Antitumor agent

Abstract

The present invention addresses the problem of providing a useful technique for treating cancer with an antitumor agent that contains a plurality of bacteria.　The present invention can provide an antitumor agent which contains a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium and is used in combination with a side effect suppressing agent. The present invention can provide a method for treating a tumor, the method comprising administering, in combination with a side effect suppressing agent, (a) a bacteria combination comprising a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium or (b) an antitumor agent containing these bacteria.

Description

antitumor agents

[0001] The present invention relates to a bacteria-containing antitumor agent that can be administered in high doses.

[0002] Various treatment methods are used to treat cancer, such as surgical therapy, drug therapy, and radiation therapy. These may be used alone or in combination depending on the type and condition of the disease. Drug therapy, such as anticancer drugs, is used. Anticancer drugs used in drug therapy can also have adverse effects on normal cells and can be a significant burden for patients. For example, anticancer drugs often accumulate in patients with long-term administration, causing significant side effects. For this reason, various useful techniques for cancer treatment are being investigated.

[0003] For example, an antitumor agent containing a purple non-sulfur bacterium isolated from a tumor has been proposed (Patent Document 1). For example, a bacterium isolated from a tumor and having antitumor activity has been proposed (Patent Document 2).

[0004] WO2023 / 223869WO2023 / 223870

[0005] An objective of the present invention is to provide a useful technology for cancer treatment using an antitumor agent containing multiple bacteria.

[0006] The present inventors have discovered that an antitumor agent containing a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium can be used in combination with an agent for suppressing side effects to provide an antitumor agent that is expected to be highly effective, and have completed the present invention. The present invention encompasses any one of the following aspects.

[0007] [1] An antitumor agent containing purple non-sulfur bacteria and bacteria other than the purple non-sulfur bacteria, used in combination with a side effect suppressor. [2] The antitumor agent according to [1], wherein the side effect suppressor is a bacteria-containing agent or a bacteria-free agent. [3] The antitumor agent according to [2], wherein the bacteria-containing agent contains the same bacteria as or a different bacteria from the bacteria contained in the antitumor agent. [4] The antitumor agent according to any one of [1] to [3], wherein the side effect suppressor is an antithrombotic agent and / or an anti-inflammatory agent. [5] The antitumor agent according to [4], wherein the antithrombotic agent is an anticoagulant. [6] The antitumor agent according to [5], wherein the anticoagulant is heparin or a salt thereof. [7] The antitumor agent according to [4], wherein the anti-inflammatory agent is a steroidal anti-inflammatory agent. [8] The antitumor agent according to [7], wherein the steroidal anti-inflammatory agent is dexamethasone. [9] The antitumor agent according to any one of [1] to [8], which is administered after the administration of the side effect suppressor.

[10] The antitumor agent according to any one of [1] to [9], which is administered multiple times after the administration of the side effect suppressor.

[11] The antitumor agent according to any one of [1] to

[10] , which is administered 26 hours or more after the prior administration of the side effect suppressor.

[12] The antitumor agent according to any one of [1] to

[11] , wherein the purple non-sulfur bacterium is a bacterium of the genus Rhodopseudomonas, and the bacterium other than the purple non-sulfur bacterium is a bacterium of the genus Proteus.

[13] The antitumor agent according to any one of [1] to

[12] , wherein the purple non-sulfur bacterium and the bacterium other than the purple non-sulfur bacterium are a complex bacterium identified by accession number: NITE BP-0362 7.

[14] The antitumor agent according to any one of [1] to

[13] above, which is included in a combination pharmaceutical with the side effect suppressor.

[15] The antitumor agent according to any one of [1] to

[14] above, which is administered in an amount equal to or greater than the dose required for tumor regression when administered alone in a single dose.

[16] The antitumor agent according to any one of [1] to

[15] above, which is administered in an amount equal to or greater than the dose required for tumor regression when administered alone in a single dose.

[0008] The present invention may also be in the following other embodiments.

[17] A combination drug comprising an antitumor agent containing a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium, and the side effect suppressor described in any one of [1] to

[16] above. Preferably, the combination drug further comprises an instruction manual describing any one of the methods described in

[20] to

[36] below.

[18] A complex bacterium comprising a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium, or use thereof, in the manufacture of the antitumor agent described in any one of [1] to

[16] above, to be used in combination with a side effect suppressor. The combination drug may further comprise a method described in any one of

[20] to

[36] below.

[19] A complex bacterium comprising a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium, or use thereof, for use in tumor treatment or antitumor treatment, in combination with a side effect suppressor. The complex bacterium may further comprise a method described in any one of

[20] to

[36] below.

[0009]

[20] A method for treating tumors, comprising administering (a) a bacterial complex composed of purple non-sulfur bacteria and bacteria other than the purple non-sulfur bacteria, or (b) an antitumor bacterial complex containing these bacteria, in combination with a side effect suppressor.

[21] The method according to

[20] , wherein the side effect suppressor comprises a bacteria-containing agent and / or a bacteria-free agent.

[22] The method according to

[20] or

[21] , wherein the side effect suppressor is at least a bacteria-containing agent, and the bacteria-containing agent contains the same or a different bacterium as the bacterium contained in the antitumor agent.

[23] The method according to any one of

[20] to

[22] , wherein the side effect suppressor is at least a bacteria-containing agent, and the dosage of the bacteria-containing agent is lower than the dosage of the tumor agent.

[24] The method according to any one of

[20] to

[23] , wherein the side effect suppressor is a bacteria-free agent or a agent containing the same, and the bacteria-free agent contains an antithrombotic agent and / or an anti-inflammatory agent.

[25] The method according to any one of

[20] to

[24] , wherein the side effect suppressor is an antithrombotic agent selected from at least bacteria-free agents, and the antithrombotic agent is an anticoagulant.

[26] The method according to any one of

[20] to

[25] , wherein the side effect suppressor is an anticoagulant selected from at least bacteria-free agents, and the anticoagulant is heparin or a salt thereof.

[27] The method according to any one of

[20] to

[26] , wherein the side effect suppressor is an anti-inflammatory agent selected from at least bacteria-free agents, and the anti-inflammatory agent is a steroidal anti-inflammatory agent.

[28] The method according to any one of

[20] to

[27] , wherein the side effect suppressor is a steroidal anti-inflammatory agent selected from at least bacteria-free agents, and the steroidal anti-inflammatory agent is dexamethasone.

[29] The method according to any one of

[20] to

[28] , wherein the administration is performed after the administration of the side effect suppressor.

[30] The method according to any one of

[20] to

[29] , wherein the compound is administered multiple times after the administration of the side effect suppressor.

[31] The method according to any one of

[20] to

[30] , wherein the compound is administered 26 hours or more after the previous administration of the side effect suppressor.

[32] The method according to any one of

[20] to

[31] , wherein the anti-inflammatory agent is administered once or multiple times after the administration of the side effect suppressor, and the side effect suppressor is one or more selected from a bacteria-containing agent, an anticoagulant, and a steroidal anti-inflammatory agent.

[33] The method according to any one of

[20] to

[32] , wherein the dose of the complex bacteria or the antitumor agent is equal to or greater than the dose required for tumor regression when administered alone in a single dose.

[34] The method according to any one of

[20] to

[33] , wherein the dose of the complex bacteria or the antitumor agent is equal to or greater than the amount required for tumor regression when administered alone in a single dose.

[35] The method according to any one of

[20] to

[34] , wherein the purple non-sulfur bacteria is a bacterium of the genus Rhodopseudomonas, and the bacterium other than the purple non-sulfur bacteria is a bacterium of the genus Proteus.

[36] The method according to any one of

[20] to

[35] , wherein the complex bacteria is a complex bacteria identified by accession number: NITE BP-03627.

[0010] The present invention can provide a useful technique for tumor treatment with a combined bacterial agent.

[0011] Figure 1 shows the results of minimum inhibitory concentration tests of various antibiotics against the complex bacterial strain AUN. The antibiotics used, from top to bottom, are ampicillin, cefotaxime, imipenem, aztreonam, amoxicillin, gentamicin, erythrosine, minocycline, clindamycin, levofloxacin, and vancomycin. Figure 2A shows the in vivo antitumor effect of AUN. AUN suspension was intravenously injected once or twice into BALB / c-nu / nu mice implanted with Colon 26. A control experiment (single intravenous administration of PBS) was also performed. Data are expressed as mean ± standard error of mean (SEM); n = 5 mice. Statistical significance of the double-administration group was calculated by comparison with the PBS group. ****, p < 0.0001, based on a two-tailed Student's t-test. Figure 2B shows the complete remission (CR) rate after 60 days of AUN (single or double doses) or PBS injection in BALB / c-nu / nu mice (n = 5) implanted with Colon26 tumors. ****, p < 0.0001, Student's two-tailed t-test. "Non-treatment" indicates no treatment, "single dose" indicates a single dose, and "double dose" indicates two doses (the same applies below). Figure 2C shows the Kaplan-Meier survival curves (60 days) for BALB / c-nu / nu mice (n = 5) implanted with Colon26 tumors. Statistical significance was calculated by comparison with the PBS group. ****, p < 0.0001, Log-rank (Mantel-Cox) test. Figure 2D shows the changes in body weight measured after each treatment (one dose of AUN, two doses of AUN, or one dose of PBS) in BALB / c-nu / nu mice implanted with Colon26 tumors. Data are presented as mean ± SEM. Statistical significance of the two-dose group was calculated by comparison with the PBS group. ****, p < 0.0001, Student's two-tailed t-test. Figure 2E shows the in vivo antitumor effect of two doses of AUN on SCID mice (left) and NOD-SCID mice (right) implanted with Colon26 tumors. PBS or AUN suspension was intravenously injected into the mice. Data are presented as mean ± SEM. Statistical significance was calculated by comparison with the PBS group.****, p < 0.0001, Student's two-tailed t-test. Figure 2F shows the CR rate of Colon-26 tumor-inoculated SCID and NOD-SCID mice (n = 5 each) after 30 days of AUN or PBS injection. Statistical significance was calculated compared to the PBS group. ****, p < 0.0001, Student's two-tailed t-test. Figure 2G shows Kaplan-Meier survival curves of Colon-26 tumor-inoculated SCID and NOD-SCID mice (n = 5) (30 days). Statistical significance was calculated compared to the PBS group. ****, p < 0.0001, Log-rank (Mantel-Cox) test. Figure 2H shows the weight changes measured after various treatments in Colon-26 tumor-inoculated SCID and NOD-SCID mice. Data are expressed as mean ± SEM (n = 5). ns indicates not significant by Student's two-tailed t-test. Statistical significance was calculated by comparison with the PBS group. ****, p < 0.0001, by two-way analysis of variance. Figure 3A shows the results of colony assays of blood samples administered with AUN at 5 minutes (left) and 6 hours (right). The concentration of administered AUN was 1 × 10. 9 CFU / mL. ns indicates not statistically significant, ** indicates p<0.01, Student's two-tailed t-test. Figure 3B shows the results of flow cytometry analysis of macrophages (left) and neutrophils (right) in the spleen 48 hours after administration of PBS or AUN. The concentration of administered AUN was 1 × 10 7 CFU / mL. Data are expressed as mean ± SEM. ****, p < 0.0001, Student's two-tailed t-test. Figure 3C shows the results of cytokine assays in the blood to measure IFN-γ after a single (left) or two (right) doses of AUN. The concentration of AUN administered in a single dose was 7.8 × 10 9 Figure 3D shows the results of a cytokine assay in the blood after a single (left) or two (right) doses of AUN to measure TNF-α. The concentration of AUN administered in a single dose was 7.8 × 10 9Figure 3E shows the results of a cytokine assay in the blood for measuring IL-6 after a single (left) or two (right) doses of AUN. The concentration of AUN administered in a single dose was 7.8 × 10 9 CFU / mL. Figure 3F shows the results of measuring the white blood cell (WBC) count after a single (left) or two (right) doses of AUN. The concentrations of AUN administered in the first and second doses were 1 × 10 7 CFU / mL and 7.8 × 10 9 The CFU / mL was measured. Data are expressed as mean ± SEM (n = 5 mice). Figure 3G shows the platelet (PLT) count after a single (left) or two (right) doses of AUN. The concentrations of AUN administered in the first and second doses were 1 × 10 7 CFU / mL and 7.8 × 10 9The expression levels were measured in CFU / mL. Data are presented as mean ± SEM (n = 5 mice). Figure 4 shows the results of measuring immune cell-related markers and cytokines 24 hours after intravenous administration of AUN to mice. Figure 4 (left) shows the results of quantitative PCR (qPCR), and Figure 4 (right) shows the results of flow cytometry analysis. Quantification by qPCR shows the mRNA expression of CD19, CD3, CXCR4, F4 / 80, NKp46, and cytokines (IFN-γ and TNF-α) as fold changes in log 10 relative quantification (RQ) compared to the control group (untreated). GAPDH gene expression was used as an internal control. Statistical significance was calculated compared to the untreated group. ns, **, p < 0.01, and ****, p < 0.0001 by Student's two-tailed t-test. Data are presented as mean ± SEM. n = 3 independent tumor samples. Figure 4 (right) shows the results of flow cytometry analysis of the expression of CD19, CD3, CXCR4, F4 / 80, and NKp46-positive cells in tumors 24 hours after intravenous injection of the combined bacterial AUN. Data are expressed as SEM ± mean. n = 3 independent tumor tissues. Statistical significance was calculated compared with the untreated group. ns, **, p < 0.01, and ***, p < 0.001, by two-tailed Student's t-test. Figure 5A shows the time course of serum factor VII specific activity after intravenous injection of AUN into mice (single dose). Factor VII specific activity was calculated by comparison with the untreated group. Data are expressed as mean ± SEM; n = 4 independent experiments. The administered concentration of AUN was 7.8 × 10 9The mean values ​​were CFU / mL. Figure 5B shows the statistical analysis results of TUNEL-positive staining of tumor tissues and IHC (TNF-α and fibrinogen). Data are presented as mean ± SEM; n = 10 independent regions (regions of interest) in each tumor tissue sampled from mice on day 1 after PBS and AUN treatment. Statistical significance was calculated compared to the PBS group. ****, p < 0.0001, Student's two-tailed t-test. Figure 5C shows the cytotoxicity of AUN. After 24 hours of treatment with AUN at different bacterial concentrations, the viability of Colon26 and MRC5 cells was measured. Data are presented as mean ± SEM. Statistical significance was calculated compared to the untreated group. ***, p < 0.001 and ****, p < 0.0001, Student's two-tailed t-test. Figure 6A shows the in vivo antitumor effect of two doses of AUN on BALB / c-nu / nu mice bearing HT29, SKOV3, and BxPC3 tumors. PBS or AUN suspension was intravenously injected into the mice. Data are presented as mean ± SEM. Statistical significance was calculated by comparison with the PBS group. ****, p < 0.0001 by two-way analysis of variance (ANOVA). Figure 6B shows the pancreas weight after each treatment. Data are presented as mean ± SEM. Statistical significance was calculated by comparison with the placebo group. ns: not significant, **: p < 0.01, ***: p < 0.0001 by Student's two-tailed t-test. Figure 6C shows the statistical analysis results of IHC (Ca19-9). Data are presented as mean ± SEM. Independent regions (regions of interest) of tumor tissues (n = 10) were collected from each mouse group on day 10 after placebo administration. Statistical significance was calculated by comparison with the placebo group. ns indicates no significance, **** indicates p<0.0001, based on Student's two-tailed t-test. Figure 7A shows the in vivo antitumor effect of AUN on BALB / c-nu-nu mice implanted with Colon26 after heparin (5 mg / head) administration. AUN suspension (15 × 10 9CFU / mL) was intravenously injected once into BALB / c-nu / nu mice implanted with Colon26 tumors. A control experiment (single intravenous administration of PBS) was also performed. Data are expressed as mean ± SEM. Statistical significance was calculated by comparison with the PBS group. ****, p < 0.0001, Student's two-tailed t-test. Figure 7B shows the CR rate 60 days after administration of heparin and AUN or PBS to BALB / c-nu / nu mice (n = 5) implanted with Colon26 tumors. ****, p < 0.0001, Student's two-tailed t-test. Figure 7C shows the effect of each treatment [heparin 5 mg / head administration followed by AUN (15 × 10 9 Figure 7 shows the changes in body weight measured after a single administration of AUN (10 × 10 CFU / mL) or a single administration of PBS. Data are expressed as mean ± SEM. Statistical significance was calculated by comparison with the PBS group. ****, p < 0.0001, by two-way analysis of variance. Figure 7D shows the Kaplan-Meier survival curves of BALB / c-nu / nu mice (n = 5 per treatment) implanted with Colon26 tumors. Statistical significance was calculated by comparison with the PBS group. ****, p < 0.0001, by the Log-rank (Mantel-Cox) test. Figure 7E shows the results of pre-administration of various antithrombotic agents to BALB / c mice (5-week-old, female, N = 5 per group) followed by administration of a high-dose AUN (10 × 10 CFU / mL). 9 Figure 8 shows the results of measuring the daily changes in body weight after intravenous injection of 200 μL of CFU / mL. Warfarin sodium (FUJIFILM-Wako): Oral administration (1 mg / head) 48 hours before AUN administration. Argatroban monohydrate (Tokyo Kasei): Oral administration (1 mg / head) 1 hour before AUN administration. Heparin sodium (Nacalai): Oral administration (5 mg / head) 1 hour before AUN administration. Figure 8A shows the changes in body weight before and after administration in mice that received intravenous administration of high-dose AUN in combination with an anti-inflammatory agent. Figure 8B shows the changes in body weight before and after administration in mice that received intravenous administration of high-dose AUN (5 × 10 9Figure 9A(a) shows the time course of blood TNF-α, IFN-γ, IL-6, IL-7A, IL-1β, and IL-10 concentrations in mice injected with 200 μL of medium containing 1000 CFU / mL of TNF-α, IFN-γ, IL-6, IL-7A, IL-1β, and IL-10 via the tail vein. Figure 9A(a) shows the antitumor effect of AUN on BALB / c mice (n = 5) implanted with EMT6 / AR1 tumors. Data are expressed as mean ± SEM. Statistical significance of the attenuated AUN group cultured on ceramic scaffolds was calculated compared with the untreated group. ****, p < 0.0001, by two-way analysis of variance (ANOVA). Figure 9A(b) shows the Kaplan-Meier survival curves of survival rates in BALB / c mice (n = 5) implanted with EMT6 / AR1 tumors and treated with AUN cultured on various scaffolds (W / O scaffold means no scaffold; the same applies to other figures). Data are presented as mean ± SEM, and n = 5 mice were used. Statistical significance was calculated compared to the untreated group. ****, p < 0.0001, log-rank (Mantel-Cox) test. Figure 9B shows the weight change after administration of AUN cultured on various scaffolds to BALB / c mice (n = 5) bearing EMT6 / AR1 tumors. Data are presented as mean ± SEM. Statistical significance was calculated compared to the untreated group. ****, p < 0.0001, two-way analysis of variance (ANOVA) test. Figure 10(a) shows the antitumor effect of AUN cultured on TiO2-PDMS scaffolds (AUN@TiO2-PDMS; similar results in other figures) on BALB / c mice (n = 5) bearing EMT6 / AR1 tumors. A control experiment without bacterial injection was also performed. Data are presented as mean ± SEM. Statistical significance of the AUN@TiO2-PDMS group was calculated compared to the untreated group. ****, p < 0.0001, by two-way analysis of variance (ANOVA). Figure 10(b) shows the Kaplan-Meier survival curves of BALB / c mice (n = 5) bearing EMT6 / AR1 tumors and treated with AUN cultured on various scaffolds. Statistical significance was calculated compared to the untreated group. ****, p < 0.0001, by log-rank (Mantel-Cox) test.Figure 10(c) shows the weight change after administration of AUN cultured on various scaffolds in BALB / c mice (n = 5) implanted with EMT6 / AR1 tumors. Data are presented as mean ± SEM. Statistical significance of the AUN@TiO2-PDMS group was calculated compared with the untreated group. ns, non-significant, by two-way analysis of variance (ANOVA). Figures 11(a) and 11(b) show the complete blood counts of white blood cells (WBC) and platelets (PLT) in the AUN@TiO2-PDMS or scaffold-free groups (AUN w / o scaffold). The concentration of the administered high-dose AUN was 5.0 × 10. 9 CFU / mL. Data are expressed as mean ± SEM, and n = 5 mice were used. Figure 11 (c) to (h) show the results of cytokine assays for IFN-γ, TNF-α, IL-6, IL-1β, IL-17A, and IL-10 to measure the blood cytokine concentrations in the AUN@TiO2-PDMS, no AUN@scaffold, or no AUN groups (Time: 0, 3, 6, and 24 hours). The concentration of the administered high-dose AUN was 6.5 × 10 9The CFU / mL was measured. Data are presented as mean ± SEM and were analyzed using n = 5 mice. Note that "Sham" refers to untreated AUNs, "AUN@TiO2-PDMS" refers to AUNs cultured on TiO2-PDMS, and "AUN w / o scaffold" refers to AUNs cultured without a scaffold. Figure 12 shows the results of flow cytometry analysis of intratumoral expression of (a) CD3 (T cells), (b) NKp46 (NK cells), (c) F4 / 80 (macrophages), (d) CXCR4 (neutrophils), and (e) CD19-positive cells (B cells). Data are presented as mean ± SEM and were analyzed using three independent tumor tissues. Statistical significance was calculated compared to the untreated group. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, Student's two-tailed t-test. "Non-treatment" and "Sample" refer to non-treatment and specimen, respectively. Figure 13(a) shows the antitumor effect of AUN on BALB / c mice (n = 5) implanted with EMT6 / AR1 tumors. A control experiment without any treatment was also performed. Data are expressed as mean ± standard error of mean (SEM). Statistical significance of the AUN@TiO2-PDMS group was calculated compared to the untreated group. ****, p < 0.0001, by two-way analysis of variance (ANOVA). Figure 13(b) shows the Kaplan-Meier survival curve of the survival rate of BALB / c mice (n = 5) implanted with EMT / 6AR1 tumors. Statistical significance was calculated compared to the untreated group. ****, p < 0.0001, by log-rank (Mantel-Cox) test. Figure 13(c) shows the weight change of BALB / c mice implanted with EMT / 6AR1 tumors after bacterial administration. Data are expressed as mean ± standard error (SEM), n = 5 mice. Statistical significance of the AUN@TiO2-PDMS group was calculated compared with the untreated group. ns, non-significant, by two-way analysis of variance (ANOVA) test.

[0012] Preferred embodiments for carrying out the present invention are described below. The embodiments described below are merely representative examples of the present invention, and should not be construed as narrowing the scope of the present invention. The upper limit (or less) and lower limit (or more) of each numerical range (to ) can be arbitrarily combined as desired. In the following descriptions of "1." to "2.", overlapping descriptions of technical features, configurations, definitions, terms, methods, etc., such as high dose, low dose, complex bacteria, and side effect suppressors, may be omitted as appropriate. The descriptions of "1." to "2." can be applied to any of the technologies or embodiments of "1." to "2.", and the technical features can be appropriately adopted in each of the technologies or embodiments.

[0013] 1. Antitumor agent according to the present invention 1-1. Bacteria complex or bacteria-contained agent 1-1-1. Purple non-sulfur bacteria 1-1-2. Bacteria other than the purple non-sulfur bacteria (the other bacteria mentioned above) 1-2. Side effect suppressor 1-2-1. Bacteria-containing agent (1. Low-dose bacteria-containing agent) 1-2-2. Bacteria-free agent <1. ​​Antithrombotic agent> <2. Immunosuppressant (preferably anti-inflammatory agent)> 1-3. Method of use of high-dose bacteria-containing agent 1-3-1. Method of use of high-dose bacteria-containing agent 1-3-2. Concomitant use with a side effect suppressor <1. Predetermined period between administration of a side effect suppressor and administration of a high-dose bacteria-containing agent> <2. Administration interval between a high-dose bacteria-containing agent and a high-dose bacteria-containing agent> <3. Example 1 of a preferred embodiment: Side effect suppressor (preferably a bacteria-containing agent)> <4. Preferred embodiment example 2: Side effect suppressor (preferably, bacteria-free agent)> 1-4. Constitution of the antitumor agent according to the present invention 1-5. Method for producing the bacteria-containing complex agent according to the present invention

[0014] 1. Antitumor agent according to the present invention

[0015] The present inventors have discovered that bacteria isolated from tumors have excellent antitumor activity. Furthermore, the present inventors have discovered a bacterium comprising a combination of purple non-sulfur bacteria and bacteria other than the purple non-sulfur bacteria, which has even stronger antitumor activity. That is, the present inventors have provided an antitumor agent containing purple non-sulfur bacteria and bacteria other than the purple non-sulfur bacteria (also referred to as "the other bacteria"). Furthermore, as a result of extensive research, the present inventors have discovered a technology that enables the administration of high doses of this antitumor agent by combining it with a side effect-reducing agent. That is, the present invention provides an antitumor agent containing purple non-sulfur bacteria and bacteria other than the purple non-sulfur bacteria, which is used in combination with a side effect-reducing agent.

[0016] In one embodiment of the present invention, a complex bacterium composed of a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium has the properties of absorbing and decomposing hemoglobin from vascular cells in the tumor microenvironment, and / or of inducing tumor-specific hemolysis and vascular blockage. Furthermore, since it has been reported that cancerous tumors use iron for cancer development, tumor growth, and metastasis, a complex bacterium composed of a purple non-sulfur bacterium and the other bacterium may have iron-requiring properties in both or either of the bacteria.

[0017] In one embodiment of the present invention, the other bacteria present in the complex bacteria may have a gene expression pattern different from that of the other bacteria of a purified single strain. Furthermore, the other bacteria coexisting with the purple non-sulfur bacteria at the tumor site may be flagellated bacteria that lack cilia but have some flagella remaining on their surface. In one embodiment, the other bacteria contained in the complex bacteria may be bacteria that have a transformation function that transforms from short swimmers before administration to elongated swarmer cells within the blood vessels at the tumor site.

[0018] Furthermore, it is preferable that either or both of the bacteria contained in the complex bacteria are bacteria that have the property of gathering in tumors or bacteria that have tumor-lytic activity, and it is more preferable that they have both of these properties.

[0019] Furthermore, the bacterial complex used in the present invention preferably has the property of not causing toxicity or having low toxicity to animals when administered (particularly parenterally) to the animals. It is generally believed that when bacteria are administered (particularly parenterally) to animals, the bacteria may often cause toxicity to the animals.

[0020] Furthermore, the complex bacteria contained in the antitumor agent of the present invention is preferably a complex bacteria that does not exist in the animal's body for a long period of time, in order to reduce concerns about adverse effects of the complex bacteria.

[0021] Furthermore, the complex bacteria are preferably bacteria that can pass through blood vessels to reach tumors without dying at the oxygen concentration in the blood and can grow without dying even at low oxygen concentrations in the tumor (e.g., O2 concentrations of 0.5% or 1% or more and / or O2 concentrations of 2%, 5%, or 8% or less).Furthermore, complex bacteria that normally reside in tumors are considered preferable because they can grow by taking advantage of immune privilege within the tumor and can more effectively exert antitumor effects.

[0022] Furthermore, the bacterial complex used in the present invention is a combination of multiple bacteria, and more preferably a combination of bacteria from two different genera, and more preferably a combination of one genus from the purple non-sulfur bacteria and one genus from the other bacteria. The purple non-sulfur bacteria are preferably bacteria of the genus Rhodopseudomonas, and / or the other bacteria are preferably bacteria of the genus Proteus. The bacterial complex is particularly preferably a bacterial complex of bacteria of the genus Rhodopseudomonas and bacteria of the genus Proteus, identified by accession number NITE BP-03627.

[0023] Furthermore, the complex bacteria used in the present invention preferably have the property that the number of complex bacteria can be easily controlled after administration using an antibiotic. Antibiotics used in the complex bacteria are not particularly limited, but examples include ampicillin, cefotaxime, imipenem, aztreonam, amoxicillin, gentamicin, erythrosine, minocycline, clindamycin, levofloxacin, and vancomycin. Antibiotics that have a strong effect on complex bacteria include ampicillin, cefotaxime, imipenem, amoxicillin, gentamicin, minocycline, and levofloxacin. Antibiotics that have a weak effect on complex bacteria include aztreonam, erythrosine, clindamycin, and vancomycin. One or more antibiotics selected from these can be used.

[0024] Furthermore, the present inventors have newly discovered from the Examples described below that when a single high dose of the bacterial complex is administered to an animal, side effects associated with high dose administration (e.g., thrombosis, weight loss, etc.) occur. As a result of extensive research, the present inventors have found that when a high dose of the bacterial complex is administered to an animal, side effects, particularly those related to thrombosis or weight loss, can be reduced by concomitantly using a side effect-reducing agent, enabling the administration of a dose higher than that of a single administration, thereby exerting a more excellent antitumor effect.

[0025] Therefore, the present invention can provide an antitumor agent containing a bacteria complex that is used in combination with a side effect reducer. The present invention can also provide an antitumor agent containing a bacteria complex that is included in a combination medicine with a side effect suppressor. The present invention can also provide a combination medicine that contains an antitumor agent containing a bacteria complex and a side effect suppressor. The high dose in the present invention is preferably an amount equal to or greater than the dose required for tumor regression when the antitumor agent is administered alone in a single dose, or is preferably an amount equal to or greater than the excessive amount when the antitumor agent is administered alone in a single dose.

[0026] Furthermore, the inventors have found from the Examples described below that administration of a low dose of a bacterial complex causes mild thrombus formation in blood vessels, but also consumes platelets and causes proliferation of phagocytes. Based on this finding, the inventors have found that it is desirable to at least reduce the number of platelets in the blood before or at the same time as administering a high dose of a bacterial complex. Furthermore, the inventors believe that administering a substance that can reduce or suppress platelets in the blood (such as a low-dose agent containing a bacterial complex or an antithrombotic agent) can suppress severe thrombus formation in organs other than the tumor site, thereby reducing the incidence of thrombosis in organs other than the tumor site.

[0027] Therefore, in a preferred embodiment of the present invention, it is preferable to use at least a platelet-reducing agent (preferably a low-dose agent containing a bacterial complex and / or an antithrombotic agent) in combination, thereby preventing or suppressing severe thrombus formation outside the tumor site due to high-dose administration of the bacterial complex-containing antitumor agent. Furthermore, it is more preferable to use a high-dose antitumor agent containing a bacterial complex simultaneously with or after the administration of at least a platelet-reducing agent. Furthermore, after administration of the high-dose antitumor agent containing a bacterial complex, it is even more preferable to administer the high dose intermittently, or to administer the high-dose antitumor agent containing a bacterial complex in single or multiple doses at predetermined intervals. For these reasons, the number of administrations of the side effect suppressing agent may be a single administration in a preferred embodiment of the present invention, but is not particularly limited and may be either single or multiple, depending on the condition of the tumor and the animal, etc. The side effect suppressing agent used may be a single type, or multiple types may be used in combination. In addition, a preferred embodiment of the present invention may be a double administration (e.g., one low dose and one high dose) or multiple administrations (e.g., one low dose and multiple high doses) of the complex bacteria or the complex bacteria-containing agent.

[0028] The present invention can provide a useful technology for tumor treatment using a bacterial complex. For example, the present invention allows for the administration of a bacterial complex at high doses, thereby achieving a stronger antitumor effect than a single administration of the bacterial complex alone. Furthermore, the antitumor agent used in the present invention has the advantage that multiple high-dose administrations can achieve a stronger antitumor effect over a long period of time, and that side effects associated with high-dose administration are suppressed even when multiple high-dose administrations are performed. Furthermore, by adjusting the timing of administration of the side-effect suppressor and the antitumor agent, the present technology can maintain a stronger antitumor effect while better suppressing side effects.

[0029] The antitumor agent of the present invention may be the complex bacteria itself or a culture containing the complex bacteria.

[0030] In this specification, the term "tumor" may refer to either a malignant tumor or a benign tumor, and the present technology is preferably used to treat a malignant tumor, but may also be used to treat cancer.

[0031] The side effect reducing agent may be a bacteria-containing agent, a bacteria-free agent, or a combination thereof. In the case of a bacteria-containing agent, the bacteria may be the same or different from the bacteria contained in the bacteria complex, for example, the same bacteria complex as the bacteria used in the bacteria-containing antitumor agent, such as BCG.

[0032] When a bacterial complex-containing agent is used as a side effect-reducing agent, the bacterial complex-containing agent is preferably administered at a low dose that is equal to or less than the dose required to cause tumor regression when the bacterial complex-containing agent is administered alone in a single dose. The bacterial complex-containing agent used in the present invention is preferably administered multiple times, with a low dose followed by a high dose administered twice or more times. The bacterial complex-containing agent used in the present invention is preferably a bacterial complex-containing agent containing the same type of bacteria (e.g., bacterial complex NITE BP-03627) for both low and high doses, and more preferably, a low-dose bacterial complex-containing agent is administered, followed by a high-dose bacterial complex-containing antitumor agent after a predetermined period of time.

[0033] When the side effect reducing agent is a bacteria-containing agent other than the bacteria-containing agent (a single bacteria-containing agent or another bacteria-containing agent), it is preferably administered before, after, or simultaneously with the bacteria-containing antitumor agent used in the present invention. The bacteria-containing agent used as the side effect reducing agent is more preferably administered before a high-dose bacteria-containing agent, and even more preferably administered after a predetermined period of time. In such cases, it is also called a pre-administered agent, a pre-administered agent, or a preliminary administered agent.

[0034] When the side effect reducing agent is a bacteria-free agent, it is preferable to use a drug other than an agent containing live bacteria, such as an antithrombotic agent or an anti-inflammatory agent. When used in combination with a bacteria-free agent, the bacteria-free agent is preferably administered before, after, or simultaneously with the high-dose bacteria-containing complex agent used in the present invention. The bacteria-free agent is more preferably administered before the high-dose bacteria-containing complex agent, and even more preferably administered after a predetermined period of time. In such cases, it is also called a pre-administered agent, a pre-administered agent, or a preliminary administered agent.

[0035] The antithrombotic agent is preferably, for example, an antiplatelet agent, an anticoagulant (e.g., heparin or a salt thereof), etc. The anti-inflammatory agent is preferably, for example, a steroid (e.g., dexamethasone), etc.

[0036] The high-dose administration of the bacteria-containing complex agent in the present invention is preferably carried out after a predetermined period of time has elapsed since the side effect-reducing agent was previously administered, more preferably after two or more days have elapsed. In addition, the number of high-dose administrations in the present invention may be either single or multiple. In the present invention, since the side effects are significantly reduced in the second or subsequent administrations of the bacteria-containing complex agent, if the tumor does not regress or grows after the predetermined period of time, the agent can be administered again after a further predetermined period of time.

[0037] The present invention is applicable to animals in general, and more preferably to humans and non-human animals (e.g., mammals), etc. Among these, humans and pets (dogs, cats, rodents (mice, rats, etc.)), etc., are preferred, and humans are more preferred.

[0038] The administration route used in the present invention is not particularly limited, and includes oral administration and parenteral administration (e.g., intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, intrathecal injection, and intravascular administration), and one or more selected from these may be used. Intravascular administration includes intraarterial injection, intravenous injection, and drip injection. Of these, parenteral administration is preferred as the preferred administration route.

[0039] As used herein, "prevention" refers to preventing or delaying the onset of a symptom or disease in a subject, or reducing the risk of developing a symptom or disease in a subject, etc. As used herein, "amelioration" refers to improving or maintaining a disease, symptom, or condition in a subject; preventing or delaying deterioration; or reversing, preventing, or delaying progression.

[0040] Each component will be described in more detail below.

[0041] 1-1. Bacterial Complex or Bacterial Complex-Containing Agent The present invention preferably uses a bacterial complex or a bacterial complex-containing agent. The bacterial complex contained in the bacterial complex-containing agent is preferably live. The bacterial complex may be a culture obtained by culturing the bacteria used together or separately, and is more preferably a bacterial complex culture obtained by culturing a mixture of different bacteria together. The bacterial complex may be derived from a combination of bacteria isolated from tumors, or from bacterial complexes isolated together. The bacterial complex may be a mixture of bacteria cultured separately or together after isolation, a bacterial complex cultured together, or a culture containing such bacterial complexes. The bacterial complex may also be a mixture of bacteria subcultured separately or subcultured together, or a culture containing such bacterial complexes. Of these, bacterial complexes cultured or subcultured in a bacterial complex state are more preferred.

[0042] It is more preferable that the complex bacteria used in the present invention contain purple non-sulfur bacteria and bacteria other than the purple non-sulfur bacteria (hereinafter also referred to as ``the other bacteria''), and even more preferably, each of the purple non-sulfur bacteria and the other bacteria is a live bacterium.

[0043] The complex bacteria is preferably a combination of Rhodopseudomonas bacteria and Proteus bacteria, and even more preferably a complex bacteria composed of Rhodopseudomonas Palustris and Proteus mirabilis. An even more preferred complex bacteria is a complex bacteria of Rhodopseudomonas Palustris and Proteus mirabilis, NITE BP-03627 (identification: Musashi), which was deposited internationally with the National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number NITE BP-03627 on March 23, 2022, and the composition ratio of Rhodopseudomonas Palustris to Proteus mirabilis in the complex bacteria is 97:3 based on CFU.

[0044] The ratio of purple non-sulfur bacteria to the other bacteria in the bacterial complex may be, for example, 99:1 to 1:99, with the purple non-sulfur bacteria preferably being present in a higher proportion. In a preferred embodiment, the ratio of purple non-sulfur bacteria to the other bacteria in the bacterial complex may be, for example, 99:1 to 50:50, more preferably 99:1 to 55:45. The ratio may be, for example, 80:20 to 55:45, or 70:30 to 60:40. These ratios may be based on the CFU of each bacterium. In this ratio, the purple non-sulfur bacteria is preferably a bacterium of the genus Rhodopseudomonas, more preferably Rhodopseudomonas palustris. In this ratio, the other bacteria is preferably a bacterium of the genus Proteus, more preferably Proteus mirabilis. With such a ratio, particularly with a higher content of purple non-sulfur bacteria than the other bacteria, excellent antitumor activity is exhibited. When the bacterial complex is composed of Rhodopseudomonas Palustris and Proteus mirabilis, the composition ratio based on CFU is preferably 95-99:5-1, more preferably 96-98:4-2, and particularly preferably 97:3. It is desirable that the composition ratio can be maintained within a certain range even after subculture, from the viewpoint of better suppressing side effects caused by the bacterial complex and exhibiting better antitumor effects.

[0045] The CFU-based composition ratio can be determined by seeding the complex bacteria in the antitumor agent onto a medium in a petri dish and counting the number of colonies formed after a predetermined culture period. Alternatively, the CFU-based composition ratio may be determined by performing quantitative PCR specific to the gene sequence of each cell. For example, Proteus mirabilis, listed as one of the other bacteria, forms colonies 2 to 3 days after the start of culture, while Rhodopseudomonas palustris, one of the purple non-sulfur bacteria, forms colonies 7 to 10 days after the start of culture. Therefore, the colony counts of Proteus mirabilis and Rhodopseudomonas palustris can be obtained by counting the number of colonies formed 2 to 3 days after the start of culture and then counting the number of colonies formed 7 to 10 days after the start of culture. The composition ratio can be determined based on these colony counts. In this way, when a complex bacteria contains both purple non-sulfur bacteria and the other bacteria, the difference in the time it takes for each bacterium to form a colony can be used to determine the composition ratio of these bacteria.

[0046] Alternatively, if the time required for colony formation is approximately the same, the number of colonies may be counted based on the optical properties of the purple non-sulfur bacteria and the other bacteria. Purple non-sulfur bacteria have specific optical properties (e.g., absorbance at 808 nm or 865 nm, fluorescence spectrum (fluorescence intensity at excitation wavelengths of 805 nm and 888 nm)), while the other bacteria do not have these optical properties. Therefore, colonies formed on the culture medium in the Petri dish may be identified as bacteria based on the presence or absence of these optical properties, and the number of colonies of each bacteria may then be counted.

[0047] 1-1-1. purple non-sulfur bacteria

[0048] The purple non-sulfur bacteria used in the present invention preferably have the following properties, which may be exerted at a tumor site. Alternatively, the following properties may be exerted in combination with bacteria other than purple non-sulfur bacteria (also referred to as "the other bacteria"), or may be exerted in the coexistence of the other bacteria at a tumor site. The purple non-sulfur bacteria preferably have the property of regulating the biological activity of the other bacteria in a bacterial complex. When in a bacterial complex, the purple non-sulfur bacteria preferably have the property of generating inter-bacterial interactions, such as biochemical reactions and inter-bacterial signaling, compared to when they are purified single strains. The purple non-sulfur bacteria may have the property of being iron-requiring. The purple non-sulfur bacteria preferably have the property of concentrating in tumors and / or have oncolytic activity, and preferably exert an anti-tumor effect even without exerting an immunostimulatory effect. The purple non-sulfur bacteria are preferably capable of growing even under low oxygen concentrations (e.g., 0.5% or 1% or more, 2% or 5% or less) such as those found in tumor sites. The purple non-sulfur bacteria are preferably non-pathogenic. The purple non-sulfur bacteria are preferably non-toxic or have low toxicity. The other bacteria are preferably those whose bacterial numbers decrease with tumor regression. The purple non-sulfur bacteria are preferably bacteria isolated from a tumor site, more preferably bacteria isolated from a tumor site after purple non-sulfur bacteria that do not normally reside in the body are inoculated into the tumor site and allowed to reside in the tumor site. The purple non-sulfur bacteria are even more preferably bacteria that form a consortium with the other bacteria normally present in the tumor after the purple non-sulfur bacteria are inoculated into the tumor site.

[0049] Examples of purple non-sulfur bacteria used in the present invention include, but are not limited to: Rhodopseudomonas bacteria, such as Rhodopseudomonas palustris and Rhodopseudomonas pseudopalustris; Blastochloris bacteria, such as Blastochloris viridis and Blastochloris sulfoviridis; Afifella bacteria, such as Afifella marina; Rhodobacter bacteria, such as Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter sphaeroides; Rubrivivax bacteria, such as Rubrivivax gelatinosus; Pararhodospirillum bacteria, such as Pararhodospirillum oryzae and Pararhodospirillum sulfurexigens; Rhodocista bacteria, such as Rhodocista centenaria; Marichromatium bacteria, such as Marichromatium litoris; Phaeochromatium bacteria, such as Phaeochromatium fluminis; Rhodoferax bacteria, such as Rhodoferax fermentans; Rhodomicrobium bacteria, such as Rhodomicrobium udaipurense and Rhodomicrobium vannielii; and Rhodovulum bacteria, such as Rhodovulum sulfidophilum.

[0050] The purple non-sulfur bacteria used in the present invention may be, for example, any one of the genera of bacteria listed above, or a combination of two or more thereof.Furthermore, the purple non-sulfur bacteria isolated from tumors contained in the antitumor agent of the present invention may be, for example, any one of the species of bacteria listed above, or a combination of two or more thereof.

[0051] The purple non-sulfur bacteria are preferably those isolated from tumors, and more preferably those isolated and cultured, or isolated and cultured and passaged. For methods of isolating and culturing purple non-sulfur bacteria, see Patent Documents 1 and 2. Among the purple non-sulfur bacteria, Rhodopseudomonas bacteria, Blastochloris bacteria, or both are preferred. Their antitumor activity is excellent and they are effective against tumors in various organs or tissues. For example, the bacteria may be any one, two, three, or four of Rhodopseudomonas palustris, Rhodopseudomonas pseudopalustris, Blastochloris viridis, and Blastochloris sulfoviridis.

[0052] In a more preferred embodiment, the purple non-sulfur bacterium is a bacterium of the genus Rhodopseudomonas. Rhodopseudomonas bacteria isolated from tumors can exhibit particularly excellent antitumor activity. For example, the purple non-sulfur bacterium contained in the antitumor agent of the present invention may be Rhodopseudomonas Palustris, Rhodopseudomonas pseudopalustris, or both. More preferably, the agent may contain at least Rhodopseudomonas Palustris.

[0053] 1-1-2. Bacteria other than the aforementioned purple non-sulfur bacteria (the aforementioned other bacteria)

[0054] Bacteria other than purple non-sulfur bacteria (also referred to as "the other bacteria") used in the present invention preferably have the following properties, which may be exhibited at the tumor site. Alternatively, the following properties may be exhibited in combination with the other bacteria, or may be exhibited in the coexistence of the other bacteria at the tumor site.

[0055] The other bacteria preferably have the characteristic of completely lacking virulence factors such as cilia and adhesins. The other bacteria preferably have one or more flagella when in a complex bacterial state, and more preferably have motility via these flagella. The other bacteria preferably have the ability to change shape upon contact with a tumor in a complex bacterial state. For example, more preferably, they have the ability to change shape from short swimmer cells to elongated filamentous swarmer cells several times longer, and more preferably, they become filamentous cells with a length 3, 5, or 10 times or more and / or 15, 20, or 30 times or less the length of the short swimmer cells. The other bacteria preferably have the characteristic of requiring iron. The other bacteria preferably have the ability to gather in tumors and / or have oncolytic activity, and preferably exhibit antitumor activity even without immunostimulatory activity. The purple non-sulfur bacteria are preferably capable of growing even under low oxygen concentrations such as those found in tumor sites (e.g., 0.5% or 1% or more, 2% or 5% or less). The other bacteria are preferably non-pathogenic. The other bacteria are preferably non-toxic or have low toxicity. The number of the other bacteria is preferably reduced with tumor regression. The other bacteria are preferably native to the tumor site, and more preferably form a consortium with the purple non-sulfur bacteria that are not native to the body after intravenous inoculation.

[0056] Examples of the other bacteria used in the present invention include, but are not limited to, bacteria of the genus Proteus, Lactococcus, Enterococcus, Acinetobacter, Bacillus, or Cutibacterium, and are preferably one or more species selected from these. Among these, bacteria of the genus Proteus are preferred, and Proteus mirabilis is more preferred.

[0057] (1. Proteus Bacteria) The Proteus bacterium used in the present invention may be, for example, any one, two, or three of Proteus mirabilis, Proteus vulgaris, and Proteus myxofaciens, and may preferably be Proteus mirabilis. Proteus mirabilis isolated (particularly isolated) from tumors exhibits particularly excellent antitumor activity. The Proteus mirabilis may be, for example, the bacterium deposited under accession number NITE BP-03626. The bacterium was internationally deposited with the National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on March 23, 2022 (Reiwa 4).

[0058] (2. Lactococcus Bacteria) The Lactococcus bacterium used in the present invention may have a shape similar to a spherical or ovoid cell. The Lactococcus bacterium can grow individually, in pairs, or in chains. The Lactococcus bacterium may also be non-spore-forming and non-motile. Examples of Lactococcus bacteria having such sequence homology include Lactococcus formosensis, Lactococcus garvieae, and Lactococcus garvieae subsp. Garvieae, and the Lactococcus bacterium may be any one, two, or three of these. The Lactococcus bacterium may be, for example, the bacterium deposited under accession number NITE BP-03694. The bacterium in question was deposited internationally at the National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on August 2, 2022.

[0059] (3. Enterococcus bacteria) The Enterococcus bacteria may be, for example, any one, two, or three of Enterococcus faecalis, Enterococcus faecium, Enterococcus alcedinis, Enterococcus bulliens, Enterococcus caccae, Enterococcus devriesei, Enterococcus eurekensis, Enterococcus rivorum, Enterococcus saccharolyticus, and Enterococcus termitis, and preferably Enterococcus faecalis. Enterococcus faecalis isolated (particularly isolated) from tumors exhibits particularly excellent antitumor activity. The Enterococcus faecalis may be, for example, the bacterium deposited under accession number: NITE BP-03690. The bacterium was deposited internationally at the National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Depositary (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on July 19, 2022.

[0060] (4. Acinetobacter Bacteria) The Acinetobacter bacterium used in the present invention may be, for example, any one, two, or three of Acinetobacter radioresistens, Acinetobacter albensis, and Acinetobacter baumannii, and is preferably Acinetobacter radioresistens. Acinetobacter radioresistens isolated (particularly isolated) from a tumor exhibits particularly excellent antitumor activity.

[0061] (5. Bacillus Bacteria) The Bacillus bacteria used in the present invention may be, for example, any one, two, or three of Bacillus thuringiensis, Bacillus agri, and Bacillus badius, and may be preferably Bacillus thuringiensis.

[0062] (6. Cutibacterium) The Cutibacterium bacterium used in the present invention may be, for example, any one, two, or three of Cutibacterium acnes, Cutibacterium avidum, and Cutibacterium granulosum, and is preferably Cutibacterium acnes. Cutibacterium acnes isolated (particularly isolated) from a tumor exhibits particularly excellent antitumor activity.

[0063] 1-2. Side effect suppression drugs

[0064] The side effect suppressant used in the present invention is not particularly limited, but is preferably, for example, a bacteria-containing agent and / or a bacteria-free agent. The bacteria-containing agent used in the present invention is preferably a live bacterium capable of proliferation or an agent containing the same, and may be a culture containing live bacteria capable of proliferation. On the other hand, the bacteria-free agent used in the present invention may contain dead bacteria that cannot proliferate, and in the case of bacteria that cannot proliferate or a culture containing bacteria that cannot proliferate, or a substance separated or purified from the culture, it is preferable to include it in the bacteria-free agent. The bacteria-free agent used in the present invention is preferably subjected to a sterilization or disinfection treatment during the manufacturing process of the agent to prevent bacteria from proliferating.

[0065] The side effect suppressant used in the present invention may be a side effect suppressant or a substance for suppressing side effects that can be used as a pharmaceutical, etc., and may be a pharmaceutical, etc. that is available on the market, whether known or new, or may be manufactured in consideration of a known manufacturing method.

[0066] The side effect suppressant used in the present invention preferably can reduce side effects (e.g., thrombosis, weight loss, etc.) caused by administration of the bacteria-complex-containing agent used in the present invention (particularly, a single high-dose administration of the bacteria-complex-containing agent alone), and the bacteria-complex-containing agent may be the bacteria-complex itself, a culture of the bacteria-complex, or a formulation containing the bacteria-complex. Furthermore, the administration form of the side effect suppressant used in the present invention is not particularly limited, and may be either oral or parenteral administration, with parenteral administration being preferred, and injection administration being more preferred.

[0067] 1-2-1. Bacteria-containing agent The bacteria-containing agent used in the present invention is not particularly limited as long as it uses bacteria (particularly preferably live bacteria), but for example, it may contain the same or a different bacterium as one of the bacteria contained in the above-mentioned bacteria-containing agent. Furthermore, it may contain the same or a different bacterium complex as the bacteria complex contained in the above-mentioned bacteria-containing agent.

[0068] Preferred embodiments of the bacteria-containing agent include, for example, the above-mentioned bacteria complex or the agent thereof prepared for low-dose administration (hereinafter also referred to as "low-dose bacteria complex or low-dose bacteria complex agent"), and bacteria other than those used in the bacteria-containing agent (e.g., BCG, etc.). One or more selected from these can be used. Furthermore, the bacteria-containing agent may contain bacteria from the bacteria complex not used in the above-mentioned high-dose bacteria-containing agent, either singly or in combination. Examples of bacteria-containing agents other than the above-mentioned bacteria complex include BCG vaccine. The "bacteria" used in the bacteria-containing agent are preferably bacteria capable of reducing blood platelets. The bacteria-containing agent used in the present invention is preferably the above-mentioned low-dose bacteria-containing complex agent. It is more preferable that the low-dose bacteria complex has the effect of reducing blood platelets and / or an immune-inducing effect, and particularly preferably has at least the effect of reducing blood platelets. Furthermore, the administration form of the bacteria-containing agent used in the present invention is not particularly limited and may be either oral or parenteral administration, but parenteral administration is preferred, injection administration is more preferred, and intravenous injection administration is even more preferred.

[0069] <1. Low-Dose Bacterial Complex Preparation> The bacterial complex used in the low-dose bacterial complex-containing preparation may be selected from the bacterial complexes described above, purple non-sulfur bacteria, and other bacteria. Preferably, the bacterial complex is a bacterium of the genus Rhodopseudomonas and a bacterium of the genus Proteus. Even more preferably, the bacterial complex is Rhodopseudomonas Palustris and Proteus mirabilis. NITE BP-03627 (identification: Musashi) is a bacterial complex of Rhodopseudomonas Palustris and Proteus mirabilis. This bacterial complex, NITE BP-03627, can effectively reduce blood platelets when administered at low doses. Administration of the bacterial complex at low doses can prevent or suppress the occurrence of thrombosis outside the tumor site. The bacterial complex-containing preparation for low-dose use may be the bacterial complex itself or a culture containing the bacterial complex. The bacterial complex used in this low dose may be either live or dead bacteria, but is preferably viable bacteria that can grow. The administration form of the bacterial complex agent for low doses used in the present invention is not particularly limited and may be either oral or parenteral administration, but parenteral administration is preferred, and injection administration is more preferred.

[0070] The "low dose" in the low-dose bacteria-containing complex agent used in the present invention preferably refers to an amount equal to or less than the dose required for tumor regression when the bacteria-containing complex agent is administered alone in a single dose. The "required dose" in the "dosage required for tumor regression" will be explained in detail in "1-3. Use of high-dose bacteria-containing complex agent" below.

[0071] The low dose in the present invention is a suitable upper limit of, for example, 5 x 10 complex bacteria per administration. 8 CFU / kg body weight or less, more preferably 1 x 10 8 CFU / kg body weight or less, more preferably 0.5 x 10 8 CFU / kg body weight or less, more preferably 1 x 10 7 CFU / kg body weight or less, more preferably 5 x 10 6CFU / kg body weight or less, more preferably 1 x 10 6 CFU / kg body weight or less, more preferably 5 x 10 5 CFU / kg body weight or less, and a suitable lower limit is, for example, 1 x 10 2 CFU / kg body weight or more, preferably 1 x 10 3 CFU / kg body weight or more, more preferably 1 x 10 4 CFU / kg body weight or more, more preferably 5 x 10 4 CFU / kg body weight or more, more preferably 1 x 10 5 The dose may be administered to achieve a CFU / kg body weight or more.

[0072] When the low-dose complex bacteria-containing agent is a liquid agent, the low-dose complex bacteria-containing agent may contain, for example, complex bacteria in a concentration of, for example, 5 x 10 8 CFU / mL or less, more preferably 1 x 10 8 CFU / mL or less, more preferably 5 x 10 7 CFU / mL or less, more preferably 1 x 10 7 CFU / mL or less, more preferably 5 x 10 6 CFU / mL or less, and a suitable lower limit is, for example, 1 x 10 3 CFU / mL or more, preferably 5 x 10 3 CFU / mL or more, more preferably 1 x 10 4 CFU / mL or more, more preferably 5 x 10 4 CFU / mL or more, more preferably 1 x 10 5 It may be contained at a content of CFU / mL or more.

[0073] 1-2-2. Bacteria-Free Agent The bacteria-free agent is not particularly limited, but may be a commercially available pharmaceutical product or the like, and may be produced by a known production method. The bacteria-free agent is preferably a substance other than live bacteria, more preferably a substance other than bacteria that can proliferate. The bacteria-free agent may be, for example, one or more selected from antithrombotic agents, immunosuppressants, anti-inflammatory agents, etc., and may be a combination of one, two, or three of these. Examples of combinations include a combination of an antithrombotic agent and an anti-inflammatory agent, or an antithrombotic agent and / or an anti-inflammatory agent. The administration route may be either oral or parenteral, with parenteral administration being preferred. The dosage of the bacteria-free agent is not particularly limited, and may be, for example, the dosage prescribed.

[0074] <1. Antithrombotic Agent> The antithrombotic agent used in the present invention is not particularly limited, but examples include anticoagulants, antiplatelet agents, vitamin K inhibitors, etc., and one or two of these can be selected. In the present invention, the antitumor agent containing a complex of bacteria is used in combination with an antithrombotic agent to reduce side effects such as thrombus formation in organs other than the tumor site, the onset of thrombosis, and death that occur when an excessive amount of the antitumor agent containing a complex of bacteria is administered alone in a single dose. Furthermore, by administering the antithrombotic agent at least once, a high-dose complex of bacteria-containing agent may be administered multiple times at predetermined intervals.

[0075] The anticoagulant compounds used as the anticoagulant in the present invention are not particularly limited, and examples thereof include heparin, heparinoids or their salts (e.g., sodium), warfarin or its salts (e.g., sodium), argatroban monohydrate, dabigatran, edoxaban, apixaban, rivaroxaban, aspirin, clopidogrel, prasugrel, and ticlopidine. Heparinoids are substances with a chemical structure similar to that of heparin and preferably have blood anticoagulant activity. Among these, one or more selected from warfarin, argatroban, heparin, and salts thereof are more preferred, and heparin or a salt thereof is even more preferred. Examples of antiplatelet agents used in the present invention include aspirin, clopidogrel, and ticlopidine. Examples of vitamin K inhibitors used in the present invention include warfarin. Of these, warfarin is preferred. One or more selected from these can be used. The salt is not particularly limited, but examples thereof include alkali metal salts such as sodium, potassium, and lithium, and alkaline earth metal salts such as calcium and magnesium, and one or more of these can be used.

[0076] Anticoagulants include factor Xa inhibitors and thrombin inhibitors. Factor Xa inhibitors bind directly to factor Xa and inhibit the conversion of prothrombin to thrombin. Thrombin inhibitors bind directly to thrombin and inhibit thrombin-mediated fibrinogen activation. Factor Xa inhibitors used in the present invention include, but are not limited to, rivaroxaban, apixaban, edoxaban, betrixaban, otamixaban, razaxaban, darexaban, retaxaban, elibaxaban, and antistasin. Thrombin inhibitors used in the present invention include, but are not limited to, dabigatran, bivalirudin, hirudin, lepirudin, desirudin, argatroban, melagatran, and ximelagatran. One or more selected from these may be used.

[0077] Among anticoagulants, one or more selected from heparin, heparinoids, warfarin, argatroban, and salts thereof are more preferred, and heparin or a salt thereof is even more preferred. Since heparin is known to have anticoagulant effects through thrombin inhibition and factor Xa inhibition, thrombin inhibitors and / or factor Xa inhibitors may be selected from among anticoagulants.

[0078] The dosage of the antithrombotic agent is not particularly limited, and when using commercially available pharmaceuticals, the dosage may be according to the prescription for the antithrombotic agent used, but a dosage that can suppress thrombus formation in organs other than the tumor site by high-dose administration of the complex bacteria-containing agent is preferred, and a dosage that can reduce platelets in the blood is more preferred. The administration form of the antithrombotic agent is not particularly limited, and may be either oral or parenteral administration, but parenteral administration is preferred, injection administration is more preferred, and intravenous injection administration is even more preferred.

[0079] The anticoagulant may be administered so that the upper limit per administration is, for example, 500 mg or less, more preferably 250 mg or less, even more preferably 125 mg or less, and even more preferably 50 mg or less, and the lower limit per administration is, for example, 0.1 mg or more, preferably 0.25 mg or more, more preferably 0.5 mg or more, and even more preferably 1 mg or more.

[0080] A suitable upper limit per administration of heparin or a salt thereof is, for example, 500 mg or less, more preferably 250 mg or less, even more preferably 125 mg or less, and more preferably 50 mg or less, and a suitable lower limit per administration is, for example, 0.1 mg or more, preferably 0.25 mg or more, more preferably 0.5 mg or more, and even more preferably 1 mg or more.

[0081] 2. Immunosuppressant (preferably anti-inflammatory agent) Among the side effect suppressants used in the present invention, it is preferable to use an immunosuppressant. Examples of immunosuppressants include, but are not limited to, agents that suppress excessive immune responses and agents that suppress excessive inflammatory responses, among which anti-inflammatory agents are preferred. In the present invention, by using the high-dose bacteria-complex-containing agent in combination with an immunosuppressant (preferably an anti-inflammatory agent), it is possible to reduce side effects such as weight loss due to loss of appetite, which occurs when a single administration of the bacteria-complex-containing antitumor agent alone is administered at the required dose for tumor regression.

[0082] By administering an immunosuppressant (e.g., prednisolone, hydrocortisone, dexamethasone, etc.) orally (p.o.) or subcutaneously (s.c.) as a premedication a predetermined period (e.g., 26 hours) before the administration of the complex bacteria-containing agent, followed by subcutaneous administration of an anti-inflammatory agent such as dexamethasone, it is possible to more effectively suppress side effects such as weight loss due to loss of appetite caused by the administration of the complex bacteria-containing agent.

[0083] The immunosuppressant used in the present invention is not particularly limited, but examples thereof include hydrocortisone, cortisone, dexamethasone, glucocorticoids, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cyclosporine, tacrolimus, cyclophosphamide, azathioprine, everolimus, mizovirin, mycophenolate mofetil, and the like, and one or more selected from these may be used.

[0084] The anti-inflammatory agent used in the present invention is not particularly limited, but examples thereof include steroidal and non-steroidal agents.

[0085] Examples of steroid compounds used as steroid anti-inflammatory agents include prednisolone, hydrocortisone, cortisone, dexamethasone, triamcinolone, triamcinolone acetonide, difluprednade, mometasone, diflucortolone, fluonicid, beclomethasone, deprodone, alclometasone, flumethasone, amcinonide, clobetasone, diflorasone, and derivatives thereof. The steroid compounds may be hydrates, hemihydrates, or anhydrous forms. One or more of these compounds may be selected and used.

[0086] Examples of the derivative include esters (particularly esters with organic or inorganic acids), salts, and salts of esters, and one or more selected from these can be used. Examples of esters include esters with organic acids such as valeric acid, acetic acid, succinic acid, butyric acid (butanoic acid), propionic acid, sulfobenzoic acid, cypesic acid, palmitic acid, furancarboxylic acid, and pivalic acid; and esters with inorganic acids such as phosphoric acid. Furthermore, one molecule may contain multiple esters. When one molecule contains multiple esters, it may contain multiple esters with one type of acid, or it may contain esters with two or more types of acids. The salt is not particularly limited as long as it can be used as a pharmaceutical or the like, and examples thereof include salts with organic bases (organic amine salts such as methylamine salt, triethylamine salt, triethanolamine salt, morpholine salt, piperazine salt, pyrrolidine salt, tripyridine salt, and picoline salt), and salts with inorganic bases (ammonium salt; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; and metal salts such as zinc salt and aluminum salt). Of these, alkali metal salts such as sodium salt and potassium salt are preferred. One or more of these can be used.

[0087] Non-steroidal compounds used as non-steroidal anti-inflammatory agents are not particularly limited, but examples thereof include allantoin, glycyrrhizinic acid, methyl glycyrrhizinate, stearyl glycyrrhizinate, glycyrrhetinic acid, stearyl glycyrrhetinate, acetaminophen, epsilon-aminocaproic acid, berberine, azulene, bromelain, zinc, etc.; plant extracts such as licorice extract, sage extract, rosemary extract, etc.; enzyme compounds such as lysozyme, serrapeptase, semi-alkaline proteinase, etc.; fenamic acid compounds such as mefenamic acid, flufenamic acid, tolfenamic acid, etc.; acemetacin, indomethacin, indomethacin, Examples of suitable aryl acetic acid compounds include endomethacin farnesyl, edotolac, diclofenac, sulindac, nabutomene, fenbufen, proglumetacin, and mofezolac; propionic acid compounds include aminoprofen, ibuprofen, oxaprozin, ketoprofen, zaltoprofen, tiaprofenic acid, naproxen, flurbiprofen, zaltoprofen, ibuprofen piconol, flurbiprofen axetil, fenoprofen, pranoprofen, and loxoprofen; and oxicam compounds such as ampiroxicam, tenoxicam, piroxicam, meloxicam, and lornoxicam. These compounds may also be salts. One or more of these compounds may be selected and used. The salt may be any of the salts described above.

[0088] The dosage of the immunosuppressant is not particularly limited, and examples include the dosage according to the prescription of the immunosuppressant used, but an amount that can suppress weight loss due to high-dose administration of the complex bacteria-containing agent is preferable, and more preferably an amount that can suppress appetite loss.

[0089] The anti-inflammatory agent may be administered so that the preferred upper limit per administration is, for example, 2000 mg or less, more preferably 1000 mg or less, even more preferably 500 mg or less, and more preferably 200 mg or less, and the preferred lower limit is, for example, 0.1 mg or more, preferably 0.25 mg or more, more preferably 0.5 mg or more, and even more preferably 1 mg or more.

[0090] The preferred upper limit of dexamethasone per administration is, for example, 200 mg or less, more preferably 100 mg or less, even more preferably 50 mg or less, and more preferably 20 mg or less, and the preferred lower limit is, for example, 0.1 mg or more, preferably 0.25 mg or more, more preferably 0.5 mg or more, and even more preferably 1 mg or more. The immunosuppressant or anti-inflammatory agent may be administered orally or parenterally, although parenteral administration is preferred, and in the case of dexamethasone, subcutaneous injection is more preferred.

[0091] 1-3. Dosage of High-Dose Bacteria-Containing Agents 1-3-1. Dosage of High-Dose Bacteria-Containing Agents The "high dose" of a high-dose bacteria-containing agent is preferably equal to or greater than the dose required for tumor regression when the antitumor agent containing the bacteria-containing complex is administered alone in a single dose. A higher dosage of the bacteria-containing complex agent is preferable from the perspective of better exerting antitumor activity. However, higher dosages are more likely to cause side effects due to the bacteria-containing complex when administered at high doses. Therefore, to maintain the effects of the bacteria-containing complex while reducing side effects, the dosage of the bacteria-containing complex agent may be lower. Furthermore, a high-dose bacteria-containing complex agent may be a culture containing the bacteria-containing complex as is, or a culture containing the bacteria-containing complex. It is more preferable that the bacteria-containing complex be viable and capable of proliferation. The administration form of the high-dose bacteria-containing complex agent is not particularly limited and may be either oral or parenteral administration, with parenteral administration being preferred, injection administration being more preferred, and intravenous injection being even more preferred.

[0092] Here, the "required dose" for tumor regression is preferably 10 x 10 per administration of the complex bacteria. 9 ~10 x 10 13 CFU / kg or a dose below that range, more preferably 5 x 10 9 ~5 x 10 13 CFU / kg or a dose below that range, more preferably 1 x 10 9 ~1 x 10 13 CFU / kg or a dose below this range, particularly preferably 0.5 x 10 9 ~0.5 x 10 13Dosage in CFU / kg.

[0093] The upper and lower limits of the "required dose" for tumor regression are, for example, 0.5 x 10 9 CFU / kg body weight or more, 1 x 10 9 CFU / kg body weight or more, 5 x 10 9 CFU / kg body weight or more, or 10 x 10 9 CFU / kg body weight or more, and a suitable upper limit is, for example, 10 x 10 13 CFU / kg body weight or less, preferably 5 x 10 13 CFU / kg body weight or less, more preferably 1 x 10 13 CFU / kg body weight or less, more preferably 0.5 x 10 13 The dose may be administered to achieve up to 100 CFU / kg body weight.

[0094] When the agent containing the complex bacteria is a liquid agent, the "required dose" (preferably the required concentration of the complex bacteria in a single administration) for the tumor to regress is preferably 10 x 10 8 ~10 x 10 12 CFU / mL or a dose below that range, more preferably 5 x 10 8 ~5 x 10 12 CFU / mL or a dose below that range, more preferably 1 x 10 8 ~1 x 10 12 CFU / mL or a dose below this range, particularly preferably 0.5 x 10 8 ~0.5 x 10 12 The dose is in CFU / mL.

[0095] The upper and lower limits of the "required dose (preferably the required concentration of the complex bacteria at one time of administration)" for tumor regression (bacterial concentration) are, for example, 0.5 × 10 as a preferred lower limit. 8 CFU / mL or more, 1×10 8 CFU / mL or more, 5 x 10 8 CFU / mL or more, or 10 x 10 8 CFU / mL or more, and a suitable upper limit is, for example, 10 x 10 12CFU / mL or less, preferably 5 x 10 12 CFU / mL or less, more preferably 1 x 10 12 CFU / mL or less, more preferably 0.5 x 10 12 It may contain complex bacteria at a content rate of CFU / mL or less.

[0096] Furthermore, the "high dose" of the antitumor bacteria-containing agent is preferably an amount greater than or equal to an excessive amount when the antitumor bacteria-containing agent is administered alone in a single dose. The more excessive the dosage of the bacteria-containing agent, the better the antitumor effect can be exhibited. However, since excessive dosages are more likely to cause side effects due to the bacteria-containing agent, the dosage of the bacteria-containing agent may be lowered to reduce side effects while still exhibiting the effects of the bacteria-containing agent. Furthermore, a high-dose bacteria-containing agent may be a culture containing the bacteria-containing agent, or the bacteria-containing agent may be a viable bacterium capable of proliferation.

[0097] Here, an "overdose" is preferably 50 x 10 per single administration of the complex bacteria. 9 ~50 x 10 13 CFU / kg or a dose below that range, more preferably 25 x 10 9 ~25 x 10 13 CFU / kg or a dose below that range, more preferably 5 x 10 9 ~5 x 10 13 CFU / kg or a dose below this range, particularly preferably 2.5 x 10 9 ~2.5 x 10 13 The upper and lower limits of the "overdose" are the dosages in terms of the number of bacteria in a single dose, and a suitable lower limit is, for example, 2.5 x 10 9 CFU / kg body weight or more, 5 x 10 9 CFU / kg body weight or more, 25 x 10 9 CFU / kg body weight or more, or 50 x 10 9 CFU / kg body weight or more, and a suitable upper limit is, for example, 50 x 10 13 CFU / kg body weight or less, preferably 25 x 10 13CFU / kg body weight or less, more preferably 5 x 10 13 CFU / kg body weight or less, more preferably 2.5 x 10 13 The dose may be administered to achieve up to 100 CFU / kg body weight.

[0098] When the high-dose agent containing the complex bacteria is a liquid, the "excess dose" (preferably the excess concentration of the complex bacteria in a single administration) is preferably 50 x 10 8 ~50 x 10 12 CFU / mL or a dose below that range, more preferably 25 x 10 8 ~25 x 10 12 CFU / mL or a dose below that range, more preferably 5 x 10 8 ~5 x 10 12 CFU / mL or a dose below this range, particularly preferably 2.5 x 10 8 ~2.5 x 10 12 The dose is in CFU / mL.

[0099] The upper and lower limits of the "overdose amount (preferably the overdose concentration of the complex bacteria at one time)" are, for example, 2.5 x 10 as a preferable lower limit. 8 CFU / mL or more, 5 x 10 8 CFU / mL or more, 25×10 8 CFU / mL or more, or 50 x 10 8 CFU / mL or more, and a suitable upper limit is, for example, 50 x 10 12 CFU / mL or less, preferably 25 x 10 12 CFU / mL or less, more preferably 5 x 10 12 CFU / mL or less, more preferably 2.5 x 10 12 It may contain complex bacteria at a content rate of CFU / mL or less.

[0100] 1-3-2. Use in Combination with Side Effect Suppressors The antitumor agent containing a bacterial complex used in the present invention is preferably administered in combination with a bacterial side effect suppressor. This provides a useful technology for tumor treatment using a bacterial complex. Compared to a single administration of the bacterial complex alone, the bacterial complex can be administered in a higher dose than the amount used in a single administration of the bacterial complex, thereby achieving a more effective antitumor effect.

[0101] In a preferred embodiment of the present invention, the use of a side effect suppressor in combination can suppress side effects (e.g., severe thrombus formation, onset of thrombosis, weight loss, infections such as sepsis, fever, nausea, etc.) caused by high-dose administration of the bacterial complex, making it possible to administer higher doses than were previously possible using the bacterial complex. Suitable side effect suppressors are one or more selected from low-dose bacterial complex-containing agents, antithrombotic agents, and anti-inflammatory agents.

[0102] In a preferred embodiment of the present invention, the side effect suppressant is preferably administered at the same time as or before the administration of the high-dose complex bacteria-containing agent. In a preferred embodiment of the present invention, after the prior administration of the side effect suppressant, the high-dose complex bacteria-containing agent is more preferably administered multiple times, and even more preferably, the high-dose complex bacteria-containing agent is administered multiple times at predetermined intervals.

[0103] A preferred embodiment of the present invention is a multiple-administration method in which a low-dose bacteria-containing complex agent or an antithrombotic agent is administered in combination with a high-dose bacteria-containing complex agent, and the administration of a high-dose bacteria-containing complex agent can be repeated multiple times. This multiple-administration method has the advantage of allowing the high-dose bacteria-containing complex agent to be administered multiple times depending on the tumor condition, such as tumor recurrence, thereby exerting a more effective antitumor effect. This multiple-administration method also has the advantage of reducing the risk of thrombosis and allowing a more effective antitumor effect to be exerted more quickly. Furthermore, this multiple-administration method also has the advantage that after the administration of a low-dose bacteria-containing complex agent, the high-dose bacteria-containing complex agent can be repeatedly administered over a long period of time, thereby exerting a more effective antitumor effect over a long period of time.

[0104] According to the present invention, the high dose can be an amount equal to or greater than the dose required for tumor regression when the agent containing the complex is administered alone in a single dose. Furthermore, according to the present invention, the high dose can be an amount equal to or greater than the excessive amount when the agent containing the complex is administered alone in a single dose. When the high dose range in the present invention is set to be greater than the amount used in a single administration of the complex, the upper or lower limit can be appropriately set taking into account the antitumor effect and side effects (e.g., thrombosis, weight loss, etc.) caused by the complex.

[0105] <1. Predetermined period between administration of side effect suppressant and administration of high-dose complex bacteria-containing agent> The side effect suppressant used in combination may be administered a predetermined period before administration of the high-dose complex bacteria-containing agent, at the same time as administration of the high-dose complex bacteria-containing agent, or a predetermined period after administration of the high-dose complex bacteria-containing agent. The side effect suppressant may be an auxiliary agent or a preliminary agent. The timing of administration of the side effect suppressant is preferably before administration of the high-dose complex bacteria-containing agent, and it is preferable to administer the high-dose complex bacteria-containing agent a predetermined period after administration of the side effect suppressant. In addition, the complex bacteria-containing agent may be referred to as the main agent.

[0106] In a preferred embodiment of the present invention, the high-dose bacteria-containing complex agent is preferably administered after a predetermined period of time has elapsed since the side effect suppressor was pre-administered. The predetermined period of time in a preferred embodiment of the present invention is not particularly limited, but its preferred lower limit is, for example, 0 hours (simultaneous use), preferably 0.5 hours or more, more preferably 1 hour or 2 hours or more, more preferably 24 hours or more, and even more preferably 26 hours or more. The preferred upper limit is, for example, 96 hours or less or 84 hours or less, preferably 72 hours or less, more preferably 60 hours or less or 48 hours or less. For example, the high-dose bacteria-containing complex agent is preferably administered after 26 hours or more since the side effect suppressor was pre-administered.

[0107] In a more preferred embodiment, the low-dose complex bacteria-containing agent is preferably administered simultaneously with or prior to the administration of the high-dose complex bacteria-containing agent, and can be administered prior to the high-dose complex bacteria-containing agent. The preferred predetermined period is preferably 26 hours or more and 72 hours or less, and the predetermined period described in <Embodiment Example 1> below can be appropriately adopted. The dosage can be an excessive amount or more. The low-dose complex bacteria-containing agent and the high-dose complex bacteria-containing agent may have different bacterial compositions and content ratios, but preferably have the same bacterial composition and content ratio. For example, when using a low-dose complex bacteria-containing agent, a more preferred embodiment is to administer the high-dose complex bacteria-containing agent 26 hours or more after the prior administration of the low-dose complex bacteria-containing agent.

[0108] In a more preferred embodiment, an antithrombotic agent is preferably used at the same time as or before the administration of the high-dose bacterial complex-containing agent, and can be administered before the high-dose agent. The preferred predetermined period is preferably 0 hours or more and 72 hours or less, and the predetermined period described in <Embodiment Example 2> below can be appropriately adopted. The dosage can be an excessive amount or more. For example, when an antithrombotic agent is used, it is preferred that the high-dose bacterial complex-containing agent be administered 0 hours or more after the antithrombotic agent is pre-administered.

[0109] In a more preferred embodiment, an anti-inflammatory agent is preferably used at the same time as or prior to the administration of the high-dose bacterial complex-containing agent, and can be administered prior to the high-dose agent. The preferred predetermined period is preferably 26 hours or more and 72 hours or less, and the predetermined period described in <Embodiment 2> below can be appropriately adopted. The dosage can be greater than the required amount. For example, when an anti-inflammatory agent is used, in a more preferred embodiment, the high-dose bacterial complex-containing agent is preferably administered 26 hours or more after the anti-inflammatory agent is administered in advance.

[0110] 2. Administration Interval Between High-Dose Bacteria-Containing Complex Agent and High-Dose Bacteria-Containing Complex Agent The present invention allows for the administration of a high-dose bacteria-containing complex agent at least once after the administration of a side effect suppressor. It is believed that the present invention also allows for the administration of a high-dose bacteria-containing complex agent multiple times by administering a side effect suppressor once. Furthermore, the present invention has the great advantage of allowing for multiple intermittent administrations, such as a second or third or more administrations after the first administration of a high-dose bacteria-containing complex agent. The present invention also allows for the administration of a high dose of a bacteria-containing complex agent in response to the tumor condition over a long period of time, thereby achieving better antitumor or tumor regression effects over a long period of time. Furthermore, a side effect suppressor may be administered as appropriate during the intermittent administration of a high-dose bacteria-containing complex agent.

[0111] When a high-dose complex bacteria-containing agent is administered two or more times, the interval between the previous high-dose administration and the next high-dose complex bacteria-containing agent is not particularly limited, but the preferred lower limit is, for example, 12 or 18 hours or more, preferably 24 hours or more, preferably 26 hours or more, more preferably 30 hours or more, more preferably 36 hours or more, more preferably 48 hours or more, and the preferred upper limit is, for example, 120 hours or less, preferably 84 hours or less, more preferably 96 hours or less, even more preferably 72 hours or less, and even more preferably 60 hours or less. The preferred numerical range is preferably 26 hours or more and 96 hours or less, more preferably 48 hours or more and 72 hours or less. The previous administration may be either the administration of a side effect suppressor or a high-dose complex bacteria-containing agent.

[0112] 3. Preferred Embodiment Example 1: Side Effect Suppressant (Preferably, Bacteria-Containing Agent) As a preferred embodiment example 1 of the administration timing of the present invention, a high-dose bacteria-containing complex agent is preferably administered after a side effect suppressant (preferably, a bacteria-containing agent) is administered in advance as a pre-administered agent. By providing a predetermined period as in the preferred embodiment example 1 of the administration timing, the administration of a side effect suppressant such as a bacteria-containing agent is more likely to exert its immune-inducing effect in the body, allowing for the subsequent administration of a higher dose of the bacteria-containing complex agent. Furthermore, even in the case of an agent that delays the in vivo platelet reduction effect, providing a predetermined period allows for the subsequent administration of a higher dose of the bacteria-containing complex agent. This allows for the exertion of an extremely excellent antitumor effect.

[0113] The side effect suppressant used in Example 1 of the preferred embodiment of the administration timing is preferably a bacteria-containing agent. The bacteria used in the bacteria-containing agent may be the same as or different from either one or both of the bacteria used in the high-dose bacteria-containing agent, but is preferably the same bacteria complex as the high-dose bacteria-containing agent. The bacteria-containing agent is more preferably a low-dose bacteria-containing agent, and the bacteria complex is preferably a bacteria complex of bacteria of the genus Rhodopseudomonas and bacteria of the genus Proteus, particularly preferably a bacteria complex consisting of Rhodopseudomonas Palustris and Proteus mirabilis (preferably, bacteria complex NITE BP-03627 (identification: Musashi)). The low dose of the low-dose bacteria-containing agent can be appropriately configured as described above, and preferably 1 x 10 per administration of the bacteria complex. 8 CFU / kg body weight or less, or preferably a bacterial complex dose concentration of 1 x 10 8 CFU / mL or less.

[0114] The high-dose bacteria-containing agent used in Example 1 of the preferred embodiment of the administration timing can appropriately adopt the composition of the high-dose bacteria-containing agent described above. The bacteria-containing agent is particularly preferably the bacteria-containing agent NITE BP-03627 (identification: Musashi). The high dose may be the "required dose" for the tumor to regress, as described above, or an "excessive dose," and the composition described above can be appropriately adopted. The "high dose" is preferably an amount that is greater than or equal to an excessive amount when the antitumor agent containing the bacteria-containing agent is administered alone in a single dose, and is preferably 2.5 x 10 per single administration of the bacteria-containing agent. 9 ~2.5 x 10 13 A dose of 100 CFU / kg is preferred, or a single dose of 2.5 x 10 bacterial overdose. 9 ~2.5 x 10 13 A dosage of CFU / kg is preferred.

[0115] As a preferred predetermined period in Example 1 of the preferred embodiment of the administration timing, it is preferable that the high-dose complex bacteria-containing agent is administered at a high dose after at least a predetermined period (e.g., more than 24 hours) has elapsed since the prior administration of the bacteria-containing agent (preferably a low-dose complex bacteria-containing agent). The predetermined period from the prior administration of the bacteria-containing agent in Example 1 of the preferred embodiment of the administration timing to the administration of the high-dose complex bacteria-containing agent is not particularly limited, but its preferred lower limit is, for example, more than 24 hours, preferably 26 hours or more, more preferably 30 hours or more, more preferably 36 hours or more, more preferably 48 hours or more, and its preferred upper limit is, for example, 120 hours or less, preferably 84 hours or less, more preferably 96 hours or less, even more preferably 72 hours or less, and even more preferably 60 hours or less. The preferred numerical range is preferably 26 hours or more and 96 hours or less, more preferably 48 hours or more and 72 hours or less.

[0116] The number of doses and intervals of the high-dose bacteria-containing agent after the administration of the pre-administration agent can be appropriately determined as described above. For example, the administration can be performed once, twice, or three or more times. The administration interval between the previous administration of the high-dose bacteria-containing agent and the next administration of the high-dose bacteria-containing agent is preferably 48 hours or more and 72 hours or less.

[0117] 4. Preferred Embodiment Example 2: Side Effect Suppressant (Preferably, Bacteria-Free Agent) As a preferred embodiment example 2 of the present invention, a high-dose bacteria-containing complex agent is preferably administered simultaneously with a side effect suppressant (preferably, a bacteria-free agent) or after the side effect suppressant has been administered in advance. By administering a side effect suppressant (preferably, a bacteria-free agent) and obtaining an antithrombotic effect or a weight loss suppression effect in the body, it becomes possible to administer a higher dose of the bacteria complex, thereby achieving an excellent antitumor effect.

[0118] The side effect suppressant used in Example 2 of the preferred embodiment of the administration period is preferably a bacteria-free agent. The bacteria-free agent is preferably an antithrombotic agent and / or an anti-inflammatory agent. When administering an antithrombotic agent, it is preferable to adopt an "excessive amount" of the complex bacteria-containing agent, which allows for the administration of a higher dose of the complex bacteria-containing agent, thereby enabling a better antitumor effect. When administering an anti-inflammatory agent, it is preferable to adopt a "required dose" of the complex bacteria-containing agent, which can suppress weight loss due to loss of appetite and lethargy caused by the required dose of the complex bacteria, leading to the maintenance of physical strength and allowing tumor treatment to proceed smoothly.

[0119] The high-dose bacteria-containing agent used in Example 2 of the preferred embodiment of the administration timing can appropriately adopt the composition of the high-dose bacteria-containing agent described above. The bacteria-containing agent is particularly preferably the bacteria-containing agent NITE BP-03627 (identification: Musashi). The high dose may be the "required dose" for tumor regression as described above, or an "excessive dose," and the composition described above can be appropriately adopted. The "high dose" is preferably an amount equal to or greater than the required dose for tumor regression when the antitumor agent containing the bacteria-containing agent is administered alone in a single dose, and the required dose is 0.5 x 10 per administration of the bacteria-containing agent. 9 ~0.5 x 10 13 A dose of 0.5 x 10 CFU / kg is preferred, or the required dose concentration of the complex bacteria per dose is 0.5 x 10 8 ~0.5 x 10 12Furthermore, the "high dose" is preferably an amount that is an excessive amount or more when the antitumor agent containing the complex bacteria is administered alone in a single dose, and in the case of an excessive amount, it is preferably 2.5 x 10 CFU / mL per single administration of the complex bacteria. 9 ~2.5 x 10 13 A dose of 100 CFU / kg is preferred, or a single dose of 2.5 x 10 bacterial overdose. 9 ~2.5 x 10 13 A dosage of CFU / kg is preferred.

[0120] As a preferred predetermined period in Example 2 of a preferred embodiment of the administration timing, a high-dose complex bacteria-containing agent may be administered in high doses at the same time as a side effect suppressor (preferably an antithrombotic agent), and it is preferable that the high dose be administered at least a predetermined period (e.g., 1 hour or 24 hours) after the side effect suppressor (preferably an antithrombotic agent or anti-inflammatory agent) has been previously administered.

[0121] In Example 2 of a preferred embodiment of the administration timing, the specified period from the prior administration of the antithrombotic agent (preferably heparin or a salt thereof) to the administration of the high-dose complex bacteria-containing agent is not particularly limited, but a suitable lower limit is, for example, 0 hours (simultaneous combination), preferably 0.5 hours or more, and more preferably 1 hour or 2 hours or more, and a suitable upper limit is, for example, 24 hours or less, preferably 12 hours or less, more preferably 6 hours or less, and more preferably 3 hours or 2 hours or less, and more preferably 0 hours or more but 3 hours or less.

[0122] In Example 2 of the preferred embodiment of the administration timing, the predetermined period from the prior administration of the anti-inflammatory agent (preferably dexamethasone) to the administration of the high-dose complex bacteria-containing agent is not particularly limited, but a suitable lower limit is, for example, 6 hours or more, preferably 12 hours or more, and more preferably 18 hours or more, and a suitable upper limit is, for example, 48 hours or less, preferably 36 hours or less, more preferably 32 hours or less, and more preferably 12 hours or more but 24 hours or less.

[0123] The number of doses and intervals of the high-dose bacteria-containing agent after the administration of the pre-administration agent can be appropriately determined as described above. For example, the administration can be performed once, twice, or three or more times. The administration interval between the previous administration of the high-dose bacteria-containing agent and the next administration of the high-dose bacteria-containing agent is preferably 48 hours or more and 72 hours or less.

[0124] 1-4. Composition of the Antitumor Agent of the Present Invention The bacterial conjugate used in the present invention exhibits excellent antitumor activity when used in combination with a side effect suppressor, and can therefore be incorporated into or used in an antitumor agent. Furthermore, since the present invention allows for high-dose administration, it can also provide a high-dose bacterial conjugate-containing agent used as an antitumor agent in combination with a side effect suppressor. Furthermore, the bacterial conjugate used in the present invention can be used for tumor prevention, treatment, or amelioration when used in combination with a side effect suppressor, and can even be used in high-dose applications. The present invention also provides a bacterial conjugate or use thereof for tumor treatment or antitumor use in combination with a side effect suppressor, and can also provide a bacterial conjugate or use thereof that can be administered in high doses. Furthermore, it can also provide a method for preventing, treating, or ameliorating tumors in which the bacterial conjugate used in the present invention is used in combination with a side effect suppressor, and can be administered in high doses to further enhance the antitumor effect. The bacterial conjugate used in the present invention can also be used to produce or in the production of an antitumor agent. The present invention also provides the bacterial conjugate or the use of the bacterial conjugate in the production of an antitumor agent in combination with a side effect suppressor. The agent may be an antitumor agent or a high-dose or low-dose agent containing a complex bacteria, which may also be called an anticancer agent, a cancer treatment composition, or a cancer therapeutic composition.

[0125] The agent containing a complex of bacteria may contain bacteria isolated from a tumor in a viable state, preferably in a proliferative state, but may also contain bacteria isolated from the tumor in a dead state.

[0126] In the present invention, "anti-tumor agents" include agents used to treat tumors that have developed in animals. "Tumor treatment" may mean, for example, one or more of reducing tumor size, inhibiting tumor growth, killing or reducing tumor cells, or inhibiting tumor cell proliferation. "Antineoplastic agents" may also mean agents used to treat or prevent tumor-bearing animals. The animals may be, for example, mammals, particularly humans, but may also be non-human animals. The non-human animals may be, for example, agricultural animals or pet animals, such as cows, horses, sheep, goats, pigs, dogs, cats, or rabbits.

[0127] The "agent" may be an agent consisting of one component, or may contain two or more components. At least one of these components is the bacterium isolated from the tumor described above. The agent may contain two or more components, in which case the antitumor agent of the present invention may be called an antitumor composition or even an antitumor pharmaceutical composition. That is, the antitumor agent of the present invention may contain other components in addition to the bacterium isolated from the tumor. The other components may be appropriately selected by those skilled in the art depending on factors such as the administration method or administration site of the antitumor agent.

[0128] The antitumor agent of the present invention may be used to treat malignant tumors or benign tumors, and is particularly preferably used to treat malignant tumors. The malignant tumors are also called "cancers." Cancers can be classified into solid cancers and blood cancers. Solid cancers can be further classified into carcinomas and sarcomas. The antitumor agent may also be called an anticancer agent, or a composition for treating cancer or a composition for treating cancer.

[0129] The antitumor agent of the present invention may be used to treat solid cancers or blood cancers, particularly solid cancers, such as carcinomas or sarcomas. The antitumor agent of the present invention can be used to treat various cancers. One of the mechanisms of the antitumor activity of the antitumor agent of the present invention is thought to be the activation of immune cells, as described below. That is, the antitumor agent is thought to exert its antitumor activity via the activated immune cells. Therefore, the antitumor agent of the present invention is thought to be effective against not only one specific type of cancer, but also various cancers, and its effectiveness against various cancers is actually demonstrated in the examples described below.

[0130] In one embodiment, the antitumor agent of the present invention may be used to treat carcinoma, for example, any one or more of head and neck cancer (e.g., pharyngeal cancer, laryngeal cancer, tongue cancer, etc.), esophageal cancer, gastric cancer, duodenal cancer, colorectal cancer (e.g., colon cancer, rectal cancer, etc.), liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer, kidney cancer, bladder cancer, prostate cancer, uterine cancer (e.g., cervical cancer, endometrial cancer), and ovarian cancer.

[0131] In another embodiment, the antitumor agent of the present invention may be used to treat sarcoma, for example, any one of osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma.

[0132] In one embodiment, the antitumor agent of the present invention may be formulated as a liquid. That is, the liquid may be a liquid containing the bacteria isolated from a tumor according to the present invention (particularly a dispersion of the bacteria). The liquid component other than the bacteria in the liquid may be, for example, an injection or infusion solution used in the pharmaceutical field, and more specifically, may be an isotonic solution, a hypotonic solution, or a hypertonic solution. For example, the liquid may be saline (physiological saline), a sugar solution, or a buffer solution. Alternatively, the liquid may be phosphate-buffered saline. Liquid formulations are particularly suitable for allowing the antitumor agent of the present invention to reach the tumor while maintaining its antitumor activity.

[0133] The antitumor agent of the present invention is preferably administered parenterally. For example, the antitumor agent of the present invention may be administered intravascularly (e.g., intravenously or intraarterially), subcutaneously, intramuscularly, or intrathecally. Preferably, the antitumor agent of the present invention is administered intravascularly (e.g., intravenously or intraarterially).

[0134] The antitumor agent of the present invention may also be administered directly to the tumor site or near the tumor site using, for example, a syringe or other tube.

[0135] The antitumor agent of the present invention may be administered only once, or may be administered two or more times. The antitumor agent of the present invention can exert its effect with a single administration, but may be administered two or more times as necessary. The antitumor agent of the present invention may be administered once a day, or two or more times (for example, two or three times) a day. When the antitumor agent of the present invention is administered multiple times, the antitumor agent may be administered daily, or every other day or every third day. The antitumor agent may also be administered weekly, every two weeks, every three weeks, or every four weeks.

[0136] The antitumor agent of the present invention may contain additives used in pharmaceutical formulations, such as pH adjusters and colorants. Furthermore, the antitumor agent of the present invention may contain known or future pharmaceutical ingredients for tumor treatment, provided that the effects of the present invention are not impaired. The antitumor agent of the present invention may be formulated by known methods, as appropriate, depending on the dosage form.

[0137] In addition to antitumor agents, the uses of the bacteria-containing complex agent used in the present invention include, for example, diagnostic reagents, detection or measurement reagents (e.g., light absorbers, fluorescent agents, etc.), and combination products (e.g., combination drugs). The bacteria-containing complex agent used in the present invention can also be used as an angiogenesis inhibitor and is effective in preventing, treating, or ameliorating symptoms or diseases caused by angiogenesis, such as choroidal neovascularization, neovascular glaucoma, and tumor angiogenesis, from which one or more can be selected. The form of the bacteria-containing complex agent or antitumor agent used in the present invention is not particularly limited and may be in any form, such as liquid, paste, gel, solid, or powder. The bacteria-containing complex agent used in the present invention may be administered orally or parenterally, including parenteral administration such as injection and infusion.

[0138] The bacterium complex used in the present invention can be contained in or used in a bacterium complex-containing combination product (preferably a combination drug) that uses a bacterium complex, the combination product comprising a bacterium complex-containing agent and a side effect suppressor. The combination product may be a product for administering a high dose of the bacterium complex. Furthermore, a combination product using a bacterium complex-containing agent can be used for the prevention, treatment, or amelioration of tumors, and can also be used in a method for preventing, treating, or ameliorating tumors by administering the bacterium complex in combination with a side effect suppressor. The bacterium complex used in the present invention can also be used to manufacture a combination product for use in a bacterium complex-containing agent or in the manufacture of such a combination product. The use of such a combination product can appropriately adopt the above-described components, dosage, and other configurations. That is, as another preferred embodiment, a combination drug may be provided that is used for high-dose administration and is composed of a bacterium complex-containing agent containing a purple non-sulfur bacterium and a bacterium other than the purple non-sulfur bacterium, and a side effect suppressor. The combination may be a set or a kit. Although a multi-dose combination drug is preferred, it may also be a single-dose drug containing the bacterium complex and a side effect suppressor.

[0139] 1-5. Method for Producing the Bacterial Composite Agent of the Present Invention The above-described bacterial composite is used to produce the antitumor agent of the present invention. Patent Documents 1 and 2 can be referenced for the method for producing the bacterial composite agent used in the present invention. Since this production method also includes a method for producing a bacterial composite, it can provide not only a method for producing an antitumor agent, but also a method for producing a bacterial composite or a bacterial culture, and a low-dose bacterial composite agent for suppressing the side effects of a high-dose bacterial composite agent. The concentration of the bacterial composite in the agent or the dosage of the bacterial composite can be determined and adjusted using CFU (Colony Forming Unit). Furthermore, steps can be replaced with other methods, means, etc. Furthermore, if the bacterial composite to be used for producing the antitumor agent already exists, the tumor recovery step and bacterial isolation step can be omitted, and the antitumor agent production method can include a bacterial culture step and a formulation step. Furthermore, it is preferable that the bacterial composite production method includes at least a bacterial culture step. The obtained bacterial culture can be used as is or after being concentrated, diluted, lyophilized, or the like, for the intended use, such as an antitumor agent.

[0140] (Tumor harvesting step) In harvesting a tumor, the tumor is harvested from a tumor-bearing animal. The harvesting may be performed, for example, by biopsy. The tumor may be, for example, a tumor formed from mammalian tumor cells, particularly a tumor formed from human tumor cells. The tumor-bearing animal may be, for example, a mammal, particularly a rodent, but may also be a primate, particularly a human.

[0141] Before the recovery, the tumor-bearing animal may be administered purple non-sulfur bacteria and / or the other bacteria at the tumor site, and the administered bacteria may be commercially available. More preferably, the purple non-sulfur bacteria are administered to the tumor site, and a consortium of bacteria formed with the other bacteria normally present at the tumor site is recovered. The administered purple non-sulfur bacteria are preferably bacteria that do not normally reside at the tumor site or in the living body. The consortium of bacteria may preferably be Rhodopseudomonas bacteria or Proteus bacteria.

[0142] The administration is, for example, parenteral, particularly intravenous, administration. After a predetermined period of time, for example, 1 to 10 days, particularly 2 to 5 days, following the administration, the tumor is recovered as described above. It is believed that the administered bacteria reach the tumor after such a period of time has elapsed.

[0143] The bacterial administration may not be performed, but may be performed. If the bacterial administration is not performed, bacteria originally present in the tumor may be collected. If the bacterial administration is performed, bacteria originally present in the tumor may be collected, or bacteria administered by the bacterial administration and delivered to the tumor may be collected.

[0144] (Bacteria isolation step) In isolating bacteria, bacteria are isolated from the tumor collected in the tumor collection step. For example, the tumor obtained by biopsy is added to a predetermined liquid (e.g., a buffer solution) and homogenized. This homogenization results in a liquid in which tumor cells and bacteria are suspended. By shaking the liquid, for example, at a predetermined speed, the tumor cells are precipitated and the bacteria are present in the supernatant. In this manner, bacteria may be separated from tumor cells, but the separation method is not limited thereto. Separation may also be performed by other methods known in the art. It is believed that the presence of bacteria in tumors and their isolation from the tumor contribute to the bacteria acquiring antitumor activity.

[0145] (Bacteria Culturing Step) In culturing bacteria, the bacteria isolated from the tumor in the bacteria isolation step are cultured. Culturing the bacteria isolated from the tumor is also thought to contribute to the bacteria acquiring antitumor activity.

[0146] For example, in the bacterial culture step, the supernatant may be added to a general-purpose agar medium in a Petri dish, and culture may then be carried out. This results in the formation of colonies on the medium. These colonies may be used as the bacteria isolated from tumors in the present invention. The temperature of the culture conditions is not particularly limited, but is preferably about 26 to 30°C, and the number of days for culture is preferably about 3 to 20 days or about 4 to 15 days. In the case of bacteria or complex bacteria containing bacteria capable of photosynthesis, the culture is preferably carried out under light irradiation (e.g., under a tungsten lamp). When culturing complex bacteria NITE BP-03627, the culture is preferably carried out under light irradiation.

[0147] The general-purpose agar medium may be, for example, a peptone-containing medium or a peptone-free medium. The peptone-free medium may be, for example, ATCC 543 medium. The general-purpose agar medium may be appropriately selected by those skilled in the art depending on the bacterium to be isolated. Furthermore, a medium containing or not containing cysteine ​​may be used. In the case of adding cysteine, the ratio (%) of the cysteine ​​content (g) in the agar medium to the amount (g) of the agar medium may be, for example, 0.1% to 10%, more preferably 1% to 5%, and even more preferably 2% to 4%.

[0148] Preferably, the colonies (particularly bacteria in the colonies) formed on the agar medium are further cultured in a liquid medium. The liquid medium may be a general-purpose liquid medium. As described above, the general-purpose liquid medium may be, for example, a peptone-containing medium or a peptone-free medium. The peptones and extracts contained in these media are as described above, and the same description also applies to the liquid medium. The liquid medium may be appropriately selected by those skilled in the art depending on the bacteria to be isolated.

[0149] (Culturing in a medium without added cysteine) In one embodiment, the liquid medium may be a liquid medium without added cysteine. The liquid medium may be, for example, a liquid medium without added cysteine, containing peptone, and containing an extract (particularly, yeast extract). The liquid medium may be, for example, an LB medium or a polypeptone medium without added cysteine. Alternatively, the liquid medium may be a liquid medium without added cysteine, without added peptone, and containing an extract (particularly, yeast extract). An example of such a medium is ATCC 543 medium without added cysteine. The bacteria cultured in the liquid medium may be further cultured on a general-purpose agar medium to form colonies. The general-purpose agar medium may be, for example, a general-purpose agar medium supplemented with deoxycholic acid. The ratio (%) of the content (g) of deoxycholic acid in the agar medium to the amount (g) of the agar medium may be, for example, 0.01% to 1%, preferably 0.03% to 0.5%, and more preferably 0.05% to 0.3%. The general-purpose agar medium may be the same agar medium (fresh medium) as the general-purpose agar medium described at the beginning of this step, except for the addition of deoxycholic acid. By this cultivation, colonies are formed on the medium. The formed colonies may be further cultured in a liquid medium. The liquid medium may be the same medium (fresh medium) as that used in the previous liquid medium cultivation. As described above, bacteria cultured in a medium to which cysteine ​​has not been added may be used as an active ingredient of the antitumor agent of the present invention.

[0150] (Culturing in a medium supplemented with cysteine) In another embodiment, the liquid medium may be a liquid medium supplemented with cysteine.

[0151] The present invention will be described in further detail below based on examples, etc. Note that the examples, etc. described below are examples of typical examples, etc. of the present technology, and the scope of the present invention should not be construed as being narrow.

[0152] Example 1

[0153] 1. Bacterial Culture The bacterial strains A-gyo, UN-gyo, and AUN used in this study were isolated from a tumor derived from a mouse colon cancer (Colon26, JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) (see Patent Documents 1 and 2 for isolation methods). A-gyo was cultured anaerobically at 26–30°C under a tungsten lamp in ATCC 543 medium containing 0.06% L-cysteine. UN-gyo, AUN, and commercially available Rhodopseudomonas palustris (ca-RP) (NBRC 16661) were cultured anaerobically at 26–30°C under a tungsten lamp in ATCC 543 liquid medium without cysteine. Commercially available Proteus mirabilis (ca-PM) (ATCC 35659) was cultured anaerobically at 30°C in NBRC802 medium in an incubator (i-CUBE FCI-280HG; AS ONE). ATCC 543 medium and NBRC 802 medium were prepared according to the cell bank preparation method. Bacterial counts and viability were confirmed by colony assays in addition to measurements using a bacterial counter (CASY Cell Counter & Analyzer; OMNI Life Science). Bacterial culture reagents were obtained from Nacalai Tesque, Tokyo Chemical Industry Co., Ltd., and Fujifilm Wako Pure Chemical Industries, Ltd.

[0154] The bacterial strain UN-gyo was Rhodopseudomonas Palustris, a purple non-sulfur bacterium of the genus Rhodopseudomonas. The bacterial strain A-gyo was Proteus mirabilis, a Proteus genus bacterium. The complex bacterium AUN was a complex bacterium consisting of the bacterial strains A-gyo and UN-gyo, and was composed of Rhodopseudomonas Palustris and Proteus mirabilis (CFU composition ratio of 97:3) (identification: Musashi) (accession number: NITE BP-03627): date of deposit: March 23, 2022, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD). Proteus mirabilis (ca-PM) (ATCC 35659) is publicly available from the American Type Culture Collection (ATCC).

[0155] 2. Characterization of bacteria The structures of A-gyo and ca-PM were negatively stained with 2% uranyl acetate and then observed using a high-resolution transmission electron microscope (JEM-1400Flash; JEOL) at an accelerating voltage of 100 kV. Microscopic observations were performed by Hanaichi Electron Microscopy Technology Institute Co., Ltd. On the other hand, optical microscopic observations of bacteria were performed using A-gyo or ca-PM suspensions (20 μL, 5 × 10 8 CFU / mL) were placed on a cover glass (AGC Technoglass) and performed at 20°C using a fluorescence microscope system (IX73, Olympus) equipped with an objective lens (×60, 1.35; UPLSAPO60X, Olympus or ×100, 0.95; PLFLN100X, Olympus). Bacterial motility assays were performed using A-gyo or ca-PM suspensions (10 μL, 4 × 10 6CFU (CFU) were plated onto ATCC 543 agar plates and colony morphology was observed. After 5 days of incubation under anaerobic conditions and a tungsten lamp, bacterial colonies derived from A-gyo or ca-PM were formed on the agar plates. A-gyo and ca-PM kinetic analysis was performed using an optical microscope system (BZ-X800, Keyence) and dynamic analysis application software (Keyence). Comparative bacterial genome and transcriptome analyses were performed by Relixa Co., Ltd. and Seibu Giken Co., Ltd. The minimum inhibitory concentrations of antibiotics against A-gyo, UN-gyo, and AUN were determined by assessing viable bacterial concentrations (OD600) using a microplate reader (Infinite M200 PRO; Tecan) at different antibiotic concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, and 50 μg / mL).

[0156] 3. Cell Culture. Colon26 and human normal diploid fibroblasts (MRC5) were obtained from the JCRB Cell Bank of the National Institutes of Biomedical Research and Development (NIBIO). The human colon adenocarcinoma cell line (HT29) was purchased from DS Pharma Biomedical. The human ovarian cancer cell line (SKOV3) and human pancreatic cancer cell line (BxPC3) were obtained from KAC Co., Ltd. All cell lines except MRC5 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, gentamicin, and penicillin-streptomycin (100 IU / mL). MRC5 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific) containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, gentamicin, penicillin-streptomycin (100 IU / mL), and Hank's Balanced Salt Solution (Thermo Fisher Scientific).

[0157] 4. Tumor spheroid 3D culture spheroid plate (Cell-able R Colon26 cells (1 × 10 cells) were cultured according to the instructions provided with the product (BP-96-R800; Toyo Gosei). 4 The spheroids were cultured at 37°C in a 5% CO2 atmosphere for 5 days in a humidified incubator. The medium was changed every two days. The spheroids were then inoculated with the complex bacteria AUN (1 × 10 7 A mixed medium (cell culture medium:bacterial culture medium = 1:1) containing 100 CFU was added, and the cells were co-cultured for 0, 24, 48, 72, 96, and 120 hours at 37°C in a humidified incubator containing 5% CO2. Observations were performed at 20°C using a fluorescence microscope system (IX73) equipped with a fluorescence mirror unit (IRDYE800-33LP-A-U01; Semrock) and an objective lens (magnification ×20, 0.75; UPLSAPO20X; Olympus). A control experiment without AUN was also performed.

[0158] 5. Antitumor test Colon26-bearing BALB / c-nu / nu mice (female, approximately 7 weeks old, n = 5, average body weight = 20 g, average tumor size = 200 mm 3 BALB / cCrSIc-nu / nu; Japan SLC) and complex bacterial AUN (1×10 7 CFU / mL or 7.8 × 10 9 A medium (200 μL) containing AUN (15 × 10 CFU / mL) was then administered into the tail vein. 9 The medium (200 μL) containing 1 × 10 CFU / mL was mixed with the complex bacterial AUN (1 × 10 7 Two days later, the mice were intravenously injected with 200 μL of PBS (200 μL). A control experiment using PBS (200 μL) was also performed. Tumor formation and overall health (survival rate and body weight) were monitored daily. Tumor volume was calculated as V = L × W 2 The survival rate of Colon26 tumor-bearing mice (n = 5) was also measured for 60 days after treatment. ... 3Mice that exceeded this time were considered to have reached the endpoint and were euthanized. PBS is an abbreviation for phosphate buffered saline.

[0159] To investigate various immunostimulants that enhance the immune system, such as polyinosinic:polycytidylic acid (Poly I:C), granulocyte-macrophage colony-stimulating factor (GM-CSF), vitamin D3, and imiquimod, BALB / c-nu / nu mice (female, approximately 6 weeks old, n = 5, average body weight = 18 g; BALB / cCrSIc-nu / nu; SLC, Japan) were injected via the tail vein with 200 μL of D-PBS (Nacalai Tesque) containing Poly I:C (Fujifilm Wako Pure Chemical Industries, Ltd.) (200 μg / head) or E. coli-derived recombinant mouse GM-CSF (Fujifilm Wako Pure Chemical Industries, Ltd.) (1 μg / head), or 200 μL of D-PBS containing 1% Cremophor EL (Nacalai Tesque) and vitamin D3 (Nacalai Tesque) (200 μg / head). Imiquimod hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) (400 μg / head) in D-PBS (Nacalai Tesque) (400 μL) was administered intraperitoneally to mice. 9 ATCC543 medium (200 μL) containing 100 CFU / mL of 1000 mg ...

[0160] The anticoagulant heparin was used to determine whether thrombosis caused by complex bacterial AUN could affect mouse survival. AUN (200 μL, 15 × 10 9 CFU / mL) were intravenously administered to BALB / c-nu / nu mice (female; approximately 6 weeks old; n = 5; average body weight = 18 g; BALB / cCrSIc-nu / nu; Japan SLC) 1 hour after the injection of saline (200 μL) containing heparin sodium salt (Nacalai Tesque) (5 mg / head). To study the effect of heparin on tumor efficacy, Colon26 tumor-bearing BALB / c-nu / nu mice (female; approximately 8 weeks old; n = 5; average body weight = 20 g; average tumor size 200 mm) were injected. 3BALB / cCrSIc-nu / nu; Japan SLC) were administered saline (200 μL) containing heparin (5 mg / head) 1 hour after AUN (15 × 10 9 Culture medium (200 μL) containing 1000 CFU / mL was administered into the tail vein.

[0161] To investigate the in vivo antitumor effect in human colon cancer, human ovarian cancer, and human pancreatic cancer models, 1 × 10 6 The cells were implanted in a 100 μL mixture of culture medium and Matrigel (Dow Corning, Corning) (v / v = 1:1) into the dorsal surface of nude mice (female; 5 weeks old; n = 5; average body weight = 18 g; BALB / cCrSIc-nu / nu; Japan SLC). Approximately 20, 30, or 60 days later (for HT29, SKOV3, or BxPC3-implanted model mice, respectively), tumors grew to a volume of approximately 400 mm. 3 (HT29) or 200 mm 3 (SKOV3 and BxPC3) reached AUN (1 × 10 7 The subjects were intravenously administered 200 μL of ATCC 543 medium containing 15 × 10 CFU / mL of microbial colonies. 9 A second dose of medium (200 μL) containing AUN (1 × 10 CFU / mL) was administered. 7 The mice were then intravenously administered 2 days later at 100 μL of PBS (200 μL). Control mice were administered 200 μL of PBS. Tumor volume and health status (survival rate and body weight) were assessed in the same manner as in the antitumor study of Colon26-inoculated BALB / c-nu / nu mice.

[0162] To generate a severely immunodeficient tumor-bearing mouse model, female severe combined immunodeficient (SCID) mice (5 weeks old; n = 5; average body weight = 18 g; CB-17 / Icr-scid / scidJcl; CLEA Japan) or non-obese diabetic (NOD)-SCID mice (5 weeks old; n = 5; average body weight = 18 g; NOD / ShiJic-scid; CLEA Japan) were inoculated with 1.0 × 10 IgG into the right dorsal side. 6Colon26 cells were inoculated into the mice with a 100 μL mixture of medium and Matrigel (Dow Corning) (v / v = 1:1). Ten days after cell transplantation, the first administration was administered with complex bacterial AUN (1 × 10 for SCID). 7 CFU / mL, 1 × 10 for NOD-SCID 8 Two days later, a second injection of mixed bacterial AUN (SCID 7 × 10 9 CFU / mL, NOD-SCID was 15 × 10 9 To evaluate the antitumor activity of a single dose of AUN, the complex bacterial AUN was administered intravenously at different concentrations (4.5 × 10 9 CFU / mL, 3.0 × 10 for NOD-SCID 9 A control experiment using PBS (200 μL) was also performed.

[0163] To investigate the efficacy of AUN in an orthotopic human pancreatic cancer model, BxPC3 (5 × 10 6 50 μL of medium containing AUN cells was surgically inoculated into the pancreas of mice (male; 8 weeks old; n = 5; average body weight = 30 g; BALB / cAJcl-nu / nu; CLEA Japan). The orthotopic human pancreatic cancer model was produced by Unitech Co., Ltd. Forty days later, the mice were inoculated with 200 μL of medium containing the complex bacteria AUN (1 × 10 cells in the double-administration group). 9 CFU / mL in the single-dose group, 7.8 × 10 9 Two days later, mice in the double-dose group were intravenously administered AUN (20 × 10 9 Culture medium (200 μL) containing 1000 CFU / mL of PBS was intravenously administered to the mice. The animals were euthanized on day 10, and the pancreas was harvested. The pancreas weight of each mouse treated with PBS or AUN was measured on the same day. An untreated control experiment (no cancer cells or bacteria inoculated) was also performed.

[0164] 6. Histological Evaluation of Tumors 9Colon26 tumor-bearing BALB / c-nu / nu mice (female; approximately 8 weeks old; n = 3; average body weight = 20 g; average tumor size: 400 mm) were intravenously administered with 1000 mg of 10 ... 3 BxPC3 orthotopic pancreatic tumor model mice (male; 11 weeks; n = 5; average body weight = 34 g; BALB / cAJcl-nu / nu; Japan SLC) were euthanized the following day or on day 10 of administration. BxPC3 orthotopic pancreatic tumor model mice (male; 11 weeks; n = 5; average body weight = 34 g; BALB / cAJcl-nu / nu; Japan SLC) were euthanized with a single dose of PBS (200 μL) or two doses of complex bacterial AUN (first dose: 200 μL, 1.0 × 10 9 CFU / mL, 2nd time: 200 μL, 20×10 9 The mice were euthanized 10 days after administration (CFU / mL, both intravenous administration). Tumor and vital organ tissues were then collected for immunohistochemical staining. Staining and specimen preparation were performed at Biopathology Research Institute Co., Ltd. according to standard protocols. Primary tumors were surgically excised, fixed in 4% formalin, embedded in paraffin, and sectioned at 3-4 μm thickness. After incubation with primary antibodies (Table 1), sections were stained with hematoxylin and examined under light microscopy (IX73). Positively stained areas in tumor tissue were analyzed using a light microscope system (BZ-X800) and Hybrid Cell Count and Micro Cell Count software (Keyence).

[0165]

[0166] 7. Flow Cytometry PBS (200 μL) or complex bacterial AUN (200 μL, 7.8 × 10 9 Colon26 tumor-bearing mice (female; approximately 8 weeks old; n = 3; average body weight = 18 g; average tumor size = 400 mm) were intravenously injected with 1000 mg / mL of IgG4-10 ... 3Mice were euthanized the day after administration of either BALB / cCrSIc-nu / nu or BALB / cCrSIc (Japan SLC). To analyze immune cells in tumors, tumors were harvested from mice in different groups 24 hours after intravenous injection and homogenized into single-cell suspensions (Handy Homogenizer; Thermo Fisher Scientific). Red blood cells were then removed with ACK lysis buffer (Thermo Fisher Scientific), and contaminants were removed using a cell strainer (mesh size = 40 μm, SureStrain; MTC Bio). 1 × 10 cells were collected. 6 Samples containing 4 × 10 cells were stained with antibodies conjugated with KIRAVIA Blue 520, Alexa Fluor 488, or BD Horizon BB515 (Table 1) according to the manufacturer's protocol, with 4 × 10 cells per sample. 4 cells were analyzed by flow cytometry (CyFlow Cube 6; Sysmex).

[0167] 8. Quantitative qPCR (PCR) PBS (200 μL) or complex bacterial AUN (200 μL, 7.8 × 10 9 Colon26 tumor-bearing mice (female; approximately 8 weeks old; n = 3; average body weight = 18 g; average tumor size = 400 mm) were intravenously injected with 1000 mg / mL of IgG4-10 ... 3Mice were euthanized the day after administration of either BALB / cCrSIc-nu / nu or BALB / cCrSIc; Japan SLC. To analyze immune cells and cytokines in tumors, tumors were collected from mice 24 hours after intravenous administration and the tissue was homogenized using a homogenizer (Thermo Fisher Scientific). qPCR was performed using a QuantStudio 1 PCR System (Thermo Fisher Scientific) to examine the relative gene expression of CD3, CD19, CXCR4, F4 / 80, NK, IFN-γ, and TNF-α using gene-specific primer-probe combinations (Thermo Fisher Scientific) via TaqMan chemistry (Table 2). Endogenous controls were determined using a 96-well TaqMan Array Mouse Endogenous Control Plate (Thermo Fisher Scientific). Reactions were performed in triplicate using GAPDH as an endogenous control. The thermal cycling parameters that yielded optimal amplification were 40 cycles of 50°C for 2 minutes for AmpErase UNG activation, 95°C for 2 minutes for AmpliTaq Gold DNA Pol. activation, 95°C for 1 second for denaturation / melting, and 60°C for 20 seconds for annealing and extension. Ten-fold serial dilutions were performed for the test genes and endogenous control standard to estimate PCR detection efficiencies, which ranged from 90 to 100%. Results were analyzed and presented as fold changes relative to the control (log10 relative quantification).

[0168]

[0169] 9. Blood Tests. Complete blood counts were measured using a Celltac α automated clinical hematology analyzer (MEK6558; Nihon Kohden), and biochemical parameters were evaluated by Oriental Yeast Co. Cytokines were measured using the Bio-Plex Multiplex Immunoassay System (Bio-Rad) and the Bio-Plex Pro Mouse Th17 Panel 6-Plex (Bio-Rad). Factor VII activity was measured using the Facteur VII Chromogenic Assay BIOPHEN FVII kit (Hyphen-BioMed). BALB / cCrSlc-nu / nu mice (female; 6 weeks old; n = 5; average body weight = 18 g; Japan SLC) were inoculated with complex bacterial AUN (200 μL, 7.8 × 10 6 CFU / mL or 1.0 × 10 7 Mice were given a second injection of AUN-containing medium (200 μL, 15 × 10 CFU / mL) or PBS (200 μL) via the tail vein. After 48 hours, mice were given a second injection of AUN-containing medium (200 μL, 15 × 10 CFU / mL). 9 CFU / mL) were intravenously administered. Blood samples were collected from the inferior vena cava of mice 3, 6, 24, 48, 72, and 240 hours after the second administration. A control experiment without bacterial administration was also performed as time 0. Plasma immunoglobulin (IgG and IgM) concentrations were measured by Oriental Yeast Co. using a Mouse IgG ELISA kit (Bethyl Laboratories) and a Mouse IgM ELISA kit (Bethyl Laboratories). In this study, BALB / cCrSlc-nu / nu mice (female; 6 weeks old; n = 5; average body weight = 18 g; CLEA Japan) were inoculated with the complex bacterial AUN (200 μL, 1.0 × 10 7 Blood samples were collected 48 hours after administration of the IgG-1 antibody (CFU / mL). Plasma was then separated by centrifugation (MX-310; TOMY) at 3500 rpm for 5 minutes at 4°C. Control blood samples were obtained from untreated BALB / cCrSlc-nu / nu mice (female, 6 weeks old, n = 5, average body weight = 18 g; Japan SLC).

[0170] 10. Colony Assay. BALB / c-nu / nu mice (female; 6 weeks old; n = 3; average body weight = 18 g; BALB / cCrSIc-nu / nu; Japan SLC) were inoculated with complex bacterial AUN (1 × 10 7 CFU / mL or 1 × 10 9 Culture medium (200 μL) containing 100 CFU / mL of IgG was administered intravenously through the tail vein. A control experiment using PBS (200 μL) was also performed. Blood samples were collected from the inferior vena cava of the mice 5 minutes and 6 hours later. Each blood sample (100 μL) was plated on an agar plate. After 7 days of anaerobically culture, the formed bacterial colonies were imaged. To count the bacterial colonies, the supernatant was diluted 10, 100, and 1000 times with PBS, and then 5 μL of the undiluted supernatant and diluted samples were plated on agar plates in the same manner as above. The formed bacterial colonies were manually counted.

[0171] 11. In vitro anticancer activity To evaluate the in vitro anticancer activity of the complexed bacteria AUN, Colon26 cells were cultured in a 96-well plate at 1 × 10 4 After 24 hours, the plates were seeded with the complex bacterial AUN (5 × 10 5 , 5×10 6 , 5×10 7 , 5×10 8 , and 5 × 10 9 CFU / mL) were added and co-cultured with these adherent cells in medium (RPMI 1640:ATCC 543 = 1:1) at 37°C in a humidified incubator containing 5% CO2 for 24 hours. Afterwards, the cells were washed with fresh medium, and viability was measured using a cell assay kit (CCK-8, DOJINDO).

[0172] 12. AUN (A-gyo) structural transformation Colon26 cells were cultured in a 96-well plate at 1 x 10 4 Cells were seeded at a density of 5 × 10 cells / 100 μL and allowed to adhere overnight. Cells were then cultured in a humidified incubator containing 5% CO2. 7The cells were exposed to a medium containing a complex bacterial AUN at 0.575 cells / mL for 24 hours at 37°C. The culture medium consisted of a 1:1 volumetric mixture of cell culture medium and bacterial culture solution. After thorough washing with PBS, the behavior of the bacteria and Colon26 cells was observed at 20°C using an optical microscope system (BZ-X800). In control experiments, cells were cultured in a 1:1 volumetric mixture of cell and bacterial culture medium in a humidified incubator containing 5% CO2 for 24 hours at 37°C, and the size and shape of the cells were observed using an optical microscope.

[0173] To observe A-gyo swarmer and thrombus formation in tumor tissue, Colon26 tumor-bearing BALB / c-nu / nu mice (female; approximately 7 weeks old; n = 3; average body weight = 20 g; average tumor size = 200 mm) were cultured. 3 BALB / cCrSIc-nu / nu; Japan SLC) in PBS (200 μL) or complex bacterial AUN (200 μL, 7.8 × 10 9 The mice were euthanized 24 hours after intravenous administration of 1000 CFU / mL of the microbial colonies. Tumor and blood samples were collected from the mice treated with complex bacterial AUN or PBS and from the untreated mice, respectively, and observed at 20°C using an optical microscope system (BZ-X800).

[0174] 13. Statistical Analysis. All experiments, with some exceptions, were repeated three or more times. Quantitative values ​​are expressed as the mean ± standard error of the mean from at least three independent experiments. Statistical differences were determined by two-tailed Student's t-test, two-way analysis of variance (ANOVA), or the log-rank test (Mantel-Cox) using GraphPad Prism, version 9.4.0 (GraphPad Software). A p-value of less than 0.05 was considered statistically significant.

[0175] 14. Steroids. Various steroids [prednisolone (Fujifilm Wako Pure Chemical Industries), hydrocortisone (Fujifilm Wako Pure Chemical Industries), or dexamethasone (Fujifilm Wako Pure Chemical Industries)] were dispersed in an injection solution containing 10% DMSO. BALB / c mice (female; 5 weeks old; n = 5; average body weight = 18 g; BALB / cCrSIc-nu / nu; Japan SLC) were orally or subcutaneously administered 400 μL of this steroid dispersion [prednisolone (0.1 mg / head), hydrocortisone (0.08 mg / head), dexamethasone (2 mg / head)]. 24 hours after administration of the various steroids, AUN (5 × 10 9 Culture medium (200 μL) containing 100 CFU / mL of HIV-1 was administered intravenously to the tail vein, and changes in mouse weight were measured daily for 5 days.

[0176] <Results of Example 1> 1-1. Microbial Characteristics of the Complex Bacterium AUN To clarify the genomic characteristics of the complex bacterium AUN, a comparative whole-genome analysis of A-gyo and UN-gyo, which constitute the complex bacterium AUN, was performed, comparing them with commercially available Proteus mirabilis (ca-PM) and Rhodopseudomonas palustris (ca-RP) strains. A-gyo completely lacked virulence factors such as cilia and adhesins. In contrast, UN-gyo was originally isolated from a solid tumor after intravenous administration of ca-RP, and its genomic characteristics were completely consistent with those of ca-RP. These results indicate that the gene function of UN-gyo is essentially identical to that of ca-RP, and that genetic mutations in ca-RP did not occur when the bacterium was physically separated from the tumor after administration to mice. Transmission electron microscopy confirmed that although several long flagella were observed on the surface of A-gyo, cilia were clearly absent. Ca-PM showed a characteristic wavy pattern on agar plates due to swarming, whereas tumor-resident A-gyo failed to form such a pattern due to the lack of genetic factors for cilia and adhesins. Similarly, in vitro bacterial motility assays demonstrated that A-gyo moved slower than ca-PM, likely due to a genetic defect in the motility function of A-gyo's fimbriae. Using fluorescence microscopy and colony assays, we confirmed that the ratio of A-gyo to UN-gyo in complex AUN was consistently 3:97, even after repeated subcultures. Transcriptome analysis revealed that complex AUN exhibited a completely different gene expression pattern from purified single-strain UN-gyo, despite the majority of bacteria in the AUN being UN-gyo. Furthermore, the bioactivity of A-gyo was significantly suppressed by the presence of UN-gyo in complex AUN compared to that of monocultured A-gyo. Even more surprisingly, compared with the single strain UN-gyo, the complex AUN specifically increased the expression of genes related to extracellular iron acquisition for siderophore and heme metabolism, likely through crosstalk between bacteria, such as biochemical reactions and bacterial signaling.Cancerous tumors typically use iron for cancer development, tumor growth, and metastasis. The complex bacterial AUN absorbs and degrades hemoglobin from blood cells in the tumor microenvironment, potentially causing tumor-specific hemolysis and thrombosis. Therefore, we believe that the increased iron requirement of the complex bacterial AUN is one of the key bacterial characteristics that induces potent anti-cancer regression. Furthermore, the viability of the complex bacterial AUN, consisting of A-gyo and UN-gyo, which has potent anti-cancer activity, can be completely controlled by antibiotics (Figure 1).

[0177] Figure 1 shows the results of minimum inhibitory concentration tests of various antibiotics against complex bacterial AUN. The antibiotics used were ampicillin, cefotaxime, imipenem, aztreonam, amoxicillin, gentamicin, erythrosine, minocycline, clindamycin, levofloxacin, and vancomycin. Antibiotics that have strong effects on complex bacterial AUN include ampicillin, cefotaxime, imipenem, amoxicillin, gentamicin, minocycline, and levofloxacin. Antibiotics that have weak effects on complex bacterial AUN include aztreonam, erythrosine, clindamycin, and vancomycin.

[0178] 1-2. Antitumor Effects of AUN in Various Immunodeficiency Model Mice. At the start of this study, we believed that cytotoxic immune cells play a major role in achieving the antitumor effects of AUN, as in conventional bacteria-based cancer therapies. Therefore, we did not expect that the bacterial AUN complex would exert a sufficient antitumor effect in the bodies of immune cell-deficient mouse models. Figure 2 shows the antitumor effects of AUN in various immunodeficiency models. We examined the antitumor effects of AUN administered intravenously once or twice in BALB / c-nu / nu mice bearing murine Colon26 tumors. Specifically, we generated BALB / c-nu / nu mice bearing Colon26 tumors and examined the time course of treatment. After tumor formation, mice were intravenously injected with the bacterial AUN complex once or twice over time. In the case of a single administration, AUN (7.8 × 109 CFU / mL) were intravenously administered (starting from day 0) and observed for 50 days. In the case of two-dose administration, a low dose of AUN (1 × 10 7 CFU / mL) was administered intravenously (starting from day 0), and then 2 days later, a high dose of AUN (15 × 10 9 CFU / mL) was administered intravenously and the mice were observed for 48 days.

[0179] Representative results from this study are shown in Figures 2A to 2D. The reason for choosing BALB / c-nu / nu mice as the initial experiment is that athymic BALB / c-nu / nu mice lack cytotoxic T lymphocytes, which are essential for the expression of antitumor effects.

[0180] Contrary to our expectations, the complex bacterial AUN was 7.8 × 10 9 CFU / mL (7.8 × 10 10 CFU / kg) or a combination of low and high doses of bacteria AUN (1 × 10 7 CFU / mL (1 × 10 8 CFU / kg) then 15 x 10 9 CFU / mL (15 × 10 10 Two doses of a bacterial suspension containing a combination of AUN (1000 mg / kg, 1000 mg / kg CFU / kg) (also called "double dose") were found to exhibit dramatic antitumor effects. Tumors turned black within 24 hours after intravenous injection of a single dose of AUN and after the second injection of a double dose of AUN. This color change is thought to be due to tumor-specific thrombosis.

[0181] The group receiving a single dose of the complex AUN bacteria initially showed significant tumor suppression, but cancer recurrence was observed by day 13. In contrast, the control group receiving PBS showed no tumor regression at all. 9 Because mice died within 1–2 days after intravenous administration when administered with complex bacterial AUNs exceeding CFU / mL, we determined that the maximum single dose of complex bacterial AUNs was 7.8 × 10 9 CFU / mL (7.8 × 1010 CFU / kg. We used a low dose of complex bacterial AUN (1 × 10 7 CFU / mL (1 × 10 8 Two days after intravenous injection of 100 mg ... 9 We found by chance that mice did not die even when they were administered 1 × 10 CFU / mL of the complex bacterial AUN (Fig. 2A-D). 7 CFU / mL (1 × 10 8 CFU / kg), 2nd dose: 15 x 10 9 CFU / mL (15 × 10 10 The combined bacterial AUN (CFU / kg) achieved a 100% complete tumor response (CR) rate and significantly prolonged mouse survival. Furthermore, no mice showed significant weight loss (>20%), a sig- nificant ethical endpoint, after single or double administration of the combined bacterial AUN. These results suggest that the combined bacterial AUN itself has low toxicity. Furthermore, colony assay results indicated that the combined bacterial AUN exhibited high tumor targeting activity while also being completely eliminated from the body within 240 hours after tumor disappearance in mice (Table 3).

[0182] We further investigated the potential anticancer effects of complex bacterial AUN, which are not mediated by the systemic immune system, using severe combined immunodeficient (SCID) mice and non-obese diabetic (NOD-SCID) mice as other immunodeficiency models.

[0183] Specifically, we generated Colon26 tumor-bearing SCID and NOD-SCID mice and examined the time course of treatment. After tumor formation, the mice were intravenously injected with two doses of the complex bacterial AUN. In the SCID mouse model, 10 days after Colon26 inoculation, a low dose of AUN (1 × 10 7 CFU / mL) (starting from day 0) was intravenously administered, and then 2 days later, a high dose of AUN (15 × 10 9 In the NOD-SCID mouse model, a low dose of AUN (1 × 10 CFU / mL) was administered intravenously 10 days after inoculation with Colon26.8 CFU / mL) (starting from day 0) was intravenously administered, and then 2 days later, a high dose of AUN (15 × 10 9 CFU / mL) was administered intravenously and the mice were observed for 48 days.

[0184] Representative results from this study are shown in Figures 2E-2H. SCID mice are characterized by a lack of functional T and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment. In contrast, NOD-SCID mice exhibit the same immune dysfunction as SCID mice, as well as significantly reduced natural killer (NK) cell, macrophage, and complement activity. Consequently, a single dose of complex bacterial AUN (4.5 × 10 in SCID mice) significantly reduced the number of NK cells, macrophages, and complement activity. 9 CFU / mL in NOD-SCID mice, 3.0 × 10 9 CFU / mL) achieved partial response in both mice to Colon26 tumors. 7 CFU / mL (1 × 10 8 CFU / kg), 2nd dose: 7 x 10 9 CFU / mL (7 × 10 10 CFU / kg), NOD-SCID, 1st dose: 1 x 10 8 CFU / mL (1 × 10 9 CFU / kg) 2nd dose: 15 x 10 9 CFU / mL (15 × 10 10 In both immunodeficient mouse models, the combined bacterial AUN (CFU / kg) achieved a 100% CR rate and a complete survival rate of at least 30 days. The control PBS group showed no antitumor effect. Furthermore, single and double administrations of the combined bacterial AUN did not induce severe weight loss in either immunodeficient model. These results clearly demonstrate that the combined bacterial AUN may serve as a useful therapeutic agent for effective tumor targeting and elimination in various immunodeficiency models.

[0185]

[0186] We also investigated the bacterial count of complex bacterial AUN within tumors (Table 4). As shown in Table 4, we found that the bacterial count of complex bacterial AUN within tumors decreased with tumor regression. We also investigated the growth performance of complex bacterial AUN in a tumor-mimicking environment (Table 5). As shown in Table 5, complex bacterial AUN was able to grow at all oxygen concentrations, suggesting that it could grow sufficiently within tumors. Based on this, we believe that complex bacterial AUN can grow in the intratumor environment, but that the bacterial count decreases and disappears with tumor regression.

[0187]

[0188]

[0189] 1-3. Response to Two-Dose Administration Improves Mortality Rate. Next, by examining the systemic immune response and intravascular cell behavior, the inventors clarified why two-dose administration of the conjugated bacterial AUN still exhibited a potent anti-cancer effect without causing lethal toxicity in mice. Representative results from this study are shown in Figures 3A to 3G. Figures 3A to 3G show the biocompatibility of a lethal dose of the conjugated bacterial AUN administered two times. Figures 7A to 7D show the results of combined administration of heparin and conjugated bacterial AUN. Figure 7 examines the generation of BALB / c-nu / nu mice implanted with Colon26 tumors and the time course of treatment. After tumor formation, mice were intravenously injected with heparin and conjugated bacterial AUN over time. Ten days after inoculation with Colon26, heparin (5 mg / head) was intravenously administered (starting at 0 hours), followed one hour later by a high-dose AUN (15 × 10 9 CFU / mL) was administered intravenously and the mice were observed for 50 days.

[0190] First, bacterial colonies of the complex bacteria AUN were quantified by a single dose of complex bacteria AUN (7.8 × 10 9 CFU / mL (7.8 × 10 10 CFU / kg) or two doses (1 × 10 7 CFU / mL (1 × 10 8 CFU / kg) then 7.8 × 10 9 CFU / mL (7.8 × 10 10The number of colonies obtained from blood samples collected 5 minutes and 6 hours after administration of the complex bacterial AUN was confirmed by colony assay of blood samples collected 5 minutes and 6 hours after administration. After two doses of the complex bacterial AUN, the number of colonies obtained from blood was significantly reduced compared with that after a single dose, especially at 6 hours. This result suggests that the complex bacterial AUN is effectively eliminated by immune activation through repeated administration. In fact, flow cytometry analysis showed that phagocytes such as macrophages and neutrophils were able to effectively eliminate the complex bacterial AUN at a low dose (1 × 10 7 CFU / mL (1 × 10 8 CFU / kg) administration, it was shown to be actively induced in the spleen 2 days after administration.

[0191] In addition, various inflammatory cytokines, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-17A (IL-17A), and anti-inflammatory cytokine, interleukin-10 (IL-10), were significantly reduced by a single administration of the complex bacterial AUN (7.8 × 10 9 CFU / mL) and two doses (first dose: 1 × 10 7 CFU / mL (1 × 10 8 CFU / kg), 2nd dose: 7.8×10 9 CFU / mL (7.8 × 10 10 CFU / kg), or 1st dose: 1×10 7 CFU / mL (1 × 10 8 CFU / kg), 2nd dose: 15 × 10 9 CFU / mL (15 × 10 10 Interestingly, the expression of inflammatory cytokines after the second administration of the complex bacterial AUN (7.8 × 10 9 CFU / mL (7.8 × 10 10 CFU / kg) was 0.01 mg / mL, and 0.01 mg / mL was 0.01 mg / mL. 9 CFU / mL (7.8 × 10 10 The cytokine expression levels after immunization were suppressed compared with those after immunization.

[0192] On the other hand, the levels of pathogen-susceptible immunoglobulins (e.g., IgG and IgM) in blood samples were significantly increased by a low dose of complex bacterial AUN (1 × 10 7 There were no significant changes two days after a single bacterial injection of 1000 CFU / mL. This suggests that adaptive immune responses, typically driven by B cells, are not initiated within such a short period (only 2 days). It is also known that the effects of a typical vaccination (adaptive immune response) take approximately 2 months. In contrast, white blood cell (WBC) and platelet (PLT) counts were significantly altered by single or double administration of the complex bacterial AUN, but other hematological parameters, such as hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and red blood cell count (RBC), were completely unchanged. Notably, after the first administration of two doses of complex bacterial AUN, the white blood cell count increased compared to the baseline value (approximately 4,700 / μL) obtained at time 0, whereas the platelet (PLT) count rapidly decreased over time. These results suggest that the populations of phagocytic immune cells, such as macrophages and neutrophils, increased, whereas the blood coagulation factor PLTs were consumed to eliminate the complex bacterial AUN after administration of a low dose of complex bacterial AUN. Therefore, we believe that the trade-off between phagocyte activation and thrombogenic PLTs is a key factor in maintaining potent antitumor efficacy while avoiding lethality in mice.

[0193] We investigated whether the toxicity that caused lethality was due to the direct pathogenicity of the complex bacterial AUN itself or indirect thrombosis mediated by the complex bacterial AUN (Table 6, Figures 7A-7E). We prepared and tested various drugs that could actively eliminate complex bacterial AUN as follows. We then tested lethal doses of complex bacterial AUN (>7.8 × 10 cells) in the presence of immunostimulants, polyinosinic acid:polycytidylic acid (poly i:c), granulocyte-macrophage colony-stimulating factor (GM-CSF), vitamin D3, imiquimod, and the anticoagulant heparin to prevent thrombosis. 9 CFU / mL (> 7.8 × 10 10 The survival rate of mice administered 1000 CFU / kg was examined.

[0194] The results showed that only the heparin-treated mice were able to tolerate a single high dose of complex bacterial AUN (15 × 10 9 CFU / mL (15 × 10 10 CFU / kg). On the other hand, all mice injected with the immunostimulant died within 48 hours after a single high-dose administration of the complex bacterial AUN. These results clearly demonstrate that the cause of death in mice caused by a lethal high dose of complex bacterial AUN is not the pathogenicity of the complex bacterial AUN, but rather thrombosis mediated by the complex bacterial AUN.

[0195] Clinical hematology and immunohistochemical (IHC) staining of vital organs confirmed that two doses of complex bacterial AUN were safe in all mouse models evaluated (BALB / c-nu / nu, SCID, and NOD-SCID). However, biochemical parameters such as alanine transaminase (ALT), aspartate aminotransferase (AST), amylase (AMY), and lactate dehydrogenase (LDH) in blood were likely somewhat affected by two doses of complex bacterial AUN, especially in NOD-SCID mice, which are inherently more vulnerable to bacterial infection due to their severe immunodeficiency.

[0196] In summary, the two-dose regimen, consisting of a low dose followed by a high dose of complex bacterial AUN, and the multi-dose regimen, consisting of a low dose followed by multiple high doses of complex bacterial AUN, are thought to have demonstrated potent antitumor effects and high biocompatibility through moderate consumption of PLTs and proliferation of intravascular phagocytes.

[0197] Although low-dose administration of the complex bacteria AUN causes mild thrombus formation in blood vessels, it also consumes platelets and induces proliferation of phagocytes. This platelet consumption prevents or suppresses thrombosis due to subsequent high-dose administration, making high-dose administration possible. Furthermore, proliferation of phagocytes contributes to antitumor activity. Furthermore, administering a high-dose at a predetermined interval after a high-dose administration does not result in lethality, but only produces mild thrombus, making it possible to administer the next high-dose. In other words, this multi-administration method allows for intermittent high-dose administration or repeated high-dose administration at predetermined intervals.

[0198] Furthermore, we have newly discovered that high-dose administration of complex bacterial AUN can cause thrombosis, a side effect. We believe that this side effect can be suppressed by administering an antithrombotic drug such as heparin at the same time or beforehand. It is believed that the administration method of high-dose complex bacterial AUN in combination with this side-effect suppressor also showed strong antitumor effects and high biocompatibility. Furthermore, intermittent administration of high doses or repeated administration of high doses at specified intervals by simultaneous or prior administration of an antithrombotic drug is also possible.

[0199] In particular, a multi-administration method in which low-dose administration or administration of an antithrombotic agent is combined with repeated high-dose administration allows for administration of a high dose after low-dose administration depending on the tumor condition, such as when the tumor begins to grow again. Therefore, this multi-administration method has the advantage of reducing the risk of thrombosis and quickly exerting a strong antitumor effect. Furthermore, this multi-administration method also has the advantage that, after low-dose administration, high doses can be repeatedly administered over a long period of time, thereby exerting a strong antitumor effect over a long period of time.

[0200]

[0201] 1-4. Mechanism of tumor suppression Next, the factors behind the potent antitumor effect of the complex bacterial AUN in immunodeficient mice bearing Colon26 tumors were investigated from multiple angles using IHC staining, quantitative polymerase chain reaction assays, flow cytometry analysis, and other methods. The key results of this study are shown in Figures 4 to 8. Figure 4 shows the expression levels of cytokine-related biomarkers.

[0202] Figure 5 shows the mechanism of tumor suppression by AUN. Figure 6 shows the antitumor effect of AUN on model mice transplanted with various human cancer cells. Figure 6A shows the generation of BALB / c-nu / nu mice bearing various human tumors (HT29, SKOV3, BxPC3) and the time course of treatment. After tumor formation, the mice were intravenously injected with two doses of PBS or complex bacterial AUN. In the HT29 model, 20 days after inoculation of HT29, a low dose of AUN (1 × 10 7 CFU / mL) (starting from day 0) was intravenously administered, followed 2 days later by a high dose of AUN (15 × 10 9 CFU / mL) were intravenously administered and observed from the start to 38 days later. In the SKOV3 model, a low dose of AUN (1 × 10 7 CFU / mL) (starting from day 0) was intravenously administered, followed 2 days later by a high dose of AUN (15 × 10 9 In the BxPC3 model, a low dose of AUN (1 × 10 CFU / mL) was administered intravenously 60 days after inoculation of BxPC3. 9 CFU / mL) (starting from day 0) was intravenously administered, and then 2 days later, a high dose of AUN (20 × 10 9 CFU / mL) was administered intravenously and observations were conducted for 10 days after the initial administration.

[0203] Figures 6B and 6C show the generation of BALB / c-nu / nu mice orthotopically implanted with BxPC3 tumors and the time course of treatment. Mice were intravenously administered PBS or complexed bacterial AUN once or twice. (In the case of single administration, AUN (7.8 × 10 9 CFU / mL) (starting from day 0) was intravenously administered, and observation was conducted from the start until 10 days later. In the case of two-time administration, a low dose of AUN (1 × 10 9 CFU / mL) (starting from day 0) was intravenously administered, and then 2 days later, a high dose of AUN (20 × 10 9 CFU / mL) was administered intravenously and observations were conducted from the start of the study up to 10 days later.

[0204] Figure 7 shows the antitumor effect of AUN in heparin-treated mice, as described in "1-3. Response to two doses to improve mortality rate."

[0205] FIG. 8 shows the changes in body weight of mice before and after administration of complex bacterial AUN with anti-inflammatory drug treatment.

[0206] Surprisingly, all three methods, IHC staining, qPCR, and flow cytometry analysis, demonstrated that the complex bacterial AUN (7.8 × 10 9 CFU / mL (7.8 × 10 10 Twenty-four hours after administration of 1000 mg of AUN (1000 CFU / kg), the number of tumor-infiltrating immune cells was not increased within solid tumors (Colon26) in BALB / c-nu / nu immunodeficient mice, and the expression of inflammatory cytokines such as TNF-α and IFN-γ was observed. The expression of inflammatory cytokines is likely due to vascular damage caused by the invasion of complex bacterial AUN into intratumoral blood vessels (Figure 4). These results clearly demonstrate that the expression of immune cells does not play a crucial role in the potent antitumor effect of complex bacterial AUN.

[0207] On the other hand, the combined bacterial AUN administration (7.8 × 10 9 The tumors excised 24 hours after administration of the complex bacterial AUN were clearly discolored dark red due to the advanced tumor-selective thrombosis mediated by the complex bacterial AUN. The specific activity of factor VII, a blood coagulation protein in the coagulation molecular cascade, was measured over time. The factor VII activity was 7.8 × 10 at 24 hours after administration of the complex bacterial AUN (7.8 × 10 ). 9 CFU / mL) reached a peak (Fig. 5A).

[0208] To further explore the mechanism by which bacterial administration leads to tumor suppression and tumor-specific thrombosis, we examined tumors stained with hematoxylin and eosin (H&E) and IHC. H&E staining revealed clear tumor damage with cellular structural destruction in mice treated with the combined bacterial AUN. TUNEL and TNF-α staining revealed widespread apoptotic cell death and a strong inflammatory response (Figure 5B). We also found that the expression of the blood coagulation protein fibrinogen was significantly increased in tumor sections from mice treated with the combined bacterial AUN. In the control experiment (PBS-treated group), no staining for TUNEL, TNF-α, or fibrinogen was observed, but tumors from the PBS-treated group also showed typical tumor histological features, such as dense cellular organization and nuclear atypia.

[0209] All of the above cytokines, including TNF-α and IFN-γ, work together with factor VII, PLTs, and fibrinogen to expand intratumoral vascular porosity, potentially leading to blood inflow and tumor-specific thrombosis. Tumor-specific thrombosis induced by complex bacterial AUNs may block the blood supply to tumors, which could be an effective strategy for inhibiting tumor angiogenesis. Furthermore, complex bacterial AUN-mediated tumor-specific thrombosis may promote intratumoral hypoxia, potentially increasing the proliferation and therapeutic efficacy of complex bacterial AUNs.

[0210] At the very least, these results may provide a new technique demonstrating that natural complex bacteria without any genetic engineering techniques can achieve complete tumor remission (CR) through tumor-specific thrombosis. Interestingly, heparin (5 mg / head) was administered to Colon26-bearing BALB / c-nu / nu mice, and 1 hour later, a high dose of complex bacterial AUN (15 × 10 9 CFU / mL (15 × 10 10Administration of AUN at high doses (1000 CFU / kg) caused obvious tumor discoloration, but suppressed the effects of systemic thrombosis and prevented mouse death (Fig. 7A-7E). All mice survived despite high-dose administration of AUN, regardless of antithrombotic agents such as warfarin, argatroban, or heparin (Fig. 7E). This suggests that tumor-specific thrombosis induced by platelets (PLTs) contributes in part to the potent antitumor effect of the complex bacterial AUN.

[0211] Next, we further examined the oncolytic potential of AUN in a Colon26 cancer cell-derived spheroid model using optical microscopy, which allows observation of the near-infrared (NIR) fluorescence characteristics of the complex bacterial AUN. This revealed that after incubation with AUN, the tumor spheroids formed extracellular and intracellular biofilms derived from the complex bacterial AUN within and around the spheroids, leading to the destruction of the spheroid structure over time. On the other hand, after 24 h of coculture with Colon26 cells, the complex bacterial AUN exhibited significant cytotoxicity at several bacterial concentrations. Conversely, human normal diploid fibroblasts (MRC5) showed some resistance to AUN. The positive control RIPA buffer showed strong cytotoxicity to both cell lines (Fig. 5C). After 24 hours of incubation, A-gyo and UN-gyo bacteria in any concentration of complex bacterial AUN were observed under an optical microscope. Surprisingly, A-gyo transformed from short swimming cells (swimmers) (approximately 2.5 μm) to elongated, filamentous cells (swarmers) (approximately 50 μm). By utilizing flagella, ca-PMs exhibit the same unique transformation ability on solid surfaces, and the differentiation of their motile swarmer cells parallels the increased expression of several virulence factors. Notably, the transformation of A-gyo was specifically observed only when complex bacterial AUNs were cocultured with Colon26 cells, but not with MRC5 cells, possibly due to the bacteria's selective sensitivity to chemical signals emitted by cancer cells. Meanwhile, A-gyo itself nonspecifically formed filamentous structures in both Colon26 and MRC5 cells, although A-gyo swarmers were more abundant in Colon26 than in MRC5. Complex bacteria AUN showed more filamentous swarmers after co-culture with Colon26 cells, but not after treatment with A-gyo. In contrast to complex bacteria AUN and A-gyo, UN-gyo did not show any distinct clumped, long filaments after co-culture with both Colon26 and MRC5 cells.

[0212] These results clearly demonstrate that the complex bacterial AUN has the ability to transform into a cancer-selective A-gyo swimmer, more potently than the pure bacterial strains A-gyo and UN-gyo. Furthermore, 24 hours after intravenous administration of the complex bacterial AUN, numerous fibrous structures derived from A-gyo and clumped thrombosis were observed in blood vessels of tumor tissues from Colon26 tumor-bearing nude mice. In contrast, the PBS group showed no fibers and no severe thrombosis. Clotted blood samples did not show fibrous structures. Therefore, we believe that the fibrous A-gyo swimmer not only contributes to the effective elimination of cancer cells in the immunocompromised tumor microenvironment, but also contributes to tumor-specific thrombosis in blood vessels, even after treatment with the anticoagulant heparin.

[0213] Summarizing these results, we conclude that the potent antitumor activity of the complex bacterial AUN is primarily due to 1) iron depletion in the tumor environment due to increased iron requirement of AUN resulting from bacterial interactions (suggested by transcriptome analysis), 2) promotion of hypoxia through cancer cell starvation and tumor-specific thrombosis, and 3) physical destruction of tumor cells through the oncolytic activity of the complex bacterial AUN.

[0214] We also examined the changes in body weight before and after bacterial administration with an anti-inflammatory agent (Figures 8A and 8B). Specifically, BALB / c mice (5-week-old, female, N = 5 per group) were treated with AUN at a concentration of 5 × 10 9 CFU / mL (200 μL), prednisolone (FUJIFILM-Wako): Oral or subcutaneous administration (0.1 mg / head) 24 hours before Musashi administration, hydrocortisone (FUJIFILM-Wako): Oral or subcutaneous administration (0.08 mg / head) 24 hours before AUN administration, dexamethasone (FUJIFILM-Wako): Oral or subcutaneous administration (2 mg / head) 24 hours before AUN administration. After AUN administration, mice's body temperature did not increase and was lower than before administration. However, subcutaneous injection of dexamethasone after AUN administration suppressed weight loss. Furthermore, AUN, when used in combination with dexamethasone, suppressed the increase in various cytokines.

[0215] Example 2

[0216] 1. Preparation of TiO2-PDMS Composites. TiO2 (titanium(IV) oxide, anatase form) (2 g) (Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) base (20 g). A cationic curing agent (2 g) was then mixed into the TiO2 dispersion. The TiO2-containing PDMS prepolymer was placed in a polypropylene (PP) mold (Bio Legend UR 1 L, Kansai Kako) and cured at 100°C for 3 hours to prepare a TiO2-containing PDMS (TiO2-PDMS) composite. A control PDMS scaffold without TiO2 was also prepared in the same manner, except that no TiO2 was added.

[0217] 2. Scaffold Characterization The morphology and surface structure of various scaffolds were observed using a scanning electron microscope (SEM) (TM3030; Hitachi) at an accelerating voltage of 15 kV. Elemental analysis of the scaffolds was performed using X-ray fluorescence spectroscopy (XRF) (S2 PUMA; Bruker). The concentrations of elements dissolved from the scaffolds were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Optima8300; PerkinElmer Japan GK). XRF and ICP-AES measurements were performed at Tosoh Analysis Center, Ltd. To measure the concentrations of elements leaching from the scaffolds, each scaffold (ceramics [Able ring S size 1 L; Charm], glass [Glass ring S size 1 L; Charm], polypropylene [PP] [Bio legend UR 1 L; Kansai Kako], TiO2-PDMS, or PDMS) or bakuhan stone (approximately 5 g) (Sone Chemiphor) was cultured in ATCC 543 medium (50 mL) under a tungsten lamp at 26-30°C for 5 days, and the supernatant was collected and analyzed by ICP-AES.

[0218] 3. Reactive Oxygen Species (ROS) Detection. ROS analysis was performed using Single Oxygen Sensor Green (SOSG) (Invitrogen) in a 96-well plate with black walls and a clear bottom (Thermo Fisher Scientific). Each scaffold or bakuhan stone (approximately 5 g) was added to a sample containing 1.0 μM SOSG (20 mL) in ATCC 543 medium and incubated under a tungsten lamp at 26–30°C for 3 hours. Green fluorescence, indicating ROS generation, was detected using a fluorometric plate reader (Infinite 200 PRO M Plex; Tecan). Measurements were taken at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. A control (ATCC 543 medium without scaffolds) was also measured after 3 hours of irradiation with a tungsten lamp at 26–30°C.

[0219] 4. Bacterial Strains and Culture. The AUN used in this study was isolated from a tumor derived from a mouse colon cancer cell line (Colon26, JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition). (For isolation methods, see Patent Documents 1 and 2.) AUN were typically grown anaerobically in ATCC 543 medium with each scaffold or bakuhan stone (approximately 5 g) in liquid culture at 26–30°C under a tungsten lamp in cysteine-free ATCC 543 medium. ATCC 543 medium was prepared according to the cell bank preparation method. Bacterial counts and viability were confirmed by colony assays using a bacterial counter (CASY Cell Counter & Analyzer; OMNI Life Science). Reagents for bacterial culture were obtained from Nacalai Tesque, Tokyo Chemical Industry, and Fujifilm Wako Pure Chemical Industries. The bacterial pellet was centrifuged at 3000 rpm / min for 5 minutes at 25°C and weighed using a balance (GR-202; A&D Co., Ltd., Tokyo, Japan). All reagents for bacterial culture were obtained from Nacalai Tesque (Kyoto, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and FUJIFILM Wako Pure Chemical. Bacterial transcriptome analysis was performed by Bioengineering Lab Co., Ltd. (Kanagawa, Japan) after culturing each scaffold or bakuhan stone (approximately 5 g) in ATCC 543 medium under a tungsten lamp at 26–30°C for 5 days. All scaffolds and bakuhan stones were thoroughly washed with Milli-Q water (1 L) and autoclaved at 121°C for 20 minutes before use.

[0220] 5. Cell Culture Drug-resistant mouse breast cancer (EMT6 / AR1) cells were obtained from KAC Co., Ltd. EMT6 / AR1 cells were cultured in Cell growth medium No. 104 (KAC Co., Ltd.) containing doxorubicin (1 μg / mL) at 37°C in a humidified incubator containing 5% CO2.

[0221] 6. Tumor spheroids EMT6 / AR1 cells (1 x 10 4 Cell-able spheroid plates (3D culture cells / well)R The cells were seeded onto a BP-96-R800 plate (Toyo Synthetic). Following the manufacturer's instructions, the cells were cultured at 37°C in a humidity-controlled incubator for 5 days. The medium was changed after 3 days. The prepared spheroids were incubated in a humidity-controlled incubator at 37°C with 1.0 × 10 AUN or PBS. 6 The spheroids were exposed to EMT6 / AR1 cells for 0, 24, 48, 72, 96, and 120 hours. The spheroids were observed at 20°C using a fluorescence microscope (BZ-X800, Keyence).

[0222] 7. Antitumor Test: EMT6 / AR1 tumor-implanted BALB / c mice (female; approximately 8 weeks; n = 5; average body weight = 20 g; average tumor size ~ 200 mm 3 BALB / cCrSIc; Japan SLC), AUN (7.5×10 9 Culture medium (200 μL) containing 1000 cells / mL of bacteria was administered intravenously through the tail vein. A control experiment without bacterial administration was also performed. Tumor formation and health status (survival rate and body weight) were monitored daily. Tumor volume was calculated as V = L × W. 2 The tumor volume was calculated using the formula: V / 2, where V, L, and W are the estimated tumor volume, longest diameter, and shortest diameter of the tumor, respectively. The survival rate of mice (n = 5) was also measured daily during the 40-day treatment. Note that the survival rate was 100% for tumors with a tumor volume of 1500 mm. 3 When the number of cells exceeded 100, the mice were euthanized according to the guidelines of the Animal Care and Use Committee. To investigate the efficacy of AUN against an orthotopic transplant model of anticancer drug-resistant mouse breast cancer, EMT6 / AR1 cells (5 × 10 5 The AUNs (6.5 × 10 cells) were administered into the mammary adipose tissue of mice (female; 6 weeks; n = 5; average body weight = 18 g; BALB / cCrSIc; Japan Clea, Tokyo, Japan). After approximately 14 days, AUNs (6.5 × 10 cells) cultured on TiO2-PDMS (AUN@TiO2-PDMS) were added to 200 μL of medium. 9 A control experiment without treatment was also performed.

[0223] 8. Flow Cytometry: EMT6 / AR1 tumor-implanted mice (female; approximately 8 weeks old; n = 3; average body weight = 18 g; average tumor size = 200 mm 3 BALB / cCrSIc; Japan SLC) is AUN@TiO2-PDMS (200 μL, 5.0 × 10 9 The mice were euthanized the day after intravenous administration of 1000 cells / mL of IgG4. To analyze the immune cells in the tumors, tumors were harvested from different groups of mice and single-cell suspensions were prepared using a handheld homogenizer (Thermo Fisher Scientific). Red blood cells were removed with ACK hemolysis buffer (Thermo Fisher Scientific), and debris was removed using a cell strainer (mesh size = 40 μm, SureStrain). TM The 1.0 × 10 6 The cells were stained with KIRAVIA Blue 520™, Alexa Fluor R The cells were stained with 488 or BD Horizon™ BB515-conjugated antibodies (listed in Table S3, Supplementary Information) and analyzed by flow cytometry (CyFlow Cube 6, Sysmex). 4 Each cell was analyzed.

[0224] 9. Tumor Immunohistochemistry (IHC) EMT6 / AR1 tumor-implanted BALB / c mice (female; approximately 8 weeks old; n = 3; average body weight = 20 g; average tumor size = 200 mm) 3 BALB / cCrSIc; Japan SLC) is AUN@TiO2-PDMS (200 μL, 6.5×10 9The day after intravenous administration of 1000 cells / mL of IgG, the animals were euthanized and tumors were harvested for IHC staining. Tumor tissue specimens were prepared by Biopathology Research Institute, Inc. using standard protocols. After surgical removal, tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, and 3-4 μm-thick sections were prepared and stained with antibodies (Table S3, listed in the Supporting Information) or hematoxylin. The specimens were observed under a light microscope (BZ-X800). Positive staining in the tumor tissue was analyzed using Hybrid Cell Count and Micro Cell Count software (Keyence) installed on the light microscope system.

[0225] 10. qPCR EMT6 / AR1 tumor-implanted mice (female; approximately 8 weeks old; n = 3; average body weight = 18 g; average tumor size = 200 mm 3 BALB / cCrSIc; Japan SLC) is AUN@TiO2-PDMS (200 μL, 5.0×10 9Mice were euthanized the day after administration of 1000 cells / mL of IgG. To analyze immune cells and cytokines in the tumors, tumors were harvested from the mice 24 hours after intravenous administration and the tissues were homogenized using a homogenizer (Thermo Fisher Scientific). qPCR was performed using a QuantStudio 1 PCR System (Thermo Fisher Scientific) to examine the relative gene expression of CD3, CD19, CXCR4, F4 / 80, NK, IFN-γ, and TNF-α using gene-specific primer-probe combinations (Thermo Fisher Scientific) with TaqMan chemistry (Table 2). Endogenous controls were determined using a 96-well TaqMan Array Mouse Endogenous Control Plate (Thermo Fisher Scientific). Reactions were performed in triplicate using GAPDH as an endogenous control. The thermal cycling parameters that yielded optimal amplification were 40 cycles of 50°C for 2 minutes for AmpErase UNG activation, 95°C for 2 minutes for AmpliTaq Gold DNA Pol. activation, 95°C for 1 second for denaturation / melting, and 60°C for 20 seconds for annealing and extension. Ten-fold serial dilutions were performed for the test genes and endogenous control standard to estimate PCR efficiency, which ranged from 90 to 100%. Results were analyzed and presented as fold change relative to the control (log10 relative quantification).

[0226] 11. Blood Tests Complete blood counts (CBCs) were measured using a Celltac α blood cell counter (MEK6558; Nihon Kohden), and plasma biochemical parameters were examined by Oriental Yeast Co., Ltd. Cytokines were measured using the Bio-Plex Multiplex immunoassay system (Bio-Rad) and Bio-Plex Pro Mouse Th17 Panel 6-Plex (Bio-Rad). BALB / cCrSlc mice (female; 6 weeks; n = 5; average body weight = 18 g; Japan SLC, Tokyo, Japan) were cultured without a scaffold and were given normal AUN (200 μL, 5.0 × 10 9 cells / mL) or AUN@TiO2-PDMS (200 μL, 5.0 × 10 9 Culture medium containing 168 cells / mL of BALF-168 cells was administered intravenously into the tail vein. Blood samples were collected from the inferior vena cava of each mouse at 3, 6, 24, and 168 hours. A control experiment without bacterial administration was also performed as the 0-hour treatment.

[0227] 12. Statistical Analysis. All experiments, with some exceptions, were repeated three or more times. Quantitative values ​​are expressed as the mean ± standard error of the mean from at least three independent experiments. Statistical differences were identified by two-tailed Student's t-test, two-way analysis of variance (ANOVA), or log-rank test (Mantel-Cox) using GraphPad Prism, version 9.4.0 (GraphPad Software). A p-value of less than 0.05 was considered statistically significant.

[0228] Results of Example 2: Microbial metabolism and function are regulated by the surrounding microenvironment, but functional microporous materials capable of modulating the anticancer activity of complex bacterial systems were unknown. This study confirmed the usefulness of scaffold-mediated bacterial culture to improve the anticancer activity of a microbial consortium called complex bacterial AUN. As described above, complex bacterial AUN (NITE BP-03627) is composed of the intratumoral bacterium Proteus mirabilis (A-gyo) and the purple photosynthetic bacterium Rhodopseudomonas palustris (UN-gyo). The discovery of the mechanism by which microporous scaffolds affect the bacterial activity of complex bacterial AUN will facilitate the further design of artificial scaffold materials for the effective treatment of anticancer drug-resistant triple-negative breast cancer models. The inventors found that polydimethylsiloxane composite artificial scaffolds, particularly those encapsulating photoactivated TiO2, could improve the therapeutic outcomes of complex bacterial AUN in both allogeneic and orthotopic tumor transplant models. We believe that these potent anti-cancer effects are exerted synergistically by the potent anti-tumor ability of the complex bacterial AUN within tumors with the help of various immune cells.

[0229] Representative results from Example 2 are shown in Figures 9 to 13. Figure 9 shows the antitumor effect of AUN attenuated by the scaffold. Figure 10 shows the antitumor effect and body weight change of the attenuated complex AUN bacteria in a photoinduced TiO2-PDMS composite. Figure 11 shows the changes in leukocytes, platelets, and cytokines, suggesting the high biocompatibility of AUN using TiO2-PDMS. Figure 12 shows the infiltration of immune system cells into tumors by AUN@TiO2-PDMS. Figure 13 shows the tumor-suppressing effect of AUN@TiO2-PDMS.

[0230] To further improve the antitumor efficacy and biocompatibility of the composite bacterial AUN, we tested an effective and simple bacterial culture system using commercially available porous scaffolds made of ceramic, glass, bakuhan stone, and polypropylene (PP). The culture substrates, each containing culture medium containing the composite bacterial AUN, were placed in a glass container, sealed, and cultured under a tungsten lamp. After culture, the composite bacterial AUN was harvested from each culture substrate. Next, the anticancer effect of the composite bacterial AUN was examined in a drug-resistant triple-negative breast cancer (TNBC) model. BALB / c mice bearing EMT6 / AR1 tumors were intravenously administered with AUN cultured on each scaffold. The EMT6 / AR1-derived tumor used in this study is an immunologically cold tumor highly resistant to antitumor drugs and is difficult to treat. Surprisingly, AUN cultured on the ceramic scaffolds demonstrated dramatic anticancer efficacy and a significant survival rate (Figure 9A). AUN cultured on Maifan stone, glass, or PP scaffolds, as well as conventional AUN without a scaffold, caused the death of mice (Figure 9A). Obvious tumor growth was observed over time in the untreated group, and all mice died within 13 days (Figure 9A). No significant weight loss (>20%) was observed in the ceramic scaffold-mediated AUN group, indicating no side effects associated with bacterial administration (Figure 9B). These results clearly demonstrate that the ceramic scaffold influences the therapeutic efficacy of AUN.

[0231] (Preparation of Photoactivated Functional Polymer Composites) Next, to elucidate the mechanism by which the ceramic scaffold influences the potent anticancer effect of AUN, elemental analysis of the four scaffolds was performed using X-ray fluorescence analysis. The elemental distributions in the matrix of the inorganic scaffolds (ceramic, bakuhanite, and glass) were very similar, and the major component of these scaffolds was SiO2. The PP scaffold was composed of 91% PP and contained trace impurities of MgO (4.4%) and SiO2 (4.3%). However, comparing only these major components did not clarify why the ceramic scaffolds significantly affected the biocompatibility and anticancer effect of AUN, as compared with the elemental distributions of the other scaffolds. The concentrations of elements eluted from each scaffold during the three-day incubation period were measured. However, although these scaffolds eluted various minerals into Milli-Q water, the concentrations of the eluted elements were very small, and all elements were already present in the culture medium.

[0232] After extensive investigation, we discovered that ceramics and bakuhan stone contain trace amounts of titanium oxide (TiO2), a known photocatalyst. We hypothesized that TiO2 may contribute to improving the therapeutic function of the complex bacteria AUN. To test this hypothesis, we used a PP mold to prepare porous polydimethylsiloxane (PDMS) (TiO2-PDMS) composites encapsulating TiO2 (titanium [IV] oxide, anatase form) by a simple thermal curing method. The prepared TiO2-PDMS composites were white, freestanding, and soft. The surface of the TiO2-PDMS composites contained numerous pores within the PDMS matrix and microscopic protrusions derived from TiO2. The control PDMS material also contained numerous pores, but the surface was smooth due to the absence of TiO2.

[0233] Next, we investigated the antitumor effect of the composite bacterial AUN (hereinafter referred to as "AUN@TiO2-PDMS"), which was cultured in a TiO2-PDMS composite and recovered from the composite, using a drug-resistant TNBC allogeneic model. Interestingly, AUN@TiO2-PDMS inhibited the antitumor effect of the composite bacterial AUN (7.5 × 10 9 A single administration of AUN@TiO2-PDMS at a concentration of 7.5 × 10 CFU / mL showed a dramatic antitumor effect (Fig. 9B). This effect was similar to that obtained with the composite bacterial AUN cultured and recovered from the ceramic scaffold described above. The excellent therapeutic ability of the AUN@TiO2-PDMS sample also significantly extended the survival rate of mice (Fig. 10). On the other hand, the composite bacterial AUN cultured and recovered from the control PDMS scaffold showed a dramatic antitumor effect at an AUN concentration (7.5 × 10 9 A single dose of AUN@TiO2-PDMS at 1000 CFU / mL resulted in death of the mice within 2 days. There was no effect on tumor progression or survival rate in the untreated group. There were no serious side effects, as no significant weight loss (>20%) was observed after treatment with AUN@TiO2-PDMS (Figure 10). These results suggest that the use of photocatalytic TiO2-embedded polymer composites in a simple scaffold culture method under light irradiation may enhance the anticancer therapeutic efficacy of the combined bacterial AUN.

[0234] To further clarify the cause of the improved biocompatibility when the complex bacterial-AUNs were cultured on the TiO2-PDMS composite, we measured complete blood counts (WBC, PLT, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, and red blood cells) from blood samples 24 h after intravenous administration of AUN@TiO2-PDMS, as well as various pro-inflammatory cytokines (interferon-γ [IFN-γ], tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6], interleukin-1β [IL-1β], and interleukin-17A [IL-17A]) and anti-inflammatory cytokines (interleukin-10 [IL-10]) (Figure 11). The AUN@TiO2-PDMS group showed higher concentrations of WBC and PLT compared with cultures of normal complex bacterial-AUNs without the scaffold (Figure 11). Other complete blood counts were not significantly different between the groups treated with AUN@TiO2-PDMS or scaffold-free composite bacterial AUN. The expression of proinflammatory cytokines in the AUN@TiO2-PDMS group tended to be lower than that in the scaffold-free AUN group (Figure 11). The expression of anti-inflammatory cytokines (IL-10) in the AUN@TiO2-PDMS group was higher than that in the conventional AUN group (Figure 11). CBC and biochemical parameters showed safe values ​​7 days after treatment with AUN@TiO2-PDMS. These results clearly demonstrate that composite bacterial AUN can be effectively inhibited by the TiO2-PDMS composite, making it a useful therapeutic agent for cancer treatment.

[0235] (Anti-cancer mechanism of AUN@TiO2-PDMS) Next, we clarified the anti-cancer mechanism of AUN@TiO2-PDMS by examining its cell destruction behavior and intratumor immune response (Figure 12). The tumor-destructive ability of AUN@TiO2-PDMS in a tumor spheroid model was examined using an optical microscope, utilizing the near-infrared (NIR) fluorescence properties of AUN. Spheroids co-cultured with AUN@TiO2-PDMS dramatically disappeared over time. As the spheroids were destroyed, the intensity of near-infrared fluorescence (FL) from AUN increased both inside and outside the spheroids. Such destructive behavior and NIR fluorescence were not observed in the untreated spheroids.

[0236] Quantitative PCR (qPCR) assays, flow cytometry analysis, and immunohistochemical staining (IHC) comprehensively revealed that 24 h after intravenous administration of AUN@TiO2-PDMS, the inflammatory cytokine TNF-α increased in solid tumors, particularly activating T cells, NK cells, and macrophages (Figure 12). Hematoxylin and eosin (H&E) staining also confirmed the destruction of tumor tissue due to the strong anticancer effect of AUN@TiO2-PDMS compared with the untreated group (Figure 12). Apoptosis markers (caspase-3 staining and TUNEL) and TNF-α staining in tumor slices after treatment with AUN@TiO2-PDMS indicated massive apoptotic cell death and a strong inflammatory response.

[0237] In summary, we believe that the antitumor activity of AUN itself plays an important role in the therapeutic effect, and that the AUN@TiO2-PDMS sample actively guides bacteria into the tumor microenvironment, enabling it to effectively destroy the targeted cancer tumors.

[0238] (Antitumor effect of the AUN@TiO2-PDMS sample on a drug-resistant TNBC orthotopic transplantation model) A drug-resistant TNBC orthotopic transplantation model was created by injecting EMT6 / AR1 (5 × 105 cells) into the mammary fat pad of mice. The tumor volume was 200 mm 3 When the concentration reached 6.5 × 10, the AUN@TiO2-PDMS (concentration 6.5 × 10) 9AUN@TiO2-PDMS was administered intravenously to mice at a dose of 1000 cells / mL. Complete tumor response and prolonged survival were achieved in a drug-resistant TNBC orthotopic transplantation model without side effects or severe weight loss (Figure 13). The tumors in the mice turned black the day after bacterial administration, similar to the subcutaneous tumors in the allograft model. Subsequently, the AUN@TiO2-PDMS-treated tumors dramatically suppressed tumor growth. Meanwhile, rapid tumor growth was observed in the untreated group. These results demonstrate that AUN@TiO2-PDMS is highly effective in both allograft and orthotopic transplantation models.

[0239] - A complex bacterium consisting of Rhodopseudomonas Palustris and Proteus mirabilis (composition ratio based on CFU: 97:3) (identification: Musashi) (accession number: NITE BP-03627): Deposited on March 23, 2022, at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD). - Proteus mirabilis (identification: Super Bac) (accession number: NITE BP-03626): Deposited on March 23, 2022, at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD). Lactococcus genus bacteria (identification: Super Lacto) (accession number: NITE BP-03694) Deposited on August 2, 2022, at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD). Enterococcus faecalis (identification: Super Entero) (accession number: NITE BP-03690): Deposited on July 19, 2022, at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD). Proteus mirabilis Hauser 35659 TM (ATCC 35659) (American Type Culture Collection)

Claims

1. An antitumor agent containing purple non-sulfur bacteria and bacteria other than said purple non-sulfur bacteria, which is used in combination with a side effect suppressant.

2. The antitumor agent according to claim 1, wherein the side effect suppressing agent is a bacteria-containing agent or a bacteria-free agent.

3. The antitumor agent according to claim 2, wherein the bacteria-containing agent contains the same bacteria as or different from the bacteria contained in the antitumor agent.

4. An antitumor agent according to any one of claims 1 to 3, wherein the side effect suppressor comprises an antithrombotic agent and / or an anti-inflammatory agent.

5. The antitumor agent according to claim 4, wherein the antithrombotic agent is an anticoagulant.

6. The antitumor agent according to claim 5, wherein the anticoagulant is heparin or a salt thereof.

7. The antitumor agent according to claim 4, wherein the anti-inflammatory agent is a steroidal anti-inflammatory agent.

8. The antitumor agent according to claim 7, wherein the steroidal anti-inflammatory agent is dexamethasone.

9. An antitumor agent according to any one of claims 1 to 8, which is administered after the administration of the side effect suppressant.

10. An antitumor agent according to any one of claims 1 to 9, which is administered multiple times after the administration of the side effect suppressant.

11. An antitumor agent according to any one of claims 1 to 10, which is administered at least 26 hours after the side effect suppressant has been previously administered.

12. An antitumor agent according to any one of claims 1 to 11, wherein the purple non-sulfur bacteria are bacteria of the genus Rhodopseudomonas, and the bacteria other than the purple non-sulfur bacteria are bacteria of the genus Proteus.

13. An antitumor agent according to any one of claims 1 to 12, wherein the purple non-sulfur bacterium and the bacterium other than the purple non-sulfur bacterium are a complex bacterium identified by accession number: NITE BP-03627.

14. An antitumor agent according to any one of claims 1 to 13, which is included in a combination pharmaceutical with the side effect suppressor.

15. An antitumor agent according to any one of claims 1 to 14, which is administered in a single dose at a dose equal to or greater than the dose required for tumor regression.

16. An antitumor agent according to any one of claims 1 to 15, which is administered in an amount greater than or equal to an excessive amount when administered alone in a single dose.

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