KR101685034B1 - Solid cancer tissue targeting bacterium and use thereof - Google Patents

Abstract

The present invention relates to cancer tissue targeting bacteria, and more particularly to solid cancer tissue targeting bacteria and uses thereof.

Description

Solid cancer tissue targeting bacteria and uses thereof Solid cancer tissue targeting bacterium and use thereof

The present invention relates to cancer tissue targeting bacteria, and more particularly to solid cancer tissue targeting bacteria and uses thereof.

Most cancers are treated primarily through one of three treatments: surgical surgery, radiation therapy, and chemotherapy, or a combination of these. Surgical operations that remove most diseased tissue are effective at removing tumors located at specific sites, such as breast, colon, and skin, but they can be used to treat tumors in some areas, such as the vertebrae, or to treat disseminated tumor diseases such as leukemia Can not be used. Chemotherapy also disrupts cell replication or cellular metabolism and is often used to treat cancers of the breast, lungs, and testes. The side effects of systemic chemotherapy used to treat tumor diseases are the most problematic . Side effects caused by chemotherapy can have a significant impact on the patient's life and can dramatically change the patient's compliance with treatment. In addition, since conventional anticancer drugs frequently kill normal cells, it is urgent to develop a tumor-targeting therapy for effective cancer therapy that does not act on normal cells but can act only on cancer cells.

The present inventors have developed a technique for selectively delivering a drug to a bacterial and infarct tissue specifically targeted against a infarct tissue or selectively infarcted tissue using the attenuated Salmonella strain (Korean Patent Publication No. 2012-0030391 number). In addition, cytolysin A, a tumor suppressor protein, and a luminescent gene were inserted into the attenuated salmonella strain vector, and then the tumor was specifically recognized by treatment with the body, and a cancer cell targeting treatment method capable of confirming this process through molecular imaging was developed (Korean Patent Publication No. 2011-0095528). During tumor development, angiogenesis and cell growth progress rapidly in the body, resulting in incomplete angiogenesis in the tumor, slowing blood flow and reducing oxygen and nutrient supply. Therefore, the inside of the tumor is hypoxic or oxygen-deficient, and it provides an environment suitable for the growth of tuberous anaerobic bacteria such as Salmonella or E. coli. For this reason, attenuated Salmonella strains are suitably used as vectors for delivering target tumor therapeutic agents. However, the above-mentioned tumor-targeting bacteria have the ability to target and treat some types of solid cancer such as colon cancer, but anti-cancer bacteria having the target ability and therapeutic ability for other solid cancer tissues have not been developed so far.

The present invention has been made to solve the above-mentioned problems and it is an object of the present invention to provide a bacteria targeting solid cancer tissue which can effectively treat cancer. 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 therapeutic composition for treating solid tumors overexpressing integrin, which comprises a genetically engineered attenuated salmonella strain to present a peptide comprising a bacterial-derived RGD motif operably linked to a promoter on its surface.

In the above composition, the salmonella strain is Salmonella typhimurium, Salmonella choleraesuis, or may be a Salmonella enteritidis, 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, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, It can be achieved by guaB, fliD, flgK, flgL, failure of one or more genes selected from the group consisting of relA and spoA.

The composition may be a strain engineered to express a fluorescent protein or a luminescent enzyme. The fluorescent protein may be a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, or a near-infrared fluorescent protein, and is well known in the art. The luminescent enzyme may be a luciferase gene derived from prokaryotic or eukaryotic cells and may be a luciferase gene derived from bacteria or Photinus pyralis luciferase, Renilla luciferase, Metridia luciferase, Luciferase derived from the bacteria can be obtained from a variety of bacteria including Vibrio harveyi (Belas et al ., Science , 218: 791-793, 1982), Vibrio fischeri (Engebrecht et al ., Cell , 32 (3): 773-781, 1983), Photobacterium phosphoreum (Mancini et al, J. Biol Chem , 263 (28):... 14308-14314, 1988), P. leiognathi (Delong et al, Gene, 54 (2-3):. 203-210, 1987), P. luminescens (Frackman et al ., J. Bacteriol ., 172: 5767-5773, 1990) or Aliivibrio fisheri (Mel'kina et al ., Mol. Biol (Mosk.), 45 (3): 524-528, 2011).

In the above composition, the integrin may be a solid cancer which is overexpressed integrin alpha _V beta ₃ integrin. The solid cancer over-expressing intergreen may be breast cancer, melanoma, glioblastoma.

In the above composition, the promoter may be an inducible promoter, and the inducible promoter may be one or more of tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL? Promoter, pR? Promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, a trp promoter, a pBAD promoter, a Tet promoter, a trc promoter, a pepT promoter, a sulA promoter, a pol11 (dinA) promoter, a ruv promoter, an uvrA promoter, an uvrB promoter, an uvrD promoter, umuDC promoter, lexA promoter, cea promoter, caa Promoter, recN promoter, pagC promoter, hip promoter, ansB promoter, or pflE promoter.

1 is a cleavage map of a bacterial expression plasmid (pBAD-ompA-RGD) containing OmpA with an RGD peptide inserted according to an embodiment of the present invention.
FIG. 2 is a schematic view showing a structure in which an RGD-4C peptide is inserted into S. typhimurium strain (SL? PpGpp) ompA (outer membrane protein A) according to an embodiment of the present invention.
FIG. 3 is a photograph showing Western blotting results of SL? PpGpp-RGD according to an embodiment of the present invention.
4 is a graph showing growth curves of S. typhimurium strains according to an embodiment of the present invention.
FIG. 5 is a micrograph showing the targeting efficiency of MCF7 and MDA-MB-231 cells, which are breast cancer cells, by OmpA-RGD bacteria according to an embodiment of the present invention.
Figure 6 is a micrograph showing the analysis of integrin [alpha] v [beta] 3-RGD interactions in breast cancer cell MDA-MB-231 cells according to an embodiment of the present invention.
FIG. 7 is a photomicrograph showing adhesion and competition analysis of MCF7 and MDA-MB-231 cells, which are breast cancer cells, according to an embodiment of the present invention.
FIG. 8 is a micrograph showing the adhesion and competition analysis of melanoma cells M21 and M21L cells according to an embodiment of the present invention. FIG.
FIG. 9 is a graph showing the results of quantitative analysis of adhesion capacity and competition analysis according to an attenuated S. typhimurium strain of MCF7 and MDA-MB-231 cells or melanoma cells M21 and M21L cells according to an embodiment of the present invention FIG.
10 is a photograph showing an image of a tumor under the mouse according to the therapeutic effect of the transformed attenuated S. typhimurium (SL? PpGpp-RGD) in a breast cancer animal model according to an embodiment of the present invention.
Figure 11 shows an image of a tumor subcutaneously under the influence of the therapeutic effect of transformed S. typhimurium (SL? PpGpp-RGD) transformed into melanoma (MDA-MB-435) animal model according to an embodiment of the present invention This is the picture shown.
12 is a graph showing a change in size of a tumor in an animal model of breast cancer according to an embodiment of the present invention.
13 is a graph showing a change in size of a tumor in a melanoma animal model according to an embodiment of the present invention.
14 is a graph showing the survival rate of breast cancer mice according to an embodiment of the present invention.
15 is a graph showing melanoma survival rate according to an embodiment of the present invention.
16 is a bacterium impermeable bioluminescence image of a tumor subcutaneously following the therapeutic effect of the transformed attenuated S. typhimurium (SL? PpGpp-RGD) in a breast cancer animal model according to an embodiment of the present invention.
FIG. 17 is a bacterial impermeable bioluminescent image of a tumor subcutaneously following treatment of a transformed attenuated S. typhimurium (SL? PpGpp-RGD) in a melanoma animal model according to an embodiment of the present invention.
18 is a graph showing the in vivo bacterial distribution of transgenic attenuated S. typhimurium (SL? PpGpp-RGD) according to an embodiment of the present invention after injection into a glioblastoma mouse.
FIG. 19 is a graph showing the in vivo bacterial distribution of transformed attenuated S. typhimurium (SL? PpGpp-RGD) according to an embodiment of the present invention over time after injection into melanoma mice.
FIG. 20 is a graph showing the in vivo bacterial distribution of transformed attenuated S. typhimurium (SL? PpGpp-RGD) according to an embodiment of the present invention after injection into a breast cancer mouse.
FIG. 21 is a graph showing the in vivo bacterial distribution of transformed attenuated S. typhimurium (SL? PpGpp-RGD) according to an embodiment of the present invention over time after injection into melanoma mice.

Definition of Terms:

The terms used in this document are defined as follows.

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. Nat . Acad. Sci. USA 91: 489-493), diphenxin resistant 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,. 1988 , 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.

As used herein, "bacterial outer membrane protein" refers to a protein (transmembrane protein) present in the outer membrane of Gram-negative bacteria. Bacterial outer membrane proteins include OmpA (Freudl et al ., 1986, J. Biol. Chem ., 261 (24): 11355-11361), OmpC (Inokuchi et al ., 2005, Microbiol ., 147: 2981-2989) (Inokuchi et al, 2005, Microbiol , 147:.. 2981-2989), OmpG (Subbarao et al, 2006, J. Mol Biol, 360 (4):... 170-159), OmpT (Stumpe et al. , J. Bacteriol ., 1998, 170 (12): 5625-5632), OmpX (Riley et al ., 2006, Nucleic Acids Res ., 34: 1-6O) omp31 (Cassataro et al ., 2005, Infect Immun ., 73 (12): 8079-8088 ), YfiO (Onufryk et al, 2005, J. Bacteriol, 187 (13..):.. 4552-4561), OpcA (Zhu et al, 2003, J. Clin Microbiol , 41 (1): 273-278), HCP1 (Zhou et al ., 2012, Infect Immun ., 80 (3): 1243-1251), PulD (d'Enfert et al ., 1989, J. Biol. Chem ., 264 (29): 17462-17468) and TolC (Murholm et al ., PLos One , 2009, 4 (12): e8458). Among them, phorin- As a transmembrane protein with a barrel structure, It has an extracellular loop.

As used herein, the "RGD motif" is the amino acid motif Arg-Gly-Asp, identified as a motif in fibronectin that mediates cell attachment. Interleukin and cell surface protein families, which serve as receptors for cell adhesion proteins. Some of the integrins are recognized as RGD motifs in their ligands, and their binding mediates both cell-substrate (cell-substratum) and cell-cell interactions.

DETAILED DESCRIPTION OF THE INVENTION [

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a therapeutic composition for treating solid tumors overexpressing integrin, which comprises a genetically engineered attenuated salmonella strain to present a peptide comprising a bacterial-derived RGD motif operably linked to a promoter on its surface.

In the above composition, the salmonella strain is Salmonella typhimurium, Salmonella choleraesuis, or may be a Salmonella enteritidis, 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, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, It can be achieved by guaB, fliD, flgK, flgL, failure of one or more genes selected from the group consisting of relA and spoA.

The composition may be a strain engineered to express a fluorescent protein or a luminescent enzyme. The fluorescent protein may be a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, or a near-infrared fluorescent protein, and is well known in the art. The luminescent enzyme may be a luciferase gene derived from prokaryotic or eukaryotic cells and may be a luciferase gene derived from bacteria or Photinus pyralis luciferase, Renilla luciferase, Metridia luciferase, Luciferase derived from the bacteria can be obtained from a variety of bacteria including Vibrio harveyi (Belas et al ., Science , 218: 791-793, 1982), Vibrio fischeri (Engebrecht et al ., Cell , 32 (3): 773-781, 1983), Photobacterium phosphoreum (Mancini et al, J. Biol Chem , 263 (28):... 14308-14314, 1988), P. leiognathi (Delong et al, Gene, 54 (2-3):. 203-210, 1987), P. luminescens (Frackman et al ., J. Bacteriol ., 172: 5767-5773, 1990) or Aliivibrio fisheri (Mel'kina et al ., Mol. Biol (Mosk.), 45 (3): 524-528, 2011).

In the above composition, the integrin may be a solid cancer which is overexpressed integrin alpha _V beta ₃ integrin. The solid cancer over-expressing intergreen may be breast cancer, melanoma, or glioblastoma.

The anticancer protein may be a protein toxin, an antibody specific for cancer antigen, or a fragment of the antibody, or a tumor suppressor gene. The protein toxin may be selected from the group consisting of botulinum toxin, Tetanus toxin, Shiga toxin, Diphtheria toxin, DT, ricin, Pseudomonas exotoxin, PE ), Cytolysin A (ClyA), and r-Gelonin. The tumor suppressor gene is a gene that inhibits tumor development. Typically, the tumor suppressor gene is VHL (von Hippel-Lindau), APC (Adenomatous polyposis coli) CD95 (cluster of differentiation 95), ST5 (Suppression of tumorigenicity 5), YPEL3 (Yippee like 3), ST7 (Suppression of tumorigenicity 7) and ST14 (Suppression of tumorigenicity 14).

In the above composition, the promoter may be an inducible promoter, and the inducible promoter may be one or more of tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL? Promoter, pR? Promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, a trp promoter, a pBAD promoter, a Tet promoter, a trc promoter, a pepT promoter, a sulA promoter, a pol11 (dinA) promoter, a ruv promoter, an uvrA promoter, an uvrB promoter, an uvrD promoter, umuDC promoter, lexA promoter, cea promoter, caa Promoter, recN promoter, pagC promoter, hip promoter, ansB promoter, or pflE promoter.

The composition of the present invention is preferably administered parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, or the like.

Suitable dosages of the compositions of the present invention will vary depending on factors such as the formulation method, the manner of administration, the age, weight, sex, pathology, food, time of administration, route of administration, excretion rate and responsiveness of the patient, A skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the invention, a suitable daily dosage is 0.0001-100 mg / kg body weight. The administration may be carried out once a day or divided into several doses.

The 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 those having ordinary skill in the art to which the present invention belongs. 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.

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

Example 1 Construction of ompA-RGD Expression

1-1: The bacterial strain used in the present invention

In the present invention, the attenuated S. typhimurium strain (ΔppGpp) SHJ2037 lacking the ability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) by deletion of the relA gene and the spoT gene is deposited under the accession number KCTC 10787BP, We used an attenuated strain of S. typhimurium (SLΔppGpp), SHJ2037 (relA :: cat, spoT :: kn) deposited at the Myung Bank (Song et al., The Journal of Biological Chemistry, 279: 34183-341902, 2004).

1-2: Preparation of pBAD-ompA-RGD plasmid

In the present invention, a recombinant OmpA (RGD-OmpA) in which an RGD-4C peptide (ACDCRGDCFCG-SEQ ID NO: 3) is inserted into a third outer loop of the outer membrane protein OmpA was designed (RGD-OmpA) to prepare a polynucleotide encoding the recombinant protein, The fusion gene was constructed by replacing RLuc8 with pBAD-RLuc8 plasmid to construct pBAD-OmpA-RGD plasmid (FIGS. 1 and 2). The pBAD-OmpA-RGD plasmid was transformed into the attenuated S. typhimurium ΔppGpp strain (SLΔppGpp) by electroporation (SLΔppGpp-RGD). Specifically, the ompA gene was first amplified with the following primers using S. typhimurium genomic DNA as a template:

5'-CCATGGCAATGAAAAAGACAGCTATCGCGATTGCAGTG-3 '(SEQ ID NO: 1) and

5'-GTTTAAACTTAAGCCTGCGGCTGAGTTACAAC-3 '(SEQ ID NO: 2).

Then, to construct a plasmid in which RGD was inserted into OmpA, Eco RI and Xho I restriction enzyme recognition sites were inserted in the third loop portion of the ompA gene using a polymerase chain reaction. Then, RGD-4C synthetic oligomer (ACDCRGDCFCG; RGD sequence, SEQ ID NO: 3) was digested with Eco RI and Xho I and inserted. The synthetic oligomer AAA-4C (ACDCAAACFCG, SEQ ID NO: 4), which is scrambled peptides, was used as a control for RGD-4C. The polymerase chain reaction was pre-treated at 94 ° C for 5 minutes, Sec and 72 [deg.] C for 1 min and 30 [deg.] C for 10 min. The recombinant DNA was digested with restriction enzymes Nco I and Pme I and inserted into the pBAD-pelB-RLuc8 (pBP-Rluc8) plasmid previously described in the present invention in place of the RLuc8 gene to obtain pBAD-OmpA-RGD plasmid (Loening AM et al., Protein engineering, design & selection: PEDS, 19: 391-400, 2006) (Figure 1). To prepare pBAD-OmpA and pBAD-OmpA-AAA plasmids for use as a control group of the plasmid, Rluc8 was removed from pBAD-Rluc8 using restriction enzymes Nco I and Pme I and the ompA gene and ompA-AAA were inserted . Bacterial concentration was used at 3 x 10 ⁷ cfu / mouse in 100 μl of PBS.

1-3: Western blotting

Expression of RGD-OmpA in the attenuated S. typhimurium strain (SL? PpGpp) was confirmed by Western blotting, and the reaction conditions were as follows. The bacterial pellet was electrophoresed on a 12% linear gradient-SDS-gel and then transferred to a nitrocellulose membrane (BioRad, USA). The membrane was incubated overnight at 4 ° C with anti-MOMP monoclonal antibody (1: 2000 dilution), washed three times with phosphate buffered saline, and incubated with HRP-conjugated anti-mouse secondary antibody (1: 2,000 dilution, Amersham, UK) The reaction was carried out at room temperature for 2 hours and then washed three times with phosphoric acid buffer solution. Then, immunologically active proteins were detected using luminol reagent (SantaCruz Biotech, USA). As a result, the expression of OmpA of 37 kD was confirmed using an anti-MOMP antibody as shown in Fig. Furthermore, it was confirmed that the OmpA-RGD fusion protein after L-arabinose induction was successfully overexpressed in the upper part of the intrinsic OmpA protein (37-kd), indicating that OmpA-RGD protein overexpression was specifically induced.

1-4: Growth curve of bacteria

In the present invention, the growth of wild-type S. typhimurium (SL WT), attenuated S. typhimurium ΔppGpp strain (SLΔppGpp) and RGD-OmpA expression-induced transformed attenuated S. typhimurium ΔppGpp strain (SLΔppGpp-RGD) were compared. To induce the pBAD system to the cultured bacteria, 0.2% L-arabinose was added to the culture (2 hours after the start of culture) to the mid-log phase, and every 1 or 1.5 hours for 10 hours Absorbance was measured at 600 nm. As a result, it was confirmed that the growth of SL? PpGpp-RGD was not affected by RGD-OmpA induction through the growth curve as shown in Fig.

Example 2: Targeting of MDA-MB-231 cells by SL? PpGpp-RGD

2-1: Cell culture

M21 and M21L (α _V- negative) human melanoma carcinoma cells (provided by HaSo Choi Choi, Beth Israel Deaconess Medical Center, Harvard Medical School) were treated with 10% fetal bovine serum and 1% penicillin-streptomycin Were cultured in RPMI 1640 medium (GIBCO, USA) at 5% CO _{2 and} 37 ° C. MDA-MB-231 and MCF7 human breast cancer cells were purchased from HTB-26, HTB-22, ATCC, USA, and the MDA-MB-231 and MCF7 human breast cancer cells were treated with 10% fetal bovine serum and 1 And cultured in MEM medium (Hyclone, USA) containing 5% penicillin-streptomycin at 5% CO _{2 and} 37 ° C. Cells were resuspended in 6-well plates (3 × 10 ⁵ cells / well) or cell culture plates (1 × 10 ⁶ cells / dish) to seed cells. The medium was replaced every 80% of the cells (approximately every 2-3 days).

2-2: Infection of tumor cells

In the present invention, human tumor cells are seeded in 6 wells with a cover slip for microscopic observation. After induction with L-arabinose for 4 hours for expression of RGD-ompA in the transformed attenuated S. typhimurium strain (SLΔppGpp-RGD), the bacteria were collected and resuspended in phosphate buffer (pH 7.4) . The collected bacteria were again washed twice with serum-free medium and diluted to 3 × 10 ⁸ / mL with serum-free medium. SLΔppGpp-RGD infection of tumor cells was then performed at a multiplicity of infection (MOI) of 100: 1 for 2 hours. Bacteria diluted in serum-free medium were added at 3 × 10 ⁷ / well to infect with a MOI of 1: 100, 5% CO ₂ , And cultured at 37 占 폚 for 2 hours in a wet state.

2-3: Targeting efficiency of tumor cells by RGD-OmpA expression bacteria by immunofluorescence staining

After bacterial infection, the cells were washed three times with phosphate buffer solution to remove unbound bacteria, and the infected tumor cells were then subjected to the following immunofluorescent staining. The cells were first fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. The fixed cells were blocked with TBS-T mixed solution containing 0.1% Triton X-100 (Sigma-Aldrich, USA) and 5% BSA at room temperature for 1 hour. Then, the nucleus (blue) of the cancer tissue was treated with 5 / / ml of phalloidin-555 (1: 200 dilution, Molecular Probes, USA) using 1 쨉 g / ml of DAPI (1: 1000 dilution, Molecular Probes, USA) ) Were used to stain the cells. BAS blocking cells were stained overnight at 4 ° C with anti-α _V β ₃ primary antibody (1: 100 dilution; ab7166, Abcam, USA). The cells were washed three times with PBS-T and reacted with a secondary antibody conjugated with Alexa Fluor 488 (1: 500, Invitrogen, USA) for 2 hours at room temperature. Pro-long ^ㄾ Gold antifade (P36930, Invitrogen, USA) reagent was added thereto. The stained cells were subsequently washed with PBS, sealed on glass slides, and analyzed for stained tissue using a FV-1000 confocal optical microscope (Fluoview-1000 laser scanning confocal microscope, Olympus, Japan). A 20-slice synthetic image was obtained by moving 0.5 [mu] m on the z-axis for each sample and combined with the FV10-ASW 2.0 viewer. The experiment was performed three times independently (Fig. 5). Blue in Figure 5 is a nucleus stained by DAPI, red is the integrin α _V β _3, green visualized by Alexa Fluor 488 indicates that the dye using the Salmonella antibody, the right panel is a three image overlay . As a result, as shown in Fig. 5, the integrin [alpha] _{v [} beta] ₃ -positive MDA-MB-231 cells infected by SLΔppGpp-RGD showed stronger fluorescence signals than MCF7 cells. Previously, MDA-MB-231 cells were known to overexpress integrin [alpha] _{v [} beta] ₃ in human breast cancer cells (Wang H et al., 2008, Mol. Cancer Ther. , 7: 1044-1053) _V ? ₃ is deficient. FIG. 6 shows the integrin? _V ? ₃ -RGD interaction in MDA-MB-231 cells, confirming the presence of SL? PpGpp-RGD at the same position as integrin? _V ? ₃ on the surface of MDA-MB-231 cells. These results support that the transgenic attenuated S. typhimurium (SLΔppGpp-RGD) can target cells expressing the integrin α _V β ₃ integrin.

Example 3: Binding efficiency of RGD-expressing bacteria by αVβ3-positive cells

The function of RGD presented on the bacterial surface was further investigated. The example was carried out the binding assay (binding assay) The RGD bacteria to determine the adhesion level not to stick well to the α _V β ₃ expressing cells. First, breast cancer cells MCF7 and MDA-MB-231 or melanoma cells M21 and M21L were incubated with SLΔppGpp, SLΔppGpp-AAA or SLΔppGpp-RGD at 37 ° C. for 2 hours. Fluorescence images were obtained by fluorescence staining (Figs. 7 and 8). At this time, the SLΔppGpp-RGD strain was cultured in the same manner as in Example 2, and OmpA-RGD expression was induced by L-arabinose. Instead of RGD, SLΔppGpp and SLΔppGpp-AAA were cultured as controls and AAA-OmpA expression was also induced by L-arabinose. In Figures 7 and 8, the blue color indicates the nucleus stained with DAPI, the red indicates the intracellular stain stained with phalloidin, the green stained with Salmonella antibody, and the right panel shows overlay of the three images . As a result, as shown in Figs. 7 and 8, in breast cancer cell MCF7 and melanoma cell M21, the relative low binding efficiency of SL? PpGpp-RGD in relation to SL? PpGpp-AAA and SL? PpGpp was shown, whereas breast cancer cells MDA-MB- In the cell M21L, the binding efficiency was significantly increased after incubation with SL? PpGpp-RGD as compared with after culturing with SL? PpGpp and SL? PpGpp-AAA.

Next, the inventors counted the number of bacteria attached per cell in order to evaluate the degree of attachment of the cancer cells to the bacteria. Specifically, the coefficient indicates the average number of bacteria attached to one cell at an MOI of 100: 1 after tumor cell infections of strain SL? PpGpp, SL? PpGpp-AAA or SL? PpGpp-AAA at a magnification of 100 × magnification in a 320 μm × 240 μm area And counting the number of bacteria attached. The exact total cell count was calculated based on the total size of the cover slip and the quantification area, repeated three times for each condition, and at least three separate experiments were performed. As a result, the present inventors conducted a competition analysis to investigate whether the increase in the adhesion of the bacteria to integrin α _V β ₃ positive cancer cells was due to the interaction between the integrin α _V β ₃ and the RGD peptide. Specifically, 1 mM of the free synthetic RGD peptide (amino acid sequence ACDCRGDCFCG, SEQ ID NO: 1) was first treated with the four cancer cells and treated with SLΔppGpp-RGD after 2 hours (FIG. As a result, as shown in Fig. 9, binding of SLΔppGpp-RGD to breast cancer cells MDA-MB-231 and melanoma cell M21L was inhibited in pretreatment of the free synthetic RGD peptide. This supports the preferential homing of SL? PpGpp-RGD to breast cancer cells MDA-MB-231 and melanoma cell M21L by RGD.

Example 4: Production of animal model for cancer

5-6 week old male nude (nu / nu) mice (20-30 g, Central laboratory animals, Korea) were used for the experiments. All animal breeding, experiments and euthanasia were conducted in accordance with the protocol approved by the Animal Research Association of Chonnam National University and the Guidance on the Rehabilitation and Use of Laboratory Animals (published by the National Institute of Health) (Announcement 85-23, Rev. 1985). 1x10 ⁷ of the breast cancer cells (MDA-MB-231) cultured in Example 1 was harvested, resuspended in 100 μl of PBS, and injected into the right thigh of the mouse. (Mm ³ ) was measured using the equation (length x height x width) / 2.

Experimental Example 1: Anticancer effect analysis

1-1: Cancer volume change

MDA-MB-231 cells (10 ⁷ cells / 100 μl per mouse) and MDA-MB-435 cells (10 ⁷ cells / 100 μl) was subcutaneously injected. All bacterial strains were transformed with the bacterial luciferase (Lux) operon using p22 bacterial phage (SL? PpGpp-lux, and SL? PpGpp-RGDlux). After the volume of the tumor reached 150 mm ³ , tumor-bearing mice were administered SLΔppGpp-lux and SLΔppGpp-RGDlux. Then, changes in the size of breast and melanoma tumors were visually confirmed over time (FIGS. 10 and 11) and the volume of tumor tissues extracted from the cancer model mice was measured (FIGS. 12 and 13). As a result, it was confirmed that the tumor growth was significantly reduced in the mice treated with the genetically modified attenuated Salmonella with RGD peptide on the surface, and these bacteria were selectively accumulated in the tumor tissues rather than other normal tissues 10 to 15). 14 and 15 are graphs showing the mouse survival rate.

1-2: Reading bioluminescence images

For bioluminescent image acquisition of the bacteria, the transformed SL? PpGpp-RGD was injected into tumor-transplanted mice and the bacterial in vivo luminescence was read. Anesthetized laboratory animals were placed in a non-illuminated chamber of an IVIS imaging system (CaliperLife Sciences) equipped with a cooled imaging device (CCD) camera. Bioluminescent images were acquired using the IVIS imaging system 100 according to the manufacturer's instructions and data analysis was performed using Living Image 2.50.1 software (Caliper Life Sciences). As a result, photons emitted from the bacteria expressing luciferase were collected and integrated for 1 minute. Pseudo-color images representing the number of photons The images of the mice were superimposed using the Living Image 2.50.1 software. Areas of manually selected intensity based on signal strength were recorded with maximum radiance in the region of each intensity. As a result, it was confirmed that the tumor scale of SLΔppGpp-RGDlux was remarkable in MDA-MB-231 and MDA-MB-435-bearing mice compared with SLΔppGpp-lux through bioluminescence images (FIGS. 16 and 17). In addition, the end tumors of SL? PpGpp-RGDlux treatment were no longer detected in 3 out of 9 mice bearing MDA-MB-231. Photon fluxes in tumor tissues were approximately 1000-fold higher in animals treated with SLΔppGpp-RGDlux compared to SLΔppGpp-lux. These results suggest that treatment with surface modified S. typhimurium is due to tumor growth inhibition. It also indicates that the targeting efficiency of the bacterial mediation therapy is enhanced in the MDA-MB-231 tumor model.

1-3: Biodistribution study in mice after bacterial infection

In accordance with one embodiment of the present invention, the following tumor tissues were homogenized and then plated on an LB agar medium containing ampicillin (50 μg ml ^-1 ), cultured overnight at 37 ° C., and the resulting colonies were counted to obtain CFU / mg Tissues were calculated (n = 5 for each group, Fig. 18 to Fig. 21). Figure 18 shows the results for glioblastoma (U87MG), Figure 19 for melanoma (M21), Figure 20 for breast cancer cells (MDA-MB-231), and Figure 21 for melanoma (MDA-MB-435) cells . At this time, in order to compare the degree of targeting of bacteria to other tissues other than tumor tissues, spleen and liver tissues were homogenized as above to count bacteria. As a result, the results of FIGS. 18 and 19 show that the glioblastoma (U87MG) and melanoma (M21) are well targeted but do not show any decrease in tumor growth. On the other hand, as shown in Figs. 20 and 21, in the mice treated with the genetically engineered attenuated salmonella with the RGD peptide presented on the surface, the tumor growth was markedly reduced as compared with the other groups, and these bacteria were more selective As shown in Fig.

The two-tailed t-test determined statistically significant differences in primary tumor growth between the control and treatment groups. P <0.05 was considered significant for all analyzes. Survival analysis was performed using the Kaplan-Meier curve and log-rank test. All data are expressed as mean SD.

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 exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. I will understand. 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 tissue targeting bacterium and use thereof <130> PD13-1031 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Salmonella ompA forward <400> 1 ccatggcaat gaaaaagaca gctatcgcga ttgcagtg 38 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Salmonella ompA reverse <400> 2 gtttaaactt aagcctgcgg ctgagttaca ac 32 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> RGD peptide <400> 3 Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly   1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> scrambled peptide <400> 4 Ala Cys Asp Cys Ala Ala Ala Cys Phe Cys Gly   1 5 10

Claims (13)

The RGD motif consisting of SEQ ID NO: 3 of the bacteria derived operably linked to a promoter is inserted into the third external loop of OmpA containing a genetically engineered attenuated Salmonella strain such that the RGD motif is present on the surface, integrin α _V β ₃ overexpressed breast cancer. The method according to claim 1,
Wherein the Salmonella strain is Salmonella typhimurium , Salmonella choleraesuis, or Salmonella enteritidis , wherein the integrin [alpha] _{v [} beta] ₃ is overexpressed. 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 A composition for treating breast cancer, wherein the integrin [alpha] _{v [} beta] ₃ is overexpressed, which is achieved by loss of function of the gene. The method according to claim 1,
Wherein the Salmonella strain is genetically engineered to express a fluorescent protein or a luciferase, wherein the integrin [alpha] _{v [} beta] ₃ is overexpressed. 5. The method of claim 4,
Wherein said fluorescent protein is overexpressed integrin? _V ? _{3 which} is a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, or a near-infrared fluorescent protein. 5. The method of claim 4,
Wherein said luminescent enzyme is a bacterial-derived luciferase gene or a composition for the treatment of solid tumors overexpressed with Photinus pyralis luciferase, Renilla luciferase, Metridia luciferase, integrin. The method according to claim 6,
Luciferase in the bacteria is derived from Vibrio harveyi, Vibrio fischeri, Photobacterium phosphoreum , P. leiognathi, P. luminescens or Aliivibrio la Lucifer fisheri derived agent, integrin α _V β ₃ The composition for the treatment of an over-expressing breast cancer. delete delete The method according to claim 1,
Wherein said Enterococcus Salmonella strain is an additional genetically engineered strain to express an anti-cancer protein selected from the group consisting of a protein toxin, an antibody specific for cancer antigen or a fragment of said antibody, and a tumor suppressor gene, A composition for treating breast cancer in which? _V ? ₃ is overexpressed. delete The method according to claim 1,
Wherein the promoter is an inducible promoter, wherein integrin [alpha] _{v [} beta] ₃ is overexpressed. 13. The method of claim 12, wherein the inducible promoter is 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 , The Tet promoter, the 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, wherein the integrin &lt; _{RTI ID = 0.0} &gt; _V &lt; / _RTI &gt; ₃ is overexpressed as an &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; hyp promoter, ansB promoter or pflE promoter.

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