WO2017138660A1 - 免疫機能の検査方法、がん患者の選別方法、がんの治療効果予測方法、細胞内カルシウムイオン濃度上昇剤、腫瘍組織におけるエフェクター・メモリー(EM)とエフェクター(eff)の選択的機能向上剤、がん治療薬の効果のモニタリング方法 - Google Patents
免疫機能の検査方法、がん患者の選別方法、がんの治療効果予測方法、細胞内カルシウムイオン濃度上昇剤、腫瘍組織におけるエフェクター・メモリー(EM)とエフェクター(eff)の選択的機能向上剤、がん治療薬の効果のモニタリング方法 Download PDFInfo
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Definitions
- the present invention relates to a method for examining immune function, a method for screening cancer patients, a method for predicting the therapeutic effect of cancer, an agent for increasing intracellular calcium ion concentration, and selective effector memory (EM) and effector (eff) in tumor tissue.
- the present invention relates to a method for monitoring the effects of a function improver and a cancer therapeutic agent.
- this method requires a culture period of 1 to 2 weeks, it does not necessarily reflect the real-time lymphocyte function in the patient.
- the process of stimulating culture with an antigenic peptide often induces nonspecific lymphocyte proliferation and often gives false positive results.
- Patent Document 1 discloses that metformin has an effect enhancing action of a chemotherapeutic agent, but it is shown in the Examples that metformin itself does not have a therapeutic effect on cancer.
- Non-Patent Document 1 discloses the mechanism of action of metformin, which is a therapeutic agent for type 2 diabetes. *
- Non-patent document 2 suggests that metformin has an anticancer effect.
- Patent Document 2 discloses that a biguanide antidiabetic drug containing metformin improves the function of immune exhausted CD8 + T cells.
- the main object of the present invention is to provide a technique effective for activation and evaluation of the immune system.
- the inventor has the following immunological function testing method, cancer patient screening method, cancer therapeutic effect prediction method, intracellular calcium ion concentration increasing agent, effector memory (EM) and effector (eff) in tumor tissue It is intended to provide a method for monitoring the effects of selective function improvers and cancer therapeutic agents.
- Item 1 Peripheral blood of a human subject is collected, and after applying an immunostimulant, the transition of calcium ion concentration in peripheral blood mononuclear cells (PBMC) or CD8 T cells is examined.
- PBMC peripheral blood mononuclear cells
- combination therapy of at least one selected from the group consisting of phenformin, buformin and metformin and another cancer therapeutic agent A method for screening cancer patients to which a combination therapy of at least one selected from the group consisting of phenformin, buformin and metformin and another cancer therapeutic agent is applied as a cancer patient to be performed.
- Item 3. Item 3. The method for selecting a patient according to Item 2, wherein the other cancer therapeutic agent is an anti-PD-1 antibody, aspirin, statin, curcumin, berberine, royal jelly or propolis.
- Item 4 A method for predicting the therapeutic effect on a cancer patient of a combination therapy of at least one selected from the group consisting of phenformin, buformin and metformin and another cancer therapeutic agent, comprising the following steps (1) to (2): (1) Evaluate whether intracellular calcium ion concentration increases transiently or continuously when mononuclear cells (PBMC) contained in peripheral blood collected from cancer patients are immunostimulated And (2) when the intracellular calcium ion concentration of PBMC at the time of immune stimulation rises transiently and then falls rapidly, at least one selected from the group consisting of phenformin, buformin, and metformin and other The process of predicting that a combination therapy of cancer drugs is likely to have a therapeutic effect.
- PBMC mononuclear cells
- a method for treating cancer patients comprising the following steps (1) to (2): (1) Evaluate whether intracellular calcium ion concentration increases transiently or continuously when mononuclear cells (PBMC) contained in peripheral blood collected from cancer patients are immunostimulated Process, (2) At least one selected from the group consisting of phenformin, buformin, and metformin and other cancer therapeutic agents when the intracellular calcium ion concentration of PBMC at the time of immune stimulation decreases transiently and then decreases rapidly (3) from the group consisting of phenformin, buformin, and metformin to predict that the combination therapy is likely to have a therapeutic effect, and (3) patients predicted to have a high therapeutic effect in step (2) Administering at least one selected as well as other cancer therapeutic agents. Item 6.
- PBMC mononuclear cells
- An agent for increasing intracellular calcium ion concentration in CD8 T cells by immunostimulation comprising at least one selected from the group consisting of phenformin, buformin and metformin.
- Item 7. The agent for increasing the intracellular calcium ion concentration of CD8 T cells by immunostimulation according to Item 6, which selectively increases the intracellular calcium ion concentration of CD8 T cells in the tumor tissue.
- Item 8 A selective function enhancer for effector memory (EM) and effector (eff) in tumor tissue, comprising at least one member selected from the group consisting of phenformin, buformin and metformin.
- Item 9 A cancer therapeutic agent administered to a cancer patient having a glucose concentration in a tumor tissue of 0.5 to 1.5 mM, comprising at least one selected from the group consisting of phenformin, buformin and metformin.
- a method for monitoring the effect of a cancer therapeutic agent comprising the following steps 1) to 3): Step 1) Separating intratumoral lymphocytes from cancer tissue, Step 2) Analyzing the glucose transporter (Glut1) expression level of the isolated tumor infiltrating lymphocytes, and Step 3) Cancer treatment that has already been administered to the cancer patient based on the analyzed expression level of Glut1 A step of determining / evaluating whether or not the drug is effective (where the cancer tissue is extracted from a cancer patient after administration of the cancer therapeutic agent).
- Item 11 Item 10. The method for monitoring the effect of a cancer therapeutic agent according to Item 9, wherein the cancer therapeutic agent is selected from the group consisting of a cancer vaccine, an immune checkpoint inhibitor, phenformin, buformin, and metformin.
- Item 12 The method for monitoring the effect of a cancer therapeutic agent according to Item 10 or 11, wherein the Glut1 expression level is analyzed by a flow cytometer.
- Item 13. The method for monitoring the effect of the cancer therapeutic agent according to any one of Items 10 to 12, wherein the tumor infiltrating lymphocytes are CD8 T cells.
- Item 14. Any one of Items 10 to 13, wherein the step 3 comprises culturing the isolated tumor infiltrating lymphocytes in a medium having a glucose concentration of 0.1 mM or more for 2 hours or more, and then analyzing a glucose transporter (Glut1) expression level.
- Glut1 glucose transporter
- Cancer is confined to a part of the body and there are a small number of immune cells in the tumor tissue, but even if peripheral blood is collected, immune cells that have come into contact with the cancer are diluted significantly to normal values or Although it seems to show a close figure, in fact, it was shown that the immune cells in the peripheral blood of cancer patients are totally impaired, including antigens not related to cancer. Decreased immune cell function is promoted by radiation therapy, aging, genetic factors, a biased diet, lack of sleep, etc., but measuring the intracellular calcium concentration of peripheral blood immune cells improves the overall immune status. It will be easy to evaluate.
- phenformin, buformin and metformin preferably a combination therapy of these and other cancer therapeutic agents such as anti-PD-1 antibody is applied.
- the intracellular calcium ion concentration of PBMC including CD8 T cells when receiving immune stimulation is considerably lower than that of healthy subjects. It leads to success. Since the calcium ion concentration does not significantly exceed the level of healthy individuals, side effects such as inflammation and allergies due to excessive activation of the immune system are not a problem.
- At least one selected from the group consisting of phenformin, buformin and metformin and an anti-PD-1 antibody each greatly increase the number of immune cells (eff and EM) required for complete remission of the cancer, and Among the three cytokines (IL-2, TNF ⁇ , IFN ⁇ ), it is possible to selectively increase the proportion of CD8 T cells that are highly active in producing two or more cytokines and that can produce one or less cytokines. The number rather decreases. As a result, immunity against cancer cells can be significantly increased. Furthermore, when at least one selected from the group consisting of phenformin, buformin and metformin is combined with an anti-PD-1 antibody, the number of immune cells can be increased synergistically.
- Effector T cells (eff) and effector memory T cells (EM) are important for cancer immunotherapy, and central memory T cells (CM) are not important unless a cancer vaccine is applied.
- the present invention that selectively increases the number and function of EM and EM is effective for improving the therapeutic results of cancer immunotherapy.
- a tumor tissue having a glucose concentration as low as 1 mM is known and the immune function is activated even in such a low glucose environment, a cancer having such a low glucose state cancer Particularly suitable for patient treatment.
- the monitoring of the effect of a cancer drug is performed by administering a cancer drug to a cancer patient, then removing the tumor tissue from the cancer patient and separating the tumor-infiltrating lymphocytes (particularly tumor-infiltrating CD8T) from the removed cancer tissue.
- (Lymphocyte) glucose transporter expression level can be examined to determine whether the cancer drug is effective or to evaluate the effectiveness of the cancer drug.
- CM central memory T cells
- EM effector memory T cells
- eff effector T
- 2-NBDG effector memory T cells
- peripheral blood immune cells peripheral blood immune cells (mononuclear cells (PBMC), especially CD8 T cells)
- PBMC peripheral blood immune cells
- an immunostimulant such as PMA (phorbol myristate acetate) or ionomycin
- the immune cells of healthy individuals gradually increase the intracellular calcium concentration for 50 to 150 seconds, and the necessary cytokines It can be released and it can be seen that normal immune function can be maintained.
- the immune cells of cancer patients are stimulated with an immunostimulant, the intracellular calcium concentration increases transiently, but then rapidly decreases.
- the intracellular calcium concentration can be determined by staining with an anti-CD8 antibody, Fluo4, FuraRed, and measuring with a flow cytometer after washing.
- the intracellular calcium concentration can be determined by staining with an anti-CD8 antibody, Fluo4, FuraRed, and measuring with a flow cytometer after washing.
- Examples of immune cells for evaluating the expression level of glucose transporter include tumor infiltrating lymphocytes, preferably tumor infiltrating CD8T lymphocytes.
- Peripheral blood mononuclear cells (PBMC) unlike tumor tissues, have a sufficiently high blood glucose concentration, so the expression level of the glucose transporter is already high and is not suitable for this test.
- the expression level of the glucose transporter can be determined by staining with an anti-glucose transporter antibody, anti-CD3 antibody, anti-CD8 antibody, etc., and analyzing with a flow cytometer after washing.
- cancer therapeutic agents that can be used in combination with the biguanide antidiabetic agents phenformin, buformin, and metformin are not particularly limited.
- immune checkpoint inhibitors such as anti-PD-1 antibodies, aspirin, statins , Curcumin, berberine, royal jelly, propolis, cancer vaccine and the like, and anti-PD-1 antibody and aspirin are more preferable.
- an anticancer drug with an elevated glucose transporter is also included in the cancer therapeutics that can be used in combination.
- the cancer therapeutic agent subject to the monitoring method of the present invention may be only one type, or a combination (concomitant drug) of two or more types of cancer therapeutic agents.
- the cancer therapeutic agent that is the target of the monitoring method is not particularly limited as long as it is a cancer therapeutic agent that acts on the immune system, for example, single administration of a biguanide antidiabetic such as phenformin, buformin, metformin, or Combination of phenformin, buformin or metformin and at least one selected from the group consisting of immune checkpoint inhibitors such as anti-PD-1 antibodies, aspirin, statins, curcumin, berberine, royal jelly, propolis, cancer vaccine And a combination drug of an anti-PD-1 antibody and a biguanide antidiabetic drug and a combination drug of aspirin and a biguanide antidiabetic drug are preferable.
- cancer therapeutic agents to be selected may be administered in combination with appropriate pharmaceutical carriers, or may be administered in combination with different formulations.
- Combinations and separate preparations may be oral or parenteral, and examples include tablets, capsules, powders, inhalants, liquids, drinks, injections, suppositories and the like. Such preparations may further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers. It can also be administered as a suspension.
- solid preparations such as tablets, pills, powders, granules, and fine granules, for example, sodium bicarbonate, calcium carbonate, starch, sucrose, mannitol, carboxymethylcellulose and other carriers, calcium stearate, stearic acid
- additives such as magnesium and glycerin
- enteric coatings are sprayed by spraying organic solvents or water solutions of enteric substances such as cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl alcohol phthalate, styrene-maleic anhydride copolymer, methacrylic acid-methyl methacrylate copolymer, etc.
- enteric preparation can contain other auxiliary agents, fragrances, stabilizers or preservatives which are usually used as necessary.
- cancer therapeutic agents can activate the immune function, but are more effective for patients with a decreased immune function. Therefore, when cancer chemotherapy is performed using the biguanide antidiabetic drugs phenformin, buformin, metformin and other cancer drugs, the intracellular calcium concentration does not increase much due to immune stimulation of peripheral blood immune cells, It can be expected to be effective for cancer patients whose immune function has declined.
- phenformin When phenformin is used as an anti-diabetic drug, side effects such as lactic acidosis are problematic, but when used for cancer treatment, side effects are not a problem because there are a dosing period and a drug holiday.
- the biguanide antidiabetic drugs phenformin, buformin, and metformin can increase the calcium concentration in CD8 T cells when immune stimulation is performed (FIG. 1).
- immune stimulation is not performed, the intracellular calcium concentration does not change regardless of the presence or absence of a biguanide antidiabetic agent.
- the immune system is not significantly activated as compared with a healthy person. That is, biguanide antidiabetics have the effect of increasing the potential ability to increase calcium levels when the immune system needs to work.
- CD8 T cells present in tumor tissue is lower than that in lymph nodes (LN) and hardly increased by immunostimulation, but in the presence of biguanide antidiabetic drugs phenformin, buformin, and metformin Then, the calcium concentration in CD8 T cells can be greatly increased in response to immune stimulation (FIG. 1).
- the dosage of phenformin, buformin, and metformin, which are biguanide antidiabetic drugs necessary for this purpose, is about 100 to 150 mg for adult phenformin and buformin, and about 500 to 2250 mg for metformin.
- the biguanide antidiabetics phenformin, buformin, and metformin can enhance the uptake of effector (eff) and effector memory (EM) sugars that may be present locally in tumor tissues and are present in secondary lymphoid tissues
- eff effector
- EM effector memory
- CM central memory
- the dosage of phenformin and buformin, which are biguanide anti-diabetic drugs necessary for eff and EM activation, is about 100 to 150 mg per day for adults, and the dosage of metformin is about 500 to 2250 mg per day for adults.
- the tumor tissue has a glucose concentration of about 1 mM or less (Cell 162: 1217-1228, 2015).
- the biguanide antidiabetic drugs phenformin, buformin, and metformin cannot promote the cytokine production ability of CD8 T cells when the glucose concentration in the tumor tissue is 0.1 mM. It is possible to promote the cytokine production performance of CD8 T cells at 1 mM, which is similar to the glucose concentration of CD8 T cells, increase the activity of CD8 T cells in malnourished tumor tissues, and kill cancer cells.
- phenformin, buformin, and metformin which are biguanide antidiabetics, can be used to treat tumor tissue having a glucose concentration of about 0.5 to 1.5 mM, preferably about 0.8 to 1.2 mM, and particularly preferably about 1 mM. It is useful as a cancer treatment for cancer patients.
- the dosage of other cancer therapeutic agents can be appropriately selected based on the clinically used dose.
- Monitoring of a cancer drug can be performed by the following steps 1) to 3). Step 1) Separating intratumoral lymphocytes from cancer tissue, Step 2) Analyzing the glucose transporter (Glut1) expression level of the isolated tumor infiltrating lymphocytes, and Step 3) Cancer treatment that has already been administered to the cancer patient based on the analyzed expression level of Glut1 A step of determining / evaluating whether or not the drug is effective (where the cancer tissue is extracted from a cancer patient after administration of the cancer therapeutic agent).
- Step 1) Separating intratumoral lymphocytes from cancer tissue Step 2) Analyzing the glucose transporter (Glut1) expression level of the isolated tumor infiltrating lymphocytes, and Step 3) Cancer treatment that has already been administered to the cancer patient based on the analyzed expression level of Glut1
- a step of determining / evaluating whether or not the drug is effective where the cancer tissue is extracted from a cancer patient after administration of the cancer therapeutic agent).
- monitoring of cancer therapeutic agents can be performed by detecting the production amounts of three types of cytokines (IL-2, TNF ⁇ , IFN ⁇ ), detection of the production amounts of these cytokines is complicated. Since the present inventor has found that the glucose transporter (Glut1) expression level correlates with the production amounts of three kinds of cytokines, the cancer therapeutic agent is monitored by detecting the glucose transporter (Glut1) expression level. be able to. The removal of cancer tissue may be performed by surgery or by biopsy. The analysis of the glucose transporter (Glut1) expression level is preferably performed after culturing tumor-infiltrating lymphocytes separated from cancer patients in a medium having a glucose concentration of 0.1 mM or more for 2 hours or more.
- Glut1 expression level is not necessarily high in tumor-infiltrating lymphocytes immediately after separation from a cancer patient, and the Glut1 expression level is increased by culturing in a medium having a glucose concentration of 0.1 mM or more, thereby facilitating analysis of the Glut1 expression level. be able to.
- the glucose concentration in the medium is about 0.1 mM or more, preferably about 1 mM or more, more preferably about 5 mM or more, further preferably about 5 to 25 mM, particularly preferably about 10 to 25 mM, particularly about 15 to 25 mM.
- the culture time is 1 hour or longer, preferably 2 hours or longer, more preferably 2 to 24 hours, further preferably about 3 to 12 hours, particularly preferably about 4 to 8 hours. The culture time is set so that the Glut1 expression level is high, and may be shorter when the glucose concentration is high, and longer culture is desirable when the glucose concentration is low.
- the administration period of the cancer therapeutic agent to be monitored may be one day or longer, and is not particularly limited, and examples thereof include 1 to 50 days, preferably 2 to 30 days.
- lymphocytes obtained from tumor tissues particularly CD8 T cells, can be preferably used.
- Cancer drugs that are evaluated for therapeutic effects include cancer vaccines, immune checkpoint inhibitors, phenformin, buformin, and metformin.
- cancers to be treated with cancer therapeutic agents include solid cancers with low glucose concentration, such as melanoma, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder / bile duct cancer, Examples include biliary tract cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, prostate cancer and the like.
- CD8 T cells Although the number of CD8 T cells is very small in the local cancer area, phenformin, buformin, metformin, and anti-PD-1 antibodies, which are biguanide antidiabetics, all greatly increase the number of CD8 T cells in the local cancer area. be able to. Furthermore, when the biguanide antidiabetic drugs phenformin, buformin, metformin and anti-PD-1 antibody are used in combination, not only can the number of CD8 T cells be increased, but also two cytokines (IL-2, TNF ⁇ , IFN ⁇ ) 2 The proportion of CD8 T cells producing more than a species can be greatly increased.
- IL-2, TNF ⁇ , IFN ⁇ two cytokines
- the dosage of phenformin and buformin, which are biguanide antidiabetics for obtaining such effects, is about 100 to 150 mg per day for adults, and the dosage of metformin is about 500 to 2250 mg.
- Anti-PD-1 antibody The dose per adult is about 2 mg / kg.
- the present invention further provides the following inventions.
- An agent for increasing the number of CD8 T cells in tumor tissue comprising an anti-PD-1 antibody as an active ingredient and administered in combination with at least one selected from the group consisting of phenformin, buformin and metformin.
- a combination of at least one selected from the group consisting of an anti-PD-1 antibody and phenformin, buformin and metformin for increasing the number of CD8 T cells in tumor tissue.
- An agent for increasing the number of CD8 T cells in a tumor tissue containing at least one selected from the group consisting of phenformin, buformin and metformin for use in combination with an anti-PD-1 antibody.
- An agent for increasing the number of CD8 T cells in a tumor tissue containing an anti-PD-1 antibody for use in combination with at least one selected from the group consisting of phenformin, buformin and metformin is used in combination.
- Example 1 2 ⁇ 10 5 MO-5 (OVA-expressing B16 melanoma cell line) was transplanted intradermally into C57BL / 6 mice, and oral administration of 5 mg / ml metformin was started 7 days after transplantation by free drinking. On day 7 after the start of administration (14 days after transplantation), lymph node (LN) and tumor tissue (Tumor) were excised, and lymph node lymphocytes and tumor infiltrating lymphocytes were collected. The cells were washed with FCS ( ⁇ ) RPMI and then stained with anti-mouse CD3 antibody BV510, anti-mouse CD8 antibody APC-Cy7, 1 ⁇ M Fluo4, 1 ⁇ M Fura Red for 30 minutes at 37 ° C.
- FCS ⁇
- the cells were washed with FCS ( ⁇ ) RPMI kept at 37 ° C., and after washing, the background of intracellular calcium ions was measured with a flow cytometer for 30 seconds.
- stimulation with 100 ng / ml PMA and 5 ⁇ M ionomycin was performed, and an increase in calcium ion in mouse CD8 T cells due to stimulation was measured (FIG. 1).
- the intracellular calcium concentration in the metformin non-administered group ( ⁇ ) hardly increased by PMA / ionomycin stimulation, but in the metformin administered group (+), the intracellular calcium concentration increased by PMA / ionomycin stimulation. It was confirmed to rise.
- Lymph nodes have high intracellular calcium levels even before PMA / ionomycin stimulation, and both the metformin administration group (+) and the non-administration group ( ⁇ ) increased intracellular calcium concentration due to PMA / ionomycin stimulation. Although it increased, the degree was larger in the metformin administration group (+).
- metformin has an effect of increasing intracellular calcium concentration by immune stimulation, and that the action is particularly remarkable in CD8 T cells in tumor tissue.
- Example 2 Detection of memory traits of antigen-specific tumor-infiltrating CD8 T cells and evaluation of glucose uptake in each cell fraction 2x10 5 MO-5 was transplanted intradermally into C57BL / 6 mice, and 5 mg by free drinking 7 days after transplantation Oral administration of / ml metformin was started. The tumor tissue was excised 7 days after the start of administration (14 days after transplantation), and tumor infiltrating lymphocytes were separated.
- the cells were washed with 0.1% BSA / PBS, anti-mouse CD8 antibody APC-Cy7, anti-mouse CD62L antibody BV421, anti-mouse CD44 antibody PerCP, anti-mouse KLRG1 antibody APC, and 400 ⁇ M 2-NBDG (2- [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] -2-deoxy-d-glucose) was stained for 30 minutes at 4 ° C.
- FIG. 2 Washed with 0.1% BSA / PBS and analyzed the frequency of antigen-specific tumor-infiltrating CD8 T cells, memory traits (CD44, CD62L, KLRG1) and deoxyglucose (2-NBDG) uptake in each cell fraction using a flow cytometer (FIG. 2).
- metformin increases the number and proportion of effectors (eff) and effector memories (EM) and activates the glycolytic system with respect to the memory traits of tumor-infiltrating CD8 T cells. It has been found that the glycolysis system is inactivated by decreasing the number and proportion of memory.
- Example 3 1x10 6 after thawing human PBMC (human peripheral blood mononuclear cells) cryopreserved from healthy people and cancer patients, washed with FCS (-) RPMI PBMC were stained with anti-human CD8 antibody APC-Cy7, 1 ⁇ M Fluo4, 1 ⁇ M Fura Red for 30 minutes at 37 ° C. After staining, the cells were washed with FCS ( ⁇ ) RPMI kept at 37 ° C., and after washing, the background of intracellular calcium ions was measured with a flow cytometer for 30 seconds.
- Human PBMC can be easily measured by collecting peripheral blood, and metformin, or the combined effect of metformin and other drugs, is high in the cancer patient type of FIG. 3, but not in healthy individuals. Patients can be selected by measuring the calcium concentration of human PBMC. This result is unexpected because peripheral blood is thought to have a very low proportion of mononuclear cells in contact with cancer cells.
- Example 4 The cryopreserved human PBMC of a cancer patient was thawed, cultured for 6 hours in the presence of FCS ( ⁇ ) RPMI 10 ⁇ M metformin, separated after culture, and washed with FCS ( ⁇ ) Glc ( ⁇ ) RPMI. After washing, the cells were stimulated with 50 ng / ml PMA and 2 ⁇ M ionomycin in the presence of 1 ⁇ M monensin for 6 hours using RPMI containing 0.1, 1, 10 mM Glc.
- CD8 T cells producing three cytokines depend on the glucose concentration, and are the lowest at 0.1 mM, and there is no difference depending on the presence or absence of metformin, but the glucose concentration is 1 mM. In this case, it was revealed that the number of CD8 T cells producing three cytokines (IL-2, TNF ⁇ , IFN ⁇ ) by metformin was significantly increased.
- Example 5 Tumor transplantation experiment (phenformin, aspirin treatment) OVA-expressing B16 melanoma cell line MO5 (2.5 ⁇ 10 5 ) was inoculated intradermally on the back of C57BL / 6 mice.
- Four groups of 5 mice were prepared, including an untreated group (C), aspirin alone, phenformin (Phen) alone, and a combination of aspirin and phenformin starting 10 days after tumor cell inoculation.
- Phenformin is a biguanide drug similar to metformin. Phenformin was given as a diet containing 0.5% from the 10th day after tumor cell inoculation. In addition, the food was replaced with normal food every 4 days.
- the phenformin-containing diet was given only from the 10th day to the 14th day and from the 18th day to the 22nd day. Normal diets without phenformin were given between days 14-18 and 22-26.
- the reason for selecting such an administration method is that phenformin daily administration results in death of mice due to side effects.
- mice were given as food to stop drinking water.
- Example 6 Intradermal injection of 2 x 10 6 tumors (MethA) into BALB / c at 8 weeks of age, followed by continuous intake of metformin (5 mg / mL) with free drinking from day 7, 3 days after treatment (day 10) and 6 days later On day 13, tumor infiltrating T cells (TIL) were isolated and stained with anti-CD8 antibody and anti-glucose transporter (Glut1) antibody. In addition, the cells were stimulated for 6 hours in the presence of PMA / Ionomycin / Monencin, stained with anti-CD8 antibody and anti-glucose transporter (Glut1) antibody, and analyzed with a flow cytometer. The results are shown in FIG. Although the expression of Glut1 tended to increase by PMA stimulation, a clear increase in the Glut1-positive population was observed in the group to which metformin had been administered in advance, including PMA stimulation (-).
- Example 7 The multifunctionality of the Glut1-positive population in FIG. 6 was analyzed. Multifunctional CD8 T cells are the most powerful effector T cells.
- TIL tumor-infiltrating T cells
- Example 8 Eight-week-old BALB / c mice were injected intradermally with MethA (2 x 10 6 ) syngeneic tumors, and metformin (5 mg / mL) and N-acetylcysteine (NAC, 10 mg / mL) were administered from day 7 Started. Three days after treatment, tumor-infiltrating T cells (TIL) were isolated and cultured at 37 ° C for 0, 1, 3, and 6 hours, and then stained with various antibodies such as CD3, CD8, and glucose transporter (Glut1). Analysis was performed with a cytometer (FIG. 8).
- TIL tumor-infiltrating T cells
- CD8TIL immediately after separation from the tumor, GLUT-1 expression was stained with antibodies immediately after separation according to previous methods, and analyzed by a flow cytometer. Is difficult to detect. However, as shown by the arrows in FIG. 8, it was found that detection was possible by culturing in vitro for 3 to 6 hours. That is, in CD8TIL of a mouse that had been administered with metformin, a very high level of GLUT-1 expression was observed after 6 hours of culture. On the other hand, no increase in GLUT-1 expression was observed in the metformin non-administered group.
- GLUT-1 is a receptor for glucose uptake
- an increase in the expression of GLUT-1 in the cell membrane means an increase in glycolysis. Since the increase in cytoplasmic calcium ion concentration associated with T cell activation is maintained by enhanced glycolysis, the increase in GLUT-1 expression in this case can be considered synonymous with the increase in cytoplasmic calcium ion concentration.
- Example 9 Syngeneic tumor MethA (2 x 10 6 ) was injected intradermally into 8-week-old BALB / c mice, and metformin (5 mg / mL) was started on day 7.
- Tumor infiltrating T cells (TIL) 3 days after treatment
- TIL Tumor infiltrating T cells
- Glut1 glucose transporter
- CD8TIL isolated from the tumor showed increased expression of GLUT-1 by culturing in vitro for 6 hours, which was found to be proportional to the glucose concentration in the culture medium.
- the highest level of Glut-1 was observed at 6.1 mM glucose, ideally 25 mM.
- Glucose at 25 mM is comparable to the glucose concentration in normal culture.
- CD8TILT expressed Glut-1 even at a glucose concentration of 0.1 mM0.1 comparable to the glucose concentration in the tumor.
- a glucose concentration of 0.1 mM is comparable to the glucose concentration in the tumor. Therefore, it is speculated that by administration of metformin, the glycolytic system of CD8TIL is enhanced even within the tumor and can attack the tumor.
- CD8TIL-glut-1 when no glucose is present (glucose concentration 0 mM), CD8TIL-glut-1 is not expressed.
- CD8TIL isolated from metformin-treated mice was significantly increased in Glut-1 expression after 6 hours of in vitro culture, which was found to be proportional to the glucose concentration in the culture medium. . It was found that the highest increase in Glut-1 expression was observed when the glucose concentration in the culture was 6.1 mM or more and 25 mM.
- CD8TIL tumor-infiltrating CD8T cells
- This method ie, the method of observing the expression level of Glut-1 with a flow cytometer after culturing CD8TIL in the presence of 25 mMmglucose for 6 hours is excellent in that it can detect the presence or absence of glycolytic system elevation of CD8TIL very easily and sensitively. It can be concluded that this is an inspection method.
- This method is not limited to metformin, but is considered to be a universal method that can monitor the effects of any cancer immunotherapy, such as cancer vaccines and immune checkpoint inhibitors.
- Tumor infiltrating lymphocyte (TIL) collection and culture protocol Tumor mass (3-4mm diameter) was removed from cancer patients by biopsy, etc., and mechanically disrupted with Medimachine (manufactured by ASONE Corporation) for 2 minutes. Collect and centrifuge at 1200 rpm for 5-10 minutes. Then, discard the supernatant and prepare the cell solution in RPMI medium (fetal calf serum may agglutinate so do not enter here) so that the concentration becomes 5 ⁇ 10 5 / mL. Add 5 ⁇ 10 5 cells / mL / well to the cells and incubate for 6 hours. After 6 hours, the cells are collected and transferred to a process of staining with antibodies.
- Medimachine manufactured by ASONE Corporation
- Example 11 In patients with stage I cancer, stage IIa cancer, and stage IV cancer, changes in calcium concentration enhancement due to PMA stimulation before and after metformin administration were measured.
- Human PBMC human peripheral blood mononuclear cells
- PMA human peripheral blood mononuclear cells
- peripheral blood mononuclear cells PBMC
- 1x10 6 lymphocytes were isolated at 37 ° C in the presence of 1 ⁇ g / ml anti-human CD8 antibody and 1 ⁇ M Fluo4 / FuraRed. Incubated for 30 minutes. Then, it is washed with AIM-V medium. After washing, AIM-V was added, and unstimulated (pre-stimulated) lymphocytes were captured by FACSCanto II for 20 seconds to capture baseline data. Thereafter, 100 ng / ml PMA / 2 ⁇ M ionomycin was added to stimulate, and immediately taken in by FACSCantoII and taken as post-stimulation data. Thereby, a waveform corresponding to the cytoplasmic calcium concentration was detected.
- PBMC peripheral blood mononuclear cells
- Example 12 Tumor transplantation experiment (Met, aspirin, abashimib and two drugs) OVA-expressing B16 melanoma cell line MO5 (2.5 ⁇ 10 5 ) was inoculated intradermally on the back of C57BL / 6 mice. Seven days after tumor cell inoculation, metformin (0.5%) was given to the mice by diet, aspirin (600 ⁇ g / mL) by free drinking, and abashimib (15 mg / kg) by intraperitoneal administration every other day. The results are shown in FIG. After the start of these treatments, the major axis and minor axis of the tumor on the sixth day were measured, and the tumor volume (mm 3 ) was calculated. It can be seen that the combined use of metformin and aspirin, or the combined use of metformin and abashimib has a higher tumor suppressing effect than the single agent.
- Example 13 Tumor transplantation experiment (combination of 3 drugs, anti-PD-1 antibody, Met, aspirin) 3LL cells (2.0 ⁇ 10 5 ) were inoculated intradermally on the back of C57BL / 6 mice.
- Metformin (0.5%) was treated with feed and aspirin (600 ⁇ g / mL) with free drinking. After the treatment was started, the major axis and minor axis of the tumor were measured, and the tumor volume (mm 3 ) was calculated. The results are shown in FIG. The combination of aspirin was found to increase the tumor suppressive effect of anti-PD-1 antibody + metformin.
- Example 14 Tumor transplantation experiment (triple combination, anti-PD-1 antibody, Met, NDGA) 3LL cells (2.0 ⁇ 10 5 ) were inoculated intradermally on the back of C57BL / 6 mice. Five days after tumor cell inoculation, anti-PD-1 antibody (10 mg / kg) was administered intraperitoneally every 6 days after the first dose, so that the total was 4 times. Metformin and NDGA (Nordihydroguaiaretic acid) were treated with metformin (0.5%) feed or metformin (0.5%) and NDGA (0.1%) mixed feed. After the treatment was started, the major axis and minor axis of the tumor were measured, and the tumor volume (mm 3 ) was calculated. Survival was also observed. The results are shown in FIG. The combination of NDGA was found to increase the tumor suppressive effect of anti-PD-1 antibody + metformin and prolong survival.
- Example 15 Tumor transplantation experiment (4,5 combined use) OVA-expressing B16 melanoma cell line MO5 (2.5 ⁇ 10 5 ) was inoculated intradermally on the back of C57BL / 6 mice. Seven days after tumor cell inoculation, metformin (0.5%) and NDGA (0.1%) were treated with mixed feed and aspirin (600 ⁇ g / mL) with free drinking. Furthermore, anti-PD-1 antibody (10 mg / kg) was administered by intraperitoneal administration every 6 days after the first administration, so that a total of 4 treatments were made. Abashimib (15 mg / kg) was administered intraperitoneally every 2 days after the first dose until the end of measurement.
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Abstract
Description
本出願は、2016年2月12日に出願された日本国特許出願第2016-24363号明細書及び2016年10月18日に出願された日本国特許出願第2016-204284号明細書(その開示全体が参照により本明細書中に援用される)に基づく優先権を主張する。
項1. ヒト被験者の末梢血を採取し、免疫刺激剤を適用後、末梢血単核球(PBMC)又はCD8T細胞内のカルシウムイオン濃度の推移を検査し、免疫刺激剤の適用後に一過性にPBMC又はCD8T細胞内のカルシウムイオン濃度が上昇し、その後刺激前の状態に戻る場合に全身の免疫機能が低下していると判断し、免疫刺激剤の適用後にPBMC又はCD8T細胞内のカルシウムイオン濃度が上昇基調にある場合に全身の免疫機能は正常であると判断される、免疫機能の検査方法。
項2. 項1の検査方法で全身の免疫機能が低下していると判断されたがん患者を、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法を行うがん患者として選別、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法を適用するがん患者の選別方法。
項3. 他のがん治療薬が、抗PD-1抗体、アスピリン、スタチン、クルクミン、ベルベリン、ロイヤルゼリー又はプロポリスである、項2に記載の患者の選別方法。
(1)がん患者から採取された末梢血に含まれる単核球(PBMC)を免疫刺激したときに細胞内カルシウムイオン濃度が一過性に上昇するか、持続的に上昇するかを評価する工程、及び
(2)免疫刺激時のPBMCの細胞内カルシウムイオン濃度が一過性に上昇した後速やかに下降する場合、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法が治療効果を示す可能性が高いと予測する工程。
項5. 下記工程(1)~(2)を含む、がん患者の治療方法:
(1)がん患者から採取された末梢血に含まれる単核球(PBMC)を免疫刺激したときに細胞内カルシウムイオン濃度が一過性に上昇するか、持続的に上昇するかを評価する工程、
(2)免疫刺激時のPBMCの細胞内カルシウムイオン濃度が一過性に上昇した後速やかに下降する場合、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法が治療効果を示す可能性が高いと予測する工程、及び
(3)工程(2)において治療効果を示す可能性が高いと予測した患者に、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種並びに他のがん治療薬を投与する工程。
項6. フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を含む、免疫刺激によるCD8T細胞の細胞内カルシウムイオン濃度上昇剤。
項7. 腫瘍組織内のCD8T細胞の細胞内カルシウムイオン濃度を選択的に上昇させる、項6に記載の免疫刺激によるCD8T細胞の細胞内カルシウムイオン濃度上昇剤。
項9. フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を含む、腫瘍組織内のグルコース濃度が0.5~1.5mMであるがん患者に投与されるがん治療薬。
項10. 以下の工程1)~工程3)を含む、がん治療薬の効果のモニタリング方法:
工程1)がん組織から腫瘍内リンパ球を分離する工程、
工程2)分離された腫瘍浸潤リンパ球のグルコーストランスポーター(Glut1)発現レベルを解析する工程、及び
工程3)解析されたGlut1の発現レベルに基づき、がん患者に既に投与していたがん治療薬が有効か否かを判定/評価する工程
(ここで、がん組織は、がん治療薬を投与後にがん患者から摘出されたものである。)。
項11. がん治療薬が、がんワクチン、免疫チェックポイント阻害薬、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる、項9に記載のがん治療薬の効果のモニタリング方法。
項12. Glut1発現レベルをフローサイトメーターにより解析する、項10又は11に記載のがん治療薬の効果のモニタリング方法。
項13. 腫瘍浸潤リンパ球がCD8T細胞である、項10~12のいずれか1項に記載のがん治療薬の効果のモニタリング方法。
項14. 工程3が、分離された腫瘍浸潤リンパ球を2時間以上、0.1mM以上のグルコース濃度の培地中で培養し、その後グルコーストランスポーター(Glut1)発現レベルを解析する、項10~13のいずれか1項に記載のがん治療薬の効果のモニタリング方法。
工程1)がん組織から腫瘍内リンパ球を分離する工程、
工程2)分離された腫瘍浸潤リンパ球のグルコーストランスポーター(Glut1)発現レベルを解析する工程、及び
工程3)解析されたGlut1の発現レベルに基づき、がん患者に既に投与していたがん治療薬が有効か否かを判定/評価する工程
(ここで、がん組織は、がん治療薬を投与後にがん患者から摘出されたものである。)。
・抗PD-1抗体を有効成分として含み、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と組み合わせて投与することを特徴とする、腫瘍組織でのCD8T細胞数増加剤。
・腫瘍組織でのCD8T細胞数を増加するための抗PD-1抗体とフェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種の組み合わせ。
・抗PD-1抗体と併用するための、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を含む腫瘍組織でのCD8T細胞数増加剤。
・フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と併用するための、抗PD-1抗体を含む腫瘍組織でのCD8T細胞数増加剤。
・抗PD-1抗体とフェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を併用することを記載した腫瘍組織でのCD8T細胞数を増加するための手順書。
2x105 MO-5(OVA発現B16メラノーマ細胞株)をC57BL/6マウスに皮内移植し、移植7日後に自由飲水により5 mg/mlメトホルミンの経口投与を開始した。投与開始後7日目(移植後14日目)にリンパ節(LN)及び腫瘍組織(Tumor)を切除し、リンパ節のリンパ球及び腫瘍浸潤リンパ球を回収した。細胞をFCS(-)RPMIで洗浄後、抗マウスCD3抗体BV510、抗マウスCD8抗体APC-Cy7、1 μM Fluo4、1 μM Fura Redで30分、37℃で染色した。染色後、37℃で保温したFCS(-)RPMIで洗浄し、洗浄後フローサイトメーターにより細胞内カルシウムイオンのバックグラウンドを30秒間測定した。バックグラウンド測定後、ただちに100 ng/ml PMAおよび5 μM ionomycinにより刺激し、刺激によるマウスCD8 T細胞内のカルシウムイオンの上昇を測定した(図1)。腫瘍組織(Tumor)では、メトホルミン非投与群(-)にはPMA/ ionomycin刺激によっても細胞内カルシウム濃度はほとんど上昇しないが、メトホルミン投与群(+)ではPMA/ ionomycin刺激により細胞内カルシウム濃度は大きく上昇することが確認された。
抗原特異的腫瘍浸潤CD8 T細胞のメモリー形質の検出および各細胞分画におけるグルコース取り込みの評価
2x105 MO-5をC57BL/6マウスに皮内に移植し、移植後7日後に自由飲水により5 mg/mlメトホルミンの経口投与を開始した。投与開始後7日目(移植後14日目)に腫瘍組織を切除し、腫瘍浸潤リンパ球を分離した。分離後、0.1%BSA/PBSで洗浄し、抗マウスCD8抗体APC-Cy7、抗マウスCD62L抗体BV421、抗マウスCD44抗体PerCP、抗マウスKLRG1抗体APC、および400 μM 2-NBDG(2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose)を30分、4℃で染色した。0.1%BSA/PBSで洗浄し、フローサイトメーターにより抗原特異的腫瘍浸潤CD8 T細胞の頻度、メモリー形質(CD44, CD62L, KLRG1)および各細胞分画におけるデオキシグルコース(2-NBDG)の取り込みを解析した(図2)。図2に示すように、メトホルミンは、腫瘍浸潤CD8 T細胞のメモリー形質に関し、エフェクター(eff)とエフェクター・メモリー(EM)の数及び割合を増加して解糖系を活性化させるが、セントラル・メモリーの数を及び割合を減少して解糖系を不活性化させることが明らかになった。
健常人とがん患者の凍結保存されたヒトPBMC(ヒト末梢血単核球)を解凍し、FCS(-)RPMIで洗浄後、1x106 PBMCを抗ヒトCD8抗体APC-Cy7、1 μM Fluo4、1 μM Fura Redで30分、37℃で染色した。染色後、37℃で保温したFCS(-)RPMIで洗浄し、洗浄後フローサイトメーターにより細胞内カルシウムイオンのバックグラウンドを30秒間測定した。バックグラウンド測定後、ただちに100 ng/ml PMAおよび5 μM イオノマイシンにより刺激し、刺激によるヒトCD8 T細胞内のカルシウムイオンの上昇を測定した。結果を図3に示す。健常人とがん患者のPBMCのPMA/イオノマイシンによる刺激前のレベルと刺激直後の上昇については、差異はないが、その後の経過は、健常人では持続的に上昇し、がん患者では急速に下降する点で大きく異なる。
がん患者の凍結保存されたヒトPBMCを解凍し、FCS(-)RPMI 10 μMメトホルミン存在下で6時間培養し、培養後分離し、FCS(-)Glc(-)RPMIで洗浄した。洗浄後、0.1、1、10 mM Glcの含有したRPMIを用いて、1 μM モネンシン存在下で50 ng/ml PMAおよび2 μM イオノマイシンにより6時間刺激培養した。細胞を分離し、抗ヒトCD8抗体APC-Cy7で細胞表面分子を染色し、細胞透過処理を行い、抗ヒトIL-2 抗体APCおよび、抗ヒトTNFα抗体BV510、抗マウスIFNγ抗体FITCにより細胞内サイトカインを染色し、フローサイトメーターにより解析した(図4)。3つのサイトカイン(IL-2、TNFα、IFNγ)を産生しているCD8T細胞は、グルコース濃度に依存し、グルコース濃度が0.1mMでは最も低く、メトホルミンの有無により差異はないが、グルコース濃度が1mMの場合には、メトホルミンにより3つのサイトカイン(IL-2、TNFα、IFNγ)を産生しているCD8T細胞の数は有意に上昇することが明らかになった。
腫瘍移植実験(フェンホルミン,アスピリン処置)
OVA発現B16メラノーマ細胞株MO5 (2.5×105) をC57BL/6マウスの背部に皮内接種した。一群5匹のマウスで、未処置の群(C)をはじめ、腫瘍細胞接種10 日後からアスピリン単独、フェンホルミン(Phen)単独、アスピリンとフェンホルミンの併用投与群の4群を作製した。フェンホルミンは、メトホルミンと同様ビグアナイド系の薬物である。フェンホルミンは、腫瘍細胞接種10 日目から0.5%含有食餌として与えた。さらに4 日ごとに餌を通常の餌と交換した。即ち、10日目から14日目、18日目から22日目のみフェンホルミン含有食餌を与えた。 14日目から18日目と22日目から26日目の間はフェンホルミンを含まない通常の食餌を与えた。このような投与法を選択した理由は、フェンホルミンの連日投与では副作用によりマウスの死亡例が出るからである。また、フェンホルミンは自由飲水で与えた場合、マウスは水を飲まなくなるために食餌として与えた。
8週齢のBALB/c に腫瘍(MethA) 2 x 106個を皮内注射し、day7からメトホルミン(5 mg/mL) を自由飲水によって継続摂取させ,治療後3日(day10)および6 日後(day13)に腫瘍浸潤 T 細胞(TIL)を分離し, 抗CD8抗体、抗グルコーストランスポーター(Glut1) 抗体で染色した。また、PMA/Ionomycin/Monencin存在下で6 時間刺激培養後に抗CD8抗体、抗グルコーストランスポーター(Glut1) 抗体で染色し、フローサイトメーターにより解析した。結果を図6に示す。PMA刺激によりGlut1の発現は増加する傾向にあるものの、PMA刺激(-)を含め、あらかじめメトホルミンを投与しておいた群ではGlut1陽性集団の明らかな増加が認められた。
図6におけるGlut1陽性集団の多機能性について解析を行った。多機能性のあるCD8T細胞は、最も強力なエフェクターT細胞である。
8週齢のBALB/cマウスに同系腫瘍 MethA(2 x 106個)を皮内注射し,day7からメトホルミン(5 mg/mL), N-アセチルシステイン(NAC,10 mg/mL) の投与を開始した。治療3日後に腫瘍浸潤 T 細胞(TIL)を分離し、37 ℃で0,1,3,6 時間培養を行い, その後, CD3,CD8,グルコーストランスポーター(Glut1)の各種抗体で染色し,フローサイトメーターにより解析した(図8)。
8週齢のBALB/cマウスに同系腫瘍 MethA(2 x 106個)を皮内注射し, day7からメトホルミン(5 mg/mL)の投与を開始した. 治療3日後に腫瘍浸潤 T 細胞(TIL)を分離し、グルコース濃度 0, 0.1, 1.2, 6.1, 12.5, 25 mM の培養液下で 37 ℃,0,1,3,6 時間培養を行い, その後, CD3,CD8,グルコーストランスポーター(Glut1)の各種抗体で染色し,フローサイトメーターにより解析した(図9)。
腫瘍浸潤リンパ球(TIL) 回収と培養プロトコル
バイオプシー等によりがん患者から腫瘍塊(3~4mm径)を摘出し, Medimachine(アズワン株式会社製)にて機械的に2分間破砕後, 細胞液を回収し, 1200rpm, 5~10 分間遠心する。その後, 上清を捨てて, RPMI培養液にて(牛胎児血清は凝集を生じる場合があるのでここでは入れない), 5×105 /mL となるように細胞液を調製し, 24 well plateに5×105 /mL/well の細胞数を入れて6時間培養する。6時間後に細胞を回収し、抗体で染色する過程に移行する。
ステージ Iのがん患者、ステージIIaのがん患者及びステージIVのがん患者において、メトホルミンの投与前後におけるPMA刺激によるカルシウム濃度亢進の変化を測定した。メトホルミンの投与前及び図示した投与スケジュールによるメトホルミン投与後にヒトPBMC(ヒト末梢血単核球)を採取し、実施例3と同様にして100 ng/ml PMAおよび5 μM イオノマイシンにより刺激し、刺激によるヒトCD8 T細胞内のカルシウムイオンの上昇を測定した。具体的には、末梢血から比重分離法により末梢血単核球(PBMC)を分離し、1x106 個のリンパ球を 1 μg/ml 抗ヒトCD8抗体および1 μM Fluo4/FuraRed存在下で37℃30分間インキュベートした。その後、AIM-V培地により洗浄する。洗浄後AIM-Vを加え、未刺激(刺激前)のリンパ球を20秒間FACSCantoIIにより取り込みベースライン・データを取り込んだ。その後、100 ng/ml PMA/ 2 μM ionomycinを加えて刺激を入れ、直ちにFACSCantoIIにより取り込み、刺激後のデータとして取り込んだ。これにより細胞質カルシウム濃度に応じた波形を検出した。
腫瘍移植実験(Met,アスピリン,アバシミブ及び2剤併用)
OVA発現B16メラノーマ細胞株MO5 (2.5×105) をC57BL/6マウスの背部に皮内接種した。腫瘍細胞接種7 日後にメトホルミン(0.5%)は飼料によって、アスピリン(600μg/mL)は自由飲水によって、アバシミブ(15 mg/kg)は隔日腹腔内投与によってマウスに与えた。
結果を図11に示す。これらの処置開始後、6日目の腫瘍の長径、短径を計測し、腫瘍の体積(mm3)を算出した。メトホルミンとアスピリンの併用、あるいはメトホルミンとアバシミブの併用は、単剤よりも腫瘍抑制効果が高いことが分かる。
腫瘍移植実験(3剤併用,抗PD-1抗体, Met, アスピリン)
3LL 細胞(2.0×105) をC57BL/6マウスの背部に皮内接種した。腫瘍細胞接種5 日後に抗PD-1抗体 (10 mg/kg) を腹腔内投与によって1 回目投与後も6 日ごとに投与し合計4 回となるよう処置をした。メトホルミン(0.5%)は飼料によって、アスピリン(600μg/mL)は自由飲水によって処置をした。処置開始後、腫瘍の長径、短径を計測し、腫瘍の体積(mm3)を算出した。
結果を図12に示す。アスピリンの併用は、抗PD-1抗体+メトホルミンの腫瘍抑制効果を高めることが分かった。
腫瘍移植実験(3剤併用,抗PD-1抗体, Met, NDGA)
3LL 細胞(2.0×105) をC57BL/6マウスの背部に皮内接種した。腫瘍細胞接種5 日後に抗PD-1抗体 (10 mg/kg) を腹腔内投与によって1 回目投与後も6 日ごとに投与し合計4 回となるよう処置をした。また、メトホルミンとNDGA (Nordihydroguaiaretic acid)はメトホルミン(0.5%)飼料あるいはメトホルミン(0.5%)とNDGA(0.1%) 混合飼料によって処置をした。処置開始後、腫瘍の長径、短径を計測し、腫瘍の体積(mm3)を算出した。また、生存期間を観察した。
結果を図13に示す。NDGAの併用は、抗PD-1抗体+メトホルミンの腫瘍抑制効果を高め、生存期間を延長することが分かった。
腫瘍移植実験(4,5剤併用)
OVA発現B16メラノーマ細胞株MO5 (2.5×105) をC57BL/6マウスの背部に皮内接種した。腫瘍細胞接種7 日後にメトホルミン(0.5%)とNDGA(0.1%) は混合飼料によってそしてアスピリン(600μg/mL)は自由飲水によって処置をした。さらに抗PD-1抗体 (10 mg/kg) は腹腔内投与によって1 回目投与後も6 日ごとに投与し合計4 回となるよう処置をした。またアバシミブ(15 mg/kg)は腹腔内投与によって1 回目投与後も2 日ごとに計測終了日まで処置を行った。処置開始後、腫瘍の長径、短径を計測し、腫瘍の体積(mm3)を算出した。また、生存期間を観察した。
結果を図14に示す。メトホルミン+NDGA+アスピリン+アバシミブは、腫瘍増大を強く抑制し、生存期間を延長することが分かった。また、メトホルミン+NDGA+アスピリン+アバシミブ+抗PD-1抗体は、さらに強く腫瘍増大を強く抑制し、生存期間を延長(5匹中2匹は腫瘍の完全退縮をみた)することが分かった。
Claims (14)
- ヒト被験者の末梢血を採取し、免疫刺激剤を適用後、末梢血単核球(PBMC)又はCD8T細胞内のカルシウムイオン濃度の推移を検査し、免疫刺激剤の適用後に一過性にPBMC又はCD8T細胞内のカルシウムイオン濃度が上昇し、その後刺激前の状態に戻る場合に全身の免疫機能が低下していると判断し、免疫刺激剤の適用後にPBMC又はCD8T細胞内のカルシウムイオン濃度が上昇基調にある場合に全身の免疫機能は正常であると判断される、免疫機能の検査方法。
- 請求項1の検査方法で全身の免疫機能が低下していると判断されたがん患者に対してフェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法を行う、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法を適用するがん患者の選別方法。
- 他のがん治療薬が、抗PD-1抗体、アスピリン、スタチン、クルクミン、ベルベリン、ロイヤルゼリー又はプロポリスである、請求項2に記載の患者の選別方法。
- 下記工程(1)~(2)を含む、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法のがん患者に対する治療効果を予測する方法:
(1)がん患者から採取された末梢血に含まれる単核球(PBMC)を免疫刺激したときに細胞内カルシウムイオン濃度が一過性に上昇するか、持続的に上昇するかを評価する工程、及び
(2)免疫刺激時のPBMCの細胞内カルシウムイオン濃度が一過性に上昇した後速やかに下降する場合、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法が治療効果を示す可能性が高いと予測する工程。 - 下記工程(1)~(2)を含む、がん患者の治療方法:
(1)がん患者から採取された末梢血に含まれる単核球(PBMC)を免疫刺激したときに細胞内カルシウムイオン濃度が一過性に上昇するか、持続的に上昇するかを評価する工程、
(2)免疫刺激時のPBMCの細胞内カルシウムイオン濃度が一過性に上昇した後速やかに下降する場合、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種と他のがん治療薬の併用療法が治療効果を示す可能性が高いと予測する工程、及び
(3)工程(2)において治療効果を示す可能性が高いと予測した患者に、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種並びに他のがん治療薬を投与する工程。 - フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を含む、免疫刺激によるCD8T細胞の細胞内カルシウムイオン濃度上昇剤。
- 腫瘍組織内のCD8T細胞の細胞内カルシウムイオン濃度を選択的に上昇させる、請求項6に記載の免疫刺激によるCD8T細胞の細胞内カルシウムイオン濃度上昇剤。
- フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を含む、腫瘍組織におけるエフェクター・メモリー(EM)とエフェクター(eff)の選択的機能向上剤。
- フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる少なくとも1種を含む、腫瘍組織内のグルコース濃度が0.5~1.5mMであるがん患者に投与されるがん治療薬。
- 以下の工程1)~工程3)を含む、がん治療薬の効果のモニタリング方法:
工程1)がん組織から腫瘍内リンパ球を分離する工程、
工程2)分離された腫瘍浸潤リンパ球のグルコーストランスポーター(Glut1)発現レベルを解析する工程、
工程3)解析されたGlut1の発現レベルに基づき、がん患者に既に投与していたがん治療薬が有効か否かを判定/評価する工程
(ここで、がん組織は、がん治療薬を投与後にがん患者から摘出されたものである。)。 - がん治療薬が、がんワクチン、免疫チェックポイント阻害薬、フェンホルミン、ブホルミン及びメトホルミンからなる群から選ばれる、請求項9に記載のがん治療薬の効果のモニタリング方法。
- Glut1発現レベルをフローサイトメーターにより解析する、請求項10又は11に記載のがん治療薬の効果のモニタリング方法。
- 腫瘍浸潤リンパ球がCD8T細胞である、請求項10~12のいずれか1項に記載のがん治療薬の効果のモニタリング方法。
- 工程3が、分離された腫瘍浸潤リンパ球を2時間以上、0.1mM以上のグルコース濃度の培地中で培養し、その後グルコーストランスポーター(Glut1)発現レベルを解析する、請求項10~13のいずれか1項に記載のがん治療薬の効果のモニタリング方法。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP17750381.0A EP3415908A4 (en) | 2016-02-12 | 2017-02-10 | IMMUNE FUNCTION TEST PROCEDURE, PROCEDURE FOR CATEGORIZATION OF CANCER PATIENTS PREDICTING THE EFFECTIVENESS OF CANCER THERAPY, AGENT FOR INCREASING INTRACELLULAR CALCIUM ION CONCENTRATION, MEDIUM TO IMPROVE THE SELECTIVE OPERATION OF EFFEKTORGEDÄCHTNISSES (EM) AND eFFeCtor (EFF) IN TUMOR TISSUE AND METHOD FOR MONITORING THE EFFECTIVENESS OF CANCER AGENT |
JP2017567023A JP6860922B2 (ja) | 2016-02-12 | 2017-02-10 | 免疫機能の検査方法、がん患者の選別方法、がんの治療効果予測方法、細胞内カルシウムイオン濃度上昇剤、腫瘍組織におけるエフェクター・メモリー(EM)とエフェクター(eff)の選択的機能向上剤、がん治療薬の効果のモニタリング方法 |
US16/076,275 US20210223229A1 (en) | 2016-02-12 | 2017-02-10 | Immune function inspection method, cancer patient categorization method, cancer treatment efficacy prediction method, agent for increasing intracellular calcium ion concentration, agent for increasing selective function of effector memory (em) and effector (eff) in tumor tissue, and method for monitoring efficacy of cancer drug |
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JP2016204284 | 2016-10-18 |
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US (1) | US20210223229A1 (ja) |
EP (1) | EP3415908A4 (ja) |
JP (2) | JP6860922B2 (ja) |
WO (1) | WO2017138660A1 (ja) |
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IL264068B2 (en) | 2018-01-05 | 2023-07-01 | Great Novel Therapeutics Biotech & Medicals Corp | Aggregate and pharmaceutical preparation containing an hdac inhibitor and an nsaid aggregate together with an immune checkpoint inhibitor for use in a method for removing immunosuppression in the microenvironment of a tumor or stimulating an immune system against cancer cells |
CN109224071A (zh) * | 2018-11-06 | 2019-01-18 | 广东美赛尔细胞生物科技有限公司 | 含有盐酸小檗碱和pd1-抗体的预防和/或治疗肿瘤的联用药物 |
CN116236468A (zh) * | 2021-12-07 | 2023-06-09 | 深圳先进技术研究院 | 二甲双胍在制备增强免疫检查点抑制剂效果的抗肿瘤药物中的应用 |
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JP2021099375A (ja) | 2021-07-01 |
JPWO2017138660A1 (ja) | 2018-12-27 |
JP6860922B2 (ja) | 2021-04-21 |
EP3415908A4 (en) | 2019-08-21 |
US20210223229A1 (en) | 2021-07-22 |
EP3415908A1 (en) | 2018-12-19 |
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