JP3642755B2 - Drugs for gene therapy using anaerobic bacteria - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
  The present invention relates to an anaerobic bacterium belonging to the genus Bifidobacterium that is useful for gene therapy of solid tumors, and a medicine containing the same.To medicineRelated.
[0002]
[Prior art]
  Hypoxic sites are characteristic of solid tumors in animals (Int J Radiat Oncol Biol Phys. 1984; 10: 695-712) and occur frequently in various human solid tumors (New York: Fischer-Verlag Stuttgart; 1994: 219-232). In the tissue oxygen electrode (membrane test device that can measure dissolved oxygen) measurements performed in cancer patients, the median oxygen partial pressure in normal tissues is 24-66 mmHg, whereas in tumors the median is The oxygen partial pressure was less than 2.5 mmHg in a considerable proportion. Thus, gene therapy for solid tumors aimed at gene expression in hypoxic tumor cells has recently been studied (Nat Med. 1997; 3: 515-520). As a result, certain anaerobic bacteria, such as Clostridium or Bifidobacterium, are known to grow selectively in hypoxic sites of solid tumors when injected into veins. (Cancer Res. 1980; 40: 2061-2068, 1955; 15: 473-478).
[0003]
  Moreover, the effectiveness as a gene transport carrier is examined about microorganisms, such as Clostridia (Clostridia) and Salmonella (Gene Ther. 1997; 4: 791-796,1996; 3: 173-178, FEMS Microbiol Rev. 1995; 17: 357-364, Cancer Biother Radio.1996; 11: 145-153, Nat Biotechnol.1999; 17: 37-41). However, since these microorganisms are pathogenic in the human body, they are not necessarily safe gene transport carriers for gene therapy of solid tumors. In fact, Clostridium butyricum (Clostridium butyricum) Spore injection or Salmonella typhi (Salmonella typhi. ) Has been reported as an adverse reaction after oral administration (Eur J Cancer. 1967; 3: 37-41, J Clin Invest. 1992; 90: 412-420, Infect Immun. 1992; 60 : 536-541).
[0004]
  On the other hand, Bifidobacterium and Lactobacillus are Gram-positive bacteria found in the downstream or large intestine of humans and other animals, and are resident and non-pathogenic microorganisms. . In particular, Bifidobacterium is widely used in many Asian and Western countries for formulating fermentation of dairy products, and it is now generally accepted that the microorganism is not pathogenic. In addition, it is also known that the microorganisms have properties that promote host health as well as pathogenicity. Such beneficial properties include, for example, enhanced immune response (J Dairy Sci. 1991; 74: l187-1195), suppression of carcinogenesis (CancerRes. 1993; 53: 3914-3918), protecting the host from viral infections. (Lancet. 1994; 344: 1046-1049).
[0005]
  Thus, although the Bifidobacterium has attracted a great deal of attention in food science, medicine and industry, it has not been used much in gene therapy. In order to be able to take advantage of the potential of these microorganisms for gene therapy, details about basic biological phenomena such as cell biosynthesis, gene expression, protein secretion or genetics But because of the lack of efficient and reproducible systems for gene transfer and sufficient selection markers, little was known about the genetic characteristics of Bifidobacterium. is there.
[0006]
  However, in recent years, an easy-to-use and reproducible genetic transformation system of Bifidobacterium has been developed (Microbiology 1996; 142: 109-114, Biosci Biotechnol Biocem. 1997; 61: 1211-1212). However, the development of regulatory sequences including a promoter for expressing a protein encoded by the introduced gene at a high concentration has not been sufficient.
[0007]
[Problems to be solved by the invention]
  An object of the present invention is to contain an anaerobic bacterium belonging to the genus Bifidobacterium as a gene transport carrier that is effective for gene therapy of solid tumors and safer for humans, and the anaerobic bacterium described above. It is to provide a pharmaceutical product.
  Another object of the present invention is to provide a gene transport method for transporting DNA effective for gene therapy of solid tumors in an anaerobic environment using the anaerobic bacteria as a gene transport carrier, and the method It is another object of the present invention to provide a method for treating a solid tumor by expressing a protein encoded by the DNA using
[0008]
[Means for Solving the Problems]
  The inventors have (a) that human and animal solid tumors are hypoxic, and (b) Bifidobacterium is anaerobic and is difficult to grow in normal tissues. Known facts such as that it grows in tumor tissues in a natural environment, and (c) Bifidobacterium is less pathogenic than microorganisms such as Clostridia and Salmonella that have been conventionally used as gene transport carriers Based on the above, it was found that Bifidobacterium is used as a gene transport carrier. Furthermore, the present inventors have repeatedly investigated a genetic transformation system of microorganisms belonging to the genus Bifidobacterium, and as a result, microorganisms belonging to the genus Bifidobacterium, particularly Bifidobacterium longum (Bifidobacterium longumHistone-like DNA-binding protein (Biochimie 1990;72207-212) [hereinafter abbreviated as HU protein. The present inventors have found that the introduced gene can be efficiently expressed by incorporating a promoter and a terminator involved in the expression of the gene encoding] into the vector. The inventors of the present invention have further studied earnestly to achieve the above object, and have completed the present invention.
[0009]
  That is, the present invention provides (1) tumor tissue specific under anaerobic environment.A gene transport carrier capable of transporting (a) DNA encoding a protein having antitumor activity, or (b) DNA encoding a protein having activity of converting an antitumor substance precursor into an antitumor substance, , Bifidobacterium longum ( Bifidobacterium longum ) Having a promoter related to the expression of a gene encoding histone-like DNA binding protein (HU protein) derived from, and using the DNA represented by base numbers 1 to 192 of the base sequence described in SEQ ID NO: 1 as the promoter, Expression in which (a) DNA encoding a protein having antitumor activity is incorporated downstream of the promoter, or (b) DNA encoding a protein having activity to convert an antitumor substance precursor into an antitumor substance It is transformed with a vector and encodes the protein (a) DNA encoding the protein having antitumor activity in the tumor tissue, or (b) the protein having activity of converting the antitumor substance precursor into the antitumor substance. Gene transport carrier comprising Bifidobacterium longum capable of expressing DNA,
(2)The expression vector isInvolved in the expression of a gene encoding a histone-like DNA binding protein (HU protein) derived from Bifidobacterium longumRuta-MinatorHave(1)The gene transport carrier according to,
(3) The protein according to (1) or (2) above, wherein the protein having antitumor activity is interleukin-2.Gene transport carriers,
(4) a protein having antitumor activity isEndostatinAs described in (1) or (2) above,Gene transport carriers,
(5) A protein having an activity of converting an antitumor substance precursor into an antitumor substance is cytosine deaminator.InIt is characterized by beingThe gene transport carrier according to (1) or (2),
(6)(A) DNA encoding a protein having antitumor activity, or (b) Bifidobacterium longum capable of expressing a DNA encoding a protein having an activity of converting an antitumor substance precursor into an antitumor substanceIs Bifidobacterium longum 105-A / pBLES100-S-eCD (FERM BP-7274),The gene transport carrier according to,
(7) Said (1)-(6)The gene transport according to any one ofA medicine comprising
About.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
  The present invention has a gene encoding a substance having antitumor activity, and a microorganism belonging to the genus Bifidobacterium (hereinafter referred to as Bifidobacterium genus) whose activity of the substance having antitumor activity is higher than that of a parent strain. Abbreviated as fungus). The activity of the substance having antitumor activity is higher than that of the parent strain, for example, when the expression level of the substance is higher than that of the parent strain, when the Km value is improved compared to the enzyme that is the substance expressed in the parent strain, or in the parent strain Examples include a case where it is less decomposed than the substance to be expressed and consequently shows high activity. Whether or not the activity of the substance having antitumor activity is higher than that of the parent strain can be easily examined using a screening method known per se. For example, a method of culturing Bifidobacterium in an appropriate medium and measuring the activity (expressed amount, enzyme activity, etc.) of the produced substance having antitumor activity by a known method can be mentioned.
[0011]
  The substance having antitumor activity may be a known substance regardless of its mechanism as long as it has antitumor activity. Here, examples of the anti-tumor activity include an activity of preventing or suppressing the generation, maturation, proliferation or spread of tumor cells or tissues, or an activity capable of degenerating tumor cells or tissues. The tumor includes, for example, carcinoma or sarcoma. However, the substance having antitumor activity in the present invention is usually a polypeptide or a protein whose structure can be encoded by a DNA base sequence.
[0012]
  Specific examples of the substance having antitumor activity in the present invention include cytokines. Specifically, cytokines having antitumor activity include interferon (IFN) -α, β, γ, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL) -1α, IL-1β, IL- 2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, tumor necrosis factor (TNF) -α, lymphotoxin (LT ) -Β, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), macrophage migration inhibitory factor (MIF), leukemia inhibitory factor (LIF), T cell activation costimulator B7 ( CD80) and B7-2 (CD86), kit ligand, oncostatin M and the like. Of these, IL-2 is preferred. Two or more of these may be combined. For example, a combination of IL-6 and TNF-α, IFN-α, IFN-β or IFN-γ, a combination of TNF-α and IFN-γ, or a combination of anti-Fas and IFN-γ is preferable. Alternatively, substances having antitumor activity include angiogenesis inhibitors such as Endostatin, Angiostatin, Kringles 1,2,3,4,5 (Kringles 1,2,3,4,5). It is done.
[0013]
  The present invention has a gene encoding an enzyme capable of converting an antitumor substance precursor having low toxicity to human livestock into an antitumor substance (hereinafter abbreviated as a convertase), and is a tumor in a substantially anaerobic environment. Provided is a microorganism belonging to the genus Bifidobacterium that can express the enzyme only in tissues. As the antitumor substance, a known substance having antitumor activity may be used. Antitumor activity is the same as described above. However, the antitumor substance precursor needs to have low toxicity to human livestock. The antitumor substance precursor may be inactive. An inactivated form means a substance that is converted into an active form by a converting enzyme and exhibits antitumor activity. The converting enzyme can be appropriately selected depending on the combination of the antitumor substance precursor and the antitumor substance. The converting enzyme may be one kind of enzyme or a plurality of kinds of enzymes, but preferably one kind of enzyme.
[0014]
  Any combination of an antitumor substance precursor, an antitumor substance and a convertase used in the present invention can be used in the present invention as long as it is a known combination. For example, a combination using 5-fluorocytosine (5-FC) as an antitumor substance precursor, 5-fluorouracil (5-FU) as an antitumor substance, and cytosine deaminase as a converting enzyme is specifically mentioned. In addition, the precursor of an antitumor substance is 5-aziridino-2,4-dinitrobenzamide (5-aziridino-2,4-dinitrobenzamide) [CB1954], An alkylating agent in which an antitumor substance is known to cause a bridge-like bond to double-stranded DNA, and a combination using nitroreductase as a converting enzyme. In addition, a combination using ganciclovir as an antitumor substance precursor, its metabolite as an antitumor substance, and herpes simplex virus type 1 thymidine kinase [HSV1-TK] as a converting enzyme can be mentioned. . Furthermore, an anti-tumor substance is modified with glucuronidation, glycine conjugation, or lysine conjugation to form a precursor (including an inactivated form) that is less toxic to the human body, and an enzyme that demodifies the precursor as a converting enzyme. It can also be used. As the enzyme for demodifying the precursor, a known enzyme may be used. For example, a combination of a glucuronidated antitumor substance precursor and β-glucuronidase as a converting enzyme may be mentioned.
[0015]
  Any known strain belonging to the genus may be used as the genus Bifidobacterium used in the present invention as long as it is anaerobic. Specifically, Bifidobacterium addressensetis (Bifidobacterium adolescentis), Bifidobacterium longum (B.longum), Bifidobacterium bifidum (B.bifidum), Bifidobacterium pseudolongum (B.pseudolongum), Bifidobacterium thermophilum (B.thermophirum), Bifidobacterium brive (B.breve), Bifidobacterium infantis (B.infantis) And the like. Among them, Bifidobacterium adrescentis (which is known to be resident in the human intestine regardless of age)B.adolescentis), Bifidobacterium longum (B.longum), Bifidobacterium bifidum (B.bifidum), Bifidobacterium infantis (B.infantis), Preferably Bifidobacterium longum (B.longum)〔Less than,B.longumIt may be abbreviated as fungus]. These resistant strains, mutant strains, and the like may also be used. These bacteria are either commercially available or easily available from depositary institutions. For example, the deposit number is Bifidobacterium longum (B.lomgum) ATCC-15707, Bifidobacterium bifidum (B.bifidum) ATCC-11863, Bifidobacterium infantis (B.infantis) Is ATCC-15697.
[0016]
  Among the above-mentioned Bifidobacterium, there are strains that can produce the above-mentioned substances having antitumor activity or convertases, and such strains can be suitably used as a gene transport carrier in the present invention. Specifically, for example, it produces a cytosine deaminase that can convert 5-FC to 5-FU.B.longumExamples include bacteria. Whether the above-mentioned substance having antitumor activity or a strain capable of producing a converting enzyme is determined by detecting a substance having antitumor activity or a converting enzyme or adding an antitumor substance precursor using a known screening method. It can be easily determined by detecting whether an antitumor substance is detected when the bacteria are cultured in the prepared medium.
[0017]
  The strain that cannot produce the substance having the antitumor activity or the converting enzyme is preferably used as a gene transport carrier in the present invention by introducing the DNA encoding the substance having the antitumor activity or the converting enzyme as follows. Can be used. In addition, the following basic operations of genetic engineering or bioengineering are described in commercial experiments such as Gene Manual Kodansha, Takagi Yasutaka edited by Gene Manipulation Experiment Method, Molecular Cloning, Cold Spring Harbor Laboratory ( Cold Spring Harbor Laboratory (1982), Molecular Cloning 2nd Edition (Molecular C1oning, 2nd ed.) Cold Spring Harbor Laboratory (1989), Methods in Enzymolology (Methodsin Enzymol) .),194(1991), a separate experiment in experimental medicine, a genetic experiment method using yeast Yodosha (1994) and the like.
[0018]
  First of all, it is necessary to obtain a DNA encoding the substance having the antitumor activity or the convertase. The above DNA can be easily obtained from known base sequence information. For example, it can be obtained from known base sequence information by chemical synthesis using a known method. Examples of the chemical synthesis method include a method of chemically synthesizing with a DNA synthesizer such as a DNA synthesizer model 392 (manufactured by Perkin Elmer Co., Ltd.) using the phosphoramidite method. In addition, primers based on the 5 ′ end and 3 ′ end base sequences of the base sequence are prepared, and cDNAs synthesized from mRNAs contained in tissues or cells of various organisms or cDNA selected from a cDNA library are used as templates. Said DNA can also be acquired by amplifying DNA using PCR method [PCRProtocols, AcademicPress (1990)]. Furthermore, based on the known nucleotide sequence information, colony high is applied to cDNA or cDNA library synthesized from mRNA contained in tissues or cells of various organisms using DNA or polynucleotide chemically synthesized or partially synthesized as a probe. The above DNA can also be obtained by performing hybridization or plaque hybridization (Molecular Cloning Second Edition).
[0019]
  The above DNA can also be easily obtained from known amino sequence information. As a method of obtaining the above DNA from known amino sequence information, a method known per se may be used. Specifically, for example, a method for amplifying a target DNA from the cDNA library or the like by a PCR method using a synthetic DNA primer having a partial base sequence of a DNA encoding a known amino sequence, or an appropriate vector And a method of screening by hybridization between the DNA incorporated in the above and a substance (antiprotumor activity) or a DNA fragment encoding a part or the entire region of the converting enzyme or a synthetic DNA labeled (probe).
[0020]
  Antitumor activity or convertase activity is known, but neither the substance having the antitumor activity or the amino acid sequence of the convertase nor the base sequence of the DNA encoding the substance having the antitumor activity or the convertase is known. In this case, as a method for obtaining the substance having the antitumor activity or the DNA encoding the enzyme, an expression cDNA library is prepared from an organism in which the antitumor activity or the enzyme activity is confirmed according to a known method or the like. And a method of screening individual cells constituting the library using the antitumor activity or the enzyme activity as an index to obtain a cell having a substance having the antitumor activity or a DNA encoding the convertase. It is done. Further, the substance having the antitumor activity or the converting enzyme is purified by a combination of methods known per se, and the amino acid sequence at the N-terminal of the substance having the antitumor activity or the converting enzyme is analyzed according to a known method, Using a synthetic DNA having a DNA base sequence encoding an amino acid sequence as a probe, and obtaining a substance or convertase having antitumor activity also by performing hybridization with a cDNA library or the like as described above Can do.
[0021]
  More specifically, the DNA encoding cytosine deaminase encodes plasmid pAdex1CSCD (RIKEN Genebank RDB No. 1591) containing DNA encoding cytosine deaminase derived from E. coli, or also encodes cytosine deaminase derived from E. coli. It is preferable to use one isolated from plasmid pMK116 containing DNA (DA Mead et al., Protein Engineering 1: 67-74 (1986)). Nitroreductase isE.coliIt is preferable to use one isolated from B. The amino acid sequence is described in Biochem Pharmacol; 44: 2289-2295, and DNA encoding nitroreductase can be easily obtained by the above method based on the amino acid sequence.
[0022]
  In the present invention, in addition to DNA encoding a substance having an antitumor activity or a convertase obtained based on the above-mentioned known nucleotide sequence information or amino acid sequence information, DNA that hybridizes with the DNA under stringent conditions Can be used. That is, since there are generally multiple types of genetic code for one amino acid, even a DNA having a base sequence different from a known base sequence or a base sequence based on a known amino acid sequence may be a substance having antitumor activity or If the convertase can be expressed, it can be used in the present invention.
[0023]
  DNA that can hybridize under stringent conditions means DNA obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using the above DNA as a probe. Specifically, using a filter on which colony or plaque-derived DNA is immobilized, about 0.7 to 1. After hybridization at about 65 ° C in the presence of about OM sodium chloride, about 0.1 to 2 times the concentration of SSC solution (the composition of the 1 time concentration of SSC solution is 150 mM sodium chloride, 15 mM citric acid DNA which can be identified by washing the filter under about 65 ° C. condition. Hybridization conforms to the method described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, DNA Cloningl: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. Can be done. Specifically, the hybridizable DNA is at least about 60% or more homologous to the base sequence of the DNA encoding the substance having the antitumor activity or the convertase obtained based on the known base sequence information or amino acid sequence information. DNA having a sexiness, preferably a DNA having a homology of about 80% or more, more preferably a DNA having a homology of about 95% or more.
[0024]
  The present invention has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the substance having the antitumor activity or the convertase, and the precursor of the antitumor activity or antitumor substance is anti-tumored. A protein or polypeptide having an action capable of being converted into a tumor substance can also be used. Such proteins or polypeptides are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. , USA,79, 6409 (1982), Gene,34, 315 (1985), Nucleic Acids Research,13, 4431 (1985), Proc. Natl. Acad. Sci USA,82, 488 (1985), etc., using a site-directed mutagenesis method described above, a deletion that can be carried out by introducing site-directed mutagenesis into DNA encoding the substance having the antitumor activity or the convertase, The number of amino acids to be substituted or added is not particularly limited, but is preferably 1 to several tens, particularly 1 to several amino acids.
[0025]
  Secondly, a recombinant DNA having the above-described substance having antitumor activity obtained as described above or a DNA encoding a convertase is prepared. In the present invention, the recombinant DNA is preferably an expression vector. An expression vector can be produced, for example, by cutting out a target DNA fragment and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. As a DNA fragment to be inserted into an expression vector, for example, the above-mentioned substance having antitumor activity or DNA encoding a convertase may be used as it is or after digestion with a restriction enzyme or adding a linker as desired. Can do. The DNA fragment may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation stop codon on the 3' end side. These translation initiation codon and translation termination codon can be added to the above-mentioned substance having antitumor activity or DNA encoding the convertase using an appropriate synthetic DNA adapter.
[0026]
  The above expression vector is usually one obtained by adding a regulatory sequence to the cloning vector described below so that the substance having the antitumor activity or the converting enzyme according to the present invention is expressed or is advantageous for the expression. Each regulatory sequence may be endogenous or foreign to the cloning vector. Such regulatory sequences include, but are not limited to, promoters, leaders, propeptide sequences, enhancers, signal sequences, selectable markers, and terminators. Among them, the regulatory sequences preferably include at least a promoter and evening light. The regulatory sequence may be provided with a linker (restriction enzyme cleavage site) so as to facilitate ligation with a substance having antitumor activity or DNA encoding a convertase, and ligation between the regulatory sequences.
[0027]
  In the present invention, the promoter and terminator may be Bifidobacterium, particularlyB.longumIt is particularly preferable to use a promoter and terminator that are involved in the expression of the HU gene (SEQ ID NO: 1) that is originally highly expressed in bacteria. Specifically, the DNA represented by each of the base numbers 1 to 192 of the base sequence described in SEQ ID NO: 1 as a promoter, and the base numbers of 472 to 600 of the base sequence described in SEQ ID NO: 1 as an evening terminator, respectively. It is preferred to use the DNA represented. An expression vector having a promoter and terminator involved in the expression of the HU gene isB.longumThe HU gene is excised from the DNA of the fungus with a restriction enzyme, incorporated into the cloning vector described below, and further, the substance having the antitumor activity or the convertase is encoded downstream of the promoter involved in the expression of the HU gene. It is particularly preferable to prepare by incorporating DNA or the like. By using the promoter and terminator involved in the expression of the HU gene, the substance having the antitumor activity or the convertase can be efficiently expressed. The HU gene isolation method isB.longumRestriction enzyme of chromosomal DNA of fungusHinThe method of digesting with dIII is mentioned.
[0028]
  More specifically, the following aspects are mentioned.B.longumRestriction enzyme of chromosomal DNA of fungusHinDigest with dIII and purify with phenol and ethanol precipitation. In addition, pBR322 (Takara Shuzo)HinDigest with dIII, purify in the same way after dephosphorylation. Each DNA is ligated to obtain recombinant DNA. Next, using the recombinant DNAE.colimH3 [Gene,45, 37 (1986)] is transformed according to a conventional method to obtain a transformant resistant to ampicillin and exhibiting sensitivity to tetracycline. Plasmid DNA is extracted from the obtained transformant according to a conventional method, and the plasmid DNA is extracted according to a conventional method.E.coliYK2741 strain [Gene,89, 133 (1990)] and transformation of the strain. Since the YK2741 strain lacks the HU gene and the IHF (Integration Host Factor) gene, it is a strain that exhibits low-temperature sensitivity, and is applied to ampicillin-containing agar medium and cultured at 27 ° C. A converter can be selected. Next, the transformant of the YK2741 strain obtained above is further cultured, a plasmid retained by the strain is extracted by a conventional method, and the plasmid DNA is extracted according to a conventional method.E.coliYK1340 [J. Mol. Biol. ,204, 581 (1988)] and transformation of the strain. The obtained transformant is subjected to Mu phage infection test according to a conventional method. The YK1340 strain is a HU gene-deficient strain, and Mu phage requires HU protein for its growth.B.longumIt can be a strong candidate for a strain carrying the HU gene derived from a fungus. Therefore, by selecting a plasmid that exhibits ampicillin resistance, and Mu strains infect and proliferate and possess lysis,B.longumPlasmid pBLHU15 carrying the promoter and terminator involved in the expression of the HU gene derived from bacteria can be obtained.
[0029]
  Further, by incorporating a signal sequence, for example, the above-mentioned substance having antitumor activity or a convertase produced in the host cell can be actively secreted outside the host cell. That is, the signal sequence is expressed in a form in which a signal peptide is added in front of the substance or convertase having the antitumor activity of the present invention, and as a result, the substance or convertase having the antitumor activity is expressed in a host cell. It can be actively secreted outside. As a method for adding a signal peptide, for example, the method of Paulson et al. [J. Bio1. Chem. ,264, 17619 (1989)], Law et al. [Proc. Natl. Acad. Sci. , USA,86, 8227 (1989), Genes Develop. ,Four1288 (1990)], or methods described in JP-A-5-336963, WO94 / 23021, and the like.
[0030]
  The selectable marker is used to select only transformed Bifidobacterium. For example, drug resistance markers such as ampicillin resistance, tetracycline resistance, neomycin or kanamycin resistance; auxotrophy; selection by medium such as selection by HAT medium. When the cloning vector described below has a selection marker, it is not necessary to incorporate a selection marker.
[0031]
  The cloning vector that can be used in the present invention can easily produce a recombinant in vitro with (a) a DNA having the above-mentioned antitumor activity or DNA encoding a convertase, and (b) pifidobacteria (C) can be introduced into Bifidobacterium, and (d) specifically detects Bifidobacterium transformed with a cloning vector. If it can, it can be used in the present invention.
[0032]
  Specific examples of the cloning vector include plasmid pBLESlOO, which is preferably used in the present invention. The plasmid schematic is shown in FIG. As can be seen from FIG. 1, the Escherichia coli vector pBR322 [the part represented by a single line in FIG. 1] andB.longumIn a composite plasmid with pTB6 plasmid (3.6 kb) derived from a fungus (the part shown in bold in FIG. 1), a 1.1 kb derived from Enterococcus FaecalisHindIIIEcoThe RI fragment (the portion represented by the bold solid line in FIG. 1) is incorporated. The fragment contains a region exhibiting spectinomycin resistance, in other words, a region encoding spectinomycin adenyltransferase.
[0033]
  For example,B.longumA plasmid pBL595 having a restriction enzyme recognition site represented by FIG. 2 and having a size of about 2.9 kb is derived from the strain SBT595 (Institute of Biotechnology, Industrial Technology Institute, Accession No. FERM P-14162). The plasmid pBL595 and pBR329 derived from E. coliAvaI ・Hinof the dIII fragment and pAMβ1 from EnterococcuS faecalisHindIIIAvaA plasmid pBLEM100 (FIG. 3) consisting of the I fragment can be mentioned. In addition, Escherichia coli carrying the plasmid pBLEM100 has been deposited under the accession number FERM P-14102 of the Biotechnology Institute of Industrial Technology.
[0034]
  Further, a composite plasmid obtained by combining a plasmid derived from a microorganism belonging to the genus Streptococcus and a plasmid derived from E. coli,B.longumA plasmid vector obtained by ligating a plasmid pBL67 or pBL78 derived from a bacterium may be used (Japanese Patent Laid-Open No. 5-13076). Plasmid pBL67 isB.longumFungus MO9101 strain (FERM P-12167) orB.longumThis is a plasmid having a size of about 3.7 kb derived from the strain MO9102 (FERM P-12168) and having the restriction enzyme recognition site shown in FIG. The plasmid pBL78 isB.longumThis is a plasmid having a size of about 8.5 kb derived from the strain MO9103 (FERM P-12169) and having the restriction enzyme recognition site shown in FIG.
[0035]
  In addition, the plasmid pBR322 derived from E. coli,B.longumA plasmid in which a plasmid pTB4, pTB6, or pTB10 derived from a bacterium is bound may also be mentioned. Moreover, the plasmid which combined all or the chloramphenicol resistance gene region of pC194 derived from Staphilococcus oureus to this plasmid is also mentioned. Furthermore, each of the above two plasmids,B.longumA plasmid to which a fungal tryptophan synthesis gene is bound may be used (Japanese Patent Laid-Open No. 63-123384). In addition, Escherichia coli carrying these plasmids has been deposited with the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (9040, 9041, 9042, 9043, 9044, 9045, 9046, 9047, 9048). Plasmid pTB4 or pTB10 isB.longumThis is a plasmid derived from the strain BK25 (Microtechnological Bacteria 9049). Plasmid pTB6 isB.longumThis is a plasmid derived from the strain BK51 (Mikkenken Bunkyo 9050).
[0036]
  As a preferred embodiment of the expression vector according to the present invention, the above-described vector pBLES100, the above-mentioned promoter and terminator involved in the expression of the HU gene, and cytosine deaminase (hereinafter abbreviated as CD) capable of converting 5-FC to 5-FU. And an expression vector into which a gene encoding the gene (hereinafter abbreviated as CD gene) is incorporated. As a more specific embodiment, there is an expression vector shown in the schematic diagram of FIG. One embodiment of a specific method for preparing the expression vector is shown below. Using recombinant DNA with CD gene derived from E. coli inserted into TOPO vector (Funakoshi)E.coliJM109 is transformed, and plasmid DNA is extracted from the resulting transformant. Restriction enzyme to target plasmid pTOPO-eCDNspV andHpaDigest with I and purify the DNA fragment encoding about 1.3 kb CD. Similarly, obtained as aboveB.longumPlasmid pBLHU15, which has a promoter and terminator involved in the expression of the HU gene derived from fungiNspV andHpaDigest with I and purify a 6.7 kb DNA fragment. The 1.3 kb and 6.7 kb DNA fragments obtained above are ligated using a conventional method to obtain recombinant DNA, and the recombinant DNA is used.E.coliJM109 is transformed according to a conventional method. Next, plasmid DNA is extracted from the transformant by a conventional method, and the plasmid DNA is extracted.HinAfter digestion with dIII, a 3.6 kb DNA fragment containing the promoter and terminator involved in the expression of the HU gene and the CD gene is separated and purified by a conventional method such as agarose gel electrophoresis. Also mentioned aboveEscherichia−BifidobacteriumPBLES100 which is the shuttle vector ofHinDigest with dIII and dephosphorylate. The above 3.6 kb DNA fragment and pBLES100HinA recombinant DNA is prepared by ligating dIII digests by a conventional method, and the recombinant DNA is used.E.coliJM109 is transformed according to a conventional method. Transformants can be selected by spectinomycin resistance. Having a gene encoding CD downstream of the promoter of the HU gene produced in this wayEscherichia−BifidobacteriumThe shuttle vector pBLES100-S-eCD can be obtained.
[0037]
  Third, recombinant DNA, preferably an expression vector, is introduced into the host Bifidobacterium. As the introduction method, any method known per se can be used. Specifically, for example, electroporation method [Cytotechnology,Three ,133 (1990)], calcium phosphate method (JP-A-2-227075), lipofection method [Proc. Natl. Acad. Sci. , USA,847413 (1987)], a method using calcium ions [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-2483942), or Gene,17, 107 (1982), Molecular & General Genetics,168111 (1979). In the present invention, it is preferable to use an electroporation method. The electroporation is preferably performed for about 4.1 to 4.5 ms under the conditions of about 10.0 kV / cm, about 200Ω, and about 25 μF.
[0038]
  The combination of the recombinant DNA to be introduced, preferably the expression vector and the host Bifidobacterium is not particularly limited, but the plasmid pBLES100 isB.longumIt is preferably introduced into the strain 105-A or 108-A (Biosci. Biotech. Biochem. 1997, 61 (7), 1211-1212). The plasmid pBLES100-S-eCD in which the promoter and terminator involved in the expression of the HU gene shown in FIG. 6 and the CD gene were incorporated was transformed.B.longumIt is fungus 105-A strainB.longum105-A / pBLES100-S-eCD is deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (FERM BP-7274).
[0039]
  The Bifidobacterium genus introduced with the expression vector is cultured using a known medium, and only transformed bacteria are selected. As the medium, a known medium suitable for the strain can be appropriately selected. In addition, in order to select a transformed Bifidobacterium genus, various drugs or amino acids are usually added to the medium according to the selection marker. As the medium, for example,B.longumThe strain BK25 or BK51 is preferably cultured in a Briggs medium having the following composition.
Briggs medium tomato juice extract (* 1) 400ml
  20% 2% glucose
  Soluble starch 0.5g
  Yeast extract 6g
  Peptone 15g
  Glutamic acid monosodium salt 2g
  Tween 80 1g Sodium acetate trihydrate 10g
  4g potassium dihydrogen phosphate
  Sodium chloride 5g
  600ml distilled water
  pH 6.8
  (* 1) An equivalent amount of distilled water was added to commercially available tomato juice, kept at 60 ° C. for 1 hour, then kept at 100 ° C. for 5 minutes, and then the residue was filtered.
[0040]
  B.longumThe strain SBT0595 is preferably cultured using a TGAM medium having the composition shown below.
Composition of TGAM medium
Tomato juice extract 400ml
Peptone 10g
Yeast extract 5g
Liver extract powder 1.2g
Glucose 3g
5g soluble starch
Sodium chloride 3g
Tween80 1g
L-cysteine-HCl · H₂O 0.3g
Soybean peptone 3g
Proteose peptone 10g
Digested serum powder 13.5g
Meat extract 2.2g
[0041]
  For example,B.longumBacteria 105-A strain or 108-A strain is prepared by replacing glucose in Briggs medium having the above composition with 2% lactose and adding 0.2 g / LL-cysteine and 3.4 g / L-sodium ascorbate. It is preferable to culture in a different medium.B.longumThe bacteria MO9101, Strain MO9102, and MO9103 are preferably cultured using 1GAM bouillon liquid medium (manufactured by Nissui Pharmaceutical).
[0042]
  Culture of microorganisms belonging to the genus Bifidobacterium can be performed as follows. When culturing in the liquid medium, after inoculating a sufficient amount of the microorganism belonging to the genus Bifidobacterium in the liquid medium, about 30-40 ° C, preferably at about 37 ° C, about 12 hours or more, The culture is preferably carried out under anaerobic conditions by standing until the middle of the logarithmic growth phase. The anaerobic condition is a sealed container that can maintain an anaerobic degree to which microorganisms belonging to the genus Bifidobacterium can grow, and examples include conditions that are possible in an anaerobic chamber or an anaerobic box.
[0043]
  The transformed Bifidobacterium can grow only in a tumor tissue in an anaerobic environment, and can express a substance or a convertase having antitumor activity in the tumor tissue. Therefore, such transformed Bifidobacterium is used as a pharmaceutical effective for the treatment of tumors having an anaerobic environment, preferably solid tumors. The administration route of the medicament of the present invention is not particularly limited, and includes oral administration or parenteral administration, and parenteral administration is particularly preferable. Parenteral administration can include administration routes such as intratracheal, rectal, subcutaneous, intramuscular and intravenous. Examples of formulations suitable for oral administration include, for example, tablets, granules, fine granules, powders, syrups, solutions, capsules or suspensions, and examples of formulations suitable for parenteral administration. Examples thereof include injections, drops, inhalants, sprays, suppositories, transdermal absorbents, transmucosal absorbents, and the like. In the present invention, it is preferably used as an injection, particularly as an intravenous injection.
[0044]
  The transformed Bifidobacterium may be subjected to a known post-treatment. For example, rough purification may be performed by centrifugation or the like. In addition, if desired, after rough purification, it may be dissolved or suspended in a solvent conventionally used in the art such as physiological saline, PBS (phosphate buffered physiological saline) or lactic acid-containing Ringer's solution. Moreover, you may freeze-dry or spray-dry as needed, and you may make it a powdery thing or a granular material.
[0045]
  As the medicament of the present invention, the transformed Bifidobacterium solution or suspension, or a granular or powdery dried product may be administered as it is. However, in general, it is desirable to administer in the form of a pharmaceutical composition comprising the above-mentioned substance as an active ingredient and one or more pharmaceutical additives. Such a pharmaceutical composition itself can be produced according to a method well known or commonly used in the field of pharmaceutics.
[0046]
  For the production of liquid preparations suitable for oral administration, for example, sugars such as water, sucrose, sorbit, fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; p-hydroxy Pharmaceutical additives such as preservatives such as benzoates can be used. For the production of solid preparations such as capsules, tablets, powders, or granules, for example, excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium alginate; magnesium stearate Lubricants such as talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; plasticizers such as glycerin can be used.
[0047]
  Among preparations suitable for parenteral administration, preparations for intravascular administration such as injections and infusions can be prepared preferably using an aqueous medium isotonic with human blood. For example, an injection can be prepared as a solution, suspension or dispersion with an appropriate auxiliary agent using an aqueous medium selected from a salt solution, a glucose solution, or a mixture of a salt solution and a glucose solution according to a conventional method. . Suppositories for enteral administration can be prepared using carriers such as cocoa butter, hydrogenated fat or hydrogenated carboxylic acid.
  The spray is prepared using a carrier that does not irritate human oral cavity and airway mucosa and can disperse Bifidobacterium according to the present invention as an active ingredient as fine particles to promote absorption. can do. As such a carrier, for example, lactose or glycerin can be used. Depending on the properties of the genus Bifidobacterium according to the present invention and the carrier used, it can be prepared as a preparation in the form of an aerosol or dry powder. For the production of a parenteral preparation, for example, one or more selected from a diluent, a fragrance, an antiseptic, an excipient, a disintegrant, a lubricant, a binder, a surfactant, a plasticizer, and the like. Pharmaceutical additives can be used.
  Note that the pharmaceutical form of the present invention and the method for producing the same are not limited to those specifically described above.
[0048]
  The dose and frequency of administration of the medicament of the present invention are not particularly limited, and the type of gene possessed by the genus Bifidobacterium, the type of pathological condition to be treated, the route of administration, the age and weight of the patient, the symptoms, and the severity of the disease It is possible to select appropriately according to various conditions such as severity. For example, for systemic administration by intravenous injection, about 2 × 10 per adult day⁶~ 2 × 10⁷It is preferable to administer about 1 body / body. In the case of intratumoral administration, about 5 × 10 5 per tumor.⁸It is preferable to administer about one. However, the dosage is not limited to this particular example.
[0049]
  The medicament according to the present invention can be applied to tumors having an anaerobic environment, preferably various solid cancers. Examples of solid cancer include colon cancer, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, islet cell cancer, choriocarcinoma, colon cancer, renal cell cancer, adrenal cortex Cancer, bladder cancer, testicular cancer, prostate cancer, testicular tumor, ovarian cancer, uterine cancer, choriocarcinoma, thyroid cancer, malignant carcinoid tumor, skin cancer, malignant melanoma, osteosarcoma, soft tissue sarcoma, neuroblastoma, Wilms Tumor, retinoblastoma, melanoma, squamous cell carcinoma and the like.
[0050]
  The medicament of the present invention may be used in combination with other medicaments. In particular, when a Bifidobacterium bacterium into which a gene encoding a convertase has been introduced is administered, administration of an antitumor substance precursor is essential. However, such an anti-cancer substance precursor may form one preparation with a Bifidobacterium genus into which a gene encoding a convertase has been introduced, or may be a separate preparation at the same time or at intervals. It may be administered after emptying. Further, 20% lactulose is preferably used in combination. Lactulose is a nutrient source for Bifidobacterium, and humans, mice, and pigs cannot metabolize, so by administering lactulose, the number of Bifidobacterium specifically in the tumor tissue This is because of the increase. The dosage is preferably about 24 to 48 g / body per adult day, regardless of the frequency of administration.
[0051]
  The medicament of the present invention can be used in combination with other antitumor agents and the like. In general, it is preferably used in combination with several kinds of antitumor agents having different mechanisms of action. Other antitumor agents include, for example, alkylating agents, various antimetabolites, antitumor antibiotics, other antitumor agents, antitumor plant components, BRM (biological response control substances), angiogenesis Inhibitors, cell adhesion inhibitors, matrix metalloprotease inhibitors, hormones, vitamins, antibacterial antibiotics or chemotherapeutic agents. More specifically, as the alkylating agent, for example, an alkylating agent such as nitrogen mustard, nitrogen mustard N-oxide, chlorambutyl; for example, an aziridine-based alkylating agent such as carbocon, thiotepa; Epoxide alkylating agents such as bromodalcitol; for example, carmustine, lomustine, semustine, nimustine hydrochloride, streptozocin, chlorozotocin, ranimustine and other nitrosourea alkylating agents; busulfan; toprolic acid toprosulphan; dacarbazine and the like Can be mentioned. Examples of various antimetabolites include, for example, purine antimetabolites such as 6-mercaptopurine, 6-thioguanine, thioinosine, and pyrimidine metabolism antagonists such as fluorouracil, tegafur, tegafur uracil, carmofur, doxyfluridine, broxuridine, cytarabine, and enocytabine. Agents, antifolates such as methotrexate, trimethrexate, and the like, and salts or complexes thereof.
[0052]
  Antitumor antibiotics include, for example, anthracycline antibiotic antitumor agents such as mitomycin C, bleomycin, peplomycin, daunorubicin, aclarubicin, doxorubicin, pirarubicin, THP-adriamycin, 4'-epidoxorubicin, epirubicin, chromomycin A_Three, Actinomycin D and the like and salts or complexes thereof. Other antitumor agents include, for example, cisplatin, carboplatin, tamoxifen, camptothecin, ifosfamide, cyclophosphamide, melphalan, L-asparaginase, acecraton, schizophyllan, picibanil, ubenimex, krestin, and salts or complexes thereof Is mentioned. In addition, procarbazine, pipbloman, neocartinostatin, hydroxyurea and the like can also be mentioned. Examples of the antitumor plant component include vinca alkaloids such as bisidecin, vincristine and vinblastine, epipodophyllotoxins such as etoposide and teniposide, and salts or complexes thereof. Examples of BRM include tumor necrosis factor, indomethacin, and salts or complexes thereof. Examples of the angiogenesis inhibitor include fumagillol derivatives and salts or complexes thereof. Examples of the cell adhesion inhibitor include substances having an RGD sequence, and salts or complexes thereof. Examples of the matrix metalloprotease inhibitor include marimastat, batimastat, and salts or complexes thereof. Examples of hormones include hydrocortisone, dexamethasone, methylprednisolone, prednisolone, plasterone, betamethasone, triamcinolone, oxymetholone, nandrolone, methanolone, phosphaterol, ethinyl estradiol, chlormadinone, medroxyprogesterone, and salts or complexes thereof. Is mentioned. Examples of vitamins include vitamin C, vitamin A, and salts or complexes thereof.
[0053]
  Bifidobacterium administered to patients according to the present invention can be easily killed with antibiotics. This is important for further improving the safety of the gene delivery system according to the present invention.
[0054]
【Example】
  Examples are shown below, but the present invention is not limited to the following examples. In the Examples, unless otherwise specified, the method described in Molecular Cloning 2nd edition was used as a method for handling DNA and the like.
Example 1 in tumor tissueB.longumConfirmation of accumulation and proliferation
(1) For cancer-bearing animalsB.longumSolution preparation
B.longum105-A orB.longum108-A [Biosci. Biotech. Biochem. ,61, 1211 (1997)] for the administration of cancer-bearing animalsB.longumThe solution was prepared as follows. In addition,B.longum105-A was cultured as follows under non-selective conditions (in the absence of spectinomycin) and then agar-containing modified Briggs broth (modified Briggs broth supplemented with 75 μg / ml spectinomycin). In addition, agar was added to 1.5%), and the plasmid was dropped, so that it can be obtained as a strain showing spectinomycin sensitivity.B.longum105-A orB.longum108-A is a modified Briggs medium [solution A [0.5 g / 1 soluble starch, 6.0 g / 1 Bacto Yeast extract (Difco), 15.0 g / 1 polypeptone, 2.0 g / l sodium glutamate, 10.0 g / 1 sodium acetate trihydrate, 4.0g / 1 potassium dihydrogen phosphate, 5.0g / 1 sodium chloride, 1.0g / 1 tween80, 400ml / 1 tomato juice extract (tomato juice extract is canned tomato juice ( (400 ml of Delmonte) Add 400 ml of water to 400 ml, incubate at 60 ° C for 1 hour, hold at 100 ° C for 5 minutes, add a small amount of High Flow Supercell (Wako Pure Chemical Industries, Ltd.), stir, and filter with an aspirator ), Adjusted to pH 6.8 with sodium hydroxide and autoclaved], B solution [20% lactose aqueous solution autoclaved], C solution [20.0 g / 1 L-cysteine, 340 g / 1 Sodium ascorbate Sterilized by filtration) and inoculated into a mixture of A solution 100: B solution 10: C solution 1], and allowed to grow to mid-logarithmic growth by static culture at 37 ° C under anaerobic conditions. It was. The obtained culture solution was centrifuged to precipitate the cells and the supernatant was removed. Then, PBS (phosphate buffered saline, 8 g / 1 sodium chloride, 0.2 g / 1 potassium chloride, 1. 44 g / 1 disodium hydrogen phosphate, 0.24 g / 1 potassium dihydrogen phosphate, PH 7.4) were added and suspended, and the centrifugation was repeated twice as above to wash the cells. After centrifuging for the second time, by adding 10 times the volume of the culture medium first subjected to centrifugation or 1/50 volume of PBS to the cells after removing the supernatant,B.longumA solution was prepared.
[0055]
(2) To cancer-bearing animalsB.longumAdministrationB.longumTwo types of tumor-bearing mice in which B16-F10 melanoma cells or Luwis lung cancer cells were transplanted into 6-8 week old male C57BL / 6 mice (purchased from Japan SLC), and Tumor-bearing rats prepared by administering 7,12-dimethylbenz [a] anthracene (DMBA) to 6-week-old male Spraque-Dawley rats (purchased from SLC, Japan) were used.
B16-F10 melanoma cells and Luwis lung cancer cells used for mouse inoculation were each Dulbecco's modified Eagle medium containing 10% fetal bovine serum [Virology,8, 396 (1959), Virology,12, 185 (1960)] at 37 ° C, 5% CO₂It was prepared in a single layer culture under the conditions described above. The cultured cancer cells are 5 x 10^FiveAs a tumor-bearing mouse that inoculates the right thigh muscle of C57BL / 6 mice and has a solid tumor in the right extremity 2 weeks after inoculationB.longumIt was used for the administration test. For chemically induced breast cancer rats, 6-week-old male Spraque-Dawley rats should be administered 1 ml of DABA (10 mg / ml) dissolved in sesame oil intragastrically using a sonde, and one week later, an equal amount of DMBA should be administered. It was produced by. As a chemically-induced breast cancer rat after the second dose, when the tumor size reached 5 mm in diameter from one and a half to two monthsB.longumIt was used for the administration test. To cancer-bearing animalsB.longumWas prepared in (1) above.B.longum0.5 ml of the bacterial cell solution or 10-fold diluted solution for miceB.longum 5-6 × 10⁶Cell) and 0.5ml (2 x 10) of 1/50 concentrated solution in rats.⁸Cell fraction) was performed by systemic administration once via the tail vein.
[0056]
(3)B.longumOf selective accumulation in tumor tissue and selective growth in tumor tissue
  B.longum6 to 8 tumor-bearing mice were administered and were sacrificed over time, 1, 24, 48, 72, 96, and 168 hours after administration, and 6 cancer-bearing rats were sacrificed and 168 hours after administration. The tumor tissue and normal tissue are removed, and each tissue extract is anaerobically cultured.B.longumAccumulation and proliferation were analyzed. The normal tissue was lung, liver, spleen, kidney and heart, the mouse tumor tissue was a tumor tissue formed in the right thigh, and the rat tumor tissue was a breast cancer tissue. The tissue extract was obtained by measuring the weight of the tissue in a sterile environment, crushing sufficiently after cutting, adding 10 times the amount of ice-cold PBS to the tissue, homogenizing, and filtering. . In each organizationB.longumThe distribution of was analyzed as follows. Dissolve the tissue extract prepared above (diluted to contain 0.01 g of tissue per 100 μl) in 100 μl aliquots in two dishes and incubate at 55 ° C in advance to dissolve. Pour 20g / l L-cysteine and 340mg / l sodium ascorbate Briggs agar medium (Briggs medium with 1.5% agar) into the above-mentioned sheare, stir well, and leave at room temperature Solidified. The petri dish has been grown in an air sealed desiccator under anaerobic conditions at 37 ° C for 3 days.B.longumBy counting colonies ofB.longumEach tissue distribution was analyzed. as a result,B.longumBoth Lewis lung cancer cell-inoculated bile cancer mice systemically administered with 105-A or 108-A have 60,000 cells per gram of tumor tissue.B.longumColonies were observed. In contrast, in normal tissues such as lung, liver, spleen, kidney and heart, 108-A was not observed after 96 hours and 105-A was not observed after 168 hours (FIG. 7).
  B.longumSimilar results as described above were obtained in tumor-bearing mice inoculated with B16-F10 melanoma cells administered with 105-A or 108-A.B.longum In chemically induced breast cancer rats administered systemically with 105-A, 10,000 rats per gram of tumor tissue.B.longumWere observed in normal tissues such as lung, liver, spleen and kidney after 168 hours (FIG. 8). From the above results,B.longumWas confirmed to accumulate and proliferate specifically in the tumor tissue.
[0057]
(4) By lactulose (1acturose; 4-0-β-D-galactopyranosyl-D-fructofuranose) administrationB.longumIn tumor tissue
  Lactulose (sold by Nikken Chemical) is a synthetic sugar that does not exist in nature, and it is known that humans, mice and pigs cannot metabolize 1acturose [Biochem. Biophys. Acta,110635 (1965), Pediatrics,32, 1033 (1963), Gastroenterology,47, 26 (1964), Die Nahrung,1139 (1967)]. on the other hand,B.longumCan grow using 1acturose as a carbon source. there,B.longumIn 6 to 8 Lewis lung cancer cell bearing mice administered 105-A,B.longumAfter the administration, 1 ml of 20% lactose solution is intraperitoneally administered for 8 days every day, and the mice are sacrificed on the 9th day and are present in each tissue.B.longumAs a result of the analysis of the number of bacteria, it is present in the tumor tissue of the lactose-administered tumor-bearing mouse group compared to the control of the lactulose-non-administered groupB.longumThe number of bacteria was 200 times. Based on the above results, administration of lactulose resulted in tumor tissue.B.longumHas been shown to be capable of selective growth.
[0058]
Example 2 Recombination with plasmid DNA in tumor tissueB.longumSpecific accumulation and proliferation
(1) Recombination with plasmid DNAB.longumCreate
By the method described in Example 1 (1) under anaerobic conditionsB.longum 105-A was cultured and then placed at 4 ° C. Next, the culture solution was centrifuged to precipitate the cells, the supernatant was removed, and then ice-cooled 10% glycerol solution was added and suspended. By returning the above operation over 3 lines,B.longumThe cells were thoroughly washed. After the final wash, the supernatant is removed, and 1/10 volume of the ice-cooled 10% glycerol solution is added and suspended in the first centrifugation, and the cells are subjected to transformation by electroporation. A body sample (2 × 10⁸~ 2 × 10⁹Colony forming unit (CFU) / 50 μl). The plasmid DNA used for transformation, pBLES100 [Biosci. Biotech. Biochem. ,61, 1211 (1977)], Biosci. Biotech. Biochem. ,61, 1211 (1997), a plasmid can be extracted from FERM BP-7274 deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology by a conventional method, and the DNA can be used as a restriction enzyme.HinAfter digestion with dIII (Takara Shuzo), phenol treatment and ethanol precipitation, DNA is dissolved in water, etc., and T4DNA ligase (Takara Shuzo) is used according to the instructions attached to the product. It can also be obtained by binding by a chemical reaction.B.longumPBLES100 used for the transformation of was prepared as follows. Using pBLES100 obtained as described aboveEscherichiacoli(E.coli) A transformant of JM109 was obtained, and the transformant was cultured in the presence of 75 μ / ml spectinomycin. By extracting the plasmid pBLES100 from the culture obtained by the culture according to a conventional method and purifying by cesium chloride density gradient ultracentrifugation (Molecular Cloning 2nd Edition),B.longumPBLES100 was obtained for transformation. 50 μl prepared aboveB.longum The 105-A cell sample and 4 μl pBLES100 (1 μg DNA / 4 μ1) were mixed in a 0.2 cm-wide electroporation cuvette (manufactured by Bio-Rad), and placed on ice for 5 minutes. The cuvette was set in Gene Pulser (manufactured by Bio-Rad), and transformation by electroporation was performed under the conditions of 2.0 KV, 25 μF capacitor, and 200 Ω parallel resistance. After applying the electric pulse, 1 ml Briggs medium was quickly added to the cuvette, and the cuvette was allowed to stand at 37 ° C. for 3 hours, and then applied to a Briggs agar plate containing 75 μg / ml spectinomycin. The plate was cultured at 37 ° C. for 3 to 4 days under anaerobic conditions using Gas Pack Anaerobic Systems (BBL). Pick up several colonies that appeared, culture them by the method described in Example 1 (1), add QIAGEN Plasmid MiniKit (Qiagen) to the P1 solution in which the cells were dissolved, add lysozyme at 37 ° C. The plasmid DNA retained by the colony was extracted by using it according to the method of use attached to the kit except that it was kept warm for 40 minutes. The extracted plasmid DNA was digested with several types of restriction enzymes, and then its structure was confirmed by agarose gel electrophoresis, and it was confirmed that the colony retained pBLES100. The recombinant obtained aboveB.longumIt was named 105-A / pBLES100.
[0059]
(2) Recombination into cancer-bearing animalsB.longumStrain administration
For cancer-bearing animalsB.longumThe 105-A / pBLES100 solution was prepared in the same manner as in Example 1 (1) except that 75 μg / ml spectinomycin was added to the modified Briggs medium and cultured. Administration of the solution to tumor-bearing mice was performed using tumor-bearing mice transplanted with B16-F10 melanoma cells in tumor-bearing mice, and administration to tumor-bearing rats was carried out using chemically-induced breast cancer rats. The same procedure as in Example 1 (2) was performed.
(3) RecombinationB.longum Selective accumulation and growth of tumor lines in tumor tissueB.longum Six to eight tumor-bearing mice and 6 tumor-bearing rats administered with the 105-A / pBLES100 solution were sacrificed on the 4th day, and the tumor tissue and each of the tumor-bearing mice were measured according to the method described in Example 1 (3). In normal tissueB.longumThe distribution of 105-A / pBLES100 was analyzed. However, 75 μg / ml spectinomycin was added to the medium mixed with the tissue extract. As a result, the tumor-bearing mice inoculated with B16-F10 melanoma cells and Luwis lung cancer cells were also non-recombinant.B.longum Distributed in the tumor tissue of the control group treated with 105-AB.longumCompared to the number of cellsB.longum Distributed in tumor tissue of tumor-bearing mice treated with 105-A / pBLES100B.longumThe number of cells of 105-A / pBLES100 did not decrease (FIG. 9). Similar results were obtained in chemically induced breast cancer rats (FIG. 8). From the above results, in the tumor tissue,B.longum105-A was found to be able to stably maintain plasmid pBLES100.
Next, non-recombinantB.longum ,andB.longumEach day from the next day of administration, 200 mg / kg of spectinomycin was intraperitoneally administered to tumor-bearing mice administered with 105-A / pBLES100, and the mice were sacrificed on the fourth day.B.longumEach tissue distribution was analyzed. Non-recombinant compared to the control group that received intraperitoneal PBS daily instead of spectinomycinB.longumIn tumor-bearing mice treated with pectin, distributed in tumor tissue by administration of spectinomycinB.longum The number of bacteria decreased to 1% or less.B.longumIn tumor-bearing mice administered with 105-A / pBLES100, even when spectinomycin is administered, it is distributed in the tumor tissueB.longum The number of 105-A / pBLES100 bacteria was 81% of that in the control group (FIG. 9). From the above results, it was confirmed that the spectinomycin resistance gene was expressed specifically in the tumor tissue.
[0060]
Example 3 Recombination that highly expresses cytosine deaminase (CD)B.longum Containing an antitumor agent
(1)B.longum Acquisition of highly expressed genes in cells
B.longum HU gene known as a highly expressed gene in cells [HU protein: histone-like DNA binding protein, Biochimie,72, 207 (1990)] was obtained by the following method.B.longumThe ATCC15707 strain was cultured in Briggs medium according to the method described in Example 1 (1), and chromosomal DNA was extracted and purified from the obtained cells according to the method described in Molecular Cloning 2nd edition. 1 μg of the chromosomal DNA is used as a restriction enzymeHinDigested with dIII and purified by phenol treatment and ethanol precipitation. Also, PBR322 (purchased from Takara Shuzo)HinThe product was digested with dIII and purified in the same manner after dephosphorylation. 100 ng of each DNA was ligated by using T4 DNA ligase (Takara Shuzo) according to the instructions attached to the product to obtain recombinant DNA.
Next, using the recombinant DNAE.coli mH3 [Gene,45, 37 (1986)] was transformed according to a conventional method to obtain a transformant resistant to ampicillin and sensitive to tetracycline. Plasmid DNA was extracted from the obtained approximately 2000 transformants according to a conventional method. coli YK2741 [Gene,89, 133 (1990)]. The YK2741 strain is deficient in HU gene and IHF (Integration Host Factor) gene, and thus exhibits low temperature sensitivity. Therefore, transformants that can grow at low temperatures areB.longumThere is a possibility of a strain carrying the HU gene derived from. Transformation was carried out according to a conventional method, applied to ampicillin-containing agar medium, cultured at 27 ° C., and the grown transformant was used for later experiments.
Next, the transformant of YK2741 strain obtained above was cultured, and the plasmids retained by each strain were extracted by the above method,E.coliYK1340 [J. Mol. Biol. ,204, 581 (1988)]. The resulting transformant was subjected to Mu phage infection test according to the method of Molecular Cloning 2nd edition. The YK1340 strain is a HU gene-deficient strain, and Mu phage requires HU protein for its growth.B.longumIt is a promising candidate for a strain carrying the HU gene. One of the plasmids possessed by ampicillin resistance and capable of infecting and growing Mu phages and possessing a lytic strain was named pBLHU15, and its structure and properties were analyzed.B.longumThe plasmid was confirmed to have the derived HU gene (FIG. 10).
[0061]
(2) Preparation of cytosine deaminase (CD) high expression plasmid
The gene encoding CD is a DNA having the nucleotide sequence represented by SEQ ID NO: 2 using the plasmid pAdex1CSCD (RIKEN Genebank RDB No. 1591) containing the gene encoding CD derived from E. coli as a template and SEQ ID NO: The DNA having the base sequence represented by 3 was obtained as a primer set by PCR. PCR is template DNA 125ng / l, each primer 0.5 / μmol / l, Pfu DNA polymerase (Stratagene) 2.5units, Pfu DNA polymerase x10 buffer (Stratagene) 4μl, deoxy Using 40 μl of the reaction solution containing 200 μmol / l of NTP, the process was repeated 30 times at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute, and then kept at 72 ° C. for 15 minutes . A portion of the reaction solution was subjected to agarose gel electrophoresis, and it was confirmed that a fragment of about 1.3 kb was amplified. The remaining reaction solution was purified by phenol treatment / ethanol precipitation, and then TOPO vector (Funakoshi) Manufactured by the above-mentioned kit. Using the recombinant DNA obtained by ligation,E.coli JM109 was transformed, plasmid DNA was extracted from the obtained transformant, and it was confirmed by subjecting various restriction enzyme digests to agarose gel electrophoresis that the target plasmid pTOPO-eCD was constructed. . pTOPO-eCD is a restriction enzymeNspV (Takara Shuzo), andHpaAfter digestion with I (Takara Shuzo), agarose gel electrophoresis was performed, and a DNA fragment encoding about 1.3 kb CD was purified using Gene clean kit (Funakoshi) according to the instruction manual. Similarly, plasmid pBLHU15 obtained in Example 3 (1)NspV andHpaDigested with I and purified a 6.7 kb DNA fragment. The 1.3 kb and 6.7 kb DNA fragments obtained above were ligated using the kit described above to obtain recombinant DNA, and the recombinant DNA was used.E.coli JM109 was transformed according to a conventional method. Among the obtained transformants, several strains were cultured, plasmids were extracted from the culture, and DNA digested with various restriction enzymes was analyzed by agarose gel electrophoresis, so that the downstream of the HU gene promoter. It was confirmed that plasmid DNA incorporating the CD gene could be obtained.
Next, the plasmid DNA isHinA 3.6 kb DNA fragment containing the gene encoding HU gene and CD was separated by digestion with dIII and agarose gel electrophoresis, and purified using Gene clean kit. Also mentioned aboveEscherichia-BifidobacteriumPBLES100 is also a shuttle vectorHinIt was digested with dIII and dephosphorylated. The above 3.6 kb DNA fragment and pBLES100HinThe dIII digest is ligated using the above kit to produce recombinant DNA, and the recombinant DNA is used.E.coli JM109 was transformed according to a conventional method. After picking up several strains of transformants showing resistance to sectinomycin, extracting the plasmid DNA of the transformants according to a conventional method, digesting them with various restriction enzymes, and then constructing the desired plasmid by agarose gel electrophoresis. Confirmed that. Having a gene encoding CD downstream of the promoter of the HU gene, prepared as described above,Esherichia-BifidobacteriumWas designated as pBLES100-S-eCD (FIG. 11). Also, have pBLESlOO-S-eCD obtained aboveE.Coli From JMlO9,B.longumThe plasmid for transformation was prepared by cesium chloride density gradient centrifugation according to the method described in Example 2 (1). Using the pBLESlOO-S-eCD prepared as described above, according to the method described in Example 2 (1),B.longum105-A was transformed, and the resulting transformed strain wasB.longum It was named 105-A / pBLESlOO-S-eCD.
B.longum The 105-A / pBLESlOO-S-eCD is based on the Budapest Treaty, and the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, August 15, 2000, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan No. (postal code 305-8586) is deposited under the accession number FERM BP-7274.
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Example 4 Recombination that highly expresses CDB.longumContaining an antitumor agent
(1) To cancer-bearing animalsB.longum Administration of 105-A / pBLESlOO-S-eCD Used to administer cancer-bearing animalsB.longumThe 105-A / pBLES100-S-eCD solution was prepared according to the method described in Example 2 (2). The administration of the solution to a tumor-bearing mouse was performed using a tumor-bearing mouse in which B16-FlOmelanoma cells were transplanted into the right thigh muscle in a tumor-bearing mouse, and 1 × 10⁷Private note.
(2) Specific conversion in tumor tissue from 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU)
In Example 4 (1),B.longum To 6-8 tumor-bearing mice administered with 105-A / pBLES100-S-eCD, 500 mg / kg of 5-FC was administered intraperitoneally every day, together with 1 ml of 20% 1acturose solution.B.longum From the next day of administration of 105-A / pBLESlOO-S-eCD, intraperitoneal administration was performed every day. The administration was performed until the cancer-bearing mouse was sacrificed. Also, for controlB.longum5-FC was administered every day in the same manner as described above using tumor-bearing mice that did not receive 105-A / pBLESlOO-S-eCD locally.B.longumOn day 8 after administration of 105-A / pBLESlOO-S-eCD, the tumor-bearing mice were sacrificed, and the 5-FU concentration in the thigh tumor tissue was measured. The measurement of 5-FU concentration isB.longumA tumor tissue locally injected with 105-A / pBLESlOO-S-eCD and a tumor tissue not locally injected were removed, and 5-FU concentration measurement in the tumor tissue [GC-MS method, J. Biol. Chromatography,564, 137 (1991)] was commissioned to Otsuka Assay Laboratory. as a result,B.longum Compared to the ability to detect only 10-Ong / g of 5-FU in the tumor tissue without local injection of 105-A / pBLESlOO-S-eCD,B.longumIn the tumor tissue locally injected with 105-A / pBLESlOO-S-eCD, 588.8 ng / g of 5-FU was detected. From the above results, it was confirmed that 5-FC administered systemically was converted into 5-FU specifically in the tumor tissue.
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【The invention's effect】
  The present invention uses a part of Bifidobacterium genus Bifidobacterium, which is a part of human intestinal resident bacteria, as a gene transport carrier in a tumor tissue in an anaerobic environment. Provided are a method for expressing a substance having specific antitumor activity or a convertase, and a transformed or mutated microorganism belonging to the genus Bifidobacterium used in the method. By using this method mainly for the treatment of solid tumors, it is possible to selectively treat tumors and to reduce the side effects of conventional chemotherapeutic agents on tumors. In addition, there is an effect that a drug that has been effective for cancer but could not be used due to side effects may be used again. Another object of the present invention is to provide an expression vector for highly expressing a protein encoded by DNA introduced into a microorganism belonging to the genus Bifidobacterium. Thereby, the treatment of the tumor tissue in an anaerobic environment, mainly a solid tumor, can be performed efficiently.
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[Brief description of the drawings]
FIG. 1 shows a schematic diagram of a plasmid of pBLESlOO. The part represented by a single line represents the Escherichia coli vector pBR322, and the part represented by the bold line isB.longumPTB6 plasmid (3.6 kb) derived from fungi, the part indicated by the outlined line is a 1.1 kb derived from Enterococcus faecalisHindIIIEcoRepresents RI fragment. Also, Spec^rRepresents the spectinomycin resistance gene and Ori represents the origin of replication.
FIG. 2 shows a plasmid schematic diagram of pBL595.
FIG. 3 shows a schematic diagram of a plasmid of pBLEMlOO. In addition, the thickly painted partB.longumThis represents the plasmid pBL595 derived from the strain SBT595.AvaI ・HinIt represents the dIII fragment, and the thin line part is that of pAMβ1 derived from Enterococcus faecalisHindIIIAvaRepresents the I fragment.
FIG. 4 shows a schematic diagram of a plasmid for pBL67.
FIG. 5 shows a schematic diagram of a plasmid for pBL78.
FIG. 6 shows a schematic diagram of a plasmid of pBLESlOO incorporating HU gene and cytosine deaminase. The part represented by the single line represents the Escherichia coli vector pBR322, and the part represented by the bold line isB.longumPTB6 plasmid (3.6 kb) derived from a fungus, and the portion represented by a hollow line is a 1.1 kb derived from Enterococcus Faecalis.HindIIIEcoRepresents the RI fragment,B.longumFungal geneHinRepresents the dIII-treated fragment, the part indicated by the outlined line with an arrow in it represents the CD gene derived from E. coli, and the shaded part in front of it (closer to Ori) is the HU gene The region containing the promoter, and the hatched part behind it represents the region containing the terminator. Also, Spec^rRepresents the spectinomycin resistance gene and Ori represents the origin of replication.
[Fig. 7] Cancer-bearing miceB.longumIt is the figure which showed the colony number of this microorganisms which exist in various organ tissue and tumor tissue after intravenously administering a microbe. ○B.longum105-A, □B.longumThe result at the time of giving 108-A is shown.
[Fig. 8] To cancer-bearing ratsB.longum105-A (shown in shaded) or transformed with pBLESlOOB.longumFIG. 10 is a graph showing the number of colonies of the microorganisms present in various organ tissues and tumor tissues 168 hours after intravenous administration of 105-A (shown in white).
[Fig. 9] In tumor-bearing miceB.longum105-A, orB.longumIt is a figure which shows the colony number of each microorganisms which exist in a tumor cell when 105-A / pBLESlOO is administered intravenously and spectinomycin is administered. The outline isB.longumlO5-A, shadedB.longum105-A / pBLESlOO represents the administration group, control is the non-administration of spectinomycin, and spectinomycin is the administration group of spectinomycin.
FIG. 10B.longumThe schematic diagram of plasmid pBLHU15 containing DNA which codes HU protein derived from is shown. The dotted pattern isB.longumFungal geneHinRepresenting the dIII-treated fragment, the part represented by the single line and the hollow line represents the pBR322 plasmid, and the part represented by the bold line isB.longumRepresents the HU gene derived from. Also Amp^rShows the ampicillin resistance gene, Ter^rRepresents the tetracycline resistance gene and Ori represents the origin of replication.
[Fig. 11] E. coli-derived CD incorporatedB.longumIt is a figure which shows the construction process of the expression plasmid vector pBLESlOO-S-eCD for use.
[Sequence Listing]
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Claims (7)

Specific to tumor tissue under anaerobic environment (a) DNA encoding a protein having antitumor activity, or (b) DNA encoding a protein having activity of converting an antitumor substance precursor into an antitumor substance A gene transporter that can be transported , having a promoter for expression of a gene encoding a histone-like DNA-binding protein (HU protein) derived from Bifidobacterium longum , SEQ ID NO: A DNA encoding the protein having antitumor activity, or (b) a precursor of an antitumor substance, wherein the DNA represented by the base number 1 to 192 of the base sequence according to 1 is used and downstream of the promoter Transformed with an expression vector incorporating a DNA encoding a protein having an activity of converting the body into an antitumor substance, A bifido capable of expressing in the tissue (a) a DNA encoding a protein having antitumor activity, or (b) a DNA encoding a protein having an activity of converting an antitumor substance precursor into an antitumor substance Gene transport carrier consisting of bacteria longum. Expression vector, according to claim 1, characterized in that it comprises a Bifidobacterium longum (Bifidobacterium longum) engaging filter terminator for expression of genes encoding histone-like DNA-binding protein (HU protein) genes from Transport carrier. The gene transport carrier according to claim 1 or 2, wherein the protein having antitumor activity is interleukin-2. The gene transport carrier according to claim 1 or 2, wherein the protein having antitumor activity is endostatin . Protein having an activity of converting an antitumor substance precursor to an antitumor substance is a gene delivery carrier according to claim 1 or 2, characterized in that cytosine der Greg peptidase. (A) DNA encoding a protein having antitumor activity, or (b) Bifidobacterium longum capable of expressing a DNA encoding a protein having an activity of converting an antitumor substance precursor into an antitumor substance but Bifidobacterium longum 105-a / pBLES100-S- eCD (FERM BP-7274) according to claim 4, wherein the gene delivery carrier, which is a. A medicament comprising the gene transport carrier according to any one of claims 1 to 6 .

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