin into glioblastoma cells as compared to azurin alone, but H. fused to the C-terminus of GST. 8 had no effect. Further, H.C. 8 and H. fused to the N-terminus of GST. Both 8 enhanced azurin cytotoxicity against glioblastoma cells when co-administered with azurin. See Example 5. It is also contemplated that this AAEAP (SEQ ID NO: 25) repeat unit can be used to design peptides that will kill leukemia cells.

  Azurin and laz are involved in multiple pathways of cancer progression, including induction of apoptosis through complex formation and stabilization of p53, inhibition of angiogenesis, and inhibition of cell signaling mediated by receptor tyrosine kinases such as EphB2 / EphrinB2. It is known to target this step, thereby minimizing the possibility of resistance development. See, eg, US Pat. No. 7,381,701, the entire disclosure of which is incorporated herein by reference. Azurin and Laz are known to exhibit entry specificity for melanoma or breast cancer cells compared to normal cells.

  Surprisingly, azurin and Laz proteins are fluid mediated such as leukemia, as demonstrated by the examples described herein, in addition to being vesicular toxic to solid types of cancer cells. It is also effective against cancer. In this example, it has been demonstrated that azurin and Laz can each enter a leukemia cell line where they can exhibit a cytotoxic effect. Furthermore, as discussed in Examples 9 and 11 and demonstrated in FIGS. 8-11, Laz exerts a cytotoxic effect on leukemia cell lines, but normal peripheral blood mononuclear cells (PBMCs). ) Has little effect and the approach is very limited. These bacterial proteins show entry specificity for leukemia cells but not entry specificity in normal PBMC (FIG. 10, panel 1).

  In addition to azurin and Laz, asparaginase and arginine deiminase (ADI) have been shown to be active against solid tumors including breast cancer, melanoma, renal cell carcinoma, hepatocellular carcinoma, etc., derived from Mycoplasma arginini ADI (Ma-ADI) has also been reported to inhibit the growth of human leukemia cells. (Yamada T et al., Proc Natl Acad Sci USA 2002; 99: 14098-103; Punj V et al., Oncogene 23: 2367-78 (2004); Ni Y et al., Cancer Lett. 261: 1- 11 (2008); Fialho AM, Chakrabarty AM, Anticancer Drug Discovery 2: 224-34 (2007); Ensor CM et al., Cancer Res 62: 5443-50 (2002); Yoon CY et al., Int. J Cancer 120: 897-905 (2007); Gong H et al., Leukemia 14: 826-9 (2000)). The catalytic triad of Ma-ADI is Cys398-His269-Glu213. However, there is only one report on the effect of Ma-ADI on leukemia cells. Apart from that, little has been known about the role of bacterial proteins such as ADI in possible therapeutic applications against leukemia. .

  Pseudomonas aeruginosa also has Cys406-His278-Asp166 in the catalytic triad and produces ADI (Pa-ADI) having primary sequence homology with Ma-ADI. By using a secondary structure matching (SSM) program, a caspase recruitment domain (“CARD”)-like domain is revealed in the N-terminal region of Pa-ADI (Protein Data Bank, PDB ID code 1RXX), which is a CARD domain. Identifiable structural homology to the caspase-9 prodomain (PDB ID code 3YGS) which retains As shown in FIG. 14D, among the members of the guanidine group superfamily, ADI is unique in that it retains the CARD domain. The CARD-like domain of the bacterial protein is the M. Other than the CARD-like domain of Arginini ADI (PDB ID code 1LXY), it is not known. Surprisingly, as disclosed herein, the CARD domain of Pa-ADI (Pa-CARD) has potent anticancer activity. Briefly, the CARD domain polypeptide exhibits significant anticancer activity but exhibits little cytotoxicity against normal cells.

  Mammalian CARD-containing proteins are usually composed of approximately 95 amino acid residues and have sequence similarity to the death domain (DD) and death effector domain (DED). The crystal structure of CARD suggests that CARD is composed of approximately six closely packed alpha-helices surrounding a hydrophobic pocket. Similarly, Pa-CARD is composed of 85 amino acid residues that form five packed alpha-helices that take the form of a “clip-on-fan” moiety (FIGS. 14B and 14C). The possibility that bacterial CARD contributes to the inhibition of mammalian CARD activity and thereby contributes to the anticancer activity of Pa-ADI containing a putative CARD-like domain because some mammalian CARD domains promote cancer growth It is interesting that there is. The Pa-CARD domain may be recruited from a mammalian CARD protein-bearing protein, and Pa-CARD is at least partly protein-protein reciprocal with a mammalian CARD protein known to be highly expressed in cancer cells. It is contemplated that the action can exert its cytotoxicity on cancer cells. It is further contemplated that this interaction may be responsible for the action of ADI in regulating cancer growth.

  Pa-CARD (SEQ ID NO: 27) exhibits unexpected cytotoxic activity against leukemia cells and ovarian cancer cells. In particular, Pa-CARD polypeptides have the ability to inhibit leukemia cell proliferation. In the leukemic cell lines HL60 and K562, the anticancer activity of Laz and Pa-CARD is phosphorylated AKT-Ser, an active form of serine / threonine kinase that is often dysregulated in Weel protein stabilization and many cancer types. Mediated by cell cycle arrest at G2 / M phase with -473 depletion. See Examples 11 and 12 and FIG. In particular, Pa-CARD and Laz inhibit the cell cycle of leukemia cells by up-regulation of Weel protein in K562 cells and down-regulation of active AKT P-Ser473 in HL60 cells (FIG. 12).

  Both Weel and phosphorylated AKT-Ser473 (AKT-P-Ser473) are known to be involved in cell cycle arrest in the G2 / M phase, which in turn is known to lead to cell death. For example, activation of the mitogenic kinase CDC2, also known as CDK1, is required for eukaryotic G2 to M phase transitions at Thr-14 and Tyr-15 residues. The phosphorylation of CDC2 is important. Inhibitory Tyr-15 phosphorylation is mediated by Weel protein kinase and, therefore, enhanced levels mediate inhibition of such M phase transitions. Viral proteins are known to mimic Laz / Pa-CARD-mediated Wee protein kinase levels. Human papillomavirus type 1 (HPV-1) E4 protein is activated in the cell cycle by forming an inactive cyclin B1-CDK1 complex by inhibitory Tyr-15 phosphorylation catalyzed by elevated levels of Weel. Inhibits the transition from G2 phase to M phase. Thus, overexpression of Weel enhances cell cycle arrest at G2 phase, while depletion of Wel by small interfering RNA (siRNA) mitigates E4 induced inhibition of G2 phase transition. Similarly, the human homologue of AKT (protein kinase B), the viral oncogene v-akt, plays a role in the phosphatidylinositol 3-kinase (PI3K) pathway. Phosphorylation at the Ser-473 residue of AKT-1 by PDK2 is important for cell cycle progression and, as revealed by both Laz and Pa-CARD in HL60 cells, phosphorylated Akt Ser-473 ( Reduction of Akt-P-S473) levels enhances G2 / M phase arrest.

  Pa-CARD shows an effect on SKOV-3 (ovarian cancer) cells by transcriptional stimulation of a gene encoding GM-CSF. Both GM-CSF and IL-12, whose genes are stimulated approximately 3-fold, are potent inducers of anti-tumor immunity in many tumor models, and are associated with granulocytes, macrophages, and dendritic cells at the tumor site. It is known to induce invasion and thereby greatly enhance tumor antigen presentation. Thus, it is believed that Pa-CARD induced apoptosis (FIG. 18) can significantly increase the anti-tumor response of any GM-CSF that is overproduced in the presence of Pa-CARD. The gene expression pattern showed stimulation of many cytokine genes, but the chemokine CCL2 (CC motif ligand 2) gene, also known as monocyte chemotactic protein-1 (MCP-1), is marked (17 times) suppression was shown (Table 3). CCL2 has been shown to promote malignant transformation of breast cancer, often by increasing tumor monocyte recruitment and harmful tumor-associated macrophages (TAM). CCL2 is also known to promote angiogenesis and metastasis, possibly by a novel transcription factor MCPIP that activates transcription of the cadherin genes cdhl2 and cdhl9. Therefore, reduction of CCL2 levels by Pa-CARD can allow tumor regression by interfering with angiogenesis and tumor growth progression.

  In summary, the examples and disclosures herein show that bacterial proteins such as azurin, Laz, and Pa-CARD can enter leukemia cells, the CDC2-cyclin B inhibitory pathway and the cellular PI3K / AKT pathway. We demonstrate that cell cycle arrest at G2 / M phase, which is thought to be triggered by modulation of cell signaling mediated by, can be induced. Furthermore, ADI can exhibit anticancer activity in several ways, including by enzymatic depletion of arginine and by an N-terminal putative CARD-like domain without ADI activity (FIGS. 14-16). Pa-cARD protein mainly shows an inhibitory role on cancer cells but not on normal cells (FIG. 18). Furthermore, anti-cancer activity may be mediated by caspase activity (FIG. 19) and can induce apoptosis in ovarian cancer cells.

Compositions of the invention The present invention relates to cytotoxic peptides and / or variants, derivatives, truncated forms, or structural equivalents of such peptides, alone or in combination with one or more other cytotoxic agents. To provide.

  In some embodiments, the cytotoxic peptide is cupredoxin or a truncated form thereof. In some embodiments, the cytotoxic peptide is azurin or a truncated form thereof. In other such embodiments, the cytotoxic peptide is Laz or a truncated form thereof. In other embodiments, the cytotoxic peptides are Laz H.P. There are 8 areas. In some embodiments, the cytotoxic peptide comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 22-24.

  In other embodiments, the cytotoxic peptide comprises a CARD domain. In some embodiments, the cytotoxic peptide is a CARD-bearing protein. In some embodiments, the CARD carrying protein is from a bacterium. In another embodiment, the bacterium is P. aeruginosa. Elginosa. In one embodiment, the cytotoxic peptide is Pa-CARD. In another such embodiment, the cytotoxic peptide comprises SEQ ID NO: 27.

  In some embodiments, the cytotoxic peptide is fused to another peptide that imparts a greater cytotoxic effect to it using techniques well known in the art. In one such embodiment, the other peptide is Laz's H.P. There are 8 areas. In another peptide, the other peptide has a sequence comprising SEQ ID NO: 24. In one embodiment, the cytotoxic peptide is a fusion protein Azu-H. 8. In another embodiment, the cytotoxic peptide is a fusion protein H.I. 8-Azu. FIG.

  One or more other cytotoxic agents that can be administered in combination with the cytotoxic peptides described herein include, but are not limited to, cisplatin, Gleevec®, retinoic acid, 5′-aza. It may be a therapeutic agent for cancer comprising -2'-deoxycytidine ("DAC") and / or a combination of arsenic trioxide and retinoic acid. Cancer therapeutics other than Gleevec®, retinoic acid, DAC, and / or a combination of arsenic trioxide and retinoic acid include the following: cell cycle control proteins such as p53; p16, p21, or cyclin-dependent kinase inhibitors such as p27; suicide proteins such as thymidine kinase or nitroreductase; cytokines such as interleukin 1, interleukin 2, or granulocyte macrophage colony stimulating factor (GM-CSF) or other immunoregulatory proteins; Toxins such as Pseudomonas aeruginosa exotoxin A; 5-fluorouracil; interferon alpha; methotrexate; tamoxifen; raloxifene; vincristine; nitrogen mustard, alkyl sulfonate, nitroso Alkylating agents such as iodine, ethyleneimine, and triazene; antimetabolites such as folic acid antagonists, purine analogs, and pyrimidine analogs; antibiotics such as anthracyclines, bleomycin, mitomycin, dactinomycin, and pricamycin; L Enzymes such as asparaginase; farnesyl-protein transferase inhibitors; 5 alpha-reductase inhibitors; 17 beta-hydroxysteroid dehydrogenase type 3 inhibitors; glucocorticoids, estrogens / antiestrogens, androgens / antiandrogens, progestins, and corpus luteum Hormone agents such as forming hormone-releasing hormone antagonists, octreotide acetate; microtubule disrupting agents such as echinasaidin or analogs and derivatives thereof; taxanes such as paclitaxel (Taxol) ), Docetaxel (Taxotere ™), and analogs thereof, and microtubule stabilizers such as epothilones such as epothilone AF and their analogs; vinca alkaloids, epipodophyllotoxins, taxanes Plant derived products such as; and topoisomerase inhibitors; prenyl protein transferase inhibitors; and various agents such as platinum coordination complexes such as hydroxyurea, procarbazine, mitotane, hexamethylmelamine, cisplatin and carboplatin; Other agents used as anticancer agents and cytotoxic agents such as biological response modifiers, growth factors; immunomodulators and monoclonal antibodies; mechloretamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan Carmustine, lomustine, semestine, streptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin C, mitomycin C Saframycin, quinocarcine, discodermolide, vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate, teniposide, paclitaxel, estramustine, estramustine phosphate sodium, flutamide, buserelin, leuprolide, pteridine, diynes (dines) Levamisole, aflacon, a Interferon, interleukin, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride, betamethasone (betamethosone), gemcitabine hydrochloride, altretamine, and Topoteka (topoteca), and any analogs or derivatives thereof.

  Examples of other cytotoxic agents also include: German Patent 413842.8; WO 97/19086, WO 98/22461, WO 98/25929, International Publication No. 98/38192, International Publication No. 99/01124, International Publication No. 99/02224, International Publication No. 99/02514, International Publication No. 99/03848, International Publication No. 99/07692, International Publication No. No. 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/543 As found in 65913, WO 99/67252, WO 99/67253, and WO 00/00485. Potillone derivatives; cyclin dependent kinase inhibitors as found in WO 99/24416 (see also US Pat. No. 6,040,321); and WO 97/30992 and WO 98/54966 Prenylprotein transferase inhibitors as found in US Pat. No. 6,011,029; and agents such as those genetically and specifically described in US Pat. No. 6,011,029; particularly in the treatment of cancer, AR modulators; A compound that can be used with an LHRH modulator together with any NHR modulator such as an ER modulator.

  The invention further provides a cytotoxic peptide that is a variant, derivative, truncated or structural equivalent of a cupredoxin and / or CARD-bearing protein. In some embodiments, the cytotoxic peptide is isolated. In some embodiments, the cytotoxic peptide is substantially pure or pharmaceutical grade. In other embodiments, the cytotoxic peptide is in a composition comprising or consisting essentially of the cytotoxic peptide. In other embodiments, the cytotoxic peptide is in a composition comprising both the cytotoxic peptide and at least one other cytotoxic agent. In another specific embodiment, the cytotoxic peptide is non-antigenic and does not elicit an immune response in mammals and more particularly in humans. In some embodiments, the cytotoxic peptide is shorter than the full length cupredoxin or CARD carrying protein and retains some of the pharmacological activity of the full length protein. In particular, in some embodiments, cytotoxic peptides can retain the ability to kill cancer cells, particularly leukemia cells.

  Due to the high structural homology between cupredoxins, it is contemplated that other cupredoxins will have the same cytotoxic properties as azurin and Laz, particularly with respect to leukemia. In some embodiments, the cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin, rusticyanin, auracyanin, stellacyanin, cucumber basic protein, or Laz. In particular, in certain embodiments, azurin or Laz is a Pseudomonas aeruginosa, Algarigenes faecalis, Achromobacter xylos oxidans species Denitificans I, Achromobacter xylosoxidans sspicdens ssf. Bronchiseptica (Bordetella bronchiseptica), Methylomonas sp., Neisseria meningitidis (Neiseria meningitidis), Neisseria gonorrhea (P), Pseudomonas fluorescens (Pseudomonas fluorescens) eudomonas chlororaphis), Kishirera-file Institut de-author (Xylella fastidiosa), Uruba pertussis (Ulva pertussis), or derived from Vibrio parahaemolyticus (Vibrio parahaemolyticus). In certain embodiments, azurin is derived from Pseudomonas aeruginosa.

  The present invention provides a cytotoxic peptide that is an amino acid sequence variant of a cupredoxin or CARD-carrying protein with amino acid substitutions, deletions, or insertions compared to a wild-type cupredoxin or CARD-carrying protein. The mutant of the present invention may be a truncated form of a wild type protein. In some embodiments, the cytotoxic peptides of the invention comprise a region of a cupredoxin or CARD-bearing protein that is shorter than the full-length wild-type polypeptide.

  The cytotoxic peptides of the present invention can also include peptides made with non-naturally occurring synthetic amino acids. For example, unnatural amino acids can be incorporated into chemopreventive agents to extend or optimize the half-life of the composition in the bloodstream. Such chemopreventive agents include, but are not limited to: D, L-peptides (diastereomers) (eg, Futaki et al., J. Biol. Chem. 276 (8)). : 5836-40 (2001); Papo et al., Cancer Res. 64 (16): 5779-86 (2004); Miller et al., Biochem. Pharmacol. 36 (1): 169-76 (1987); peptides containing unconventional amino acids (eg, Lee et al., J. Pept. Res. 63 (2): 69-84 (2004)), followed by hydrocarbon stapling. Olefin-containing unnatural amino acids (eg, Schafmeister et al., J. Am. Ch. em.Soc.122: 5891-5892 (2000); Peptide.

  In other embodiments, the cytotoxic peptides of the present invention are cupredoxins or derivatives of CARD carrying proteins. A cupredoxin or CARD-bearing protein derivative is a chemical modification of a peptide such that the peptide still retains some of its fundamental activity. For example, a “derivative” of azurin, Laz, or Pa-CARD is a chemically modified protein that retains its ability to kill cancer cells, particularly leukemia cells and / or ovarian cancer cells. Another derivative may have an increased or optimized blood half-life. Chemical modifications include, but are not limited to, those disclosed in US patent application Ser. No. 12 / 389,120, the entire disclosure of which is incorporated herein by reference.

  In some embodiments, cytotoxic peptides include, but are not limited to, methods of reducing peptide hydrolysis, methods of reducing peptide deamidation, methods of reducing oxidation, methods of reducing immunogenicity. And / or may be modified using methods, including methods that increase the structural stability of peptides. It is contemplated that two or more of the modifications described or incorporated herein by reference can be combined in one modified cupredoxin derived peptide, as well as one or more modifications described herein. It is well known to those skilled in the art to improve the pharmacokinetic properties in combination with other modifications. Many methods for designing such variants and derivatives are well known in the art.

Methods of Use The present invention provides methods for killing mammalian, particularly human, cancer cells, particularly leukemia cells and / or ovarian cancer cells. The method includes contacting the cancer cell with an isolated peptide that is cytotoxic to the cancer cell, or a variant, derivative, truncated form, or structural equivalent thereof. The isolated peptide may be a cupredoxin or CARD-carrying protein as described herein. In one embodiment, the isolated peptide is azurin. In another embodiment, the isolated peptide is Laz. In yet another embodiment, the isolated peptide is Pa-CARD. In a further embodiment, the isolated peptide can be obtained from H. Laz. It is a fusion protein containing 8 regions. In one such embodiment, the fusion protein is H.264. 8-Azu. In another such embodiment, the fusion protein is Azu-H. 8. Cytotoxic peptides may be used alone or in combination with another cytotoxic agent described herein or from Laz H.P. It can be administered to cancer cells in combination with 8 regions. In some embodiments, the cytotoxic peptide is Laz's H.P. It is administered at the same time or almost at the same time in the 8 regions. In other embodiments, the cytotoxic peptide is administered simultaneously or nearly simultaneously with the sequence comprising SEQ ID NO: 24.

  The present invention relates to a method of treating a mammalian patient with cancer by administering an isolated peptide having a cytotoxic effect on cancer cells, or a variant, derivative, truncated form, or structural equivalent thereof, or the like. There is also provided a method for killing cancer cells in a mammalian patient. The isolated peptide may be a cupredoxin or CARD-carrying protein as described herein. In one embodiment, the isolated peptide is azurin. In another embodiment, the isolated peptide is Laz. In yet another embodiment, the isolated peptide is Pa-CARD. In a further embodiment, the isolated peptide can be obtained from H. Laz. It is a fusion protein containing 8 regions. In one such embodiment, the fusion protein is H.264. 8-Azu. In another such embodiment, the fusion protein is Azu-H. 8. Cytotoxic peptides may be used alone or in combination with another cytotoxic agent described herein or from Laz H.P. It can be administered in combination with 8 regions. In some embodiments, the cytotoxic peptide is Laz's H.P. It is administered at the same time or almost at the same time in the 8 regions. In other embodiments, the cytotoxic peptide is administered simultaneously or nearly simultaneously with the sequence comprising SEQ ID NO: 24.

  The present invention also provides a method of inducing death of leukemia cells and / or ovarian cancer cells by cell differentiation by administering one or more cytotoxic peptides to leukemia cells and / or ovarian cancer cells. The cytotoxic peptide may be a cupredoxin or CARD-bearing protein as described herein. In one embodiment, the cytotoxic peptide is azurin. In another embodiment, the cytotoxic peptide is Laz. In another embodiment, the cytotoxic peptide is a fusion protein H.I. 8-Azu. In yet another embodiment, the cytotoxic peptide is a fusion protein Azu-H. 8.

  The present invention relates to a method of selectively entering leukemia cells and / or ovarian cancer cells by administering one or more cytotoxic peptides to leukemia cells and / or ovarian cancer cells, and exhibiting a cytotoxic effect there. Also provide. The cytotoxic peptide may be a cupredoxin or CARD-bearing protein as described herein. In one embodiment, the cytotoxic peptide is azurin. In another embodiment, the cytotoxic peptide is Laz. In yet another embodiment, the cytotoxic peptide is Pa-CARD. In a further embodiment, the cytotoxic peptide is Laz's H.P. It is a fusion protein containing 8 regions. In one such embodiment, the fusion protein is H.264. 8-Azu. In another such embodiment, the fusion protein is Azu-H. 8.

  The present invention further includes a method of killing leukemia cells and / or ovarian cancer cells by causing cell cycle arrest at G2 / M phase, comprising administering one or more cytotoxic peptides. . In further embodiments, the cytotoxic peptide stabilizes the Weel protein and / or increases the level of the Weel protein in a cell, including but not limited to the cytoplasm and / or nucleus of the cell. In yet another embodiment, the cytotoxic peptide depletes phosphorylated AKT-Ser-473. In yet another embodiment, the cytotoxic peptide stabilizes the Weel protein / increases Weel protein levels and depletes phosphorylated AKT-Ser-473. In a further embodiment, the cytotoxic peptide is azurin. In another embodiment, the cytotoxic peptide is Laz. In yet another embodiment, the cytotoxic peptide is Pa-CARD. In yet another embodiment, the cytotoxic peptide is a fusion protein H. coli. 8-Azu and Azu-H. One or more of eight.

Nucleic Acids Encoding Cytotoxic Peptides and Expression Vectors In another aspect, the present invention provides nucleic acid molecules encoding the cytotoxic peptides described herein, and variants, derivatives, and / or structural equivalents thereof. provide. Nucleic acid molecules according to the present invention can be prepared by a combination of techniques known in the art. The coding sequences used for these nucleic acids may be those found in natural genomic DNA encoding a particular peptide, or may be designed from known codons. These coding sequences can also be designed taking into account alternating codon usage and the preferred codon usage of the organism in which the peptide is expressed. The nucleic acid sequences of cytotoxic peptides may be prepared separately by chemical synthesis or cloning. The nucleic acid sequences can then be ligated together with ligase to obtain the desired nucleic acid molecule.

  Vectors used to transfer genetic material between organisms can be divided into two general classes: cloning vectors have regions essential for growth in suitable host cells and are foreign A replication plasmid or phage into which sex DNA can be inserted, and foreign DNA is replicated and propagated as if it were a component of the vector. Expression vectors (such as plasmids, yeast, or animal virus genomes) are available from Laz, azurin, Pa-CARD, H. 8-Azu, or Azu-H. In order to transcribe and translate foreign DNA, such as cytotoxic peptide DNA such as 8, it is used to introduce foreign genetic material into a host cell or tissue. In an expression vector, the introduced DNA is operably linked to an element such as a promoter that sends a signal to the host cell to highly transcribe the inserted DNA. Some promoters are very useful, such as inducible promoters that control gene transcription in response to specific factors. By operably linking the cytotoxic peptide and variants and derivatives thereof to an inducible promoter, the expression of the cytotoxic peptide and variants and derivatives thereof can be controlled in response to specific factors. Examples of classical inducible promoters include steroids such as alpha-interferon, heat shock, heavy metal ions, and glucocorticoids (Kaufman, Methods Enzymol. 185: 487-511 (1990)), and tetracycline. Includes responding. Other desirable inducible promoters include those that are not endogenous to the cells into which the construct is introduced, but are responsive in those cells when the inducing agent is supplied exogenously. In general, useful expression vectors are often plasmids. However, other forms of expression vectors are contemplated, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses).

  The choice of vector is determined by the organism or cell being used and the desired fate of the vector. In general, a vector includes a signal sequence, an origin of replication, a marker gene, a polylinker site, an enhancer element, a promoter, and a transcription termination sequence.

The present invention relates to at least one cytotoxic peptide that is a cupredoxin or CARD carrying protein, or a variant, derivative, truncated or structural equivalent of a cupredoxin or CARD carrying protein, particularly a pharmaceutical. Also provided is a composition comprising in the composition alone or in combination with at least one other cytotoxic agent. In certain embodiments, the pharmaceutical composition is designed for a particular method of administration, such as, but not limited to, oral, intraperitoneal, or intravenous. Such compositions may be hydrated in water or may be dried for later hydration (such as by lyophilization). Such a composition is not limited to this, but may be in a solvent other than water, such as alcohol.

  The pharmaceutical composition of the present invention containing a cytotoxic peptide can be in any conventional manner, such as a conventional mixing process, dissolution process, granulation process, dragee manufacturing process, emulsification process, encapsulation process, encapsulation process, or It can be produced by a lyophilization process. The cytotoxic peptide can be readily mixed with pharmaceutically acceptable carriers well known in the art. With such carriers, preparations can be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and the like. Suitable excipients also include, for example, fillers and cellulose preparations. Other excipients may include, for example, fragrances, colorants, anti-tacking agents, thickeners, and other acceptable additives, adjuvants, or binders.

  In various embodiments, the composition includes the following: carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, (Including bacteriostatic agents, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils, saline, dextrose and glycerin aqueous solutions, buffers, isotonic agents, and wetting agents Other pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions such as It will be appreciated that the compositions of the invention can be administered using any suitable carrier known to those of skill in the art, and the type of carrier will vary depending on the method of administration. The compound can also be encapsulated within the liposome using well known techniques. Biodegradable microspheres can also be used as carriers for the compositions of the present invention. Suitable biodegradable microspheres are, for example, U.S. Pat. Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820, 883, 5,853,763, 5,814,344, and 5,942,252. As used herein, “compound” includes peptides, amino acid sequences, cargo compounds and complexes of the present invention, and nucleic acids.

  Intravenous fluids used to prepare pharmaceutical compositions for administering the cytotoxic peptides and nucleic acids disclosed herein may consist of a crystalline or colloidal solution. A crystalline liquid, as used herein, is an aqueous solution of mineral salts or other water soluble molecules. A colloidal liquid, as used herein, contains larger insoluble molecules such as gelatin. Intravenous fluids may be sterile.

  Crystalline solutions that can be used for intravenous administration include, but are not limited to, normal saline (0.9% strength sodium chloride solution), lactated Ringer's solution, as described in Table 1 Or Ringer's solution and 5% dextrose aqueous solution, sometimes referred to as D5W.

  The blood half-life of the compositions of the present invention can be extended or optimized by several methods well known to those skilled in the art, including but not limited to: cyclic Peptide (Monk et al., BioDrugs 19 (4): 261-78 (2005); DeFreest et al., J. Pept.Res. 63 (5): 409-19 (2004)), D , L-peptide (diastereomer) (Futaki et al., J. Biol. Chem. Feb. 23; 276 (8): 5836-40 (2001); Papo et al., Cancer Res. 64 (16). No.): 5779-86 (2004); Miller et al., Biochem. Pharmacol. 36 (1): 169-76 (1987)); Peptides containing mino acids (Lee et al., J. Pept. Res. 63 (2): 69-84 (2004)), and N- and C-terminal modifications (Labrie et al., Clin. Invest. Med) 13 (5): 275-8 (1990)). Of particular interest are d-isomerization (substitution) by D-substitution or L-amino acid substitution and modification of peptide stability.

  Where administration is by injection, the composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulation agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the composition may be in powder form for constitution with a suitable medium prior to use, such as pyrogen-free sterilized water.

  When administration is by inhalation, the composition is delivered in the form of an aerosol spray from a pressurized container or nebulizer using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide, or other suitable gas. can do. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver the measured amount. Gelatin capsules and cartridges can be formulated containing, for example, an inhaler or insufflator, containing a powder mixture of protein and a suitable powder base such as lactose or starch.

  Where administration is by topical administration, the composition can be formulated as solutions, gels, ointments, creams, suspensions, and the like, as is well known in the art. In some embodiments, administration is by a transdermal patch. Where administration is by suppository (eg, rectal or vaginal), the composition can also be formulated in a composition containing a conventional suppository base. When administered orally, the composition can be readily formulated in combination with pharmaceutically acceptable carriers well known in the art. Solid carriers such as mannitol, lactose, and magnesium stearate can be used, such as tablets, pills, dragees, capsules for ingestion of chemotaxin by the subject to be treated. , Liquids, gels, syrups, slurries, suspensions, and the like. For example, for oral solid preparations such as powders, capsules and tablets, suitable excipients include fillers such as sugar, cellulose preparations, granulating agents, and binders.

  Other convenient carriers also include multivalent carriers such as bacterial capsular polysaccharides, dextrans, or genetically engineered vectors, as is well known in the art. In addition, a sustained release formulation comprising the composition allows for release of the composition over an extended period of time, otherwise the composition may be subject to the subject's system prior to inducing or enhancing the therapeutic effect. And / or degraded by, for example, protease and simple hydrolysis.

Kits Comprising Cytotoxic Peptides In another aspect, the present invention provides a kit containing one or more of the following in a package or container: (1) one or more cells described herein A reagent containing a toxic peptide; (2) a reagent containing a pharmaceutically acceptable adjuvant or excipient; (3) a medium for administration such as a syringe; (4) instructions for administration. Embodiments in which two or more of components (1)-(4) are found in the same container are also contemplated. In other embodiments, the kit components can also include one or more additional cytotoxic agents. In other embodiments, the reagent is formulated for intravenous administration and / or the medium of administration is suitable for intravenous administration.

  When the kit is supplied, the different components of the composition are packaged in separate containers and may be mixed immediately before use. Such separate packaging of the ingredients may allow long term storage without losing the function of the active ingredient.

  The reagents included in the kit can be supplied in any type of container that preserves the lifetime of the various components and is not adsorbed or altered by the material of the container. For example, a sealed glass ampoule can contain a lyophilized polypeptide or polynucleotide or buffer packaged under a neutral inert gas such as nitrogen. The ampoule may be made of any suitable material such as glass, an organic polymer such as polycarbonate, polystyrene, ceramic, metal, or any other material typically used to contain similar reagents. . Other examples of suitable containers include simple bottles that may be made of similar materials as ampoules and medicine bags that may have a foil-lined interior such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container may have a sterile outlet, such as a bottle with a stopper that can be penetrated with a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane that allows mixing of the components upon removal. The removable membrane may be glass, plastic, rubber or the like.

  The kit may be supplied with instructions. The instructions may be printed on paper or other substrate and / or electronically read such as floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, flash memory device, etc. It may be supplied as a possible medium. The detailed instructions may not be physically attached to the kit, but instead may direct the user to an internet website specified by the kit manufacturer or distributor, or provided as an email. May be.

  A more complete understanding of the invention can be obtained by reference to the following specific examples. The examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Variations in form and substitution of equivalents are contemplated so that the situation can suggest or confer convenience. Although specific terms are used herein, such terms are intended in a descriptive sense and not for limitation. Modifications and variations of the present invention as set forth above may be made without departing from the spirit and scope thereof, and are thus only limited as shown by the appended embodiments. Should.

Examples Example 1 Laz and H.C. Cloning and expression of 8-azurin fusion gene The laz gene from Neisseria gonorrhoeae was cloned based on its known sequence (SEQ ID NO: 1) (Figure 1A). p. Elginosa azurin gene (SEQ ID NO: 2) (FIG. 1B), and The laz H. cerevisiae derived from N. gonorrhoeae. Using the 8 epitope sequence (SEQ ID NO: 3), H. The 8 epitope gene was cloned in frame, 8-paz (FIG. 1C) was generated and H. The 8 epitope gene was cloned in frame, and the paz-H. 8 (FIG. 1D) was produced.

Cell lines and reagents. Human cancer cells, bacterial strains, and plasmids are listed in Table 2. Human breast cancer MCF-7 cells and brain tumor LN-229 cells are derived from the preserved culture collection of the Department of Surgical Oncology at the University of Illinois at Chicago (UIC). Cells in MEM with eagle salt containing 2 mM L-glutamine, 0.1 mM MEM essential amino acids supplemented with 10% heat inactivated fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin In culture. All cells were grown at 37 ° C. in 5% CO ₂ . (Yamada et al., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002); Punj et al., Oncogene 23: 2367-2378 (2004)).

Cloning and expression of paz and laz genes. Cloning and high expression of the azurin gene has been described. (Yamada et al., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002); Punj et al., Oncogene 23: 2367-2378 (2004)). The Neisseria gonorrhoeae Laz coding gene (laz) PCR was performed using genomic DNA of Gonorrhoeae strain F62 as template DNA. The forward and reverse primers were 5′-CCG GAATTC CGGCAGGGATGTTGTTAAATATCCG-3 ′ (SEQ ID NO: 4) and 5′-GG GGTACC GCCGTGGCAGGCATACAGCATTTCAATCGGG-3 ′ (SEQ ID NO: 5), with additional restriction on EcoRI and Kp sites Are underlined. A 1.0 kb amplified DNA fragment digested with EcoRI and KpnI was transferred to a pUC18 vector (Yanisch-Perron et al., Gene 33: 103-119 (1985)) so that the laz gene was located downstream of the lac promoter. Was inserted into the corresponding site to obtain an expression plasmid pUC18-laz (Table 2, FIG. 1).

N. Gonolea Laz H.C. 8 and P.I. A plasmid expressing an aeruginosa azurin (Paz) fusion was constructed by PCR using pUC19-paz and pUC18-laz as templates. H. In the case of 8-Paz fusion, using pUC18-laz as a template and using primers, 5 ′-(phosphorylated) GGCAGCAGGGGCTCTCGCAGAGCATCTGC-3 ′ (SEQ ID NO: 6) and 5′-CTGCAG GTCGAC TCTAGAGGATCCCG-3 ′ (SEQ ID NO: 7) A 3.1 kb fragment was amplified. The SalI site is underlined. Fragment of 0.4kb amplified by PCR is, pUC19-paz as template, and primers 5 '- (phosphorylated) GCCGAGTGCTCGGTGGACATCCAGG-3' (SEQ ID NO: 8) and 5'-TA CTCGAG TCACTTCAGGGTCAGGGTG-3 ' ( SEQ No. 9). The XhoI site is underlined. The PCR fragment digested with SalI derived from pUC18-laz and the PCR fragment digested with XhoI derived from pUC19-paz were cloned, and the expression plasmid pUC18-H. 8-paz (Table 2, FIG. 1) was obtained.

Paz-H. In the case of 8 fusions, 3.3 kb using pUC19-paz as a template and using primers 5′-CTTCAGGGTCAGGGTGCCCCTCATC-3 ′ (SEQ ID NO: 10) and 5′-CTGCAGGTCGAACTCTAGA GGATCC CG-3 ′ (SEQ ID NO: 11) The fragment was amplified. The BamHI site is underlined. Using pUC18-laz as a template and a primer, 5 ′-(phosphorylated) TGCTCTCAAGAAACCTGCCCGCGCCTGC-3 ′ (SEQ ID NO: 12) and 5′-TA GGATCC TTAGGCAGCAGGGGCTTCGGCAGGCATCTGC-3 ′ (SEQ ID NO: 13), a 0.13 kb fragment Amplified. The BamHI site is underlined and the additionally introduced TTA corresponding to the bacterial gene stop codon is shown in italics. Two BamHI digested PCR fragments were cloned and the expression plasmid pUC19paz-H. 8 was obtained (Table 2).

E. coli JM109 was used as a host strain for expression of azurin and its derivative genes. The recombinant E. coli strain was cultured in 2 × YT medium containing 100 μg / ml ampicillin, 0.1 mM IPTG, and 0.5 mM CuSO ₄ for 16 hours at 37 ° C. to produce azurin protein.

Plasmid construction of the fused GST protein. The glutathione S-transferase (GST) -encoding gene was amplified by PCR using pGEX-5X-3 (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) as template DNA. The forward and reverse primers used were 5'-CG AGCTCA TGTCCCCCTATACTAGGTTTATTGG-3 '(SEQ ID NO: 14) and 5'-CCC AAGCTT TCAGGGGATCCCACGACCTCTCGATCAGATCC-3' (SEQ ID NO: 15), additionally introduced SacI and HindIII Each restriction site is underlined and the additionally introduced TCA corresponding to the bacterial gene stop codon is shown in italics. The 1.0 kb amplified DNA fragment digested with SacI and HindIII was inserted into the corresponding site of the pET29a vector to obtain the expression plasmid pET29a-gst (Table 2).

H. In the case of the 8-GST fusion, pUC18-laz was used as the template DNA, and the signal peptide of laz and H.P. The 8 coding region was amplified by PCR. The forward and reverse primers used were 5′-GGAATT CATATG AAAGCTTATCTGGC-3 ′ (SEQ ID NO: 16) and 5′-CC GGAATT CGGCAGCGGGGCTTCCGC-3 ′ (SEQ ID NO: 17), and additionally introduced NdeI and EcoRI sites. Each restriction site is underlined. A 0.14 kb amplified DNA fragment digested with NdeI and EcoRI was inserted into the corresponding site of the pET29a-gst vector, and the expression plasmid pET29a-H. 8-gst was obtained (Table 2).

GST-H. For the 8 fusion, pUC18-laz was used as template DNA to The 8 coding region was amplified by PCR. The forward and reverse primers used were 5′-CG GGATCC CCTGCTCTCAAGAAACCTGCCCGCGCC-3 ′ (SEQ ID NO: 18) and 5′-CG GAATTC TTAGGCAGCAGGGGCTTCGGCAGGCATCTGCAGGG-3 ′ (SEQ ID NO: 19), additionally introduced Bam Restriction sites are underlined and the introduced bacterial gene stop codon TTA is shown in italics. A 0.14 kb amplified DNA fragment digested with BamHI and EcoRI was inserted into the corresponding site of the pGEX-5X-3 vector, and pGEX-5X-3-H. 8 was obtained. Thereafter, pGEX-5X-3-H. 8 as template DNA, GST-H. Eight fusion regions were amplified by PCR. The forward and reverse primers used were 5′-CG AGCTCA TGTCCCCCTATACTAGGTTTATTGG-3 ′ (SEQ ID NO: 20) and 5′-CCG CTCGAG TCAGGCAGCAGGGGCTTCGGCAG-3 ′ (SEQ ID NO: 21), additionally introduced SacI and XhoI sites The restriction sites are underlined and the additionally introduced TCA corresponding to the bacterial gene stop codon is shown in italics. The 1.1 kb amplified DNA fragment digested with SacI and XhoI was inserted into the corresponding site of the pET29a vector and the expression plasmid pET29a-gst-H. 8 was obtained (Table 2).

  E. coli BL21 (DE3) was used as a host strain for expression of gst and its functional derivatives. E. coli strains carrying these plasmids are grown in the presence of IPTG, the cells are lysed, and the protein is purified as described for azurin (Yamada et al., Proc. Natl. Acad. Sci. USA 99. Volume: 14098-14103 (2002); Punj et al., Oncogene 23: 2367-2378 (2004); Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)), various azurins The derivative migrated as a single component (FIG. 1E) by SDS-PAGE. The 8-containing protein (approximately 17 kDa) showed anomalous migration as previously described (Cannon et al., Ibid .; Fistette et al., Ibid.).

Example 2 H. 8 shows P. cerevisiae for glioblastoma cells. Preferential entry of Paz to cancer cells (Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)), which enhances cytotoxicity of Erginosaazurin but not breast cancer cells, and Its cytotoxicity against human melanoma (Yamada et al., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002)) and breast cancer (Punj et al., Oncogene 23: 2367-2378 (2004)). Has been reported both in vitro and in vivo. See, for example, US patent application Ser. No. 12 / 338,480, the entire disclosure of which is incorporated herein by reference. However, the effect of Paz or Laz on brain tumors such as glioblastoma is not known. As used herein, Paz, Laz, H. et al. For both glioblastoma (LN-229 cell line) and breast cancer (MCF-7 cell line) cells. 8-Paz (H.8 epitope is at the N-terminus of Paz) and Paz-H. The effect of 8 (H.8 epitope is at the C-terminus of Paz) was studied.

  Protein preparation. P. Elginosa azurin (Paz), N.I. Gonorea Laz, Paz-H. 8 and H.I. 8-Paz was purified as previously described. (Yamada et al., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002); Punj et al., Oncogene 23: 2367-2378 (2004); Yamada et al., Cell. Microbiol. 7 : 1418-1431 (2005)). All recombinant GST fusion derivatives were purified as previously described. (Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)). A chemically synthesized 39 amino acid H.P. 8 peptides were purchased.

Cytotoxicity assay. A 3- (4,5-dimethylthiazol-2-yl-2,5-diphenyl) tetrazolium bromide (MTT) assay was performed to determine cytotoxicity against cancer cells. Cells (5 × 10 ³ per well) were seeded in 96-well culture dishes of 100: 1 medium at 37 ° C., 5% CO ₂ . After overnight incubation, the supernatant was removed and fresh media containing the various specified concentrations of protein was added to the adherent cells. After incubating these cells for various designated times, 10 μl of 5 mg / ml MTT (Sigma-Aldrich, St. Louis, MO) solution was added to the culture and incubated for 2 hours at 37 ° C. The number of viable cells was determined by assay. The MTT reaction was terminated by adding 100 μl of 40 mM HCl in isopropanol. The MTT formazan formed was measured spectrophotometrically according to the method described by Mosmann (J. Immunol. Methods 65: 55-63 (1983)).

  Synthesis H. The 8 peptide showed little cytotoxicity to either glioblastoma LN-229 (FIG. 2A) or breast cancer MCF-7 (FIG. 2B) cells. Although the effect of azurin (Paz) is low, it is dose-dependent in glioblastoma (FIG. 2A), increasing azurin concentration from 10 μM to 40 μM and increasing cytotoxicity, but breast cancer (FIG. 2B). It was not the case with cells. After a 6 hour incubation period, the cytotoxicity increased only slightly. Most notably, the glioblastoma and breast cancer cells Paz, Paz-H. 8, H. There was a difference in the cytotoxicity of 8-Paz and Laz. Paz, Paz-H. 8, H. Although 8-Paz and Laz showed essentially the same cytotoxicity against MCF-7 cells at all doses and at various incubation periods (FIG. 2B), Paz was particularly short for incubation periods. (6 hours) against glioblastoma cells, Paz-H. 8, H. It showed much lower cytotoxicity than 8-Paz or Laz. Therefore, H.I. The 8 part was thought to enhance Paz cytotoxicity only against glioblastoma and not breast cancer cells, although it itself lacks cytotoxicity.

Example 3. H. present in Paz or Laz. The 8 epitope promotes azurin uptake in glioblastoma cells, compared to Paz-P. 8, H. Due to the enhanced cytotoxicity of 8-Paz and Laz against glioblastoma cells, The question arises as to whether the eight parts somehow facilitated azurin uptake in glioblastoma cells. Alexa fluor® 568 labeled red fluorescent protein (Invitrogen-Molecular Probes Corp., Carlsbad, Calif.) Was used to determine the internalization of these proteins inside glioblastoma and breast cancer cells. This technique was previously used to demonstrate azurin internalization in MCF-7 cells (Punj et al., Oncogene 23: 2367-2378 (2004); Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)).

Confocal microscope. To prepare the microscopic sample, the cells were cultured overnight at 37 ° C. on a cover glass under 5% CO ₂ . Pre-warmed fresh medium at 37 ° C. was mixed with red fluorescent label (Alexa fluor® 568) azurin or GST fusion derivative and incubated with the cells for the indicated time. Cells were washed with PBS and fixed with methanol at −20 ° C. for 5 minutes. Washed 3 times with PBS and contains 1.5 mg / ml 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining (VECTASHIELD®, Vector Laboratories, Burlingame, Calif.) After adding the encapsulant, images were taken by using an LSM510 laser scanning confocal microscope from Carl Zeiss. (Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)).

  Azurin (Paz) is a Paz-H. 8, H. Internalizing with reduced efficiency than 8-Paz and Laz, it was demonstrated that glioblastoma LN-229 cells have a barrier to Paz entry (FIGS. 3A and 4A). In contrast, Paz, as previously reported in breast cancer MCF-7 cells, is Paz-H. 8, H. Efficiently internalized with the same or slightly higher efficiency than 8-Paz or Laz (FIGS. 3B and 4B). (Punj et al., Oncogene 23: 2367-2378 (2004); Yamada et al., Cell. Microbiol. 7: 1418-1431 (2005)). The dose dependence of Laz entry in LN-229 cells was approximately 16 μM at the optimal concentration during the 30 minute incubation period at 37 ° C. (FIGS. 3C and 4C), with no further enhancement thereafter (data not shown) Proved that. At 10 μM concentration, most Laz was internalized into LN-229 cells in about 10-20 minutes (FIGS. 3D and 4D), but Paz internalization was minimal under such conditions. (FIG. 3E), suggesting that Paz internalization was essentially inefficient in LN-229 cells. Paz-H. 8 and H.I. The marked internalization of 8-Paz is similar to Laz, but in contrast to Paz in LN-229 cells (FIGS. 3A and 4A), H.P. The relative position of the 8 parts did not affect its ability to promote internalization of the Paz part in glioblastoma cells.

Example 4 H. Part 8 promotes Paz entry in glioblastoma but not breast cancer cells. To promote Paz entry into glioblastoma cells, To determine whether the 8 epitope needs to be part of Paz as well as Laz or can function alone, H. et al. In addition to 8 alone, various H.P. Eight fusion proteins were used. Synthesis of 39 amino acids Since small peptides such as 8 moieties have low stability in solution, the inventors have described Paz-H. 8 or H.I. Similar to 8-Paz, H.P. H. 8 is incorporated into the N-terminus of GST (H.8-GST) or the C-terminus of GST (GST-H.8). A fusion of 8 parts and glutathione S-transferase (GST) was constructed. The construction of GST fusion peptides is described in Example 1.

  Alexa fluor® 568 conjugated Paz, which emits red fluorescence, is added to phosphate buffered saline (PBS) as a control to add unlabeled synthetic H.P. 8 peptide, GST, GST-H. 8 and H.I. After incubating separately with 8-GST fusion protein and incubating at 37 ° C. for 30 minutes, internalization of the 20 μM Paz mixture in LN-229 cells was determined. Synthesis H. When the 8 peptides were introduced separately with Paz, PBS (FIG. 5E), GST (FIG. 5B), or GST-H. Compared to 8 (FIG. 5C), Paz internalization was enhanced (FIG. 5A). By quantifying the fluorescence, Eight peptides were shown to stimulate Paz entry 2.1-fold. However, H.C. The presence of 8-GST significantly enhanced Paz internalization (> 3-fold) (FIG. 5D). On the other hand, GST-H. 8 showed only slight irritation (FIG. 5C). Paz itself only slowly entered glioblastoma cells (FIG. 5E), and entry in brain tumor cells is H.264. 8 has been demonstrated to be mediated. H. 8 alone did not enter glioblastoma cells (FIG. 3A), but its ability to stimulate internalization of Paz (FIG. 5A) reflects its ability to promote protein entry into brain tumor cells. is doing.

Example 5 FIG. H. glioblastoma cells Enhanced internalization of Paz in the presence of 8-GST leads to higher cytotoxicity in such cells.
We have synthesized H.P. 8 peptide, GST, GST-H. 8 and H.I. Incubating 8-GST proteins (20 μM each) with LN-229 cells in the absence or presence of 20 μM Paz for 24 hours, and then measuring glioblastoma cells that can grow by MTT assay after 24 hours Was used to measure the degree of cytotoxicity. In the absence of Paz, H.P. None of the 8 peptides, GST, or GST fusion proteins showed any significant cytotoxicity (FIG. 5F, -Paz). H. In the presence of 20 μM Paz showing low cytotoxicity in the presence of 8 peptides or PBS (FIG. 5F, + Paz), GST itself or GST-H. 8 showed some enhancement of cytotoxicity (FIG. 5F, + Paz), but a significant enhancement of cytotoxicity was observed in H.P. Only observed in the presence of 8-GST (FIG. 5F, + Paz). In summary, these data indicate that H.C. It is suggested that the 8 moiety, when present as part of or in the presence of a protein such as Paz, promotes Paz transport into such cells and results in enhanced cytotoxicity.

Example 6 H. 8 mediates the passage of the BBB and allows entry into the brain. The ability of the 8 epitope to allow enhancement of fusion or individual protein internalization in glioblastoma LN-229 cells (FIGS. 3A, 4A, and 5D) H. as a part of the N-terminus of 8-Paz or Laz. The question raised whether 8 facilitated the passage of the BBB and allowed the transport of these proteins from the peripheral circulation to the cerebral venules.

  Odyssey® assay. All proteins were labeled using IRDye® 800CW (LI-COR Biosciences, Lincoln, Nebra) under conditions recommended by the manufacturer. 500 μg Paz bound to IRdye® 800 CW, H.P. 8-Paz and Laz were injected intraperitoneally into nude mice. After 24 hours, the mice were sacrificed, the brains were removed, and brain images were detected with a LI-COR Odyssey® infrared imaging system (resolution 84 μm, offset 1 mm). Thereafter, the mouse brain was cut horizontally and a cranial midbrain region image was taken to detect the presence of the labeled protein.

  Quantification of azurin protein fluorescence. Quantification of fluorescence was measured by using Adobe® Photoshop® as follows: one cell was selected with the Photoshop® lasso tool (Lasso Tool), Average values were obtained from the red histogram in the image menu. At least three different cells were measured for one sample and the standard deviation was calculated.

  500 μg Paz labeled with infrared dye IRdy® 800 CW (manufactured by LI-COR Bioscience), H.P. 8-Paz and Laz proteins were injected intraperitoneally into living nude mice. After 24 hours, the mice were sacrificed, the brains were isolated, and images were taken using the LI-COR Odyssey® infrared imaging system. Paz has been found to enter small amounts of cerebral venules, but even more Laz and especially H. 8-Paz (> 4 times) is detected inside the brain under such conditions (FIG. 6). It has been demonstrated that the 8 epitope plays a clear role in allowing the fusion protein to enter the brain.

Example 7. H. The 8 epitope, when at the N-terminus, allows bacterial surface display of periplasmic proteins.
Laz H.C. To study whether the location of the 8 epitope at the N-terminus contributes to its surface presentation, H. et al. Eight fusion derivatives were constructed at the N-terminus and C-terminus of GST (FIG. 5) and Paz (FIGS. 2 and 3A / B and 4A / B) as described in Example 1.

  Localization of surface exposed proteins in E. coli. pET29a-gst, pET29a-H. 8). gst, or pET29a-gst-H. E. coli strain BL21 (DE3) carrying 8 and pUC19-paz, pUC19-paz-H. 8, pUC18-H. E. coli strain JM109 carrying 8-paz or pUC18-laz was cultured at 37 ° C. with 0.4 mM isopropyl β-D-thiogalactoside (IPTG). 1 ml of each of these bacterial cultures was centrifuged and the resulting pellet collected. After washing twice with PBS, 1 ml of 1% FBS-PBS containing anti-GST antibody against GST derivative (1: 2000) or anti-azurin antibody against azurin derivative (1: 500) was added. The cell suspension was incubated on ice for 1 hour and then washed twice with PBS. FITC-conjugated anti-rabbit IgG against GST derivative or FITC-conjugated anti-rabbit antibody against azurin derivative was added and incubated on ice for 30 minutes. Cells were washed twice with PBS to remove unbound antibody and fixed with ethanol on ice. Thereafter, E. coli samples treated with DAPI were observed with a confocal microscope.

  H. Eight fusion proteins were purified (FIGS. 1E and 7A). Cell localization of GST, and two H.C. Eight fusions (H.8-GST and GST-H.8) are shown in FIG. When detected by Western blotting using anti-GST antibody, all three proteins were highly expressed in E. coli and were present in whole cell lysates of E. coli (FIG. 7B). Periplasmic fractions were isolated from E. coli and examined for the presence of the three proteins, GST and GST-H. 8 protein was detected in large quantities (FIG. 7B, lanes 1 and 3 below the periplasmic fraction). 8-GST was only detectable in such periplasmic fractions (FIG. 7B, lane 2 under periplasmic fraction).

  The remaining H. To investigate whether the 8-GST fusion protein may have been transported to the surface of E. coli cells, cells that highly express the three proteins were grown, harvested, washed and treated with anti-GST antibody Any surface exposed GST was then bound, washed again and treated with a FITC-conjugated secondary antibody. If GST is exposed on the surface, anti-GST antibodies will bind to them, which can then be detected by a FITC-conjugated secondary antibody. In fact, H. Only E. coli cells carrying 8-GST show green fluorescence generated by FITC (FIG. 7C, H.8-GST). Due to the presence of the 8 epitope at the N-terminus of GST, H. It suggested that the transport of 8-GST was promoted. At the C-terminus of GST The 8 part present (GST-H.8), and GST itself, remained mostly in the periplasm and cells, without surface presentation (FIG. 7C, GST, and GST-H.8). .

  Using the same technique as described above, Paz and Paz-H. 8 remains intracellular (FIG. 7D, Paz and Paz-H.8). It was determined that both 8-Paz and Laz showed surface presentation, and H. The presence of 8 was confirmed to be important for transporting the fusion protein to penetrate the outer membrane and reach the surface, probably because free cysteine is required for lipidation.

Example 8. Preparation of azurin, Laz, and Pa-CARD peptides for leukemia studies Wild-type (wt) azurin and mutant azurin were prepared using the method of Yamada T et al., Proc Natl Acad Sci USA 99: 14098-103 (2002) and Punj. Purified as described in V et al., Oncogene 23: 2367-78 (2004). Laz was purified using the same protocol as described in Hong CS et al., Cell Cycle 5: 1633-41 (2006). Briefly, Laz was expressed in E. coli using the pUC18 vector. The cells were incubated for 24 hours, centrifuged, washed twice with PBS, and then lysed to isolate the periplasmic fraction. Periplasmic fractions are collected for Q Sepharose exchange for protein purification. Fractions are concentrated and FPLC is performed to isolate purified protein.

  Cloning, expression and purification of Pa-CARD. A pET-SUMO expression vector having a SUMO fusion protein in its N82 terminal region was introduced into P. a. It was used for high level expression of soluble arginine deiminase from Elginosa ADI and a caspase recruitment domain (CARD) containing domain. In order to clone the CARD motif of the ADI gene into the pET-SUMO vector (manufactured by Invitrogen), P.P. Genes were amplified from genomic DNA of an aeruginosa clinical isolate: F, forward: 5′-ATGCACAATCTGCTGACCGAGACCATCCAG-3 ′ (SEQ ID NO: 28) and R, reverse: 5′-TCAGGTCGAGGAGCCGTGGCTCTGTC-3 ′ (SEQ ID NO: 29) . The PCR product was directly ligated into the pET-SUMO TA cloning vector. The sequence of the resulting expression vector was confirmed and transformed into E. coli 90 strain BL21 (DE3).

The overnight culture was grown in LB medium at 37 ° C. containing 50 μg / ml kanamycin. When OD ₅₉₅ nm was approximately 0.5, IPTG was added to the culture at a final concentration of 0.5 mM and incubated at 37 ° C. for 5 hours. Cells were collected by centrifuging at 5000 rpm for 15 minutes. The cell pellet was lysed with lysis buffer, pH 8.0, containing 50 mM Tris-Cl, 100 mM NaCl, and 25% (weight / volume) sucrose. After addition of lysis buffer, 20 mg / ml lysozyme was added to the cell suspension and incubated at 4 ° C. for 20 minutes. Triton X-100 [final concentration 0.01% (volume / volume)] was added and incubated for 5 minutes. 100 μl of DNase and RNase was added per 500 ml of the culture and incubated at 37 ° C. for 30 minutes. The cell suspension was centrifuged at 15,000 rpm for 35 minutes at 4 ° C. The supernatant was loaded onto a 1 ml Ni-NTA column that had been equilibrated in advance. After loading, the column was washed with buffer (50 mM Tris-Cl, 300 mM NaCl, 10% glycerol, 10 mM imidazole). SUMO-CARD was eluted with a 25 ml step gradient of 50-500 mM imidazole in elution buffer (100 mM Tris-Cl, 500 mM NaCl, 20 mM imidazole, pH 8.9). The purified SUMO-CARD fraction was pooled, and the buffer solution was exchanged with a buffer solution for digesting SUMO protease (20 mM Tris-Cl, 150 mM NaCl, pH 8.0). Thereafter, the SUMO-CARD protein was concentrated to 1.5-2 ml. Thereafter, DTT was added at a final concentration of 1 mM. Following the addition of SUMO protease, the digestion mixture was incubated at 30 ° C. for 2-3 hours and the CARD polypeptide called Pa-CARD was purified by using nickel resin. The final protein concentration was measured with a protein reagent (Pierce) using BSA as a standard. P. CARD domain was derived from it. The Aeruginosa ADI enzyme was purified from the SUMO-ADI fusion protein using the same procedure (Pa-ADI).

Cell culture. All cell lines were cultured in RPMI 1640 medium (Gibco / Life Technologies Inc.) supplemented with 10% fetal bovine serum (FBS), 2% penicillin / streptomycin, and 2% glutamine. All cells were grown in a humidified 37 ° C. incubator with 5% CO ₂ .

Cytotoxicity assay. The MTT [3- (4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide)] assay was performed by Yamada T et al., Proc Natl Acad Sci USA 99: 14098-103 (2002) and Punj V et al., Oncogene 23: 2367-78 (2004) was used to measure the cytotoxicity of wt and mutant azurin. Briefly, the water-soluble tetrazolium salt, [3- (4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide)] is metabolized to water-insoluble formazan by intact mitochondrial dehydrogenase. The formazan is then solubilized by adding 2-propanol + 40 mM HCl and incubating for 1 hour. 3 × 10 ⁴ cells were treated with various concentrations of azurin, Laz, or Pa-CARD, and 5-aza-2-deoxycytidine (DAC) as a positive control. Cell viability was assessed based on formazan formation detected spectrophotometrically with an optical density of 570 nm.

Cell cycle analysis. HL60 and K562 cells (3 × 10 ⁵ cells per well were seeded in a 24 well plate) were treated with wild type azurin, Pa-CARD, or Laz (5 and 10 μM) for 48 hours. Cells were washed twice with PBS and fixed with 70% methanol for 24 hours at -20 ° C. The fixed cells were washed twice with PBS, stained with 50 μg / ml propidium iodide in PBS containing 20 μg / ml RNase A for 30 minutes in the dark, and by flow cytometry (Becton Dickinson). analyzed. The percentage of cells in various phases of the cell cycle was determined with the MODFIT LT software.

  Western blotting. In the case of immunoblotting, the cytoplasmic fraction and the nuclear fraction are isolated using NE-PER extraction reagent (Pierce) using complete protease and phosphatase inhibitor (Sigma) according to the manufacturer's protocol. Released. The protein concentration of the cell lysate was measured using the Bradford Bio-Rad assay. Cell lysate proteins were separated by SDS-PAGE and transferred to a PVDF membrane for immunoblotting. Block the membrane in Tris buffered saline (0.15 M NaCl, 0.05 M Tris-HCl [pH 8.0], 0.05% Tween 20) containing 5% nonfat dry milk (Difco) Incubated with antibody (TBST, recommended dilution in 5% nonfat dry milk) at 4 ° C overnight with gentle agitation. After washing 3 times with TBST for 5 minutes each time, with horseradish peroxidase-labeled goat anti-rabbit or rabbit anti-mouse antibody (TBST, 1: 3000 in 5% nonfat dry milk) purchased from Zymed Laboratories (San Francisco, Calif.) The membrane was probed for 1 hour at room temperature. After an additional washing step, the membrane was incubated with chemiluminescent substrate for 1 minute at room temperature. To remove unbound antibody between each antibody incubation, the membrane was incubated in Restore Western blot strip buffer (Pierce) according to the manufacturer's protocol and probed again. Anti-B actin was from Sigma. AKT, Weel, and anti-phospho-AKT-S473 antibodies were from Cell Signaling Technology (Beverly, Mass.). Secondary antibodies, horseradish peroxidase labeled goat anti-rabbit and rabbit anti-mouse were from Zymed Laboratories (San Francisco, CA).

  Confocal microscope. Azurin and Laz proteins were conjugated with the fluorescent chemical AlexaFluor 568 (Molecular Probes) and incubated with HL60, K562, or normal peripheral blood mononuclear cells for 1 hour. The entry of fluorescent chemically labeled peptides or proteins into cells is described in Yamada T et al., Cell Microbiol. 7: 1418-31 (2005), observed with a confocal microscope (LC510, manufactured by Carl Zeiss).

Example 9 Effects of azurin and Laz on leukemia cells The bacterial proteins Laz and azurin induce cytotoxicity, thereby reducing the viability of two leukemia cell lines, the AML cell line HL60 and the CML cell line K562. We investigated the ability to do. Viability of the cell lines HL60 and K562 was measured by their ability to metabolically reduce MTT to a purple formazan product after 24, 48 and 72 hours of treatment with 10 μM Laz or azurin (FIG. 1A and B). 10 μM protein was found to be very cytotoxic and reduced cell viability by more than 90%. Laz is a Neisseria protein having an azurin moiety. Has substantial sequence identity with Erginosaazurin, but has an additional 39 amino acid peptide called the H8 epitope.

  To determine whether this H8 epitope modulates the anticancer activity of azurin, H. et al. The 8 epitope Cloning at the N-terminus and C-terminus of Erginosaazurin yielded H8 azurin (H8-Azu) and Azu-H8. Hong CS et al., Cell Cycle 5: 1633-41 (2006). These two Laz-like proteins were also tested against leukemia cell lines. To determine a lower effective dose, K562 cells were treated with various concentrations of protein at a final concentration of 3.75 nM or higher, and HL60 cells were treated with a final concentration of 1-10 μM. All bacterial proteins showed a dose-dependent cytotoxic effect on the two leukemia cell lines. Significant cell viability reduction was observed even at nanomolar (nM) protein concentration, and the viability peak was reduced with 5 μM protein (FIGS. 8C and D). This cytotoxic effect was evident after 24 hours of treatment and all proteins exerted a cytotoxic effect in both HL60 and K562 cells (FIGS. 8A and B).

  The cytotoxic effects of Laz and azurin were also compared with a known leukemia drug, 5-aza-2'-deoxycytidine (DAC). 5-Aza-2'-deoxycytidine is a DNA methylation inhibitor that acts by reactivating tumor suppressor genes that are silenced by DNA hypermethylation. Reactivation of tumor suppressor genes leads to cell cycle arrest and apoptosis. The viability reduction effect of each bacterial protein was significantly better than that of DAC (FIGS. 8C and D). Azurin or Laz is cytotoxic to cancer cells and not cytotoxic to normal cells. This cytotoxicity was shown not to be due to cellular contaminants such as endotoxin, but to the peptide itself.

  In order to identify possible mechanisms of cell death induced by Laz and azurin, a clear global morphological change was sought with a fluorescence microscope. We looked for changes that could help identify cell necrosis, apoptosis, and cell differentiation, processes that ultimately lead to cell death. Changes such as granulation or cell hypertrophy are indicators of possible differentiation or necrosis and ultimate cell death in the case of both acute and chronic myeloid leukemia cells. Fluorescence microscopy results show that Laz and azurin can induce cell differentiation of HL60 and K562 cells after a 210-hour incubation 210 at a concentration of 10 μM (FIG. 9).

Example 10 Laz and azurin entry into HL60 and K562 cells The ability of azurin and Laz to enter HL60 and K562 cells was investigated using confocal microscopy and AlexaFluor 568-conjugated red fluorescent azurin or Laz. These two proteins were thought not to enter normal PBMC (FIGS. 10A and B, panel 1), but Laz and azurin were K562 cells (FIGS. 10C and D, panel 1) and HL60 cells (FIGS. 10E and 10). F, both panel 1) entered significantly. Azurin entry was shown to be less (FIGS. 10D and F, panel 1). The cytotoxic level of azurin from 1.0 to 2.5 μM (FIGS. 8C and D) may be related to its entry level into HL60 cells or K562 cells.

  Investigate the entry of H8-bound azurin, Azu-H8, and H8-Azu, especially at lower concentrations such as 1.0-2.5 μM, but similar or somewhat higher than azurin, but with cytotoxicity comparable to Laz It has been demonstrated (Figures 8C and D). Both Azu-H8 and H8-Azu show higher levels of entry than azurin, comparable to the entry of Laz into K562 cells (Figure 10, panel 2), allowing entry of azurin or Laz into leukemia cells It was demonstrated that the H8 epitope plays a role.

Example 11 Laz causes cell cycle arrest in K562 cells by increasing Weel protein levels and decreasing phosphorylated AKT of Laz, azurin, and / or 5-aza-2-deoxycytidine (DAC) on cell cycle progression The effect was assessed using flow cytometric analysis of K562 cells, and K562 cells were considered very sensitive. The effects of Laz, azurin, and DAC on K562 cell cycle arrest were observed (FIG. 11). Laz, azurin, and DAC can arrest K562 cells in the G2 / M phase. Cell cycle arrest at the G2 / M phase is often associated with induction of apoptosis and cell death, as observed with azurin and Laz. It is conceivable that proteins such as Laz and azurin can affect the level and / or activity of G2 cell cycle regulators of leukemia cells.

  The effects of Laz on cell cycle regulators that mediate G2 phase arrest, including Weel and phosphorylated AKT, were analyzed. The Weel protein is known to be a mediator of cell cycle arrest in the G2 / M phase. Many viral proteins have been shown to be able to arrest the cell cycle in the G2 phase by interacting with and affecting Weel activity in vivo. Treatment of K562 cells with 10 μM Laz led to increased Weel protein levels in both the cytoplasm and nucleus where Weel is known to exert its effects (FIG. 12). In contrast to K562 cells, HL60 cells showed a marked reduction in Weel protein levels. Inhibition of AKT phosphorylation at serine 473, and thus AKT activity, is also associated with G2 / M cell cycle arrest. Laz treatment was also associated with a marked decrease in AKT activity in HL60 cells, as evidenced by a decrease in phosphorylated AKT serine 473 levels (FIG. 12). Interestingly, the decrease in AKT-P-Ser473 levels was more prominent in HL60 cells than in K562 cells, and the change in nuclear Weel protein levels by Laz was more prominent in K562 cells.

Example 12 Pa-CARD, an anti-cancer bacterial protein, shows activity against leukemia cells.
M.M. Arginini ADI has been reported to inhibit the growth of cultured human lymphocytic leukemia cells by causing growth arrest in the G1 / S phase and leading to apoptosis. Gong H et al., Leukemia 14: 826-9 (2000). ADI is believed to exert its action by depleting arginine and converting arginine to citrulline and ammonia. Arginine is a non-essential amino acid because mammalian cells can re-synthesize arginine from citrulline. However, certain cancers such as hepatocellular carcinoma, melanoma, or renal cell carcinoma do not express argininosuccinate synthetase, an enzyme important for arginine synthesis from citrulline, in vivo, and as such The cells are characterized by sensitivity to arginine deficiency due to ADI action.

  M.M. An interesting feature of Arginini ADI is the identifiable structural similarity of its N-terminal portion to the mammalian CARD protein. Eukaryotic, mainly mammalian, CARD proteins possess a domain called the caspase recruitment domain that is a protein-protein interaction motif. The presence of the CARD domain in the CARD-bearing protein allows for the formation of complexes through CARD interactions, leading to activation / inhibition of processes that lead to cell signaling or cell death mediated by NF-kβ. CARD-carrying proteins such as caspases are not only involved in the induction of apoptosis, but are also involved in the production of inflammatory cytokines. Thus, caspases such as caspase 1 are involved in proteolytic processing of cytokines such as IL-1B, allowing it to be released from cells in an activated form. Since IL-1B promotes angiogenesis and is overproduced in many tumors, CARD-bearing proteins such as caspase 1 are considered major targets for the development of inhibitors as anticancer agents.

  M.M. called Ma-ADI and Pa-ADI, respectively. Arginini and P.I. The gene sequence and crystal structure of ADI from Aeruginosa are known, exhibit about 27% sequence identity, and have a common CARD motif at the N-terminus (FIG. 13A). ADI is a guanidine modifying enzyme (GME) that has the ability to catalyze nucleophilic substitution reactions at guanidinium carbon atoms, similar to other family members such as dimethylarginine dimethylaminohydrolase (DDAH) and arginine: glycine amidinotransferase (AGAT). ) Super family member. However, by aligning the structural features of GME superfamily members, it was shown that the CARD motif is present only in ADI and not in other members (FIG. 13A), indicating mobilization of the CARD domain by ADI, Suggests its potential role as a binding anticancer agent.

  P. CARD having the structural characteristics of CARD (amino acids 75-225). The region from Aeruginosa ADI was cloned in-frame with the SUMO portion derived from the plasmid pET-SUMO to obtain a SUMO-CARD fusion protein. The fusion protein was purified using a Ni-NTA column and the CARD polypeptide was isolated as a homogeneous 17 kDa protein (FIG. 13B). Thereafter, P.I. The N-terminal CARD domain of Aeruginosa ADI; Because it is derived from ADI derived from Aeruginosa (Pa-ADI, 46 kDa, FIG. 13B), the activity of this protein, called Pa-CARD, was reduced to solid tumor cell lines, fibrosarcoma HT-1080, breast cancer MCF-7, and leukemia cells. Determined using system HL-60.

  Pa-CARD showed marked cytotoxic activity against HT-1080, MCF-7, and leukemia HL60, and other cancers (FIG. 13C), but cells against normal breast cells such as normal fibroblasts and MCF-10A Toxic activity was lower (data not shown). M.M. Since Arginini ADI has been reported to inhibit human leukemia cell proliferation by inducing cell cycle arrest, the ability of Pa-CARD to induce cell cycle arrest of cancer cells was also measured. As previously reported for Laz and azurin (Figure 11), Pa-CARD showed a marked arrest of the cell cycle (data not shown). Similar to Laz, Pa-CARD enhanced Weel protein levels in the nuclear fraction of K562 cells, but did not show such activity in HL60 cells (FIG. 12). However, in the nuclear fraction of HL60 cells, and the cytoplasmic fraction of K562 and HL60 cells, AKT-P-S473 levels are significantly reduced by Pa-CARD (FIG. 12), and cell cycle arrest of HL60 cells by Pa-CARD. May be mediated by reduced phosphorylation of serine at position 473 of AKT, whereas in K562 cells such an effect is also to some extent phosphorylation of serine at position 473 of cytosolic AKT. Affected but suggested that it is mainly mediated by enhancement of nuclear Weel protein levels. Laz mimics Pa-CARD in mediating such effects (enhancement of nuclear Weel at K562 and cytoplasmic AKT-S473 depletion in HL60 cells), suggesting a common mechanism of action.

Example 13 Substances and Methods for Ovarian Cancer Research Chemicals, Reagents, and Cell Culture: MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) and propidium iodide are from Sigma Purchased from. Pre-stained gels were obtained from Biorad. The pET SUMO kit and SUMO protease were purchased from Invitrogen. Human cancer and normal cell lines SK-OV3, HOSE6-3, MCF-7, and HT1080 cells are all derived from the University of Illinois Chicago (UIC) Department of Surgical Oncology, Chicago's Conservative Culture Collection. Cells in MEM with eagle salt containing 2 mM L-glutamine, 0.1 mM MEM essential amino acids supplemented with 10% heat inactivated fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin In culture. All cells were grown at 37 ° C. with 5% CO ₂ .

  Protein cloning, expression, and purification: were performed as described in Example 8 herein.

  MTT-cytotoxicity assay: A 3- (4,5-dimethylthiazol-2-yl-2,5-diphenyl) tetrazolium bromide (MTT) assay is performed as described in Example 8 to produce cells against cancer cells. Toxicity was determined.

  Caspase-3 / 7 activity assay: The test was performed by Yamada, T .; Et al., Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 14098-14103, using a caspase-Glo3 / 7 (Promega) reagent in a 96-well plate.

Cell cycle arrest by flow cytometry: SKOV3 cells were seeded in 6-well plates at a volume of 2 ml per well with 10 ⁶ cells per well. Cell pellet (> 1 × 10 ⁶ cells) was recovered, fixed with 1 mL of 70% ethanol for 60 minutes at 4 ° C., washed with 1 mL of PBS, and containing 0.5 mg of RNAse A (Sigma) Resuspend in 400 □ L PBS. After gently mixing, 100 μL equivalent of propidium iodide (1 g / L PBS) (manufactured by Sigma) was added. Cells were incubated in the dark for 15 minutes at room temperature and then stored in the dark at 4 ° C. for flow cytometric analysis. For each sample, at least 1 × 10 ⁴ cells were analyzed for DNA content using a Becton-Dickinson FACS Calibur flow cytometer. The distribution of cells in sub-G1, G1, S, and G2-M phases was determined using Multi Cycle AV software.

  Microarray of TLR pathway and NF-kB signaling genes: SKOV3 cells (100,000 cells per well in a volume of 2 ml per well) were seeded in 6-well plates. After treatment with protein, cells were harvested with lysis buffer. Samples were sent to Superarray Biosciences for microarray for toll-like receptor genes and NF-kB signaling genes.

  TUNEL assay: This technique is described in Yamada, T .; Et al., Proc. Natl. Acad. Sci. U. S. A. , 2002, 99, 14098-14103, by using a terminal deoxynucleotide transferase to enzymatically incorporate fluorescein-dUTP at the 3 ′ end of the DNA fragment, Identify the contained nuclei.

Example 14 ADI has a putative CARD-like domain Secondary structure comparison showed that there was 27% sequence identity between Pa-ADI and Ma-ADI. Structural alignment using the MolMol program and the VAST algorithm clearly demonstrates the presence of both the putative CARD-like and catalytic domains of Pa-ADI and Ma-ADI (FIGS. 14A and B). Both Pa-ADI and Ma-ADI are characteristic 5 found in all proteins of the guanidine-modifying enzyme superfamily of which dimethylarginine dimethylaminohydrolase (DDAH) and arginine: glycine amidinotransferase (AGAT) proteins are members. It has two ββαβ subunits (FIG. 14B). However, unlike DDAH and AGAT, both Pa-ADI and Ma-ADI are correctly inserted between the first and second ββαβ subdomains and contain a typical “Clip-on-fan” portion. It has a unique 85-residue 5-alpha helix bundle domain that results (FIGS. 14B and 14C). It is this region that shows structural similarity to mammalian CARD containing proteins. Since CARD-like domains are present only in Pa-ADI and Ma-ADI, but not in other members (FIG. 14B), mobilization of CARD domains by ADI is not mobilized by other members of the family. It is likely that ADI has been given its unique anti-cancer properties.

Example 15. Pa-CARD exhibits anticancer activity CARD-containing mammalian protein has various functions such as helicase, kinase, and caspase activity. The CARD domain itself is not known for any other activity other than mediating the formation of larger protein complexes through CARD interactions. Assuming that bacteria compete with cancer cells for the same nutrients inside the body as a source of their survival and growth, it is possible to assume that the bacteria acquire a CARD domain from a mammalian source and interfere with cancer growth. . To test this hypothesis, a putative CARD domain (amino acids 75-225) with a hydrophobic pocket derived from Pa-ADI was cloned and purified to investigate its function as an anticancer agent and any possible ADI-like activity. The gene sequence of Pa-ADI including amino acids 75-225 and full-length ADI protein was cloned in-frame with the SUMO part derived from plasmid pET-SUMO (Invitrogen) as described in Example 8. , SUMOCARD fusion and SUMO-ADI protein were obtained. These recombinant proteins were purified in a single step using a Ni-NTA column and then cleaved with SUMO protease. The homogeneity and SDS-PAGE migration pattern of these two bacterial proteins are shown in FIG. 15A. As expected, purified Pa-ADI migrated as a single band of Mw 46 kDa, while Pa-CARD migrated as a 17 kDa protein.

  Next, the arginine deiminase activity of these two proteins was determined. As expected, Pa-ADI showed strong enzyme activity, but the important amino acids Cys-406 and His-278 of the catalytic triad of Pa-ADI are deleted in Pa-CARD, so Pa- CARD did not show such activity (FIG. 15B). The presence of such activity has not been evaluated with Pa-ADI or Pa-CARD. Arginini ADI (Ma-ADI) is known to have anti-cancer activity. The growth inhibitory activity of Pa-ADI and Pa-CARD was investigated against a series of human cancer cells such as fibrosarcoma HT-1080, breast cancer MCF-7, and ovarian cancer SKOV-3 (FIG. 16A). Both Pa-ADI and Pa-CARD showed significant inhibitory activity against all cancer cell types. Most interestingly, Pa-CARD showed higher growth inhibitory activity than Pa-ADI on these cancer cells (FIG. 16A). Since Pa-ADI and Pa-CARD showed somewhat higher activity against ovarian cancer SKOV-3 cells, the cytotoxicity of various concentrations of Pa-CARD (FIG. 16B) and Pa-ADI (FIG. 16C) was It was determined as a function of the incubation time with these cells. Cytotoxic activity was evident during the 24 hour incubation, but the activity increased significantly at higher concentrations (10-20 μM) during the 48 hour incubation. Similarly, Pa-CARD showed higher cytotoxicity in a concentration and time dependent manner compared to Pa-ADI.

Example 16 Pa-CARD has little inhibitory effect on normal cells. The cytotoxicity of Pa-ADI and Pa-CARD at a concentration of 20 μM was investigated against both ovarian cancer SKOV-3 and normal ovarian HOSE6-3 cells. In these studies, 20 μM azurin was used as a control because azurin exhibits anticancer activity against a range of cancers but cannot enter normal cells and exhibits little cytotoxicity against such cells. Added. The known anticancer agent “cisplatin” (cis-diamminedichloroplatinum (II); CDDP) was used as a positive control. The cytotoxic activity of these agents against HOSE6-3 normal ovary and SKOV-3 ovarian cancer cells is shown in FIG. 17A. Both Pa-ADI and Pa-CARD showed significant cytotoxic activity against SKOV-3, but showed little cytotoxicity against normal ovarian cells. Azurin also showed similar cell death characteristics. Both Pa-CARD and azurin showed lower cytotoxicity against normal HOSE6-3 cells than cisplatin, a commonly used drug. Furthermore, SKOV-3 cells treated with Pa-CARD and cisplatin show a somewhat higher cytotoxic effect (FIG. 17B) and some degree of additive effect compared to those treated individually.

Example 17. Pa-CARD induces apoptosis of cancer cells by caspase activation To determine the nature of cell death induced by Pa-CARD, and other bacterial proteins such as azurin and Laz are The degree of induction of apoptosis by Pa-CARD, Pa-ADI, and azurin in both ovarian cancer SKOV-3 and normal ovarian HOSE6-3 cells is measured in situ cell death. It was determined by TUNEL assay using a detection fluorescein kit (Roche). When SKOV-3 or HOSE6-3 cells were treated with DNase I, essentially all cells emitted green fluorescence (positive control, FIG. 18). When both SKOV-3 and HOSE6-3 cells were treated with 10 μM Pa-CARD, Pa-ADI, and azurin, apoptosis was clearly enhanced selectively in SKOV-3 cells. Also, when SKOV-3 cells were treated with Pa-CARD, the highest percentage of apoptosis was observed compared to Pa-ADI and azurin. Apoptotic cells tend to impair adhesion properties, so fewer cells are found on slides with a higher percentage of apoptotic cells (FIG. 18). HOSE6-3 cells only showed a green fluorescence under all conditions (Figure 18) and Pa-CARD induces apoptotic cell death mainly in cancer cells, but in normal cells Confirmed not to induce. Treatment with bovine serum albumin (BSA) or protein-free treatment (control) showed that there were few cells emitting green fluorescence in the field of view and many were blue (non-apoptotic cells) (FIG. 18, bottom row). ), Cancer or normal cells showed less spontaneous apoptosis unless treated with Pa-CARD, Pa-ADI, or azurin.

  To determine whether Pa-CARD induces cell death in ovarian cancer cells rather than normal ovarian cells by caspase 3 activation, the levels of caspase 3 / caspase 7 in SKOV-3 and HOSE6-3 cells Was measured in the absence or presence of Pa-ADI, Pa-CARD, cisplatin, and azurin. Caspase enzyme activity was determined with a caspase-Glo ™ 3/7 assay kit (Promega). Pa-ADI and Pa-CARD were used at two different concentrations, 10 and 20 μM. Both Pa-ADI and Pa-CARD induced caspase 3/7 activation in a dose-dependent manner, but only in ovarian cancer SKOV-3 cells. In normal ovary cells HOSE6-3, almost no activation was observed (FIG. 19).

Example 18 Pa-CARD Inhibits Cell Cycle at G2 / M Phase Mycoplasma arginini ADI (Ma-ADI) has previously been shown to inhibit hepatocellular carcinoma growth and induce cell cycle arrest and apoptosis of leukemic cells It is shown. It is known that the induction of apoptosis by caspase activation, including induction in liver cancer, leukemia, and other cancers, is often mediated by cell cycle arrest at the G2 / M phase.

  G1, S, and G2 levels were examined in cancer cells treated with Pa-ADI and Pa-CARD or untreated. When SKOV-3 cells were treated with Pa-ADI at a concentration of 10 μM for 24 hours, G1 phase cells decreased from 86.0% to 69.1%, while S and G2 phase cells were 7. It rose from 9 to 19.7 and from 6.1 to 11.2%. However, treatment with Pa-CARD under similar conditions reduced the G1 phase level of cells from 86.0 to 19.1%, whereas the S and G2 phases of the cells were 7.9 to 43, respectively. It increased to 5% and from 6.1 to 37.4% (data not shown), clearly showing that Pa-CARD significantly inhibited the cell cycle at G2 / M phase.

Example 19. Pa-CARD regulates the expression of NF-kB signaling pathway genes The effect of Pa-CARD in SKOV-3 cells was investigated by measuring the expression of several genes involved in the NF-kB signaling pathway. The goal was to identify candidate genes that could play a critical role in determining the fate of SKOV-3 cells after treatment with Pa-CARD protein. For this study, SKOV-3 cells were incubated with 10 μM Pa-CARD and azurin, respectively, for 48 hours, and controls were incubated without any treatment. Total RNA was isolated and quantitative real-time PCR microarray was performed. Multiples of control were calculated by normalizing with the expression of the housekeeping gene. Only changes greater than twice the control level are reported.

  A striking observation in the gene expression profile is that when SKOV-3 cells are treated with Pa-CARD, most genes in the NF-kB pathway, including Toll-like receptor (TLR), are overall compared to azurin. It was up-regulated (Table 3). This is due to Pa-CARD-mammalian CARD interaction in SKOV-3 cells. Mammalian CARD-bearing proteins are known to modulate the NF-kB signaling pathway and any interaction with Pa-CARD is likely to alter such gene expression characteristics as observed. . On the other hand, azurin is known to inhibit cancer cell growth through protein-protein interactions between p53 and receptor tyrosine kinases, rather than through regulation of the NF-kB pathway. Thus, these two bacterial anticancer proteins have two different mechanisms of action.

  The most marked up-regulation of genes by Pa-CARD in SKOV-3 cells is a 55-fold increase in expression of the gene encoding colony stimulating factor CSF2, also known as granulocyte macrophage colony stimulating factor (GM-CSF) Is accompanied. IL-12 gene expression is also stimulated approximately 3-fold under such conditions (Table 3). Western blotting data (not shown) confirmed that GM-CSF was overproduced (approximately 5 times) under such conditions. The level was lower than a 55-fold increase, presumably because GM-CSF is a secretable protein that leaves the cell. Other cytokines whose expression is upregulated during Pa-CARD treatment are IL-2, IL-8, IL-10, IL-1alpha, and IL-1beta (Table 3).

Table 3. Measurement of mRNA levels of Toll-like receptor (TLR) and NF-kB pathway genes using real-time PCR microarray: SKOV-3 cells were incubated for 48 hours with 10 μM CARD and azurin, respectively. As a control, one sample of SKOV-3 cells was left untreated. After incubation, cells were harvested and total RNA was extracted. Samples were analyzed using real-time PCR microarray analysis (Superarray Bioscience). Results were normalized to housekeeping genes. Bold numbers indicate up-regulation compared to untreated control samples and numbers with an asterisk ( ^* ) indicate down-regulation. A decrease in expression is indicated by a minus sign.

Claims (47)

  An isolated peptide comprising a caspase mobilization (CARD) -like domain capable of killing cancer cells.   The isolated peptide according to claim 1, which is derived from bacteria.   The isolated peptide according to claim 2, wherein the bacterium is Pseudomonas aeruginosa.   The isolated peptide according to claim 1, which is Pa-CARD.   2. The isolated peptide of claim 1 comprising SEQ ID NO: 27.   6. The isolated peptide of claim 5, consisting of SEQ ID NO: 27.   2. The isolated peptide of claim 1, wherein the cancer is selected from the group consisting of leukemia, ovarian cancer, fibrosarcoma, and breast cancer.   2. The isolated peptide of claim 1, which is chemically modified to increase or optimize its blood half-life.   Killing cancer cells by contacting the cells with one or more proteins selected from the group consisting of the isolated peptide of claim 1, Laz, H8-Azu, and Azu-H8. Method.   10. The method of claim 9, wherein the cancer cell is selected from the group consisting of leukemia cells, fibrosarcoma cells, ovarian cancer cells, and breast cancer cells.   10. The isolated peptide of claim 9, which also comprises contacting the cell with one or more cytotoxic agents capable of killing cancer cells.   12. The one or more cytotoxic agents are selected from the group consisting of cisplatin, Gleevec®, retinoic acid, 5′-aza-2′-deoxycytidine, and arsenic trioxide. the method of.   13. The method of claim 12, wherein the one or more cytotoxic agent is cisplatin.   12. The method of claim 11, wherein the cancer cell is contacted with the one or more cytotoxic agents at about the same time as the one or more proteins.   A method comprising administering to a mammalian patient suffering from cancer one or more proteins selected from the group consisting of the isolated peptide of claim 1, Laz, H8-Azu, and Azu-H8. .   16. The method of claim 15, wherein the cancer is selected from the group consisting of leukemia, fibrosarcoma, ovarian cancer, and breast cancer.   16. The method of claim 15, further comprising administering to the patient one or more cytotoxic agents capable of killing cancer cells.   18. The one or more cytotoxic agents are selected from the group consisting of cisplatin, Gleevec®, retinoic acid, 5′-aza-2′-deoxycytidine, and arsenic trioxide. the method of.   The method of claim 18, wherein the one or more cytotoxic agent is cisplatin.   18. The method of claim 17, wherein the one or more cytotoxic agents are administered at about the same time as the one or more proteins.   The cells were treated with azurin and Laz H. et al. A method comprising killing leukemia cells by contacting with a peptide comprising 8 regions.   The leukemic cells were treated with the azurin and Laz H. 24. The method of claim 21, wherein the peptides comprising the 8 regions are contacted simultaneously or nearly simultaneously.   In mammalian patients suffering from leukemia, azurin and Laz H. et al. Administering a peptide comprising 8 regions.   The patient was treated with the azurin and Laz H.H. 24. The method of claim 23, wherein peptides comprising 8 regions are contacted simultaneously or nearly simultaneously.   Laz, azurin, H.M. 8-Azu, Azu-H. 8. A method comprising inducing cell differentiation of a leukemia cell by contacting the leukemia cell with one or more proteins selected from the group consisting of the isolated peptide of claim 8 and claim 1.   Laz, azurin, H.M. 8-Azu, Azu-H. 8 and selectively entering the cancer cell by contacting the cancer cell with one or more proteins selected from the group consisting of the isolated peptides of claim 1, Is selected from the group consisting of leukemia cells and ovarian cancer cells.   Laz, azurin, H.M. 8-Azu, Azu-H. 8. A method comprising inducing cell cycle arrest of a cancer cell by contacting the cancer cell with one or more proteins selected from the group consisting of the isolated peptide of claim 8 and claim 1.   28. The method of claim 27, wherein the cancer cells are selected from the group consisting of leukemia cells, fibrosarcoma cells, breast cancer cells, and ovarian cancer cells.   28. The method of claim 27, wherein the protein increases Weel protein levels in the cell.   28. The method of claim 27, wherein the protein causes depletion of phosphorylated AKT-Ser-473.   28. The method of claim 27, wherein the protein increases Weel protein levels in the cell and causes depletion of phosphorylated AKT-Ser-473.   A method comprising inducing apoptosis of the cancer cell by caspase 3 activation by contacting the cancer cell with the peptide of claim 1.   34. The method of claim 32, wherein the cancer cell is an ovarian cancer cell.   A method comprising modulating expression of a NF-kB signaling pathway gene in the cancer cell by contacting the cancer cell with the peptide of claim 1.   35. The method of claim 34, wherein the cancer cell is an ovarian cancer cell.   An expression vector encoding the isolated peptide according to claim 1.   The expression vector according to claim 36, which encodes the isolated peptide according to claim 4.   A pharmaceutical composition comprising the isolated peptide of claim 1.   40. The pharmaceutical composition according to claim 38, further comprising a pharmaceutically acceptable carrier.   Azurin, Laz, H.M. 8-Azu, and Azu-H. 39. The pharmaceutical composition according to claim 38, further comprising a protein selected from the group consisting of 8.   40. The pharmaceutical composition of claim 38, further comprising one or more cytotoxic agents capable of killing cancer cells.   39. The pharmaceutical composition according to claim 38, wherein the pharmaceutically acceptable carrier is suitable for intravenous injection.   40. A method comprising administering to a patient suffering from leukemia a therapeutically effective amount of the pharmaceutical composition of claim 38.   44. The method of claim 43, wherein the pharmaceutical composition is administered to the patient in a manner selected from the group consisting of intravenous, topical, subcutaneous, intramuscular, oral, and intratumoral.   A kit comprising the pharmaceutical composition according to claim 38.   A nucleic acid molecule encoding the isolated peptide of claim 1. A nucleic acid molecule encoding the isolated peptide of claim 4.

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