WO2004110138A1

WO2004110138A1 - Model mouse

Info

Publication number: WO2004110138A1
Application number: PCT/JP2004/008231
Authority: WO
Inventors: Akikuni Yagita
Original assignee: Orient Cancer Therapy Co.,Ltd.
Priority date: 2003-06-13
Filing date: 2004-06-11
Publication date: 2004-12-23
Also published as: JP2006340602A

Abstract

It is intended to provide means for ascertaining the valid effect of molecular target curative medicine per se or used in combination with immunotherapy under nonclinical conditions closely related to human. In particular, the usefulness of antitumor agent acting on EGFR combined with an immunotherapeutic agent can be nonclinically evaluated by establishing an experimental system wherein an EGFR-positive mouse tumor strain is transplanted into a nonimmunodeficient mouse as means for nonclinically ascertaining the attainment of superior synergistic effect of joint use of tyrosine kinase inhibitor and immunotherapy such as IL-12 production inducer on cancer treatment.

Description

Technical field

[0001] The present invention relates to the provision of a novel model animal system for determining the efficacy of an antitumor agent. In other words, to determine the usefulness of the therapeutic effect of an antitumor agent that targets EGFR (vascular endothelial cell growth factor receptor), which is attracting attention as a novel cancer therapy, for example, a tyrosine kinase inhibitor alone or in combination with other therapies Statement on system provision

You.

Rice field

Background art

[0002] Direct selection of a substance useful for the prevention or treatment of cancer (malignant neoplasms) (cancer) has conventionally focused on its direct action on cancer cells. However, it is thought that there is a tug of war between cancer cells and antitumor immunity in living organisms. The mechanism by which low-dose anticancer drugs, which by themselves have little or no direct effect on cancer cells, have a small but effective effect in the clinic, is that low-dose anticancer drugs produce immunosuppressive T cells. It has long been said that it is indirect by holding down. Although immunostimulants were found to be useful for cancer treatment, none of the compounds obtained as immunostimulants had weak anticancer effects, and were treated with immunotherapy alone or in combination with chemotherapy. However, sufficient therapeutic effects of cancer have not been achieved.

[0003] The present inventor's medical doctor, Yagida, first focused on the usefulness of a substance that induces interleukin 12 (IL-12) in vivo as a revolutionary method in cancer treatment, He discovered that the processed product had that function and established a novel cancer therapy with Novel Immunotherapy for cancer (NITC). Conventionally, IL-12 has an anticancer effect, but there is a fact that if IL-12 itself is directly administered into a living body, patients will not be able to tolerate the treatment due to side effects. I couldn't use it. 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, Yagita achieved the goal of treating cancer by administering an effective amount of processed mushroom mycelium that could induce IL-12 in vivo. (Patent Document 1).

[0004] IL-12 has a TNFa → IFNy → IL_12 → CTL activity and recruitment effect to activate and enhance killer T cells. In other words, IL-12 production enhancement is expected to have an anticancer effect by activating and enhancing killer T cells.

[0005] Yagida reports that activation of NKT cells is useful for anticancer effects separately from a system for enhancing IL-12 production. Taniguchi et al. Discovered a specific glycolipid antigen recognized by a unique T cell antigen receptor (TCR) called Vh 24V / 311 that NKT cells possess, and developed this antigenic potential with galactosinoleceramide. Report that there is. Furthermore, it was proved that NKT cells were activated in cancer-bearing mice to which higalactosinoreceramide was administered, and that metastasis was suppressed although cancer disappearance was not observed.

It has been reported that NKT cells include NK 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 activation of NKT cells and that the anticancer effect by this activation is more superior (Patent Document 2).

[0006] 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, tyrosine kinase inhibitors have attracted attention as agents having a signal transduction inhibitory effect. Gefitinib (ZD1839) (Iletsa: AstraZene®) has a competitive effect with ATP at the ATP-binding site of EGFR (Epidermal Growth Factor Receptor) tyrosine kinase, and suppresses tyrosine kinase autophosphorylation, thereby inhibiting tyrosine kinase. Inhibits kinase activity. As a result, signal transduction related to proliferation, invasion, differentiation, and metastasis of EGFR [binding of ligand such as epidermal growth factor (EGF) to extracellular domain of EGFR causes EGFR tyrosine kinase in intracellular domain to become active. Activates and triggers autophosphorylation of EGFR and phosphorylation of various intracellular target proteins to transmit growth signals from the cancer cell surface to the nucleus, causing cancer cell growth, invasion, metastasis, and angiogenesis.) It exerts an anti-cancer effect by blocking it. IMC-C225 (EGFR-targeted monoclonal antibody) recognizes a part of the EGFR receptor on the cell membrane surface and inhibits tyrosine kinase activity by suppressing autophosphorylation of EGFR. Herceptin is a monoclonal for Her2 / Neu with homology to EGFR STI-571 (Daribec ^) is an antibody and has the ability to inhibit the tyrosine kinase activity of BCR-Abl and the tyrosine kinase activity of c-kit (Non-patent Document 2).

[0007] Such a molecular target therapeutic agent is attracting attention as a cancer therapeutic drug of a new mechanism. Its effect is still not revolutionary. For example, ZD1839 (Iressa ^B¾ ) is a powerful and selective inhibitor of the tyrosine kinase of EGFR newly developed by AstraZeneca, and its usefulness has been found in humans. However, clinical results for non-small cell lung cancer and prostate cancer have PR (partial remission) of 10 to 20%, and CR (complete remission) is very rare if at all. It took more than four months. Therefore, ZA183 9 (although combination therapy with Iressa and various anticancer agents has been attempted, at this time, an additive or synergistic effect has been obtained.

[0008] Doctor of Medicine Yagida found the usefulness of a combination of an antitumor agent and an immunotherapeutic agent acting on EGFR, and has actively tried this combination in clinical practice. In spite of its remarkable effects, clinical trials have shown tremendous effects, but there was no means to evaluate its usefulness at a non-clinical level. As an animal test system for EGFR-positive tumors, an animal test system in which a human EGFR-positive tumor cell transplant or tumor cell suspension is transplanted and grown subcutaneously in nude mice has been known. However, nude mice are immunodeficient (T-cell deficient) mice, a model far from real human patients.

[0009] In the prior art, there are scattered literatures that examine EGFR expression using a technique such as RT-PCR extracted from solid tumors, but endothelial cells of blood vessels that invade solid tumors are naturally EGFR positive. Therefore, it is possible that EGFR signals from endothelial cells, but not tumor cells, are spread. For example, in Non-Patent Document 3, EGFR was not detected by immunohistochemistry at the local tumor where human lung cancer strain LX-1 was transplanted and grown in nude mice, and EGFR was detected by 28 cycles of RT-PCR. Slightly detected, and 32 cycles of RT-PCR detect EGFR. There is also a report in the prior art that cancer cell lines are grown in vitro (in vitro) and EGFR expression is examined by RT-PCR or other techniques, and EGFR expression in tumors after transplantation into animals is not examined. Force was expressed in vitro. Force, rattle, and in vivo after transplantation into a living body, are not always expressed at the protein level. Patent Document 1: JP-A-10-139670

Patent Document 2: US2002-0010149A1

Non-patent document 1: Special issue: Fundamentals and clinics of NKT cells: New Medicine 55 Vol. 4 2000 818—823 ぺ¹

Non-patent document 2: Blood 'immune' tumor Vol. 7, No. 3, 2002-7

Non-Patent Document 3: Clinical cancer research 6; 4885-4892 (2000)

Disclosure of the invention

Problems the invention is trying to solve

[0010] An object of the present invention is to provide a means for confirming a non-clinical IJ effect by a molecular target therapeutic agent alone or in combination with immunotherapy non-clinically under conditions closer to humans as described above. Means to solve the problem

[0011] The present invention provides a means for non-clinically confirming that a combination of a tyrosine kinase inhibitor and an immunotherapy such as an IL-12 production inducer achieves a superior synergistic effect in cancer treatment. Established an experimental system in which a mouse tumor strain was transplanted into a nonimmune-deficient mouse, and found that the usefulness of a combination of an antitumor agent acting on EGFR and an immunotherapeutic agent could be non-clinically evaluated. Thus, the present invention has been completed.

That is, the present invention includes the following.

1. A model mouse for confirming the antitumor effect, which is prepared by transplanting a mouse tumor strain positive for EGFR (vascular endothelial growth factor receptor) into a mouse without immunodeficiency.

2. A method for determining the effect of an anti-tumor agent targeting EGFR using a model mouse for confirming the anti-tumor effect prepared by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency.

3. A method for determining the combined effect of an anti-tumor agent targeting EGFR and another therapy using a model mouse for confirming the anti-tumor effect prepared by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency.

4. Using an anti-tumor effect model mouse prepared by transplanting an EGFR-positive mouse tumor strain into a non-immunodeficient mouse. Anti-tumor agent targeting EGFR and immunity A method for determining the effect of combination with a therapeutic agent.

5. A method for determining the effect of an immunotherapeutic agent using an anti-tumor effect confirmation model mouse prepared by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency.

6. Antitumor agent ability targeting EGFR The determination method according to item 24 above, which is a tyrosine kinase inhibitor.

7. The method according to 6 above, wherein the tyrosine kinase inhibitor has a selective target action on at least one of the following receptors;

HER2 / neu, HER3, HER4, c_kit, PDGFR, bcr-abl, EGFR.

8. The method according to the above item 6, wherein the tyrosine kinase inhibitor selectively has an EGFR or c-kit target action.

9. The method according to 4 or 5 above, wherein the immunotherapeutic agent is an IL-12 production inducer.

10. The determination method according to the above item 9, wherein the IL-12 production inducer is a substance having a 3 / 1,6 glucan structure.

11. The method according to the above item 10, wherein the IL-12 production inducer is a mushroom mycelium-derived component or a yeast-derived component having a βΐ, 3/1, 6 glucan structure.

12. A method for determining the EGFR positivity of a mouse tumor strain through the following steps;

1) transplanting the mouse tumor strain into a non-immunodeficient mouse,

2) A step of collecting local tumor and detecting the presence or absence of EGFR positivity by immunohistochemistry.

13.Immune deficiency transplanted with a mouse tumor strain that has been confirmed to be EGFR-positive by the method described in 12 above, using a model mouse system, growth of the transplanted tumor by administration of an EGFR-related inhibitor A screening method for an EGFR-related inhibitor, which determines the effect of an EGFR-related inhibitor alone by observing and determining suppression of EGFR-related inhibitors.

14. Use of a non-immunodeficient model mouse transplanted with a mouse tumor strain that has been confirmed to be EGFR-positive by the method described in 12 above, and suppress tumor growth by co-administration of an EGFR-related inhibitor and other therapeutic agents A screening method for the combination use of an EGFR-related inhibitor and another therapeutic agent to observe the effect of EGFR-related inhibitor and another therapeutic agent.

15. The screening method according to the above 13 or 14, wherein the EGFR-related inhibitor is a tyrosine kinase inhibitor. 16. The screening method according to the above item 14 or 15, wherein the other therapeutic agent is an immunotherapy agent.

17. The screening method described in 16 above, wherein the influence on various immune markers is also used as a judgment factor.

The invention's effect

[0013] The present invention has confirmed the usefulness of a combination of a drug targeting EGFR and other therapies, specifically, a tyrosine kinase inhibitor and an IL-12 production inducer (enhancement of Thl site force-in production). It has established an animal model system for judging and enabling epoch-making results in cancer treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a staining diagram of a 3LL tumor transplantation blocking test.

[Figure 2] 3LL tumor transplant EGF-R staining.

[Fig. 3] HE staining of 3LL tumor transplantation.

[FIG. 4] Staining diagram of colon26 tumor transplantation blocking test.

[Fig. 5] EGF-R staining of colon26 tumor transplantation.

[Figure 6] HE staining of colon26 tumor transplantation.

FIG. 7 shows the results of the survival rate when an antitumor test using a combination of a BRM formulation and Iressa (trade name) (gefitinib) was examined using a model animal of the present invention.

FIG. 8 shows the change in tumor size (mm 3) in a model animal when an antitumor test using a BRM formulation and Iressa (trade name) (gefitinib) was examined using the model animal of the present invention. In the figure, a) showed a significant difference at p = 0.05. b) was significantly different at p = 0.02

[Figure 9] A graph of the change in tumor size (mm 3) in a model animal when an antitumor test using a BRM formulation and Iressa (trade name) (gefitinib) in combination was studied using the model animal of the present invention FIG.

FIG. 10 shows the results of the survival rate when an antitumor test using a BRM formulation and Iressa (trade name) (gefitinib) was examined using the model animal of the present invention.

[Figure 11] Changes in IL-12 production in a model animal when an IL-12 production test using a BRM formulation and Iressa (trade name) (gefitinib) was studied using the model animal of the present invention (pg / ml). In the figure b), there was a significant difference at ρ = 02.

FIG. 12 shows a change in tumor size (mm 3) in a model animal when an antitumor test using a BRM formulation and Iressa (trade name) (gefitinib) was examined using the model animal of the present invention. In the figure, a) showed a significant difference at p = 0.05.

[Figure 13] Changes in IL-12 production in the model animal when the IL-12 production test using the BRM formulation and Iressa (trade name) (gefitinib) was studied using the model animal of the present invention (pg / ml) FIG.

[Figure 14] A graph of the change in tumor size (mm 3) in a model animal when an antitumor test using a BRM formulation and Iressa (trade name) (gefitinib) was examined using the model animal of the present invention. FIG.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] 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.

As the immunotherapeutic agent of the present invention, widely known immunotherapeutic agents can be applied. In particular, the immunotherapeutic agent used in the present inventor's medical doctor, Dr. Yagita's cancer new immunotherapy (NITC) is used. Is mentioned. Here, NITC is a powerful treatment that combines four different mechanisms of action. Hereinafter, this NITC will be described as a typical application method of the immunotherapy agent according to the present invention.

[0017] The first mechanism of action is a method of reducing cancer by administering an antiangiogenic substance (bettershark) to impair blood flow to the cancer. In this method, the effect can be determined by measuring vascular endothelial cell growth factor (VEGF). The angiogenesis inhibitory effect can be evaluated by a minus (negative) value of VEGF (one VEGF). Instead of this VEGF value, other vascular growth factors such as FGF and HGF can be used to evaluate the ability to inhibit angiogenesis. In addition, instead of VEGF, the positive value of an angiogenesis inhibitor can be evaluated (eg, an endostatin value).

[0018] The second mechanism of action is to activate a CTL by administering a compound having a βΐ, 3 glucan structure to induce Thl site force-in (TNFα, IFNγ, IL-12) It is. CTL activity Sex can be determined by the ability to produce CD8 (+) perforin. However, CD8 (+) perforin levels include cytotoxic T cells (CTLs) and immunosuppressive T cells (STCs), and the former impairs cancer cells and activates the latter. Conversion eventually results in the proliferation of gan. Therefore, its absolute value cannot be used to evaluate CTL activity. However, in the clinical setting, the former is a CTL if the IFN o / value is lOIUZml or more, or the IL-12 value is 7.8 pgZml or more, and is a STC if the IFN o / and IL-12 are low. Therefore, CTL activity can be evaluated based on the ability to produce IFN y (IFN y value) or the ability to produce IL-12 (IL-12 value). By the way, these figures are based on Yagida's measurement system, and it is a well-known fact that even small differences in the measurement system can cause different measurement values.

[0019] The effector cells activated by the administration of a compound having a 1,3-glucan structure, which is the third and fourth mechanism of action, are NK cells and NKT cells. These NK and NKT cells share NKR-P1 (NK cell receptor CD161 (+) in humans, NK cell surface receptor stained with NKR-P1 antibodies such as ΝΚ1.1 in mice). In clinical practice, the former NK cells can be measured for their cell number or cell ratio using the surface marker of CD3 (-) CD161 (+), and their activation can be determined by their ability to produce CD3 (-) CD161 (+) perforin. It is possible. On the other hand, the latter NKT cells can be measured with CD3 (+) CD161 (+), and the cell number or cell ratio can be measured, and NKT cell activation can be measured by its perforin-producing ability (abbreviated as NKTP).

[0020] Therefore, regardless of whether it is a new immunotherapy (NITC) for cancer treatment or a therapy using a general immunotherapeutic agent, each effector cell or angiogenesis inhibitory effect is evaluated by the following measurement items. It is possible. Specifically, CTL activity can be evaluated based on its ability to induce the production of IL-112. NK cell activation can also be assessed by CD3 (-) CD161 (+) or CD3 (-) CD161 (+) perforin levels. Activation of NKT cells can also be assessed by CD3 (+) CD161 (+) or CD3 (+) CD161 (+) perforin levels (NKTP levels).

[0021] The present invention provides a means for non-clinically evaluating the effect of using a tyrosine kinase inhibitor in combination with the above immunotherapeutic agent. And immunological measurements and anti- It has established a system for reliably evaluating cancer effects in biological systems.

[0022] The IL-12 production inducer, which is one of the immunotherapeutic agents, used in the present invention is not particularly limited and can be widely used. For example, a mushroom mycelium composition preparation having a ^β1,3 glucan structure (eg, ILX ^ffin¾ : Tozai Pharmaceutical Research Institute, ILY ^ffiB¾ *: a ^seishin company, AHCC: amino up), or various yeasts having a 1,3 glucan structure ( Marine yeast, baker's yeast, NBG ™) can be used. In addition, by combining the measurement of CD8 perforin-producing ability with a novel IL-12 production inducer, those skilled in the art can easily identify the IL-12 production inducer (CTL activator). The CTL activator has the same meaning as the IL-12 production inducer used in the present invention.

[0023] The system of the present invention is extremely useful for judging the usefulness of the combined use of the IL-12 production inducer and the EGFR-related inhibitor. A typical example of an EGFR-related inhibitor is tyrosine kinase. Examples of a tyrosine kinase inhibitor, or ZD1839 (Iressa ^ ^[pi) is with STI571 (Daribekku ^¾), various tyrosine kinase inhibitors can be effectively utilized. They include HER2 / neu, HER3, HER4, c_kit, PDGFR, bcr-abl, EGFR and the like as target molecules. The most effective molecules are EGFR or c_kit. The clinical dose of the tyrosine kinase inhibitor should be in accordance with the recommended dose of each molecular target compound. For ZD1839, the oral dose is 10-500 mg / day.

[0024] The combination of the IL-12 production inducer and the tyrosine kinase inhibitor is not particularly limited, but it does not matter which one is prior to the initial treatment. In a specific example, a dramatic clinical effect was confirmed when a tyrosine kinase inhibitor was used in combination after a certain period of administration of an NITC therapy, particularly an IL-12 production inducer.

[0025] In the system of the present invention, it is possible to evaluate the effect of the combined use of an NK activator or an NKT activator as an immunotherapeutic agent in addition to an IL-12 production inducer. A composition preparation of a compound having an a1,3 glucan structure, such as a nig-mouth oligosaccharide or fucoidan, is useful as an NK activator or NKT activator. Various compounds having a 1,3 gnorecan structure are known, and this known structure and the ability to produce CD3 (_) CD161 (+), CD3 (_) CD161 (+) perforin, CD3 (+) CD161 (+ ) And CD3 (+) CD161 (+) perforin-producing ability, a person skilled in the art can easily specify the NK activator. In addition, it means that CD3 (+) CD161 (+) acts on NK T cell receptor NKR-P1. [0026] Examples of the saccharide substance having an a1,3 glucan structure include nigerooligosaccharide (TSO), fucoidan, and oligosaccharide sulfate.

Nigerooligosaccharides are saccharides containing 3_0_a_D_dalcoviranosyl-D-glucose as a constituent unit. Typical examples include nigerose, nigerosyl glucose, and nigerosyl maltose.

[0027] Examples of commercially available nig-mouth oligosaccharides include nig-mouth oligosaccharide liquid sugar (seller: Takeda Shokuhin Kogyo Co., Ltd.), and the main nig-mouth oligosaccharides contained therein are (1) ) Nigguchi H-D—Glc p— (1,3) —D—Glc, (2) Nigerosylglucose H—D—Glc p— (1,3) -a-D-Glc p— (1 , 4) —D—Glc, (3) Nigerosyl maltose, D—Glc p— (1, 3) -a-D-Glc p_ (l, 4) _hi _D_Glc p_ (l, 4) _D_Glc (note that , Glc is glucose, and p is an abbreviation for pyranose).

[0028] In a narrow sense, fucoidan is a sulfated fucose-containing polysaccharide in which one to two sulfuric acids are bonded to two to six molecules of fucose, and a fucoidan-like polysaccharide containing xylose or peronic acid is added at the food level. It is called "Fucoidan". Fucoidan, for example, is obtained by crushing kelp, forming chips, extracting an aqueous solution component, removing the extraction residue by centrifugation, removing low molecular substances such as eodo and sodium chloride by ultrafiltration, and freeze-drying. It is made into a formulation.

Examples of the fucoidan include fucoidan derived from brown algae, for example, fucoidan derived from gagome kelp, fucoidan derived from Okinawa mozuku, and the like. Fucoidans derived from brown algae Laminariaceae such as gagome kelp include at least three types of fucoidan, F-fucoidan (aL-fucosic polymer), and U-fucoidan (_D-glucuronic acid and a_D-mannose, G-fucoidan (having / 3 _D_galactose as the main chain and side chain having L-fucose) exists, and both fucoidans have fucoidan. Sulfated.

[0030] Examples of the oligosulfate sulfate include an extract derived from Susabinori (Poryphyra Yezaens is) manufactured by Shiroko Co., Ltd. The main components of the extract are a 1,3-linked galactan sulfate oligosaccharide and a galactan sulfate oligosaccharide composed of 1,3 and j3 1,4 bonds.

[0031] The system of the present invention comprises a tyrosine kinase inhibitor and a CTL activator (IL-12 production inducer, IF (誘導 γ-production inducer), and also useful in evaluating the usefulness of combined use with ΝΚactivators, ΝΚΤactivators and neovascular inhibitors. By using the system of the present invention and selecting the applicable method, lung cancer (lung squamous cell carcinoma, lung adenocarcinoma, small cell lung carcinoma), thymoma, thyroid cancer, prostate cancer, kidney cancer, bladder cancer, colon cancer Cancer, rectal cancer, esophageal cancer, cecal cancer, ureteral cancer, breast cancer, cervical cancer, brain cancer, tongue cancer, pharyngeal cancer, nasal cavity cancer, larynx cancer, gastric cancer, liver cancer, bile duct cancer, testicular cancer, ovarian cancer , Endometrial cancer, metastatic bone cancer, melanoma, osteosarcoma, malignant lymphoma, plasmacytoma, liposarcoma, etc.

[0032] The system according to the present invention comprises a combination of a tyrosine kinase inhibitor and an immunotherapeutic agent, particularly a CTL activator (IL-12 production inducer, IFNo / production inducer), furthermore, a NK activator, NKT activity The combined use with a chemical or a neovascular inhibitor is useful for inducing or enhancing its activation and also for judging the usefulness of a therapeutic method used in a formulation that can maintain the activation. That is, the system of the present invention is used to select or use a dose capable of inducing or enhancing its activation and maintaining the activation, and a period of administration. Specifically, the clinical dose of the compound having a glucan structure of NK activator or NKT activator is about lg-40 g / day, preferably about 5-20 g / day. The compound having a β-1,3 gnorecan structure, which is (IL-12 production inducer, IFNy production inducer), is about lg-10 g / day, preferably about 3-6 g / day. In addition, the clinical administration period is generally 10 days to 24 months, and the administration frequency is every other day or 113 times / day, preferably daily or every other day. The CTL activator (IL-12 production inducer, IFNy production inducer), NK activator, and NKT activator are preferably taken orally. Of course, parenteral ingestion (including intravenous or intramuscular administration) is also possible by reducing the dosage and preparing them to a parenteral quality. The system of the present invention is also effective for evaluating the effective dose and administration method as described above.

[0033] (Model animal)

The model mouse for confirming the antitumor effect prepared by transplanting the EGFR (vascular endothelial cell growth factor receptor) -positive mouse tumor strain of the present invention into a mouse without immunodeficiency is, for example, a colon 26 tumor strain derived from mouse colon cancer. A 3LL (also known as Lewis Lung Cancer) tumor line derived from mouse lung cancer was transplanted into a syngeneic BALBZc mouse. A specific example is a system that is transplanted into a C57BL / 10 (also called BIO) mouse that matches.

Although the presence or absence of EGFR positivity may be confirmed in advance by immunohistochemical staining of tumor cells in vitro, more preferably, a mouse tumor line is transplanted into a non-immunodeficient mouse, and a local tumor is then collected. More preferably, EGFR is detected by a chemical method to confirm that the transplanted tumor is EGFR-positive. For example, a system transplanted with the same mouse tumor strain as a control may be used as a control, and the same system in which EGFR positivity has been confirmed may be identified as a model animal.

The following are examples of preparation techniques for such a system.

In one embodiment of the present invention, it was confirmed that EGFR expression was found at the protein level in the tumor cells by immunohistochemistry at a solid tumor site after transplanting the mouse tumor into a non-immunodeficient mouse and growing it. And model animals were identified. In the preparation procedure, colon 26 tumors were transplanted into BALB / c mice, and 21 days later, local tumors were collected. 3LL tumors were implanted into C57BL / 10 mice, and 21 days later, tumor localities were collected. The collected tissues were fixed with neutral buffered formalin solution, and then cut into paraffin-embedded sections by a standard method. The sections were deparaffinized, treated with 0.5 mg / ml protease type XXIV (SIGMA) at room temperature for 10 minutes, and washed with deionized water. Treated with 3% aqueous hydrogen peroxide for 5 minutes to block endogenous peroxidase and washed with deionized water. To block nonspecific staining, the cells were treated with 10% normal pig serum (Kohjin Bio # 12180910) in PBS for 10 minutes at room temperature. EGFR antibody (Santa Cruz Biotechnology, Code # SC-03G) was diluted 50-fold and used as a primary antibody, and incubated at 4 ° C. After washing with PBS, Biotinylated Rabbit Antibody (Goat immunoglobulin obulins, ode # E0466 from DAKO) was diluted 500-fold and incubated at room temperature for 30 minutes. After washing with PBS, peroxidase-conjugated streptavidin (Peroxidase-Conjugated Strepta vidin, Code # P0397 from DAK〇) was diluted 500-fold and incubated at room temperature for 30 minutes. After washing with PBS, the plate was incubated with a DAB solution (100 ml PBS, 20 mg 3, 3'-diaminobenzidine tetrahydrochloride, and 20 µl 30% hydrogen peroxide) at room temperature for 5 minutes. After washing with water, the cells were stained with hematoxylin for 1 minute. After washing with water, it was dehydrated and mounted on a slide. [0035] As a result, as shown in Fig. 16, EGFR was clearly stained brown at the site confirmed to be vascular endothelium and tumor cells by conventional HE staining (EGF-R immunostaining: EGF R. Goat LSAB was used for the detection reaction, and the appropriate primary antibody concentration was 1:50). In addition, a blocking test was attempted to rule out non-specific staining. The blocking test was performed by adding a blocking reagent (Santa Cruz Biotechnology Code # SC-03P) to the diluted EGFR antibody so that the amount of protein became 5 times, and incubating at 4 ° C for 1 hour. The antibody was treated in the same manner as described above. As a result of the blocking test, it was confirmed that the brown color seen by EGFR staining was not seen as shown in the photograph, so non-specific staining was denied, and EGFR-positive images were seen in vascular endothelial cells and tumor cells. confirmed. The photographs were taken at almost the same site for the HE, EGF-R and blocking tests with the objective lenses X10 and X20. Figure 13 shows the blocking test, EGF-R test, and HE in the case of 3LL tumor cell transplantation. Fig. 46 shows each blocking test, EGF-R test, and HE in the case of colon26 tumor cell transplantation.

(Determination of usefulness of drug using model animal)

In the present invention, colon cancer-derived colon 26 tumor cells (EGFR-positive) are transplanted subcutaneously into the back of 2 million BALB / c mouse mice (immune-deficient), for example, to prepare a model animal. . To this model animal, for example, the next day, a potential drug, for example, an antitumor agent targeting EGFR, for example, gefitinib, for example, is suspended in an aqueous solution such as a 0.5% Tween 20 aqueous solution (excipient) and orally administered daily, Examining the change in increase in tumor volume can confirm the efficacy of this candidate drug. The tumor volume was calculated by, for example, tumor major axis X (tumor minor axis) ² ÷ 2. In the specific experimental example, the tumor volume (mean + SD, mm ³ ) 15 days after transplantation was 1922 + 575 in the vehicle (0.5% Tween 20) administration group, 1879 + 401, lOOmgZkg in the 50 mg Zkg administration group. The dose was 2050 + 743 in the administration group and 1099 + 438 in the 200 mg / kg administration group, and a significant suppression of tumor growth was observed for the first time at 200 mg / kg administration. However, the body weight (mean + SD, g) from day 1 to day 15 after transplantation was 24.3 + 1.4 → 23.8 + 1.5 in the vehicle administration group and 23.7 in the 50 mg Zkg group in the vehicle administration group. + 0.9 → 23.0 + 1.3, 100 mg / kg group 23.9 + 1.6 → 23.3 + 1.1, 200 mg / kg group 26.3 + 1.0 → 20 9 + 0.7, and the weight loss of the gefitinib 200 mg / kg group was particularly marked at 200 mg / kg. Discontinued gefitinib administration in 15 days because eyelid edema developed in all individuals. Even in experimental systems in which human tumors of the prior art are transplanted into nude mice, there are many reports that the dose of gefitieb, at which tumor growth is significantly suppressed, is set at 100 mg / kg to 200 mg / kg. This model suggests that this model can be used as a model to see the direct effects of anticancer agents targeting GFR-positive cancer cells on candidate compounds, especially EGFR.

Conventional models that cannot be evaluated without using 200 mg / kg or 100 mg / kg gefitinib are models that are performed at the side effect onset at which weight loss and eyelid edema occur. The model was an experimental system with a dose that was too far from human patients. If the clinical dose of gefitinib is high and 500 mgZ days, it will be about 1 OmgZkg in terms of body weight. What is needed is an experimental system that mirrors real human patients, an "animal experimental system that is not immunodeficient and can be evaluated at lower doses of gefitinib." Gefitinib (trade name: Iressa, development code: ZD1839), approved by the Ministry of Health, Labor and Welfare in the name of the director of the National Institute of Health Sciences for Pharmaceutical Sciences It has been reported that oral administration of / kg / day causes a decrease in food consumption, suppression of weight gain, and corneal epithelial atrophy. That is, in rats, oral administration of gefitinib at 40 mg / kg / day suppresses weight gain but does not cause weight loss. Also, due to the difference in the ratio of body surface area to body weight between mice and humans, it is common in pharmaceutical research that mice are ineffective unless given about 5 times more drug than humans in terms of body weight. Therefore, when an anti-tumor agent targeting EGFR, such as Gefutieb, is evaluated in mice, it is preferable that the evaluation can be performed per 50 mg / kg / day oral administration. The model mouse system of the present invention enables evaluation at such a low dose.

Here, the model mouse provided in the present invention is obtained by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency. As a result, for example, the Colon26 tumor-B ALBZc mouse system and the 3LL tumor-C57BLZ10 mouse system are mice that are not immunodeficient and that can be analyzed immunologically. Therefore, unlike the conventional technology, which analyzes only tumor size as the main index, the immune functions important in anti-tumor immunity, such as the dynamics of T cells, NK cells, NKT cells, and the ability to produce IL-112, are simultaneously analyzed as indicators. That It is possible. For example, in this model animal, for example, the next day, a potential drug, for example, an antitumor agent targeting EGFR, for example, gefitinib, and another therapeutic agent, particularly an immunotherapy agent, for example, krestin (trade name: Sankyo), An IL-12 inducer such as ILX (Tozai Pharmaceutical) or ILY (Tozai Pharmaceutical) is suspended in an aqueous solution such as a 0.5% Tween20 aqueous solution (vehicle) and orally administered daily, and orally administered every day. Examining the changes can confirm the efficacy of this candidate drug. Confirmation of such effects can be achieved by screening for new anti-tumor agents targeting EGFR or novel immunotherapeutic agents, or by using new or known combinations of anti-tumor agents targeting EGFR and immunotherapeutic agents. Applicable to sex script.

[0039] Examples of the method for measuring an immune marker include the following, which describe a clinical test method as a representative example. It can be modified and applied as appropriate for the animal model of the present invention.

[0040] The method of 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 a cell surface antigen (CD3 and / or CD161) specifically present on the cell surface of NKT cells. Specifically, the lymphocytes in the peripheral blood of the clinical sample are tested for CD3 positive and CD161 positive (CD3 (+) CD161 (+)) cells. That is, CD3 and CD161 which are cell surface antigens of NKT cells are measured by a test using a flow cytometry using a monoclonal antibody. Here, that the NKT cells are activated means that the proportion of CD3 (+) CD161 (+) NKT cells in the lymphocytes is 10% or more, more preferably 16% or more. The NKT cell activating ability means a function of increasing the proportion of NKT cells to 10% or more, more preferably 16% or more, or a function of further increasing the proportion of NKT cells before administration of a certain substance.

Similarly, (CD3 (_) CD161 (+)) is an assay for cells that are negative for CD3 and positive for CD161. This method is useful for measuring NK cells. By the way, these figures are based on Yagida's measurement system, and it is a well-known fact that even small differences in measurement systems can cause different measurement values. Furthermore, CD8 (+) refers to the assay of CD8-positive cells. This method is useful for measuring CTL activity.

(Measurement of perforin-producing cells)

For lymphocytes in the peripheral blood of clinical specimens, two of cell surface antigens, CD3, CD161 and CD8, and perforin are measured by flow cytometry using a routine method. Specifically, a fixative is added to the collected blood to fix the cells, a membrane permeate is added, and then an anti-perforin antibody (Pharmingen) is added to react the cells. Further, a PRE-Cy5-labeled secondary antibody (manufactured by DAKO) is added for reaction, and an anti-CD3-PE (Coulter 6604627) antibody and an anti-CD161_FITC (B_D) antibody are added for reaction. Measure by flow cytometry.

(Preparation of Sample for Measuring Site Force In)

First, a mononuclear cell fraction is separated and prepared from clinical sample blood. Heparinized peripheral blood is diluted 2-fold with Phosphate Buffered Saline (PBS) and mixed, and then layered on Ficoll-Conray solution (specific gravity: 1077). After centrifugation at 400G for 20 minutes, collect the mononuclear cell fraction. After washing, RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) is added to adjust the cell number to 1 × 10 ⁶ . Phytohemagglutinin (manufactured by DIFCO) was added to 200 μl of the obtained cell suspension at a concentration of 20 μg / ml, and the solution was incubated at 37 ° C in a 96-well microplate in the presence of 5% CO. 2 in C

Two

After culturing for 4 hours, it is used as a sample for measuring cytoforce in the cultured cell solution.

(Measurement of IL-12)

Although the clinical and biochemical tests known per se can be used for the measurement of the amount of IL-12, a measurement kit by enzyme immunoassay (ELISA) available from R & D SYS TEMS or MBL is used. For clinical samples, a measurement kit from R & D SYSTEMS was used. In practice, 50 µl of the assay diluent RD1F was used in each well of a 96-well microplate, and 200 µl of the standard solution (standard) or the sample obtained from the sample preparation for measuring the cytodynamic force was used. After dispensing μ μ, the mixture was allowed to stand at room temperature and reacted for 2 hours. Thereafter, horseradish peroxidase (HRP) -labeled anti-IL-l2 antibody was dispensed in 200 µl portions, and allowed to stand at room temperature for 2 hours. After removing the reaction solution from each well and washing three times, The chromogenic substrate solution was dispensed at 200 / il each, and allowed to stand at room temperature for 20 minutes, and then the enzyme reaction stop solution was dispensed at 50 μl each. Using 550 nm as a control, the absorbance of each well at 450 nm was measured by Emax (manufactured by Kako Pure Chemical Industries, Ltd.). IL 12 levels are expressed as pg / ml. Here, the ability to induce IL 12 production refers to the function of enhancing the amount of IL-12 produced by stimulation of the peripheral blood mononuclear cell fraction of a clinical sample to 7.8 pg / ml or more, or the ability to increase the level before administering a certain substance. It means a function that enhances the production of IL-12. By the way, the figures are based on Yagida's measurement system, and it is a well-known fact that even small differences in the measurement system will result in different measured values.

In the mouse model animals used in the experimental system of the present invention, IL-12 in blood (in serum or plasma) can be directly measured using an ELISA kit.

(Measurement of IFN y)

The IFN y was measured by enzyme immunoassay (EIA method) using IFN y EASIA kit of Bio Source Europe S. for clinical samples. Actually, in each well of a 96-well microplate, dispense 50 μl of a standard solution (standard) or a 2-fold dilution of the sample prepared above, and dispense 50 μl of HRP-labeled anti-IFN γ antibody. Then, the mixture was reacted at room temperature for 2 hours with shaking. After removing the reaction solution in each well and washing three times, the chromogenic substrate solution was dispensed in 200 / l portions, reacted at room temperature for 15 minutes while shaking, and dispensed 50 μl of the enzyme reaction termination solution. Using 630 nm as a control, the absorbance of each well at 450 nm and 490 nm was measured by Emax (manufactured by Wako Pure Chemical Industries, Ltd.). IFNγ content is expressed as IU / ml.

(Measurement of angiogenesis inhibitory ability)

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

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

Example

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

Example 1

Two million BLL mice (confirmed non-immunodeficient mice) were transplanted subcutaneously with 2 million BLL mice (confirmed to be non-immunodeficient) of 3LL tumor cells derived from mouse lung cancer (confirmed to be an EGFR-positive tumor strain in the control). the 0. 5% Tween20 aqueous solution case of daily suspended in (vehicle) orally, tumor volume 20 days after transplantation (mean + SD, mm ^3), the excipient (0. 5% Tween20) ¾ 1947 + 1648, 1874 + 947 in the gefitinib 50 mgZkg alone group The dose of gefitinib 50 mg / kg and ILX (IL_12 production inducer) lOOOOmgZkg in the oral administration group shows a tendency to suppress tumor volume to 1437 + 870 Was. Spleens of the mice 21 days after tumor implantation were collected, and T cells, NK cells, and NKT cells were analyzed using a flow cytometer FACS Calibur manufactured by Betaton Dickinson. The analysis of Example 1 is absolutely impossible in a conventional experimental system in which a human tumor is transplanted into a nude mouse having no T cells.

[0048] The spleen was ground with a homogenizer vessel in PBS containing 2% FCS and 0.05% sodium azide (all subsequent operations were performed by suspending splenocytes in this solution), and then passed through a cell strainer. Free cell. After washing by centrifugation, hemolysis was carried out by a conventional Shii-Dani ammonium method to remove red blood cells. After centrifugation, add FITC-conjugated CD3e antibody (clone 145 — 2C11) (Betaton Dickinson Cat. # 553062) and APC-conjugated NK1.1 antibody (clon e PK136) (Betaton Dickinson Cat. # 550627). The mixture was incubated on ice for 30 minutes, washed by centrifugation, and subjected to flow cytometer measurement. Propidium iodide (PI) staining was also performed to remove PI-positive cells as dead cells from the analysis. CD3e-positive cells are T cells, NK1.1-positive cells are NK cells, and both CD3e and NK1.1-positive cells are NKT cells.

[0049] les transplanted tumor, a Eaux circle mouse spleen Niore Te,, T, NK, NKT cells, respectively of the i-rate ^{_{(mean + SD, 0/0}} ) f or, 27.93 + 2.04 , 3.02 + 0.21, and 1.45 + 0.37. On the other hand, in the spleen of the vehicle-administered group 21 days after transplantation of 3LL tumor, T, Ν, Ν ΚΤItoda Tsuzuki]; It was 78 + 0.17. It can be seen that the ratio of T, NK, and NKT cells decreases at the end of cancer-bearing stage. [0050] In the spleen of the gefitinib 50 mg / kg single administration group, the T, ΝΚ, and ΝΚΤ cell ratio was 15.89 + 4.96, 2.08 + 0.41, 0.41 + 0.08 Met. As a result, the ratio was slightly higher than that of the vehicle-administered group, and it was found that the 、, 、, ΝΚΤ cell ratio did not decrease when gefitinib 50 mg / kg was administered.

[0051] Unfortunately, the data point 21 days after tumor implantation was too late to determine the effect of the combined use of the IL-12 production inducers ILX and ILY on the spleen Τ, ΝΚ, and 比率 cell ratios. However, this is an experiment supporting the finding that Gefitinib is a type of anticancer drug that does not reduce anticancer immunity, and that Yagida has already obtained clinical findings and applied for it. It can be said that this is a useful animal experiment system.

Example 2

In Example 1, blood IL-12 was measured 21 days after transplantation. Serum was prepared from mouse blood by a conventional method and stored frozen until the time of measurement. The concentration of IL-12 in mouse serum was measured using an ELISA kit (Interleukin-12 total [(m) IL-12], (p40 and p70) mouse ELISA Biotrak (TM) system, Code # RPN2702) manufactured by Amersham Armasia. The results were 3947 + 1709, 4641 + 861, 5771 + 819 (mean + SD, pg / ml), respectively.In the ILX combination group, the Mann-Whitney U test was p = 0.0192 compared to the vehicle administration group, and gefitinib Even in the single administration group, ρ = 0.1922 showed a mathematically significantly higher IL-12 value.

[0053] A group in which oral administration of 50 mg / kg of gefitinib and 1000 mg / kg of ILY (trade name) (IL12 production inducer) was also tested. The blood IL-12 concentration 7 days after transplantation was 2761 + 416 in the vehicle group, 2813 + 620 in the gefitinib 50 mg / kg alone group, and 3709 + 461 in the gefitinib and ILY combination group. The ILY combination group was mathematically significantly higher in the Mann-Whitney U test, with ρ = 0.0001 for the vehicle-administered group and p = 0.0023 for the gefitinib-only group, IL-12 value It was shown.

[0054] A test was also conducted in a group in which three drugs of gefitinib 50 mg / kg and ILX (IL-12 production inducer) l OOOmgZkg and ILY (trade name) (IL-12 production inducer) 1000 mg / kg were administered orally in combination. On day 14 after transplantation, the blood IL-12 concentration was 3129 + 867 in the vehicle group, 3005 + 819 in the gefitinib 50 mg alone group, and 4019 + 702 in the group with the combination of three drugs. In the Mann-Whitney U test, p = 0.0126 for the vehicle administration group and p = 0.0112 for the gefitinib alone administration group showed a mathematically significantly higher IL-12 value. Furthermore, the tumor volume (mean + SD, mm ³ ) 20 days after transplantation was 1947 + 1648 in the vehicle (0.5% Tween 20) group and 1874 + 947 in the gefitinib 50 mgZkg alone group. In the group administered orally, the tumor volume tended to be suppressed to 1301 + 735.

[0055] These facts indicate that the efficacy of gefitinib, for which Yagida has already filed application, in combination with IL-12 production inducers such as ILX and ILY in humans, and that gefitinib lowers the anticancer immunity ability. This is an experiment that supports the finding that it is an intractable type of anticancer drug, and is a useful animal experimental system in terms of immunological analysis and characterization.

Example 3

Colon26 tumor cells derived from mouse colorectal cancer (confirmed to be EGFR-positive tumor strain in control) were subcutaneously applied to the back of 2 million BALB / c mice (confirmed to be immunodeficient and mice). After transplantation, gefitinib was suspended in a 0.5% Tween20 aqueous solution (vehicle) and administered orally every day.The gefitinib 50 mg / kg single oral administration group, gefitinib 50 mg / kg and ILY1000 mg / kg The group receiving oral administration of kg was also tested. On day 14 after transplantation, blood IL-12 levels were 2987 + 377 in the vehicle group, 3126 + 376 in the gefitinib 5011¾ / 1¾ alone group, and 38 18 + 528 in the gefitinib and ILY combination group. In the ILY combination group, p = 0.0006 for the vehicle group and p = 0.0039 for the gefitinib alone group in the Mann-Whitney U test. It was shown.

A group was also tested for oral administration of a combination of 50 mg / kg gefitinib, ILX lOOOOmgZkg, and ILY 1000 mg / kg. The blood IL-12 concentration on day 14 after transplantation was 3350 + 562 in the group that received the combination of the three drugs, and the group that received the drug in the combination of the three drugs was p = 0 compared to the vehicle-treated group by the Mann-Whitney U test. 0333 showed a mathematically significant increase in IL-12 value.

[0057] In the gefitinib single administration group, the blood IL-12 concentration after 21 days was 2648 + 404, and p = 0 in the Mann-Whitney U test for the vehicle administration group of 2101 + 258. The result was mathematically significantly higher at 0164, indicating an IL_12 value. [0058] These facts indicate that the efficacy of gefitieb, for which Yagida has already filed application, with ILX and ILY, which are IL-12 production inducers, has been applied in humans, and that gefitinib has reduced anticancer immunity. This is an experiment that supports the finding that it is an unconventional type of anticancer agent, and is a useful animal experimental system in terms of immunological analysis and characterization.

Example 4

2 million LLC. Ca. (also known as Lewis lung cancer, also known as 3LL) tumor cells derived from mouse lung cancer (confirmed to be an EGFR-positive tumor strain by control) B10 (also known as C57BL / 10) mice (not immunodeficient) (10 mice) were implanted subcutaneously in the left axillary region, and gefitinib (trade name Iressa) 50 mgZkg was orally administered in a system in which gefitinib was suspended daily in water and administered orally every day. Treatment group, gefitinib 50 mgZkg and IL X1000 mg / kg combined oral administration, gefitinib 50 mg / kg and PSK (trade name Krestin, an existing BRM (biologic response modifier) used as a drug in Japan, IL 12 It is known to have an immunostimulatory effect such as induction of production.) A group in which 1000 mg / kg was orally administered in combination was tested. Tumor volume ^{(mea n + SD, mm 3} ) 10 days after transplantation, with water administration group 3115 + 1162, a combined administration group Gefuichinibu 50 mg / kg in single administration group 2641 + 994 since there force Gefuichiebu and ILX 1999 + 1074 And tumor volume were suppressed with a significant difference (p <0.05), and even in the group treated with gefitinib and PSK, the tumor volume was suppressed with a significant difference (p <0.05) to 1664 + 1147. The tumor volume on day 14 after transplantation was 5450 + 1064 in the water group and 5653 + 1279 in the gefitinib 50 mg / kg alone group.The tumor volume was 3510 + 1546 in the combination group of gefitinib and PSK, a significant difference (P 0.02). In addition, 10 out of 10 mice died of cancer in the water-administered group, whereas 1 in 10 animals survived and healed in the group administered gefitinib and PSK. FIG. 7 shows the survival rate of each group, and FIGS. 8 and 9 show changes in tumor size.

Example 5

The test was repeated in the same system as in Example 4. In other words, 2 million LLC. Ca. tumor cells were implanted subcutaneously in the left axillary region of 10 B10 (also known as C57BLZ10) mice, and gefitinib was suspended in water from the next day and administered orally every day. , Gefitinib 50 mg / kg alone orally, gefitinib 50 mg / kg and ILXlOOOOmgZkg orally, gefitinib A group of oral administration of nib 50 mg / kg and PSKlOOOOmg / kg was tested. Blood IL-12 concentration was measured on days 7, 10, and 14 after transplantation, and serum IL-12 was measured by ELISA Kit. Tumor volume (mean + SD, mm ³⁾ 10 days after transplantation, with water administration group 25 17 + 946, in the combined administration group Gefuichinibu 50 mg / kg in single administration group 2155 + 780 since there force Gefi Chinibu and ILX 1744 +586 and the tumor volume were suppressed with a significant difference (p <0.05). The blood IL-12 concentration (mean + SD, pg / ml) 14 days after transplantation is 1229 + 428 in the water administration group and 1598 + 776 in the gefitinib 50 mgZkg alone administration group.S Combined administration of gefitinib and ILX In the group, 1853 + 858 and blood IL-12 levels showed an increasing trend, and in the group treated with gefitinib and PSK, the blood IL-12 level increased with a significant difference (p <0.02) to 1992 + 326 . FIG. 10 shows the survival rate of each group, FIGS. 11 and 13 show the amount of IL-12 in each group, and FIGS. 12 and 14 show changes in tumor size of each group.

These facts indicate that the combination of gefitinib, for which Yagida has already filed application, with IL-12 production inducers such as ILX and PSK in humans, and that gefitinib does not lower the anti-cancer immunity ability This is an experiment that supports the finding that it is an anticancer drug, and is a useful animal experimental system in terms of immunological analysis and characterization.

Claims

The scope of the claims

[I] A model mouse for confirming the antitumor effect prepared by transplanting an EGFR (vascular endothelial cell growth factor receptor) positive mouse tumor strain into a non-immunodeficient mouse.

[2] A method for determining the effect of an anti-tumor agent targeting EGFR using a model mouse for confirming the anti-tumor effect prepared by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency.

[3] Method of determining the combined effect of an anti-tumor agent targeting EGFR and other therapies using an anti-tumor effect confirmation model mouse prepared by transplanting an EGFR-positive mouse tumor strain into a non-immunodeficient mouse .

[4] Method of determining the combined effect of an anti-tumor agent targeting EGFR and an immunotherapy using a model mouse for confirming the anti-tumor effect prepared by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency .

[5] A method for determining the effect of an immunotherapeutic agent using an antitumor effect confirmation model mouse prepared by transplanting an EGFR-positive mouse tumor strain into a non-immunodeficient mouse.

[6] The determination method according to any one of claims 2 to 4, which is an antitumor agent tyrosine kinase inhibitor targeting EGFR.

[7] The method according to claim 5, wherein the tyrosine kinase inhibitor has a selective target action on at least one of the following receptors;

HER2 / neu, HER3, HER4, c_kit, PDGFR, bcr-abl, EGFR.

[8] The method according to claim 6, wherein the tyrosine kinase inhibitor selectively has an EGFR or c-kit target action.

[9] The method according to claim 4 or 5, wherein the immunotherapeutic agent is an IL-12 production inducer.

[10] The method according to claim 9, wherein the IL-12 production inducer is a substance having a β1,3 / 1,6 glucan structure.

[II] The method according to claim 10, wherein the IL-12 production inducer is a component derived from a mushroom mycelium or a component derived from yeast having a β1,3 / 1,6 glucan structure.

[12] A method for determining EGFR positivity of a mouse tumor strain through the following steps;

1) transplanting the mouse tumor strain into a non-immunodeficient mouse, 2) A step of collecting local tumor and detecting the presence or absence of EGFR positivity by immunohistochemistry.

[13] Suppression of the growth of the transplanted tumor by administration of an EGFR-related inhibitor using a non-immunodeficient model mouse system transplanted with a mouse tumor strain that has been confirmed to be EGFR-positive by the method of claim 12 A method for screening an EGFR-related inhibitor to determine the sole effect of an EGFR-related inhibitor.

[14] A method using a non-immune-deficient model mouse system transplanted with a mouse tumor strain that has been confirmed to be EGFR-positive by the method according to claim 12, wherein the tumor is caused by co-administration of an EGFR-related inhibitor and another therapeutic agent. A screening method for a combination use of an EGFR-related inhibitor and another therapeutic agent to determine the effect of a combination use of an EGFR-related inhibitor and another therapeutic agent by observing suppression of proliferation.

15. The method according to claim 13, wherein the EGFR-related inhibitor is a tyrosine kinase inhibitor.

[16] The screening method according to claim 14 or 15, wherein the other therapeutic agent is an immunotherapy agent.

17. The screening method according to claim 16, wherein the influence on various immune markers is also used as a judgment factor.