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WO1999061592A1 - Nouvelle endonuclease de cellule immunitaire, son procede de production et adjuvant immunitaire obtenu au moyen de ladite enzyme - Google Patents

Nouvelle endonuclease de cellule immunitaire, son procede de production et adjuvant immunitaire obtenu au moyen de ladite enzyme Download PDF

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Publication number
WO1999061592A1
WO1999061592A1 PCT/KR1998/000136 KR9800136W WO9961592A1 WO 1999061592 A1 WO1999061592 A1 WO 1999061592A1 KR 9800136 W KR9800136 W KR 9800136W WO 9961592 A1 WO9961592 A1 WO 9961592A1
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Prior art keywords
endonuclease
dna
cell
activity
cells
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PCT/KR1998/000136
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English (en)
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Yeong Joong Jeon
Wan Je Park
Na Gyong Lee
Sang Bo Jung
Doo Sik Kim
Hyung Joo Kwon
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Cheil Jedang Corporation
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Priority to JP2000550977A priority Critical patent/JP2002516086A/ja
Priority to AU77889/98A priority patent/AU7788998A/en
Publication of WO1999061592A1 publication Critical patent/WO1999061592A1/fr
Priority to US09/722,776 priority patent/US6881561B1/en
Priority to US10/860,844 priority patent/US20070173468A9/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants

Definitions

  • the present invention relates to a novel endonuclease enzyme which is secreted from immune cells and recognizes bacterial DNA as foreign substance and processes it to generate approximately 10 bases single-stranded oligonucleotide including CpG motif known to involve immune response.
  • the present invention relates to an immune adjuvant comprising the said single-stranded oligonucleotides of approximately 10 bases generated by the said endonuclease enzyme.
  • Mammalian animals develop immune systems to defend against foreign agents.
  • the immune systems is classified into natural (nonspecific) immunity or acquired
  • the innate or nonspecific immunity is a primary resistance against diseases caused by one species, and creates defence barrier as four types such as structural, physiological, endocytic and phagocytic, and inflammatory response.
  • Representative examples of the structural defence barrier include skin and mucus.
  • the physiological defence barrier include, for example, temperature, pH, oxygen pressure, and various aqueous soluble factors.
  • the endocytic and phagocytic defence barrier refers to endocytosis and phagocytosis degradation systems in which foreign macromolecules are incorporated into and subsequently degraded by certain cells.
  • the inflammation defence barrier is an inflammatory response which is evolved by various vasoactive and chemotactic agents generated by penetration of bacteria and followed by skin damage.
  • the acquired immunity is different from the innate immunity in that the former possesses specificity, diversity, memory and self and/or non-self recognition.
  • the properties of the acquired immunity Eire derived from the humoral and cellular immunities which respond by B lymphocytes, T lymphocytes, antibody, cytokine, etc.
  • the immune response by penetration of microorganisms is generated by the innate mechanism which rapidly recognizes certain molecules of the microorganisms at the initiation stage of the penetration.
  • the proteins and lipids present in microorganisms are well known as agents inducing immune systems which specifically respond to antigen.
  • LPS, formyl methionine, lipoarabinomannan, peptidoglycan, etc. are well known as agents which directly activate the complement system (Marrack, P., and Kapple, J. W. (1994) Cell 76, 323-332).
  • humoral and cellular immunities are activated by distinguishing their intrinsic DNA from bacterial DNA and recognizing the bacterial DNA as foreign agent and that also such bacterial DNA involves innate immunity.
  • DNA possesses potent immunological properties which activate polyclonal B cell and produce antibodies having specificity in mouse (Gilkeson, G. S., et al (1995) J. Clin. Invest. 95, 1398-1402; and Gilkeson, G. S., et al (1991) Clin. Immunol. Immunopathol. 59, 288-300).
  • the activity degree is due to the fact that the base sequence motif present in bacterial DNA is different from the base sequence motif of mammalian DNA and may be recognized as foreign agent, i.e., non-self (Messina, J. P. et al (1993) Cell. Immunol. 147, 148-157; Krieg, A. M.
  • a production of antibody by stimulating and activating B cell with protein antigen is well known as the process in which protein antigen is processed by antigen presentation cell (APC) and is bound to major histocompatibility complex (MHC) to induce a presentation of antigen so that MHC- restricted T cell is activated and the activated T cell secrets cytokine to activate B cell (Parker, D. C.(1993) Annu. Rev. Immunol. 11, 331-360; and Clark, E. A., and Ledbetter, J.A.(1994) Nature 367, 425-428).
  • APC antigen presentation cell
  • MHC major histocompatibility complex
  • lipoarabinomannan lipoglycans mycolic acid lipids, processed form of constitutive element of mycobacterial cell wall which are distinct from protein antigen can be presented by hCDlb (Beckman, E. M. et al (1994) Nature 372, 691-694; Bendelac, A.(1995) Science 269, 185-186; Sieling, P.
  • CDl family is nonpolymorphic cell surface glycoprotein which is encoded at the different sites from MHC molecule.
  • CDlb is recognized by CD4 " and CD8 " T cells (Bendelac, A.(1995) Science 269, 185-186). It is thus assumed that CDl is involved in presentation of various antigens other than proteins found in pathogenic microorganisms. Many studies reported that DNA is involved in anti-DNA-specific
  • the bacterial DNA recognized as non-self by vertebrate animals is characterized by generating nonmethylated CpG dinucleotide at high level.
  • the extraordinary difference between bacterial DNA and vertebral DNA may be summarized as follows. First, bacterial DNA generates CpG dinucleotide of 16 dinucleotides at most level, but vertebral DNA generates 1/4 of bacterial DNA. This means that CpG suppression exists in vertebral DNA. Second, methylation frequency of CpG dinucleotide present in bacterial DNA is low.
  • bacterial DNA is higher than vertebral DNA in the frequency of flanking two 5'-purines and two 3'-pyrimidines at both ends of CpG dinucleotide (Razin, A., and
  • CpG motif The specific structure of the bacterial DNA called "CpG motif was reported to activate immune response. That is, the activation of immune cell when two 5'-purines and two 3'- pyrimidines were flanked at both ends of CpG dinucleotide (mitogenic CpGs) is much higher compared to when other bases are flanked at both ends of CpG dinucleotide
  • ODN oligodeoxyribonucleotide
  • Yamamoto and other researchers showed that bacterial DNA increased lytic activation of NK cell and induced the production of interferon- ⁇ (IFN- ⁇ ) (Yamamoto, S., et al (1992) J. Immunol. 148, 4072-4076; Cowdery, J. S., et al (1996) J. Immunol. 156, 4570-4575; and Ballas, Z. K., et al (1996) J. Immunol. 157, 1840- 1845).
  • Kuramoto reported that such effects are associated with palindromic base sequence of CpG motif included in bacterial DNA ( Kuramoto, E., et al (1992) J.
  • Cytokines generated therefrom include IL-6 which plays a role in activating T cell and B cell (Uyttenhove, C, et al (1988) J. Exp. Med 167, 1417-1427; Muraguchi, A., et al (1988) J. Exp. Med. 167, 332-344; Le, J. M., and
  • IFN- ⁇ which promotes the function of macrophage to eliminate intra- and extra-cellular pathogenic bacteria
  • IL- 12 which regulates production of IFN- ⁇ and activates NK cell
  • IL-12 and IFN- ⁇ take an important role to eliminate human pathogenic bacteria by increasing type 1 cytokine (Klinman, D. M., et al (1996) Proc. Natl. Acad. Sci. USA 93, 2879-2883; Zhan, Y, and Cheers, C.(1995) Infect. Immun. 63, 1387-1390; Bohn, E., et al (1994) Infect. Immun. 62, 3027-3032; and Heinzel, F. P., et al (1991) Proc. Natl. Acad. Sci.
  • IL-6 stimulates the production of antibody by promoting growth and differentiation of T cell and B cell by type 2 cytokine (Uyttenhove, C, et al (1988) J. Exp. Med 167, 1417-1427; Muraguchi, A., et al (1988) J. Exp. Med. 167, 332-344; Le, J. M., and Vilcek, J.(1989) Lab, Invest. 61, 588-602; and Hirano, T., et al (1990) Immunol. Today 11, 443-449).
  • IFN- ⁇ and IL-12 are increased. This result indicates that if plasmid including ISS is transfected into bone marrow stem cell, then surrounding macrophage and T cell are activated and in vivo rearrangement of the stem cell may be mistakenly occurred. Thus, vector for somatic or stem cell-replacing therapy should be designed not to include ISS . As contrast, one approach to improve the efficacy of vaccine is to design plasmid DNA so as to include many repetitive ISS.
  • Bacterial DNA has been so far understood to take a critical role in the immune system. It has been known that autoimmune disease SLE is occurred by the generation of anti- DNA antibody by bacterial DNA and that CpG motif of bacterial DNA is incorporated into immune cell to activate the cell and thereby promote secretion of cytokine and IgM. However, there is no report as to what mechanism enables such critical bacterial DNA to produce antibody and how oligonucleotide having CpG motif is made in cell.
  • a novel endonuclease was identified by the inventors from human B-lymphoblastic
  • the present invention provides novel endonuclease enzyme which is secreted from immune cell and which recognizes bacterial DNA as foreign agent and processes it to produce about 10 bp single-stranded oligonucleotide having CpG motif which is involved in immune response.
  • the present invention provides a process for producing the endonuclease of the present invention which comprises culturing human B- lymphoblastic IM9 or TPA-treated myelogeneous U937 cell line on an appropriate medium to produce the said endonuclease and isolating the said endonuclease from cell lysates or culture medium.
  • the present invention provides an immune adjuvant comprising about 10 bp single-stranded oligonucleotide having CpG motif which is produced by treating bacterial DNA with the endonuclease of the present invention.
  • Fig.1 shows the endonuclease activity of IM9 cells according to the present invention analyzed by DNA-native-PAGE.
  • the endonuclease activity in IM9 cell lysates (A), medium (B) and medium cultured in serum-free (C) was detected by in-gel system.
  • Bovine DNase 1 (lane) and culture medium endonuclease activity (lane2) were compared (D).
  • Fig. 2 shows the endonuclease activity of DNase 1 (A) and IM9 culture medium (B) analyzed by DNA-native-PAGE.
  • Fig. 3 shows the immunoprecipitation of endonuclease activity by anti-bovine DNase
  • IP immunoprecipitation
  • Fig. 4 shows the pretreatment of IM9 cells with actinomycin D and secretion of the endonuclease.
  • the endonuclease activity in cell lysates (A) and culture medium (B) according to the present invention was analyzed at indicated culture periods after pretreatment.
  • Fig. 5 shows the comparative analysis of endonuclease activity in immune cell lines according to the present invention.
  • the endonuclease activity in lysates (A) and culture medium (B) of each cell line was analyzed by the DNA-native-PAGE.
  • Fig. 6 shows the endonuclease activity of IFN- ⁇ or IL-l ⁇ treated IM9 cell culture medium and cell lysates according to the present invention analyzed by DNA-native-PAGE.
  • IM9 cells were treated with 10 units/ml of IFN- ⁇ or IL-l ⁇ for the indicated culture times.
  • IM9 culture medium in RPM 16-40 medium containing 10% FBS for 12 hours Control, IM9 culture medium in RPM 16-40 medium containing 10% FBS for 12 hours.
  • Fig. 7 shows the optimal pH for the endonuclease activity according to the present invention.
  • Fig. 8 shows the requirement of divalent cation for the endonuclease activity according to the present invention.
  • the enzyme was reacted with 100 ng of plasmid DNA for the enzyme.
  • Fig. 9 shows the growth curve of TPA, LPS, and CHX treated U937 cells.
  • U937 cells were treated with TPA (10 ng/ml), LPS (1 ng/ml), and CHX (10 /ml) as the indicated times. Cell number and viability were esteminated by trypan blue exclusion in a hemocytometer during culture time.
  • Fig 10 shows the TPA-concentration dependent synthesis and secretion of endonuclease in U937 cells were analyzed by DNA-native-PAGE.
  • Fig. 11 shows the endonuclease activity of TPA treated U937 cell lysates and culture medium analyzed by in-gel system.
  • U937 cells were treated with 10 ng/ml TPA for the indicated culture times.
  • Fig. 12 shows the endonuclease activity of LPS treated U937 cell lysates and culture medium.
  • the endonuclease activity in cell lysates (A) and medium (B) was analyzed at the indicated culture periods after 1 ng/ml LPS treatment by DNA-native-PAGE.
  • Fig. 13 shows the endonuclease activity of U937 cells treated with stimulatory factors (lane 1 , U937 cell culture in RPML1640 medium containing 10% FBS for 48 hr; lane 2, TPA(10 ng/ml) treatment for 48 hr; lane 3, LPS(lng/ml) treatment for 24 hr; and lane 4, CHX(10/ml) treatment for 12 hr).
  • stimulatory factors lane 1 , U937 cell culture in RPML1640 medium containing 10% FBS for 48 hr; lane 2, TPA(10 ng/ml) treatment for 48 hr; lane 3, LPS(lng/ml) treatment for 24 hr; and lane 4, CHX(10/ml) treatment for 12 hr).
  • Fig. 14 shows the endonuclease activity in nuclei isolated from IM9 cells by autodigestion method (A, Nuclei were incubated for 2 hr at 37 ° C either alone or on the presence of the indicated concentration of Ca 2+ and/or Mg 2+ ; B, Inhibition of internucleosomal DNA fragmentation; C and D, Nuclei were incubated at 37 ° C in the presence of 10 mM Mg 2+ (C) or 10 mM Ca 2+ (D) for 0-240 min as indicated; and Marker, lkb ladder)
  • Fig. 15 shows the apoptotic cell death of IM9 cells by CHX treatment (A, 1.8% agarose gel electrophoreis of DNA from IM9 cells treated with CHX (10/ml); Untreated (B) and treated (C) with CHX of IM9 cells were cultured for 24 hr and cytocentrifuge preparations stained with Wright-
  • Fig 16 shows the endonuclease activity of IM9 cell lysates and nuclei analyzed by DNA-native-PAGE according to the present invention.
  • the endonuclease activity was detected in IM9 cell lysates during culture time (A), cell lysates (B) and nuclei (C) treated with 10 ug/ml CHX for the indicated times.
  • Control FBS DNase 1.
  • Fig 17 shows the requirement of divalent cation for the endonuclease activity according the present invention.
  • the endonuclease was isolated from nuclei and the enzyme activity was determined by resolving the reaction products on 1 % agarose gel.
  • Fig. 18 shows the time-course degradation of plasmid DNA by the endonuclease of the present invention.
  • the endonuclease activity of RPMI medium containing 10% FBS (A), IM9 cell culture medium in serum free (B), and IM9 cell culture containing 10% FBS (C) was estimated by resolving the reaction products on 1% agarose gel.
  • Fig. 19 shows the products of endonuclease reaction of the present invention on E. coli DNA, IM9 cells DNA, and salmon sperm DNA.
  • Fig. 20 shows the southern blot analysis of foreign DNA incorpotated into IM9 cells.
  • Fig. 21 shows the construction scheme for the cloning and sequencing of the DNA fragments obtained by endonuclease reaction according to the present invention.
  • Fig. 22 shows the DNA fragments produced by processing of bacterial DNA in IM9 cells according to the present invention.
  • Fig 23 shows the DNA fragments produced by processing of bacterial DNA in U937 cells accirding to the present invention (A, labelled DNA incorporation after U937 cells culture in RPMI1640 medium containing 10% FBS for 48 hr; and B, labelled DNA incorporation after TPA (10 ng/ml) treatment for 12 hr).
  • Fig. 24 shows the detection of endonuclease reaction products sequence of the present invention.
  • the endonuclease reaction products were hybridized with synthetic oligonucleotides as described under "Materials and Methods".
  • Fig. 25 shows the induction of IgM secretion by CpG motifs in bacterial DNA or oligonucleotides.
  • IM9 cells were stimulated with oligonucleotides (25/ml), or E. coli DNA (25/ml) for 24 hr.
  • Fig. 26 shows the products of endonuclease reaction using ECl PCR product as a substrate according to the present invention.
  • Fig. 27 shows the product of endonuclease reaction using EC2 PCR product as a substrate according to the present invention.
  • Fig. 28 shows the product of endonuclease reaction using HC1 PCR product as a substrate according to the present invention.
  • Fig. 29 shows the inhibition of endonuclease activity by Zn 2+ and EDTA.
  • Fig. 30 shows the product of endonuclease reaction from ECl PCR product reacted with indicated IM9 cell culture medium amounts according to the present invention.
  • Fig. 31 shows the product from endonuclease reacted with ECl PCR product for the indicated times according to the present invention.
  • Fig. 32 shows the endonuclease activity comparing EC2 PCR product (157 bp) with short DNA fragment cleaved by Alu I (lane 1 , 32 p-labelled PCR product; lane 2, endonuclease digested product of lane 1; lane 3, 32 p-labelled short DNA fragment cleaved by Alu I; lane 4, 30 min reaction of lane 3; lane 5, 1 hr reaction of lane 3; lane 6, 2 hr reaction of lane 3).
  • Fig. 33 shows the identification of 3'-exonuclease activity in IM9 cell secreted endonuclease (lane 1 , labelled PCR product; lane 2, products of endonuclease reaction on labelled double stranded DNA; lane 3, products of endonulease reaction on labelled single stranded DNA).
  • Fig. 34 shows the comparison between endonuclease reaction product and processed product by IM9 cells with DNase I reaction product (lane 1 , labelled PCR product; lane 2, reaction product of IM9 cell culture medium; lane 3, processed product in IM9 cells; lane 4, DNase I reaction product; Panel A, 20% native-PAGE in TBE buffer; Panel B, denatured-urea (8.3 M)-PAGE in TBE buffer)
  • Fig. 35 shows the identification of single stranded fragments derived from endonuclease reaction by S 1 nuclease reaction (A, 20% native-PAGE in TBE buffer; B, 20% denatured-Urea (8.3 M)-PAGE in TBE buffer).
  • Fig. 36 shows the chromatographic fractionation of IM9 cell culture medium by
  • Fig. 37 shows the purfication of endonuclease by RESOURCE PHE hydrophobic interaction chromatography (A, hydrophobic interaction profile of activity fraction obtained from ion exchange chromatography; B, the enzyme activity was determined at the peak fractions by resolving the reacction products on 1% agarose gel.
  • Fig. 38 shows the SDS-PAGE of purified endonuclease by ion exchange chromatography and hydrophobic interaction chromatography.
  • Fig. 39 shows the molecular weight determination of purified endonuclease by SDS-PAGE (marker proteins are myosin (200 kD), ⁇ -galactosidase (116.3 kD), phosphorylase B (97.4 kD), bovine serum albumin (66.2 kD) and ovalbumin (45 kD)).
  • marker proteins are myosin (200 kD), ⁇ -galactosidase (116.3 kD), phosphorylase B (97.4 kD), bovine serum albumin (66.2 kD) and ovalbumin (45 kD)).
  • Fig. 40 shows the native-pore gradient gel electrophoreis (4-15%) of Mono S chromatography fraction containing activity (B) and endonuclease activity of eluted protein from gel band of panel B on agarose gel electrophoresis (A).
  • Fig. 41 shows the SDS polyacrylamide gel electrophoresis of purified endonuclease by ion exchange chromatography and native-gradient PAGE gel elution of activity band.
  • Fig. 42 shows the effects of cation on purified endonuclease activity in isolated U937 cell nuclei.
  • the novel endonuclease was identified from IM9 cell lysates and culture medium using DNA-native-PAGE nuclease assay system.
  • the molecular weight of the endonuclease was determined as 72.4 kD by SDS-PAGE.
  • the endonuclease activity of the present invention was detected in IM9 cell nuclei during culture time and accumulation of the enzyme activity was shown in the IM9 cell nuclei protein extracts of the apoptotic cells.
  • the signals for proliferation and differentiation of myelogeneous U937 cells are provided by extracellular stimuli such as lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbil 13-acetate (TPA).
  • LPS lipopolysaccharide
  • TPA 12-O-tetradecanoylphorbil 13-acetate
  • TPA has significant effect on the degree of endonuclease secretion.
  • the enzyme activity was induced in U937 cells by LPS treatment, while the secretion of the enzyme was not detected in the culture medium.
  • the endonuclease activity determined with the enzyme isolated from the cell culture medium.
  • the endonuclease, with Mg 2+ alone, was able to catalyze the conversion of the plasmid DNA into linear form followed by further degradation,
  • the pH optimum required for the catalytic activity was determined to be in the range of pH 6.6-7.4.
  • the foreign DNA antigen partially processed in cell culture medium appears to be bound to the cell surface followed by incorporation into the cell.
  • radiolabelled DNA fragments as a foreign antigen
  • the further processing of DNA antigen in the immune cell lines was demonstrated by autoradiography.
  • Experimental results showed that the single-stranded DNA fragments of approximately 10 bases that are generated by the endonuclease were degraded by SI nuclease reaction.
  • the short single-stranded DNA sequence was successful to be hybridized with complementary synthetic oligonucleotide containing a CpG motif with unmethylated CpG dinucleotide flanked by two 5'-purines and two 3'-pyrimidines.
  • the present invention demonstrates the presence and characteristics of a novel endonuclease that exists both in human immune cell lines and in their culture media. Also, the present invention shows that the endonuclease from immune cell recognizes bacterial DNA as a foreign substance and carries out immunological process by generating DNA fragments containing a CpG motif.
  • DNase I is known to cleave internucleosomal DNA during apoptosis (Peitsch M. C, et al (1993) EMBO J. 12, 371-377). The presence of DNase I in human serum and the biochemical properties thereof were reported (Love J. D., and Hewitt R. R.(1979) J.
  • IM9 and RPMI1788 cell line Human B-lymphoblastic (IM9 and RPMI1788) cell line, T-lymphoblastic (Molt-4 and Jurkat) cell line and myelogeneous (U937) cell line were purchased from American Type Culture Collection. Cells were cultured on RPMI 1640 containing heated fetal bovine serum (FBS, Gibco BRL) 10% while maintaining 4-5 x 10 5 cells/ml. Cell culture was carried out in incubator (Forma) including 5% CO 2 at 37 ° C. The number of cells and the viability of cells during culture were periodically measured by trypan blue exclusion method using hemocytometer. The viability of cells was kept at 95% or more over whole experiment. IM9 cell line was pretreated with actinomycin D
  • IM9 cell line was treated with ACD, cultured for 30 minutes and washed. Then, the enzymatic activity of the endonuclease was measured at regular intervals while the cells were cultured for 48 hours on RPMI1640 containing 10% FBS.
  • DNA-native-PAGE nuclease activity assay was performed to detect endonuclease enzyme activity in cell culture solution and cell lysate.
  • the activity of the secreted endonuclease in tested cell cultures was observed only in IM9 cell line (Fig. 5B, lane 2).
  • the endonuclease activity was always detected in cell lysate of human T-lymphoblastic Molt-4 cell line (Fig 5A, lane 4) but not in cell culture solution.
  • myelogeneous U937 cell line, B-lymphoblastic RPMI1788 cell line and T- lymphoblastic Jurkat cell line the endonuclease activity was detected in neither cell lysate nor cell culture solution.
  • Cytokine involving immune response was treated with interferon- ⁇ (IFN- ⁇ , 10 units/ml, Genetech Inc.) and interleukine-l ⁇ (IL-l ⁇ , 10 units/ml, Genetech Inc.) to confirm the effects of cytikine on the biosynthesis and secretion of endonuclease.
  • DNA-native-PAGE assay was conducted while culturing cells for 24 hours on medium containing interleukine. As shown in Fig. 6, such cytokines did not significantly influence on the secretion of endonuclease. Also, it was observed that neither lipopolysaccharide (LPS) nor tetradecanoylphorbol 13-acetate (TPA, Sigma) influenced on the secretion of endonuclease.
  • LPS lipopolysaccharide
  • TPA tetradecanoylphorbol 13-acetate
  • U937 cells were treated with TPA at different concentrations over indicated hours.
  • U937 cell treated by cycloheximide (CHX, 10 ug/ml, Sigma) and LPS (1 ng/ml, Sigma) was compared with U937 cell treated by TPA.
  • Cells were washed by phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM Kcl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) and stained by Wright-
  • Giemsa (Sigma) over cytocentrifuge slides to observe cell shape following the treatment of cells by various agents.
  • the cell shape of differentiated human myelogeneous leukemia cell line with TPA stimuli was takes to be mature and growth was stopped, but the proliferation of cell following stimulation of LPS was taken place.
  • the growth curve of cell stimulated by such mitogens is shown in Fig. 9. It can be seen from Fig. 9 that U937 cell lines were differentiated by TPA to change the cell shape and ultimately growth was stopped and that the cells were proliferated by LPS. In addition, it was observed that the cells were killed by treatment of CHX, apoptosis- occurring agent.
  • Fig. 1 OA shows that the biosynthesis of endonuclease in cell was increased in line with increased TPA concentration.
  • Fig. 10B shows that endonuclease enzyme was secreted from cell at the same time that the biosynthesis of endonuclease in cell was increased.
  • Fig. 10A shows that the biosynthesis of endonuclease was rapidly increased at 10 ng/ml of TPA or more.
  • Fig. 11 shows the results obtained when endonuclease activity was observed at regular intervals following treatment of U937 cell culture solution by 10 ng/ml of TPA.
  • Fig. 12 shows the results obtained when endonuclease activity was determined at regular intervals after treatment of cell by 1 ng/ml of LPS. The results indicate that endonuclease activity was detected in cell lysate at 12 hours after treatment of LPS and any endonuclease activity was not detected in cell culture solution over 24 hours. It was microscopically observed that U937 cell line was killed upon treatment by CHX, apoptosis-inducing agent, and such a treatment did not effect on the biosynthesis of endonuclease (Fig. 13, lane 4). The endonuclease activity after treatment of U937 cell line by TPA, LPS and CHX was shown in Fig. 13.
  • the cell culture was centrifuged at 1 ,500 rpm for five(5) minutes and supernatant was removed.
  • the centrifuged cells were washed twice with cold PBS and resuspended in 0.5% Nonidet P-40(NP-40) buffer solution (lysis buffer solution) containing 150 mM
  • the gel was washed three times with distilled water and reacted in a reaction buffer solution containing 20 mM Tris-HCl, pH 7.0, 1 mM CaCl 2 and 10 mM MgCl 2 (TCM buffer) at 37°C for 4 hours while stirring.
  • An enzyme activity of the endonuclease enzyme was observed while changing the reaction time to identify the reaction specificity of the enzyme on DNA-native-PGAE.
  • the reacted gel was stained with TCM buffer solution comprising 1/ml ethidium bromide at 37 ° C for 30 minutes and photographed with 302nm transilluminator. The sites exhibiting nuclease activity on the gel were observed as black band on orange background.
  • the standard for endonuclease activity was bovine pancreatic DNase I
  • Enzyme activity in cell culture and cell lysate can be determined by DNA-native-PAGE nuclease assay system designed for the present invention (Fig. 1). Intensive reaction bands were observed at the site exhibiting endonuclease activity in cell culture solution and cell lysate containing 10 ug of protein. The analyzed endonuclease biosynthesis and secretion of IM9 cells for indicated times are shown in Fig. 1. The endonuclease activity in cell lysate was constantly detected during 48 hour culture (Fig. 1A), but under the same reaction conditions, the endonuclease activity in cell culture solution was considerably accumulated (Fig. IB).
  • Fig. IB When IM9 cell culture was washed several times with PBS and then transferred to serum-free medium, the major band for endonuclease activity shown in Fig. IB was detected as it was, but any DNase I enzyme activity was not detected (Fig. C). This result indicates that the endonuclease detected in IM9 cell culture and cell lysate was not derived from FBS which is an ingredient of medium composition on which the cells were cultured, but was synthesized in the cell and secreted into the medium.
  • the weak band of nuclease activity rapidly migrated on the electrophoresis in the cell culture of Fig. IB was identified as DNase I by comparing with bovine DNase I obtained from Boehringer Mannheim.
  • Figure 2 shows the enzyme activity for 1 hour and 4 hours detected by DNA-native- PAGE under the conditions of different reaction solutions.
  • a reaction was performed in 20mM Tris-HCl, pH 7.0, buffer solution containing lOmM Mg 2+ for 1 hour, the activity was detected only in the endonuclease secreted by IM9 cell as shown in Fig. 2A.
  • DNase I present in FBS as well as purchased DNase I showed the enzyme activity on the same site. This result shows that the migration distance of the endonuclease synthesized and secreted by IM9 cell is different from the migration distance of DNase I on native-PAGE and the enzyme reactivity is also different from each other under a given reaction condition.
  • Anti-DNase I antibodies in the serum containing DNase I were purified by using Immunopure plus protein A/G IgG purification kit (Pierce) and Separose-CL 4B bound by bovine DNase I. 50 ml of cell culture or 50 ml of human serum 10 times diluted to PBS was immunoprecipitated by beads to which the purified anti-DNase I antibodies and Protein A-Sepharose CL 4B were bound. The immunoprecipitation was performed at 4°C for 6 hours while stirring. Immunoprecipitated supernatant was collected and the enzyme activity of endonuclease was detected on DNA-native- PAGE.
  • Figure 3 A shows that the prepared antibodies recognize the DNase I derived from FBS and are cross-reactive with DNase I present in human serum.
  • the preimmune serum did not recognize FBS and human serum DNase I.
  • the cross reactivity of the secreted endonuclease to each DNase I was detected.
  • the supernatant immunoprecipitated from IM9 cell culture solution by anti-DNase I antibodies showed rapidly mobile DNase I enzyme activity, but the enzyme activity of the secreted endonuclease was not immunoprecipitated an recovered as it was (Fig 3B). This result demonstrates that the endonuclease secreted from IM9 cell line is immunologically distinct from DNase I.
  • Example 1 -2 was eluted to partially purify the endonuclease from the culture solution of IM9 cell.
  • the protein band exhibiting nuclease activity was cut, fragmented into small pieces and transferred to eppendorf microtubes. Then, protein elution was carried out by using 20mM Tris-HCl, pH 7.0, buffer solution at 4°C for 10 hours while stirring. The sample was centrifuged at 4°C, 14,000 rpm for 10 minutes and the supernatant was divided by 20 ul. 20 ng of eluted protein sample was reacted with supercoiled plasmid DNA in 20 mM Tris-HCl buffer solution (pH 7.0) at 37 ° C for 10 minutes.
  • the enzyme activity was determined in 20mM MOPS buffer solutions containing 1 mM CaCl 2 and 10 mM MgCl 2 having each different pH.
  • enzyme reaction was observed in 20 mM Tris-HCl buffer solution (pH 7.0) containing lOmM MgCl, at 37°C for 180 minutes at regular intervals.
  • TE lOmM Tris-HCl, pH 7.6, 1 mM EDTA
  • DNA sample buffer solution (30% glycerol, 0.5% Bromophenol Blue and 0.5% xylene cynol
  • the reaction product was identified by conducting an electrophoresis on 1% agarose gel containing ethidium bromide (0.5 ng/ml).
  • the isolated enzyme was determined as having the optimal activity at pH 7. However, the catalytic activity was detected at relatively broad range of pH (Fig. 7).
  • the enzyme activity of endonuclease in 20 mM Tris-HCl (pH 7.0) buffer solution was dependent on Mg 2 " and was not affected by Ca 2+ (Fig 8A).
  • the enzyme was not activated in the range of 1 to 1 OmM Ca 2+ , but linear DNA was formed from plasmid by the enzyme activity depending on the concentration of Mg 2+ a formation of linear DNA by endonuclease for indicated reaction time was observed.
  • the present invention showed that the Mg 2+ -depending endonuclease activity is produced in a constant amount and consistently secreted into the cell culture. Also, the fact that the endonuclease is different from the DNase I present in FBS and human serum was confirmed by the difference of migrating distance on native-PAGE and the immunoprecipitation result obtained by using anti-DNase I antibody. These indicate that the endonuclease of the present invention is distinct from the endonuclease reported so far to digest DNA in the process of apoptosis, in aspects of various biochemical properties such as cation-dependence for enzyme activity, mobility distance in native-PAGE, optimal pH required for activity, etc.
  • the fact that the enconuclease was produced and secreted at the time that U937 cell line was differentiated confirms that the endonuclease has an very important biological function which can recognize foreign DNA in immune reaction and digest it into a suitable size.
  • the function of the endonuclease according to the present invention supports the report of Stacey that macrophage is activated when bacterial DNA is incorporated into the cell and the report of Higashi that cell toxicity mediated by mononuclear cell/macrophage may be occurred by nuclease (Stacey, K.J. et al.(1986) J. Immunol 157, 2116-2122; and Higashi, N. et al.(1993) Cell. Immunol. 150, 333-342).
  • Apoptosis is defined as specific type of "cell death” such as chromatin condensation, membrane blebbing or chromatin fragmentation as various nucleosome sizes by endonuclease activity (Wyllie, A. H., et al (1984) J. Pathol. 142, 67-77; Wyllie, A. H. (1980) Nature 284, 555-556; and Kerr, J. F. R., et al (1972) Cancer. 26, 239-257). Endonuclease activation is significantly responsible for apoptosis process (Arends, M. J., and Wyllie, A, H. (1990) J. Pathol. 136, 593-608).
  • Example 1 is identical with the enzyme present in nucleus of IM9 cell line. It is assumed that the endonuclease is closely related to Mg 2+ -dependent endonuclease reported by many reasearchers to be present in various tissues and cells (Anzai N., et al (1995) Blood 86, 917-923; Kawabata H., et al (1993) Biochem. Biophys. Res. Commun. 191, 247-254; Sun X. M., and Cohen G. M. (1994) J. Biol. Chem. 269,
  • the endonuclease identified by the inventors is distinct from enzymes involving internucleosomal fragementation of DNA reported so far, for example, Ca 2 7Mg 2+ - dependent endonuclease (Stratling W. H. et al (1984) J. Biol. Chem.259, 5893-5898; Pandey S. et al (1997) Biochemistry 36, 711-720; and Ribeiro J. M., and Carson D.
  • IM9 Human B-lymphoblastic (IM9) cells were cultured and then apoptosis was induced by treating the cells with 10 ug/ml CHX(Sigma) and then culturing them for 24 hours (Chow, S.C. et at. (1995) Exp. Cell. Res. 216, 149-159).
  • CHX Human B-lymphoblastic
  • Figure 15A shows that the treatment of IM9 cells with CHX(10/ml) for 24 hours provides the DNA fragments as a characteristic form of internucleosomal DNA digestion. After 24 hours, the effect of CHX was exhibited and after further 24 hours large amount of DNA fragments were formed. The cell was stained with Wright-Giemsa dye to observe the cell shape during the cell death caused by apoptosis. The result is shown in Fig. 15C. Many apoptosis cells were generated by CHX treatment, and the cell shape characterized by distortion, chromosomes aggregation and nuclei fragmentation was distinct from that of normally growing cells.
  • IM9 cell lines were cultured to 5 x 10 5 cells/ml and treated with 10 ug/ml CHX (Sigma). While culture was performed for 24 hours, 1 x 10 6 cells were collected at regular intervals and then DNA was extracted according to modified method of Blin and Stafford (Blin, N., and Stafford, D.W. (1976) Nucleic Acids Res. 3, 2303-2308.
  • IM9 cells treated with CHX were collected at regular intervals and cell lysate and nuclei were prepared. After the cell lysate and nuclei were dissolved, a gel assay of
  • DNA-native-PAGE nuclease activity was performed to identify the enzyme activity of the endonuclease present in nucleus.
  • Fig. 16A shows that when IM9 cells were cultured for 24 hours, the enzyme activity of the endonuclease was constantly detected in cell lysate over the period. However, the enzyme activity in the cell lysate treated by CHX was reduced for 6 to 12 hours (Fig. 16B), whereas the enzyme activity in the nucleus was accumulated (Fig. 16C).
  • 1 x 10 7 cells was washed three times with cold PBS and dissolved in 0.5 ml cold buffer solution comprising 50 ml Tris-HCl, pH8.0, 5mM MgCl 2 and 0.9 M sucrose at 40 ° C for 20 minutes to isolate nucleus of cells.
  • the prepared cell lysate was placed in 0.5 ml of 1.2 M sucrose solution and centrifuged at 800 g for 40 minutes. The resulting precipitate was suspended in 20 mM Tris-HCl, pH 7.0, buffer solution to obtain nuclei.
  • DNA fragmentation was carried out at the constant concentration of Mg 2+ (lOmM) and Ca 2+ (lOmM) for 0-4 hours. As results, 30 minutes after the reaction was initiated in the presence of Mg 2 ⁇ alone, the DNA fragments was produced, and at 60 to 240 minutes, the DNA fragments were accumulated (Fig. 14C). But, in the presence of Ca 2+ , the formation of DNA fragments was not observed for 4 hours (Fig. 14D). These results indicate that Mg 2 ⁇ is required for the endonuclease activity present on the nucleus of IM9 cell to form DNA fragments.
  • the endonuclease present in the nucleus of cells was partially purified by dissolving the nucleus and eluting the protein band exhibiting the endonuclease activity as described in the above Examples 1 -4.
  • the enzyme activity was measured by using the supercoiled plasmid DNA as a substrate and the divalent ion dependence was observed according to the above Examples 1-4. Also, the change of enzyme activity by treatment of ZnCl 2 , apoptosis-inhibiting substance, and EDTA, chelating agent was observed.
  • the enzyme was isolated and eluted by native-PAGE to characterize the enzyme activity of enconuclease present in the nucleus of IM9 cell which was identified by DNA-native-PAGE.
  • the endonuclease activity was completely inhibited by Zn 2+ , apoptosis-inhibiting substance, and EDTA, chelating agent .
  • the results indicate that Mg 2* is required to convert the supercoiled plasmid DNA into the linear DNA and is consistent with the result shown in Fig. 8.
  • Halpern reported that CpG motif directly activates B cell to promote secretion of IL-6 and IL-12 for short period (Halpern, M. D. et al (1996) Cell. Immunol. 167, 72-78; Yi. A.-K. et al (1996) J. Immunol. 157, 5394-5402; and Klinman, D. M. et al (1996) Proc. Natl. Acad. Sci. USA 93, 2879-2883).
  • Bird showed that CpG motif weakly act for NK cell to induce IFN- ⁇ from CD4 4 (Bird, A. P. (1995) Trends Genet. 11, 94-100; aid Yamamoto, S. et al (1992) Microbiol.
  • IM9 cell The culture solution of IM9 cell was used as enzyme source to analyse the specificity of the enzyme activity of the endonuclease and the property of the final reaction product.
  • IM9 cells were cultured in RPMI 1640 medium containing 10% FBS in 5%
  • the culture solution was used as enzyme source. Also, the culture solution obtained by culturing the cells in FBS-free medium for 36 hours was used as enzyme source including only endonucleases secreted by IN9 cell without DNase I.
  • the plasmids (pGEM-T vector, 3.0Kb) used as the substrate of enzyme were obtained by disrupting the E. coli by the alkaline lysis method (Birboim, H.C., and Doly, J.(1979) Nucleic Acids Res. 7, 1513-1523), followed by extracting twice with phenol/chloroform and precipitating with ethanol. Genomic DNA of E.coli and DNA of IM9 cell were extracted by the modified method of Blin and Stafford. Cells were twice washed with PBS and was floated by TE buffer solution.
  • DNA extraction buffer solution (10 mM Tris-HCl, pH 7.6, 10 mM EDTA, 50 mM NaCl, 0.2% SDS, 20 ug/ml Rnase A) was added and the solution was stood at 37°C for 10 minutes. Proteinase K was added to 100 ug/ml and the mixture solution was reacted at 50 " C for 8 hours. The reaction solution was extracted three times with phenol/chloroform to remove protein and ethanol precipitation reaction was carried out to genomic DNA. Salmon sperm DNA was also extracted and used as described above. The amount of LPS present in DNA was measured by Limulus amebocyte lysate assay (Sullivan, J. D. et al (1976) in Mechanisms in Bacterial Toxicity, A. W. Bernheimer, ed. Wiley, New York, p. 217) and was 2.5 ng/ml or less.
  • RPMI 1640 medium containing 10% FBS and IM9 cell culture was directely reacted with bacterial DNA to observe the digestion degree of bacteria DNA in vitro.
  • Fig 18A shows the digestion degree of plasmid DNA in RPMI 1640 medium containing 10%
  • Fig. 19 shows that all of E. coli DNA, IM9 cell DNA and salmon sperm DNA was degraded by endonuclease.
  • IM9 cell line was cultured on a 1 :1 mixed medium of RPMI 1640 medium containing heated 10% FBS and IM9 cell culture solution which was cultured for 48 hours.
  • the bacterial DNA 25 ug/ml was added to the culture solution. While the IM9 cell line was cultured in cell density of 1 x 10 6 cells/ml, the cells were collected at regular intervals and were used to extract the DNAs incorporated into the cells.
  • IM9 cell line was treated with the bacterial DNA
  • the cells were collected at regular intervals and washed three times with PBS.
  • the cells were resuspended in a cold lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 0.5% NP-40), and the cell lysates were obtained by the method described in the above Example 1-2.
  • the same volume of DNA extraction buffer was added to the cell lysates and placed at 42 °C for 3 hours and then treated twice with phenol/chloroform to remove protein.
  • the DNA present in the cell lysates was extracted by ethanol precipitation. The precipitate was dissolved in TE buffer and treated with RNase A and used for a sample of southern blotting.
  • the DNA extracted from the cell lysates was separated by electrophoresis on a 1.8% agarose gel and southern transfer was carried out. After electrophoresis, the agarose gel was shaken for 20 minutes in about 200 ml of alkaline solution (1.5 M NaCl and 0.5 M NaOH), washed with distilled water, and placed for 20 minutes in about 200 ml of the neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.5). Subsequently, the agarose gel was shaken in a fresh neutralization solution.
  • alkaline solution 1.5 M NaCl and 0.5 M NaOH
  • the neutralization solution 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.5
  • the agarose gel was reversely placed on the paper, and Hybond * nylon membrane was placed on the agarose gel to prevent air bubbles from being generated.
  • the bacterial DNA was reacted with the endonuclease at 37 ° C for 30 minutes and 100 to 200 bp of the resulting product was electrophoresed on 1% agarose gel. Using the Gene Clean Kit (Promega Inc.), the DNA was recovered from the gel and was used as a DNA probe. 50 to 100 ng of the DNA was heated to 100° C in water for 3 minutes, and cooled rapidly on ice to separate the DNA fragments and then labeled with 32 P.
  • DNA labeling reaction was conducted according to the random priming method in which 20 ul of a mixture consisting of 10 ng of random primer, 4 ul of buffer solution (50 mM Tris-HCl, pH 80, 5 mM MgCl 2 , 2 mM DTT, 0.5 mM HEPES, pH 6.6), 25 uM dATP, 25 uM dGTP, 25 uM dTTP, 60 uCi [c - 32 P]dCTP, and 5 ul of Klenow enzyme was reacted at 37° C for 2 hours.
  • buffer solution 50 mM Tris-HCl, pH 80, 5 mM MgCl 2 , 2 mM DTT, 0.5 mM HEPES, pH 6.6
  • 25 uM dATP 25 uM dGTP
  • 25 uM dTTP 25 uM dTTP
  • 60 uCi [c - 32 P]dCTP 60 uCi [c - 32
  • UV-crosslinked Hybond * nylon membrane was added to a prehybridization solution (6 x SSC, 5 x Denhardt's solution, 0.05% sodium pyrophosphate, 0.5% SDS, and 100 ug/ml of salmon sperm DNA) prebathed at 68 °C, and shaken at 68 °C for at least 1 hour.
  • the labeled probe prepared by the random priming method and cooled on ice, was added to be hybridized for about 12 hours.
  • the filter was transferred to the washing solution I (2 x SSC, 0.1% SDS) and washed for about 10 minutes at ambient temperature. The filter was then transferred to the washing solution I prebathed at 68 °C, and shaken and washed about 25 minutes.
  • the filter was transferred to the washing solution II (0.2 x SSC, 0.1% SDS) prebathed at 68 °C and washed while the radioactivity of the filter was measured by Geiger counter.
  • the filter was inserted to a cassette fit with intensifying screen and X-ray film. After 12 to 24 hours at -70 °C. the filter was developed.
  • the bacterial DNA or the plasmid DNA were added during cell culture, and incubated for 1 hour at 37 °C.
  • the DNA fragments present in the cell lysates were electrophoresed on a 1.8% agarose gel. Then, the gel of 50 - 200 bp sites by southern transfer was recovered using Gene Clean Kit (Promega Inc.).
  • the DNA fragments were introduced into the pGEM-T vector (Promega Inc.) as shown in Fig. 21 to identify base sequences.
  • the resulting vector was cloned in E. coli and analyzed by a SEQUENASETM version 2.0 DNA sequencing kit (USB), according to the Sanger dideoxyribonucleotide chain termination method (Sanger, F. et al.
  • the test results show that when 25 ug/ml of bacterial DNA was incubated with IM9 cell, the DNA was properly processed in the cell culture and incorporated into the cells. This was confirmed by southern hybridization (Fig. 20). The presence of 100- 200 bp DNA was found 30 minutes after the cell culture. The amount of 100-200 bp DNA in the cells was decreased with the lapse of the culture time. The results suggest that properly processed DNA was incorporated into IM9 cell line and the processing was continued in cells.
  • the bacterial DNA incorporated into cells was isolated and its DNA sequence was analyzed as shown in Fig.21.
  • the reaction product of the endonuclease had a characteristic base sequence, i.e., CpG motif carrying two purine bases at 5' end and two pyrimidine bases at 3' end. It is known that CpG motif activates B cell or macrophage in an immune system and promotes the secretion of cytosine and IgM and is present in bacterial DNA at high frequency. Accordingly, it was now found that the endonuclease present in IM9 cell culture solution and the DNA processing by the enzyme activity in the cell generate CpG motif which functions to activate immune cells. to t o Us o o
  • the PCR reactive solution contained 0.2 mM dNTP, 10 pmole of primer, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , and 2.5 units of Taq polymerase. 5'- CTCCCGGCCGCCATG-3' and 5'-TTGGGAGCTCTCC-3' (Table I) were synthesized and used as a PCR primer.
  • the PCR reaction was repeated 35 times under the following condition: denaturation (at 94°C for 30 sec); primer annealing (at 42 °C for
  • PCR product was run on 8% native-PAGE in a TBE buffer solution and was stained with ethidium bromide. Subsequently. DNA was separated from the gel of the PCR reaction product. The separated DNA was precipitated by ethanol, and was dissolved in PBS for cell culture or was dissolved in TE buffer solution for the detection of the activity of the endonuclease.
  • DNA fragments were labeled with 32 P by a random-priming method using random primer and Klenow enzyme. 100 ng/ml of the DNA fragments labeled with 2 P were incorporated into IG9 cell line, U937 cell line, and U937 cell line treated with TPA. Whether DNA was incorporated into cells was confirmed at regular intervals. The endonuclease was treated with 0.2 mM ZnSO 4 (Sigma) to terminate its enzyme activity. The DNA incorporated into cells was recovered in a cell lysate and run on 20% native-PAGE in the TBE buffer solution. The gel was dried and autoradiographed to confirm the processing of DNA within cells and the end product obtained by the endonuclease reaction.
  • the ECl DNA fragment labeled with 32 P was incorporated into IM9 cell line.
  • the DNA incorporated into cells were extracted from cell lysates and 20% native-PAGE was carried out.
  • the processed DNA labeled with 32 P was extracted from the gel and annealed with a various synthesized oligonucleotides to compare their base sequences with each another.
  • a predetermined amount of the extracted DNA labeled with 32 P and 10 ng of each of oligonucleotides were mixed with 6 x SSC. The mixture was boiled at a temperature of 100 ° C for 5 minutes, and annealed at temperatures lowered by 1 ° C per 30 seconds.
  • oligonucleotides complimentarily conjugated to the DNA incorporated into cells were confirmed.
  • standards a mixture of DNA and oligonucleotides which were not annealed, and a DNA denaturated and annealed with only the DNA incorporated into cells were used.
  • Fig. 23 shows the incorporation of exterior DNA observed in U937 cell line and U937 cell line differentiated by TPA treatment and the processing of the DNA in the cells.
  • Fig. 23 B shows the incorporation of exterior DNA and the processing in cells by U937 cell line differentiated by the treatment of TPA. As shown in Fig. 23 B, the DNA processing in the cells was inhibited by 0.2mM of Zn 2+ .
  • the test results demonstrate that the immune cells produce the end product of 10 bases by processing the exterior DNA.
  • the properties of the base sequence of the DNA fragments were also confirmed.
  • ECl DNA products amplified by PCR were labeled with 32 P and were incorporated into cells
  • the DNA fragments of 10 bases processed by cellular endonucleases were annealed with several types of synthetic oligonucleotides consistent with partial sequences of ECl to identify the base sequences bound complementarily with the oligonucleotides (Fig. 24).
  • the oligonucleotides As a result of annealing, the oligonucleotides, the most bound complementarily with the base sequences, were oligo A sequences of Table 1, i.e., the base sequences which have AACGTT motif and are present in ECl DNA. From the above results, it was clearly confirmed that CpG motif known as the base sequences of bacterial DNA which activates immune cells by enzymatic activity of endonuclease in cells was produced.
  • the oligonucleotides having CpG motif which is known to secrete cytokines and IgM were synthesized.
  • the oligo A present in ECl and oligo B present in EC2 and oligo E which does not have CpG motif were synthesized (Table 1).
  • ECl or EC2 PCR products containing several CpG motifs, and several types of DNA were also used in this experiment.
  • To methylate CpG motif bacterial DNA was digested with endonuclease therey obtaiing the fragments having 100-200bp.
  • the methylation was carried out by mixing 30 ⁇ g of DNA with the buffer solution (10 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , 50 mM NaCl, and 1 mM DTT) containing CpG methylase and 160 uM of S-adenosylmethionine, and reacting for 3 hours at 37 ° C .
  • CpG methylation was confirmed by digesting with Hpa II.
  • IM9 cell line human B-lymphoblastic cell line
  • RPMI 1640 medium containing 10%o FBS with the cell counts being 2 x 10 5 /ml at the beginning of culture.
  • the culture was treated with several types of preprepared oligonucleotides and DNA in 25 ug/ml and was cultured for 24 hours, the cell culture was obtained.
  • Anti-human- ⁇ -chain-specific IgM (5 ug/ml, Sigma) in 0.1 M carbonate buffer solution (pH 9.6) was incorporated into flat bottom plate in 100 ul/well and then the plate was placed for 16 hours at 4°C. The plate was washed three times with PBS and placed for 2 hours at room temperature. Subsequently, the plate was washed three times with TPBS (0.05% Tween - 20 in PBS). Approximately diluted cell culture or purified human IgM (Sigma) in 100 ul/well was introduced. After being placed for 2 hours at room temperature, it was washed three times with TPBS.
  • Horseradish peroxidase- linked anti -human Ig diluted in 1/4,000 in PBS containing 1% BSA was introduced, and placed for 1 hour at room temperature, and then washed three times with TPBS.
  • the plate was subsequently treated with O-phenylenediamine dihidrochloride in 0.05M phosphate buffer solution (pH 5.0) for 30 minutes.
  • the plate was treated with 0.67 N H 2 S0 4 to terminate the reaction and IgM was quantified using microplate reader.
  • DNA increased the secretion of IgM by activating IM9 cell line and it was consistent with the results of study manifested by using CpG motif synthesized by previous several researchers.
  • ECl, EC2, and HC1 DNA amplified by PCR were labeled with 32 P random-priming method employing random primer and Klenow enzyme.
  • EC2 DNA was digested with Alu 1 (Promega Inc.) and the fragments whose 5'-end was labeled with 32 P were used as substrate.
  • ECl DNA was 5'-end or 3'-end labeled and utilized.5'-end labeling was carried out by mixing 10 ng EC 1 DNA, [ ⁇ - 32 P] ATP 50 uCi, kinase buffer solution and 5 units of polynucleotide kinase (Promega Inc.) and reacting form 1 hour at 37°C. 3'-end labeling was carried out by mixing lOng ECl DNA, [cc- 32 P]CTP 50 uCi, TdT buffer solution and 5 units of terminal deoxytransferase (Boehringer Mannheim) and reacting for 1 hour at 37 °C and utilized.
  • ECl, EC2, and HC1 DNA listed in Table 2 were labeled with 32 P by random-priming method and utilized as substrates. It was confirmed that the reaction of these substrates with IM9 cell line cultured in FBS-free- medium resulted in DNA reaction products having about 10 bases as shown in Fig. 26, 27 and 28. The results show that the endonuclease does not recognize specifically the base sequence of DNA. The endonuclease acted on foreign DNA regardless of base sequences, thereby resulting in DNA fragments having about 10 bases.
  • the endonuclease manifested by this invention does not exert any activity on the DNA fragments having less than about 10 bases.
  • Fig. 32 shows several DNA fragments, i.e., endonuclease reaction products formed before EC2 DNA product amplified by PCR was digested with Alu I and the resulting
  • DNA fragments were reacted with endonuclease to produce the end product.
  • the substrates employed in this experiment were labeled with 32 P in 5'-end of DNA reacted with Alu I.
  • the labeled enzymatic reaction resultant product was DNA fragment having about 10 bases. From this fact, it is confirmed that the enzymatic activity of 5'-exonuclease for producing single stranded DNA is not present.
  • EC2 DNA labeled with 32 P at 5'- or 3 '-end was employed as substrate and reacted with endonuclease for 1 hour at 37 °C.
  • the reaction resultant products were compared by autoradiography by the same method as stated above.
  • labeled DNA was boiled for 5 minutes at 100°C, cooled on inc, and reacted with endonuclease as stated above.
  • Fig. 33 shows that the DNA fragments having about 10 bases are formed by the action of endonuclease on the substrate labeled in 5'-end (5'-end label, lane 2).
  • the results are the same as shown in Figure 32. That is, 5'-exonuclease activity was not present in endonuclease. However, the DNA fragments having about 10 bases, i.e., the enzymatic reaction resultant products, were not formed in the substrates labeled with 32 P in 3'-end. After the substrates labeled with 32 P were boiled for 10 minutes at
  • reaction resultant products produced by enzymatic activity of endonuclease present in cell cultures with the products produced by processing in cell and the reaction end product produced by the action of DNase I, 5 units of bovine pancreatic DNase I (Boehringer Mannheim) and ECl DNA labeled with 32 P by random-priming method were reacted in 1 OmM Tris-HCl, pH7.6, 1 OmM MgCl 2 buffer solution for 1 hour at 37 °C.
  • the reaction resultant was run on 20% of natural-PAGE and 20%) of denatured-PAGE (8.3M urea), autoradiographed, and compared with the enzymatic reaction resultant product of endonuclease.
  • the DNA reaction products processed by the endonuclease of cell cultures (lane 2) and cellular endonuclease (lane 3) are all the DNA fragments having about 10 bases.
  • the DNA fragments were degraded into less than 10 bases. The fragments were subsequently degraded into mononucleotides.
  • the products were treated with SI nuclease and the enzymatic reaction products were observed.
  • IM9 cell line synthesized and secreted Mg 2+ -dependent endonuclease which is distinct from nuclease known so far. It was also found that the endonuclease is present in the nucleus of IM9 cell line and participates in the apoptosis process.
  • the endonucleases involved in the apoptosis include Mg 2+ - dependent endonuclease (Anzai N. etal (1995) Blood 86, 917-923; Kawabata H. et al (1993) Biochem. Biophys. Res. Commun. 191, 247-254; Sun X. M., and Cohen G. M. (1994) J. Biol. Chem. 269, 14857-14860; and Kawabata, H. et al (1997) Biochem.
  • IM9 cell line secreting the endonuclease was massively cultured in RPMI 1640 medium containing heated 10% FBS and the resulting cell culture was used as enzyme source. After the cell culture was centrifuged at 1 ,500 x g for 5 minutes, IM9 cell line was discarded and the cell culture solution was recovered. 101 of cell culture solution was centrifuged at 14,000 x g for 30 minutes at 4°C, and the resulting supernatant was used as enzyme source.
  • the enzyme activity was measured as the degree of the formation and digestion of linear DNA from supercoiled plasmid DNA used as a substrate. 100 ng of plasmid DNA was added to 20 mM Tris-HCl, pH 7.0, buffer solution containing 10 mM MgCl 2 , and the mixture was reacted with 20 ul of a sample obtained during the protein purification at 37 °C for 10 minutes. The enzyme reaction was terminated with the DNA sample buffer and the enzyme activity was assayed by electrophoresis on a ⁇ % agarose gel.
  • the cell culture solution was saturated to a concentration of 80%) by slowly adding NH 4 SO 4 , and then centrifuged at 14,000 x g. The resulting precipitate was dialyzed overnight in 20 mM sodium acetate buffer solution, pH 5.2. A 5 ml aliquot of the enzyme solution was loaded on Mono-S column (0.5 x 5.0 cm, Pharmacia LKB) preequilibrated with 5 ml of the same buffer solution. The protein was first eluted by a linear concentration gradient of 0 - 0.08 M NaCl (15 ml) in the same buffer solution and then was passed over 15 ml of the same buffer solution containing 0.08 M NaCl.
  • the protein was again eluted with a linear concentration gradient of 0.08 - 0.2 M NaCl (20ml). The volume of each fraction was 1 ml and the flow rate was 0.5 ml/min. The above procedure was repeated and the fraction containing the endonuclease was pooled and concentrated using Centricon, and then equilibrated with 50 mM of sodium phosphate buffer solution, pH 7.0, containing 1.5 M (NH 4 ) 2 S0 4 . This enzyme source was loaded on a RESOURCE PHE column (0.64 x 30 mm, 1 ml. Pharmacia LKB) and the hydrophobic interaction chromatography was carried out.
  • the column was washed with 15 ul of the same buffer solution and the protein was eluted with a linear concentration gradient of 1.5 - 0 M (NH 4 ) 2 S0 4 (25 ml).
  • the fraction with the enzyme activity was concentrated in Centricon and equilibrated with 20 mM Tris-HCl buffer solution, pH 7.0.
  • IM9 cell culture solution was concentrated over ammonium sulfate and used for purification.
  • the differential precipitation over ammonium sulfate was conducted depending on the concentration difference, but did not significantly effect on the isolation of the enzyme.
  • culture solution was concentrated to 80%> and the pretein was precipitated for the purification.
  • the enzyme activity in the sample passed over Mono S column was recovered by a linear concentration gradient of 0.08 - 0.2 M NaCl (Fig. 36).
  • the fraction exhibiting the activity on cation-exchange resin was pooled and passed over RESOURCE PHE column in a hydrophobic interaction chromatography.
  • the fraction exhibiting the enzyme activity on the cation-exchange resin chromatography was pooled and concentrated in Centricon.
  • the concentrated solution was loaded on 4 - 15%> linear gradient of acrylamide gradient gel and electrophoresis was performed with 4 - 5 mA for 18 hours at 4°C.
  • the gel was stained with coomassie brilliant blue R-250 to detect the protein band.
  • the gel portion of the protein band was cut to make a small piece prior to the staining and was eluted with
  • the enzyme activity among the eluted protein fraction was detected using the supercoiled plasmid DNA as a substrate.
  • the enzyme activity fraction was concentrated and separated by SDS- polyacrylamide gel electrophoresis. The concentrations of the staking gel and the running gel were 4%> and 1%, respectively.
  • the standard protein in the native porous gradient PAGE was a mixture of ferritin (440 kD), catalase (232 kD), lactate dehydrogenase (140 kD) and bovine serum albumin (87 kD).
  • the standard protein in the SDS-PAGE was a mixture of myosin (200 kD), ⁇ -galactosidase (116.3 kD), phosphorylase B (97.4 kD), bovine serum albumin (66.2 kD) and ovalbumin (45 kD).
  • the enzyme activity fraction eluted on Mono S column was concentrated and 4 - 15% native porous gradient gel electrophoresis was carried out to detect the protein band showing the enzyme activity (Fig. 40).
  • the protein band exhibiting the enzyme activity was not shown as clear single band and was spread around 140 kD as compared with the standard protein.
  • the protein band exhibiting the enzyme activity was eluted.
  • electrophoresis was conducted on SDS-PAGE (Fig 41).
  • the purified protein band was shown at the same site as the endonuclease purified by chromatography and the molecular weight of the protein was determined as about 72.4 kD (Fig. 39).
  • a comparison of the results of the native porous gradient gel electrophoresis and SDS-PAGE revealed that the endonuclease exhibits the enzyme activity in the form of homodimer.
  • Nuclei were isolated from U937 cell from which any endonuclease enzyme activity was not detected for 4 hours, and were used as substrate. The specificity of the enzyme activity was confirmed by adding 1 mM Ca 2+ , 1 mM Mg 2+ , 1 mM Zn 2+ , and EDTA to 20 mM Tris-HCl buffer solution, pH 7.0 containing the isolated nuclei and the reaction was allowed for 10 minutes at 37°C.
  • the activity of the purified enzyme was exhibited in the presence of Mg 2+ and was completely inhibited by Zn 2+ , apoptosis inhibitor, and EDTA, chelating agent (Fig. 42).
  • the characteristic enzyme activity was consistent with that of the above enzymatic activity.
  • the endonuclease of the present invention is able to degrade foreign bacterial DNA and incorporate the DNA fragments into cells.
  • the DNA incorporated into cells is processed by the intracellular endonuclease to produce oligonucleotide including CpG motif and then the immune cell is activated by the CpG motif to promote the secretion of antibody. Accordingly, the endonuclease of the present invention is industrially valuable as an pharmaceutical immune adjuvant.

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Abstract

L'invention concerne une nouvelle endonucléase qui est sécrétée par une cellule immunitaire et reconnaît l'ADN bactérien en tant qu'agent étranger et le traite pour produire un oligonucléotide monocaténaire à environ 10 paires de bases comprenant le motif CpG qui intervient dans la réponse immunitaire. L'invention concerne également un procédé pour produire ladite endonucléase, consistant à mettre en culture une lignée cellulaire IM9 lymphoblastique B humaine ou une lignée cellulaire U937 myélogène traitée par TPA dans un milieu approprié pour produire ladite endonucléase, puis à isoler cette dernière du lysat cellulaire ou du milieu de culture. En outre, l'invention concerne un adjuvant immunitaire comprenant un oligonucléotide monocaténaire à environ 10 paires de bases comportant le motif CpG, produit par traitement de l'ADN bactérien avec ladite endonucléase.
PCT/KR1998/000136 1998-05-27 1998-05-30 Nouvelle endonuclease de cellule immunitaire, son procede de production et adjuvant immunitaire obtenu au moyen de ladite enzyme WO1999061592A1 (fr)

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JP2000550977A JP2002516086A (ja) 1998-05-27 1998-05-30 免疫細胞の新規エンドヌクレアーゼ、これを製造するための方法およびこれを用いる免疫アジュバント
AU77889/98A AU7788998A (en) 1998-05-27 1998-05-30 Novel endonuclease of immune cell, process for producing the same and immune adjuvant using the same
US09/722,776 US6881561B1 (en) 1998-05-27 2000-11-27 Endonuclease of immune cell, process for producing the same and immune adjuvant using the same
US10/860,844 US20070173468A9 (en) 1998-05-27 2004-06-04 Novel endonuclease of immune cell, process for producing the same and immune adjuvant using the same

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WO2000045849A3 (fr) * 1999-02-05 2000-12-07 Genzyme Corp Utilisation de lipides cationiques pour generer une immunite anti-tumorale
JP2011236246A (ja) * 2000-08-18 2011-11-24 Shire Human Genetic Therapies Inc 高マンノースタンパク質および高マンノースタンパク質の製造方法
US9453847B2 (en) 2010-07-19 2016-09-27 Shire Human Genetic Therapies, Inc. Mannose receptor C type 1 (MRC1) codon optimized cell line and uses thereof
US9623090B2 (en) 2012-03-02 2017-04-18 Shire Human Genetic Therapies, Inc. Compositions and methods for treating type III gaucher disease
US9694057B2 (en) 2006-02-07 2017-07-04 Shire Huma Genetic Therapies, Inc. Stabilized compositions of proteins having a free thiol moiety
US11571466B2 (en) 2009-07-28 2023-02-07 Takeda Pharmaceutical Company Limited Compositions and methods for treating Gaucher disease

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KR19990086271A (ko) * 1998-05-27 1999-12-15 손경식 면역세포의 신규한 엔도뉴클레아제 및 이를 사용한 면역보조제

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KR19990086271A (ko) * 1998-05-27 1999-12-15 손경식 면역세포의 신규한 엔도뉴클레아제 및 이를 사용한 면역보조제

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000045849A3 (fr) * 1999-02-05 2000-12-07 Genzyme Corp Utilisation de lipides cationiques pour generer une immunite anti-tumorale
JP2011236246A (ja) * 2000-08-18 2011-11-24 Shire Human Genetic Therapies Inc 高マンノースタンパク質および高マンノースタンパク質の製造方法
US10041137B2 (en) 2000-08-18 2018-08-07 Shire Human Genetic Therapies, Inc. High mannose proteins and methods of making high mannose proteins
US9694057B2 (en) 2006-02-07 2017-07-04 Shire Huma Genetic Therapies, Inc. Stabilized compositions of proteins having a free thiol moiety
US11571466B2 (en) 2009-07-28 2023-02-07 Takeda Pharmaceutical Company Limited Compositions and methods for treating Gaucher disease
US9453847B2 (en) 2010-07-19 2016-09-27 Shire Human Genetic Therapies, Inc. Mannose receptor C type 1 (MRC1) codon optimized cell line and uses thereof
US9623090B2 (en) 2012-03-02 2017-04-18 Shire Human Genetic Therapies, Inc. Compositions and methods for treating type III gaucher disease

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