Medicine for infectious disease

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

[Technical field to which the invention belongs]

The present invention relates to an infectious disease therapeutic agent for treating an infectious disease such as tuberculosis.

【0002】

[Conventional technology]

It goes without saying that the presence of therapeutic agents for infectious diseases is indispensable in medical care. Currently, a large number of therapeutic agents for infectious diseases such as antibiotics and synthetic antibacterial agents are provided and used in the medical field.

【0003】

However, it is also a fact that the antibacterial agents mainly provided as therapeutic agents for infectious diseases are accompanied by the unavoidable problem of the emergence of resistant bacteria. In other words, the ironic state that new resistant bacteria are being created by providing new antibacterial agents continues.

【0004】

For example, tuberculosis, which has the highest mortality rate among single infectious diseases, has become a global problem due to the recent increasing trend. Furthermore, the existence of resistant bacteria resistant to most antibiotics has been confirmed, and this problem is becoming apparent.

【0005】

[Problems to be Solved by the Invention]

Recently, granulysin and call barrel molecule expressed on NK cells and CTL is, bacteria such as Mycobacterium tuberculosis, direct is found to have a lethal [Stenger, S. et al ., Science 282, 121-125 (1998)].

【0006】

Granularisin is made as a precursor of 15K and then processed to 9K in cytotoxic granules, and this 9K granularisin is known to have antibacterial activity (Pena, SV et al., J). .Immunol, 158,2680-2688 (1997)). Furthermore, it has been reported that the same cytotoxic intragranular molecule, perforin, punctures target cells, from which granulaicin enters the cells and kills the infecting bacteria inside the cells [ Stenger, S. et al., Science 282, 121-125 (1998)].

【0007】

Thus, granuleicin is considered to play an important role in infection protection. It is unlikely that the active form of 9K granuleicin will acquire bacterial resistance to it, and it is possible that it will be applied to therapeutic agents for infectious diseases that have characteristics different from those of antibiotics. Also, recently, the existence of a perforin pathway for the elimination of intracellular infectious agents by 9K granuleicin has been questioned (David, HC et al., J. Immunol, 167, 2734-2742 (2001)). Moreover, this molecule has been shown to be cytotoxic (Pena, SVet al., J. Immunol, 158, 2680-2688 (1997)) and, when administered as is, has considerable side effects. I am concerned.

【0008】

An object to be solved by the present invention is to study granularisin in more detail and to provide a means for using it as a therapeutic agent for infectious diseases.

【0009】

[Means for solving problems]

The present inventor has conducted diligent studies in order to solve this problem. As a result, they found that there is a new pathway in which 15K granuleicin is taken up by macrophages and then activated to kill the bacteria taken up in macrophages.

【0010】

That is, it was found that by using 15K granuleicin, which does not show cytotoxicity by itself, as an active ingredient, it is possible to provide an effective therapeutic agent for infectious diseases, which has few side effects and it is difficult for bacteria to acquire resistance. ..

【0011】

The present invention is an invention for providing an infectious disease therapeutic agent (hereinafter, also referred to as the present therapeutic agent) containing 15K granuleicin as an active ingredient. In the present invention, 15K granuleicin is a protein having a molecular weight of 15,000 (15k), which is localized in cytotoxic granules and consists of 145 amino acids, as described above. It is partially cleaved in cytotoxic granules and exists as a protein with a molecular weight of 9,000 (9k) (Pena, SV, et al., J.Immunol., 158, 2680-2688 (1997), Stenger, S., et al., Science, 282, 121-125 (1998)).

【0012】

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described. A. Active ingredient of this therapeutic agent 15K granuleicin used as the active ingredient of this therapeutic agent can be used separately from the living body, but since it is a biotrace component, it is a recombinant protein obtained by expressing the gene encoding 15K granuleicin. It is preferable to use as. In addition, it is also a preferable means to produce 15K granularisin in the body by using an in-vivo expression vector of 15K granularisin in which a gene encoding 15K granulysin is incorporated as an active ingredient.

【0013】

Recombinant protein of 15K granuleicin The gene sequence encoding 15K granulaicin has already been reported (Jongstra, et al., J.Exp.Med, 165,601). It is a 15K granulaicin protein with the corresponding amino acid sequence. Based on this, it is possible to efficiently prepare a gene encoding 15K granularisin and express this gene to prepare a recombinant protein of 15K granularisin.

【0014】

Specifically, a gene amplification product of a gene encoding 15K granuleicin is obtained by a gene amplification method such as PCR by using a nucleotide chain complementary to both ends of the gene sequence encoding 15K granuleicin as a primer for amplification. To prepare.

【0015】

This can be incorporated into an appropriate gene expression vector to obtain the desired 15K granulaicin from an appropriate host such as Escherichia coli, Bacillus subtilis, yeast, insect cells, etc. transformed with such a recombinant vector.

【0016】

As the gene expression vector used here, it is preferable to use a vector having a promoter, enhancer, and a transcription termination sequence in the downstream region of the gene to be normally expressed.

【0017】

The expression of the 15K granulaicin gene is not limited to the direct expression system, but can also be a fusion protein expression system using, for example, a β-galactosidase gene, a glutathione-S-transferase gene, or a thioredoxin gene.

【0018】

Examples of the gene expression vector include pQE, pGEX, pT7-7, pMAL, pTrxFus, pET, and pNT26CII as the host of Escherichia coli. Moreover, as a host using Bacillus subtilis, pPL608, pNC3, pSM23, pKH80 and the like can be exemplified.

【0019】

Moreover, as a host using yeast, pGT5, pDB248X, pART1, pREP1, YEp13, YRp7, YCp50 and the like can be exemplified.

【0020】

Examples of host cells such as mammalian cells or insect cells include p91023, pCDM8, pcDL-SRα296, pBCMGSNeo, pSV2dhfr, pSVdhfr, pAc373, pAcYM1, pRc / CMV, pREP4, and pcDNAI.

【0021】

These gene expression vectors can be selected according to the purpose for expressing 15K granuleicin. For example, when expressing 15K granuleicin, it is preferable to select a gene expression vector capable of selecting Escherichia coli, Bacillus subtilis, yeast, etc. as a host, and express 15K granuleicin so as to have reliable activity even in a small amount. In some cases, it is preferable to select a gene expression vector capable of selecting a mammalian cell or an insect cell as a host.

【0022】

Further, although it is possible to select an existing gene expression vector as described above, it is also possible to appropriately prepare a gene expression vector according to the purpose and use it.

【0023】

The introduction of the above gene expression vector incorporating the 15K granulaicin gene into a host cell and the transformation method using this method are common methods, for example, when the host cell is Escherichia coli or Bacillus subtilis, the calcium chloride method or electro. If the host is a mammalian cell or an insect cell, the poration method or the like can be carried out by means such as the calcium phosphate method, the electroporation method or the liposome method.

【0024】

By culturing the transformant thus obtained according to a conventional method, the desired 15K granuleicin is accumulated. The medium used for such culture can be appropriately selected depending on the properties of the host. For example, when the host is Escherichia coli, LB medium or TB medium is used, and when the host is mammalian cells, the medium is selected. RPMI1640 medium or the like can be used as appropriate.

【0025】

Isolation and purification of 15K granuleicin from the culture obtained by this culture can be performed according to a conventional method, for example, the culture can be obtained in various cultures utilizing the physical and / or chemical properties of 15K granularisin. It can be done by using the processing operation of.

【0026】

Specifically, treatment with a protein precipitate, ultrafiltration, gel filtration, high performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography using a specific antibody, dialysis, etc. alone or in combination of these methods Can be used.

【0027】

As described above, 15K granuleicin can be isolated and purified. 15K granulaicin causes infectious diseases that are phagocytosed by macrophages by being taken up by macrophages in the blood and activated in them by using it as an active ingredient of therapeutic agents for infectious diseases. By killing bacteria, viruses, fungi and the like, it becomes possible to treat infectious diseases caused by these microorganisms. As described above, unlike 9K granuleicin, 15K granuleicin itself is not cytotoxic and is expected to be effective as a therapeutic agent for infectious diseases with few side effects due to administration.

【0028】

15K Granularisin expression vector in the body The active ingredient of this therapeutic agent in this form is a recombinant vector in which the gene encoding 15K granuleicin used for the expression of the above-mentioned recombinant protein is incorporated into an expression vector in the body.

【0029】

Examples of the in-vivo expression vector include, but are not limited to, an adenovirus vector, a retrovirus vector, and the like. The expression vector in the recombinant body is, for example, a cosmid vector in which the above-mentioned viral gene is incorporated, and a gene capable of expressing 15K granuleicin is further incorporated into the cosmid vector, and the parent viral DNA treated with a restriction enzyme-. Transfection of TP into 293 cells results in homologous recombination within the 293 cells to produce the desired in vivo expression vector.

【0030】

B. Form of this therapeutic agent This therapeutic agent containing the recombinant protein of 15K granuleicin as an active ingredient The first form of this therapeutic agent is formulated with 15K granuleicin as an active ingredient, and it is also possible to formulate it in the form of a formulation composition by blending an appropriate pharmaceutical formulation carrier (15K granulara). Of course, it is possible to use only Ishin). As the pharmaceutical formulation carrier, for example, a filler, a bulking agent, a binder, a humidifying agent, a stabilizer, a solubilizing agent, a disintegrant, and a surface which can be appropriately used as a pharmaceutical formulation carrier depending on a specific dosage form. Excipients such as activators and diluents can be freely selected. The form of the formulation composition is not particularly limited as long as 15K granulaicin can be effectively used for the treatment of infectious diseases, and is, for example, a solid preparation such as a tablet, a powder, a granule, or a pill. It can also be in the form of an injection such as a liquid, a suspension, or an emulsion. It is also possible to make a dried product that should be liquefied at the time of use by adding an appropriate carrier to 15K granuleicin.

【0031】

The dose of the therapeutic agent thus obtained can be appropriately selected according to the administration method, administration form, patient's symptom, etc. of the agent, and is not particularly limited.

【0032】

Such various forms of pharmaceutical preparations have an appropriate route of administration depending on the form, for example, intravenous, intramuscular, intraosseous, intra-articular, subcutaneous, intradermal, and intraperitoneal in the case of injection form. By administration or the like, in the case of a solid form, it can be administered orally or by enteral administration.

【0033】

This therapeutic agent containing the in-vivo expression vector of 15K granuleicin as an active ingredient By isolating and purifying the in-vivo expression vector that can be prepared as described above and administering it to a living body, 15K granuleicin can be produced in vivo and the pharmacological effect of this 15K granuleicin can be exerted. ..

【0034】

The administration form in this case is generally an injection form, and can be administered by intravenous, intramuscular, intraosseous, intra-articular, subcutaneous, intradermal, intraperitoneal administration, or the like. The dose of such a therapeutic agent can be appropriately selected according to the administration method, administration form, patient's symptom, etc. of the agent, and is not particularly limited, but is generally an active ingredient. It is preferable to appropriately prepare an in-vivo expression vector expressing 15K granuleicin, and administer this preparation in an appropriate amount once a day or in several divided doses.

【0035】

[Example]

Examples of the present invention will be described below. [Test example] Examination of antibacterial effect against tubercle bacilli when 15K granuleicin and macrophages coexist (1) Preparation of monoclonal antibody specific for 15K granuleicin Human peripheral blood lymphocytes (2 x 10) cultured in the presence of 100-200 unit / ml IL-2⁶Cell / ml human peripheral blood lymphocytes in RPMI1640 medium containing 10% fetal bovine serum at 37 ° C. 5% CO₂RNA was extracted according to a conventional method (cultured under 10 days), and the RT-PCR method (PCR primer 1: SEQ ID NO: 2, PCR primer 2: SEQ ID NO: 3) was performed using this RNA as a template, and 15K. Gene part containing the region encoding the total length of the protein of granulaicin [Jongstra, et al., J.Exp.Med, 165,601: the part corresponding to the amino acid sequence of SEQ ID NO: 1] was synthesized as an amplification product of complementary DNA (cDNA). The cDNA encoding the full-length protein of this 15K granuleicin was incorporated into a mammalian expression vector, pRc / CMV or pcDL-SRα296, and the obtained recombinant vector was dissolved in physiological saline to be dissolved subcutaneously or intradermally in mice. Immune to. After immunization 4 to 5 times at intervals of 1 to 2 weeks, mouse splenocytes whose antibody titer was observed to increase by indirect immunofluorescence (following the method described later) were fused according to a conventional method. , Hybridomas that produce antibodies that specifically bind to granulaicin were again searched by indirect immunofluorescence. That is, cells (Cos7) expressed by transforming with the gene encoding 15K granulaicin described above were fixed with 4% paraformaldehyde, and then the membrane was solubilized with 0.5% tween20. Hybridoma culture supernatant is added and the antibody is reacted, then a fluorescently labeled anti-mouse IgG antibody is reacted, and fluorescence is detected to screen for hybridomas that produce an antibody that specifically binds to granulaicin. did. As a result, a hybridoma that produces an antibody that specifically binds to nine granulysins was obtained. Using each culture supernatant of the obtained hybridoma, ammonium sulfate precipitation and purification with a protein G column were carried out to prepare two types of monoclonal antibodies against 15K granuleicin. Hereinafter, this is bound to the monoclonal antibody RF10 (which binds to 15K granulysin but not to 9K granulysin) and the monoclonal antibody RC8 (which binds to both 15K granularisin and 9K granularisin). Also called).

【0036】

(2) Preparation of polyclonal antibody against 15K granuleicin Partial Amino Acid Sequence of Granularicin (29 Amino Acids) (J. Exp. Med. 165: 601-614 (1987), J. Exp. Med., 172: 1159-1163 (1990)): Arg Thr Gly Arg SerArg Trp Arg Asp Val Cys Arg Asn Phe Met Arg Arg Tyr Gln Ser Arg Val ThrGln Gly Leu Val Ala Gly (N5-1: SEQ ID NO: 4) in combination with the umbrella shell hemocyanin to immunize rabbits according to conventional methods and antiserum Obtained. The obtained antiserum was purified by sulfite precipitation and a protein G column, and further purified by affinity chromatography using a column to which the above synthetic peptide (N5-1) was bound, and a polyclonal antibody against granulaicin (anti-N5-) was performed. 1 antibody) was prepared.

【0037】

(3) Preparation of culture supernatant containing granulaicin A gene recombination vector was created by incorporating the nucleotide chain of the base sequence encoding the portion corresponding to the 15K granuleicin protein in the base sequence of SEQ ID NO: 1 into a pFLAG-CMV vector (manufactured by Sigma) by a conventional method. did. As a control, the above pFLAG-CMV vector without gene recombination was used. These genetically modified vectors were introduced into Cos7 cells, which were used in DMEM medium (manufactured by Gibco) at 5% CO.₂-Culturing at 37 ° C for 72 hours. After completion of the culture, the culture was centrifuged (2500 rpm, 20 minutes, 4 ° C) to obtain the culture supernatant.

【0038】

For each culture supernatant, proteins were separated by SDS-PAGE. After transferring the protein from the electrophoresed SDS-PAGE gel to a nylon membrane and then blocking the membrane with a blocking solution (1% skim milk / washing solution), this membrane is specific for 15K granulicin. The monoclonal antibody RF10 was bound to this, and an enzyme-labeled anti-mouse antibody and a chromogenic substrate were allowed to act on the monoclonal antibody RF10 to develop a band. As a result, a band showing a molecular weight of 15K appeared in the supernatant obtained by expressing the gene encoding the 15K granuleicin protein, but this band was not observed in the control. Moreover, when the same test was performed using the polyclonal antibody N5-1 that binds to 15K and 9K granulysin, a band showing 15K was observed, but a band showing 9K was not observed.

【0039】

From this result, it was clarified that 15K granulysin was specifically present in the above-mentioned granulysin culture supernatant, and that granulysin was not present in the control culture supernatant.

【0040】

(4) Examination of antibacterial effect Lymphocytes separated from human blood by conventional methods were placed in a plastic culture plate (24 wells / plate), medium (RPMI 1640, 10% human serum), 2 x 10 per 1 ml of medium.⁷Suspended to the extent of cells, dispense 1 ml into each well and leave at 37 ° C for 24 hours to allow lymphocytes to attach to the plate wall and in this attached macrophage. , The antibacterial effect of 15K granulaicin was examined.

【0041】

Next, 1 ml of RPMI1640 (10% human serum) was added to the well, and tubercle bacilli (H37Rv, 1 × 10) were added thereto.⁵~1×10⁶cfu), 37 ° C, 5% CO₂Underneath, static culture was carried out for 4 to 12 hours to infect macrophages with M. tuberculosis. After completion of infection, 1 ml each of Grn supernatant or Cont supernatant was added to the wells, and the cells were allowed to stand for 2 to 12 hours under the same conditions.

【0042】

After completion of the culture, the culture supernatant is removed, the adherent cells in the plate are washed 3 times with PBS, and the intracellular tubercle bacilli are extracted with a total of 5 ml of 1% saponin aqueous solution, and this extract is used. The cells were diluted, sown on a flat plate agar medium [7H11 medium (manufactured by Gibco)], and statically cultured at 37 ° C for 14 days, and the number of colonies was counted.

【0043】

The results are shown in Fig. 1. In FIG. 1, Cont represents the control culture supernatant and Grn represents the granulysin culture supernatant. The vertical axis shows the number of colonies. 1 on the horizontal axis shows the result of contacting macrophages, Mycobacterium tuberculosis, and culture supernatant as described above. In No. 2, in the same manner as in 1, each culture supernatant and M. tuberculosis were statically cultured in the absence of macrophages, washed, and then statically cultured on a 7H11 plate agar medium, and the effect of non-specific adsorption on the plate of M. tuberculosis. The result of confirming is shown. 3 shows the results of statically culturing 1 ml of each culture supernatant together with Mycobacterium tuberculosis at 37 ° C. for 2 hours on the above culture plate, and statically culturing this on a 7H11 plate agar medium.

【0044】

As a result, it was clarified that 15K granuleicin has an antibacterial effect against tubercle bacilli due to the intervention of macrophages. In the absence of macrophages, 15K granuleicin had no antibacterial effect against M. tuberculosis.

【0045】

From the above test examples, it was clarified that 15K granuleicin has an antibacterial effect and can be used as an active ingredient of an infectious disease therapeutic agent. When 15K granularisin was transfected into cells such as Cos7 cells, HeLa, and PC12, 15K granularisin was detected in the culture supernatant, but no damage was observed to the cells. It has been reported that the use of 9K granuleicin in place of 15K granuleicin induces cytotoxic activity and apoptosis (Pena, SVet al., J. Immunol, 158, 2680-2688 (1997)). ..

【0046】

[Effect of the invention]

INDUSTRIAL APPLICABILITY According to the present invention, an infectious disease therapeutic agent containing 15K granuleicin as an active ingredient, which has no side effects and is effective, is provided.

【0047】

[Sequence list]

SEQUENCE LISTING <110> BML, INC. National Kinki Central Hospital For Chest Diseases <120> Remedy for Infectious Diseases <130> PBM67 <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 745 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (129) .. (563) <400> 1 gtatctgtgg taaacccagt gacacggggg agatgacata caaaaagggc aggacctgag 60 aaagattaag ctgcaggctc cctgcccata aaacagggtg tgaaaggcat ctcagcggct 120 gccccacc atg gct acc tgg gcc ctc ctg ctc ctt gca gcc atg ctc ctg 170 Met Ala Thr Trp Ala Leu Leu Leu Leu Ala Ala Met Leu Leu 1 5 10 ggc aac cca ggt ctg gtc ttc tct cgt ctg agc cct gag tac tac gac 218 Gly Asn Pro Gly Leu Val Phe Ser Arg Leu Ser Pro Glu Tyr Tyr Asp 15 20 25 30 ctg gca aga gcc cac ctg cgt gat gag gag aaa tcc tgc ccg tgc ctg 266 Leu Ala Arg Ala His Leu Arg Asp Glu Glu Lys Ser Cys Pro Cys Leu 35 40 45 gcc cag gag ggc ccc cag ggt gac ctg ttg acc aaa aca cag gag ctg 314 Ala Gln Glu Gly Pro Gln Gly Asp Leu Leu Thr Lys Thr Gln Glu Leu 50 55 60 ggc cgt gac tac agg acc tgt ctg acg ata gtc caa aaa ctg aag aag 362 Gly Arg Asp Tyr Arg Thr Cys Leu Thr Ile Val Gln Lys Leu Lys Lys 65 70 75 atg gtg gat aag ccc acc cag aga agt gtt tcc aat gct gcg acc cgg 410 Met Val Asp Lys Pro Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg 80 85 90 gtg tgt agg acg ggg agg tca cga tgg cgc gac gtc tgc aga aat ttc 458 Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp Val Cys Arg Asn Phe 95 100 105 110 atg agg agg tat cag tct aga gtt acc cag ggc ctc gtg gcc gga gaa 506 Met Arg Arg Tyr Gln Ser Arg Val Thr Gln Gly Leu Val Ala Gly Glu 115 120 125 act gcc cag cag atc tgt gag gac ctc agg ttg tgt ata cct tct aca 554 Thr Ala Gln Gln Ile Cys Glu Asp Leu Arg Leu Cys Ile Pro Ser Thr 130 135 140 ggt ccc ctc tgagccctct caccttgtcc tgtggaagaa gcacaggctc 603 Gly Pro Leu 145 ctgtcctcag atcccgggaa cctcagcaac ctctgccggc tcctcgcttc ctcgatccag 663 aatccactct ccagtctccc tcccctgact ccctctgctg tcctcccctc tcacgagaat 723 aaagtgtcaa gcaagaaaaa aa 745 <210> 2 <211> 24 <212> DNA <213> Homo sapiens <400> 2 catctcagcg gctgccccac catg 24 <210> 3 <211> 27 <212> DNA <213> Homo sapiens <400> 3 tgtatacctt ctacaggtcc cctctga 27 <210> 4 <211> 29 <212> PRT <213> Homo sapiens <400> 5 Arg Thr Gly Arg Ser Arg Trp Arg Asp Val Cys Arg Asn Phe Met Arg 1 5 10 15 Arg Tyr Gln Ser Arg Val Thr Gln Gly Leu Val Ala Gly 20 twenty five

[Simple explanation of drawings]

[Figure 1]

It is a drawing which shows the result of having examined the antibacterial effect of 15K granuleicin against tubercle bacillus.