WO2005054496A1

WO2005054496A1 - Method of examining cell kinetics

Info

Publication number: WO2005054496A1
Application number: PCT/JP2004/017843
Authority: WO
Inventors: Akikuni Yagita
Original assignee: Orient Cancer Therapy Co., Ltd.
Priority date: 2003-12-02
Filing date: 2004-12-01
Publication date: 2005-06-16

Abstract

It is intended to achieve an improved efficacy of a molecule-targeting therapeutic drug by providing a means for elevating a complete remission ratio, shortening the period requiring for complete remission and establishing synergistic effects with an immunotherapy. That is to say, it is intended to achieve synergistic effects by the combined use of a novel immunotherapy focusing on CTL activity, NKT activity, NK activity, VEGF, etc. with a molecule-targeting therapeutic drug, in particular, a tyrosine kinase inhibitor. It is found out that a key factor resides in an immunological therapeutic drug such as an IL-12 production inducer efficaciously affecting perforin-producing cells. Namely, a method of examining an immunotherapy for cancer by using the kinetics of perforin-producing cells as a marker and an immunological therapeutic drug for cancer which is screened by using the kinetics as a marker.

Description

Specification

Cell dynamics testing method

Technical field

The present invention provides a new area of cancer treatment. In other words, a tyrosine kinase inhibitor, which is attracting attention as a novel cancer treatment, and NK cell activation, NKT cell activation, angiogenesis inhibitory, and IL-12 production induction developed by Dr. New immunotherapy that focuses on the dynamics of perforin-producing cells in combination with new immunotherapy that focuses on the dynamics of IFNγ and the ability to induce the production of IFNγ. .

[0002] The present application claims priority from Japanese Patent Application No. 2003-403676, which is incorporated herein by reference.

Background art

[0003] In the selection of useful substances for the prevention or treatment of cancer (malignant neoplasms) (cancer), direct action on cancer cells has been conventionally regarded as important. Although immunostimulants have been found to be useful in cancer treatment, all of the compounds obtained as immunostimulants have weak anticancer effects, indicating that immunotherapy alone or in combination with chemotherapy. Even with the combination treatment, a sufficient therapeutic effect for cancer has been achieved!

[0004] The inventor of the present invention, Dr. Yagida's medical science, first focused on the usefulness of a substance that induces interleukin 12 (IL-12) in vivo as an epoch-making technique in the treatment of cancer. He discovered that a substance has its function and established a cancer treatment method that could be called a novel immunotherapy (Novel Immunotherapy for cancer) (MTC). Conventionally, IL-12 has an anticancer effect, but there is a fact that if IL-12 itself is administered directly into a living body, patients will not be able to tolerate the treatment due to side effects. Unable to use as a power. However, the formulation containing the processed mushroom mycelium reported by Yagida achieved a significant healing / life extension effect in the treatment of cancer. In other words, Yagida achieved the goal of treating cancer by administering an effective amount of a processed mushroom mycelium capable of inducing IL-12 in vivo. [0005] IL-12 has TNF a → IFN y → IL-12 → CTL activity, and has an activating and enhancing effect on killer T cells through the root. In other words, the enhancement of IL-12 production is expected to have an anticancer effect by activating and enhancing killer T cells.

[0006] Yagida reports that the activity of NKT cells, apart from the IL-12 production enhancement system, is useful for anticancer effects. Taniguchi et al. Discovered a specific glycolipid antigen recognized by the specific -cell antigen receptor (TCR) called να24ν | 811, which NKT cells possess, and found that this antigen is α-galactosylceramide. Reporting. Furthermore, it was demonstrated that in cancer-bearing mice to which α-galactosylceramide was administered, ΝΚΤ cells were activated and the migration was suppressed although the disappearance of the cancer was not observed.

It has been reported that ΝΚΤ cells have ΝΚ cell antigen receptor (NKR-P1; natural killer receptor P1) as another receptor (Non-patent Document 1). Yagida has found that NKR-P1 is also involved in the activity of ΝΚΤ cells, and that this activity is more superior in anticancer effect (Patent Document 2).

[0007] The molecular target therapeutic agent for cancer has attracted attention as a new type of anticancer agent in comparison with the conventional cell target therapeutic agent. Among them, a tyrosine kinase inhibitor has been particularly noted as a drug having a signal transduction inhibitory action. ZD1839 (Iressa: registered trademark AstraZene Power) has a competitive action with EG at the binding site of EGFR (Epidermal Growth Factor Receptor) tyrosine kinase, and suppresses tyrosine kinase autophosphorylation by controlling tyrosine kinase. Suppress activity. As a result, signal transduction related to the proliferation, invasion, differentiation, and metastasis of EGFR (the binding of EGFR tyrosine kinase to the extracellular domain of EGFR Activating, triggering autophosphorylation of EGFR and phosphorylation of various intracellular target proteins transmit cell surface forces, proliferation signals to nuclei, and proliferate signals from cancer cell surfaces to nuclei. Is transmitted to cause cancer cell proliferation, invasion, metastasis, and angiogenesis. IMC-C225 (EGFR-targeted monoclonal antibody) recognizes the EGFR receptor on the cell membrane surface and inhibits tyrosine kinase activity by suppressing EGFR autophosphorylation. Herceptin is a monoclonal antibody against Her2 / Neu with homology to EGFR, and STI-571 (Gleevec) is a tyrosine kinase activity of BCR-Abl And the ability to inhibit the tyrosine kinase activity of c kit (Non-Patent Document 2).

[0008] Such a molecular target therapeutic agent is attracting attention as a cancer therapeutic agent of a new mechanism. Its effect is not yet revolutionary. For example, ZD1839 (Iressa) is a powerful and selective EGFR tyrosine kinase inhibitor newly developed by AstraZeneca, and its usefulness has been proven in humans. Clinical results for non-small cell lung cancer and prostate cancer have PR (partial remission) of 10 to 20% and CR (complete remission) at all, but they are very rare It took more than four months to complete remission. Therefore, combination therapy with ZA1839 (Iressa) and various anticancer drugs has been tried! /, But at the moment it is additive !, but no synergistic effect has been obtained.

[0009] CX3C-chemokine has been reported as a chemokine that induces IFN γ production from NK cells, and its receptor is CX3CR1, which is a G protein-coupled seven-transmembrane receptor {cells, monocytes and some of them). It is expressed in cells (Non-Patent Document 3). Nishimura et al. Have shown that CX3CR1 expression in Τ cells is limited to CD3 (+) CD161 (+) and granzym Β positive cells, and that cytotoxic activity is carried by CX3CR1 positive cells (Non-patent Document 4 )

Patent Document 1: JP-A-10-139670

Patent Document 2: US2002-0010149A1

Non-patent document 1: Special issue: Basics and clinical practice of NKT cells: Latest Medicine 55 Vol. 4 2000 818—823 ぺ¹ ~~

Non-Patent Document 2: Blood and Immune 'Tumor Vol. 7 No.3 2002-7

Non-Patent Document 3: Cell 1997; 91: 521

Non-Patent Document 4: J. Immunol 2002; 168: 6173

Disclosure of the invention

Problems to be solved by the invention

[0011] The present invention aims to provide a more effective effect of the above-mentioned molecular target therapeutic agent, and achieves a higher complete remission rate, a shorter time to complete remission, and a synergy with immunotherapy. It provides the means to achieve the effect. In other words, new immunotherapy focusing on CTL activity, NKT activity, NK activity, VEGF, etc., and molecular targeted therapeutics, especially tyrosine kinases The task is to achieve a synergistic effect when used in combination with an inhibitor.

Means for solving the problem

[0012] In the present invention, in order for a combined use of a tyrosine kinase inhibitor and a cancer immunotherapy to achieve an excellent synergistic effect in cancer treatment, the cancer immunotherapy effectively affects perforin-producing cells. It has been found that this is important, and the present invention has been completed.

[0013] That is, the present invention provides

"1. A method for examining cell kinetics using, as a marker, the kinetics of perforin-producing cells by a cancer immunotherapeutic agent in a combination use of a tyrosine kinase inhibitor and a cancer immunotherapy agent.

2. A cancer immunotherapeutic agent that uses the dynamics of perforin-producing cells as a marker to determine the efficacy of the cancer immunotherapeutic agent in combination with a tyrosine kinase inhibitor and a cancer immunotherapeutic agent.

3. A method for screening a cancer immunotherapeutic agent using a tyrosine kinase inhibitor and a cancer immunotherapeutic agent in combination with the kinetics of perforin-producing cells to determine the efficacy of the cancer immunotherapeutic agent.

4.The tyrosine kinase inhibitor has a selective targeting effect on at least one of the following receptors, and the function of the cancer immunotherapeutic agent is IL-12 production induction and Z or IL-21-mediated response. 3 ways.

HER2 / neu, HER3, HER4, c-kit, PDGFR, bcr-abl, EGFR

5.The tyrosine kinase inhibitor has a selective targeting effect on at least one of the following receptors, and the function of the cancer immunotherapeutic agent is IL-12 production induction and Z or IL-21-mediated response. Cancer immunotherapy.

HER2 / neu, HER3, HER4, c-kit, PDGFR, bcr-abl, EGFR

6. The method according to the above item 4, wherein the immunotherapeutic agent is a substance having a β1,3 / 1,6 glucan structure.

7. | The cancer immunotherapeutic agent according to the above item 5, wherein the cancer immunotherapeutic agent is a substance having a | 1,3 / 1,6 glucan structure.

8. The method according to the above item 4, wherein the cancer immunotherapy agent is a yeast-derived component having a β1,3 / 1,6 glucan structure.

9. The cancer immunotherapy agent according to the above item 5, wherein the cancer immunotherapy agent is a yeast-derived component having a β1,3 / 1,6 glucan structure. 10.Perforin-producing cell dynamics are determined by at least one of NKTP (+), NKT8 (+) P (+), CD8 (+) P (+) T, and Thl / Th2 ratio Any one of methods 1, 3, 4, 6, and 8 above.

11.Perforin-producing cell dynamics are determined by at least one of NKTP (+), NKT8 (+) P (+), CD8 (+) P (+) T, and Thl / Th2 ratio The cancer immunotherapeutic agent according to any one of the above items 2, 5, 7, and 9.

12. The cancer immunotherapeutic agent according to any one of the above items 2, 5, 7, 9, and 11, which is treated without combination with a chemotherapeutic agent and radiation therapy for cancer.

13. Any one of the above 2, 5, 7, 9, 11, and 12 for cancer immunotherapy which is used in combination with a substance which selectively acts on NKT-P1 of NKT cells to activate NKT cells. Agent.

14. The cancer immunotherapeutic agent according to any one of the above items 2, 5, 7, 9, 11, 12, and 13, which is used in combination with a substance capable of inhibiting angiogenesis. "

Consists of

The invention's effect

[0014] In the present invention, the clinical significance of using the effect of immunotherapeutic agents on the dynamics of perforin-producing cells as a marker was discovered, and by measuring this, the combination of a tyrosine kinase inhibitor and an immunotherapeutic agent could be more reliably determined. The effect by the above can be achieved.

Brief Description of Drawings

FIG. 1 is a multivariate analysis of the contribution of an immune marker.

FIG. 2 is a diagram in which the contribution of perforin-producing cells to each effector cell was examined.

FIG. 3 is a diagram showing the degree of contribution as a ratio.

FIG. 4 is a diagram examining the importance of immunological factors.

FIG. 5 is a diagram examining the importance of immunological factors.

FIG. 6 is a diagram examining the importance of immunological factors.

FIG. 7 is a diagram examined for NK cells.

FIG. 8 is a diagram examined with CD8 (+) cells.

FIG. 9 is a graph showing contribution to response in lung adenocarcinoma (N = 50).

FIG. 10 is a graph showing contribution to response in lung adenocarcinoma (after administration of Iressa) (N = 48). FIG. 11 is a view showing a response rate (N = 30) in lung adenocarcinoma (after administration of Iressa). BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. Unless defined otherwise, technical and scientific terms used in the present specification have ordinary knowledge in the technical field to which the present invention pertains. Has a meaning commonly understood by others.

[0017] The present inventor's medical doctor, Dr. Yagida's New Cancer Therapy for Cancer (NITC) is a therapeutic means comprising a combination of four different mechanisms of action.

The first mechanism of action is to administer an angiogenesis inhibitor (Bettershark) to impede blood flow to the cancer and to reduce the size of the cancer. The effect can be determined by measuring vascular endothelial cell growth factor (VEGF). The angiogenesis inhibitory effect can be evaluated by a negative (negative) VEGF value (-VEGF). It is possible to evaluate the angiogenesis inhibitory ability by using other vascular growth factors such as FGF and HGF instead of the VEGF value. In addition, the positive value of an angiogenesis inhibitor can be evaluated instead of VEGF (for example, endstatin value).

[0018] The first mode of action is that a compound having a β1,3 glucan structure is administered to induce Thl cytokines (TNF o; IFN y, IL-12) to activate CTLs. How to CTL activity can be determined by CD8 (+) perforin producing ability This CD8 (+) perforin level includes cytotoxic T cells (CTL) and immunosuppressive T cells (STC; Suppressor T cells). On the other hand, the former damages cancer cells, and the latter activates cancers as a result. Therefore, it cannot be evaluated with its absolute value. If the IFN y force S 10 IU / ml or more or the IL-12 value is 7.8 pg / ml or more, it is CTL, and if the IFN γ and IL-12 are low, it is judged as STC. Therefore, CTL activity can be evaluated based on IFN y production ability (IFN y value) or IL-12 production ability (IL-12 value).

[0019] The effector cells activated by the administration of the compound having the ex-1,3 glucan structure, which is the third and fourth mechanism of action, are NK cells and NKT cells. The NK and NKT cells share the NKR-P1 (NK cell receptor CD161 (+), and for the former, the number of NK cells can be measured using the CD3 (-) CD161 (+) surface marker. Activatedi can be determined by its ability to produce CD3 (-) CD161 (+) perforin, whereas the latter NKT cells are CD3 (+) CD161 (+). The cell number can be measured, and the activity of NKT cells can be measured by its perforin-producing ability (denoted as NKTP (+)).

[0020] Therefore, in the treatment of cancer, whether using new immunotherapy (NITC) or general immunotherapy, it is possible to evaluate each effector cell or angiogenesis inhibitory effect by the following measurement items. It is possible. Specifically, CTL activity can be evaluated based on its ability to induce IFNy or IL-12 production. NK cell activity is CD3 (-) CD161 (+), or

It can also be evaluated by CD3 (-) CD161 (+) perforin value. NKT cell activity

Evaluation can be performed using CD3 (+) CD161 (+) or CD3 (+) CD161 (+) perforin (NKTP).

The present invention was carried out by examining clinical results obtained by using a tyrosine kinase inhibitor in combination with the above-mentioned new immunotherapy. The present inventors have proposed a new immunotherapy (NITC) in cancer patients, a compound carrying an a1,3 glucan structure, a compound carrying a / 1,3 glucan structure, and an angiogenesis inhibitory substance (shark cartilage). In addition, IL-12, IFNy, and various other sites were measured. CD8 (+) perforin production has a strong positive correlation with IFNy and IL-12 production.As a result, measurement of CD8 (+) perforin production is significant for the evaluation of CTL activity route. Have found that there is. According to the present invention, a tyrosine kinase inhibitor is used in combination with a new immunotherapy, and tumor cytotoxic cells of an effective patient are identified.

NKTP (+) (CD3 (+) CD161 (+) perforin (+)), NKT8 (+) P (+) (CD3 (+) CD161 (+) CD8 (+) perforin (+)), CD8 (+) P (+ 丌 CD8 (+) perforin (+)] T cells and ΝΚΡ (+ 丌

CD3 (-) CD161 (+) perforin (+)]. Then, the presence of effector cells that were not activated by IFNy or IL-12 but were dependent on the Thl / Th2 ratio was confirmed. These cells have a very strong relationship with perforin, and the present inventors tentatively named Y cells as being CX3CR1-like cells. These cells were presumed to be involved in an IL-21-mediated response activated by IL-21.

[0022] Due to this significance, in the combination use of an immunotherapeutic agent and a tyrosine kinase inhibitor, at least the measurement of perforin-producing cells can be applied to a method for screening a cancer immunotherapy agent useful in combination with a tyrosine kinase inhibitor. Screening methods can be used to demonstrate useful cancer cytotoxicity in combination with tyrosine kinase inhibitors. It is possible to identify the new ι8 1,3 glucan to be carried. The cancer immunotherapeutic agent used in the present invention is, for example, a mushroom mycelium composition preparation having a ^β1,3 glucan structure (eg, ILX ^ffi ^ _: Tozai Pharmaceutical Research Institute, ILY ^ffi ^ _: Seishin Enterprise), or j8 Various yeasts with 1,3 glucan structure (marine yeast, baker's yeast, NBG ™) can be used. In particular, marine yeast is preferred. In addition, a novel cancer immunotherapeutic agent can be easily identified by those skilled in the art by combining measurement of perforin-producing cells. According to this method, in the present invention, marine yeast was most suitable as a useful cancer immunotherapeutic agent in combination with a tyrosine kinase inhibitor, and it was considered that this system also activated CX3CR1-expressing cells.

In the present invention, a combination use of this cancer immunotherapy agent and a tyrosine kinase inhibitor is essential.

In a specific example, various oral synthase inhibitors using ZD1839 (trade name of Iressa) or STI571 (trade name of Dalibec) can be effectively used. Examples of such target molecules include HER2 / neu, HER3, HER4, c-kit, PDGFR, bcr-abl, EGFR and the like. The most effective molecules are EGFR or C-kit.

The dose of the tyrosine kinase inhibitor follows the recommended dose of each molecular target compound,

Oral administration of 10-500 mg / day is performed.

[0024] The combination use of the cancer immunotherapeutic agent and the tyrosine kinase inhibitor is not particularly limited, but may be performed from the early stage of the treatment or either of them may be performed first. In a specific example, a dramatic clinical effect was confirmed using a tyrosine kinase inhibitor in combination with NITC therapy, especially a cancer immunotherapy drug, for a certain period of time.

[0025] In the present invention, an NK activator or NKT activator can be used in combination with an IL-12 production inducer as a cancer immunotherapy agent. - Geroorigo sugar, is useful as _a 1, 3-glucan composition formulations structure with compound NK or NKT activator agent such Fukoidan. a Various compounds having a 1,3 glucan structure are known, and the known structure and CD3 (-) CD161 (+), CD3 (-) CD161 (+) perforin-producing ability, CD3 (+) CD161 (+), Those skilled in the art can easily specify the NK-activating agent by combining the measurement of the ability to produce CD3 (+) CD161 (+) perforin. CD3 (+) CD161 (+) means that it acts on the NKT cell receptor NKR-P1.

A Examples of the saccharide substances having a 1,3 glucan structure include -gelooligosaccharide (TSO), fucoida , Sulfated oligosaccharides and the like.

-Gello-oligosaccharide is a saccharide containing 3-0-a D darcoviranosyl-D glucose as a constituent unit. Typical examples include -gelose, -gelosylglucose, and digerosylmaltose.

[0027] Examples of commercially available -gelooligosaccharides include -gerooligosaccharide liquid sugar (seller: Takeda Shokuhin Kogyo Co., Ltd.). Source a— D— Glcp— (1,3) — D— Glc (2) -Gerosylglucose a— D— Glcp— (1,3) — α-D- Glcp- (1,4)-D- Glc (3) -Gerosil maltose a-D- Glcp- (1,3)-a-D- Glcp- (1,4)-a-D- Glcp- (1,4)-D- Glc ( Glc is glucose, and p is an abbreviation for viranose).

Fucoidan, in a narrow sense, is a sulfated fucose-containing polysaccharide in which one to two sulfuric acid molecules are bonded to two to six molecules of fucose, and a fucoidan-like polysaccharide containing xylose or peronic acid is called `` fucoidan '' at the food level. Is called. Fucoidan, for example, is obtained by crushing kelp, chipping, extracting aqueous components, removing the extraction residue by centrifugation, removing low-molecular substances such as eodo and sodium salt by ultrafiltration, and freeze-drying. It is formulated into a formulation.

Examples of fucoidan include fucoidan derived from brown algae, such as fucoidan derived from gagome kelp, and fucoidan derived from Okinawa mozuku. Fucoidans derived from brown algae Laminariaceae, such as gagome kelp, include at least three types of fucoidan, F-fucoidan (a polymer of a L-fucos), and U-fucoidan (13D-glucuronic acid and a-D mannose as the main chain and a L-fucose) and G-fucoidan (| 8—D-galactose in the main chain and α L-fucose in the side chain), and all fucoidans are sulfated.

[0028] Examples of the oligosulfate sulfate include, for example, an extract derived from susabinori (Poryphyra Yezaensis) manufactured by Shiroko Co., Ltd. The main components of the extract are an oligosaccharide of ex-1,3 linkage galactan sulfate and a galactan sulfate oligosaccharide consisting of ex1,3 linkage and β1,4 linkage.

[0029] Use of the tyrosine kinase inhibitor of the present invention in combination with a cancer immunotherapy agent and a CTL activator (IL-12 production inducer, INFy production inducer), and furthermore, a NK activator and an NKT activator , New blood vessels In combination with inhibitors, the method of application is selected to determine whether lung cancer (lung squamous cell carcinoma, lung adenocarcinoma, small cell lung cancer), thymoma, thyroid cancer, prostate cancer, renal cancer, bladder cancer, colon cancer , Rectal cancer, esophageal cancer, cecal cancer, ureteral cancer, breast cancer, cervical cancer, brain cancer, tongue cancer, pharyngeal cancer, nasal cavity cancer, larynx cancer, stomach cancer, liver cancer, bile duct cancer, testicular cancer, ovarian cancer It is effective in treating cortical cancer, metastatic bone cancer, malignant melanoma, osteosarcoma, malignant lymphoma, plasmacytoma, liposarcoma, and the like.

[0030] A combination of the tyrosine kinase inhibitor according to the present invention and a cancer immunotherapy agent, a CTL activator (IL-12 production inducer, INFy production inducer), and further a NK activator, an NKT activator, When used in combination with a neovascular inhibitor, it is used in a manner capable of inducing or enhancing its activation and maintaining its activation. That is, the dose and the administration period that can induce or enhance the activation and maintain the activation can be selected and used. Specifically, the dose of the compound having a 1,3-glucan structure that is an NK activator or NKT activator is about lg-4 OgZ days, preferably about 5 g-20 gZ days, The compound having a β-1,3 glucan structure as an agent (IL-12 production inducer, INFy production inducer) has an lg of about 10 gZ days, preferably about 3 g to 6 gZ days. The administration period is generally 10 days to 24 months, and the administration frequency is every other day or 1 to 3 times Z days, preferably daily administration. The cancer immunotherapy agent, CTL activator (IL-12 production inducer, INFy production inducer), NK activator, and NKT activator are preferably orally ingested.

[0031] When an anticancer (chemotherapy) agent, radiation, or steroid combination therapy is performed in addition to the combination of the present invention, TNF Q; → IFN Y → IL-12 → The killer T cell lineage is severely impaired. Therefore, they are preferably not used in the present invention. However, when administering anticancer drugs, low-dose chemotherapy, which is a method that does not impair the immune system, such as low-dose chemotherapy such as 5FU, UFT, mifurol, furturon, and CDDP ^ / zg-lO / zg) It is useful to apply administration methods such as taxotere or low-concentration anticancer drugs such as taquinol, adriamycin, mitomycin, and CPT-11. Similarly, it is necessary to select low-dose irradiation for radiotherapy and low-concentration administration for steroid therapy.

[0032] A method for measuring the force of cells and each site is described below. (Measurement of NKT cells) (Measurement of NK cells) (Measurement of CD8)

The measurement of NKT cells having NKR-P1 can be performed by measuring cell surface antigens (CD3 and CD161) specifically present on the cell surface of NKT cells. Specifically, for lymphocytes in peripheral blood, cells that are positive for CD3 and positive for CD161 [CD3 (+) CD161 (+)] are assayed. That is, CD3 and CD161 which are cell surface antigens of NKT cells are measured by a two-color test using a flow cytometry using a monoclonal antibody. Here, “activated NKT cells” means that the ratio of NKT [CD3 (+) CD161 (+)] cells in lymphocytes is 10% or more, more preferably 16% or more. NKT cell activity is a function that increases the proportion of NKT cells to 10% or more, more preferably 16% or more, or a function that further enhances the proportion of NKT cells before administration of a certain substance. Similarly, [CD3 (-) CD161 (+)] refers to assaying cells that are negative for CD3 and positive for CD161. This method is useful for measuring NK cells.

Further, CD8 (+) refers to assaying for CD8-positive cells. This method is useful for measuring CTL activity.

[0033] In Examples, the blood cell of a cancer patient is used to detect cell surface antigens.

CD3, CD161, and CD8 were discriminated as positive or negative, and the proportion of each cell was measured by a two-color test using a flow cytometer as usual. At this time, monoclonal antibodies against CD3, CD161 and CD8 were manufactured by Coulter or Betaton Dickinson, respectively.

(Measurement of perforin-producing cells)

For lymphocytes in peripheral blood, two of cell surface antigens, CD3, CD161, and CD8, and perforin are measured by a three-color test using flow cytometry as usual. Specifically, cells were fixed by collecting a fixation solution into the collected blood, and after adding a membrane permeate, the reaction was performed by adding an anti-perforin antibody (manufactured by Pharmingen) and reacting, followed by PRE-Cy5 labeling. A secondary antibody (manufactured by DAKO) was added and reacted, and then an anti-CD3-PE (Coulter 6604627) antibody and an anti-CD161-FITC (B-D) antibody were added and reacted. Measure by flow cytometry. Figures · Abbreviations in the table are indicated as P or PER. (Preparation of Sample for Measuring Site Force In)

First, a mononuclear cell fraction is separated and prepared from blood. Heparin-added peripheral blood is diluted 2-fold with Phosphate Buffered Saline (PBS) and mixed, then layered on FicoU-Conray solution (specific gravity 1.077), and 400 G for 20 minutes After centrifugation, the mononuclear cell fraction is collected. After washing, prepare RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), and adjust the cell number to 1 × 10 6. Phytohemagglutinin (manufactured by DIFCO) was added to the obtained cell suspension 2001 to a concentration of gZml, and cultured in a 96-well microplate at 37 ° C for 24 hours in the presence of 5% CO. And in the cultured cell solution

2

Use as a sample for measuring the site force-in.

[0036] (Measurement of IL-12)

For the measurement of the amount of IL-12, a measurement kit by enzyme immunoassay (ELISA) available from R & D SYSTEMS or MBL is used, which is a power that can use clinical and biochemical tests known per se. Here, a measurement kit from R & D SYSTEMS was used. Actually, into each well of a 96-well microplate, dispense 50 1 of the assay diluent Assay Diluent 1¾, and 200 μl of the standard solution or 200 μl of the sample prepared by the above-mentioned method for preparing a cytoforce-in sample. After that, the reaction was left standing at room temperature for 2 hours. Thereafter, horseradish peroxidase (HRP) -labeled anti-IL-12 antibody was dispensed in 200 μl aliquots and allowed to stand at room temperature for 2 hours. After removing the reaction solution in each well and washing three times, 200 1 of the chromogenic substrate solution was dispensed at a time, and the mixture was allowed to stand at room temperature for 20 minutes. Using 550 nm as a control, the absorbance of each well at 450 nm was measured by Emax (manufactured by Wako Pure Chemical Industries, Ltd.). IL-12 levels are expressed as pgZml. Here, the ability to induce IL-12 production refers to a function that enhances the amount of IL-12 produced by stimulation of the peripheral blood mononuclear cell fraction to 7.8 pgZml or more, or IL-12 production before administration of a certain substance. A function that enhances the amount.

(Measurement of IFN y)

IFN y was measured by an enzyme immunoassay (EIA method) using an IFN y EASIA kit from BioSource Europe S. Actually, in each well of a 96-well microplate, dispense 50 1 of a standard solution or a 2-fold dilution of the sample prepared above, and dispense 50 μl of HRP-labeled anti-IFN-γ antibody. The mixture was further reacted at room temperature with shaking for 2 hours. After removing the reaction solution from each well and washing three times, 200 1 of the chromogenic substrate solution was dispensed, and the mixture was allowed to react at room temperature for 15 minutes while shaking, and 50 1 of the enzyme reaction stopping solution was dispensed. The absorbance of each well at 450 nm and 490 nm was measured by Emax (manufactured by Wako Pure Chemical Industries, Ltd.) with 630 nm as a reference. IFN y content is expressed as IUZml.

[0038] (Thl / Th2 ratio)

The Thl / Th2 ratio indicates the ratio of cells producing Νγ (Thl) to cells producing IL-4 (Th2) among helper T cells having the cell surface antigen CD4, and CD4 (+ ) IFN y (+) / CD4 (+) IL-4 (+). The Thl / Th2 cell ratio was determined by a helper T (Th) cell line Three Color analysis test by flow cytometry using a conventional method known to those skilled in the art, and specifically described in WO 02/04944. The test was carried out using the method described above.

(Measurement of angiogenesis inhibitory ability)

(Measurement of vascular endothelial cell growth factor / VEGF, basic fibroblast growth factor / bFGF, and angiogenesis inhibitor endostatin / endostatin)

Serum concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (ACCUCYTE Human VEGF, ACCUCYTE Human bFGF, ACCUCYTE Human Endostatin: CYTIMMUNE Sciences Inc.) of the sales kit.

[0040] Each marker used in the clinical test was a commercially available marker, and the measured value was shown by each recommended method. The displayed abbreviations are based on each general display method.

[0041] Patients were evaluated for the following CR (complete remission), PR (partial remission), LNC (long-term unchanged), SNC

(Short-term invariant) and PD (disease progression) were evaluated in five stages. The response rate for each type of cancer indicates the proportion of CR, PR, LNC, SNC, and PD in all cases of each type of cancer.

Example

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the Examples.

We have been treating advanced terminal cancer patients as new immunotherapy (MTC). This NITC induces endogenous TNFα, IFNγ, and IL-12 upon β-1,3 glucan administration to activate CTLs (killer T cells), and ΝΚ and で upon α-1,3 glucan administration. This is a BRM therapy that works to activate cells and also to inhibit angiogenesis by oral administration of Better Shear. For patients , A cancer immunotherapeutic agent, an IL-12 production inducer, shark cartilage (Seishin Enterprise), and saccharides having an o1,3 structure were administered according to the recommended formulations. ILX (Tozai Pharmaceutical), ILY (Seishin Pharmaceutical), Krestin (Sankyo), Immutol (NBG), etc. were administered alone or in combination as an IL-12 production inducer depending on the patient's condition.

The measurement of the amount of IL-10 in this example was performed by an ELISA method using a kit manufactured by Bio Source Europe. Actually, 50 1 of a standard solution or a sample prepared by the above-described method for preparing a site force-in sample was dispensed into each well of a 96-well microplate, and reacted at room temperature for 2 hours. After removing the reaction solution in each well, washing three times, dispensing 50 1 of peroxidase-labeled anti-human IL-10 monoclonal antibody, and reacting at room temperature for 2 hours, removing the reaction solution in each well, Washed three times. The chromogenic substrate solution was dispensed at 200 1 each, and allowed to stand at room temperature for 25 minutes, and then the enzyme reaction stop solution was dispensed at 501 each. Using 620 nm as a control, the absorbance of each well at 450 nm was measured by Emax (manufactured by Wako Pure Chemical Industries, Ltd.). The amount of IL-10 is the same as pgZml.

Iressa (gefunitib) in patients with lung adenocarcinoma who have been effective after NITC

250 mg / day was orally administered. Thirty-seven target patients underwent multivariate analysis (oral dystech analysis) for the contribution of various immune markers to response. As a result, factors with a contribution of 1.0 or more or 1.0 or less were found in NKT cells (CD3 (+) CD161 (+) cells), especially

NKT8 (+) T cells [CD3 (+) CD161 (+) CD8 (+) cells] and Thl / Th2 ratio, IFNy, IL-12, and IL-10 as a negative factor (Fig. 1) ). In the figures below, NKT (8+) is NKT8 (+ 丌 CD3 (+) CD161 (+) CD8 (+)], NKT (4+) is NKT4 (+) (CD3 (+) CD161 (+) CD4 (+)], And (P +) and P (+).

Therefore, when infecting cancer cells, it is known that each effector cell exerts cytotoxicity by injecting perforin or granzym B protein into target tumor cells. The contribution of perforin-producing cells was examined (Fig. 2). As a result, those with a contribution of 1.0 or more than 1.0 were NKT P (+) [CD3 (+) CD161 (+) perforin (+)], especially NKT8 (+) P (+) [CD3 (+) CD161 ( +) CD8 (+) perforin (+)] cells and CD8 (+) P (+) T [CD8 (+) perforin (+)] cells. The contributions of the Thl / Th2 ratio and IL-12 and IL-10 were also high. Furthermore, when the contribution is viewed as a ratio, ALL / NKT8 (+) (CD3 (+) CD161 (+) CD8 (+) perforin (+) / CD3 (+) CD161 (+) CD8 (+)), CD8 (+) P (+) / CD8 (+ 丌 CD8 (+) perforin (+) / CD8 (+)], Thl / Th2 ratio, and IL-10 were significant (FIG. 3).

[0044] Summarizing the above results, the most notable effector cells of the combination use of MTC and Iressa are CD3 (+) CD161 (+) CD8 (+) perforin-producing cells and CD8 (+) perforin T cells. It is thought that sex is strong. In addition, the factors that were noted as immunological effects at that time were thought to be the Thl / Th2 ratio and IL-10.

Next, whether the immunological factor was important in 23 effective groups and 14 ineffective groups in the combination therapy of NITC and Iressa was examined, and the results are shown in FIGS. 4, 5, and 6. In NKT8 (+) [CD3 (+) CD161 (+) CD8 (+)] cells, NKT8 (+) P (+) [CD3 (+) CD161 (+) CD8 (+) perforin (+)] cells , Significantly increased in effective cases compared to ineffective cases. Conversely, in NKT4 (+) P (+) [CD3 (+) CD161 (+) CD4 (+) perforin (+)] cells, there was no difference between effective and ineffective.

[0046] When examined in NK cells, NKP (+) / NK (CD3 (-) CD161 (+) perforin (+))

The ratio in the / CD3 (-) CD161 (+) cells increased only in the effective group (P <0.05) (FIG. 7).

[0047] In the CD8 (+) cells, the ratio of CD8 (+) P (+) / CD8 (+) [CD8 (+) perforin (+) / CD8 (+)] showed a (Figure 8).

[0048] Therefore, in the case of combined use of NITC and Iressa, the order of NKT8 (+) P (+)> CD8 (+) P (+)> NKP (+) is important, and Effector cells.

[0049] From the above analysis, NKT8 (+) P (+) [CD3 (+) CD161 (+) CD8 (+) perforin (+)] cells,

CD8 (+) P (+) (CD8 (+) perforin (+)) T cell, NK P (+) (CD3 (-) CD161 (+) perforin (+)) cell effector effector in damaging cancer cells Forces that have become cells It has recently been discovered that such cells are commonly expressed in such cells (Non-Patent Documents 3 & 4). It has been confirmed that it is a cell expressing the chemokine of CX3CR1, a perforin-granzyme B-positive cell that can exert strong cytotoxicity. These CX3CR1-expressing cells have been reported to be expressed in cells beyond a subset of NK cells and monocyte T cells (CD4, CD8, yδΤ cells) and show strong cytotoxicity. this time Among the identified effector cells, NKT8 (+) P (+) (CD3 (+) CD161 (+) CD8 (+), which is a cell induced by co-administration of NITC and Iressa and is at least a perforin-producing cell Perforin (+)] cells were the most potent. Thus, the cytotoxicity of CD8 (+) P (+) (CD8 (+) perforin (+)) T cells and NKP (+) (CD3 (-) CD161 (+) perforin (+)) cells in that order. Yes, but this cell is very similar to CX3CR1-expressing cells.

The marine yeast activates this group of cells, and the NITC or the marine yeast group is thought to activate CX3CR1-expressing cells.

A. Contribution to response in lung adenocarcinoma (N = 50)

As shown in FIG. 9, in the case of NITC alone, the effector cells at which the contribution of IFN-γ and IL-12 were high were NKT8 (+) P (+) / NKT8 (+) (CD3 (+) CD161 ( +) CD8 (+) perforin

(+) / CD3 (+) CD161 (+) CD8 (+)] cells, CD8 (+) P (+) / CD8 (+) [CD8 (+) perforin

(+) / CD8 (+)] T cells and NKP (+) / NK [CD3 (-) CD161 (+) perforin (+) / CD3 (-) CD161 (+)] cells in this order.

B. Contribution to response in lung adenocarcinoma (after Iressa) (N = 48)

As shown in FIG. 10, in the combination of NITC and Iressa, IFN-γ and IL-12 are not activated, but there are effector cells controlled by the Thl / Th2 ratio balance, and their cytotoxicity is CD8 (+ ) P (+) / CD8 (+) (CD8 (+) perforin (+) / CD8 (+)) T cells, NKT8 (+) P (+) / NKT8 (+) (CD3 (+) CD161 (+) CD8 (+) perforin (+) / CD3 (+) CD161 (+) CD8 (+)] cells, NK P (+) / NK (CD3 (-) CD161 (+) perforin (+) / CD3 (-) CD161 (+)] The activity is higher in the order of cells.

The CX3CR1-bearing cells induced by NITC of A. above have slightly lower cytotoxic potency than the Y cells (provisional name) similar to the CX3CR1-bearing cells induced by combined use of NITC and Iressa of B. it is conceivable that.

In Y cells, it is independent of IFN-γ and IL-12 but is dependent on the Thl / Th2 ratio

CD8 (+) P (+), NKT8 (+) P (+), and NKP (+) in order of decreasing cytotoxicity titer According to Fig. 11, miliary properties from No.1 to No.6 Strong cells that can exert cytotoxicity in terminal cancers that have metastasized to the lung or in other terminal cancers that have metastasized to other organs (with strong immunosuppression) (Y cells) are considered to be stimulated. These Υ cells are considered to be activated by an IL-21-mediated reaction (The Journal of Immunology, 2003, 171: 608-615). Independent of cytoin and is not affected by CD4 (+) T cells. In addition, CD8 (+) and NK cells, which are hardly damaged by immune cells, show strong cytotoxicity, and are very similar to the force Y cells known to be mediated by IL-21R. .

Y cells may be activated and cultured with IL-21 and returned to the subject, or adjusted for rejection (for example, in the case of the same HLA) so that others can administer the Y cells.

Industrial applicability

As described above, by using the method for examining cell kinetics of the present invention, it is possible to examine the efficacy of cancer treatment in a combination use of a tyrosine kinase inhibitor and a cancer immunotherapeutic agent, and more effectively. It is possible to perform cancer treatment. Further, by using the screening method of the present invention, a cancer immunotherapeutic agent having an excellent effect when used in combination with a tyrosine kinase inhibitor can be obtained, and the cancer immunotherapeutic agent can be used effectively for cancer treatment.

Claims

The scope of the claims

[I] A method for examining cell kinetics using, as a marker, the kinetics of perforin-producing cells by a cancer immunotherapeutic agent in a combination use of a tyrosine kinase inhibitor and a cancer immunotherapeutic agent.

[2] A cancer immunotherapeutic agent that uses the dynamics of perforin-producing cells as a marker to determine the efficacy of a cancer immunotherapeutic agent in combination with a tyrosine kinase inhibitor and a cancer immunotherapeutic agent.

[3] A screening method for a cancer immunotherapeutic agent that uses the dynamics of perforin-producing cells as a marker to determine the efficacy of the cancer immunotherapeutic agent in combination with a tyrosine kinase inhibitor and a cancer immunotherapeutic agent.

[4] A claim that the tyrosine kinase inhibitor has a selective targeting effect on at least one of the following receptors, and the function of the cancer immunotherapeutic agent is induction of IL-12 production and a Z or IL-21-mediated response. The method of paragraph 1 or 3.

HER2 / neu, HER3, HER4, c-kit, PDGFR, bcr-abl, EGFR

[5] A claim is that the tyrosine kinase inhibitor has a selective targeting effect on at least one of the following receptors, and the function of the cancer immunotherapeutic agent is induction of IL-12 production and Z or IL-21-mediated response. Scope 2. A cancer immunotherapy agent according to item 2.

HER2 / neu, HER3, HER4, c-kit, PDGFR, bcr-abl, EGFR

[6] The method according to claim 4, wherein the cancer immunotherapy agent is a substance having a β1,3 / 1,6 glucan structure.

[7] The cancer immunotherapeutic agent according to claim 5, wherein the cancer immunotherapeutic agent is a substance having a β1,3 / 1,6 glucan structure.

[8] The method according to claim 4, wherein the cancer immunotherapeutic agent is a yeast-derived component having a 131,3 / 1,6 glucan structure.

9. The cancer immunotherapeutic agent according to claim 5, wherein the cancer immunotherapeutic agent is a yeast-derived component having a 131,3 / 1,6 glucan structure.

[10] The dynamics of perforin-producing cells were ΝΚΤΡ (+), ΝΚΤ8 (+) Ρ (+), CD8 (+) P (+) T, and

9. The method according to any one of claims 1, 3, 4, 6, and 8, determined by at least one of the Thl / Th2 ratios.

[II] The dynamics of perforin-producing cells are determined by NKTP (+), NKT8 (+) P (+), CD8 (+) P (+) T, The cancer immunotherapeutic agent according to any one of claims 2, 5, 7, and 9, which is determined by at least one of the Thl / Th2 ratio.

[12] Claims 2, 5, and 7 treated without concomitant use with cancer chemotherapeutics and radiation therapy

The cancer immunotherapeutic agent according to any one of claims 9, 9 and 11.

13. The method according to any one of claims 2, 5, 7, 9, 11, and 12, wherein the substance is used in combination with a substance which selectively acts on NKR-P1 of NKT cells to activate NKT cells. Cancer immunotherapy.

[14] The claims 2, 5, 7, 9, 11, 12, and 1 used in combination with a substance having angiogenesis inhibitory ability

The cancer immunotherapeutic agent according to any one of the three items.