KR101452830B1 - E. coli strain for Providing Two Anti-tumor Substances - Google Patents

Abstract

The present invention relates to a non-toxic diacyl lipoyl-oligosaccharide having a stable structure and an oligodeoxynucleotide having immunostimulatory activity, a vaccine composition, and an anticancer composition. The two active ingredients contained in the composition of the present invention synergize with each other, and thus can be used as an anticancer agent having low toxicity and excellent immunogenicity. Also, according to the present invention, a safe anticancer agent comprising a purified DNA showing a drug efficacy higher than BCG at low cost and a non-toxic polymer substance derived from bacterial LPS can be conveniently produced using one strain.

Description

E. coli strains providing both anticancer substances simultaneously {E. coli strain for Providing Two Anti-tumor Substances}

The present invention relates to an Escherichia coli strain that simultaneously provides two anticancer substances.

The treatment of cancer that developed in the 1960s is the three major methods of surgery, radiation, and chemotherapy, and the gradual rise pattern of cancer mortality rates in the United States in 1973 speaks of some success of this therapy. However, since surgery and radiation therapy are topical treatments, early prognosis is limited when they are first treated with localized cancer, and chemotherapy is successful if the entire cancer cell is killed. In order to kill the entire cancer cells by such a treatment, there is a drawback that the life of the aged person is endangered because the host, that is, the normal tissues of the patient, especially the immune system tissues, is given simultaneously.

 To overcome these drawbacks, the developed method is immunotherapy, and it is the essence of immunotherapy that it tries to resist the cancer development by reinforcing immunity monitoring mechanism. Various immunotherapies have been studied for the time being, but nowadays, nonspecific immunotherapy is receiving the most attention, and they are used alone or in combination with a chemotherapeutic agent to treat almost all types of cancer. Nonspecific immunotherapy is not limited to the types of cancers as it is expressed. To date, various theories have been proposed about the essential mechanism of action, but non-specific methods stimulate the activity of reticular endothelial cells, especially lymphocytes It is thought that there is a point of action in the view of the so-called immune surveillance mechanism. Corynebacterium is the main substance used in clinical practice, and Picibanil (OK-432) has been introduced to Korea for a long time and has already been used in many patients. It has been studied mainly in Japan and has been marketed by Japanese pharmaceutical companies, and is marketed in Japan, Korea, or parts of Southeast Asia. The commercial history of these organisms in cancer has been quite long, and in 1968 Bush Fehleison et al. Found that the cancer in a patient was either stopped or had less cancer, In 1891, a surgeon in Chicago, Coley, announced the use of a so-called collie blend toxin, which has been used in many cancer patients, and has shown efficacy in a considerable number of patients. This is known as a substance extracted from the medium after cultivation of streptococcus.

One of the characteristic differences between mammalian and bacterial DNA is the fact that mammals are selectively methylated to CpG inhibition and cytosine of CpG dynucleotides. Recently, researchers have shown that CpG motifs present in bacterial DNA rapidly activate polyclonal B cells to promote IgM secretion, stop cell cycle by anti-IgM antibody, and arrest B cells where apoptosis occurs Of CpG motifs inhibit c-myc expression and increase myn, bcl2, and bcl-XL mRNA expression to protect cells from apoptosis. Another study reported that CpG motif directly activates B cells and promotes IL-6 and IL-12 secretion in a short time. CPG, a US company, is currently conducting clinical trials on immunosuppressants and asthma medications using synthetic oliginucleotides containing CpG sequences, but it has a disadvantage in that it is very expensive. However, recent studies have shown that methylation of CpG dignified to cytosine is not related to the anticancer effect, and that the antitumor effect of bacterial DNA is due to its structural factors. There is also a study that the role of unmethylated CpG in DNA anticancer drugs is not essential, and alternatives have been devised.

Lipopolysaccharide (LPS) is a typical Thymus Independent Antigen that acts directly on B cells to induce a nonspecific immune response. It is known to cause side effects such as inflammation. However, it can kill cancer cells In particular, lipid A is known to exhibit anticancer effects through the induction of various transcription factors. However, LPS is a typical endotoxin and has a strong toxicity. Moreover, the linkage between normal LPS and DNA may cause serious consequences such as sepsis.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop an adjuvant composition having safety and immunostimulatory function. As a result, oligosaccharides were obtained from (i) diacyl lipoyl oligosaccharide obtained from E. coli strain EG0023 (accession number: KCCM11218P) excavated from a human intestine and (ii) Oxynucleotide increased the activity and secretion of various cytokines related to the immunological mechanism of type 1 T helper cells (Th1) and natural killer cells (NK cells), and confirmed that the anticancer efficacy was excellent. Further, the present inventors have completed the present invention by removing the toxicity which was problematic in conventional lipopolysaccharide (LPS) in the above composition and maximizing anticancer efficacy by mixing with oligodeoxynucleotide at an optimal concentration.

Accordingly, an object of the present invention is to provide an Escherichia coli strain capable of simultaneously providing lipoyl-saccharide and oligodeoxynucleotide having excellent safety, immunostimulatory function and anticancer efficacy.

It is another object of the present invention to provide an adjuvant composition.

It is another object of the present invention to provide a vaccine composition.

It is still another object of the present invention to provide an anticancer agent or an anticancer adjuvant composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an Escherichia coli strain which produces a lipo oligosaccharide having a molecular weight of 2,000-5,000 Da, wherein the lipo oligosaccharide comprises a sugar chain chain represented by the following formula (1) ) Strain:

Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine, and P is phosphate.

The present inventors have made extensive efforts to develop an adjuvant composition having safety and immunostimulatory function. As a result, oligosaccharides were obtained from (i) diacyl lipoyl oligosaccharide obtained from E. coli strain EG0023 (accession number: KCCM11218P) excavated from a human intestine and (ii) It was confirmed that the mixed composition of oxynucleotide increases the activity and secretion of various cytokines related to the immunity mechanism of helper T cell 1 (Th1) and natural killer cell (NK cell). In addition, the present inventors have completed the present invention by eliminating the toxicity which has been problematic in conventional lipopolysaccharide in the composition and maximizing anticancer efficacy by mixing with oligodeoxynucleotide at an optimal concentration.

Although general lipopolysaccharide (LPS) can exhibit anticancer efficacy through induction of various transcription factors, it has strong toxicity, and when combined with a synthetic oligonucleotide, serious side effects such as sepsis may be caused, and urgent improvement is required . Accordingly, the present inventors obtained lipoylosaccharide from E. coli strain EG0023, which was excised from human intestine, and removed cytotoxicity through deacylation reaction. In addition, the composition was mixed with an oligodeoxynucleotide (ODN) generated from the same E. coli at an optimal concentration to develop a composition having an immunological stimulation reaction and cancer cell killing ability.

The E. coli strain EG0023 used in the present invention was an E. coli strain excavated from a human intestine. A single colony of Escherichia coli obtained from a healthy adult male was cultured and a total of 50 strains were firstly screened. Then, the human lymphocyte cell line obtained by differentiating the lipopolysaccharide (LPS) obtained from each strain with a granulocyte-macrophage colony stimulating factor (GM-CSF) was subjected to measurement of TNF-a secretion . The strain with the lowest value was selected second, and the 5 selected strains were re-cultured, and the TNF-a value was confirmed by the above method, and the strain with the lowest value was selected. This strain was deposited on Nov. 9, 2011 with the deposit number KCCM11218P in the Korean Microorganism Conservation Center, 361-221 Hongje-dong, Seodaemun-ku, South Korea.

The major feature of the E. coli strain EG0023 of the present invention is that it can simultaneously produce two anticancer substances (diacyl lipoyl-oligosaccharide and oligodeoxynucleotide) at low cost and high efficiency, unlike existing strains capable of producing only a single substance , And the diacyl lipoyl oligosaccharides and oligodeoxynucleotides produced and obtained by the strains are structurally stable, nontoxic, and can induce a specific immune response.

As used herein, the term " hexose " refers to monosaccharides containing six carbon atoms in a molecule such as ketohexose (psicose, fructose, sorbose, tagatose), aldohexose (Fucose, fuculose, rhamnose), and deoxysugars (such as alos, altrose, glucose, mannose, gulose, idose, galactose and talos). Preferably, said hexose is ketohexose, aldohexose or deoxyzide, more preferably ketohexose or aldohexose.

As used herein, the term " heptose " refers to monosaccharides containing seven carbon atoms in a molecule, and the positions of aldoheptose (position 1) and Ketoheptose (position 2). The alto heptose is, for example, L-glycero-D-manno-heptose, but is not limited thereto. The ketoheptose may be, for example, sedoheptuloses and mannoheptuloses, but is not limited thereto.

According to a preferred embodiment of the present invention, the chromosomal DNA of the E. coli strain produces oligodeoxynucleotides having immunostimulatory activity by pulverization, wherein the diacyl lipoyl oligosaccharide having a molecular weight of 2,000-5,000 Da is 3 - < / RTI > hydroxytetradecanoyltrimethylsilyl ester.

As used herein, the term " immunostimulatory " refers to inducing an initial immune response or increasing the existing immune response to an antigen to a measurable extent.

According to a preferred embodiment of the present invention, the Escherichia coli strain is Escherichia coli EG0023 (Accession No .: KCCM11218P).

According to another aspect of the present invention, there is provided an adjuvant composition comprising, as an active ingredient, diacyl lipoyl-oligosaccharide having a molecular weight of 2,000-5,000 Da and containing a sugar chain chain represented by the following formula (1)

Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine, and P is phosphate.

The greatest feature of the immunosuppressive compositions of the present invention is the use of a deacylated non-toxic LOS (Lipooligosaccharide) as an adjuvant. As used herein, the term " LOS (Lipooligosaccharide) " is a modification of LPS (lipopolysaccharide), meaning that it has a shorter molecular weight than a natural occurring LPS. The pre-deacylated LOS preferably has a molecular weight of 2,000-5,000 Da. The term " deacylated LOS "means that in this LOS the fatty acids bound to the glucosamine of lipid A by -C (O) O- bond are removed and the toxicity is greatly reduced compared to LPS. (O) O- bond and a -C (O) NH- bond. The deacylated LOS of the present invention is obtained by dissolving the fatty acid bound to the -C (O) O- bond by deacylation of lipid A Removed.

The deacylated non-toxic LOS can be prepared by various methods, but Korean Patent Registration No. 0456681, which is a prior patent of the present inventors, WO 2004/039413; Korea Patent No. 0740237; And WO 2006/121232. For example, LPS is deacylated by treatment with a strong base (e.g., 2 N NaOH) to remove some fatty acids from lipid A and then to be deoxidized.

According to a specific embodiment of the present invention, the deacylated LOS used as an adjuvant in the present invention is unoxidized by alkali treatment of LPS and deacylation. Preferred examples of the alkali are NaOH, KOH, Ba (OH) 2, CsOH, Sr (OH) 2, Ca (OH) 2, LiOH, the RbOH comprising a Mg (OH) _2, more preferably NaOH, KOH (OH) ₂ , Ca (OH) ₂ , LiOH and Mg (OH) ₂ , more preferably NaOH, KOH and Mg (OH) ₂ , and most preferably NaOH.

The degree of toxicity of LPS can be assayed through a variety of methods known in the art. For example, toxicity can be analyzed by measuring the amount of TNF-alpha (tumor necrosis factor-alpha) secreted in LPS treated THP-1 (Acute monocytic leukemia). The dedifferentating non-toxic LOS of the present invention induces a relatively small amount of TNF-a secretion compared to conventional LPS.

Another feature of the non-toxic, deacylated LOS, an adjuvant used in the present invention, is that the molecular weight is generally small compared to the LPS conventionally used. Preferably, the deacylated non-toxic LOS used in the present invention has a molecular weight of 2,000-5,000 Da, more preferably 2,000-4,000 Da, even more preferably 2,000-3,500 Da, even more preferably 2,000-3,000 Da Molecular weight. Such a molecular weight can be measured using a conventional method in the art, for example, MALDI-MASS.

The non-toxic, deacylated LOS used in the present invention is well suited for the immunosuppressive composition of the present invention, as the immunostimulatory efficacy is superior as compared to conventional immunoadjuvants, as well as a much reduced toxicity.

According to a preferred embodiment of the present invention, the deacylated LOS contained in the immunoassay composition of the present invention is Escherichia will derived from the coli), it is the most preferably derived from a proprietary gyunin E. coli EG0023 (KCCM11218P) by the present inventors.

According to a preferred embodiment of the present invention, the lipo oligosaccharide, which is an active ingredient of the above-mentioned immuno-assisting composition, comprises lipid of 3-hydroxytetradecanoyltrimethylsilyl ester, and the chromosome of the E. coli strain excavated in the present invention And may additionally include oligodeoxynucleotides.

The oligodeoxynucleotides used in the present invention are preferably 50-10,000 bp, more preferably 500-7,000 bp, even more preferably 500-4,000 bp, even more preferably 500-2,000 bp, Preferably 500-1,500 bp.

According to a preferred embodiment of the present invention, the immunoassay composition comprises a mixture of diacyl lipoyl oligosaccharide and oligodeoxynucleotide in a weight ratio of 1: 0.005 to 1: 200, preferably 1: 0.05 to 1: 150, Preferably 1: 0.5 to 1: 150, more preferably 1:10 to 1: 120, and most preferably the weight ratio of diacyl lipoyl oligosaccharide and oligodeoxynucleotide is 1: 100.

The immunoassay composition according to the present invention stimulates the secretion of the antibody by stimulating the immune cells. In particular, in relation to the Th1 cell immunity mechanism, the dendritic cell, which is an antigen presenting cell, is a priming cell of the immune response and effectively stimulates B cells and T cells (Banchereau J et al., Nature , 392: 245-53 (1998)). In general, immature dendritic cells are unable to stimulate T cells due to insufficient secretion of CD40, CD54, and CD86, which are required for T cell activation. Nevertheless, when an antigen enters the body, it recognizes the antigen, Dendritic cells are matured and actively induced. In addition, mature dendritic cells exhibit resistance to the inhibitory effect of IL-10 and synthesize IL-12, a cytokine that stimulates both innate and acquired immune responses (Koch F et al, J Exp Med. , 184: 741-7 (1996); Cella M et al., J Exp Med , 184: 747-52 (1996)). Dendritic cells secrete a number of mediators that can bind to T cell receptors, promoting cell adhesion and signal transduction, and this process begins on the day of exposure to bacterial infection or various body stresses. In the presence of mature dendritic cells and IL-12, CD4 T helper cells bound to MHC type II are transformed into Th1 cells that produce interferon-γ. Secreted IFN-γ acts as an antimicrobial activity of macrophages with IL-12 (Reis < / RTI > Sousa C et al, J Exp Med. , 186: 1819-29 (1997)).

Helper T cells (Th) are cells of Th-1 type (e.g., IL-2, IL-12, IFN- ?, TNF- ?, IL- 4, IL-6, IL- 8, is divided into TGF-β) (Lucey DR et al, Clin Microbiol Rev, 9:... 532 (1996)).

According to a preferred embodiment of the present invention, the above-mentioned immunostimulating composition increases the activity of the interleukin-8 and interleukin-12 promoters by activating NF-kB.

According to a preferred embodiment of the present invention, the immunoassay composition increases the secretion of interferon-y and interleukin-12p40 of helper T cell 1.

According to yet another aspect of the present invention, the present invention provides a kit comprising (a) an antigen; And (b) a diacyl lipoyl-oligosaccharide having a molecular weight of 2,000-5,000 Da and containing a sugar chain chain represented by the following formula (1) as an immunostimulatory component, as an active ingredient:

Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine, and P is phosphate.

The vaccine composition of the present invention induces immune responses to pathogens greatly and thus has excellent anticancer efficacy against certain alicyclic as well as deacylated non-toxic LOS used as an immunosuppressant has excellent toxicity and safety.

The vaccine composition of the present invention includes the same active ingredients as those contained in the above-mentioned " immunoadjuvant ", so that description thereof is omitted in order to avoid excessive redundancy.

As used herein, the term " antigen " is a substance that induces an immune response of a recipient. Examples of viral pathogen antigens that may be used in the present invention include Orthomyxoviruses such as influenza virus; Retroviruses such as respiratory syncytial virus (RSV), simian immunodeficiency virus (SIV) and HIV; Herpesviruses such as EBV (Epstein-Barr Virus); CMV (cytomegalovirus) or HSV (herpes simplex virus); Lentiviruses; Rhabdoviruses such as rabies; Picomoviruses such as poliovirus; Poxviruses such as Vaccinia; Rotavirus; And antigens derived from Parvoviruses. More specifically, examples of viral pathogen antigens include HPV antigen L1, L2, E6 or E7 proteins of HPV; Examples of HIV antigens include T cells and B cell epitopes of nef, p24, gp120, gp41, tat, rev, pol, env and gp120 (Palker et al, J. Immunol . , 142: 3612-3619 . Examples of surface antigens of HBV are described in Wu et al, Proc . Natl . Acad . Sci ., USA , 86: 4726-4730 (1989). Examples of antigens of rotavirus include VP4 (Mackow et al, Proc . Natl . Acad . Sci ., USA , 87: 518-522 (1990)) and VP7 (Green et al, J. Virol . , 62: 1819-1823 (1988); influenza virus antigens include hemagglutinin (HA) and nucleoprotein; HSV antigens include thymidine kinase (Whitley et al, In: New Generation Vaccines, pages 825-854); avian influenza virus antigen includes hemagglutinin; pig cholera virus antigen is envenop; foot-and-mouth virus antigen is envelope; and Newcastle virus antigen is hemagglutinin-neuraminidase (HN) .

Examples of bacterial pathogen antigens that may be used in the present invention include Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli , Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp. And Borellia and antigens derived from burgdorferi . Specifically, examples of bacterial pathogen antigens that may be used in the present invention include Shigella sonnei form-1 antigen (Formal et al, Infect . Immun . , 34: 746-750 (1981)); V. cholerae O- antigen (Forrest et al, J. Infect Dis 159:.. 145-146 (1989); E. coli FA / I ciliary antigens (fimbrial antigen) (Yamamoto et al , Infect Immun, 50: 925-.. 928 (1985)) and non-toxic B-subunits of heat sensitive toxins (Klipstein et al, Infect. Immun . , 40: 888-893 (1983)); Bordetella pertactin of pertussis (Roberts et al, Vacc. , 10: 43-48 (1992)); Adenylate cyclase- hemorajine of B. pertussis (Guiso et al, Micro. Path . , 11: 423431 (1991)); And Clostridium Tetanus toxin fragment C of tetani (Fairweather et al, Infect . Immun . , 58: 1323-1326 (1990)).

Examples of parasitic antigens that can be used in the present invention include Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Entamoeba spp., Eimeria spp., Laishmania spp., Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp. < / RTI > More specifically, examples of parasitic antigens that can be used in the present invention include circumsporozoite antigens of Plasmodium bergerii and circumsporozoite antigens of Plasmodium spp. Such as P. falciparum circumsporozoite antigens (Sadoff et al, Sci . , 240: 336-337 1988)); Plasmodium spp the merozoite surface antigen (Spetzler et al, Int J. Pept Prot Res, 43:.... 351-358 (1994)).; Galactose specific lectin of Entamoeba histolytica (Mann et al, Proc . Natl . Acad . Sci ., USA , 88: 3248-3252 (1991)); Leishmania gp63 of the spp. (Russell et al, J. Immunol ., 140: 1274-1278 (1988)); Parmaiocin of Brugia malayi (Li et al, Mol . Biochem . Parasitol . , 49: 315-323 (1991)); And Schistosoma mosoni triose -phosphate isomerase (Shoemaker et al, Proc . Natl. Acad . Sci . USA , 89: 1842-1846 (1992)).

Examples of cancer antigens that may be used in the present invention include prostate-specific antigens (Gattuso et al, Human Pathol . , 26: 123-126 (1995)), TAG-72 and carcinoembryonic antigen (Guadagni et al, Int . J. Biol . Markers , 9: 53-60 (1994)), MAGE-1 and tyrosinase (Coulie et al, J. Immunothera, 14:. 104-109 (1993)), p53 (WO 94/02167), NY-ESO1 (cancer-testis antigen), AFP (α-feto protein) and a cancer antigen 125 ( CA-125), and EPCA (Early Prostate Cancer Antigen).

According to a specific embodiment of the present invention, the antigen is HEL (hen egg lysozyme).

According to a preferred embodiment of the present invention, in the vaccine composition of the present invention, the lipo oligosaccharide comprises a lipid of 3-hydroxytetradecanoyltrimethylsilyl ester, and the vaccine composition further comprises the Escherichia coli strain Lt; RTI ID = 0.0 > oligodeoxynucleotides < / RTI > of chromosomes.

According to a preferred embodiment of the present invention, when the vaccine composition further comprises an oligodeoxynucleotide, the weight ratio of diacyl lipoyl oligosaccharide and oligodeoxynucleotide is 1: 0.005 to 1: 200, preferably 1: : 0.05 to 1: 150, more preferably 1: 0.5 to 1: 150, even more preferably 1:10 to 1: 120, and most preferably 1: 100.

On the other hand, as demonstrated in the following examples, the dedifferentiated nontoxic LOS of the present invention is much less toxic than LPS. In addition, as shown in the following examples, it can be seen that when the dedifferentating non-toxic LOS of the present invention is administered together with an antigen of various pathogens, the immune response is greatly increased. The in vivo immune response can be divided into Th1 immune response and Th2 immune response. As used herein, the term " ThI immune response " means a reaction that induces a cellular immune response.

In the present invention, the measurement of the immune response in the immunized animal can be performed by, for example, a method of collecting serum from the animal to which the immunogen is administered to identify the activity of the anti-immunogen antibody (total IgG, IgG1 and IgG2a) . For example, enzyme-linked immunosorbent assay (ELISA), lateral flow assay, magnetic immunoassay (MIA), and immunoprecipitation may be used to confirm the activity of the antibody. And may be accomplished by a variety of immunoassay methods known in the art, preferably using ELISA assays.

Administration of the vaccine composition of the present invention increases the total amount of IgG and IgG _{2a in the} serum and also increases the activity of the interleukin-8 and interleukin-12 promoters by activating NF-κB and enhances the immune mechanism of helper T cell 1 Lt; RTI ID = 0.0 > interleukin-12 < / RTI >

The vaccine composition of the present invention can induce a sufficient immune response even with the basic composition, antigens of pathogens and desialylated non-toxic LOS, and can exert the preventive effect against specific diseases. Alternatively, the vaccine composition of the invention may additionally comprise other immunosuppressive agents, for example a second group of elements selected from the group consisting of Mg, Ca, Sr, Ba and Ra, Ti, Zr, Hf and Rf A salt of a Group 4 element selected from the group consisting of aluminum, or a hydrate thereof. The salts are preferably formed with oxides, peroxides, hydroxides, carbonates, phosphates, pyrophosphates, hydrogen phosphates, dihydrogenphosphates, sulfates or silicates. For example, the adjuvants that may be further utilized in the vaccine composition of the present invention include, but are not limited to, magnesium hydroxide, magnesium carbonate hydoxideside pentahydrate, titanium diadoxides, calcium carbonate, barium oxide, barium hydexide, Barium sulfate, barium sulfate, calcium sulfate, calcium pyrophosphate, magnesium carbonate, magnesium oxide, aluminum hydroxide, aluminum phosphate and hydrated aluminum potassium sulfate (Alum). Most preferably, the adjuvant that may be further utilized in the vaccine composition of the present invention is aluminum hydroxide.

The vaccine composition of the present invention may contain a pharmaceutically acceptable carrier and is conventionally used in the present invention and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, But is not limited thereto. The vaccine composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The vaccine composition of the present invention may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, the vaccine composition may be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, .

A suitable dosage of the vaccine composition of the present invention may be variously prescribed by factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . On the other hand, the oral dose of the pharmaceutical composition of the present invention is preferably 0.0001-1000 mg / kg (body weight) per day.

The vaccine composition of the present invention may be prepared in a unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs Or into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to still another aspect of the present invention, there is provided an anticancer agent or an anticancer adjuvant composition comprising diacyl lipoyl-oligosaccharide having a molecular weight of 2,000-5,000 Da and containing a sugar chain chain represented by the following formula (1) as an active ingredient:

Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine and P is phosphate.

The composition of the present invention may be used as an anti-cancer agent or anti-cancer adjuvant as a cancer treatment or prevention composition, and may be used independently or in parallel with physical / chemotherapy. The inventors of the present invention have devised a non-toxic polymeric material (dLOS) derived from bacterial LPS as an anticancer adjuvant, and succeeded in effectively using oligodeoxynucleotide as an anticancer drug. In particular, oligodeoxynucleotides which are ineffective alone do not show any particular toxicity when used with diacyl lipoolysaccharide (dLOS), and exhibit excellent anticancer effects against cancer cells.

According to a preferred embodiment of the present invention, the diacyl lipoic acid saccharide in the anticancer agent or the anticancer adjuvant composition comprises a lipid of 3-hydroxytetradecanoyltrimethylsilyl ester.

According to another preferred embodiment of the present invention, the anticancer agent or anticancer adjuvant composition may further comprise an oligodeoxynucleotide of the E. coli strain chromosome of the present invention. When the anticancer agent or the anticancer adjuvant composition further comprises an oligodeoxynucleotide, the weight ratio of the diacyl lipoic oligosaccharide and the oligodeoxynucleotide is 1: 0.005 to 1: 200, preferably 1: 0.05 to 1: 150 More preferably from 1: 0.5 to 1: 150, even more preferably from 1:10 to 1: 120, and most preferably 1: 100.

The anticancer agent or anticancer adjuvant composition of the present invention may include a pharmaceutically acceptable carrier, may be prepared into various formulations, and may be administered through various routes. The contents thereof are the same as those for the vaccine composition, and thus are omitted as duplicated descriptions.

The composition of the present invention can be used for treating various kinds of cancer and can be used for treatment of various kinds of cancers such as fibrosarcoma, bladder cancer, pituitary adenoma, glioma, brain tumor, female cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, Pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureter cancer, renal cell cancer, prostate cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, pediatric cancer, ovarian cancer and cervical cancer But are not limited thereto. Preferably, the cancer is fibrosarcoma or bladder cancer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an immunocompromising composition, vaccine composition and anticancer composition comprising a stable non-toxic diacyl lipoic oligosaccharide and an oligodeoxynucleotide having immunostimulatory activity.

(b) The two active ingredients contained in the composition of the present invention synergize with each other, and thus can be used as an anticancer agent having low toxicity and excellent immunogenicity.

(c) According to the present invention, it is possible to conveniently produce a safe anticancer drug comprising a purified DNA showing a drug efficacy higher than BCG at low cost and a non-toxic polymer substance derived from bacterial LPS, using one strain.

FIG. 1 is a graph showing the amount of TNF-α secreted as an indicator for selecting a strain producing LPS with the lowest toxicity during screening of a strain capable of simultaneously producing bacterial DNA and dLOS, which are components of anticancer drugs.
FIG. 2 is a result of purifying the LPS produced from the strain (D) selected in FIG. 1 and confirming its size through electrophoresis and silver staining.
FIG. 3 shows the results of electrophoresis after DNA was purified from the selected strains and disrupted using an ultrasonic disintegrator to a size superior in efficacy.
FIG. 4 shows that when the separated LPS is alkali-treated, the size of the lipid A is reduced by decomposing it. Lane 1 is a purified LPS and lane 2 is a non-toxic diacyl lipo-oligosaccharide (dLOS).
Figure 5 shows that dLOS decreased about 45-fold in toxicity compared to pre-toxic LPS by TNF-a secretion assay (J5: control, LPS: LPS before toxin removal, dLOS: ).
FIG. 6 relates to an abnormal toxicity test (C: control group, S: sample administration group) among the toxicity test for dLOS produced in one strain.
FIG. 7 shows the immunoenhancing effect (a; total IgG, b; IgG _2a ) according to the mixing ratio of oligodeoxynucleotide and dLOS produced in one strain in order to confirm the immunological effect in the mouse.
FIG. 8 shows the secretion effect of (a; IFN-y, b; IL-12p40) secretion of immuno-related cytokines according to the mixing ratio of oligodeoxynucleotide and dLOS produced from one strain using whole blood of a human.
FIG. 9 shows that the treatment of RAW cells with a mixture of oligodeoxynucleotides and dLOS produced from one strain increased the activity of IL-8 (9a) and IL-12 (9b) promoters by activating NF-kB Lt; / RTI >
FIG. 10 shows the results of comparing the anticancer effect with a conventional therapeutic agent for a mixture of oligodeoxynucleotide and dLOS produced in one strain in a mouse model (FIG. 10a: survival rate, FIG. 10b: stigma size).
Fig. 11 shows the results of MLADI-TOF / MS of dLOS produced in one strain.
12 is data on the confirmed sugar structure using MS / MS and MLADI-TOF / MS analysis for dLOS produced from one strain (Hex: hexose, Hep: heptose, Kdo: Dioxy-octonate, HexNAc: N-acetylhexosamine, P: phosphate).
Figure 13 is data on the lipid structure identified using GC-MS analysis for dLOS produced in one strain.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Detection of strains capable of simultaneously producing non-toxic dLOS and oligodeoxynucleotides

Screening and excavation of mutant E. coli that has a very short lipopolysaccharide and can produce stable oligonucleotides at the same time

A strain ( E. coli EG0023) having a lipopolysaccharide chain having a very short chain of lipopolysaccharide sugar chains and capable of stably producing oligodeoxynucleotides was found from E. coli in a healthy person's field. The screening method was as follows Respectively.

A single colony of Escherichia coli from a healthy adult male was selected by liquid culture and repeated 5 times. Then, 50 strains were selected first. One of the 50 strains selected was suspended in 4 ml of 0.9% physiological saline after collecting one of the colonies on the plate, and 2 ml of DNAase 1 (DNase 1) was transferred to Eppendorf tube and treated at 37 ° C And reacted for 1 hour in an incubator. 50 μl of 10 mg / ml RNase (RNase) was treated with the DNA solution after completion of the reaction, and then reacted in an incubator at 37 ° C. for 1 hour. Then, 100 쨉 l of 20 mg / ml proteinase K was added thereto, followed by reaction at 37 째 C overnight. LPS of each strain obtained by the above procedure was treated with human granulocyte-macrophage colony stimulating factor (GM-CSF) differentiated into human lymphocyte cell line and TNF-a secretion was measured to obtain the lowest value Five strains were selected and five strains were selected for secondary selection. The TNF-a value was confirmed by the above method and the lowest strain (D) was selected (Fig. 1). LPS of the selected strain was electrophoresed and silver stained to confirm the molecular weight of the lipopolysaccharide. This attenuated strain has no morphological or bacterial characteristics and has no lipopolysaccharide ladder having a molecular weight of only 50,000 to 100,000 and a molecular weight of 2,000 to 7,000 daltons (Fig. 2), and the strain name was designated as EG0023. The oligodeoxynucleotides were also purified from the same strain in the same manner as in Example 2.

Example 2: Purification of Escherichia coli Oligodeoxynucleotide

<2-1> Oligodeoxynucleotide Purification of

E. coli EG-0023 was shake-cultured for 10 hours in a TSG (Tryptic soy broth; Difco) medium of 30 g / l at 37 占 폚. After culturing 10 L, 150 g of cells obtained by centrifugation at 8,000 G were washed in 300 ml of TE buffer (10 mM Tris, pH 8.0, 25 mM EDTA) and centrifuged. Then, 150 g of the obtained cells was dissolved in 750 ml of a dissolution solution mM Tris (pH 8.0), 25 mM EDTA, 100 g / ml lysozyme) and treated for 1 hour at 37 ° C in an incubator. One hour later, protease K (Sigma) was added at a final concentration of 100 g / ml and treated for 12 hours at 50 ° C. The process of collecting the water layer with phenol / chloroform / isoamyl alcohol (25: 24: 1) mixture 1: 1 was repeated 3 times. The obtained E. coli chromosome was precipitated with ethanol. The concentration of purified E. coli DNA was measured at 260 nm and 280 nm wavelengths of UV spectroscopy after dilution with sterile distilled water. The DNA concentration of the purified E. coli against the measured value was calculated as follows.

Equation 1

Double stranded DNA concentration ([mu] g / ml) = OD. 260 nm x dilution factor x 50

(Provided that the value of OD 260 nm / 280 nm is 1.7 to 1.8)

<2-2> Fragmentation of chromosomes

The purified chromosome was dissolved in TE (Tris-HCl, EDTA) buffer to a concentration of 0.5 mg / ml and ultrasonically disrupted in a glass beaker. The ultrasonic wave shredder was a Sonics 500 Watt sonication VCX500 and the tips were run at 20 ml at a time using 630-0220 (tip diameter: 1/2 '(13 mm)). The pulverization conditions for the production of Escherichia coli DNA of the present invention were pulsed at 20,000 J for 7 minutes.

Example 3: Removal of endotoxin from shredded E. coli DNA and determination of purity

<3-1> Removal of endotoxin

The disrupted DNA was treated with chloroform for 12 hours at 4 DEG C, and then a 3-fold volume of ethanol was added to obtain a precipitate. Triton X-114 (Sigma) was added to the resulting precipitate to a final concentration of 0.5% and then treated at 4 占 폚 for 4 hours, followed by warming at 37 占 폚 for 5 minutes and then phenol / chloroform / isoamyl alcohol (25: 24: 1) and 1: 1 to collect the water layer.

After the obtained E. coli DNA was precipitated with ethanol, E. coli DNA was dissolved in pyrogen-free water. The endotoxin-depleted DNA was subjected to a Limulus Amebocyte Lysate (LAL) assay (BioWhittaker QCL-1000) to identify residual endotoxin.

<3-2> Confirmation of purity

The amount of residual organic solvent was measured by GC / MSD (gas chromatography / mass selected detector). The instrument used was HP-5890A / HP-5870B, and the columns were 50 m.ultra-1, SIM (Selected Ion Monitoring), and ethanol, acetone, chloroform and phenol were measured. In addition, using Brad-Ford method, E. coli DNA mg protein incorporation was confirmed and the purity level was maintained at over 99%.

Example 4: Purification of lipopolysaccharide from mutant E. coli

E. coli EG-0023 was cultured for 20 hours in a culture medium of 30 g / l of TSB (Tryptic soy broth; Difco) at 37 占 폚, and cells were recovered using a centrifuge. The prepared cells were mixed with twice the volume of ethanol and centrifuged at 4,000 g to obtain a precipitate. Thereafter, 1.5 times as much acetone was added to the obtained precipitate, and the mixture was thoroughly mixed, followed by centrifugation at 4,000 g.

To the obtained precipitate, an equal amount of ethyl ether was added and mixed well, followed by centrifugation at 4,000 g. Cell pellets obtained by centrifugation were covered with aluminum foil and dried. The cell mass was weighed and then 7.5 ml per 1 g of dry weight was extracted with an extraction mixture (90% phenol: chloroform: petroleum ether = 2: 5 : 8).

The mixture of the Escherichia coli and the extract mixture was dispensed into a glass centrifuge tube, centrifuged at 25 ° C and 3000 rpm (1,200 g) for 20 minutes, and the supernatant was allowed to stand in the hood for 12 hours and dispensed into a glass centrifuge tube . Lipopolysaccharide obtained by centrifuging the above glass centrifuge tube at 25 ° C and 3000 rpm (1,200 g) for 20 minutes was dissolved in ethyl ether, and the lipopolysaccharide was transferred to an Eppendorf tube and dried in a hood, Was used to measure the dry weight, followed by the addition of ethanol and storage until just before use.

The purified E. coli lipopolysaccharide stored in ethanol was completely removed from the ethanol and the amount of KDO (2-keto-3-dioxyoctonate) in the lipopolysaccharide was measured using a lipopolysaccharide standard (List Biological Lab. , And the concentration was measured. Then, the concentration was determined by SDS-PAGE according to the size and confirmed by silver staining. The size of the lipopolysaccharide was found to be between about 2,000 and 6,000, which was very small compared to the general E. coli lipopolysaccharide (Fig. 4, lane A).

Example  5: mutant purified from E. coli Lipopolysaccharide  Toxicity elimination

<5-1> Lipopolysaccharide Lipid  Removal of toxicity by A decomposition

The purified Escherichia coli lipopolysaccharide (LPS) was adjusted to a concentration of 3 mg / ml, mixed with a 1: 1 volume of 0.2 N NaOH, and shaken for 10 minutes at 60 ° C for 140 minutes. Subsequently, about 1 / 5th of 1 N acetic acid as an initial amount of 0.2 N NaOH was added and titrated to pH 7.0. After pH titration, non-toxic lipopolysaccharide (or non-toxic lipoylosaccharide) was obtained by ethanol precipitation. Non-toxic lipopolysaccharide was measured by KDO method and its size was compared with lipopolysaccharide by SDS-PAGE before silver staining. As a result of silver staining, it was confirmed that lipid A of lipopolysaccharide was decomposed by alkali treatment to be smaller than lipopolysaccharide before alkali treatment (Fig. 4, lane B).

<5-2> Non-toxic Lipopolysaccharide  Confirm toxic removal

For the safety test of non - toxic lipopolysaccharide, inflammatory protein secretion experiment, abnormal toxicity test and pyrogenicity experiment were performed.

<5-2-1> Inflammatory protein secretion test

The amount of secreted TNF-α was measured after treatment of dLOS (diacyl lipoyl-saccharide) produced from EG0023 into human acute monocytic leukemia cell (human acute monocytic leukemia cell) THP-1 cells. Commercially available LPS (J5) was used as a control. The control LPS secretes 1 ng of TNF-α per 1 μg of lipopolysaccharide while dLOS secretes 22 pg of TNF-α per 1 μg of non-toxic lipopolysaccharide, resulting in a 45-fold increase in inflammatory response due to toxicity (Fig. 5).

<5-2-2> Abnormal toxicity test

In order to confirm the toxicity of the produced dLOS samples, a high dose of the sample was administered to the rodents according to the KFDA - ICR mice were used for about 5 weeks. One mouse was injected intraperitoneal injection of 0.5 ㎖ into the peritoneal cavity for more than 7 days. As a result of the experiment, no change in body weight was observed after administration of the sample (FIG. 6).

<5-2-3> Pyrogenicity test

Rabbits are individuals weighing more than 1.5 kg. If the rabbits used in the test are to be used again, they should be more than 3 days after the end of the previous test. Body temperature was measured at 0.1 ℃. Syringes and injection needles were pre-sterilized by heating at 250 ° C for 30 minutes or more. The animals were given no feeds other than water until the end of the study, 16 hours prior to use, and the restraint was kept as low as possible when the animals were fixed.

To investigate the toxicity of the dLOS samples, three rabbits were injected with dLOS to observe changes in rectal temperature. Rabbits were injected intravenously at a dose of 0.2 μg / ml per kg of body weight of rabbits, and then a thermometer was inserted into the rectum to measure abnormal body temperature changes. The body temperature was measured after inserting the temperature measuring part into the rectum at a constant depth within the range of 60-90 mm and after a certain period of time. The body temperature of the animal was measured before injection and the body temperature was taken as the reference body temperature. After the control body temperature was measured, the sample warmed to about 37 ° C within about 15 minutes was injected into the ear vein. Body temperature was measured at least once every three hours after injection. The difference between the measured value and the reference body temperature was obtained and referred to as difference body temperature, and the maximum value of the body temperature was regarded as an exothermic reaction of the test animal. The test was performed using three animals. When the total of three reactions was below 1.3 ° C, the test was negative for the exothermic substance, and when the temperature was above 2.5 ° C, the exothermic substance was tested for the positive. The test was carried out three times, and when the exothermic substance test was negative, it was considered to be suitable for this test. The test results confirmed that the dLOS samples produced were not toxic. The detailed results are shown in Table 1 below.

Number of times rabbit
number Body temperature by number of measurements before administration
(Unit: ℃) Post-dose body temperature
(Unit: ℃) Maximum rising body temperature Total elevated body temperature standard result 1 time Episode 2 3rd time 0.5h 1h 1.5h 2h 2.5h 3h 1 time One 39.2 39.2 39.1 39.3 39.4 39.3 39.3 39.2 39.3 0.2 0.7 ℃ 1.3 DEG C
Below Pass 2 39.1 39.2 39.1 39.2 39.3 39.4 39.3 39.1 39.2 0.3 3 39.2 39.3 39.3 39.3 39.5 39.5 39.5 39.2 39.1 0.2 Episode 2 One 39.1 39.3 39.0 39.2 39.3 39.4 39.2 39.3 39.2 0.3 1.8 DEG C 3.0 DEG C
Below Pass 2 39.1 39.0 39.2 39.4 39.5 39.4 39.3 39.3 39.2 0.4 3 39.2 39.1 39.4 39.3 39.6 39.5 39.5 39.3 39.3 0.4 3rd time One 39.3 39.1 39.1 39.5 39.4 39.5 39.3 39.2 39.3 0.4 2.9 ° C 5.0 ° C
Below Pass 2 39.2 39.3 39.3 39.6 39.6 39.4 39.2 39.3 39.4 0.3 3 39.1 39.3 39.2 39.4 39.6 39.6 39.5 39.4 39.3 0.4

* Round to the decimal place

Example  6: Oligodeoxynucleotide , Non-toxic Of lipopolysaccharide (dLOS)  Mixed and Potency  Confirm

<6-1> Oligodeoxynucleotide  And Non-toxic Lipopolysaccharide  mix

To confirm the optimum concentration of the two substances (oligodeoxynucleotide, non-toxic lipoyl-saccharide (dLOS)) produced according to each standard, shake blending was carried out at a ratio of 100: 1, 50: 1.

<6-2> Check your potency

In order to confirm the efficacy as an adjuvant in mice, 50 쨉 g of HEL (Sigma) was administered as an antigen to ICR mice (male, 4 weeks, 20 g) intraperitoneally twice at intervals of 0.1 ml per week. Seven days after the last administration, whole blood was collected and serum was separated. IgG _2a , which is a total antibody and IgG subtype in serum, was confirmed by ELISA as an antigen (FIGS. 7A and 7B).

1 μg of commercially available LPS (J5) was used as a control group, and the test group was divided into two groups of antigen, dLOS 1 μg + DNA 50 μg, and dLOS 0.5 μg + DNA 50 μg. As a result, it was confirmed that IgG _2a, which is effective for chemotherapy related to cell immunity among immunoglobin subclass as well as whole IgG, shows the highest concentration when 0.5 μg of dLOS and 50 μg of DNA (FIG. 7b).

<6-3> Whole blood analysis  Used Potency  Confirmation test

Venous blood was aseptically collected from a healthy adult male in a vacuum tube containing heparin as an anticoagulant. The whole blood was mixed at a ratio of 1: 1 with RPMI 1640 medium (2 mM L-glutamine, 1 mM sodium pyruvate, 80 g / ml gentamicin). To 1 ml of the whole blood mixed with the medium was adjusted with PBS to make a total of 20 μl of DNA 50 μg + dLOS 1 μg or DNA 50 μg + dLOS 0.5 μg, and cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours. The amount of secreted IFN-γ and IL-12p40 in culture supernatants was measured using commercially available ELISA kits (IFN-γ (R & D, DY485), IL-12p40 (R & D, DY2398) 8A and 8B.

The results showed that synergistic immunostimulating effect was obtained when oligo deoxynucleotide was administered more than when dLOS alone was administered. The dose was most favorable when DNA was 50 μg and dLOS was 0.5 μg.

<6-4> Lucifer Reyes Assay

Raw 264.7 (mouse leukocyte-like cell line) cells were first plated in 12 well plates at 5 × 10 ⁴ per well (1 ml DMEM / 10% FBS) and cultured in a CO ₂ incubator at 37 ° C. for 24 hours. (20 μg / well) and IL-12 Luciferase reporter plasmid (0.2 mg / well) and PRL-null plasmid (20 ng / well) were mixed and reacted with serum-free DMEM , Roche Cat. No. 1814443), and the mixture was allowed to stand for 5-10 minutes. The mixture was added to Raw 264.7 cells at 52 ml / well and then cultured in a CO ₂ incubator at 37 ° C for 24 hours. After incubation, 20 ㎍ of oligodeoxynucleotide per well and 200 ng of dLOS were treated in Raw 264.7 cells and cultured in a CO ₂ incubator at 37 캜 for 12 hours. Luciferase reaction was performed using Luciferase assay kit (Promega Cat. No. E1500), and luciferase activity was measured using a luminometer.

The effect of oligodeoxynucleotides and dLOS on IL-8 and IL-12 promoter activity was measured by the luciferase activity. As a result, the NF-κB binding site was common in the IL-8 and IL-12 promoters, and RAW264. 7 cell line, NF-kB was activated by DNA and dLOS, thereby increasing the activity of the promoter (FIGS. 9A and 9B).

<6-5> Oligodeoxynucleotide  + dLOS  Union Cell soluble ability  Use of chemotherapy

The cancer cell viability of oligodeoxynucleotides + dLOS was measured by the ⁵¹ Cr-release assay. Male, 5-8 week old, C3H / HeN mice were subcutaneously administered antigen alone or antigen and oligodeoxynucleotide + dLOS to the sole of the foot.

(10 mM HEPES, 100 units / ml penicillin, 100 占 퐂 / ml streptomycin, 300 占 퐂 / ml glutamine; Gibco Laboratories, Grand Island, NY) was used as a basic medium for cell line culturing. 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY), which had been inactivated by heating at 56 ° C for 30 minutes, was used. Sarcoma 180 and mouse bladder cancer cell line MBT-2 were used as target cells to measure the activity of lymphokine activated killer cell (LAK) and cancer cell mediated kill ability.

The mice in each experimental group were slaughtered with cervical disassembly, and the spleen was aseptically extracted. The microspheres were prepared by scissors on a stainless steel wire mesh having a diameter of 100 microns, and then, with PBS (phosphate buffered saline) It was passed through a wire mesh while being lightly pulverized and filtered to remove tissue debris. The cells were washed once with the RPMI-1640 basic medium and then suspended in 0.84% ammonium chloride solution at 37 째 C for 5 minutes to dissolve the red blood cells. Cells were washed twice more with the basal medium, suspended in complete medium, dispensed into culture flasks, and incubated for 1 hour at 37 ° C in a 5% CO ₂ thermostat. After the cells were cultured, cells that did not adhere to the flask were collected, and viable cells were counted by tryptophan blue dye exclusion method, and a cell suspension having a density of 5 x 10 ⁶ cells / ml was prepared with the complete medium.

After the target cell line (Sarcoma 180 and MBT-2) was cultured, the number of cells was calculated and 1 x 10 ⁶ cells were taken and centrifuged at 300 g for 3 minutes. After centrifugation, carefully remove the supernatant with a pasteur pipette to keep the precipitated cells intact. The precipitated cells were labeled with 100 ci Na ₂ ⁵¹ CrO ₄ (1 ml CI / ml, NEZ 030S, NEN, USA) for 1 hour in a shaking incubator at 37 ° C, and then washed three times with the basic medium. Then, viable cells were counted by trypival blue dye exclusion method, and the labeled target cells were resuspended in complete medium to 5 x 10 ⁴ cells / ml.

The bottom 96 well each per well of a microtiter plate labeled round target cells 5 × 10 ³ Dog 0.1 ㎖ by frequency division, and the spleen cell response cell to enter: the ratio of the target cell 100: the reaction cell to be a 1 0.1 ㎖ And then cultured in a 5% CO ₂ constant temperature and humidity chamber at 37 ° C for 4 hours. Up to 3 wells were prepared per each experiment, and after 4 hours of culture, the cells were centrifuged at 500 g for 15 minutes. Then, 0.1 ml of the supernatant was taken from each well and radioactivity was measured with a gamma counter (Packard, USA). At this time, 0.1 ml of 5% Triton X-100 (Sigma, USA) was added to the control well to induce maximum release. Only the labeled cells were cultured in the same volume of complete medium to measure the amount of spontaneous release. The cytotoxicity was calculated by the following formula.

Equation 2

Cytotoxicity (%) = (ER-SR / MR-SR) X 100

ER: Mean count (cpm)

SR: mean count (cpm) of target cells cultured but not cultured

MR: Mean count (cpm) of target cells treated with 5% Triton X-100

As a result, the Sarcoma 180 cell line showed 7.2 times higher cytolytic activity than non-immunized cells and 1.3 times superior to BCG (Bacillus Calmette-Gu) treated group. In addition, the MBT-2 cell line showed a 5.2-fold increase in cytolytic activity compared to the non-immunized cells, and was approximately 1.3-fold superior to the BCG-treated group (Table 2). This suggests the possibility of the combination of oligodeoxynucleotide + dLOS as an adjuvant treatment for BCG which is reported to have various side effects.

Sachoma 180 Day of administration 0 3 7 15 Control group 100 100 100 100 BCG 96 ± 4 108 ± 3 561 ± 16 121 ± 8 DNA + dLOS 95 ± 7 139 ± 3 721 ± 15 118 ± 7 MBT-2 Control group 100 100 100 100 BCG 101 ± 2 107 ± 6 398 ± 14 105 ± 6 DNA + dLOS 103 ± 4 118 ± 7 521 ± 16 114 ± 3

<6-5> Using bladder cancer mouse model Oligodeoxynucleotide  + dLOS  Measuring the effectiveness of combination chemotherapy

The effect of dLOS and DNA on bladder cancer was confirmed by using C3H / HeJ mouse (magnetic, 4 weeks old, 19-21 g) induced bladder cancer using MBT-2 cell line. The administration of dLOS + DNA, BCG, and PBS started on the day following the transplantation of the tumor cell line, and was administered over a total of 13 times every day for the first week and every two days for the next two weeks. The administration site was right flank and the administration route was subcutaneous injection method. The treatment effect on bladder cancer was confirmed by comparing the vertical diameter of the cancer lesion and the mortality rate by the experimental group.

In detail, the mice were transplanted into the right flank of the mouse at the concentration of 5 × 10 ⁵ cells / 100 μl of the MBT-2 bladder cancer cell line on day 0 (day 0) after one week of environmental adaptation. DLOS + DNA, BCG, or PBS was injected subcutaneously into the tumor cell line every day for 1 week from the first day after the cancer cell transplantation, and then injected every other day for 2 weeks. The vertical diameter of the lesion was measured at intervals of 2 days from the 10th day to the 40th day, and the pessary rate was confirmed (Figs. 10a and 10b). As can be seen from FIGS. 10A and 10B, it was confirmed that the mixture of dLOS and DNA showed a stronger anticancer effect than BCG.

Example  7: Mutant purified from E. coli dLOS  Stability of structure

The dLOS produced by using organic solvent from E. coli is produced in a prescribed form with a small size and a clear structure, and mass production using a mass production system is also possible.

<7-1> MALDI - TOF

1) Sample preprocessing

The DHB (2,5-dihydroxybenzoic acid) matrix was prepared at a concentration of 10 mg / ml (50% methanol and 0.1% trifluoroacetic acid), and then the sample / matrix was mixed at a ratio of 1/10 to 100/1, Lt; / RTI &gt; Samples for positive mode analysis were added 0.01 M NaCl to DHB.

2) MALDI-MS analysis

The instrument used was MALDI-TOF from Autoplex (Bruker Daltonics). Linear mode and Reflect mode can be used for sample analysis. In case of linear mode, resolution is low but sensitivity is good, which is advantageous for analyzing sample with large molecular weight. In case of symmetrical mode, sensitivity But the resolution is superior, which is advantageous for analyzing a sample having a small molecular weight. Prior to analysis, mass spectra were calibrated by selecting the peptides and protein standards provided by Bruker Daltonics to fit the analytical mass range. A laser shot of each data was obtained at 3000. The obtained data were analyzed by FlexAnalysis (Bruker Daltonics).

MALDI-TOF / MS, a high-energy ionization method, was used to analyze the fragmentation pattern of the molecules that constitute the entire structure including the sugar moiety, considering the characteristics of the dLOS sample containing sugar chains.

As a result of MALDI-TOF / MS analysis, a peak of molecular ion having a distinct dLOS at m / z 2824 was detected. On the other hand, the other fragment ions detected at m / z 1233 and 1455 showed very low intensity as compared with the base ion. From the information of these peaks, additional information on sugar and lipid constituents of dLOS was obtained. These peaks were estimated as sugar and lipid components dissociated in the ionization process.

In general, the analysis of the structure of lipid-containing structure reveals a polymer pattern that can be generated by bonding Na to COOH or phosphate present in lipids. When the analyzed peaks are magnified, the m / z value at which the polymer pattern starts is m / z 2804, from which the molecular weight increases by 22, which can be confirmed. As a result, the total molecular weight of dLOS is m / z 2804 (Fig. 11).

<7-2> And methylation ( permethylation ) On by MS / MS

A 1 ml sample was prepared by adding 2% acetic acid to a sample of purified dLOS 1 mg / ml, heated at 100 ° C for 1 hour, and then cooled to room temperature. C18 reversed phase column, eluted with 5% acetic acid to obtain a sample, and then dried with a rotary evaporator. The dried sample was hypermethylated using iodomethane, dried, dissolved in 0.1 mM NaOH, and introduced into a mass spectrometer.

The MS / MS spectrum of the mass spectrum of the introduced sample was obtained by using the total ion mapping function of the mass spectrometer to obtain tandem MS data of the mass spectrum. We then attempted a possible structural analysis of the sample by obtaining multiple fragment ions at m / z 1046. The molecular structure of the linear type was analyzed by utilizing the separation pattern of fragment ions in the core structure of general LPS, and MALDI-TOF / MS was further performed to determine the structure of the sugar chain. A base peak was detected at m / z 1842 of the linear mode analysis. For structural analysis of the detected base peak, the acyl group of dLOS was identified from the information of the base peak obtained in the symmetric mode analysis. From the above results, dLOS was identified to have the skeletal structure of sugar and lipid shown in FIG. 12, and one sugar chain structure was confirmed by several possible sugar chain structure (FIG. 12).

<7-3> GC - MS

The sample was placed in HCl under methanol and reacted at 80 ° C for 18 hours to hydrolyze and esterify it. Distilled water was added to the reaction mixture, and then derivatized acid was extracted with hexane to remove the organic solvent from the separated layer. The extracted residues were redissolved in dry pyridine (1 ml) and BSTFA-TMCS [N, O-bis (trimethylsilyl) trifluoroacetamide] -trimethylchlorosilane] (Supelco, 200 μl) Lt; 0 &gt; C for 1 hour, and the reaction sample was analyzed by GC-MS (Agilent).

The lipid structure was analyzed by using GC-MS after specific separation of lipids from dLOS samples. The lipid database (http://lipidlibrary.aocs.org/ms/arch_me/index.htm #hydroxy) As a result of a comparative analysis of the lipid analysis results specifically separated from the dLOS sample, the structure (3-hydroxytetradecanoyltrimethylsilyl ester) as shown in Fig. 13 was confirmed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Microorganism Conservation Center (overseas) KCCM11218P 20111109

Claims (22)

Wherein the lipo oligosaccharide produces a lipo oligosaccharide having a molecular weight of 2,000-5,000 Da, and the lipo oligosaccharide comprises a sugar chain chain represented by the following formula (1): Escherichia coli strain, KCCM11218P,
Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine and P is phosphate.
The Escherichia coli strain according to claim 1, wherein the chromosomal DNA of the Escherichia coli strain produces oligodeoxynucleotides having immunostimulatory activity by pulverization.
The Escherichia coli strain according to claim 1, wherein the lipo oligosaccharide comprises a lipid of 3-hydroxytetradecanoyltrimethylsilyl ester.
delete An adjuvant composition comprising a diacyl lipoyl oligosaccharide having a molecular weight of 2,000-5,000 Da and containing a sugar chain chain represented by the following formula (1) as an active ingredient:
Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine and P is phosphate.
6. The composition of claim 5, wherein the diacyl lipoic oligosaccharide comprises a lipid of 3-hydroxytetradecanoyltrimethylsilyl ester.
6. The composition of claim 5, wherein said immunoadjuvant composition further comprises an oligodeoxynucleotide of the chromosome of the E. coli strain of any one of claims 1 to 3.
8. The composition of claim 7, wherein the weight ratio of diacyl lipoic oligosaccharide and oligodeoxynucleotide is from 1: 0.005 to 1: 200.
8. The composition of claim 7, wherein said immune aid composition increases the activity of interleukin-8 and interleukin-12 promoters by activating NF-kB.
8. The composition of claim 7, wherein the composition increases secretion of interferon-y and interleukin-12p40 of helper T cell 1.
(a) an antigen; And (b) a vaccine composition comprising diacyl lipoyl oligosaccharide having a molecular weight of 2,000-5,000 Da and containing a sugar chain chain represented by the following formula (1) as an immunostimulatory component as an active ingredient:
Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine and P is phosphate.
12. The composition of claim 11, wherein the diacyl lipoic oligosaccharide comprises a lipid of 3-hydroxytetradecanoyltrimethylsilyl ester.
12. The composition of claim 11, wherein the vaccine composition further comprises an oligodeoxynucleotide of the chromosome of the E. coli strain of any one of claims 1 to 3.
14. The composition of claim 13, wherein the vaccine composition is a mixture of diacyl lipoic oligosaccharides and oligodeoxynucleotides in a weight ratio of 1: 0.005 to 1: 200.
The method of claim 13, wherein the vaccine composition is a composition, comprising a step of increasing the total amount of IgG, and IgG _2a in the serum.
14. The composition of claim 13, wherein said vaccine composition increases the activity of interleukin-8 and interleukin-12 promoters by activating NF-kB.
14. The composition of claim 13, wherein the vaccine composition increases secretion of interferon-y and interleukin-12p40 of helper T cell 1.
Diacyl lipoyl oligosaccharide having a molecular weight of 2,000-5,000 Da and containing a sugar chain chain represented by the following formula (1); And an anticancer agent or anticancer adjuvant composition comprising the oligodeoxynucleotide of the chromosome of the E. coli strain of any one of claims 1 to 3 as an active ingredient:
Formula 1

In the above formula, Hex is hexose, Hep is heptose, Kdo is 2-keto-3-deoxy-octonate, HexNAc is N-acetylhexosamine and P is phosphate.
19. The composition of claim 18, wherein the diacyl lipoic oligosaccharide comprises a lipid of 3-hydroxytetradecanoyltrimethylsilyl ester.
delete 19. The composition of claim 18, wherein the anticancer agent or anticancer adjuvant composition comprises diacyl lipoic oligosaccharide and oligodeoxynucleotide in a weight ratio of 1: 0.005 to 1: 200.
The method of claim 18, wherein the cancer is selected from the group consisting of fibrosarcoma, bladder cancer, pituitary adenoma, glioma, brain tumor, female cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, Wherein the cancer is cancer, gallbladder cancer, penile cancer, ureteral cancer, renal cell cancer, prostate cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, pediatric cancer, skin cancer, ovarian cancer or cervical cancer.

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