Anticancer strain secreting a streptavidin fusion protein with biotin-binding capacity, antineoplastic composition using the same, antineoplastic adjuvant and tumor imaging aid

Abstract

The present invention relates to a gene construct characterized in that an anti-sigma factor flgM gene and a gene encoding streptavidin are linked to encode a fusion protein of flgM and Stv with biotin-binding capacity, which enables the expression and secretion of a streptavidin fusion protein with biotin-binding capacity in an anticancer strain that targets tumor regions and can stimulate immune activity, as well as an antineoplastic adjuvant and a tumor imaging aid which not only itself has antineoplastic activity, but can also be used usefully for the diagnosis and treatment of tumor cells in conjunction with antineoplastic agents or contrast agents labeled with biotin that specifically reacts with streptavidin.

Description

Technical area

[0001] The present invention relates to a gene construction that enables the expression and secretion of a streptavidin fusion protein with biotin binding ability in an anticancer strain that targets tumor areas and can stimulate immune activity, as well as to an antineoplastic adjuvant and a tumor imaging aid that can be usefully used for the diagnosis and treatment of tumor cells, not only in conjunction with the antineoplastic activity itself, but also together with antineoplastic agents or contrast agents labeled with biotin that specifically reacts with streptavidin. State of the art

[0002] Cancer is a disease that ultimately threatens life by causing cells to proliferate uncontrollably and invade surrounding tissue, destroying normal cells. Because the body's ability to regulate itself declines with aging, making it more susceptible to cancer, cancer has been the undisputed number one cause of death in today's aging society since 2007, with a mortality rate more than twice that of heart disease, which ranks second.

[0003] The most effective response system for treating disease is to boost immunity, but cancer cells evade the body's immune response because they are not external invaders. Certain viruses or bacteria infect cancer cells more frequently than normal cells, and the infected bacteria can become targets of immune cells. Therefore, methods of cancer treatment have been proposed that deliberately infect the body with certain viruses or bacteria to stimulate the body's immune response and thus act against cancer cells. However, infectious bacteria such as Shigella, Vibrio cholera, and pathogenic E. coli only invade the intestinal cells and do not reach the liver and spleen, which are important organs for the immune response. In contrast, Salmonella can invade the spleen and liver through the lymph nodes and stimulate a systemic immune response. Specifically, Leschner et al. (J. Mol. Med.2010, 88, 763-773) investigated the route of infection over time after intravenously injecting fluorescent Salmonella into mice with CT26 tumors. The results showed that systemic infection via the blood of the mice occurred immediately after infection, Salmonella accumulated in the spleen and liver after 20 minutes, and Salmonella was concentrated only in the tumor tissue after 24 hours. This suggests that Salmonella can be used more effectively in cancer immunotherapy than other strains because it targets the cancer and activates local immunity after stimulating a systemic immune response in the spleen and liver.

[0004] However, Salmonella is a typical pathogen of food poisoning that can cause life-threatening sepsis through an abnormal host response when infecting tissues other than tumors. A research team at Yale University in the USA reported that genetically modified Salmonella can retain its tumor-attacking properties while eliminating its toxicity through attenuation. Injection of these attenuated Salmonella can induce immune stimulation and suppress tumors. Therefore, active studies are being conducted on transformed strains that enable attenuated Salmonella to express antineoplastic substances.However, attenuated Salmonella has low viability and reduced immunostimulatory ability, and there is concern that the attenuated strain could mutate into wild Salmonella and cause sepsis, so additional safety precautions are needed.

[0005] To compensate for the low immunostimulatory capacity of attenuated Salmonella, studies have been conducted to develop Salmonella strains that can express or inhibit additional substances useful in cancer treatment and to use them in cancer treatment. Korean Patent No. 10-0852687 discloses a Salmonella strain expressing tumor necrosis factor-alpha produced by transforming a tumor necrosis factor-alpha protein vector into an attenuated Salmonella strain, and an antineoplastic therapeutic composition containing it. Korean Patent No. 10-1750007 discloses a therapeutic agent for solid tumors using an attenuated Salmonella strain capable of selectively expressing L-asparaginase at the tumor site.Although the above-mentioned Salmonella strains have further enhanced antineoplastic activity by expressing antineoplastic substances, they still carry the risk of the attenuated strain transforming into wild Salmonella and causing sepsis. When antineoplastic substances are expressed in the body, the Salmonella strain itself may be killed by the physiological activity of the antineoplastic substance. Therefore, after targeting the tumor area, the Salmonella strain must be able to survive stably and continuously express and secrete the antineoplastic substance to ensure the anti-cancer efficacy of both the antineoplastic substance and Salmonella simultaneously.

[0006] Streptavidin has a very fast binding rate with biotin and a very strong binding force (K d = 10 -15M), which is little affected by pH, temperature, or organic solvents, and is therefore useful for the detection or purification of certain proteins. Korean Patent Application No. 10-2022-0058461 discloses that a gene encoding streptavidin can be introduced into a cancer-targeting host cell for use in cancer diagnosis. However, in this patent, the expression of streptavidin could only be increased by a specific structure of the gene construct while retaining the binding ability to biotin. This is because when the streptavidin gene was introduced alone, the expression was low, and when maltose-binding protein was additionally introduced to increase expression, the binding ability to biotin was weakened. In addition, after this gene construct, the streptavidin remained in the periplasmic space of the host cell and was not secreted.However, if streptavidin remains only inside the stem, it binds only to biotin-containing substances that have been taken up by the stem, which tends to reduce its usefulness. Especially when used as an antineoplastic adjuvant, the externally administered antineoplastic agent is not targeted to the tumor cells but to the interior of the host cell, making it difficult to achieve antineoplastic efficacy. Furthermore, the accumulation of expressed streptavidin in the host cell, since it cannot be secreted, can also affect host cell survival. Content of the inventionTechnical task

[0007] An object of the present invention is to provide a gene construct that enables the stable expression and secretion of a fusion protein with biotin binding ability to solve the problems of the prior art.

[0008] Another object of the present invention is to provide an antineoplastic recombinant strain and an antineoplastic composition containing the same, which is transformed by the above-mentioned construct, secretes a fusion protein having biotin binding ability and targets the tumor area, thereby itself exhibiting immunological antineoplastic activity against the tumor.

[0009] Another object of the present invention is to provide an antineoplastic adjuvant or a tumor imaging adjuvant that can improve antineoplastic efficacy or imaging efficacy by targeting externally administered biotin-linked antineoplastic agents or biotin-linked contrast agents for tumor imaging to the tumor area.

[0010] Another object of the present invention is to provide a method for providing information for cancer diagnosis using the above-mentioned tumor imaging adjuvant. Technical solution

[0011] To achieve the above-mentioned objectives, the present invention relates to a gene construct characterized in that an anti-sigma factor flgM gene and a gene encoding streptavidin (hereinafter abbreviated as 'Stv') are linked to encode a fusion protein of flgM and Stv (hereinafter abbreviated as 'flgM-Stv'). Specifically, the flgM-Stv expressed by the gene construct of the present invention is characterized in that it has biotin binding ability.

[0012] Unless otherwise defined in this specification, all scientific and technical terms used in this specification have the same meaning as commonly understood by one skilled in the art to which the invention belongs. Furthermore, in explaining the invention, a detailed explanation of the known art related to the invention is omitted if it might unnecessarily obscure the essential aspects of the invention.

[0013] The flgM-Stv expressed by the gene construct of the present invention can bind to biotin in secreted form or within the flgM-Stv expressing strain like streptavidin, thereby demonstrating the biological equivalence of streptavidin and flgM-Stv with respect to biotin binding ability.

[0014] In the gene construct of the present invention, the flgM gene and the gene encoding Stv may be directly linked or linked via a linker. In the following examples, SEQ ID NO: 1 or SEQ ID NO: 2 of the constructs are exemplified, but are not limited to, SEQ ID NO: 1 or SEQ ID NO: 2, which are linked via a linker encoding an amino acid sequence consisting of HIS*6-SGG (SEQ ID NO: 5) or GGSSSS-HIS*6-SGG (SEQ ID NO: 6). The following examples demonstrate that both strains expressed flgM-Stv regardless of the linker length, and the expression efficiency may vary depending on the linker length. Therefore, it is understood that further linker optimization may be performed to achieve better expression efficiency, but it is easy for one skilled in the art to modify the length or sequence of the linker accordingly.

[0015] To improve expression efficiency, a Shine-Dalgarno sequence can be placed at the 5' end of the gene construct of the present invention. It is also understood that suitable restriction enzymes can be placed at the 5' and 3' ends to introduce the gene construct of the present invention into a vector. In the following examples, a double His tag was expressed in the middle and a Flag tag at the end for easier detection of the fusion protein. However, this is not essential, and the type of tags can be varied as needed.

[0016] The present invention also relates to a recombinant expression vector containing the above-mentioned gene construct. In the present invention, a "vector" is a DNA construct operably linked to the gene construct of the present invention for expression in a suitable host, and may be a plasmid, a phage particle, or a potential genomic insert. In the description of the present invention, the terms plasmids and vectors are sometimes used interchangeably. It is understood that the expression vector can be selected and used with high efficiency depending on the sequence of the gene construct.

[0017] Furthermore, the present invention relates to an antineoplastic recombinant strain characterized by being transformed by the above-mentioned gene construct operably linked to an inducible promoter, expressing and secreting a flgM Stv with biotin binding ability, and targeting tumor cells. The strain of the present invention is known to target tumors and exhibit immunological antineoplastic activity, and any strain that is harmless to humans can be used. Examples of such strains include, but are not limited to, Escherichia coli, attenuated Salmonella, Shigella, Acinetobacter, Pseudomonas aeruginosa, Listeria, Clostridium, or intestinal bacteria (beneficial bacteria), probiotics, etc.

[0018] The recombinant strain of the present invention can be transformed using a recombinant expression vector containing the above-mentioned gene construct. The transformation method follows conventional methods and may use, for example, but is not limited to, the calcium chloride method, electroporation, etc. The recombinant strain of the present invention is characterized in that, through the action of flgM, it not only expresses flgM-Stv encoded by the gene construct of the present invention inside the cell but also secretes it outside the strain. Since flgM-Stv is secreted, it does not accumulate in the body above a certain concentration even if the strain continuously expresses flgM-Stv, so the survival of the strain is not endangered by the expressed flgM-Stv, and stable expression is possible.

[0019] Furthermore, the strain of the present invention can more widely utilize the binding of flgM-Stv and biotin by secreting flgM-Stv with biotin-binding ability. When flgM-Stv is expressed and present only inside the strain, the externally administered biotin-linked substance must be incorporated into the strain to bind to biotin. Therefore, the biotin-linked substance accumulates inside the strain and does not directly contact the tumor cells. On the other hand, when the strain targets the tumor and not only expresses flgM-Stv but also secretes it to the tumor cells, it can target the biotin-linked substance to the tumor and act directly on the tumor cells. Even if the secreted flgM-Stv partially spreads, it will be present at the highest concentration in the tumor targeted by the strain.Furthermore, it is known that avidin-like proteins are rapidly degraded in serum upon injection in vivo, so that flgM-Stv secreted by the stem and diffused to areas outside the tumor is also rapidly degraded and loses its biotin-binding ability, thus not affecting the targeting of the biotin-associated substance.

[0020] In this description, “functionally linked to an inducible promoter” means that the function of the linked gene can be switched by an inducer substance. The inducible promoter can, for example, be a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter, pepT promoter, sulA promoter, pol11(dinA) promoter, ruv promoter, uvrA promoter, uvrB promoter, uvrD promoter, umuDC promoter, lexA promoter, cea promoter, caa promoter, recN promoter, pagC promoter, hip promoter, ansB promoter or pflE promoter.

[0021] In particular, the strain of the present invention may be an attenuated Salmonella strain. Unlike other strains, Salmonella can invade the liver and spleen and stimulate a systemic immune response, thereby exhibiting superior immunological antineoplastic activity. "Attenuation" refers to the artificial attenuation of the toxicity of a live pathogen by mutating genes involved in the pathogen's essential metabolism, so that it cannot cause disease in the body and only stimulates the immune system to induce immunity.The genes responsible for Salmonella attenuation are well known in the art and can be achieved by loss of function of one or more genes 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, relA and spoT. To prevent reversion to wild type during in vivo application and to further mitigate toxicity, two or more of the above-mentioned genes can be functionally inactivated.

[0022] Specifically, when flgM-Stv was expressed in an attenuated Salmonella strain, it was observed that the flagella disappeared, resulting in inhibition or even loss of motility. This means that the safety aspect is significantly improved, as movement to other organs is restricted by the expression of flgM-Stv after targeting the tumor area. Specifically, the strain can be, but is not limited to, the S-flgM-streptavidin-pBAD18-ASD+ / BRD509 asd strain with the accession number KCTC15482BP, which was deposited on June 26, 2023, at the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology.

[0023] The present invention also relates to an antineoplastic composition containing the above-mentioned antineoplastic recombinant strain as an active ingredient. The antineoplastic recombinant strain of the present invention can be used as an antineoplastic composition because it targets the tumor area itself and exhibits immunological antineoplastic activity.

[0024] The composition of the present invention can be prepared for use as a medicament by methods known in the pharmaceutical field and can be used alone or in admixture with pharmaceutically acceptable carriers, excipients, diluents, etc.

[0025] The composition of the present invention can be formulated as an oral or parenteral dosage form. The dose of the active ingredient of the present invention can be appropriately selected depending on the absorption of the active component in the body, the dosage form, the patient's age, gender, and condition, the severity of symptoms, etc., and administration can be once daily or in several divided doses. The general dosage ranges from 0.001 mg / kg / day to 10 g / kg / day.

[0026] Furthermore, the present invention relates to an antineoplastic adjuvant characterized by containing the above-mentioned antineoplastic recombinant strain as an active ingredient and administered together with an antineoplastic agent labeled with a substance that specifically binds to Stv. Biotin may be mentioned as a substance that specifically binds to Stv, for example, but is not limited to it.

[0027] As described above, the strain of the present invention targets tumor cells and, upon induction by an inducer substance, secretes flgM-Stv with biotin-binding ability to the tumor cells. Thus, when co-administered with an antineoplastic agent in biotin-bound form, the antineoplastic agent can be targeted to the tumor cells. This minimizes the side effects of the antineoplastic agent on other organs and increases the antineoplastic activity. Furthermore, antineoplastic activity can be achieved even with a lower dose, thereby reducing the burden of side effects on patients. In this way, the recombinant strain of the present invention can be used as an antineoplastic adjuvant administered together with an antineoplastic agent in biotin-bound form.Antineoplastic agents that can be used include all types of nanoparticles, antibodies, antineoplastic proteins, or low-molecular-weight substances that have no or only a low ability to target tumor cells but exhibit antineoplastic activity and can be labeled with a substance that specifically binds to Stv. As can be seen from the tumor imaging results with dyes in the following examples, the recombinant strain of the present invention can inhibit the excretion of the antineoplastic agent by binding flgM-Stv and biotin, thus allowing the antineoplastic agent to act as a sustained-release drug.

[0028] The antineoplastic recombinant strain of the present invention can also be used as a tumor imaging adjuvant, which can improve the imaging efficiency of tumor tissue by co-administering it with a contrast agent in biotin-bound form and targeting the contrast agent to the tumor area. The contrast agent can be one or more selected from the group consisting of radioactive nuclides, fluorescent labels, enzyme labels, chemiluminescent markers, gold preparations, and magnetic preparations. Determining the imaging method according to the type of contrast agent will be easy for one skilled in the art. The tumor imaging adjuvant of the present invention not only improves the efficiency of tumor imaging but also exhibits antineoplastic activity itself, thus achieving even more beneficial effects for cancer patients.

[0029] The tumor imaging adjuvant of the present invention can be usefully used to provide information for cancer diagnosis. For example, providing information for cancer diagnosis can be achieved by a method comprising: (A) administering the tumor imaging adjuvant of the present invention to a patient suspected of having cancer or to a patient undergoing antineoplastic therapy to target tumor cells; (B) inducing expression of the flgM-streptavidin fusion protein in the recombinant strain of the tumor imaging adjuvant; (C) administering a contrast agent in biotin-bound form; and (D) detecting and imaging the contrast agent.However, the steps are not limited to this, and depending on the nature and properties of the strain and the contrast agent, for example, steps (B) and (C) may be performed simultaneously, or steps (A) and (C) may be performed simultaneously, or additional steps may be added, which is self-evident. Effects of the invention

[0030] As described above, the gene construct of the present invention enables the secretion of a fusion protein having biotin binding ability when introduced into an antineoplastic strain that targets tumor cells and has immunological antineoplastic activity.

[0031] Therefore, the antineoplastic recombinant strain containing the gene construct of the present invention can not only have antineoplastic activity itself, but also be used as an antineoplastic adjuvant or tumor imaging adjuvant, which can improve the antineoplastic efficacy and tumor imaging efficacy by co-administering with antineoplastic agents or contrast agents that do not have tumor targeting function but are linked to biotin, and targeting the antineoplastic agent or contrast agent to the tumor through the specific binding of biotin and streptavidin. Short description of the drawings Fig. Figure 1 is a drawing showing the amino acid sequence of monomeric streptavidin and the linker. Fig. Figure 2 is a drawing showing the DNA base sequence of an embodiment designed for the preparation of a plasmid for expressing the flgM-Stv fusion protein. Fig. Figure 3 is a drawing showing the DNA base sequence of another embodiment designed for the preparation of a plasmid for expressing the flgM-Stv fusion protein. Fig. Figure 4 is an electrophoresis photograph showing the quantification of flgM-Stv in the culture medium of the strain transformed with the above-mentioned plasmids. Fig. Figure 5 is a schematic diagram and a result graph of the HABA assay showing the biotin binding ability of the flgM-Stv secreted by an embodiment of the strain of the present invention. Fig. Figure 6 is a fluorescence microscope image and a quantification graph showing the results of the biotin uptake assay for one embodiment of the strain of the present invention. Fig. Figure 7 is a picture and graph showing the results of tumor imaging using an embodiment of the strain of the present invention. Fig. Figure 8 is a picture showing the inhibition of stem motility by inducing the expression of flgM-Stv. Fig. Figure 9 is a TEM image showing the morphological changes of the strain after induction of flgM-Stv expression. Best mode for carrying out the invention

[0032] The present invention will now be explained in more detail with reference to the accompanying drawings and exemplary embodiments. However, these drawings and exemplary embodiments serve only to illustrate the content and scope of the technical idea of ​​the present invention by way of example and are not to be understood as limiting or changing the technical scope of the present invention. Based on these examples, it will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of ​​the present invention. Embodiment ExamplesEmbodiment Example 1: Preparation of a plasmid for the expression of the streptavidin fusion protein

[0033] A plasmid was constructed that fused flgM protein, which is secreted through the basal body and hook system, to Stv.

[0034] First, a gene capable of expressing a fusion protein of flgM and Stv was designed. A Flag tag sequence (SEQ ID NO: 4) was added to the amino acid sequence of the monomeric Stv (SEQ ID NO: 3) for detection, and a 6-histidine linker (SEQ ID NO: 5) or a 6-histidine GS linker (SEQ ID NO: 6) was used as a linker for fusion to the flgM protein. Fig. 1 shows the connection of the linker with monomeric Stv and Flag tag.

[0035] The DNA base sequence of flgM of wild-type Salmonella (flgM anti-Sigma-28 factor FlgM [Salmonella enterica subsp. enterica serovar Typhimurium str. LT2] Gene ID: 1252690, Locus tagSTM1172 NC_003197.2 (1257036..1257341, complement)) was linked to the DNA sequence obtained by Salmonella codon optimization from the amino acid sequence of monomeric streptavidin (His-streptavidin-Flag or His-GS LINKER-streptavidin-Flag in Fig. 1). To increase the expression of the flgM gene, 14 Shine-Dalgarno sequences (SEQ ID NO: 7) were inserted upstream of the flgM gene, and for cloning, an NheI site was inserted at the 5' end and a SacI site was inserted after the stop codon of the Flag tag. Fig. 2 and Fig. Figure 3 shows the base sequences of flgM-his-streptavidin-flag tag and flgM-GS LINKER-his-streptavidin-flag tag, respectively, designed by this process. In Fig. 2 and Fig. Figure 3 shows the flgM coding sequence and the Stv coding sequence separately. Gene synthesis was commissioned from Cosmo Genetech Co., Ltd. The sequences without the restriction enzyme sites and Shine-Dalgarno sequence correspond to the genes (SEQ ID NOs: 1 and 2) encoding the fusion protein of flgM and Stv (hereinafter referred to as "flgM-Stv").

[0036] The synthesized flgM-his-streptavidin-flag tag and flgM-GS LINKER-his-streptavidin-flag tag were incubated with 10 U each of NheI (Takara) and SacI (Takara) at 37°C for 2 hours. The reaction products were electrophoresed, and DNA of approximately 760 bp was extracted using a Gel Extraction Kit (Qiagen) to obtain insert DNA for each flgM-Stv fusion protein.

[0037] The pBad18 asd plasmid (from Prof. Lim Hun-man, Chungnam National University) was incubated with 10 U each of NheI and SacI at 37°C for 2 hours, and the reaction product was purified using a PCR purification kit (Qiagen) to obtain vector DNA. The vector DNA was ligated with the two flgM-Stv inserts at 25°C for 30 minutes each (Invitron) and transformed into DH5a competent cells. The transformed cells were plated on LB amp solid medium and incubated at 37°C to select for antibiotic-resistant colonies. Six candidate colonies were selected and incubated with 10 U each of NheI and SacI at 37°C for 1 hour, and the formation of a 760 bp band was confirmed by electrophoresis. The nucleotide sequence of these candidates was provided by Cosmo Genetech Co., Ltd.analyzed to confirm the insertion of the genes with SEQ ID NO: 1 or 2 and to establish the plasmids flgM-his-streptavidin-flag tag and flgM-GS linker-his-streptavidin-flag tag for the secretion of flgM-Stv. Example 2: Preparation of an attenuated Salmonella strain secreting flgM-Stv with biotin binding ability1) Transformation of the attenuated Salmonella strain and confirmation of the secretion of flgM-Stv

[0038] An attenuated Salmonella strain was transformed with the flgM-his-streptavidin-flag tag and flgM-GS linker-his-streptavidin-flag tag plasmids prepared in Example 1, and expression of the flgM-Stv fusion protein was confirmed. An aroA aroD asd strain (from Prof. Lim Hun-man, Chungnam National University) was used as the attenuated Salmonella strain.

[0039] A single colony of each transformed strain was inoculated into LB amp liquid medium and incubated at 37°C for 12-16 hours with shaking. Afterwards, the cells were inoculated to OD in fresh LB amp liquid medium. 600 0.05 and incubated at 37°C for 2 hours with shaking. After reaching an OD 600 After a concentration of 0.4-0.6 w / w was adjusted by shaking incubation, arabinose was added to a final concentration of 0.2 w / w% and incubated at 37°C for a further 5 hours with shaking. As a control, incubation was performed without arabinose under identical conditions. The culture supernatant was separated by centrifugation at 3000 rpm and filtered through a 0.2 µm filter to completely remove the bacteria.

[0040] The amount of flgM-Stv in the cell-free supernatant was determined by Western blot. Fig. Figure 4 shows the electrophoresis photograph of the results, where lanes 1, 2 show the strain transformed with flgM-his-streptavidin-flag tag plasmid and lanes 3, 4 show the strain transformed with flgM-GS linker-his-streptavidin-flag tag plasmid, and lanes 1, 3 show the culture supernatant of the experimental group without arabinose treatment and lanes 2, 4 show the culture supernatant of the experimental group with arabinose treatment. Fig. Figure 4 shows that the fusion protein was detected in the culture supernatant of both strains after arabinose treatment, and the Salmonella strain transformed with flgM-GS linker-his-streptavidin-flag tag plasmid showed a significantly superior secretion ability for the flgM-Stv fusion protein compared to the Salmonella strain transformed with flgM-his-streptavidin-flag tag plasmid. Therefore, the strain with superior flgM-Stv secretion ability transformed with flgM-GS linker-his-streptavidin-flag tag plasmid was designated S-flgM-streptavidinpBAD18-ASD+ / BRD509 asd- and deposited at the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on June 26, 2023 (accession number KCTC15482BP).

[0041] Subsequently, the strain S-flgM-streptavidin-pBAD18-ASD+ / BRD509 asd- was used to test the efficiency of expression of the flgM-Stv fusion protein. 2) Confirmation of the biotin binding ability of flgM-Stv (HABA assay)

[0042] The biotin-binding ability of flgM-Stv was confirmed using HABA (2-(4'-HydroxyAzoBenzene)Benzoic Acid). The complex formed by the binding of HABA and streptavidin absorbs light at 500 nm. Therefore, if flgM-Stv secreted into the culture supernatant retains the binding ability of streptavidin, it should bind to HABA in solution and increase the absorbance at 500 nm. Furthermore, since biotin binds more strongly to streptavidin than HABA, with sufficient biotin concentration in the solution and if flgM-Stv retains the biotin-binding ability of streptavidin, flgM-Stv should bind to biotin and not form a complex with HABA, thus resulting in no change in absorbance at 500 nm.

[0043] Specifically, a single colony of the attenuated Salmonella strain expressing the flgM-Stv fusion protein prepared in 1) was added to 20 mL of LB amp liquid medium and cultured at 37°C for 16 hours. The culture was incubated in fresh LB amp liquid medium to OD 600 0.05 and incubated at 37°C for a further 2 hours until an OD 600of 0.4-0.6. Then, 200 µL of 20% (w / v) arabinose was added to achieve a final concentration of 0.2%, and 1 mL of culture samples were taken hourly at 37°C during cultivation. The same volume of PBS was added to the control. The culture supernatant was separated by centrifugation at 3000 rpm and filtered through a 0.2 µm filter to completely remove the bacteria. HABA reagent and 100 µL of biotin solution (1 mg / 50 mL) were added to 100 µL of the cell-free culture supernatant and mixed for 1 hour at 600 rpm using a vortex mixer. 100 µL of PBS was added to the control instead of biotin and mixed under identical conditions. After 1 hour, the OD 500 -value measured.

[0044] Fig. Figure 5 shows the schematic diagram and result graph of the HABA assay to confirm the biotin-binding ability of flgM-Stv. For the samples without biotin addition, there was no change in the absorbance at 500 nm until 2 hours after arabinose induction. After that, the absorbance gradually increased, indicating complex formation with HABA. This means, first, that flgM-Stv, like streptavidin, has the ability to bind to HABA, and second, that flgM-Stv begins to be secreted at the latest 2 hours after arabinose induction. When biotin was added to the solution, there was no change in the absorbance at 500 nm, proving that flgM-Stv binds strongly to biotin.

[0045] This confirmed that the secreted flgM-Stv binds strongly with biotin like streptavidin. 3) Confirmation of the biotin binding ability of the flgM-Stv expressing strain (biotin uptake assay)

[0046] The biotin-binding capacity of the transformed recombinant strain itself was assessed by biotin uptake analysis. For this purpose, the pellet obtained together with the supernatant after centrifugation of the samples collected at different time points in step 2) was used. The separated bacterial pellet was resuspended in 1 mL PBS. 100 µL of a biotin-conjugated fluorescent probe (Bioacts cat# RFP0613, excitation / emission wavelength = 749 nm / 774 nm) solution (1 mg / 5 mL) was added and mixed for 1 hour at 600 rpm on a vortex mixer. The samples were washed and observed under a fluorescence microscope. Fig. Figure 6 shows the fluorescence microscopy images and the graph quantified with ImageJ software. Fig. 6 shows that in the strain in which the expression of flgM-Stv was induced by arabinose, a fluorescence signal was measured by the binding of the fluorescent dye to biotin and the proportion increased with the induction time. 4) Tumor imaging

[0047] The utility of the strain of the present invention for targeting biotin-bound substances to tumors was confirmed by in vivo tumor imaging analysis.

[0048] Specifically, 4T1 cells with 5×10 5 Cells / 60 µL PBS were injected subcutaneously into the left thigh of female Balb / c nude mice and cultured for 2 weeks to induce tumors. These mice were treated with the S-flgM-streptavidinpBAD18-ASD+ / BRD509 asd- strain at a concentration of 5 × 10 6cfu / 60 µl PBS were injected intravenously. 72 hours after strain injection, 200 µL of 20% (w / v) arabinose was injected intraperitoneally, and 4 hours later, 20 µL of a biotin-conjugated fluorescent probe solution (20 µg / mL), identical to that used in 3), was injected subcutaneously into the tumor area. The mice were then placed under inhalation anesthesia, and the fluorescence signal was monitored at various time points using an in vivo imaging system (IVIS).

[0049] Fig. Figure 7 shows that despite injecting the same amount of fluorescent probe, the fluorescent signal not only lasts longer due to the binding of biotin and flgM-Stv, which inhibits the excretion of the dye, but also a higher overall signal is observed. This means that the same imaging sensitivity can be achieved even with a drastic reduction in the amount of contrast agent required for tumor imaging. Also, when used to target antineoplastic agents, this suggests that by targeting the antineoplastic agent, the same efficacy in the tumor area can be achieved even with a smaller amount of the antineoplastic agent. This can not only reduce side effects but also achieve a sustained-release effect by inhibiting the excretion of the antineoplastic agent. 5) Motility test of the Salmonella strain

[0050] During strain cultivation, an interesting observation was made that the expression of flgM-Stv appeared to influence strain motility. Therefore, strain motility was formally tested as a function of flgM-Stv expression using soft agar tryptone plates (per liter: 10 g Bacto Tryptone (BD:211705), 5 g NaCl (Biosesang S1009), and 3 g Bacto Agar (BD:214010)).

[0051] On soft agar tryptone plates with or without 0.2% arabinose, one colony each of the pBAD18 asd+ / aroA aroD asd plasmid-transformed Salmonella strain (1) and the S-flgM-streptavidin-pBAD18-ASD+ / BRD509 asd- strain (2) were inoculated with a sterile toothpick at distant sites. The cells were then incubated at 37°C for 5 hours to assess motility. Fig. Figure 8 shows the results demonstrating the loss of motility of the Salmonella strain by the expression of flgM-Stv.

[0052] To investigate the cause of the loss of motility, the morphology of the stem before and after arabinose induction was analyzed by TEM. The TEM images in Fig. 9 show that the flagella of the Salmonella strain disappear after induction of flgM-Stv expression by arabinose. Since the flagella of Salmonella are organs responsible for movement, their disappearance also results in the loss of motility. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] KR 10-0852687

[0005] KR 10-1750007

[0005] KR 10-2022-0058461

[0006] Cited non-patent literature

[0000] Leschner et al. (J. Mol. Med. 2010, 88, 763-773

[0003]

Claims

[1] A gene construct, characterized by that an anti-sigma factor flgM gene and a streptavidin-encoding gene are linked to encode a flgM-Stv fusion protein that has biotin-binding capacity. [2] Gene construct according to claim 1, characterized by that the flgM gene and the gene encoding streptavidin are directly linked or connected via a linker. [3] Gene construct according to claim 1, characterized by that it consists of sequence number 1 or sequence number 2. [4] A recombinant expression vector containing a gene construct according to any one of claims 1 to 3. [5] An antineoplastic recombinant strain that characterized by is that it is transformed by the gene construct according to claim 1 operably linked to an inducible promoter, expresses and secretes a fusion protein of flgM and streptavidin with biotin binding ability and targets tumor cells. [6] Antineoplastic recombinant strain according to claim 5, characterized by that the strain is an attenuated strain of Salmonella. [7] Antineoplastic recombinant strain according to claim 6, characterized by that the strain's motility is inhibited or lost when the fusion protein of flgM and streptavidin is expressed by the inducible promoter. [8] Antineoplastic recombinant strain according to claim 7, characterized by that the strain is the strain with the deposit number KCTC15482BP. [9] An antineoplastic composition comprising an antineoplastic recombinant strain according to any one of claims 5 to 8. [10] An antineoplastic adjuvant comprising a recombinant strain according to any one of claims 5 to 8 and administered together with an antineoplastic agent in biotin-bound form. [11] Antineoplastic adjuvant according to claim 10, characterized bythat the antineoplastic agent targets tumor cells and acts as a sustained-release drug. [12] A tumor imaging adjuvant comprising a recombinant strain according to any one of claims 5 to 8 and administered together with a contrast agent in biotin-bound form. [13] Tumor imaging adjuvant according to claim 12, characterized by that the contrast agent is one or more selected from the group consisting of radioactive nuclides, fluorescent labels, enzyme labels, chemiluminescent labels, gold preparations and magnetic preparations. [14] A method for providing information for cancer diagnosis, comprising: (A) administering the tumor imaging adjuvant of claim 12 to a patient suspected of having cancer or a patient undergoing antineoplastic therapy to target tumor cells; (B) Induction of expression of the fusion protein of flgM and streptavidin in the recombinant strain of the tumor imaging adjuvant; (C) administration of a contrast agent in biotin-bound form; and (D) Detection and imaging of the contrast agent.

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