WO2004110138A1 - Modele souris - Google Patents

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Publication number
WO2004110138A1
WO2004110138A1 PCT/JP2004/008231 JP2004008231W WO2004110138A1 WO 2004110138 A1 WO2004110138 A1 WO 2004110138A1 JP 2004008231 W JP2004008231 W JP 2004008231W WO 2004110138 A1 WO2004110138 A1 WO 2004110138A1
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egfr
mouse
tumor
effect
positive
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PCT/JP2004/008231
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English (en)
Japanese (ja)
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Akikuni Yagita
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Orient Cancer Therapy Co.,Ltd.
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Publication of WO2004110138A1 publication Critical patent/WO2004110138A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases

Definitions

  • the present invention relates to the provision of a novel model animal system for determining the efficacy of an antitumor agent.
  • a novel model animal system for determining the efficacy of an antitumor agent.
  • an antitumor agent that targets EGFR vascular endothelial cell growth factor receptor
  • EGFR vascular endothelial cell growth factor receptor
  • IL-12 interleukin 12
  • NITC Novel Immunotherapy for cancer
  • IL-12 has a TNFa ⁇ IFNy ⁇ IL_12 ⁇ CTL activity and recruitment effect to activate and enhance killer T cells.
  • IL-12 production enhancement is expected to have an anticancer effect by activating and enhancing killer T cells.
  • NKT cells include NK cell antigen receptor (NKR-P1; natural killer receptor P1) as another receptor (Non-patent Document 1).
  • 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).
  • 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.
  • 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.
  • 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.
  • ligand such as epidermal growth factor (EGF)
  • EGF epidermal growth factor
  • 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).
  • Such a molecular target therapeutic agent is attracting attention as a cancer therapeutic drug of a new mechanism. Its effect is still not revolutionary.
  • ZD1839 Iressa B3 ⁇ 4
  • ZD1839 is a powerful and selective inhibitor of the tyrosine kinase of EGFR newly developed by AstraZeneca, and its usefulness has been found in humans.
  • PR partial remission
  • CR complete remission
  • 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.
  • 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.
  • 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 ⁇ 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)
  • 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
  • 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.
  • 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.
  • the present invention has been completed.
  • the present invention includes the following.
  • 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.
  • EGFR vascular endothelial growth factor receptor
  • 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.
  • Antitumor agent ability targeting EGFR The determination method according to item 24 above, which is a tyrosine kinase inhibitor.
  • HER2 / neu HER3, HER4, c_kit, PDGFR, bcr-abl, EGFR.
  • IL-12 production inducer is a mushroom mycelium-derived component or a yeast-derived component having a ⁇ , 3/1, 6 glucan structure.
  • a method for determining the EGFR positivity of a mouse tumor strain through the following steps;
  • 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.
  • FIG. 1 is a staining diagram of a 3LL tumor transplantation blocking test.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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. 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.
  • NITC cancer new immunotherapy
  • the first mechanism of action is a method of reducing cancer by administering an antiangiogenic substance (bettershark) to impair blood flow to the cancer.
  • the effect can be determined by measuring vascular endothelial cell growth factor (VEGF).
  • VEGF vascular endothelial cell growth factor
  • the angiogenesis inhibitory effect can be evaluated by a minus (negative) value of VEGF (one VEGF).
  • VEGF value other vascular growth factors such as FGF and HGF can be used to evaluate the ability to inhibit angiogenesis.
  • the positive value of an angiogenesis inhibitor can be evaluated (eg, an endostatin value).
  • the second mechanism of action is to activate a CTL by administering a compound having a ⁇ , 3 glucan structure to induce Thl site force-in (TNF ⁇ , IFN ⁇ , IL-12) It is.
  • CTL activity Sex can be determined by the ability to produce CD8 (+) perforin.
  • 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.
  • 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).
  • IFN y value the ability to produce IFN y
  • IL-12 value ability to produce IL-12
  • 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.
  • NK and NKT cells share NKR-P1 (NK cell receptor CD161 (+) in humans, NK cell surface receptor stained with NKR-P1 antibodies such as ⁇ 1.1 in mice).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • a mushroom mycelium composition preparation having a ⁇ 1,3 glucan structure eg, ILX ffin3 ⁇ 4 : Tozai Pharmaceutical Research Institute, ILY ffiB3 ⁇ 4 *: a seishin company, AHCC: amino up
  • various yeasts having a 1,3 glucan structure Marine yeast, baker's yeast, NBG TM
  • CTL activator 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.
  • 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.
  • a tyrosine kinase inhibitor, or ZD1839 Iressa ⁇ [pi) is with STI571 (Daribekku 3 ⁇ 4)
  • 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.
  • 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.
  • NK activator or NKT activator As an immunotherapeutic agent in addition to an IL-12 production inducer.
  • CD3 (+) CD161 (+) acts on NK T cell receptor NKR-P1.
  • 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.
  • 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).
  • 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.
  • fucoidans examples 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.
  • 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.
  • the system of the present invention comprises a tyrosine kinase inhibitor and a CTL activator (IL-12 production inducer, IF ( ⁇ ⁇ -production inducer), and also useful in evaluating the usefulness of combined use with ⁇ activators, ⁇ activators and neovascular inhibitors.
  • IL-12 production inducer IF ( ⁇ ⁇ -production inducer)
  • IF ⁇ ⁇ -production inducer
  • 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.
  • 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
  • a CTL activator IL-12 production inducer, IFNo / production inducer
  • a NK activator a NK activator
  • NKT activity a tyrosine kinase inhibitor
  • IL-12 production inducer IFNo / production inducer
  • a NK activator a NK activator
  • 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 ⁇ -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.
  • 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.
  • parenteral ingestion including intravenous or intramuscular administration
  • the system of the present invention is also effective for evaluating the effective dose and administration method as described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SIGMA protease type XXIV
  • Biotinylated Rabbit Antibody (Goat immunoglobulin obulins, ode # E0466 from DAKO) was diluted 500-fold and incubated at room temperature for 30 minutes.
  • peroxidase-conjugated streptavidin (Peroxidase-Conjugated Strepta vidin, Code # P0397 from DAK ⁇ ) was diluted 500-fold and incubated at room temperature for 30 minutes.
  • the plate was incubated with a DAB solution (100 ml PBS, 20 mg 3, 3'-diaminobenzidine tetrahydrochloride, and 20 ⁇ l 30% hydrogen peroxide) at room temperature for 5 minutes.
  • 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.
  • colon cancer-derived colon 26 tumor cells are transplanted subcutaneously into the back of 2 million BALB / c mouse mice (immune-deficient), for example, to prepare a model animal.
  • 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
  • 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.
  • 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.
  • 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.
  • gefitinib 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.
  • 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.
  • mice 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.
  • the model mouse provided in the present invention is obtained by transplanting an EGFR-positive mouse tumor strain into a mouse without immunodeficiency.
  • 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.
  • 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.
  • aqueous solution such as a 0.5% Tween20 aqueous solution (vehicle)
  • aqueous solution such as a 0.5% Tween20 aqueous solution (vehicle)
  • 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. Applic
  • 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.
  • 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.
  • a cell surface antigen CD3 and / or CD161 specifically present on the cell surface of NKT cells.
  • 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.
  • 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.
  • CD3 (_) CD161 (+) is an assay for cells that are negative for CD3 and positive for CD161. This method is useful for measuring NK cells.
  • CD8 (+) refers to the assay of CD8-positive cells. This method is useful for measuring CTL activity.
  • 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.
  • 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 .
  • PBS Phosphate Buffered Saline
  • FBS fetal bovine serum
  • Phytohemagglutinin (manufactured by DIFCO) was added to 200 ⁇ l of the obtained cell suspension at a concentration of 20 ⁇ g / ml, and the solution was incubated at 37 ° C in a 96-well microplate in the presence of 5% CO. 2 in C
  • a measurement kit by enzyme immunoassay (ELISA) available from R & D SYS TEMS or MBL is used for clinical samples.
  • ELISA enzyme immunoassay
  • a measurement kit from R & D SYSTEMS was used.
  • 50 ⁇ l of the assay diluent RD1F was used in each well of a 96-well microplate, and 200 ⁇ l of the standard solution (standard) or the sample obtained from the sample preparation for measuring the cytodynamic force was used. After dispensing ⁇ ⁇ , the mixture was allowed to stand at room temperature and reacted for 2 hours.
  • HRP horseradish peroxidase
  • 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 ⁇ l each.
  • Emax manufactured by Kako Pure Chemical Industries, Ltd.
  • IL 12 levels are expressed as pg / ml.
  • 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.
  • 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.
  • IL-12 in blood in serum or plasma
  • IL-12 in serum in serum or plasma
  • the IFN y was measured by enzyme immunoassay (EIA method) using IFN y EASIA kit of Bio Source Europe S. for clinical samples.
  • EIA method enzyme immunoassay
  • a standard solution standard
  • HRP-labeled anti-IFN ⁇ antibody HRP-labeled anti-IFN ⁇ antibody
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • BLL mice 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).
  • Example 1 5% Tween20) 3 ⁇ 4 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.
  • 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).
  • CD3e-positive cells are T cells
  • NK1.1-positive cells are NK cells
  • both CD3e and NK1.1-positive cells are NKT cells.
  • the T, ⁇ , and ⁇ cell ratio was 15.89 + 4.96, 2.08 + 0.41, 0.41 + 0.08 Met.
  • the ratio was slightly higher than that of the vehicle-administered group, and it was found that the ⁇ , ⁇ , ⁇ cell ratio did not decrease when gefitinib 50 mg / kg was administered.
  • 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.
  • ELISA kit Interleukin-12 total [(m) IL-12], (p40 and p70) mouse ELISA Biotrak (TM) system, Code # RPN2702
  • 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 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.
  • 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.
  • the tumor volume tended to be suppressed to 1301 + 735.
  • Colon26 tumor cells derived from mouse colorectal cancer were subcutaneously applied to the back of 2 million BALB / c mice (confirmed to be immunodeficient and mice).
  • 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.
  • a group was also tested for oral administration of a combination of 50 mg / kg gefitinib, ILX lOOOOmgZkg, and ILY 1000 mg / kg.
  • 0333 showed a mathematically significant increase in IL-12 value.
  • 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 mice (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.)
  • 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.
  • 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).
  • 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
  • FIGS. 8 and 9 show changes in tumor size.
  • Example 4 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.
  • FIG. 10 shows the survival rate of each group
  • FIGS. 11 and 13 show the amount of IL-12 in each group
  • FIGS. 12 and 14 show changes in tumor size of each group.

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Abstract

L'invention concerne des moyens permettant de vérifier l'efficacité d'un médicament curatif moléculaire cible, seul ou utilisé en combinaison avec une immunothérapie, dans des conditions non cliniques étroitement apparentées à celles de l'humain. Ces moyens permettent en particulier d'évaluer de manière non clinique l'efficacité d'un agent antitumoral agissant sur EGFR, combiné à un agent immunothérapeutique, en établissant un système expérimental consistant à transplanter une souche de cellules tumorales de souris EGFR-positives dans une souris non immunodéficiente, comme moyen permettant de vérifier la réalisation d'un effet synergique de l'utilisation conjoint d'un inhibiteur de la tyrosine kinase et d'une immunothérapie telle qu'un stimulateur de la production d'IL-12 dans un traitement anticancéreux.
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JPH10139670A (ja) * 1996-11-11 1998-05-26 Terukuni Yakida インターロイキン１２誘導物質及び医薬組成物

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