Antitumor Composition

Abstract

The present invention relates to an antitumor composition comprising (A) Bifidobacterium longum and (B) an immune checkpoint inhibitor such as a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The present invention also relates to use of Bifidobacterium longum in the preparation of a medicament for treating a tumor, wherein the Bifidobacterium longum can be used in combination with an immune checkpoint inhibitor formulation.

Description

[Technical field]

[0001] The present application relates to an antitumor composition, and more specifically, to the combination of Bifidobacterium longum with an immune checkpoint inhibitor. [Background technology]

[0002] Cure of cancer has always been one of the great challenges in the medical field, and conventional treatment methods such as chemotherapy and radiotherapy all bring about great side effects and suffering to patients. In recent years, cancer immunotherapy has shown remarkable therapeutic effects on blood tumors and some solid tumors, and has little side effects on patients, providing great hope for the cure of human cancer.

[0003] Generally, the surface of tumor cells has antigens that can be recognized by human immune T cells, and the human immune system can recognize and kill tumor cells. However, in order to survive and grow, tumor cells may avoid recognition and killing by the immune system in various ways. Tumor immunotherapy recognizes and destroys such intentions of tumor cells, and restores the human body to a normal anti-tumor immune response, thereby controlling and eliminating various types of tumors. Tumor immunotherapy mainly includes five types: (1) therapeutic antibodies, (2) cancer vaccines, (3) cell therapy, i.e., CAR-T therapy, (4) immunomodulators, and (5) immune checkpoint inhibitors.

[0004] The activity of the human immune system is regulated and controlled by costimulatory molecules, which are immune checkpoints. When antigen recognition occurs, other molecules interact with the immune cells and the molecules on the surface of the target cells, which further determine the balance of the interactions. When the signal is approximately positive, the immune cells are activated and attack the antigens presented by the target cells. Conversely, when the signal is negative, the immune cells are inactivated, and such inactivation may be permanent, and the antigens will be recognized as normal antigens / self-antigens. Cancer-related immune checkpoints that have been relatively revealed by research include CTLA-4, PD-1, and PD-L1. Immune checkpoint inhibitors are monoclonal antibody drugs developed against the corresponding immune checkpoints, and their main action is to block the inhibitory effect of tumor cells on immune cells by blocking the interaction between tumor cells expressing immune checkpoints and immune cells. Currently widely used immune checkpoint inhibitors include monoclonal antibodies targeting programmed cell death protein 1 (PD-1) and its ligand (PD-L1). PD-1 antibodies are highly effective in blocking late-stage melanoma, non-small cell lung cancer, and renal cell carcinoma, and have made great strides in tumor treatment. However, due to the complex pathogenesis of tumors, the large individual differences in patients, and the influence of environmental factors, immune checkpoint inhibitors are currently only effective in about 25% of patients.

[0005] More and more studies have revealed that gut microbes are also a factor affecting the efficacy of cancer immunotherapy. In 2015, Marie Vetizou et al. found that tumors inoculated into the bodies of antibiotic-treated or germ-free mice did not respond to CTLA-4 inhibitors, but the antitumor effect of CTLA-4 inhibitors was restored after the mice were administered Bacteroides fragilis. In 2017, Routy B et al. found that the combination of anti-PD-1 inhibitors with certain gut microbiota significantly improved the response of tumor patients to anti-PD-1 inhibitors and significantly prolonged the mean progression-free survival (PFS) of patients. In summary, gut microbes have a wide application prospect in improving the therapeutic effect of immune checkpoint inhibitors. Therefore, there is a need in this field to develop new antitumor compositions that have better antitumor effects, especially those that show better therapeutic effects on certain tumors. Summary of the Invention

[0006] The inventors of the present application have found that the combination of Bifidobacterium longum and immune checkpoint inhibitors can effectively inhibit various tumors, including but not limited to colon cancer, lung cancer, breast cancer, melanoma, renal cancer, urothelial carcinoma, etc.

[0007] In one aspect of the present application, (A) Bifidobacterium longum, (B) an immune checkpoint inhibitor, and an antitumor composition comprising the same. In one embodiment of the present application, the Bifidobacterium longum is Bifidobacterium longum 6-1.

[0008] In one embodiment of the present application, the Bifidobacterium longum is combined with other bacteria (eg, Bifidobacterium, Lactobacillus, Clostridioides, Enterococcus faecium, Prevotella copreli, Ruminococcus).

[0009] In one embodiment of the present application, the Bifidobacterium longum and the immune checkpoint inhibitor are mixed together to prepare a single formulation or physically separated and used separately.

[0010] In one embodiment of the present application, the Bifidobacterium longum is in an oral or injectable form.

[0011] In one embodiment of the present application, the antitumor composition further comprises an antibiotic.

[0012] In one embodiment of the present application, the tumor is selected from colon cancer, lung cancer, gastric cancer, liver cancer, head and neck cancer, cervical cancer, breast cancer, lymphatic cancer, breast cancer, melanoma, renal cancer or urothelial cancer.

[0013] The present application also provides the use of Bifidobacterium longum in the preparation of a medicament for treating a tumor.

[0014] In one embodiment of the present application, the Bifidobacterium longum is used in combination with an immune checkpoint inhibitor formulation.

[0015] In one embodiment of the present application, the use is to improve the tumor inhibitory effect of an immune checkpoint inhibitor, and preferably, the tumor is selected from colon cancer, lung cancer, gastric cancer, liver cancer, head and neck cancer, cervical cancer, breast cancer, lymphatic cancer, breast cancer, melanoma, renal cancer, or urothelial cancer. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the effect of antibiotic-induced disruption of the intestinal flora on tumor growth and the therapeutic efficacy of anti-mPD-1 in MC38 tumor-bearing mice. [Diagram 2] FIG. 2 shows the effect of a single injection of anti-mPD-1 and a single oral administration of Bifidobacterium longum 6-1 on tumor growth in MC38 tumor-bearing mice. [Diagram 3]FIG. 3 shows the therapeutic effect of a combination of anti-mPD-1 and Bifidobacterium longum 6-1 on tumors in MC38 tumor-bearing mice. [Figure 4] FIG. 4 shows the difference in therapeutic effect of different probiotics in combination with anti-mPD-1 on tumors in MC38 tumor-bearing mice. [Diagram 5] FIG. 5 shows the average CD3 cell counts in MC38 tumor-bearing mice when anti-mPD-1 was injected alone and in combination with Bifidobacterium longum 6-1. [Figure 6] FIG. 6 shows the number of tumor-infiltrating CD8 T cells in MC38 tumor-bearing mice when anti-mPD-1 was injected alone and in combination with Bifidobacterium longum 6-1. [Figure 7] FIG. 7 shows the therapeutic effect of anti-mPD-1 injection alone and in combination with Bifidobacterium longum 6-1 on 4T1 tumors. [Figure 8] FIG. 8 shows the therapeutic effect of anti-mPD-1 injection alone and in combination with Bifidobacterium longum 6-1 on LLC1 tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In the present invention, unless otherwise specified, percentages (%) or parts all refer to percentages by weight or parts by weight of the composition. In the present invention, unless otherwise specified, each relevant component or preferred components thereof can be combined with each other to form a new technical solution.

[0018] In the present invention, unless otherwise specified, all embodiments and preferred embodiments described herein can be combined with each other to form a new technical solution.

[0019] In the present invention, unless otherwise specified, all technical features and preferred features described herein can be combined with each other to form a new technical solution.

[0020] In the present invention, unless otherwise specified, the total content of each component in a composition is 100%.

[0021] In the present invention, unless otherwise specified, the total number of parts of each component in the composition may be 100 parts by weight.

[0022] In the present invention, unless otherwise specified, the numerical range "a to b" is a shorthand expression of any combination of real numbers from a to b, where a and b are both real numbers. For example, the numerical range "0 to 5" indicates that all real numbers "0 to 5" are listed in the text, and "0 to 5" is merely a shorthand expression of the combination of these numerical values.

[0023] In the present invention, unless otherwise specified, the integer range "a to b" is a shorthand expression of any combination of integers from a to b, where a and b are both integers. For example, the integer range "1 to N" means 1, 2...N, where N is an integer.

[0024] In the present invention, unless otherwise specified, "combination thereof" refers to a multi-component mixture of each of the above elements, such as a multi-component mixture of two, three, four, and as many more components as possible.

[0025] Unless otherwise specified, the term "one" as used herein means "at least one."

[0026] Unless otherwise specified, all percentages (including weight percentages) set forth in the present invention are based on the total weight of the composition.

[0027] The "ranges" disclosed herein are defined by lower and upper limits. There may be one or more lower limits and one or more upper limits. A given range is defined by selecting one lower limit and one upper limit. The selected lower and upper limits define the limits of a particular range. All such defined ranges are inclusive and combinable, i.e., any lower limit and any upper limit can be combined to form a range. For example, if the ranges 60-120 and 80-110 are recited for a particular parameter, it is understood that the ranges 60-110 and 80-120 are also contemplated. It is noted that if the minimum range values ​​recited are 1 and 2, and the maximum range values ​​are 3, 4, and 5, then the ranges 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5 are all contemplated.

[0028] In the present specification, unless otherwise specified, the percentage or weight of each component means dry weight.

[0029] In the present specification, unless otherwise specified, "no change" means that the change is within ±10%, preferably within ±5%, more preferably within ±2%, and most preferably within ±1%.

[0030] As used herein, Bifidobacterium longum includes active ingredients derived from Bifidobacterium longum or strains that, as a result of sequence identification, have at least 99% sequence identity with Bifidobacterium longum.

[0031] Bifidobacterium longum The Bifidobacterium longum described in this application may be commercially available or may be obtained by conventional techniques, for example, the Bifidobacterium longum may be obtained according to CN103830278A, CN103131647A, CN101244090A, CN101244089A, CN1223865A or US6368591.

[0032] The Bifidobacterium longum described in the present invention is a common strain in the art and can be isolated from feces of healthy adults, as disclosed, for example, in the patent US6368591. Bifidobacterium longum 6-1 has been deposited at the China Center for Type Culture Collection (CCTCC), an international depository institution recognized under the Budapest Treaty, under the accession number CTCCC M98003. .

[0033] Bifidobacterium longum is an anaerobic bacterium, Gram-stain positive, non-uniformly stained, non-spore, non-capsule, non-flagellate, and the bacterial body is linear or curved, and can have various morphologies such as "Y" or "V" branched, rod-shaped, etc. In one preferred embodiment of the present invention, the Bifidobacterium longum is Bifidobacterium longum (6-1).

[0034] In one preferred embodiment of the present application, the Bifidobacterium longum may be in the form of Bifidobacterium longum powder. In one embodiment of the present application, the Bifidobacterium longum powder contains Bifidobacterium and a first protective agent, and the first protective agent contains 8 to 50% skim milk powder, 0.01 to 10% sodium glutamate, 8 to 50% allolactose, 0.01 to 5% Vc-Na, and 4 to 30% starch, based on the total weight of the first protective agent.

[0035] The Bifidobacterium longum powder can be in various suitable dosage forms, such as oral liquid, tablets, capsules, orally disintegrating tablets, freeze-dried powder, etc. In one preferred embodiment of the present application, the dosage form is a capsule. In another preferred embodiment of the present application, the dosage form is a tablet. The dosage form is preferably a freeze-dried bacterial agent, the viable cell count of which is 10 10 CFU / g is preferred.

[0036] In one preferred embodiment of the present application, the Bifidobacterium longum may be used in combination with other strains, including, but not limited to, Bifidobacterium, Lactobacillus, Clostridioides, Enterococcus faecium, Prevotella copril, Ruminococcus, and the like.

[0037] Immune checkpoint inhibitors The immune checkpoint inhibitor used in the present application may be any commercially available product, such as PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, etc. In one preferred embodiment, the immune checkpoint inhibitor includes Keytruda, Tecentriq, Nivolumab Injection, Bavencio (avelumab), Tuoyi (Toripalimab Injection).

[0038] Antitumor Composition In the present application, the Bifidobacterium longum and the immune checkpoint inhibitor may be mixed and prepared into a single preparation and used together, or may be physically separated and used separately. In one embodiment of the present application, the Bifidobacterium longum and the immune checkpoint inhibitor are physically separated and used separately. In another embodiment of the present application, the Bifidobacterium longum may be administered to a patient before the administration of the immune checkpoint inhibitor. In general, the Bifidobacterium longum can be administered to a patient in any suitable manner (including, but not limited to, oral, injection, etc.). The immune checkpoint inhibitor can be administered to a patient in any suitable manner (including, but not limited to, oral, injection, etc.). The method of using the Bifidobacterium longum or the immune checkpoint inhibitor is a conventional method in the art, and a person skilled in the art can directly determine it by referring to the prior art according to the description of the specification.

[0039] In the present application, the antitumor composition may further comprise an antibiotic, which may be any suitable antibiotic, including, but not limited to, quinolone antibiotics, β-lactam antibiotics, macrolides, aminoglycosides, and the like. In one preferred embodiment of the present application, the antibiotic is a β-lactam antibiotic (e.g., penicillin, ampicillin, carbenicillin, methicillin, oxacillin, dicloxacillin, flucloxacillin, cephradine, cefotaxime, cephalexin, ceftriaxone, cefpirome, cefixime, cefditoren pivoxil, cefdinir, ceftibuten, cefpodoxime proxetil, imipenem, aztreonam, cefminox, biapenem, imipenem, meropenem, etc.), a macrolide antibiotic (e.g., erythromycin, leucomycin, roxithromycin, erythromycin ethylsuccinate, azithromycin, clarithromycin, acetylspiramycin, melemycin, midecamycin, etc.), , josamycin, telithromycin, etc.), aminoglycoside antibiotics (streptomycin, gentamicin, arbekacin, amikacin, etc.), quinolone antibiotics (e.g., ciprofloxacin, levofloxacin, norfloxacin, etc.), other antibiotics and antibacterial drugs (e.g., tetracyclines, chloramphenicols, rifamycins such as lincomycin and rifapentine, polypeptides such as vancomycin, sulfonamides such as bactrim, metronidazoles, etc.), antifungal drugs (e.g., amphotericin, griseofulvin, dactarin, etc.), antitumor antibiotics (e.g., mitomycin, actinomycin D, bleomycin, doxorubicin, etc.), etc., but are not limited to these.

[0040] In this application, the antibiotic may be packaged separately from the other ingredients and administered alone.

[0041] In one preferred embodiment of the present application, the antitumor composition further comprises instructions, which state that Bifidobacterium longum is administered for a certain period of time (e.g., 1-10 days) followed by administration of a PD-1 inhibitor for a certain period of time (e.g., 10-100 days). Preferably, the dosage range of Bifidobacterium longum is 10 4 ~10 13 CFU / person / day, and the dose range of immune checkpoint inhibitors is 0.5-10 mg / kg.

[0042] The instructions also state that an antibiotic should be administered for a certain period of time (e.g., 1 to 3 days) before administering Bifidobacterium longum and a PD-1 inhibitor. Preferably, the dosage range of the antibiotic is 1 to 500 mg / kg.

[0043] The antitumor composition of the present application can effectively treat various types of tumors, particularly, but not limited to, colon cancer, lung cancer, breast cancer, melanoma, renal cancer, and urothelial cancer.

[0044] In another aspect of the present application, there is provided the use of Bifidobacterium longum in the preparation of a medicament for treating a tumor.

[0045] In the present application, the Bifidobacterium longum can be used in combination with an immune checkpoint inhibitor to inhibit the growth of tumors (e.g., colon cancer, lung cancer, breast cancer, melanoma, renal cancer, urothelial cancer, etc.). The inventors found that when an antibiotic is administered to a patient, the tumor growth inhibitory effect of the immune checkpoint inhibitor is limited, but the administration of Bifidobacterium longum before or simultaneously with the administration of the immune checkpoint inhibitor can effectively improve the tumor inhibitory effect of the immune checkpoint inhibitor.

[0046] In another aspect of the present application, there is provided a use of Bifidobacterium longum in tumor treatment. In one embodiment of the present application, the use includes inhibiting tumor growth. Preferably, the Bifidobacterium longum can be used in combination with an immune checkpoint inhibitor. In general, the tumor includes, but is not limited to, colon cancer, lung cancer, breast cancer, melanoma, renal cancer, urothelial carcinoma, etc.

[0047] The present application will be described in detail below with reference to examples, but the scope of the present application is not limited to these examples.

[0048] The raw materials used in the examples are as follows:

[0049] SPF male C57BL / 6J mice aged 6 to 8 weeks (20 to 26 g) were purchased from Jiangsu Jisui Yaokang Biotechnology Co., Ltd. with a quality certification number of 201805120. The breeding conditions were temperature controlled at (23±3)°C, humidity at 40 to 70%, and the mice were allowed to eat and drink freely.

[0050] The PD-1 inhibitor was purchased from Yikang (Beijing) Pharmaceutical Technology Co., Ltd., with a concentration of 7.09 mg / mL, and was stored in the dark at 2 to 8 ° C. An appropriate amount of phosphate buffered saline was added thereto and mixed uniformly to obtain a solution of the specified concentration.

[0051] Ampicillin was purchased from Anhui Anfengtang Animal Pharmaceutical Co., Ltd., streptomycin was purchased from Solarbio Company, and colistin sulfate soluble powder was purchased from Shandong Luxi Veterinary Medicine Co., Ltd.

[0052] MC38 tumor cells were purchased from Ruting Biological Technology (Beijing) Co., Ltd. The tumor cells were cultured in a DMEM medium containing 10% inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine in an incubator at 37°C and 5% CO2. After the cells were filled, they were transferred to a new flask and subcultured about every 3 days. The tumor cells in the logarithmic growth phase were used for inoculation of the internal tumor.

[0053] Mouse breast cancer 4T1 cells (ATCC, product number: CRL-2539), LLC1 lung cancer cells (purchased from Shanghai Institute of Life Sciences).

[0054] Bifidobacterium longum 6-1 is Bifidobacterium longum 6-1 (CCTCC M98003).

[0055] Tumor volume measurement: Tumor volume: The tumor volume was measured three times a week using a caliper, and the long and short diameters of the tumor were measured. The volume was calculated as follows: volume = 0.5 × long diameter × short diameter. 2 It is.

[0056] Example 1 Forty-eight male C57BL / 6J mice, aged 6-8 weeks, were allowed to adapt to the environment for one week, and then randomly divided into four groups based on body weight, with 12 mice per group: Group 1 (no antibiotic treatment, blank, ip), Group 2 (no antibiotic treatment, anti-mPD-1, 10 mg / kg, ip), Group 3 (antibiotic treatment, blank, ip), and Group 4 (antibiotic treatment, anti-mPD-1, 10 mg / kg, ip). For antibiotic treatment, the broad-spectrum antibiotics ampicillin (1 mg / mL) + colistin (1 mg / mL) + streptomycin (5 mg / mL) were administered in drinking water for 5 days. All groups were cultured with MC38 tumor cells resuspended in phosphate-buffered saline (PBS) at a concentration of 1 × 10 7The mice were inoculated at 100μL / mL and subcutaneously inoculated at the right rib region of the experimental animals. Mice in groups 2 and 4 were injected with anti-mPD-1 on day 4 after tumor cell inoculation, and once every 4 days for a total of 4 injections. Mice were euthanized on day 19 after tumor inoculation.

[0057] MC38 tumor cells were purchased from Ruting Biotechnology Co., Ltd., and the tumor cells were cultured in a DMEM medium containing 10% inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine in an incubator at 37°C and 5% CO2. After the cells were filled, they were transferred to a new flask and subcultured about every 3 days, and the tumor cells in the logarithmic growth phase were used for inoculation of the internal tumor.

[0058] The test results are shown in Figure 1. As can be seen from the results, antibiotic treatment destroyed the intestinal flora of the mice, which accelerated tumor growth and increased tumor volume in the mice, and had a certain impact on the therapeutic effect of anti-mPD-1. The intestinal flora affects the tumor growth and therapeutic effect of anti-mPD-1 in mice.

[0059] Example 2 Thirty-six male C57BL / 6J mice (20-26 g, SPF grade) aged 6-8 weeks were allowed to adapt to the environment for one week, and then randomly divided into three groups according to body weight: Group 1 (antibiotic treatment, blank, ip), Group 2 (antibiotic treatment, anti-mPD-1, 10 mg / kg, ip), and Group 3 (antibiotic treatment, Bifidobacterium longum 6-1, po). For antibiotic treatment, the broad-spectrum antibiotics ampicillin (1 mg / mL) + colistin (1 mg / mL) + streptomycin (5 mg / mL) were administered in drinking water for five days. Mice in Group 3 were first administered a freeze-dried sample of Bifidobacterium longum, so that the intragastric administration concentration was 1.0 × 10 8 After 2 weeks of intragastric administration, mice in all groups were administered MC38 tumor cells resuspended in PBS at a concentration of 1 × 10 7The mice were inoculated at 100μL / mL and subcutaneously inoculated at the right rib region of the experimental animals. Mice in group 2 were injected with anti-mPD-1 on day 4 after tumor cell inoculation, and once every 4 days for a total of 4 injections. Mice were euthanized on day 19 after tumor inoculation.

[0060] MC38 tumor cells were purchased from Ruting Biotechnology Co., Ltd., and the tumor cells were cultured in a DMEM medium containing 10% inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine in an incubator at 37°C and 5% CO2. After the cells were filled, they were transferred to a new flask and subcultured about every 3 days, and the tumor cells in the logarithmic growth phase were used for inoculation of the internal tumor.

[0061] The test results are shown in Figure 2. When Bifidobacterium longum 6-1 was orally administered to tumor-bearing mice with destroyed intestinal flora, the same effect was achieved as when anti-mPD-1 was injected for treatment, and Bifidobacterium longum 6-1 had a certain tumor growth inhibitory effect.

[0062] Example 3 Thirty-six male C57BL / 6J mice (20-26 g, SPF grade) aged 6-8 weeks were allowed to adapt to the environment for one week, and then randomly divided into three groups according to body weight: Group 1 (antibiotic treatment, blank, ip), Group 2 (antibiotic treatment, anti-mPD-1, 10 mg / kg, ip), and Group 3 (antibiotic treatment, Bifidobacterium longum 6-1, po, anti-mPD-1, 10 mg / kg, ip). For antibiotic treatment, the broad-spectrum antibiotics ampicillin (1 mg / mL) + colistin (1 mg / mL) + streptomycin (5 mg / mL) were administered in drinking water for 5 days. Mice in Group 3 were first administered a freeze-dried sample of Bifidobacterium longum, so that the intragastric administration concentration was 1.0 × 10 8 CFU / mouse / day and continued intragastric administration for 2 weeks. After that, mice in all groups received MC38 tumor cells resuspended in PBS at a concentration of 1 × 10 7The mice were inoculated at 100μL / mL and subcutaneously inoculated at the right rib region of the experimental animals. Mice in groups 2 and 3 were injected with anti-mPD-1 on day 4 after tumor cell inoculation, and once every 4 days for a total of 4 injections. Mice were euthanized on day 19 after tumor inoculation.

[0063] Tumor cell culture: Tumor cells are cultured in a DMEM medium containing 10% inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine in an incubator at 37°C and 5% CO2. After the cells become full, they are transferred to a new flask and subcultured about every 3 days. Tumor cells in the logarithmic growth phase are used for inoculation of internal tumors.

[0064] The results are shown in Figure 3. As can be seen from the results, oral administration of Bifidobacterium longum 6-1 in combination with injection of anti-mPD-1 can better inhibit tumor growth, reduce tumor volume, and improve the therapeutic effect on tumors.

[0065] Example 4 Forty-eight male C57BL / 6J mice (20-26 g, SPF grade) aged 6-8 weeks were allowed to adapt to the environment for one week, and then randomly divided into four groups according to body weight: Group 1 (antibiotic treatment, blank, ip), Group 2 (antibiotic treatment, Bifidobacterium longum 6-1, po, anti-mPD-1, 10 mg / kg, ip), Group 3 (antibiotic treatment, Bifidobacterium longum BL2 (fecal sample from a healthy individual), po, anti-mPD-1, 10 mg / kg, ip), and Group 4 (antibiotic treatment, Bifidobacterium longum BL3 (fecal sample from a healthy individual), po, anti-mPD-1, 10 mg / kg, ip). For antibiotic treatment, the broad-spectrum antibiotics ampicillin (1 mg / mL) + colistin (1 mg / mL) + streptomycin (5 mg / mL) were administered in drinking water for 5 days. Mice in groups 2 to 4 were first administered a freeze-dried sample of Bifidobacterium longum, with an intragastric concentration of 1.0 × 10 8CFU / mouse / day and continued intragastric administration for 2 weeks. After that, mice in all groups received MC38 tumor cells resuspended in PBS at a concentration of 1 × 10 7 The mice were inoculated at 100 μL / mL and subcutaneously inoculated at the right rib region of the experimental animals. Mice in groups 2-4 were injected with anti-mPD-1 on day 4 after tumor cell inoculation, and once every 4 days for a total of 4 injections. Mice were euthanized on day 19 after tumor inoculation.

[0066] Cultivation of MC38 tumor cells: Using DMEM medium containing 10% inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine, the tumor cells are cultured in an incubator at 37°C and 5% CO2. After the cells become full, they are transferred to a new flask and subcultured about every 3 days. The tumor cells in the logarithmic growth phase are used for inoculation of the internal tumor.

[0067] The results are shown in Figure 4. As can be seen from the results, the combined treatment of oral administration of Bifidobacterium longum 6-1 and injection of anti-mPD-1 can better inhibit tumor growth and reduce tumor volume.

[0068] Example 5 The mice in Example 3 were euthanized on the 19th day after tumor inoculation, and CD3, CD4, CD8, FOXP3, CD25, CXCR3, Gata3, Granzyme B, CD69, PD-1, CTLA-4, CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, and Gr-1 were detected in tumor cells by flow cytometry. Immune factor analysis included TNF-α, IL-17, IL-13, IL-12p70, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP-10, MIP1b, and MAC-1. The results are shown in Figure 5. As can be seen from the results, after oral administration of Bifidobacterium longum 6-1 and combined treatment with anti-mPD-1 injection, the average CD3 cell count of mice was significantly increased, and the immune system response of mice was improved. In the figure, all compared with the antibiotic treatment group, P<0.05 indicates significant difference, *P<0.05, ***P<0.001.

[0069] Example 6 The mice in Example 3 were euthanized on the 19th day after tumor inoculation, and CD3, CD4, CD8, FOXP3, CD25, CXCR3, Gata3, Granzyme B, CD69, PD-1, CTLA-4, CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, and Gr-1 were detected in tumor cells by flow cytometry. The experimental results are shown in Figure 6. As can be seen from the results, the number of tumor-infiltrating CD8 T cells was significantly increased after oral administration of Bifidobacterium longum 6-1 in combination with injection of anti-mPD-1, compared with the injection of anti-mPD-1 alone, and the immune system response of the mice was improved. In the figure, all were compared with the antibiotic treatment group, P<0.05 indicates significant difference, **P<0.01.

[0070] Example 7 Forty-eight male C57BL / 6J mice (20-26 g, SPF grade) aged 6-8 weeks were adapted to the environment for one week and then randomly divided into four groups according to body weight: Group 1 (blank, ip), Group 2 (anti-mPD-1, 10 mg / kg, ip), Group 3 (Bifidobacterium longum 6-1, po), and Group 4 (Bifidobacterium longum 6-1, po, anti-mPD-1, 10 mg / kg, ip). Mice in Groups 3 and 4 were intragastrically administered a freeze-dried sample of Bifidobacterium longum 6-1, resulting in an intragastric administration concentration of 1.0 × 10 8 After 2 weeks of intragastric administration, mice in all groups were administered 4T1 tumor cells resuspended in PBS at a concentration of 1 × 10 7 The mice were inoculated at 100μL / mL and subcutaneously inoculated at the right rib region of the experimental animals. Mice in groups 2 and 4 were injected with anti-mPD-1 on day 4 after tumor cell inoculation, and once every 4 days for a total of 4 injections. Mice were euthanized on day 19 after tumor inoculation.

[0071] The results are shown in Figure 7. In a 4T1 breast cancer model that is not sensitive to anti-mPD-1 treatment, the combined treatment of Bifidobacterium longum 6-1 and anti-mPD-1 significantly improved the therapeutic effect of anti-mPD-1 on 4T1 breast cancer and significantly reduced tumor volume (P<0.0001). As can be seen from the results, Bifidobacterium longum 6-1 can improve the body's response to anti-mPD-1 and expand the application range of anti-mPD-1 tumor treatment.

[0072] Example 8 Thirty-six male C57BL / 6J mice (20-26 g, SPF grade) aged 6-8 weeks were acclimated to the environment for 1 week and then randomly divided into three groups according to body weight: Group 1 (blank, ip), Group 2 (anti-mPD-1, 10 mg / kg, ip), and Group 3 (Bifidobacterium longum 6-1, po, anti-mPD-1, 10 mg / kg, ip). Mice in Group 3 were intragastrically administered a freeze-dried sample of Bifidobacterium longum 6-1, resulting in an intragastric administration concentration of 1.0 × 10 8After 2 weeks of intragastric administration, mice in all groups were administered LLC1 lung cancer cells resuspended in PBS at a concentration of 1 × 10 7 The mice were inoculated at 100μL / mL and subcutaneously inoculated at the right rib region of the experimental animals. Mice in groups 2 and 3 were injected with anti-mPD-1 on day 4 after tumor cell inoculation, and once every 4 days for a total of 4 injections. Mice were euthanized on day 19 after tumor inoculation.

[0073] As shown in Figure 8, in LLC1 lung cancer model that is insensitive to anti-mPD-1 treatment, the combination treatment of Bifidobacterium longum 6-1 and anti-mPD-1 effectively improved the organism's response to anti-mPD-1 treatment, significantly inhibited tumor growth, and reduced tumor volume compared with anti-mPD-1 treatment alone. In the figure, significant differences are all compared to the control, **P<0.001, ****P<0.0001.

Claims

1. An anti-tumor composition in oral dosage form comprising Bifidobacterium longum 6-1, deposited as CCTCC M98003, for use in combination with an anti-PD-1 antibody.

2. An anti-tumor composition in oral dosage form comprising Bifidobacterium longum 6-1, deposited as CCTCC M98003, for improving the effect of anti-PD-1 antibodies in inhibiting tumor growth in patients.

3. 3. The antitumor composition according to claim 1, further comprising a bacterium selected from the group consisting of Bifidobacterium, Lactobacillus, Clostridioides, Enterococcus faecium, Prevotella copril and Ruminococcus.

4. The antitumor composition according to any one of claims 1 to 3, wherein the Bifidobacterium longum 6-1 is in the form of Bifidobacterium longum 6-1 powder.

5. The antitumor composition of claim 4, wherein the Bifidobacterium longum 6-1 powder comprises Bifidobacterium longum 6-1 powder and a first protective agent.

6. 6. The antitumor composition according to claim 5, wherein the first protective agent comprises, based on a total weight of the first protective agent, 8 to 50% skim milk powder, 0.01 to 10% sodium glutamate, 8 to 50% allolactose, 0.01 to 5% Vc-Na, and 4 to 30% starch.

7. The antitumor composition according to any one of claims 1 to 3, wherein the Bifidobacterium longum 6-1 is in the form of an oral liquid, a tablet, a capsule, or a lyophilized powder.

8. The Bifidobacterium longum 6-1 is ４ ~10 １３ The antitumor composition of any one of claims 1 to 7, administered in a dose range of CFU / person / day.

9. The antitumor composition according to any one of claims 1 to 8, wherein the anti-PD-1 antibody is pembrolizumab, nivolumab, or toripalimab.

10. The antitumor composition according to any one of claims 1 to 9, wherein the tumor is selected from colon cancer, lung cancer, gastric cancer, liver cancer, head and neck cancer, cervical cancer, lymphatic cancer, breast cancer, melanoma, renal cancer, or urothelial cancer.

11. The antitumor composition according to any one of claims 1 to 10, which is administered prior to administration of the anti-PD-1 antibody.

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