Anti-tumor agent, image generation method, bacterium and preparation method thereof

Abstract

The purpose of the present invention is to provide a technique useful for the treatment or diagnosis of cancer. The present invention provides an antitumor agent comprising a red non-sulfur bacterium isolated from a tumor. Furthermore, the present invention provides an image generation method for identifying a tumor, comprising: an administration step for administering bacteria isolated from the tumor to an animal; and an image generation step in which, after the administration, the animal is imaged in a state in which the animal is irradiated with light having a predetermined wavelength range, and an image for specifying a tumor that may be present in the animal is generated. In addition, the invention also provides a bacterium separated from the tumor, and the bacterium at least comprises red non-sulfur bacteria.

Description

Technical Field

[0001] The present invention relates to an antitumor agent, an image generation method, bacteria, and a method for preparing the same. In particular, the present invention relates to an antitumor agent comprising bacteria isolated from a tumor, an image generation method using bacteria isolated from a tumor, bacteria isolated from a tumor, and a method for preparing bacteria comprising a step of isolating bacteria from a tumor. Background Art

[0002] In order to treat cancer, various treatment methods are used, such as surgical treatment, drug therapy, and radiotherapy. Depending on the type and condition of the disease, they can be used alone or in combination. In drug therapy, for example, drugs such as anticancer agents are used. Anticancer agents used in drug therapy can also have adverse effects on normal cells and even bring great burdens to patients. For example, anticancer agents often accumulate in the patient's body due to long-term use, causing great side effects to the patient.

[0003] In addition, early detection of cancer through examinations is also one of the important factors in improving patient prognosis. For example, various tumor markers, MRI, and CT are used for cancer examinations. The presence or location of cancer cannot be determined by the value of tumor markers alone. MRI and CT are very useful in determining the presence or absence of cancer, but these devices are relatively large and costly.

[0004] The use of photosynthetic bacteria for the treatment or diagnosis of cancer is disclosed in the following Patent Document 1. For example, the document describes administering photosynthetic bacteria to a subject, causing the photosynthetic bacteria to accumulate in a cancerous area, and then irradiating the area with light.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2022 / 025043 Summary of the invention

[0008] Problems to be solved by the invention

[0009] An object of the present invention is to provide a technique useful for the treatment or diagnosis of cancer.

[0010] Means for solving problems

[0011] Various bacteria exist in tumors. The present inventors have found that when bacteria existing in tumors are isolated from tumors, the isolated bacteria are useful for the treatment or diagnosis of cancer, or both.

[0012] That is, the present invention provides the following contents.

[0013] [1] An antitumor agent comprising red non-sulfur bacteria isolated from a tumor.

[0014] [2] The antitumor agent according to [1], wherein the red non-sulfur bacteria are Rhodopseudomonas bacteria or Chlorobacterium bacteria, or both.

[0015] [3] The antitumor agent according to [1] or [2], wherein the antitumor agent is administered parenterally.

[0016] [4] The antitumor agent according to any one of [1] to [3], wherein the antitumor agent further comprises other bacteria isolated from a tumor in addition to the red non-sulfur bacteria.

[0017] [5] The antitumor agent according to [4], wherein the other bacteria is a Proteus bacterium.

[0018] [6] The antitumor agent according to [4] or [5], wherein the antitumor agent contains the red non-sulfur bacteria and the other bacteria at a ratio of 1 CFU:99 CFU to 99 CFU:1 CFU.

[0019] [7] The antitumor agent according to any one of [1] to [6], wherein the bacteria contained in the antitumor agent has immune cell activating properties.

[0020] [8] The antitumor agent according to any one of [1] to [7], wherein the bacteria contained in the antitumor agent has an expression enhancing property of enhancing the expression of an antitumor biomarker in a tumor.

[0021] [9] The antitumor agent according to any one of [1] to [8], wherein the bacteria contained in the antitumor agent exhibit the following characteristics related to the absorption spectrum.

[0022] <Characteristics related to absorption spectrum>

[0023] When the bacteria contained in the antitumor agent were measured at 2×10 7 When the concentration of CFU / mL is dispersed in PBS buffer, the absorbance at 808 nm is 0.029 or more.

[0024]

[10] The antitumor agent according to any one of [1] to [9], wherein the bacteria contained in the antitumor agent exhibit the following characteristics related to the fluorescence spectrum.

[0025] <Characteristics related to fluorescence spectrum>

[0026] When the bacteria contained in the antitumor agent were measured at 2.5×10 7 When the fluorescence spectrum of the dispersion liquid with a concentration of CFU / mL dispersed in PBS buffer is taken, the fluorescence intensity at 888 nm is 4.4 or more.

[0027]

[11] A method for generating an image for determining a tumor, comprising:

[0028] an administration step of administering to the animal one or more bacteria isolated from the tumor; and

[0029] The image generation step is to photograph the animal after the administration while irradiating the animal with light in a predetermined wavelength range, thereby generating an image for identifying a tumor that may exist in the animal.

[0030]

[12] A bacterium isolated from a tumor, comprising at least red non-sulfur bacteria.

[0031]

[13] The bacteria according to

[12] , wherein the bacteria isolated from the tumor also include Proteus bacteria.

[0032]

[14] The bacterium according to

[12] or

[13] , wherein the red non-sulfur bacterium is a bacterium of the genus Rhodopseudomonas or a bacterium of the genus Chlorosporum, or both.

[0033]

[15] The bacterium according to any one of

[12] to

[14] , wherein the bacterium exhibits the following characteristics related to the absorption spectrum.

[0034] <Characteristics related to absorption spectrum>

[0035] When the bacteria were measured at 2×10 7 When the concentration of CFU / mL is dispersed in PBS buffer, the absorbance at 808 nm is 0.029 or more.

[0036]

[16] The bacterium according to any one of

[12] to

[15] , wherein the bacterium exhibits the following characteristics related to fluorescence spectrum.

[0037] <Characteristics related to fluorescence spectrum>

[0038] When the bacteria were measured at 2.5×10 7 When the fluorescence spectrum of the dispersion liquid with a concentration of CFU / mL dispersed in PBS buffer is taken, the fluorescence intensity at 888 nm is 4.4 or more.

[0039]

[17] A method for preparing bacteria, comprising:

[0040] an isolation step, a step of isolating bacteria from a tumor; and

[0041] a culturing step of culturing the bacteria separated in the separation step in a medium to which cysteine ​​is not added,

[0042] The cultured bacteria at least contain red non-sulfur bacteria and have anti-tumor activity.

[0043] Effects of the Invention

[0044] Bacteria isolated from tumors are very useful for treating tumors. For example, tumors can be treated with the anti-tumor agent according to the present invention. In addition, the anti-tumor agent according to the present invention can also be used to treat cancers of various organs or tissues.

[0045] In addition, bacteria isolated from tumors are also very useful for diagnosing tumors. For example, the method according to the present invention (particularly the image generation method) or the bacteria according to the present invention are useful for determining the presence or absence of a tumor, and further useful for determining the location or shape of the tumor.

[0046] Furthermore, according to the present invention, the treatment and diagnosis of a tumor can be performed simultaneously. For example, by administering the antitumor agent according to the present invention, in addition to treating the tumor, the location of the tumor can also be determined.

[0047] In addition, the effects of the present invention are not limited to the effects described here, and may be any effects described in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] [ Figure 1 ] Diagram illustrating the isolation and culture of intratumoral bacteria.

[0049] [ Figure 2A ] Figure used to illustrate the in vivo anticancer experiment.

[0050] [ Figure 2B ] Photographs of mice after intravenous administration of each bacteria.

[0051] [ Figure 2C ] A diagram showing the evaluation results of the antitumor activity of various bacteria.

[0052] [ Figure 2D ] A graph showing the survival rate of mice 40 days after cancer cell transplantation in each bacterial administration.

[0053] [ Figure 2E ] A graph showing the complete efficacy achievement rate of each experimental group.

[0054] [ Figure 2F ] Figure showing experimental methods for studies related to cancer immunity acquisition.

[0055] [ Figure 2G ] are photographs showing the observation results of mice after re-transplantation of Colon-26.

[0056] [ Figure 2H ] is a graph showing the measurement results of tumor volume in each treatment group.

[0057] [ Fig.2I ] is a diagram showing the measurement results of the absorption spectrum.

[0058] [ Figure 2J ] is a diagram showing the measurement results of fluorescence spectra.

[0059] [ Figure 2K ] shows a diagram of images obtained by in vivo fluorescence bioimaging.

[0060] [ Figure 3A ] A diagram showing the staining results of immune cells.

[0061] [ Figure 3B ] A diagram showing the staining results of anti-tumor biomarkers.

[0062] [ Figure 3C ] A graph showing the results of colony analysis when iR.P. was administered.

[0063] [ Figure 3D ] A graph showing the results of colony analysis when iP.M. was administered.

[0064] [ Figure 3E ] shows the results of H&E tissue staining of the above-mentioned various organs when iP.M., iR.P., or PBS was administered.

[0065] [ Figure 4A ] A diagram illustrating the method for preparing sarcoma model mice.

[0066] [ Figure 4B ] A diagram showing the time-dependent changes of tumors in sarcoma model mice.

[0067] [ Figure 4C ] A diagram illustrating the method for preparing a metastatic lung cancer model mouse.

[0068] [ Figure 4D ] Photographs of lungs removed from each mouse.

[0069] [ Figure 4E ] A graph showing the results of weight measurement of lungs removed from mice.

[0070] [ Figure 4F ] A graph showing changes in mouse body weight.

[0071] [ Figure 5 ] is a diagram showing the measurement results of the absorption spectrum and the fluorescence spectrum.

[0072] [ Figure 6 ] shows a photograph of a mouse and a diagram of a fluorescent observation image obtained by in vivo fluorescent bioimaging. DETAILED DESCRIPTION

[0073] The present invention will be described in the following order.

[0074] 1. Contents of the Invention

[0075] 1.1 Antitumor activity

[0076] 1.2 Diagnostic usefulness

[0077] 1.3 Usefulness from a toxicity perspective

[0078] 1.4 Usefulness from the perspective of cancer immunity

[0079] 1.5 Responses to Problems with Proposed Treatment Methods

[0080] 2. Implementation Method

[0081] 2.1 First Embodiment (Antitumor Agent)

[0082] 2.1.1 Antitumor agent comprising red non-sulfur bacteria isolated from tumors

[0083] 2.1.2 Antitumor agents comprising Proteus bacteria isolated from tumors

[0084] 2.1.3 Examples of other bacteria

[0085] 2.1.4 Structure of antitumor agents

[0086] 2.1.5 Preparation of antitumor agents

[0087] 2.2 Second Embodiment (Bacteria)

[0088] 2.2.1 Red non-sulfur bacteria

[0089] 2.2.2 Proteus bacteria

[0090] 2.3 Third Embodiment (Image Generating Method)

[0091] 2.4 Fourth Embodiment (Method for Preparing Bacteria)

[0092] 2.5 Fifth Embodiment (Treatment Method and Diagnosis Method)

[0093] 3. Example

[0094] Furthermore, the present invention is not limited to these embodiments and can be freely changed within the scope of the present invention.

[0095] 1. Contents of the Invention

[0096] As described above, various treatment methods such as surgical treatment, drug therapy, and radiotherapy are used to treat cancer. In addition, various proposals have been made regarding treatment methods for cancer.

[0097] As a method for treating cancer, nanomedicine, for example, has been proposed. As an example of the nanomedicine, a substance utilizing the EPR effect is listed. The following EPR effect (Enhanced Permeation and Retention Effect) is known, that is, unlike normal vascular endothelial cells, there is a wide gap of about 200nm between the vascular endothelial cells of cancer tissue and inflammatory sites, and nanoparticles whose size is controlled to be about 10 to 100nm specifically aggregate in cancer tissue. In most cases, anticancer treatment using nanotechnology is close to the performance limit due to its reliance on this EPR effect. In fact, there are several environmental factors that inhibit solid cancer, and there are also reports that the EPR effect itself is not useful (for example, Yokoyama Masayuki, Drug Delivery System, 33 (2), p.89-97 (2018).). In addition, drug carriers utilizing the EPR effect have not yet been successful in human clinical trials.

[0098] Therefore, nanomedicine relies on the EPR effect, whose mechanism is unclear, and has low selectivity for cancer. In addition, nanomedicine also has the problem of using anticancer agents with strong side effects. In addition, nanomedicine requires a lot of time and cost in synthesis. In addition, since the tumor is in a state of hypoxia, the photodynamic therapy type nanomedicine using oxygen is less effective.

[0099] In addition, antibody therapy has also been proposed as a method for treating cancer. For example, a strategy has been proposed to carry antibodies specific to cancer cells on drug carriers to improve tumor targeting performance (for example, Hisataka Kobayashi, Drug Delivery System, 29 (4), p. 274-284 (2014).). However, large-sized antibodies have difficulty penetrating the interstitial barrier surrounding cancer, and therefore do not achieve sufficient selectivity and efficacy.

[0100] As such, since antibodies cannot penetrate the cancer-stromal barrier (they only bind to the surface of the tumor), it is difficult to eliminate cancer cells deep inside the tumor even if immune cells are used. In addition, for antibody therapy, it is also necessary to make antibodies using Order (Owner) made for biomarkers whose expression levels differ between cancer patients. In addition, it takes a lot of time and cost to prepare drug monomers carrying antibodies.

[0101] In addition, as a method for treating cancer, bacterial therapy has also been proposed in recent years, especially the use of anaerobic microorganisms that can selectively aggregate, grow and proliferate inside tumors in a hypoxic state for targeted cancer therapy has attracted much attention (Shibin Zhou et al. Nature Reviews Cancer, 18, p.727-743 (2018).). However, previous cancer bacterial therapy is basically a concept of so-called drug delivery system for the transportation of anticancer agents. In addition, in order to express anticancer activity, it is necessary to manipulate or change microorganisms using genetic engineering. As a result, it is possible to produce unpredictable and uncontrollable problems such as bacterial resistance, which is said to be difficult to apply to medical treatment. In addition, the bacteria used are in most cases Salmonella or Escherichia coli that have been genetically modified and weakened, and are often accompanied by the risk of re-enforcing in vivo.

[0102] As such, regarding bacterial therapy, it has been proposed to use, for example, Salmonella, Listeria or Escherichia coli that have been attenuated by genetic modification, but they have the risk of becoming highly toxic again (reversion mutation) in vivo. In addition, there is also the possibility of acquiring drug resistance through genetic modification. In addition, tumor specificity is insufficient and it can also accumulate in normal organs or tissues. In addition, the above-mentioned bacterial therapy is passive drug delivery and requires complex genetic design.

[0103] 1.1 Antitumor activity

[0104] The present inventors have found that bacteria present in a tumor can exhibit excellent antitumor activity by isolating the bacteria from the tumor.

[0105] There are various bacteria in tumors. It is believed that these bacteria will not attack the tumor when they are present in the tumor. However, the present inventors have found that the bacteria present in the tumor exert anti-tumor activity by being separated from the tumor. That is, the present invention provides an anti-tumor agent comprising bacteria separated from the tumor.

[0106] In one embodiment, the above-mentioned bacteria separated from the tumor are red non-sulfur bacteria. The red non-sulfur bacteria separated from the tumor have excellent anti-tumor activity. In particular, the red non-sulfur bacteria separated from the tumor and cultured in the culture medium have excellent anti-tumor activity. The above-mentioned red non-sulfur bacteria can be, for example, Rhodopseudomonas bacteria or Chlorobacterium bacteria or both. The anti-tumor activity is very excellent and is effective for tumors in various organs or tissues.

[0107] In the above embodiment, preferably, the above-mentioned red non-sulfur bacteria can be used in combination with other bacteria isolated from a tumor. The combination of red non-sulfur bacteria isolated from a tumor and other bacteria isolated from a tumor exerts particularly excellent anti-tumor activity. The other bacteria can be, for example, Proteus bacteria. Two or more bacteria constituting the combination can preferably be isolated from the same tumor and cultured together in the same culture medium (co-culture).

[0108] In another embodiment, the above-mentioned bacteria isolated from the tumor are Proteus bacteria. As mentioned above, Proteus bacteria isolated from the tumor can be used in combination with red non-sulfur bacteria or alone. Proteus bacteria isolated from the tumor have excellent antitumor activity. The antitumor activity is very excellent and is effective against tumors in various organs or tissues.

[0109] 1.2 Diagnostic usefulness

[0110] In addition, the inventors have also found that the above-mentioned bacteria isolated from the tumor have the property of gathering at the tumor site. For example, the bacteria isolated from the tumor gather at the tumor site when administered non-orally to an animal. Therefore, for example, analysis using the optical properties of the bacteria is useful for judging the presence or absence of a tumor or determining the location or shape of the tumor. For example, the information (especially image information) obtained by photographing the animal using the optical properties of the bacteria is useful for the above judgment or the above determination. As such, the bacteria isolated from the tumor can be used not only for treating the tumor, but also for diagnosing the tumor, for example, as a diagnostic drug. Further, the bacteria isolated from the tumor can be used for tumor treatment and tumor diagnosis.

[0111] In one embodiment, the bacteria isolated from the tumor may be red non-sulfur bacteria. The red non-sulfur bacteria isolated from the tumor have optical properties different from those of conventional red non-sulfur bacteria (e.g., commercially available red non-sulfur bacteria, etc.). In addition to the optical properties, the presence or absence of a tumor can be determined by utilizing the properties of aggregation at the tumor site, or the location or shape of the tumor can be determined.

[0112] 1.3 Usefulness from a toxicity perspective

[0113] Furthermore, the present inventors have found that the above-mentioned bacteria isolated from tumors are also excellent in terms of toxicity.

[0114] In general, when bacteria are administered to animals (particularly parenterally), the bacteria are often toxic to the animals. For example, when commercially available red non-sulfur bacteria or Proteus bacteria are administered to animals, these bacteria may be toxic to the animals.

[0115] However, it is believed that the above-mentioned bacteria isolated from tumors do not exert toxicity to animals when administered (particularly parenterally) to the animals, or even if toxicity is exerted, the toxicity is very low.

[0116] In addition, the inventors have also found that the bacteria isolated from the tumors are present in the animal body for a period of time after administration, but cannot be confirmed in the animal body after a certain period of time. In other words, it can be considered that the bacteria isolated from the tumors will not exist in the animal body for a long time, thereby reducing concerns about the adverse effects of the bacteria.

[0117] 1.4 Usefulness from the perspective of cancer immunity

[0118] In addition, the present inventors have also found that the above-mentioned bacteria isolated from tumors impart or enhance immunity against tumors.

[0119] For example, as described above, the bacterium isolated from the tumor has antitumor activity, so the tumor in the animal can be eliminated by administering the bacterium to the animal with the tumor. And it was found that even if the animal is treated to produce a tumor after the tumor disappears, the animal will not develop a tumor. Therefore, for example, the above antitumor agent (especially the above bacterium isolated from the tumor) can be used to prevent the recurrence of the tumor.

[0120] 1.5 Responses to Problems with Proposed Treatment Methods

[0121] The antitumor agent according to the present invention can also cope with the problems associated with the above-mentioned treatment methods.

[0122] For example, as mentioned above, nanomedicines rely on the EPR effect, whose mechanism is not clear, and have low selectivity for cancer. On the other hand, the anti-tumor agent (especially bacteria) according to the present invention can express high selectivity for tumors. It can be believed that this high selectivity is caused by, for example, hypoxia, immune avoidance and chemotaxis, that is, it is exerted through a clear mechanism.

[0123] Furthermore, although nanomedicine uses an anticancer agent having strong side effects, the antitumor agent according to the present invention does not use an anticancer agent having strong side effects, and the bacteria used in the present invention are low in toxicity.

[0124] In addition, nanomedicines require a lot of time and cost to synthesize, but the bacteria contained in the antitumor agent according to the present invention can replicate themselves indefinitely, and the bait is cheap and easy to manage.

[0125] In order to perform antibody therapy, as described above, it is necessary to prepare antibodies with Order (Owner) made for biomarkers with different expression levels among cancer patients. On the other hand, the bacteria contained in the antitumor agent according to the present invention highly selectively aggregate in the microenvironment common in solid cancers, and can grow and reproduce in the microenvironment. Therefore, it is not necessary to prepare antibodies as described above.

[0126] In addition, since the active ingredients of antibody therapy cannot penetrate the cancer-stroma barrier and mainly bind to the surface of the tumor, it is difficult to eliminate cancer cells deep in the tumor even if immune cells are used. On the other hand, the bacteria contained in the anti-tumor agent according to the present invention can also gather in the deep part of the tumor, grow and reproduce there. Therefore, cancer cells can be eradicated from deep in the cancer.

[0127] In addition, the active ingredients of antibody therapy require a lot of time and cost to prepare. On the other hand, the bacteria contained in the antitumor agent according to the present invention can replicate themselves indefinitely, and the feed is cheap and easy to manage.

[0128] In the past, in bacterial therapy, for example, when using Salmonella, Listeria or Escherichia coli weakened by genetic modification, these bacteria have the risk of becoming strong again in vivo (reversion mutation), and it is possible to acquire drug resistance by genetic modification. In addition, these bacteria are also insufficient in tumor specificity and can also accumulate in, for example, normal organs or tissues. On the other hand, the bacteria included in the antitumor agent according to the present invention do not need genetic modification and can show high selectivity for tumors. In addition, the bacteria used are of low toxicity.

[0129] In addition, conventional bacterial therapies are based on passive drug delivery and require complex genetic design. On the other hand, the bacteria contained in the antitumor agent according to the present invention do not require genetic modification, and tumors can be eliminated with only a single administration of the bacteria.

[0130] The embodiments of the present invention are described in more detail below.

[0131] 2. Implementation Method

[0132] 2.1 First Embodiment (Antitumor Agent)

[0133] The present invention provides an antitumor agent comprising bacteria isolated from a tumor. The antitumor agent may comprise, for example, one or more bacteria isolated from a tumor, for example, the antitumor agent may comprise one or two bacteria isolated from a tumor. In the antitumor agent, the bacteria isolated from the tumor may be used as an effective ingredient for exerting antitumor activity.

[0134] 2.1.1 Antitumor agent comprising red non-sulfur bacteria isolated from tumors

[0135] In one embodiment of the present invention, the bacteria isolated from the tumor include red non-sulfur bacteria isolated from the tumor. As mentioned above, the red non-sulfur bacteria isolated from the tumor can exert excellent anti-tumor activity.

[0136] Furthermore, the red non-sulfur bacteria accumulate in tumors when administered to animals (particularly when administered parenterally). Therefore, the antitumor agent may not be administered directly to the tumor site, but may be administered directly to the tumor site.

[0137] In addition, the red non-sulfur bacteria have specific optical properties. Therefore, the anti-tumor agent can be administered to treat tumors based on the anti-tumor activity, but in addition to treating the tumor, it can also be used to determine the presence or absence of a tumor or determine the shape or location of a tumor.

[0138] (Type of bacteria)

[0139] As examples of the above-mentioned red non-sulfur bacteria, the following can be cited:

[0140] ・Bacteria of the genus Rhodopseudomonas, such as Rhodopseudomonas Palustri and Rhodopseudomonas pseudopalustri;

[0141] ・Blastochloris bacteria, such as Blastochloris viridis and Blastochloris sulfoviridis;

[0142] ・Afifella bacteria, such as Afifella marina;

[0143] ・Rhodobacter bacteria, such as Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter sphaeroides;

[0144] ・Rubrivivax bacteria, such as Rubrivivax gelatinosus;

[0145] ・Bacteria of the genus Pararhodospirillum, such as Pararhodospirillumoryzae and Pararhodospirillum sulfurexigens;

[0146] ・Bacteria of the genus Rhodocista, such as Rhodocista centenaria;

[0147] ・Marichromatium bacteria, such as Marichromatium litoris;

[0148] ・Bacteria of the genus Phaeochromatium, such as Phaeochromatium fluminis;

[0149] ・Bacteria of the genus Rhodoferax, such as Rhodoferax fermentans;

[0150] ・Bacteria of the genus Rhodomicrobium, such as Rhodomicrobium udaipurense and Rhodomicrobium vannielii; and

[0151] ・Bacteria of the genus Rhodovulum, such as Rhodovulumsulfidophilum.

[0152] The red non-sulfur bacteria isolated from a tumor and contained in the antitumor agent according to the present invention may be, for example, any one or a combination of two or more of the bacteria of the genera listed above.

[0153] Furthermore, the red non-sulfur bacteria isolated from a tumor and contained in the antitumor agent according to the present invention may be, for example, any one or a combination of two or more of the above-mentioned bacterial species.

[0154] In a preferred embodiment, the red non-sulfur bacteria contained in the antitumor agent according to the present invention can be a bacterium of the genus Rhodopseudomonas or a bacterium of the genus Chlorobacter or both. These bacteria isolated from tumors can exert particularly excellent antitumor activity. In addition, these bacteria isolated from tumors have the characteristic of aggregating in tumors when administered to animals (especially parenteral administration), and these bacteria have specific optical properties, so they are useful for generating tumor diagnostic images.

[0155] For example, the red non-sulfur bacteria isolated from the tumor contained in the above-mentioned antitumor agent can be any one, two, three, or four of Rhodopseudomonas palustris, Rhodopseudomonas pseudopalustris, Green Chlorobacterium and Thiochlorophyll. Particularly preferably, the red non-sulfur bacteria contained in the antitumor agent according to the present invention can at least contain Rhodopseudomonas palustris isolated from the tumor. For example, the red non-sulfur bacteria contained in the antitumor agent according to the present invention can only contain Rhodopseudomonas palustris isolated from the tumor.

[0156] In a particularly preferred embodiment, the red non-sulfur bacteria contained in the antitumor agent according to the present invention is a bacterium of the genus Rhodopseudomonas. Bacteria of the genus Rhodopseudomonas isolated from tumors can exert particularly excellent antitumor activity.

[0157] For example, the red non-sulfur bacteria contained in the antitumor agent according to the present invention can be Rhodopseudomonas palustris or Rhodopseudomonas pseudopalustris or both. Particularly preferably, the red non-sulfur bacteria contained in the antitumor agent according to the present invention can at least contain Rhodopseudomonas palustris isolated from a tumor.

[0158] In a particularly preferred embodiment, the antitumor agent according to the present invention, in addition to the above-mentioned red non-sulfur bacteria, also contains other bacteria. The combination of the red non-sulfur bacteria isolated from the tumor and the other bacteria isolated from the tumor exerts particularly excellent antitumor activity. The other bacteria can be, for example, Proteus bacteria. The two or more bacteria constituting the combination can preferably be isolated together from the same tumor and cultured together (co-cultured). That is, the antitumor agent according to the present invention can include a combination of the above-mentioned red non-sulfur bacteria (such as the above-mentioned red Pseudomonas bacteria) and the above-mentioned other bacteria (such as Proteus bacteria) isolated together with the tumor.

[0159] The Proteus bacteria as the other bacteria may be, for example, any one, two, or three of Proteus mirabilis, Proteus vulgaris, and Proteus myxofaciens, preferably Proteus mirabilis. The combination of the red non-sulfur bacteria isolated from a tumor and Proteus mirabilis isolated from a tumor exerts a particularly excellent anti-tumor effect.

[0160] In a particularly preferred embodiment, the above-mentioned antitumor agent may comprise a combination of Rhodopseudomonas palustris and Proteus mirabilis isolated from a tumor. These two composite bacteria exert particularly excellent antitumor activity. The composite bacteria may be, for example, a composite bacteria preserved with the deposit number NITEBP-03627. The deposit date of the composite bacteria is March 23, 2022, and it is internationally deposited in the National Institute for Technology Evaluation, Patent Microbial Preservation Center (NPMD, zip code 292-0818, Room 2-5-8122, Kazusa Kamata, Kisarazu City, Chiba Prefecture). In addition, in the present invention, one of the composite bacteria can be used separately from the other.

[0161] The composition ratio of the red non-sulfur bacteria to the other bacteria in the anti-tumor agent may be, for example, 99:1 to 1:99, and preferably, the content of the red non-sulfur bacteria is higher. In a preferred embodiment, the composition ratio of the red non-sulfur bacteria to the other bacteria in the anti-tumor agent may be, for example, 99:1 to 50:50, and more preferably 99:1 to 55:45. The composition ratio may be, for example, 80:20 to 55:45, or may be 70:30 to 60:40. These composition ratios may be composition ratios based on the CFU of each bacterium.

[0162] With this composition ratio, in particular, the content of the red non-sulfur bacteria is higher than the content of the other bacteria, thereby exerting excellent antitumor activity.

[0163] The composition ratio based on the above CFU can be determined by inoculating the bacteria in the above antitumor agent onto a culture medium in a culture dish and counting the number of colonies produced by culturing for a predetermined time.

[0164] For example, Proteus mirabilis listed as the other bacteria forms colonies 2 to 3 days after the start of culture, while Rhodopseudomonas palustris among the red non-sulfur bacteria forms colonies 7 to 10 days after the start of culture. Therefore, 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 colony counts of Proteus mirabilis and Rhodopseudomonas palustris can be obtained respectively. The above composition ratio can be determined based on these colony counts.

[0165] As described above, when the red non-sulfur bacteria and the other bacteria are contained in the antitumor agent, the difference in the period for each bacteria to form colonies is used to determine the composition ratio of these bacteria.

[0166] In addition, when the period for forming colonies is the same, the number of colonies can be counted based on the optical properties of the red non-sulfur bacteria and the other bacteria. The red non-sulfur bacteria have specific optical properties, but the other bacteria do not have the optical properties. Therefore, it is possible to determine which bacteria the colonies formed on the culture medium in the culture dish are based on the presence or absence of the optical properties, and count the number of colonies of each bacterium.

[0167] (Optical properties of bacteria)

[0168] Preferably, the bacteria isolated from the tumor may have the optical properties described below. For example, the bacteria isolated from the tumor have optical properties different from those of commercially available bacteria of the same species. Having such optical properties is believed to be related to exerting anti-tumor activity.

[0169] The bacteria isolated from the tumor contained in the antitumor agent of the present invention can show the following characteristics related to the absorption spectrum. The bacteria isolated from the tumor (especially the above-mentioned red non-sulfur bacteria) have higher absorption characteristics than ordinary bacteria of the same species (such as commercially available bacteria). Having this higher absorption characteristic is believed to be related to the antitumor activity of the above-mentioned bacteria (especially the above-mentioned red non-sulfur bacteria).

[0170] For example, when the bacteria (particularly the red non-sulfur bacteria) contained in the antitumor agent are measured at 2×10 7 When the concentration of CFU / mL is dispersed in the absorbance spectrum of the dispersion in PBS buffer, the absorbance at 808nm is preferably 0.029 or more, more preferably 0.030 or more, and further more preferably 0.031 or more, 0.032 or more, 0.033 or more, 0.034 or more, or 0.035 or more. Such a high absorbance is believed to be related to the antitumor activity of the above-mentioned bacteria (particularly red non-sulfur bacteria).

[0171] The upper limit of the absorbance at 808 nm may not be particularly set, and may be, for example, 0.10 or less, 0.9 or less, or 0.8 or less.

[0172] The above-mentioned absorption spectrum was measured as shown in the following Examples.

[0173] In addition, when the bacteria (especially the red non-sulfur bacteria) contained in the antitumor agent were measured at 2×10 7When the concentration of CFU / mL is dispersed in the absorbance spectrum of the dispersion in PBS buffer, the absorbance at 865nm is preferably 0.032 or more, more preferably 0.033 or more, further more preferably 0.034 or more, 0.035 or more, 0.036 or more, 0.037 or more, or 0.038 or more. Such a high absorbance is believed to be related to the antitumor activity of the above-mentioned bacteria (particularly red non-sulfur bacteria).

[0174] The upper limit of the absorbance at 865 nm may not be particularly set, and may be, for example, 0.11 or less, 0.10 or less, or 0.09 or less.

[0175] Particularly preferably, when measuring the absorption spectrum of the above dispersion, the absorbance at 808 nm is preferably 0.029 or more, more preferably 0.030 or more, further more preferably 0.031 or more, 0.032 or more, 0.033 or more, 0.034 or more, or 0.035 or more, and the absorbance at 865 nm is preferably 0.032 or more, more preferably 0.033 or more, further more preferably 0.034 or more, 0.035 or more, 0.036 or more, 0.037 or more, or 0.038 or more. Such high absorbance at these two wavelengths is believed to be related to the antitumor activity of the above bacteria (especially red non-sulfur bacteria).

[0176] The bacteria isolated from the tumor contained in the antitumor agent of the present invention can show the following characteristics related to the fluorescence spectrum. The bacteria isolated from the tumor (especially the above-mentioned red non-sulfur bacteria) have higher fluorescence characteristics than ordinary bacteria of the same species (such as commercially available bacteria). Having this higher fluorescence characteristic is believed to be related to the antitumor activity of the above-mentioned bacteria (especially the above-mentioned red non-sulfur bacteria).

[0177] For example, when the bacteria contained in the above antitumor agent are measured at 2.5×10 7 When the fluorescence spectrum of the dispersion liquid with a concentration of CFU / mL dispersed in PBS buffer (excitation wavelength is 805 nm), the fluorescence intensity at 888 nm is preferably 4.4 or more, more preferably 4.5 or more, further preferably 5.0 or more, 7.0 or more, or 9.0 or more.

[0178] The upper limit of the fluorescence intensity may not be particularly set, and may be, for example, 30 or less, 25 or less, or 20 or less.

[0179] Such high fluorescence intensity is considered to be related to the anti-tumor activity of the above bacteria (especially red non-sulfur bacteria).

[0180] The above-mentioned fluorescence spectrum was measured as shown in the following examples.

[0181] (Toxicity of bacteria)

[0182] Preferably, the bacteria isolated from the tumor contained in the antitumor agent of the present invention have no toxicity as described below, or even if toxicity is very low. The bacteria isolated from the tumor may have such toxicity characteristics, for example, different from commercially available bacteria of the same species. The bacteria isolated from the tumor have such toxicity characteristics, which further improves the usefulness of the antitumor agent.

[0183] For example, when the bacteria contained in the above antitumor agent are present at a concentration of 1×10 9 When the amount of CFU is administered once from the tail vein of the mouse, the survival rate of the mouse within 40 days after the administration is preferably 90% or more, more preferably 95% or more, for example, 100%. The above-mentioned bacteria contained in the antitumor agent of the present invention may have such toxicity characteristics, especially extremely low toxicity characteristics. Various conditions for determining the survival rate of the mouse (e.g., the culture medium of the administered bacterial suspension and its preparation method, etc.) are shown in the following examples.

[0184] (Immune Activating Properties)

[0185] The bacteria isolated from tumors contained in the antitumor agent of the present invention may be bacteria having immunogenicity. The antitumor agent can treat tumors by virtue of the immunogenicity.

[0186] The immune activation property may be, for example, a property of activated immune cells.

[0187] As the above-mentioned immune cells, for example, natural immune cells can be listed, and as the above-mentioned natural immune cells, macrophages, NK cells and neutrophils can be listed. That is, the above-mentioned bacteria isolated from the tumor can be bacteria having the characteristics of activating natural immune cells, for example, bacteria having the characteristics of activating one, two, or all three of macrophages, NK cells and neutrophils.

[0188] In addition, as the above-mentioned immune cells, for example, acquired immune cells can be listed, and as the above-mentioned acquired immune cells, T cells and B cells can be listed. That is, the above-mentioned bacteria separated from the tumor can be bacteria having the property of activating acquired immune cells, for example, can be bacteria having the property of activating T cells or B cells or both.

[0189] This immune cell-activating property is thought to contribute to the antitumor activity of the bacteria isolated from tumors.

[0190] In addition, the immune activation property may be, for example, a property of enhancing the expression of anti-tumor markers. As the anti-tumor markers, necrosis markers and apoptosis markers are listed, and as specific examples of each, TNF-α and Caspase-3 are listed. That is, the bacteria isolated from the tumor may be bacteria having the property of enhancing the expression of anti-tumor markers, for example, bacteria having the property of enhancing the expression of necrosis markers or apoptosis markers macrophages or both.

[0191] It can be considered that this anti-tumor marker expression enhancement property also contributes to the anti-tumor activity of the above-mentioned bacteria isolated from tumors.

[0192] In addition, since the above-mentioned bacteria isolated from tumors have such immunoactivation properties, they can also be used as the active ingredient of immunoactivators. That is, the present invention also provides an immunoactivator, which contains the above-mentioned bacteria isolated from tumors. The above-mentioned immunoactivator can be called an immunoactivation composition. The structure related to the above-mentioned antitumor agent recorded in this specification can be used as the structure of the above-mentioned immunoactivator.

[0193] 2.1.2 Antitumor agents comprising Proteus bacteria isolated from tumors

[0194] In another embodiment of the present invention, the above-mentioned bacteria isolated from the tumor contained in the antitumor agent of the present invention include Proteus bacteria. The Proteus bacteria isolated from the tumor can exert excellent antitumor activity as described in 1. above. In addition, when the Proteus bacteria are administered to animals, especially when administered parenterally, they accumulate in the tumor, so they can be administered directly to the tumor site instead of directly to the tumor site. In addition, the Proteus bacteria can be administered to treat the tumor based on the antitumor activity.

[0195] In a preferred embodiment, the Proteus bacteria may be bacteria isolated from a tumor. That is, the antitumor agent may contain only Proteus bacteria isolated as bacteria.

[0196] (Type of bacteria)

[0197] The above-mentioned Proteus bacteria can be, for example, any one, two, or three of Proteus mirabilis, Proteus vulgaris, and Proteus mucosa, preferably Proteus mirabilis. Proteus mirabilis isolated (especially isolated) from a tumor exerts particularly excellent anti-tumor activity. The Proteus mirabilis can be, for example, a bacterium deposited with the deposit number NITEBP-03626. The deposit date of the bacterium is March 23, 2022, and it is internationally deposited in the National Institute for Technology Evaluation, Patent Microorganism Depository (NPMD, zip code 292-0818, Room 2-5-8122, Kazusa Kamata, Kisarazu City, Chiba Prefecture).

[0198] (Toxicity of bacteria)

[0199] Preferably, the Proteus bacteria isolated from the tumor contained in the antitumor agent of the present invention have no toxicity as described below, or even if toxicity is very low. The bacteria isolated from the tumor may have such toxicity characteristics, for example, different from commercially available bacteria of the same species. The bacteria isolated from the tumor have such toxicity characteristics, which further improves the usefulness of the antitumor agent.

[0200] For example, when the above-mentioned Proteus bacteria contained in the above-mentioned antitumor agent is present at 1×10 8 When the amount of CFU is administered once from the tail vein of the mouse, the survival rate of the mouse within 40 days after the administration is preferably 90% or more, more preferably 95% or more, for example, 100%. The above-mentioned Proteus bacteria contained in the antitumor agent of the present invention may have such toxicity characteristics, especially extremely low toxicity characteristics. Various conditions for determining the survival rate of the mouse (e.g., a culture medium for the administered bacterial suspension and a preparation method thereof, etc.) are shown in the following examples.

[0201] (Immune Activating Properties)

[0202] The Proteus bacteria isolated from the tumor may be bacteria having immunostimulatory properties. The anti-tumor agent may treat the tumor by virtue of the immunostimulatory properties.

[0203] The immune activation property may be, for example, a property of activated immune cells.

[0204] As the above-mentioned immune cells, for example, natural immune cells can be listed, and as the above-mentioned natural immune cells, macrophages, NK cells and neutrophils can be listed. That is, the Proteus bacteria separated from the tumor can be bacteria having the characteristics of activating natural immune cells, for example, can be bacteria having the characteristics of activating one, two, or all three of macrophages, NK cells and neutrophils.

[0205] In addition, as the above-mentioned immune cells, for example, acquired immune cells are listed, and as the above-mentioned acquired immune cells, T cells and B cells are listed. That is, the Proteus bacteria separated from the tumor can be bacteria having the property of activating acquired immune cells, for example, can be bacteria having the property of activating T cells or B cells or both.

[0206] This immune cell-activating property is thought to contribute to the antitumor activity of the Proteus bacteria isolated from tumors.

[0207] In addition, the immune activation property may be, for example, a property of enhancing the expression of anti-tumor markers. As the anti-tumor markers, necrosis markers and apoptosis markers are listed, and as specific examples of each, TNF-α and Caspase-3 are listed. That is, the Proteus bacteria isolated from the tumor may be bacteria having the property of enhancing the expression of anti-tumor markers, for example, may be bacteria having the property of enhancing the expression of necrosis markers or apoptosis markers macrophages or both.

[0208] It can be considered that this anti-tumor marker expression enhancement property also contributes to the anti-tumor activity of the Proteus bacteria isolated from the tumor.

[0209] In addition, the Proteus bacteria isolated from the tumor have such immunoactivation properties and can also be used as an effective ingredient of an immunoactivator. That is, the present invention also provides an immunoactivator, which contains the Proteus bacteria isolated from the tumor. The above-mentioned immunoactivator can be called an immunoactivation composition. The structure related to the above-mentioned anti-tumor agent recorded in this specification can be used as the structure of the immunoactivator.

[0210] 2.1.3 Examples of other bacteria

[0211] In another embodiment of the present invention, the above-mentioned bacteria separated from the tumor can be, for example, other bacteria present in the tumor, in particular, tumor resident bacteria. As examples of such bacteria, bacteria belonging to the order Enterobacteriales, bacteria belonging to the order Pseudomonadales, bacteria belonging to the order Burkholderiales, bacteria belonging to the order Rhodobacterales, bacteria belonging to the order Aeromonadales, bacteria belonging to the order Bacillales, bacteria belonging to the order Clostridiales, bacteria belonging to the order Lactobacillales, bacteria belonging to the order Actinomycetales, and bacteria belonging to the order Actinomycetales. ctinomycetales), the bacterium belonging to Bifidobacteriales, the bacterium belonging to Bacteroidales, the bacterium belonging to Flavobacteriales, the bacterium belonging to Fusobacteriales, the bacterium belonging to Streptophyta, the bacterium belonging to Corynebacteriales, the bacterium belonging to Rhodospirillales, the bacterium belonging to Neisseriales. The bacterium used in the present invention can be any one or a combination of two or more of the purpose bacteria belonging to these enumerated ones.

[0212] More specific examples of the bacteria of each target listed above include the following.

[0213] Bacteria of the order Enterobacteriales: for example, bacteria of the family Enterobacteriaceae, in particular bacteria of the genera Proteus, Enterobacter, Klebsiella and Citrobacter, more particularly the species of Proteus listed in 2.1.2 above, as well as Enterobacter asburiae, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae, etc.;

[0214] Pseudomonadales bacteria: such as Pseudomonadaceae bacteria and Moraxellaceae bacteria, in particular Pseudomonas bacteria and Acinetobacter bacteria, more particularly Pseudomonas sargentinensis and Acinetobacter US-424, etc.;

[0215] Rhodobacterales bacteria: for example, Rhodobacteraceae bacteria, in particular, Paracoccus bacteria, and more particularly, Paracoccus marcusii;

[0216] Sphingomonadales bacteria: for example, Sphingomonadales bacteria, in particular, Sphingomonas bacteria, more particularly, Sphingomonas yunnanensis, Sphingomonas US-602, and Sphingomonas yanoikuyae, etc.;

[0217] Bacillales bacteria: for example, bacteria of the family Staphylococcus, in particular bacteria of the genus Staphylococcus, more particularly Staphylococcus aureus and Staphylococcus cohnii;

[0218] Bacteria of the order Lactobacillales: such as bacteria of the family Streptococcus and the family Lactobacillaceae, in particular bacteria of the genus Streptococcus and the genus Lactobacillus, more particularly Streptococcus infantis and Lactobacillus iners;

[0219] Actinomycetales bacteria: for example, bacteria of the family Actinomycetales, in particular bacteria of the genus Actinomyces, and more particularly Actinomyces massiliensis;

[0220] Fusobacteriales bacteria: such as Fusobacteriaceae bacteria, especially bacteria of the genus Fusobacterium, and more especially Fusobacterium nucleatum;

[0221] Corynebacteriales bacteria: for example, Corynebacteriaceae bacteria, in particular bacteria of the genus Corynebacterium, and more particularly Corynebacterium US-1715;

[0222] Rhodospirillales bacteria: such as Acetobacteraceae bacteria, especially Roseomonas bacteria, and more especially Roseomonas mucosa bacteria;

[0223] Neisseriales bacteria: for example, Neisseriaceae bacteria, particularly bacteria of the genus Neisseria, and more particularly Neisseria macacae.

[0224] In the present invention, any one or a combination of two or more of the bacteria belonging to the above-listed families or genera can be isolated from the tumor. That is, in the present invention, as the bacteria isolated from the tumor, the one or a combination of two or more can be used in the present invention.

[0225] 2.1.4 Structure of antitumor agents

[0226] The antitumor agent according to the present invention may contain the bacteria isolated from the above tumor in a living state. That is, the above bacteria isolated from the tumor can be administered to the animal in a living state. Therefore, it can be considered that the bacteria can more easily reach the tumor site, and it can be considered that the antitumor activity of the bacteria can be more effectively exerted. Reaching the tumor site is also useful for the diagnosis described later.

[0227] Furthermore, the antitumor agent according to the present invention may also contain bacteria isolated from the above-mentioned tumors in a dead state.

[0228] In the present invention, "anti-tumor agent" may refer to an agent, particularly a pharmaceutical agent, for treating a tumor generated in an animal. "Treating a tumor" may refer to, for example, reducing tumor size, inhibiting tumor growth, killing or reducing tumor cells, or inhibiting tumor cell proliferation.

[0229] "Antitumor agent" may refer to an agent used to treat or prevent an animal suffering from a tumor. The animal may be, for example, a mammal, particularly a human, but may also be a non-human animal. The non-human animal may be, for example, an agricultural animal or a pet, such as a cow, horse, sheep, goat, pig, dog, cat, rabbit, etc.

[0230] The above-mentioned "agent" may be an agent consisting of one component or an agent containing two or more components. At least one of these components is a bacterium isolated from the above-mentioned tumor. The above-mentioned agent may contain two or more components, and therefore, in this case, the antitumor agent of the present invention may be referred to as an antitumor composition or an antitumor pharmaceutical composition.

[0231] That is, the antitumor agent of the present invention may contain other components in addition to the bacteria isolated from the tumor. Those skilled in the art can appropriately select the other components according to the administration method or administration site of the antitumor agent.

[0232] The antitumor agent of the present invention can be particularly used for treating malignant tumors or benign tumors, and is particularly preferably used for treating malignant tumors. The above-mentioned malignant tumors are also called "cancers". Cancers can be divided into solid cancers and blood cancers. Solid cancers can be further divided into carcinomas and sarcomas. The above-mentioned antitumor agent can be called an anticancer agent or a composition for treating cancer or a composition for treating cancer.

[0233] The antitumor agent of the present invention can be used to treat solid cancer or blood cancer, and can be used to treat solid cancer, for example, can be used to treat carcinoma or sarcoma.

[0234] 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 believed to be the activation of immune cells described later. That is, it can be considered that the antitumor agent exerts antitumor activity through the activated immune cells. Therefore, the antitumor agent of the present invention is believed to be effective not only for a specific cancer, but also for various cancers, and in fact, the effectiveness for various cancers is shown in the examples described later.

[0235] In one embodiment, the antitumor agent of the present invention can also be used to treat cancer. The antitumor agent of the present invention can be used to treat, for example, head and neck cancer (e.g., pharyngeal cancer, laryngeal cancer, tongue cancer, etc.), esophageal cancer, gastric cancer, duodenal cancer, colon 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, uterine cancer) and ovarian cancer. Any one or more agents.

[0236] In another embodiment, the antitumor agent of the present invention can also be used to treat sarcoma. The antitumor agent of the present invention can be, for example, an agent for treating any one of osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma.

[0237] In one embodiment, the antitumor agent of the present invention can be constituted by a liquid. That is, the liquid can be a liquid containing bacteria isolated from a tumor according to the present invention (particularly the bacterial dispersion). The liquid component other than the above-mentioned bacteria in the liquid can be, for example, an injection or drip used in the medical field, more specifically, an isotonic solution, a hypotonic solution or a hypertonic solution. For example, the above-mentioned liquid can be saline (physiological saline), a sugar solution, or a buffer solution, etc. In addition, the liquid can be phosphate-buffered saline. The dosage form of the liquid is particularly suitable for allowing the antitumor agent of the present invention to reach the tumor while maintaining antitumor activity.

[0238] The antitumor agent of the present invention is preferably administered parenterally. For example, the antitumor agent of the present invention can be administered intravascularly (e.g., intravenously or intraarterially), subcutaneously, intramuscularly, or intramedullary. Preferably, the antitumor agent of the present invention is administered intravascularly (e.g., intravenously or intraarterially).

[0239] As described above, the bacteria isolated from tumors contained in the antitumor agent of the present invention have the property of gathering at the tumor site, so when it is administered intravascularly, the bacteria can reach the tumor site more easily. In addition, intravascular administration also causes less trauma.

[0240] Furthermore, by administering bacteria intravascularly, the bacteria often exhibit toxicity and adversely affect patients. However, as described above, the bacteria isolated from tumors contained in the antitumor agent of the present invention do not exhibit such toxicity, or even if they do, the degree of such toxicity is very low.

[0241] In addition, the antitumor agent of the present invention can be administered directly to the tumor site or the vicinity of the tumor site using, for example, a syringe or other tube. In addition, the antitumor agent of the present invention can be administered orally depending on the location of the tumor.

[0242] The antitumor agent of the present invention can be administered in the following manner, that is, each administration of the above-mentioned bacteria isolated from tumors is, for example, 10 4 CFU / kg body weight~10 11 CFU / kg body weight, preferably 10 5 CFU / kg body weight~10 11 CFU / kg body weight, more preferably 10 6 CFU / kg body weight~10 11 CFU / kg body weight. When the amount of bacteria administered is too small, the antitumor effect may be reduced. In addition, when the amount of bacteria administered is too large, it is not desirable from the perspective of cost.

[0243] When the antitumor agent of the present invention is a liquid, the antitumor agent can be, for example, 10 5 CFU / ml~10 12CFU / ml, preferably 10 6 CFU / ml~10 12 CFU / ml, more preferably 10 7 CFU / ml~10 12 The CFU / ml content includes the above-mentioned bacteria isolated from the tumor.

[0244] The antitumor agent of the present invention may be administered only once, or may be administered twice or more. The antitumor agent of the present invention can exert its effect by a single administration, but may be administered twice or more if necessary.

[0245] The antitumor agent of the present invention may be administered once a day, or may be administered twice or more (eg, twice or three times) a day.

[0246] When the antitumor agent of the present invention is administered multiple times, the antitumor agent may be administered every day, or may be administered every other day or every two days. In addition, the antitumor agent may be administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

[0247] The antitumor agent of the present invention may contain additives used in the formulation of the pharmaceutical field, and may contain various components, such as pH adjusters and colorants. In addition, as long as the effect of the present invention is not impaired, the antitumor agent of the present invention may contain known or future discovered drug components for treating tumors. The formulation of the antitumor agent of the present invention can be implemented by a suitable known method according to the dosage form.

[0248] As described above, the antitumor agent of the present invention can be used not only for treating tumors but also for diagnosing tumors. As described later, the bacteria isolated from tumors have the property of gathering in tumors when administered. Therefore, utilizing this property, the site where the bacteria gather can be designated as a tumor. For this diagnosis, please refer to the following description.

[0249] 2.1.5 Preparation of antitumor agents

[0250] In order to prepare the above antitumor agent, the above-mentioned bacteria can be used. The bacteria can be prepared, for example, as follows.

[0251] (Tumor Recovery Step S11)

[0252] In the above-mentioned tumor recovery step, the tumor is recovered from an animal with a tumor. The recovery can be performed, for example, by biopsy. The tumor can be, for example, a tumor formed by a tumor cell of a mammal, in particular a tumor formed by a human tumor cell. The above-mentioned animal with a tumor can be, for example, a mammal, in particular a rodent, but can also be a primate, in particular a human.

[0253] Prior to the above recovery, red non-sulfur bacteria may be administered to the above-mentioned animal with a tumor. The administered red non-sulfur bacteria may be any of the red non-sulfur bacteria listed in 2.1.1 above, preferably Rhodopseudomonas bacteria or Chlorobacter bacteria, and more preferably Rhodopseudomonas bacteria. The specific types of these bacteria are as described in 2.1.1 above, and any one or more than two of them may be administered. The administered bacteria may be commercially available.

[0254] When the red non-sulfur bacteria are administered as described above, the administration is, for example, parenteral administration, particularly intravenous administration. After a predetermined period of time after the administration, for example, 1 to 10 days, particularly 2 to 5 days, the above-mentioned tumor recovery is performed. It can be assumed that, over time, the administered red non-sulfur bacteria will reach the tumor.

[0255] The above-mentioned bacterial administration may or may not be performed. In the case where the above-mentioned bacterial administration is not performed, the bacteria originally present in the tumor can be recovered. In the case where the above-mentioned bacterial administration is performed, the bacteria originally present in the tumor can be recovered, or the bacteria that have reached the tumor through the above-mentioned bacterial administration can be recovered.

[0256] (Bacteria separation step S12)

[0257] In the above-mentioned bacteria separation process, bacteria are separated from the tumor recovered in the above-mentioned tumor recovery process. For example, the tumor obtained by the above-mentioned biopsy is added to a predetermined liquid (such as a buffer solution, etc.) and then homogenized. Through this homogenization, a liquid in which tumor cells and bacteria are suspended is obtained. The liquid is shaken, for example, at a predetermined speed, and the tumor cells are precipitated, and the bacteria are present in the supernatant. In this way, bacteria can be separated from tumor cells, but the separation method is not limited thereto. Separation can be performed by other methods known in the art.

[0258] It is believed that the presence of bacteria in tumors and the isolation of the bacteria from the tumors contribute to the anti-tumor activity of the bacteria.

[0259] (Bacteria Cultivation Step S13)

[0260] In the above-mentioned bacteria culturing step, the bacteria isolated from the tumor in the above-mentioned bacteria isolating step are cultured. Cultivating the bacteria isolated from the tumor is also considered to help the bacteria acquire anti-tumor activity.

[0261] For example, in the above-mentioned bacterial culture step, the supernatant can be added to a general agar medium in a culture dish, for example, and then cultured. Thus, colonies are formed on the culture medium. The colonies can be used as the bacteria isolated from the tumor of the present invention.

[0262] The universal agar medium may be, for example, a medium containing peptone or a medium not containing peptone.

[0263] The peptone contained in the above-mentioned peptone-containing culture medium can be, for example, one or more of casein peptone, animal peptone, gelatin peptone and soy peptone. The above-mentioned peptone-containing culture medium can be, for example, LB culture medium or polypeptone culture medium. The above-mentioned peptone-containing culture medium can also contain an extract in addition to the above-mentioned peptone. The extract can be, for example, a yeast extract or a meat extract (particularly a beef extract) or a combination thereof.

[0264] The above-mentioned culture medium without peptone contains, for example, an extract. As mentioned above, the extract can be a yeast extract or a meat extract (particularly a beef extract) or a combination thereof. The above-mentioned culture medium without peptone can be, for example, an ATCC543 culture medium.

[0265] The above-mentioned universal agar medium can be appropriately selected by those skilled in the art according to the isolated bacteria.

[0266] Preferably, the colonies formed on the above-mentioned agar medium (particularly, bacteria in the colonies) are further cultured in a liquid medium.

[0267] The above-mentioned liquid culture medium can be a liquid universal culture medium. As mentioned above, the liquid universal culture medium can be, for example, a culture medium containing peptone, or can also be a culture medium without peptone. The peptone and extract contained in these culture media are as mentioned above, and their description also applies to the above-mentioned liquid culture medium. The above-mentioned liquid culture medium can be appropriately selected by those skilled in the art according to the isolated bacterium.

[0268] (Cultivation in medium without cysteine ​​addition)

[0269] In one embodiment, the above-mentioned liquid culture medium can be a liquid culture medium without adding cysteine. The above-mentioned liquid culture medium can be, for example, a liquid culture medium without adding cysteine, and comprising peptone, comprising an extract (particularly a yeast extract). The liquid culture medium can be, for example, an LB culture medium or a polypeptone culture medium without adding cysteine. Alternatively, the above-mentioned liquid culture medium can be, for example, a liquid culture medium without adding cysteine, and without peptone, comprising an extract (particularly a yeast extract). As an example of this culture medium, the ATCC543 culture medium without adding cysteine ​​can be cited.

[0270] The bacteria cultured in the above-mentioned liquid culture medium can be further cultured on a universal agar medium to form colonies. The universal agar medium can be, for example, a universal agar medium to which deoxycholic acid has been added. The ratio (%) of the content (g) of deoxycholic acid in the above-mentioned agar medium to the amount (g) of the above-mentioned agar medium can be, for example, 0.01% to 1%, preferably 0.03% to 0.5%, and more preferably 0.05% to 0.3%. The universal agar medium can be an agar medium (fresh medium) the same as the universal agar medium described initially in this step, except for the addition of deoxycholic acid. Through this culture, colonies are formed on the above-mentioned culture medium.

[0271] The formed colonies can 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 culture.

[0272] As described above, bacteria cultured in a medium to which cysteine ​​is not added can be used as an active ingredient of the antitumor agent of the present invention.

[0273] This embodiment is suitable for obtaining, for example, red non-sulfur bacteria (especially Rhodopseudomonas bacteria) with anti-tumor activity or composite bacteria of red non-sulfur bacteria (especially Rhodopseudomonas bacteria) with anti-tumor activity and other bacteria (especially Proteus bacteria).

[0274] (Cultivation in medium supplemented with cysteine)

[0275] In another embodiment, the liquid culture medium can be a liquid culture medium to which cysteine ​​has been added. The liquid culture medium can be, for example, a liquid culture medium to which cysteine ​​has been added, and which contains peptone and an extract (particularly a yeast extract). The liquid culture medium can be, for example, an LB culture medium or a polypeptone culture medium to which cysteine ​​has been added. Alternatively, the liquid culture medium can be, for example, a liquid culture medium to which cysteine ​​has been added, and which contains no peptone and an extract (particularly a yeast extract). As an example of such a culture medium, an ATCC543 culture medium to which cysteine ​​has been added can be cited.

[0276] When the cysteine ​​content in the liquid medium to which the cysteine ​​is added is expressed as the ratio (%) of the amount of cysteine ​​(g) to the amount of the medium (g), it is, for example, 0.1% to 10%, more preferably 1% to 5%, and even more preferably 2% to 4%.

[0277] The bacteria cultured in the above-mentioned liquid culture medium can be further cultured on a universal agar medium to form colonies. The universal agar medium can be, for example, a universal agar medium to which deoxycholic acid and cysteine ​​are added. The ratio (%) of the content (g) of deoxycholic acid in the above-mentioned agar medium to the amount (g) of the above-mentioned agar medium can be, for example, 0.01% to 1%, preferably 0.03% to 0.5%, and more preferably 0.05% to 0.3%. In addition, the ratio (%) of the content (g) of cysteine ​​in the above-mentioned agar medium to the amount (g) of the above-mentioned agar medium can be, for example, 0.1% to 10%, more preferably 1% to 5%, and even more preferably 2% to 4%. The universal agar medium can be the same agar medium (fresh medium) as the universal agar medium initially described in this step, except for the addition of deoxycholic acid and cysteine. By this culture, colonies are formed on the above-mentioned culture medium.

[0278] The formed colonies can 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 culture.

[0279] The bacteria cultured as described above can be used as an effective ingredient of the antitumor agent of the present invention.

[0280] This embodiment is suitable, for example, for obtaining Proteus bacteria having antitumor activity.

[0281] (Preparation Step S14)

[0282] The antitumor agent is obtained by using the bacteria cultured in the above-mentioned bacterial culture step for formulation. The antitumor agent can be, for example, the above-mentioned liquid. The liquid can be prepared by mixing the above-mentioned bacteria with a predetermined liquid. Those skilled in the art can appropriately select the formulation method according to, for example, the dosage form, the ingredients contained, etc.

[0283] 2.2 Second Embodiment (Bacteria)

[0284] The present invention also provides a bacterium isolated from a tumor. The bacterium has the anti-tumor activity as described above. Therefore, for example, it can be used to prepare an anti-tumor agent. In addition, as described above, the bacterium has the property of aggregating in a tumor, so it can be used to generate a tumor determination image, and can also be used, for example, to prepare a diagnostic drug.

[0285] 2.2.1 Red non-sulfur bacteria

[0286] In one embodiment of the present invention, the bacteria isolated from the tumor include at least one red non-sulfur bacteria isolated from the tumor. The red non-sulfur bacteria are as described in 2.1.1 above, and the description thereof (e.g., the type of bacteria, the optical properties of bacteria, the toxicity of bacteria) is also applicable to this embodiment.

[0287] In one embodiment, the bacteria isolated from the tumor include Rhodopseudomonas bacteria or Chlorobacter bacteria or both isolated from the tumor, and particularly preferably include Rhodopseudomonas bacteria isolated from the tumor. The bacterial species belonging to these genera can be as described in 2.1.1 above.

[0288] The above-mentioned bacteria isolated from the tumor may include other bacteria isolated from the tumor in addition to the above-mentioned red non-sulfur bacteria. The other bacteria may be, for example, bacteria of the genus Proteus. That is, the above-mentioned bacteria isolated from the tumor may be a combination of the above-mentioned red non-sulfur bacteria and other bacteria, for example, a combination of red non-sulfur bacteria (especially red pseudomonas bacteria) and the above-mentioned Proteus bacteria. This combination, as described in 2.1.1 above, exerts particularly excellent anti-tumor activity.

[0289] 2.2.2 Proteus bacteria

[0290] In another embodiment of the present invention, the bacteria isolated from the tumor include at least one Proteus bacteria isolated from the tumor. The Proteus bacteria are as described in 2.1.2 above, and the description thereof (e.g., the type of bacteria and the toxicity of the bacteria) also applies to this embodiment.

[0291] 2.3 Third Embodiment (Image Generating Method)

[0292] The present invention also provides an image generation method. The method may include, for example: an administration step of administering bacteria isolated from a tumor to an animal; and an image generation step of photographing the animal after the administration under a state of being irradiated with light in a predetermined wavelength range, and generating an image for determining a tumor that may be present in the animal. The bacteria isolated from the tumor according to the present invention have specific optical properties, and also have the property of gathering at the tumor site when administered to an animal. Therefore, using these properties, the presence or absence of a tumor can be determined, or the location or shape of the tumor can be determined. In addition, the image generated in this manner can be used not only to determine the tumor, but also to determine the location where the administered bacteria are gathered.

[0293] Hereinafter, each step included in this method will be described.

[0294] (Application Step S21)

[0295] In the above-mentioned administration step, bacteria isolated from a tumor are administered to the animal. The bacteria isolated from the tumor preferably include red non-sulfur bacteria. The red non-sulfur bacteria are as described in 2.1.1 above, and the description thereof is also applicable to the present embodiment. In addition, in addition to the red non-sulfur bacteria, the bacteria may also include other bacteria described in 2.1.1 above (especially Proteus bacteria). The red non-sulfur bacteria preferably have the optical properties described in 2.1.1 above. In addition, the red non-sulfur bacteria preferably have the toxic properties described in 2.1.1 above.

[0296] In the administration step, the above-mentioned bacteria isolated from the tumor are preferably administered non-orally. For example, the above-mentioned bacteria can be administered intravascularly (e.g., intravenously or intraarterially), subcutaneously, intramuscularly, or intramedullary. Preferably, the above-mentioned bacteria are administered intravascularly (e.g., intravenously or intraarterially), particularly intravenously.

[0297] As described above, the bacteria isolated from tumors contained in the antitumor agent of the present invention have the property of gathering at the tumor site, so when it is administered intravascularly, the bacteria can reach the tumor site more easily. In addition, intravascular administration also causes less trauma.

[0298] Furthermore, by administering bacteria intravascularly, the bacteria usually exhibit toxicity and adversely affect the patient. However, as described above, the bacteria isolated from tumors contained in the antitumor agent of the present invention do not exhibit such toxicity, or even if they do, the degree of such toxicity is very low.

[0299] For the parenteral administration, the bacteria may be in the form of a liquid. The liquid may be formed in the same manner as the liquid described in 2.1.4 above, and the description thereof is also applicable to the present embodiment. The amount of bacteria contained in the liquid may also be the same as the bacterial content in the liquid described in 2.1.4 above.

[0300] (Photographing step S22)

[0301] After the administration step, the animal is photographed while being irradiated with light of a predetermined wavelength range. Through the photographing, an image is generated for identifying a tumor that may be present in the animal. The wavelength range of the light can be selected according to the optical properties of the administered bacteria.

[0302] The light may be, for example, infrared light, particularly near infrared light, mid-infrared light or far infrared light. The infrared light has little effect on the irradiated animal. In addition, the red non-sulfur bacteria isolated from the tumor emit fluorescence when irradiated with infrared light (particularly near infrared light), and the fluorescence intensity of the fluorescence is strong. Therefore, the image obtained by photographing the animal under the condition of irradiating infrared light is suitable for determining the tumor.

[0303] For the above-mentioned shooting, a device capable of observing the fluorescence generated by the above-mentioned bacteria can be used. For example, the device can be a device capable of acquiring a fluorescent image of the shooting object. As more specific examples of the device, a fluorescence imaging system, an in vivo fluorescence imaging system, and a fluorescence imager are listed, but not limited thereto.

[0304] From the image obtained by the above imaging, for example, the site where fluorescence from the above bacteria is generated can be identified.

[0305] The bacteria have the property of gathering in tumors. Therefore, if there is a part emitting fluorescence in the image, the part can be determined as the part where the tumor is present. In addition, the position or shape of the tumor can also be determined based on the image.

[0306] Furthermore, when there is no site emitting fluorescence in the image, it can be determined that there is no tumor in the subject.

[0307] As described above, the image obtained by the above-mentioned imaging is useful for determining the presence or absence of a tumor or determining the position or shape of a tumor.

[0308] 2.4 Fourth Embodiment (Method for Preparing Bacteria)

[0309] The present invention also provides a method for preparing useful bacteria. For example, in one embodiment, the bacteria prepared by the preparation method can be bacteria with anti-tumor activity. In addition, the bacteria prepared by the preparation method can be bacteria having the optical properties described in 2.1.1 above.

[0310] The preparation method of the present invention may include, for example, a bacteria separation step of isolating bacteria from a tumor and a bacteria culture step of culturing the bacteria separated in the separation step. As described in 1. above, by isolating bacteria present in a tumor from the tumor and culturing the bacteria, antitumor activity or optical properties or both are obtained. The bacteria separation step and the bacteria culture step are as described in 2.1.5 above, and the description thereof is also applicable to this embodiment.

[0311] The preparation method may further include a tumor recovery step of recovering the tumor containing bacteria before the most recent separation step. The tumor recovery step is also as described in 2.1.5 above, and the description thereof is also applicable to this embodiment.

[0312] 2.5 Fifth Embodiment (Treatment Method and Diagnosis Method)

[0313] The present invention provides a method for treating a tumor using bacteria isolated from a tumor. In the above treatment method, the above bacteria isolated from a tumor can be used as described in 2.1 above regarding the anti-tumor agent. That is, the treatment method can include administering the anti-tumor agent to an animal. The administration can also be performed as described in 2.1 above.

[0314] In addition, the present invention provides a tumor diagnosis method using bacteria isolated from a tumor. In the above diagnosis method, the above bacteria can also be used as described in the above 2.3 regarding the image generation method. Then, the tumor can be diagnosed based on the image obtained in the image generation method.

[0315] In addition, both the treatment and diagnosis of tumors can be performed by the bacteria. That is, the present invention also provides a method for treating and diagnosing tumors using bacteria isolated from tumors. In this method, the above-mentioned bacteria are administered to animals (particularly humans) to treat tumors. The administered bacteria reach the tumor and exert their anti-tumor activity, and are used in an image generation method based on their optical properties. That is, in the above-mentioned method, the above-mentioned bacteria can be administered to treat tumors, and the above-mentioned image generation method can be performed after the administration. Then, the tumor can be diagnosed based on the image obtained in the image generation method.

[0316] Example

[0317] 3. Example

[0318] <Cell culture and cytotoxicity evaluation>

[0319] Mouse colon cancer cells (Colon26) were obtained from the Japanese Collection of Research Bioresources Cell Bank. Mouse sarcoma cells and mouse melanoma cells (B16-F10) were obtained from the Institute of Ageing Medicine, Tohoku University (Medical Cell Resource Center Cell Bank). In order to culture Colon26, Meth-A, and B16-F10, Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) containing 10% fetal bovine serum, 2mMl-glutamine, 1-mM sodium pyruvate, gentamicin, and penicillin-streptomycin (100IUml-1) was used. Cells were cultured in a humidified chamber at 37°C and 5% CO2 atmosphere.

[0320] <Bacterial culture>

[0321] The bacteria used in this example were obtained from the National Institute of Technology and Evaluation Biological Resource Center (NBRC). Rhodopseudomonas palustris (hereinafter also referred to as "RP") (NBRC 16661) was cultured anaerobically at 26-30°C in 543ATCC medium while irradiated with a tungsten lamp. Proteus mirabilis (hereinafter also referred to as "PM") (NBRC 3849) was cultured anaerobically at 30°C in 802NBRC medium. The reagents used in bacterial culture were obtained from Fujifilm Wako Pure Chemical Industries, Ltd., Nacalai Tesque, and Tokyo Chemical Industry.

[0322] <Isolation and culture of intratumoral bacteria>

[0323] refer to Figure 1 The isolation and culture of bacteria within tumors are also described.

[0324] All animal experiments were performed with the approval of the Animal Experiment Committee of the Hokuriku Institute of Science and Technology. Four-week-old female wild-type mice (n = 10; average body weight = 15 g; BALB / cCrSlc) were purchased from Japan SLC Co., Ltd. and acclimated for one week. Colon26 cell dispersion (1 × 10 6 A mixed solution of 100 μL of cells and culture medium / matrigel (Corning) (v / v, 1:1) was administered to a site under the skin on the right side of the abdomen of mice to create colon cancer model mice. About two weeks later, solid cancer (~400 mm 3 ) were injected into the tail vein of mice with 9 CFU / mL) (Figure A). Two days after the administration, the presence of RP in the tumor was confirmed using VISQUE (trademark) In Vivo Smart-LF (Vieworks) (Figure B), and the tumor was removed (Figure C). The tumor was minced with a scalpel and then homogenized in PBS buffer at 4°C with a pestle (Figure D). The resulting mixture was shaken at 15°C and 380 rpm / min for 20 minutes. After the shaking, the supernatant of the above mixture was diluted 10 times with PBS buffer to obtain a bacterial sample.

[0325] The sample (100 μL) was inoculated on an agar medium composed of 543ATCC medium and cultured anaerobically for 7 days (Figure E). Through this anaerobic culture, bacterial colonies were formed on the above agar medium. Several red bacterial colonies formed were picked up with a loop (Figure F), and anaerobically cultured at 26-30°C in 543ATCC medium while irradiating with a tungsten lamp (Figure G). The culture solution obtained by the anaerobic culture was divided into two parts for the next culture.

[0326] Then, one of the above culture solutions was cultured in a common 543ATCC medium to which cysteine ​​(Cys) was added (Figure H1, the culture obtained by this culture is also referred to as "culture 1" hereinafter). When the content of the cysteine ​​is expressed as the ratio (%) of the amount (g) of the cysteine ​​relative to the amount (g) of the 543ATCC medium, it was 3%. In addition, in other culture media to which cysteine ​​was added that appeared later, the content of cysteine ​​was 3% regardless of whether the culture medium was a liquid culture medium or an agar culture medium.

[0327] The other was cultured in 543ATCC medium to which Cys was not added (Fig. H2; the culture obtained by this culture is also referred to as "culture 2" hereinafter).

[0328] Culture 1 was irradiated with a tungsten lamp on an agar medium and cultured anaerobically at a temperature of 26-30°C. The agar medium consisted of 543ATCC medium (added with 0.1% deoxycholic acid) to which Cys was added (Figure I1). The above-mentioned content of deoxycholic acid is the ratio (%) of the amount (g) of deoxycholic acid relative to the amount (g) of the agar medium. In addition, the agar medium used to culture the next culture 2 was included, and the deoxycholic acid content in other subsequent culture media with added deoxycholic acid was 0.1%.

[0329] Culture 2 was cultured anaerobically at 26-30°C on an agar medium consisting of 543ATCC medium (supplemented with 0.1% deoxycholic acid) without Cys addition, while irradiated with a tungsten lamp ( FIG. 12 ).

[0330] A white colony on the agar medium composed of 543ATCC medium was picked up with a needle (Figure I1), and cultured in 543ATCC medium (Figure J1). In this way, bacteria isolated from the tumor were obtained. Genetic identification of the bacteria was performed (BEX Co., Ltd.), and it was found that the bacteria was Proteus mirabilis. The isolated Proteus mirabilis is also referred to as "iP.M." in this manual.

[0331] The deposit date of this Proteus mirabilis is March 23, 2022, the deposit number is NITEBP-03626, and it is internationally deposited in the National Institute for Technology Evaluation, Patent Microorganism Depository (NPMD, postal code 292-0818, Room 2-5-8122, Kazusa Kamata, Kisarazu City, Chiba Prefecture).

[0332] A red colony on an agar medium composed of a 543ATCC medium without the addition of Cys was picked up with a needle (Figure I2), and cultured in a 543ATCC medium without the addition of Cys (Figure J2). In this way, bacteria isolated from the tumor were obtained. The bacteria contain two types, Rhodopseudomonas palustris and Proteus mirabilis, that is, it is a composite bacterium. The composition ratio of Rhodopseudomonas palustris and Proteus mirabilis in the composite bacteria is 97:3 based on CFU. The composite bacteria is also referred to as "iR.P." in this manual.

[0333] The deposit date of the composite bacteria is March 23, 2022, the deposit number is NITEBP-03627, and it is internationally deposited in the National Institute for Technology Evaluation, Patent Microorganism Depository (NPMD, zip code 292-0818, Room 2-5-8122, Kazusa Kamata, Kisarazu City, Chiba Prefecture).

[0334] iR.P. was further cultured on an agar medium consisting of a 543ATCC medium to which Cys was not added and to which 0.1% deoxycholic acid was added. From the colonies produced on the agar medium, red colonies were carefully picked up with a needle under microscopic observation (Fig. K2) and continued to be cultured in a 543ATCC medium to which Cys was not added (Fig. L2). In this way, bacteria in which only Rhodopseudomonas palustris was isolated were obtained from the above-mentioned composite bacteria. The bacteria are also referred to as "isp-RP" in this specification.

[0335] <In vivo anticancer experiment>

[0336] like Figure 2A As shown, 4-week-old female wild-type mice (n=4; average body weight=15 g; BALB / cCrSlc) were purchased from SLC Co., Ltd., Japan, and acclimated for one week. 6 A mixed solution of 100 μL of cells and culture medium / matrigel (Corning) (v / v, 1:1) was administered to a site under the skin of the flank of the mouse to create a colon cancer model mouse. About two weeks later, solid cancer (~400 mm 3 ) were administered (single administration) into the tail vein of mice. 200 μL R.P. (1×10 9 CFU), PM (1×10 8CFU), iR.P.(1×10 9 CFU), or iP.M.(1×10 8 CFU) of bacterial samples or PBS buffer. The behavior and traits of mice, the size of solid tumors and the changes in mouse weight were measured every day. In addition, the size of solid tumors was calculated by the following formula.

[0337] V=L×W 2 / 2

[0338] In this formula, V represents the volume of the solid cancer, L represents the length of the solid cancer, and W represents the width of the solid cancer.

[0339] In addition, the method for preparing the above four bacterial samples to be administered to mice is described below.

[0340] The iR.P. was irradiated with a tungsten lamp in ATCC543 medium without Cys added, and anaerobically cultured at a temperature of 26°C-30°C for 3 days. The culture solution obtained by the anaerobic culture was centrifuged at 3000rpm for 5 minutes. The bacteria precipitated by the centrifugation were suspended in fresh ATCC543 medium without Cys added to obtain a suspension. The number of bacteria in the suspension was measured using a microbial counter (Panasonic, model NP-BCM01-A), and according to the measurement results, fresh ATCC543 medium without Cys added was appropriately added to the suspension to reach a predetermined bacterial concentration, thereby obtaining a bacterial sample of the iR.P.

[0341] RP was irradiated with a tungsten lamp in ATCC543 medium supplemented with Cys and anaerobically cultured at 26°C-30°C for 3 days. The culture solution obtained by the anaerobic culture was centrifuged at 3000rpm for 5 minutes. The bacteria precipitated by the centrifugation were suspended in fresh ATCC543 medium supplemented with Cys to obtain a suspension. The number of bacteria in the suspension was measured using a microbial counter (Panasonic, model NP-BCM01-A), and according to the measurement results, fresh ATCC543 medium supplemented with Cys was appropriately added to the suspension to reach a predetermined bacterial concentration, thereby obtaining a bacterial sample of RP.

[0342] The bacterial sample of iP.M. was obtained by the same method as the bacterial sample of RP described above, that is, the bacterial sample of iP.M. was obtained by anaerobic culture in ATCC543 medium supplemented with Cys, centrifuging the culture solution obtained by the anaerobic culture, adjusting the suspension using the precipitate obtained by the centrifugation, and adjusting the concentration of bacteria in the suspension.

[0343] PM was cultured anaerobically in 802NBRC medium at 30°C for 3 days. The culture solution obtained by the anaerobic culture was centrifuged at 3000 rpm for 5 minutes. The bacteria precipitated by the centrifugation were suspended in fresh 802NBRC medium to produce a suspension. The number of bacteria in the suspension was measured using a microbial counter (Panasonic, model NP-BCM01-A), and fresh 802NBRC medium was appropriately added to the suspension according to the measurement results to achieve a predetermined bacterial concentration, thereby obtaining a bacterial sample of PM.

[0344] In addition, the CFU value of each of the above bacterial samples is the CFU value of bacteria in 200 μL of the bacterial sample administered by the above single administration.

[0345] In subsequent experiments, each bacterial sample to be administered to mice was prepared using the same method.

[0346] The PBS buffer solution is a product of FUJIFILM Wako Pure Chemical Industries, Ltd. (https: / / labchem-wako.fujifilm.com / jp / product / detail / W01W0116-2355.html) and has the following composition.

[0347] pH:7.1~7.3

[0348] NaCl:9000mg / L

[0349] KH 2 PO 4 :144mg / L

[0350] Na 2 HPO 4 (Anhydrous): 421mg / L

[0351] In subsequent experiments, the same PBS buffer was also used.

[0352] Figure 2B The photographs of mice after intravenous administration of each bacteria are shown in FIG.

[0353] As shown in the figure, in mice administered iR.P. or iP.M., tumor size decreased over time, and 30 days after treatment, the tumor disappeared.

[0354] On the other hand, the tumor size of mice treated with commercially available RP or PBS buffer did not decrease, but increased over time, with tumor volumes exceeding 2000mm. 3 Therefore, the experiments on these mice were stopped from a humanitarian point of view. At the same time, no photos were taken 30 days after treatment.

[0355] Figure 2C The evaluation results of the antitumor activity of various bacteria are shown (N=4). The figure shows the changes in the volume of solid cancer in each group. When PBS or RP was administered, the volume of solid cancer increased. On the other hand, when iR.P. or iP.M. was administered, the volume of solid cancer decreased, and the volume became 0 4 to 5 days after administration.

[0356] Figure 2D The survival rate of mice 40 days after cancer cell transplantation in each bacterial administration is shown (N=4). The group administered with iP.M. or iRP. confirmed 100% survival. On the other hand, the survival rate within 40 days in the group administered with PBS or RP was 0%.

[0357] Figure 2E The complete response (CR) achievement rate of each experimental group is shown. The group administered with iP.M. or iR.P. recorded a high CR achievement rate. In addition, when commercially available PM was administered, mice developed extreme weakness and walking difficulties, so the experiment was immediately stopped from a humanitarian perspective. Therefore, data for commercially available PM could not be obtained.

[0358] Figure 2F This is a diagram showing the experimental method of the study related to cancer immunity acquisition. As shown in the figure, Colon-26 was re-transplanted into mice that achieved CR by administering bacteria (iP.M. or iR.P.) 120 days after achieving the CR. The course of the disease was observed for 20 days after the re-transplantation.

[0359] Figure 2G The photographs of the above observations are shown. In the photograph, the black arrows indicate the cancer cell transplantation sites, and the black dotted lines indicate the tumor formation sites. In mice that achieved CR using iP.M. and iR.P., the re-transplanted Colon-26 did not survive and no tumors formed. On the other hand, when Colon-26 was transplanted into healthy mice administered with PBS, tumor formation was found. In addition, Figure 2H The results of measuring the tumor volume (N=3) of each treatment group are shown. In the figure, ND means that no tumor formation was observed.

[0360] Based on these results, it can be considered that the bacterial therapy of iP.M. or iR.P. is very useful for the treatment of tumors. That is, the bacteria isolated from the tumor have excellent anti-tumor activity and are very useful for the treatment of tumors. For example, the bacteria isolated from the tumor can have an anti-tumor activity that shrinks or eliminates the tumor formed in the mouse within 30 days after the bacteria are administered to the above-mentioned colon cancer model mouse, and can particularly have an anti-tumor activity that shrinks or eliminates the tumor within 10 days after administration.

[0361] In addition, based on these results, it can be considered that the toxicity of iP.M. and iR.P. is very low. For example, the bacteria isolated from the tumor have low toxicity, for example, the survival rate of the mice within 40 days after the administration of the bacteria to the above-mentioned colon cancer model mice can be, for example, 90% or more, 95% or more, or 100%.

[0362] In addition, based on these results, it is believed that cancer immunity can be obtained by implementing bacterial therapy based on iP.M. or iR.P. In other words, it is believed that by administering bacteria isolated from tumors, not only tumors can be treated, but also immunity against tumors can be obtained.

[0363] <Tissue staining>

[0364] The anti-tumor mechanism of each bacteria was analyzed by tissue immunostaining of tumor block sections after bacterial administration. Specifically, colon cancer model mice (female, 8 weeks; n = 5; average body weight = 19 g; average tumor size ~400 mm) inoculated with Colon26 were given a 4% dapoxetine-containing antibiotic. 3 ; BALB / cCrSIc; Japan SLC) were administered 200 μL of iP.M. bacterial sample (5×10 8 CFU / mL), iR.P. bacterial samples (5×10 9 CFU / mL) or PBS buffer. 24 hours after administration, the tumor was removed and paraffin sections of the tumor were made. Paraffin sections were tissue stained and observed with an optical microscope. In addition, tissue staining was performed by Biopathology Research Institute Co., Ltd.

[0365] The results of immune cell staining are as follows Figure 3A As shown in the figure, iP.M. was confirmed to be significantly activated in both natural immunity (macrophages, NK cells, neutrophils) and acquired immunity (T cells, B cells). In addition, the immune activation ability of iR.P. was not as high as that of iP.M., but it was confirmed that macrophages, T cells, and B cells were significantly activated. On the other hand, PBS and RP did not confirm such activation.

[0366] That is, bacteria isolated from tumors have an immune cell activating effect, and this effect is considered to be one of the factors that produce excellent anti-tumor activity.

[0367] In addition, the bacteria isolated from the tumor can be used to activate immune cells, in particular, can be used to activate immune cells in tissues where the tumor exists. That is, the present invention also provides an immune cell activator, which comprises the above-mentioned bacteria isolated from the tumor.

[0368] In addition, the staining results of anti-tumor biomarkers such as Figure 3BAs shown in the figure, both iP.M. and iR.P. caused the expression of necrosis marker TNF-α and apoptosis marker Caspase-3, and significant tissue damage was confirmed. iP.M. had particularly strong expression of TNF-α and Caspase-3.

[0369] That is, bacteria isolated from tumors have an effect of enhancing the expression of anti-tumor biomarkers, and this effect is considered to be one of the factors that produce excellent anti-tumor activity.

[0370] In addition, bacteria isolated from tumors can be used to enhance the expression of anti-tumor biomarkers, and in particular can be used to enhance the expression of anti-tumor biomarkers in tissues where tumors exist. That is, the present invention also provides an anti-tumor biomarker expression enhancer, which comprises bacteria isolated from tumors.

[0371] <Toxicity in vivo>

[0372] The in vivo toxicity of the bacteria (iR.P., iP.M.) was studied by blood examination of mice and tissue identification of various organs.

[0373] For mouse blood tests, 200 μL of bacterial samples (5 × 10 6 CFU / mL) or PBS buffer. 30 days after the administration, blood was collected from the abdominal aorta. Complete blood cell count (CBC) and biochemical tests were analyzed by Japan SLC Co., Ltd. and Japan Oriental Yeast Industry Co., Ltd.

[0374] In order to identify the tissues of various organs, solid cancer (~400mm 3 ) were administered into the tail vein of colon cancer model mice with 200 μL of bacterial samples (iR.P. = 5 × 10 9 CFU / mL, iP.M.=5×10 8 CFU / mL) or PBS buffer. After 30 days of administration (reaching complete response (CR)), various organs were removed from the mice. Paraffin sections of each removed organ were made. Each paraffin section was stained with hematoxylin and eosin (H&E) and observed with an optical microscope. In addition, tissue staining was performed by Biopathology Research Institute Co., Ltd.

[0375] Tables 1 and 2 below show the results of blood tests when iR.P. and iP.M. were administered.

[0376] [Table 1]

[0377]

[0378] [Table 2]

[0379]

[0380] As shown in the table, the blood test results were not significantly different from those of the case where PBS buffer was administered, regardless of whether iRP or iPM was administered. That is, the administration of iRP and iPM is not considered to have an adverse effect on blood status.

[0381] Figure 3E The results of H&E tissue staining of the above-mentioned various organs when iP.M., iR.P., or PBS were administered are shown. As shown in the figure, no damage to organ tissues was observed after bacterial administration.

[0382] Based on these results, it can be considered that administration of bacteria isolated from tumors is not toxic from the viewpoint of the state of blood and the state of various organs.

[0383] <Colony Analysis>

[0384] The bacterial persistence in various tissues after bacterial administration was investigated by the following colony analysis. Specifically, colon cancer model mice (female, 8 weeks; n = 3; average body weight = 19 g; average tumor size ~400 mm) inoculated with Colon26 were given 3 ; BALB / cCrSIc; Japan SLC) were administered 200 μL of iP.M. bacterial sample (5×10 8 CFU / mL), iR.P. bacterial samples (5×10 9 CFU / mL) or PBS buffer. After 30 days of application (reaching CR), each tissue was removed and its weight was measured after cutting. Each tissue was homogenized with a pestle in PBS buffer at a temperature of 4°C. The homogenized mixture was shaken at 15°C and 380rpm / min for 20 minutes. After the shaking, the supernatant of the above mixture was diluted 10 times with PBS buffer to obtain a sample. Each sample (100 μL) was inoculated on an agar medium and cultured anaerobically for 7 days to form bacterial colonies produced on the agar medium. The number of bacterial colonies was determined visually (manually).

[0385] Figure 3C The results of the above-mentioned colony analysis when iRP was administered are shown. This figure confirms that iRP does not remain in the organs at all after cancer treatment.

[0386] Figure 3DThe results of the above-mentioned colony analysis when iP.M. was administered are shown. This figure confirms that iP.M. does not remain in the organs at all after cancer treatment.

[0387] These results suggest that bacteria isolated from tumors do not persist in various tissues when administered.

[0388] <Effects on other cancers>

[0389] like Figure 4A As shown in the figure, sarcoma model mice were prepared by the same method as colon cancer model mice. To prepare sarcoma model mice, cells (Meth-A) from mouse sarcoma (sarcoma) were transplanted subcutaneously. 6 to 10 days after the transplantation, solid cancer (~100 mm 3 ) were injected into the tail vein of mice (n=3) with 200 μL of iP.M. bacterial sample (1×10 8 CFU / mL), and tumors were observed over 14 days.

[0390] The changes of the tumor over time are as follows Figure 4B As shown in the figure, CR was achieved after intravenous injection of iP.M. and iR.P. In addition, no cancer recurrence was found after day 8.

[0391] From this result, it can be seen that the bacteria isolated from the tumor showed antitumor activity not only against colon cancer but also against sarcoma.

[0392] like Figure 4C As shown, metastatic lung cancer model mice were prepared. That is, 4-week-old female wild-type mice (n=3; average body weight=15 g; C57BL / 6NCrSlc) were purchased from SLC Co., Ltd., Japan, and acclimated for one week. A dispersion of mouse melanoma cells (B16-F10 cells) (5×10 6 Cells) (200 μL). Metastatic lung cancer model mice were prepared in this way. One week after the administration, 200 μL of bacterial samples of various bacteria (iR.P., iP.M.) were administered into the tail vein of the metastatic lung cancer model mice (iR.P. = 5×10 9 CFU / mL, iP.M.=5×10 8 CFU / mL) or PBS buffer. After 7 days of administration, the lungs were removed, and the photographs and weights were measured. In addition, untreated mice (healthy mice) were prepared without the administration of mouse melanoma cells and various bacteria.

[0393] Figure 4DPhotos of removed lungs are shown. As shown in the figure, the lungs of untreated mice and mice administered iR.P. or iP.M. were the same size. On the other hand, tumors were confirmed in mice administered PBS. In other words, it can be seen that tumors were alleviated by administration of various bacteria.

[0394] Figure 4E The results of lung weight measurement (N=3) of the model mice after each treatment and the mice in the untreated group are shown. The lungs of the iR.P. or iP.M.-administered groups showed the same weight as the healthy lungs of untreated mice, while the lung weight of the PBS-administered group increased with the formation of tumors.

[0395] Figure 4F The weight changes of model mice and untreated mice after each treatment are shown (N=3). The arrows in the figure indicate the time of administration of PBS or bacteria. Weight gain can be observed over time in untreated mice, while significant weight loss can be observed in the PBS administration group from around the 10th day. The bacterial administration group all experienced slight weight loss on the second day after administration, but then healthy weight gain occurred.

[0396] From these results, it can be seen that bacteria isolated from tumors show antitumor activity not only against colon cancer but also against lung cancer.

[0397] <Analysis of optical properties of bacteria>

[0398] The absorbance spectra of bacterial dispersions containing iRP isolated from tumors or commercially available RP were measured at room temperature using a UV-Vis-NIR spectrophotometer (V-730BIO; Jasco). 7 The concentration of CFU / mL of the dispersion was dispersed in PBS buffer.

[0399] The dispersion was prepared as follows.

[0400] iR.P. was irradiated with a tungsten lamp in a 543ATCC medium without adding Cys, and anaerobically cultured at a temperature of 26°C-30°C for 3 days. The culture solution obtained by the anaerobic culture was centrifuged at 3000rpm for 5 minutes. The bacteria precipitated by the centrifugation were suspended in PBS buffer to obtain a bacterial suspension. The number of bacteria in the suspension was measured using a microbial counter (Panasonic, model NP-BCM01-A), and based on the measurement results, PBS buffer was appropriately added to the suspension to reach a predetermined bacterial concentration, thereby obtaining a dispersion of iR.P. of the above concentration.

[0401] RP was cultured anaerobically for 3 days in a 543ATCC medium supplemented with Cys while being irradiated with a tungsten lamp at a temperature of 26°C to 30°C. The culture solution obtained by the anaerobic culture was centrifuged at 3000 rpm for 5 minutes, and the bacteria precipitated by the centrifugation were suspended in a PBS buffer in the same manner as iRP, and then PBS buffer was appropriately added to achieve a predetermined bacterial concentration, thereby obtaining an RP dispersion of the above concentration.

[0402] The measurement conditions in the measurement using the above-mentioned UV-Vis-NIR spectrophotometer are shown in Table 3 below.

[0403] [Table 3]

[0404]

[0405] In addition, the fluorescence spectrum of the bacterial dispersion containing iRP isolated from tumor or commercially available RP was measured using a fluorescence spectrometer (FP-8600NIR spectrometer; Jasco). The excitation wavelength was 805 nm. The bacterial dispersion was prepared by 2.5×10 7 The dispersion was prepared as described above. The measurement conditions in the measurement using the above fluorescence spectrometer are shown in Table 4 below.

[0406] [Table 4]

[0407]

[0408] Fig.2I The measurement results of the above fluorescence spectrum are shown in . As shown in the figure, it can be seen that iR.P. has higher light absorption characteristics than RP. Regarding iR.P., for example, the absorbance at 808nm is, for example, above 0.029, and the absorbance at 865nm is, for example, above 0.032. The absorbance of iR.P. at these wavelengths is particularly high. It can be considered that this difference in absorbance is caused by the difference in the expression state of light absorbing substances (such as light absorbing proteins, etc.). In addition, based on the above experimental results on anti-tumor activity, it can be considered that this high absorbance can be used as an indicator of the anti-tumor activity of bacteria.

[0409] Figure 2JThe measurement results of the above fluorescence spectrum are shown in . As shown in the figure, it can be seen that iR.P. has higher fluorescence characteristics than RP. For iR.P., for example, the fluorescence intensity at 888nm is above 4.4. The fluorescence intensity of iR.P. at this wavelength is particularly high. This difference in fluorescence intensity is believed to be caused by the difference in the expression state of fluorescent substances (such as fluorescent proteins, etc.). In addition, based on the above experimental results on anti-tumor activity, it can be considered that such a high fluorescence intensity can be used as an indicator that the bacteria have anti-tumor activity.

[0410] These results indicate that bacteria isolated from tumors (particularly red non-sulfur bacteria) have optical properties that are different from those of bacteria that do not exist in tumors (e.g., commercially available bacteria). Furthermore, this optical property can be used as an indicator of anti-tumor activity. Furthermore, it is believed that this optical property can be used to determine the presence or absence of tumors or to determine the location or shape of tumors.

[0411] In addition, the absorption spectrum and fluorescence spectrum of the bacterial dispersion liquid dispersed with iP.M. isolated from tumors or commercially available PM were measured as described above. The bacterial concentration of the bacterial dispersion liquid used for measuring the absorption spectrum was 2×10 7 CFU / mL. In addition, the bacterial concentration of the bacterial dispersion used to measure the fluorescence spectrum was 2.5×10 7 CFU / mL, excitation wavelength is 805nm.

[0412] These dispersions were prepared as follows.

[0413] The iP.M. was irradiated with a tungsten lamp in a 543ATCC medium supplemented with Cys, and anaerobically cultured at a temperature of 26°C-30°C for 3 days. The culture solution obtained by the anaerobic culture was centrifuged at 3000rpm for 5 minutes. The bacteria precipitated by the centrifugation were suspended in PBS buffer to produce a bacterial suspension. The number of bacteria in the suspension was measured using a microbial counter (Panasonic, model NP-BCM01-A), and based on the measurement results, PBS buffer was appropriately added to the suspension to reach a predetermined bacterial concentration, thereby obtaining a dispersion of iP.M. of the above concentration.

[0414] PM was cultured anaerobically in 802NBRC medium at 30°C for 3 days. The culture solution obtained by the anaerobic culture was centrifuged at 3000 rpm for 5 minutes, and the bacteria precipitated by the centrifugation were suspended in PBS buffer in the same manner as iP.M., and then PBS buffer was appropriately added to achieve a predetermined bacterial concentration to obtain a PM dispersion of the above concentration.

[0415] The results of the test are as follows Figure 5As shown, Figure (a) is the measurement result of the absorption spectrum, and Figure (b) is the measurement result of the fluorescence spectrum. From these results, it can be seen that the light absorption characteristics of iP.M. and commercially available PM are the same, and the fluorescence characteristics of iP.M. and commercially available PM are the same.

[0416] <In vivo fluorescence bioimaging>

[0417] To determine the in vivo distribution of bacteria using near-infrared (NIR) fluorescence, we used a mouse colon cancer model consisting of Colon26 mice (female; 8 weeks; n = 3; mean body weight = 19 g; mean tumor size = 400 mm 3 ; BALB / cCrSIc; Japan SLC) were injected once into the tail vein of the rats. The bacterial sample (5×10 9 CFU / mL liquid 200μl, i.e. 1×10 9 CFU) or iR.P. bacterial samples (5×10 9 CFU / mL liquid 200μl, i.e. 1×10 9 CFU) or PBS buffer without bacteria. An imaging system (VISQUE (trademark) In Vivo Smart-LF, Vieworks) was used to observe near-infrared fluorescence images of the whole body of the mouse. The excitation wavelength and fluorescence wavelength used for this observation were λ ex =740-790nm and λ em = 810-860 nm. In addition, CleVue (trademark) software (Vieworks) was used for image analysis.

[0418] The result of this observation is Figure 2K The image shown in the figure was taken 4 days after the above administration. As shown in the figure, it can be seen that iR.P. exhibits higher tumor specificity and higher fluorescence characteristics than RP.

[0419] <Statistical Analysis of Data>

[0420] ± in the data indicates standard deviation, and n indicates the number of samples used. The statistical analysis of the data was performed using Student's t-test. *, **, and *** indicate p values ​​of <0.05, <0.005, and <0.001, respectively.

[0421] <Compound bacteria and isolated bacteria>

[0422] As mentioned above, "iR.P." is a composite bacterium composed of two species, Rhodopseudomonas palustris and Proteus mirabilis, isolated from tumors. In addition, "isp-RP" is only Rhodopseudomonas palustris isolated from tumors, that is, Rhodopseudomonas palustris isolated from tumors. Their antitumor activity and optical properties were evaluated.

[0423] For this evaluation, PBS buffer and commercially available RP bacterial samples were prepared in addition to the isp-RP and iRP bacterial samples. The iRP bacterial samples and RP bacterial samples were prepared as described above. The isp-RP bacterial samples were prepared by the same method as the iRP bacterial samples.

[0424] As described in the above <In vivo anticancer experiment>, colon cancer model mice were prepared and solid cancer (~400 mm 3 ) were administered once into the caudal vein of mice in the same manner. Then, the state after administration was observed, and fluorescence observation was performed using the above imaging system as described in the above <In vivo fluorescent bioimaging>.

[0425] These observations are Figure 6 This figure shows a photograph and an image of each sample obtained by fluorescence observation.

[0426] As shown in the photographs of the figure, both iRP and isp-RP have antitumor activity. On the other hand, commercially available RPs have not been confirmed to have antitumor activity such as iRP and isp-RP. In addition, PBS has not been confirmed to have antitumor activity.

[0427] In addition, as shown in the fluorescence observation image of the figure, it was confirmed that both iRP and isp-RP accumulated in the tumor. In addition, it was confirmed that both iRP and isp-RP had the above-mentioned fluorescence characteristics. On the other hand, fluorescence was also confirmed for commercially available RP, but it was weaker than the fluorescence of iRP and isp-RP. In addition, no fluorescence was confirmed for PBS.

[0428] In addition, the antitumor activity of iR.P. was stronger than that of isp-RP. In addition, the fluorescence intensity of iR.P. was stronger than that of isp-RP. Therefore, it can be considered that the composite bacteria composed of red non-sulfur bacteria and other bacteria are superior to the isolated red non-sulfur bacteria in terms of antitumor activity and optical properties.

[0429] In addition, the ratio of the composite bacteria was further verified, and it was found that in order to exert excellent anti-tumor activity, the content of Rhodopseudomonas palustris is preferably higher than that of Proteus mirabilis. For example, the composition ratio of Rhodopseudomonas palustris and Proteus mirabilis based on CFU is 99:1 to 55:45, especially 99:1 to 60:40.

[0430] <Base sequence analysis>

[0431] The base sequence of the 16S ribosomal RNA gene of isp-RP was analyzed. The base sequence is shown in the following sequence ID No. 1.

[0432]

[0433] The base sequence is 100% identical to the 16S ribosomal RNA gene of commercially available RP, and the sequence is also 100% identical, so isp-RP is identical to commercially available RP at least with respect to the above genes.

[0434] Gene mutation is not thought to occur in the above-mentioned isolation process from the tumor and culture process in the culture medium. Therefore, isp-RP is thought to be genetically identical to commercially available RP.

[0435] On the other hand, as described above, the optical properties of isp-RP and iRP are different from those of commercially available RP, that is, they are different in absorption spectrum and fluorescence spectrum. The optical properties of isp-RP and iRP that are different from those of commercially available RP are considered to be obtained by separation from tumors and cultivation in culture medium after the separation. For example, it can be considered that the optical properties are obtained by increasing the expression of light-absorbing substances and fluorescent substances (particularly light-absorbing proteins and fluorescent proteins) by the separation process from the tumor (particularly the separation process and the cultivation process in culture medium).

[0436] In addition, it is believed that isp-RP and iRP acquire excellent antitumor activity as the optical property is acquired. Therefore, the presence of the optical property can be used as an indicator of excellent antitumor activity. In addition, it is believed that the separation process from the tumor (particularly the above separation process and the above culture process) performed to obtain the optical property is a process useful for imparting antitumor activity to the bacteria.

[0437] The 16S rRNA gene sequence of iP.M. was analyzed. The base sequence is shown in the following sequence ID No. 2.

[0438]

[0439] The base sequence is 99.80% identical to the 16S rRNA gene sequence of commercially available PM, and the sequence identity is also 99%. Therefore, from a genetic point of view, the genes of iP.M. are considered to be basically the same as those of commercially available PM.

[0440] Gene mutation is not thought to occur during the isolation process from the tumor and the culture process in the culture medium performed above. Therefore, iP.M. is also thought to be genetically identical to commercially available PM.

[0441] On the other hand, as described above, iP.M. has superior antitumor activity than commercially available PM. This is believed to be obtained by the separation process from the tumor (particularly the separation process and the culture process in the culture medium). In addition, it is believed that the separation process from the tumor (particularly the above separation process and the above culture process) carried out to obtain the excellent antitumor activity optical characteristics is a process useful for imparting antitumor activity to bacteria.

[0442] In addition, the present invention also provides the following uses, products and methods. The components such as "anti-tumor agent" and "bacteria isolated from tumor" in these uses, products and methods are as described above, and the description thereof also applies to these uses, products and methods.

[0443] [1] Use of bacteria isolated from tumors in the preparation of anti-tumor agents.

[0444] [2] A bacterium isolated from a tumor, which is used for preparing an antitumor agent.

[0445] [3] A method for treating a tumor, comprising administering bacteria isolated from a tumor.

[0446] Accession number

[0447] Proteus mirabilis (Super Bac): NITE BP-03626

[0448] Bacterial complex of Rhodopseudomonas palustris and Proteus mirabilis (Musashi): NITE BP-03627.

Claims

1. An antitumor agent comprising red non-sulfur bacteria isolated from a tumor.

2. The antitumor agent according to claim 1, wherein The red non-sulfur bacteria are Rhodopseudomonas bacteria or Chlorosporum bacteria or both.

3. The antitumor agent according to claim 1 or 2, wherein The antitumor agent is administered parenterally.

4. The antitumor agent according to claim 1 or 2, wherein The anti-tumor agent contains other bacteria separated from tumors in addition to the red non-sulfur bacteria.

5. The antitumor agent according to claim 4, wherein The other bacteria are bacteria of the genus Proteus.

6. The antitumor agent according to claim 4, wherein The antitumor agent contains the red non-sulfur bacteria and the other bacteria at a ratio of 1:99 to 99:

1.

7. The antitumor agent according to claim 1 or 2, wherein The bacteria contained in the antitumor agent have immune cell activating properties.

8. The antitumor agent according to claim 1 or 2, wherein The bacteria contained in the anti-tumor agent have expression enhancing properties that enhance the expression of anti-tumor biomarkers in tumors.

9. The antitumor agent according to claim 1 or 2, wherein The bacteria contained in the antitumor agent show the following characteristics related to the absorption spectrum, <Characteristics related to absorption spectrum> When the bacteria contained in the antitumor agent were measured at 2×10 7 When the concentration of CFU / mL is dispersed in PBS buffer, the absorbance at 808 nm is 0.029 or more.

10. The antitumor agent according to claim 1 or 2, wherein The bacteria contained in the antitumor agent showed the following characteristics related to the fluorescence spectrum, <Characteristics related to fluorescence spectrum> When the bacteria contained in the antitumor agent were measured at 2.5×10 7 When the fluorescence spectrum of the dispersion liquid with a concentration of CFU / mL dispersed in PBS buffer is taken, the fluorescence intensity at 888 nm is 4.4 or more.

11. A method for generating an image for determining a tumor, comprising: an administration step of administering to the animal one or more bacteria isolated from the tumor; and The image generation step is to photograph the animal after the administration while irradiating the animal with light in a predetermined wavelength range, thereby generating an image for identifying a tumor that may exist in the animal.

12. A bacterium isolated from a tumor, comprising at least red non-sulfur bacteria.

13. The bacterium according to claim 12, wherein The bacteria isolated from the tumor further include bacteria of the genus Proteus.

14. The bacterium according to claim 12 or 13, wherein The red non-sulfur bacteria are Rhodopseudomonas bacteria or Chlorosporum bacteria or both.

15. The bacterium according to claim 12 or 13, wherein The bacteria show the following properties related to the absorbance spectrum, <Characteristics related to absorption spectrum> When the bacteria were measured at 2×10 7 When the concentration of CFU / mL is dispersed in PBS buffer, the absorbance at 808 nm is 0.029 or more.

16. The bacterium according to claim 12 or 13, wherein The bacteria show the following properties related to the fluorescence spectrum, <Characteristics related to fluorescence spectrum> When the bacteria were measured at 2.5×10 7 When the fluorescence spectrum of the dispersion liquid with a concentration of CFU / mL dispersed in PBS buffer is taken, the fluorescence intensity at 888 nm is 4.4 or more.

17. A method for preparing bacteria, comprising: The isolation process is to separate bacteria from the tumor; as well as a culturing step of culturing the bacteria separated in the separation step in a medium to which cysteine ​​is not added, The cultured bacteria at least contain red non-sulfur bacteria and have anti-tumor activity.

Images