KR20160039495A - Solid cancer agent using bacteria - Google Patents

Abstract

The present invention relates to a solid cancer therapeutic agent using bacteria. According to the present invention, provided is an attenuated Salmonella strain which selectively expresses L-asparaginase (L-ASNase) in tumor sites by being transformed to a gene construct comprising polynucleotide which encodes the L-asparaginase (L-ASNase) operably coupled to a promotor.

Description

      {Solid cancer agent using bacteria}

The present invention relates to a therapeutic agent for solid cancer, and more particularly to a therapeutic agent for solid cancer using bacteria.

Since L-aspartase (L-ASNase) was found to hydrolyze L-asparagine to catalyze the reaction of producing aspartic acid and ammonia, it rapidly and almost completely eliminated cancer cells in leukemia-induced mice, It was approved by the FDA in 1978 and was used to treat tumors mainly infected through blood, including certain types, including acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma. The reason for this is that in the asparagine synthesis, T-lymphocytic leukemia cancer cells can not synthesize asparagine due to the reduced expression of asparagine synthase, which must depend on the extracellular pool of asparagine, This is because. Thus, exposure to asparagine-degrading aspartate L-ASNase inhibits protein synthesis due to the lack of asparagine supplied as a nutrient, and T-lymphocytic leukemia cancer cells die.

Currently, cancer therapy using tumor-targeted bacteria, such as Salmonella and Clostridium spp , relies on the function of certain bacteria that can target solid tumors and proliferate in tumors, but tumorlytic proteins May be administered to the bacteria to minimize side effects that are selectively expressed at the tumor site and are toxic to normal cells.

It is an object of the present invention to solve the various problems including the above-mentioned problems and to provide a therapeutic agent for solid tumors capable of selectively killing solid tumor tissue. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a method for producing L-asparaginase (L), which is transformed with a gene construct containing a polynucleotide encoding an Escherichia coli L-ASNase operably linked to a promoter, -ASNase) is selectively expressed at the tumor site.

According to another aspect of the present invention, there is provided a therapeutic agent for solid tumor cancer, which contains the attenuated Salmonella strain as an active ingredient.

According to one embodiment of the present invention as described above, the tumor growth inhibitory effect of the attenuated Salmonella strain selectively expressing L-ASNase at the tumor site can be realized. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a diagram showing the structure of a pASN plasmid expressing L-ASNase prepared according to an embodiment of the present invention. FIG. 1 (A) shows the result of culturing a recombinant attenuated Salmonella strain and determining the amount of L-ASNase (B) Western blot gel photograph showing expression and secretion.
2 is a graph showing MTT analysis of animal tumor cells and normal cells in order to confirm the cytotoxicity of the bacterial culture solution.
FIG. 3 is a Western blot gel photograph (A) showing the observation of annexinV expression by treating a culture supernatant of a bacterial culture supernatant containing L-ASNase in the cultured cells, and FIG. 3 This is a photograph of immunohistochemistry (B). annexinV is green, DAPI is blue and F-actin is red.
FIG. 4 is a photograph showing the expression and secretion of L-ASNase from salmonella in a tumor having pASN, showing Western blot gel showing L-ASNase expression (A), immunohistochemical staining (B).
5 shows graphs and photographs showing the tumor size and survival rate of C57BL / 6 mice transplanted with MC38 tumor cells (A) and 4T-1 tumor cells (B), showing the antitumor effect of Salmonella expressing L-ASNase (B), BALB / c thymus nu- / nu-mice transplanted with AsPC-1 tumor cells and the survival rate of BALB / c mice transplanted with AsPC- The graph and the photograph are shown (C).
FIG. 6 is a Western blot gel photograph showing mutant L-ASNase expression and cytotoxicity, showing mutation and expression of wild-type L-ASNase in culture supernatant and bacterial pellet according to L-arabinose induction (A) (B) analysis of cytotoxicity of mutant L-ASNase using cultured MC38, 4T-1 and AsPC-1 cells (C) analysis of the enzyme activities of spent media containing ASNase ).
FIG. 7 is a graph showing the antitumor effect of the mutated L-ASNase expressed in the tumor, showing the change in the total morphology of the tumor tissue after the bacterial treatment (A), and the graph showing the change in the tumor size after the bacterial treatment B), Kaplan-Meier survival analysis curves of tumor-transplanted mice (C).
FIG. 8 is a photograph showing Western blot gel images showing the expression of L-ASNase and annexinV in serum and tumor tissues of tumor-transplanted mice over time in serum and tumor tissues of tumor-transplanted mice A), and L-ASNase activity using an enzyme assay kit (B).
FIG. 9 is a graph showing changes in tumor size by administering Leunase ^® in a tumor-transplanted mouse according to a concentration (A) and a Kaplan-Meier survival analysis curve of a tumor-transplanted mouse (* P = 0.003) Graph (B).
FIG. 10 is a graph showing the change in tumor size according to the concentration of Leunase ^® , which is a result of injecting Leunase ^® into a tumor and observing the antitumor effect. FIG. 10 (A) is a photograph showing the total morphology of tumor tissue, (C) Western blot gel showing the distribution of L-ASNase in post-serum and tumor tissues.

Definition of Terms:

The term " L-ASNase &quot;, as used herein, is defined in EC 3.5.1.1. As an amide hydrolyzing enzyme, it catalyzes the reaction of hydrolyzing L-asparagine to produce aspartic acid and ammonia. Some of these enzymes act on glutamine, although the substrate specificity is high. It is widely distributed in yeast, bacteria, fungi, higher plants, liver and kidneys of animals. The enzyme extracted from Torula utilis , which is a kind of yeast, is liable to lose activity in acidic condition (pH <5.5) and trace amounts of Ag ⁺ , Hg ²⁺ &Lt; / RTI &gt; Because tumor cells such as acute lymphoblastic leukemia cells require asparagine in blood for proliferation, clinically, asparagine is injected intravenously to treat leukemia.

As used herein, " attenuation "means modifying the pathogenicity of a microorganism to reduce it. Attenuation is done for the purpose of reducing toxicity and other side effects when the microorganism is administered to the patient. The attenuated bacteria may be produced through a variety of methods known in the art. For example, attenuation is achieved by destroying or destroying the virulence factor that causes the bacteria to survive in the host cell. Such deletions and deletions are well known in the art and are carried out by methods such as homologous recombination, chemical mutagenesis, irradiation mutagenesis or transposon mutagenesis. Examples of toxic agents of Salmonella that can lead to attenuation when deleted include: 5'-adenosine monophosphate (Biochenko et al. , 1987, Bull. Eksp. Biol. Med. 103: 190-192) lysine (Libby et al, 1994, Proc Nati Acad Sci USA 91:..... 489-493), defensin resistance loci (Fields et al, 1989, Science 243:. 1059-62), DNAK (Buchmeier et al ., 1990, Science 248: 730-732 ), Pim brie lover (Ernst et al, 1990, Infect Immun 58:... 2014-2016), GroEL (Buchmeier et al, 1990, Science 248:. 730-732) , Inv loci (Ginocchio et al ., 1992, Proc Natl Acad Sci USA 89: 5976-5980), lipoprotein (Stone et al ., 1992, J. Bacteriol . 174: 3945-3952), LPS . Gianella et al, 1973, J. Infect Dis 128:.. 69-75), PhoP and PhoQ (Miller et al, 1989, Proc Natl Acad Sci USA 86:..... 5054-5058), Pho activation gene (Abshiro et al, 1993, J. Bacteriol 175:.. 3734-3743), PhoP and PhoQ regulatory genes (Behlau et al, 1993, J. Bacteriol 175:.. 4475-4484), morpholine (Tufano et al,. 19 88, Eur J. Epiderniol 4: 110-114 ), toxic factors (Loos et al., 1994, Immun. Infekt. 22: 14-19; Sansonetti, 1992, Rev. Prat 42: 2263-2267). The contents of attenuated bacteria are described in detail in WO 96/40238, which is incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a method for producing a L-asparaginase gene, which comprises transforming a gene construct comprising a polynucleotide encoding L-ASNase operably linked to a promoter, A selectively attenuated Salmonella strain is provided.

The Salmonella strain may be Salmonella typhimurium , Salmonella choleraesuis or Salmonella enteritidis .

In the attenuated salmonella strain, the promoter may be an inducible promoter, and the inducible promoter may be a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL? Promoter, pR? Promoter, rac5 promoter, amp promoter, recA promoter, SP6 Promoter, uvrD promoter, uvrB promoter, umuDC promoter, lexA promoter, cea (promoter), trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter, pepT promoter, sulA promoter, pol11 Promoter, caa promoter, recN promoter, pagC promoter, hip promoter, ansB promoter or pflE promoter.

In the attenuated Salmonella strains, the attenuation is selected from the group consisting of aroA , aroC , aroD , aroE , Rpur , htrA , ompR , ompF , ompC , galE , cya , crp , cyp , phoP , phoQ , rfaY , dksA , hupA , sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, one selected from the group consisting of relA and spoA Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; In addition, the attenuated strain may be a strain lacking the ability to produce ppGpp, the strain lacking the ability to produce ppGpp may be a strain lacking the spoT and / or relA gene, and preferably all of the spoT and relA genes are deficient Lt; / RTI &gt;

The deletion of the genes relA or spoT gene refers to a variant (modifications) in the genes relA or spoT gene transcription or translation of the gene, resulting in damage (impairment) of a gene product activity. Such genetic modification may include inactivation of the ppGpp cytoset sequencing (CDS) as well as inactivation of its promoter. The specific inactivation of only the gene of interest on the bacterial genome is possible by mutation through substitution, insertion, deletion, or a combination thereof in all or at least one subdomain of the coding gene. For example, deletion of a gene and insertion of a heterogenous sequence into a gene can be accomplished by truncation of the gene, nonsense mutation, frameshift mutation, and missense mutation. . The specific inactivation of these genes can be accomplished through methods established in the art. On the other hand, gene deletion can be carried out through various mutagenesis methods known in the art. For example, deletion of the relA gene or the spoT gene can be performed by PCR mutagenesis and cassette mutagenesis (Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001) .

In addition to the relA gene and spoT gene, examples of Salmonella toxicity factors that may result in attenuation if deleted include: 5'-adenosine monophosphate (Biochenko et al., Bull. Eksp. Biol. Med. USA, 91: 489-493, 1994), dipenthine tolerance loci (Fields et al., Science 243: 1059-62, 1989), cytochromosin (Lipi et al., Proc. Nat . ), DNAK (Buchmeier et al., Science 248: 730-732. 1990), Pimbria (Ernst et al., Infect. Immun. 58: 2014-2016 1990), GroEL (Buchmeier et al., Science 248: 730-732 1990), Inv loci (Ginocchio et al., Proc. Nat. Acad Sci USA 89: 5976-5980 1992), lipoprotein (Stone et al., J. Bacteriol. 174: 3945-3952 1992) , LPS (Gianella et al., J. Infect. Dis. 128: 69-75 1973), PhoP and PhoQ (Miller et al., Proc. Nat. Acad Sci. USA 86: 5054-5058 1989) Genes (Abshiro et al., J. Bacteriol. 175: 3734-3743 1993), PhoP and PhoQ regulatory genes (Behlau et al., J. Bacteriol. 175: 4475-4484 1993), Tufano e ... t al,, Eur J. Epiderniol 4: 110-114 1988), virulence factors (Loos et al, Immun Infekt 22 :... 14-19 1994; Sansonetti et al, Rev. Prat 42:.. 2263 -2267, 1992), etc., and the attenuation is aroA, aroC, aroD, aroE ( Levine et al, J. Biotechnol, 1996. 44 (1-3..): 193-196), Rpur, htrA (Chatfield et al ., Microb. Pathog, 1992. 12 (2): . 145-151), ompR, ompF, ompC (Chatfield et al, Infect Immun 1991. 59 (1...):. 449-452), galE (Hone et al, J . Infect Dis, 1987. 156 (1 ):.. 167-174), cya, crp (Curtiss, R., 3rd and SM Kelly, Infect Immun, 1987. 55 (12..): 3035-3043), cyp , PhoP (Hohmann et al ., J. Infect. Dis ., 1996. 173 (6): 1408-1414; Miller et al ., Proc Natl Acad Sci USA , 1989. 86 (13): 5054- 5058), phoQ, rfaY (Turner et al, Infect .. Immun .. 1998. 66 (5.): 2099-2106), dksA (Turner et al, Infect .. Immun .. 1998. 66 (5.): 2099-2106), hupA (Turner et al ., Infect .. Immun .. 1998. 66 (5): 2099-2106), sipC (Turner et al ., Infect .. Immun . 1998. 66 (5): 2099-2106) or clpB (Turner et al, Infect .. Immun .. 1998. 66 (5.): 2099-2106), clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, be achieved by galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, failure of one or more genes selected from the group consisting of relA and spoA . The loss of function of the gene can be achieved by deletion, substitution, addition, or the like of a specific nucleotide sequence in the gene or by mutation of a transcriptional regulatory region (including a promoter and a terminator) of the gene.

According to another aspect of the present invention, there is provided a therapeutic agent for solid cancer, which comprises an attenuated Salmonella strain as an active ingredient.

In the solid cancer therapeutic agent, the solid cancer may be any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer and pancreatic cancer .

The therapeutic agent may be administered orally or parenterally at the time of clinical administration and may be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, And may be used in the form of a general pharmaceutical preparation.

 The solid cancer therapeutic agent according to one embodiment of the present invention further comprises an inert ingredient including a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is a term referring to a composition, specifically a component other than the active ingredient of the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, lubricants, solubilizers or emulsifiers and salts.

In practical use, the solid cancer therapeutic agent according to one embodiment of the present invention may be combined with a pharmaceutical carrier according to conventional pharmaceutical preparation techniques. The carrier may take a wide variety of forms depending upon the preparation desired, for example, for oral or parenteral administration (including intravenous administration).

In addition, the solid cancer therapeutic agent according to an embodiment of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably 0.1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

According to another aspect of the present invention, there is provided a method of treating solid cancer comprising the step of administering a therapeutically effective amount of the Enterobacteriaceae Salmonella strain to an individual suffering from solid cancer.

The attenuated salmonella strain may be administered to the subject by parenteral administration, and the parenteral administration may be performed by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous administration subcutaneous injection, intraventricular injection, intracranial injection, intracerebrospinal injection, or intratumoral injection.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Plasmid construction

DNA encoding the ASNase Ⅱ (asnB) according to one embodiment of the present invention is L-ASN1 sense primer (5'-GG GAA TTC ATGGAGTTT TTC AAA AAG-3 ', SEQ ID NO: 1) and L-ASN2 antisense primer (5 Was amplified from the genomic DNA of E. coli B strain (BL21) using '-GGG TCT AGA TTA GTA CTG ATT GAA GAT CTGCTG-3', SEQ ID NO: 2). 1,047 kb DNA fragment of the gene is asnB using the Eco RI and Xba I restriction enzyme site to produce the pASN was inserted into the vector GlmS ⁺ p. The mutant L-ASNases were amplified using a P1 sense primer (5'-GACTCCGCAACCAAATCTGGCTACACAGTGGGTAAAG-3 ', SEQ ID NO: 3) for N24G and a P2 antisense primer (5'-ACGTCTATGAGCGCAGGCGGTCCATTCAACCTGT-3', SEQ ID NO: 4) for D124G and pASN -directed mutagenesis (Stratagene, San Diego, Calif., USA). All constructs were identified by automated DNA sequencing.

As a result, 1,047 bp PCR amplified asnB open reading frames (open reading frame) the GlmS ⁺ p was cloned into a vector araBAD promoter of the E. coli arabinose operon (operon) induced by L- arabinose (P _BAD) of A pASN plasmid carrying the asnB gene of E. coli B (BL21) under control was constructed (Figure 1A). The GlmS ⁺ p vector is a balanced lethal host vector system and is dependent on the phenotype of the GlmS ^- mutation. If the mutation is not complemented by a plasmid that carries the glmS gene, lysis occurs when it grows in the absence of N-acetyl-Dglucosamine (GlcNac).

Example 2: Salmonella strains

The attenuated S. typhimurium strain (14028s) (patent application no. 2014-0002258) used in the present invention is deficient in ppGpp synthesis, has a mutation of the glmS gene (ΔppGpp S. typhimurium ), pASN Lt; / RTI &gt; plasmid. The Salmonella strains were grown in LB medium (Difco Laboratories, USA) containing 1% NaCl added with (+) or without (-) L-arabinose (0.2%) and grown in solid support medium % -bacto agar was included. In addition, all cultures were supplemented with 100 μg / ml of antibiotics, 50 μg / ml of kanamycin, and 10 μg / ml of chloramphenicol. The supernatant and bacterial pellet fractions were separated by centrifugation (5000 xg, 5 min). L-ASNase was isolated from each fraction by Western blotting using an antibody specific for L-ASNase Respectively.

As a result, it was confirmed that L-ASNase was induced by addition of L-arabinose and that a substantial portion of L-ASNase was secreted from E. coli (FIG. 1B).

Example 3: western blot

The mammalian cell lines and tumor tissues used in the present invention were inoculated into RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium chloride) containing a protease inhibitor mixture (Roche Applied Science) deoxycholate and 0.1% SDS). For Western blot analysis, the protein samples were boiled for 5 minutes and then separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences, USA). The membranes were blocked with 5% defatted milk powder and incubated with rabbit anti-L-ASNase antibody (1: 1000; Abcam), rabbit anti -annexinV antibody (1: 2000, Abcam) or mouse anti- And the reaction was allowed to proceed overnight at 4 ° C. The membrane was reacted with anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Sigma-Aldrich, UK) for 1 hour and the bound protein was visualized by ECL (Amersham Biosciences).

Example 3: Cell cytotoxicity analysis

3-1: Cell culture

Cell lines of MC38 (mouse colorectal cancer cells), 4T-1 (mouse breast cancer cells) and CT26 (mouse colorectal cancer cells) used in the present invention were cultured in Dulbecco's modified eagle medium (DMEM) containing high glucose concentration and HEK293 cells were cultured in EME culture medium AsPC-1, a human pancreatic cancer cell, and Jukat T, a leukemic cell, were grown in RPMI1640 medium containing 10% FBS and 1% penicillin-streptomycin.

3-2: MTT analysis

MTT cytotoxicity analysis of the proliferated cells was performed as follows. Cells were plated on 96-well culture plates in 100 μL of culture medium ( ³ × 10 ³ per well) and cultured overnight at 37 ° C. and 5% CO _2. The supernatant was removed and replaced with fresh culture medium. Then, it sikyeotgo holding the mother plasmid GlmS ⁺ p or pASN and exposed for 72 hours to the concentrated liquid (200 ㎍ / ml) of Salmonella culture supernatant that was analyzed for the degree of apoptotic cells by MTT assay. At this time, clinical L-ASNase (Leunase ^® , 5 units / ml, Kyowa Hakko Kirin, Japan) was used as a control. In addition, Jukat T cells sensitive to L-ASNase were used as positive control and HEK293 cells insensitive to L-ASNase were used as negative control.

As a result, all cell lines except HEK293 cells showed various grades of susceptibility (60-80%) to bacterial culture supernatant containing L-ASNase and clinical L-ASNase. Conversely, when exposed to the culture supernatant of Salmonella bearing GlmS ⁺ p , no cytotoxicity was observed. As a result, it was confirmed that L-ASNase specificity of cytotoxicity observed upon exposure to the culture supernatant of salmonella having pASN (FIG. 2) was confirmed.

Example 4: Apoptotic cell death

4-1: L-ASNase induced cell death

In order to investigate the mechanism of bacterial L-ASNase induced tumor cell death according to one embodiment of the present invention, the amount of annexinV related to apoptotic cell death was measured. Total protein was extracted from the cells exposed to the bacterial culture supernatant (0.1 mg) containing L-ASNase for 36 hours, and then subjected to electrophoresis on 10% SDS-PAGE gel, followed by the use of annexinV- Western blot analysis was performed.

As a result, it was confirmed that the level of annexinV was markedly increased in all the tumor cells tested (Fig. 3A).

4-2: Immunofluorescence

For immunofluorescence, the tumor cells were grown on sterile glass cover slips, blocked with 4% paraformaldehyde and blocked in PBS supplemented with 0.1% fetal bovine serum albumin. The cells were then stained with rabbit anti-AnnexinV antibody (1: 200, Abcam) and Alexa 488-conjugated secondary antibody (1: 500, Invitrogen). The images were stained with Texas Red-X phalloidin (1: 500, Invitrogen) and DAPI / antifade (1: 200, Invitrogen) and images were taken using a Bio-Rad confocal microscope (Bio-Rad laboratories, Hercules, CA, USA) Respectively.

As a result, it was confirmed that a considerable amount of annexinV was accumulated on the cell surface due to the bacterial supernatant containing L-ASNase. Thus, it was confirmed that the expression of L-ASNase by the Salmonella strain in the cell culture medium depleted asparagine and caused tumor cell death (FIG. 3B).

Example 5: Expression and secretion of L-ASNase in tumor

5-1: Animal model

The 5 to 6 week old male mice (20 to 30 g body weight) used in the present invention were purchased from Samtako Company (Korea) and all animal care, experiment and euthanasia were performed according to the protocol approved by Chonnam National University Animal Research Committee Respectively. Tumor implantation mice were injected subcutaneously in vitro cultured tumor cells suspended in 100 and mouse ㎕ PBS collected from the right thigh (4T-1 and CT26 is 1 x 10 ⁶ cells, ASPC1 is 1 x 10 ⁷ cells). The tumor volume (mm ³ ) was measured using mm, L was the length, W was the width, and H was the formula (L x H x W) / 2 representing the height of the tumor.

5-2: Salmonella injection

MC38 tumor transplanted mice were injected with Salmonella strains transformed into pASN plasmid. Specifically, the proliferated MC38 tumor cells (1 × 10 ⁶ ) were suspended in 100 μl of PBS, subcutaneously injected into the right thigh of C57BL / 6 mouse, and when the tumor volume was ~120 mm ³ , the pASN plasmid Salmonella strains (ΔppGpp S. typhimurium, 4.5 × 10 ⁷ ) were suspended in 100 μl PBS and injected through the tail vein of tumor-transplanted mice using a 1 cc insulin syringe. L-ASNase was induced on day 3 ( dpi ) after intraperitoneal (IP) injection of L-arabinose (60 mg). No significant partial or total inflammatory response was observed at 3 dpi following induction of cytotoxic antitumor protein in the tumor Salmonella strain. Tumor tissues were dissected and homogenized at 8 hours after induction of L-arabinose and centrifuged (5000 xg, 5 min) and then filtered to obtain the original bacterial deficient fraction (0.45 μm, filtrate) The homogenized total cell lysate (0.1 mg) and the filtrate were electrophoresed on a 12% SDS PAGE gel.

As a result, L-ASNase-specific bands (32 kDa) were observed in L-arabinose-treated animals, and L-ASNase was found in a significant amount (about 1/4) of the amount found in total cell lysates Was found in the filtrates (Fig. 4A).

5-3: Immunohistochemical staining

The tumor tissue was fixed overnight in PBS supplemented with 4% formaldehyde at room temperature and frozen using optical optimal cutting temperature compound (OCT, Tissue-Tek). The slices were then cut to a thickness of 6 mu m using a cryostat microtome and the tissue slices were obtained on slides coated with aminopropyltriethoxysilane. The slides were washed with PBS (pH 7.4) for complete OCT removal and incubated with rabbit anti-L-ASNase antibody (1: 100, Abcam) and mouse anti-Salmonella antibody (1: 100, Abcam) , And reacted overnight at 4 ° C. Alexa 568-conjugated goat anti-mouse antibody (1: 100) was used as a secondary antibody. The tumor tissue samples were mounted on DAPI / antifade (1: 200, Invitrogen) and analyzed by confocal microcopy after addition of L-ASNase-specific antibody.

As a result, it was confirmed that the level of L-ASNase rapidly increased in mice treated with salmonella strain having pASN after administration of L-arabinose. Thus, it was confirmed that Salmonella strain was proliferated in tumor-transplanted mice and L-ASNase was expressed under the control of araBAD promoter ( P _BAD ) (FIG. 4B).

Example 6: Antitumor effect of Salmonella strains in vivo

Antitumor efficacy of Salmonella strains transformed with pASN plasmid in tumor-transplanted mice according to one embodiment of the present invention was observed.

Specifically, the tumor-transplanted mice were (a) C57BL / 6 mice transplanted with MC38 tumor cells, (B) BALB / c mice transplanted with 4T-1 tumor cells, and (C) c thymus nu- / nu-mice. The tumor transplanted mice were injected into the tail vein (n = 6) only when the size of tumor was ~ 120 mm ³ , or Salmonella strains carrying empty vectors, Salmonella strains carrying GlmS ⁺ p vectors (SL / ⁺ GlmS, for n = 6), or Salmonella strains have the pASN (SL / pASN, n = 6) were injected via the tail vein (tail vain). L-arabinose (60 mg / day / mouse) was intraperitoneally injected daily from day 4 after the treatment of transformed Salmonella strain (SL / pASN (Ara +)) and induced L-ASNase Respectively.

As a result, a strong antitumor effect of Salmonella strains having L-ASNase induced by administration of L-arabinose was observed. The effect of monotherapy treated with Salmonella alone was weaker than that of the group treated with salmonella strain pASN without administration of L-arabinose (MC38), and the antitumor effect was comparable to that of the 4T-1 test group, (PBS). At the end of the experiment, the tumor sizes of the bacterial strains carrying pASN without the L-arabinose induction, the bacterial strains carrying the parental plasmid, and the PBS-treated experimental group all showed> 1,000 mm ³ whereas the L-ASNase The mean sizes of MC38 tumors (33 days), 4T-1 tumors (35 days) and AsPC-1 tumors (45 days) treated with salmonella strains expressing the Salmonella strains were 463.7, 367.4 and 331 mm ³ , respectively. In addition, survival was significantly delayed in the experimental group treated with the bacterial strain expressing L-ASNase compared to other experimental groups (P <0.001) (middle panel). Thus, it was confirmed that L-ASNase expressed and secreted from Salmonella strains was effective in inhibiting tumor proliferation in tumor-transplanted mice (FIG. 5).

Example 7 Expression of Mutant L-ASNase and Cytotoxic Activity

Since L-ASNase is expressed and secreted by Salmonella strains, it is not possible to confirm the dose-dependent effect using the conventional method, so mutant L-ASNase deletion using enzyme activity defective methods. First, two amino acid substitutions mutants with 45% and 3% activity, respectively, were selected compared to wild-type proteins bearing Gly at the Asn ²⁴ or Asp ¹²⁴ , N24G and D124G alternative mutant positions, respectively. The pASN plasmid carrying the mutation in the asnB gene was prepared and named pASN _N24G and pASN _D124G . Inactivated Salmonella strains transformed into the above plasmids were proliferated in L-arabinose-added (-) or no-added (+) LB medium, and the bacterial culture was classified into bacterial pellets and culture, and L-ASNase expression was confirmed by Western blot .

As a result, it was confirmed that the wild-type enzyme and mutant L-ASNase were induced by L-arabinose and secreted in culture medium. As a result of analysis of enzyme activity of the mutant L-ASNase-containing bacterial supernatant, mutant L-ASNase Showed about 50% and less than 10% activity, respectively, as compared to the wild type control. MTT analysis using cultured cancer cells (MC38, 4T-1 and AsPC-1) revealed that there was a significant correlation between L-ASNase activity and cytotoxicity (10.90% surviving cells) (Fig. 6).

Example 8: Antitumor effect of mutated L-ASNase expressed in tumor

To test the antitumor effect of mutant L-ASNases in vivo , Salmonella strains (1 x 10 ⁷ CFU), pASN _N24G or pASN _D124G carrying _pASN were cultured in MC38- _transfected C57BL / 6 mice (n = 5) and intraperitoneal (IP) injection of L-arabinose at 3 dpi . Tumors were extracted at 3 dpi to quantify tumor size and measure survival.

As a result, it was confirmed that there was a direct correlation between activity of L-ASNases, tumor size and prolongation of viability (FIG. 7).

Example 9: Distribution of L-ASNase in blood and tumor tissue

In order to observe the distribution of L-ASNases expressed and secreted in vivo by Salmonella Enteritidis, MC38 tumor-grafted mice were treated with Salmonella strain (SKS1002 / pASN) having Leunase ^® (400 KU) or pASN and serum And tumor tissue were analyzed. L-ASNase was induced by L-arabinose alone at 4 days after bacterial injection (t = 0). Total serum and tumor tissue (100 μg) were collected after administration of Leunase ^® or after administration of L-arabinose to Salmonella-treated mice and quantified by Western blot analysis.

As a result, Leunase ^® (400 KU) the treated experimental group, a significant amount of L-ASNases was observed thereafter decreased with the lapse of time was not observed in tumor tissue (Fig. 8A) in the serum of the initial time (4 hours). In contrast, mice treated with salmonella expressing L-ASNase were only observed in tumor tissues and observed to increase rapidly up to 48 hours over time (FIG. 8B). In addition, the tumor tissue of the intravenous injection of Leunase ^® into the MC38 tumor-transplanted mouse did not decrease, and the survival time of the experimental group treated with high concentration (400 KU) was reduced (Fig. 9).

Example 10: Leunase ^® Antitumor effect after intratumoral injection

The anti - tumor effect of the clinical Leunase ^® was observed after tumor implantation in tumor - transplanted mice. Specifically, Leunase ^® was directly injected into MC38 tumors of C57BL / 6 mice (n = 5) for 3 days at a concentration of 4 KU and 40 KU. On the fourth day of experiment, the animals were sacrificed and tumor size was measured.

Direct injection of Leunase® into the tumor resulted in a significant reduction in tumor size (Leunase ^® 40 KU vs. control, difference = 336 mm ³ , 95% CI = 220 at 451, P = 0.008; Leunase ^® 4KU vs. The control group, difference = 607 mm ³ , 95% CI = 532 to 681, P = 0.008) increased in survival. Thus, it was confirmed that L-ASNase can be used as a therapeutic agent for treating solid malignant tumors if it can be delivered to the tumor site by a carrier such as Salmonella strains and expressed in a sufficient amount of the tumor (Fig. 10).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Solid cancer agent using bacteria <130> PD14-1349 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> L-ASN1 sense primer <400> 1 gggaattcat ggagtttttc aaaaag 26 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> L-ASN2 antisense primer <400> 2 gggtctagat tagtactgat tgaagatctg ctg 33 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <220> <222> P1 sense primer <400> 3 gactccgcaa ccaaatctgg ctacacagtg ggtaaag 37 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> P2 antisense primer <400> 4 acgtctatga gcgcaggcgg tccattcaac ctgt 34

Claims (7)

Wherein said L-asparaginase is selectively expressed at a tumor site, wherein said L-asparaginase is transformed with a gene construct comprising a polynucleotide encoding L-aspartase (L-ASNase) operably linked to a promoter, Strain. The method according to claim 1,
Wherein said Salmonella strain is Salmonella typhimurium , Salmonella choleraesuis or Salmonella enteritidis . The method according to claim 1,
Wherein the promoter is an inducible promoter. The method of claim 3,
The inducible promoter may be selected from the group consisting of tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL? Promoter, pR? Promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter , the pepT promoter, the sulA promoter, the pol11 (dinA) promoter, the ruv promoter, the uvrA promoter, the uvrB promoter, the uvrD promoter, the umuDC promoter, the lexA promoter, the cea promoter, the caa promoter, the recN promoter, the pagC promoter, the hip promoter, The attenuated Salmonella strain, which is the pflE promoter. The method according to claim 1,
The attenuation is aroA , aroC , aroD , aroE , Rpur , htrA , ompR , ompF , ompC : 449-452), galE , cya , crp , cyp , phoP , phoQ , rfaY , dksA , hupA , sipC, clpB , clpP , one selected from clpX, pab, nadA, pncB, the group consisting of pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA and spoA or more Attenuated Salmonella strains, achieved by loss of function of the gene. A therapeutic agent for solid tumor cancer, comprising the attenuated Salmonella strain according to any one of claims 1 to 5 as an active ingredient. The method according to claim 6,
Wherein the solid cancer is any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer.

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