JP2007202431A - Method for assessing antitumor effect using canine interleukin 12 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for assessing antitumor effect of the administration of canine IL(interleukin) 12 supposed to be effective as a curative for canine tumor by a method other than the increase/decrease of tumor mass, i.e. by a molecular-biological method. <P>SOLUTION: The method for assessing the antitumor effect comprises the following procedure. Blood is collected from a tumor-contracted dog administered with canine interleukin 12. Then, leukocytes are isolated from the collected blood, and a cytokine gene is extracted from the leukocytes to examine the expression variation of interferon-γ gene and/or canine interleukin 10 gene. Specifically, it is judged that when a canine interferon-γ expression is found, the antitumor effect is high; on the contrary, when a canine interleukin 10 expression is found, the antitumor effect is low. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for evaluating canine anti-tumor effects and an evaluation kit thereof. More specifically, the present invention relates to a method and an evaluation kit for evaluating the anti-tumor effect by measuring changes in expression of cytokine genes of leukocytes in dog blood collected from dogs administered with canine interleukin-12.

  Interleukin (IL) 12 stimulates LAK cells, NK cells and T cells directly, promotes cytokine release from those cells, and enhances cytotoxic activity of NK cells and CTLs. It also stimulates and induces T cells to enhance cellular immunity.

Due to advances in gene manipulation technology, not only human IL12 but also IL12 of animals such as cattle, horses, cats, dogs, etc., have been cloned one after another, and as a result, development of use as therapeutic agents for viral diseases and tumors Some have been studied (Non-Patent Documents 1, 2, 3, and 4). Canine IL12 is expected to have an effect on canine tumors, but as a means of evaluating its antitumor effect, the size of the tumor mass is of course varied, but no clear indicator has been reported. It is necessary to establish an evaluation method for the presence or absence of tumor effects.
Wolf et al .: Journal of Immunology (J. Immunol). 146, 3074-3081 (1991). Schoenhaut et al .: Journal of Immunology (J. Immunol). 148, 3433-3440 (1992). Nastala et al .: Journal of Immunology (J. Immunol). 153, 1697-1706 (1994). Gubler et al .: Gene 25, 236-269 (1983).

  Dogs are known for numerous tumors, including breast tumors. Although removal of tumor lesions by surgery is the first choice, some owners do not necessarily want surgery and often want to extend their lives with pharmaceuticals. In response to these demands, human anticancer agents are often used, but the frequency of use is low in terms of side effects. In response to this situation, antitumor drugs for dogs are desired. Therefore, it is considered that administration of canine IL12 is effective as these therapeutic agents. However, depending on the type of tumor, it may be unresponsive, and no matter how much it is administered, no effect can be expected. If it can be clarified by an evaluation method using a molecular biological approach, it will be possible to determine in advance whether or not an effect can be expected in the initial stage of medication, and a reduction in burden due to medication to dogs can be expected. Further, improvement of the certainty of the antitumor effect opens the way for the development of canine interleukin 12 as a veterinary drug, and development of an antitumor effect evaluation kit is expected. Accordingly, an object of the present invention is to establish a method for evaluating the antitumor effect of canine interleukin 12, which is promising as an antitumor drug for dogs, and to develop an evaluation kit.

  In view of this situation, the present inventor administers canine interleukin 12 to tumor-affected dogs, collects blood, isolates leukocytes, extracts genes, and changes expression of canine interferon-γ and canine interleukin 10 as cytokines. When examined, there is a correlation between the antitumor effect and the expression of these genes. In the case of increased expression of canine interferon-γ and decreased expression of canine interleukin 10, the antitumor effect of canine interleukin 12 is high, On the contrary, it was found that the antitumor effect of canine interleukin 12 may be low when the expression of canine interferon-γ is decreased and the expression of canine interleukin 10 is increased. In addition, by examining the expression pattern of these genes several weeks after the administration, it was possible to determine the success or failure of canine interleukin 12 against tumors, thus completing the present invention.

 That is, the present invention has the following configuration.

  (1) A method for evaluating the canine anti-tumor effect of canine interleukin 12, which comprises measuring changes in the expression of cytokine genes in leukocytes in dog blood.

  (2) When the cytokine is canine interferon γ and / or canine interleukin 10, and when expression of canine interferon γ gene is observed, it is judged that canine interleukin 12 has an antitumor effect, and expression of canine interleukin 10 gene The method for evaluating the canine anti-tumor effect of canine interleukin 12 as described in (1) above, wherein it is determined that there is no anti-tumor effect of canine interleukin 12 when

  (3) An evaluation kit for measuring canine leukocyte cytokine gene expression fluctuations in dog leukocytes collected from dogs administered with canine interleukin 12 and evaluating canine interleukin 12 for canine anti-tumor effects.

  (4) The evaluation kit for evaluating the canine anti-tumor effect of canine interleukin 12 as described in 3 above, wherein the cytokine is canine interferon γ and canine interleukin 10.

  According to the present invention, anti-tumor effects of canine IL12 can be obtained by extracting genes from leukocytes in canine blood administered with canine IL12 and examining changes in expression of canine interferon-γ and canine interleukin 10 as cytokines. Can be evaluated. Further, by evaluating the expression of these genes at the initial stage of administration, it is possible to provide an effective treatment.

  Hereinafter, the present invention will be described in detail.

1. Cytokines Cytokines are intercellular signaling substances that play an important role in immune responses, responses during infection, hematopoiesis, viral infections and tumor cell damage. Cytokines are involved not only in constitutive hematopoiesis from hematopoietic stem cells but also in induced hematopoiesis during inflammation and the like. They are produced from all types of cells such as activated T cells, macrophages, B cells, mast cells, and vascular endothelial cells. Examples of immune system cytokines include proteins or glycoproteins, such as interleukins and interferons. Interleukin is a name given to cytokines that transmit signals between lymphocytes.

2. About Th1 and Th2 There are various types of lymphocytes, which are one type of white blood cell. Among them, helper T cells recognize antigens on antigen-presenting cells and are activated to produce various helper factors. By these helper factors, effector T cells and B cells are stimulated to differentiate and mature. According to Mosmann and Coffman, helper T cells are classified into those that secrete IL2, but not IL4, and conversely, those that do not secrete IL2, but secrete IL4. The former is Th1, the latter is Th2. be called. That is, one type of helper T cell does not activate both effector T cells and B cells, but Th1 secretes cytokines that act on effector T cells and Th2 acts on B cells. Examples of the Th1-derived cytokine include IL2, IFN-γ, TNF-α, and IL3, and examples of the Th2-derived cytokine include IL4, IL5, IL6, and IL10.

3. About Canine Interleukin (IL) 12 Like human IL12, canine IL12 consists of a heterodimer of a molecular weight of about 40 kD protein (hereinafter abbreviated as P40) and about 35 kD protein (hereinafter abbreviated as P35). It is a cytokine having a physiological activity such as activating helper T cells.

4). About interferon (IFN) -γ
IFN-γ is known to be induced from T cells or NK cells by mitogens such as ConA and PHA and IL12. In addition to its antiviral effect by induction of antiviral protein synthesis, its biological activity acts on NK cells to enhance killer activity, enhances macrophage IL-1 and TNF production, and enhances phagocytosis. Furthermore, it is involved in neutrophil activation and suppresses delayed hypersensitivity reactions. In addition, it is generally known that the antibody production of B cells acts in a suppressive manner. For T cells, it is a cytokine known to promote induction of killer T cells and enhance expression of IL2 and IL2 receptors. In addition, according to the Th1 / Th2 hypothesis proposed by Mosmann et al., Th1 is thought to suppress the proliferation and activity of Th2, and IFN-γ produced from Th1 suppresses the proliferation of Th2 and produces Th2. Inhibits the action of cytokines.

5). About Interleukin (IL) 10
IL10 is produced by the Th0 and Th2 subsets of helper T cells and is also known to produce mononuclear cells and macrophages. Biological activity inhibits accessory cell-dependent production of most cytokines from Th1 and growth of Th1 cell lines, but does not inhibit cytokine production from Th2. For example, it is said that IL10 suppresses IFN-γ production from Th1 by about 90%. Further, it is known from various studies that the action of macrophages that stimulate cytokine production of Th1 cells is suppressed, but the action of B cells is not affected. That is, IL10 is a cytokine suggested to act directly on antigen-presenting cells because IL10 exhibits its inhibitory action only in the presence of antigen-presenting cells.

6). Method for producing canine interleukin 12 A plasmid incorporating DNAs encoding the two subunits of canine IL12 protein can be produced, for example, as follows. That is, after extracting poly (A) RNA from canine cells, cDNA was synthesized and polymerase chain reaction (hereinafter referred to as “polymerase chain reaction”) using primers based on gene sequences encoding two subunits of bovine and human IL12, respectively. By abbreviating PCR, it is possible to clone two genes each encoding two subunits exhibiting canine IL12 activity. In addition, a full length of canine IL12P40 cDNA and canine IL12P35 cDNA can be cloned by preparing a phage library from the synthesized cDNA recombinant and performing plaque hybridization with two gene fragments obtained by PCR.

  As a method for obtaining RNA from dog furniture or cells, such as canine mononuclear cells or lymphocytes stimulated with mitogen, etc., conventional methods such as polysome separation, sucrose density gradient centrifugation and electrophoresis are used. The method that I did. As a method for extracting RNA from the above canine apparatus and canine cells, guanidine thiocyanate-cesium chloride method in which CsCl density gradient centrifugation is performed after guanidine / thiocyanate treatment (Reference 12) is used in the presence of a ribonuclease inhibitor in the presence of a ribonuclease inhibitor. Method of phenol extraction after treatment with activator (Reference 13), guanidine thiocyanate-hot phenol method, guanidine thiocyanate-guanidine hydrochloride method, guanidine thiocyanate-phenol chloroform method, guanidine thiocyanate and chlorination An appropriate method can be selected from the method of precipitating RNA by treatment with lithium. It can be obtained by isolating mRNA from canine mononuclear cells and lymphocytes stimulated with canine utensils, mitogens, etc. by conventional methods such as lithium chloride / urea method, guanidine isothiocyanate method, oligo dT cellulose column method, etc. MRNA from conventional methods, for example, the method of Gubler et al. CDNA is synthesized by the method of Okayama et al. In order to synthesize cDNA from the obtained mRNA, basically, a reverse transcriptase such as avian osteoblast virus (AMV) or the like may be used, or a method using a DNA polymerase or the like using a one-part primer may be combined. It is convenient to use a commercially available synthesis or cloning kit.

  By performing PCR using this cDNA as a template and primers based on human, mouse and bovine base sequences, genes encoding P40 subunit and P35 subunit exhibiting canine IL12 activity can be cloned. In addition, the synthesized cDNA is linked to a λ phage vector and then packaged by mixing with a coat protein of λ phage in vitro, and the generated phage particles are infected with a host such as E. coli. At this time, Escherichia coli infected with λ phage is lysed, and each clone is recovered as a plaque. The plaques are transferred to a filter such as nitrocellulose, and the full lengths of canine IL12P40 cDNA and canine IL12P35 cDNA can be cloned by hybridization using a gene obtained by radiolabeled PCR as a probe.

  Prokaryotes or eukaryotes can be used as the host. As prokaryotes, bacteria, particularly Escherichia coli, Bacillus bacteria, such as Bacillus subtilis, and the like can be used. Examples of eukaryotes include yeasts, e.g., yeasts of the genus Saccharomyces, e.g., eukaryotic microorganisms such as Saccharomyces cerevisiae, insect cells, e.g. Cells (Bombyx mori), animal cells such as human cells, monkey cells, mouse cells and the like can be used. In the present invention, the organism itself, for example, an insect such as a silkworm, a cabbage looper, or the like can also be used.

  As expression vectors, plasmids, phages, phagemids, viruses (baculo (insects), vaccinia (animal cells)) and the like can be used. The promoter in the expression vector is selected depending on the host bacterium. For example, a lac promoter, a trp promoter or the like is used as a bacterial promoter, and an adh1 promoter, a pqk promoter, or the like is used as a yeast promoter. Examples of the promoter for insects include baculovirus polyhedrin promoter and p10 promoter, and examples of animal cells include, but are not limited to, the early or late promoter of Simian Virus 40. Transformation of a host with an expression vector can be carried out by conventional methods well known in the art, and these methods include, for example, Current Protocols in Molecular Biology, John Willy and Sons ( John Wiley & Sons). The transformant can be cultured according to a conventional method.

  The produced canine IL12 has an apparent molecular weight of about 70-80 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reduction.

  In SDS-PAGE, the 70-80 kD band produces two subunits with molecular weights of about 40 kD and about 35 kD under reducing conditions.

7). Method for Purification of Canine Interleukin 12 As shown in Example 2 below, canine IL12 is capable of in vitro induction of interferon γ (hereinafter abbreviated as IFNγ) from canine leukocytes and phytohemaglutinin (hereinafter referred to as PHA). It is characterized mainly by the growth promoting effect of canine lymphocytes stimulated with (abbreviated). In addition, it has the activity of activating NK cells and cytotoxic T cells to thaw their target cells, such as tumor-derived cell lines or virus-infected fibroblasts.

  In the present invention, for purification of canine interleukin 12, it is desirable to select a column chromatography method. Since column chromatography can be developed with an aqueous solution in all steps, it can be used to purify a protein having an intrinsic biological activity such as interleukin-12. Examples of the column carrier used in column chromatography include an ion exchange carrier and a dye carrier.

  First, examples of the ion exchange carrier include agarose, cellulose, synthetic polymer gel, and the like such as diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, carboxymethyl, sulfone. Examples thereof include those introduced with a functional group such as propyl (Sulphopropyl) and methyl sulfonate (Methyl sulphonate). Preferably, SP Sepharose High Performance (manufactured by Amersham) or SP Sepharose Fast Flow (manufactured by Amersham) into which sulfopropyl is introduced (hereinafter abbreviated as SP Sepharose carrier) is used. Elution from the SP Sepharose carrier is determined by the pH value, ionic strength, etc. of the eluent. Proteins composed of a combination of P40 and P35 have different charges, and can be fractionated using the present carrier according to the difference in charge. For example, in the case of canine IL12 and canine P40 homodimer produced in insect cells, each can be fractionated at a pH value of 7 or less.

  Furthermore, as the dye carrier, a carrier to which a blue dye is bound (hereinafter abbreviated as blue carrier) is preferably used. The following are used as the blue carrier. The blue dye has a general name of CI Reactive Blue 2, and examples thereof include a blue dye commercially available from CIBA-GEIGY under the trade name Zibacron Blue F3GA or Zibacron Blue 3GA. As a carrier used in actual chromatography, Blue Sepharose CL-6B (Amersham), Blue Sepharose 6 First Flow (Amersham), Blue Sepharose 6 High Performance (Amersham), Matrex Gel Blue A (Amicon) ), Affigel Blue (manufactured by Biorat), and other brand names such as Blue Agarose Gel, or Blue Tris Acrylic M (manufactured by LKB) and Blue Cellulofine (manufactured by Chisso). A commercially available blue sepharose gel is suitable and can be easily obtained. Elution from the blue carrier is determined by the pH value, ionic strength, hydrophobicity, etc. of the eluent. For example, in the case of canine IL12 and canine P40 homodimer, each can be separated and eluted at a pH near neutral and purified with high purity.

  Even if the above-described purification using the column carrier is carried out independently, it is possible to increase the purity of canine IL12 and canine P40 homodimer, but it is preferable to carry out the purification in combination with each other. For example, a solution containing canine IL12 and canine P40 homodimer produced in silkworm organisms contains a large number of silkworm-derived contaminants and combinations of a plurality of canine P40 and canine P35. It is preferable to combine the carriers. In the production of canine IL12 and canine P40 homodimers produced in insect cells and silkworm organisms, it is preferable to use a sulfopropyl carrier for the first stage purification and a blue carrier for the second stage purification.

  In the purification operation using each column carrier, the composition of the eluent, the amount of liquid, etc. are not particularly limited, and optimum separation conditions include the amount of contaminating proteins, canine IL12 and canine P40 homodimers, and column It is determined appropriately depending on the dimensions of

  In addition, in order to simultaneously produce IL12 and P40 homodimer in large quantities simultaneously, a gene encoding P40 is connected under a promoter with higher expression, and a gene encoding P35 is connected under a promoter with lower expression. By producing a recombinant containing these simultaneously and expressing each gene, IL12 and P40 homodimer can be produced simultaneously. For example, in a baculovirus expression system, a gene encoding canine P40 is linked under the polyhedrin promoter of a vector containing both the polyhedrin promoter and the P10 promoter, and a gene encoding canine P35 is linked under the P10 promoter, By producing recombinant viruses and expressing them in insect cells, both canine IL12 and canine P40 homodimers can be produced in large quantities simultaneously.

8). Formulation method of canine interleukin 12 For formulation of interleukin 12, canine IL12 and various stabilizers may be added. Known methods for stabilizing useful proteins include methods of mixing with other proteins such as gelatin, serum, albumin, collagen, etc. (references 2 to 5), saccharides, particularly monosaccharides, disaccharides, dextran and hydroxy. There are a method of mixing with a polysaccharide such as ethyl starch (References 6 to 9), or a method of using cyclodextrin or polysaccharide alcohol as a stabilizer (References 10 and 11). By adding a stabilizer in addition to canine IL12 produced by the production method of the present invention, a long-term stabilization effect can be expected. There is no limitation in particular as the stabilizer added. The ingredients added to romiplostim are mainly determined depending on the mode of administration of romiplostim. When this drug is used as a solid, for example, fillers such as lactose, binders such as carboxymethylcellulose and gelatin, colorants, coating agents and the like can be used, and such agents are suitable for oral administration. Further, for example, white petrolatum, cellulose derivatives, surfactants, polyethylene glycol, silicone, olive oil etc. can be added as a carrier or activator, and it can be applied to the affected area as an external medicine in the form of cream, emulsion, lotion or the like. . In addition, when the drug is administered as a liquid, it can contain physiologically acceptable solvents, emulsifiers, and stabilizers that are commonly used. Examples of the solvent include water, PBS, isotonic saline, and the like. Examples of the emulsifier include polyoxyethylene surfactants, fatty acid surfactants, and silicones. Examples of the stabilizer include dog serum albumin. And polyols such as gelatin, or sugars such as sorbitol and trehalose. Although there is no particular limitation on the administration method of the therapeutic agent and prophylactic agent of the present invention, the most therapeutic effect can be expected by injection administration. The injection administration method is not limited to any of intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, and intrathoracic administration.

9. About Canine Tumors as Target Diseases Dog tumors include breast tumors, eosinophilic granuloma, epidermoid tumors, skin tumors, lipomas, otohematoma, pulmonary edema, cutaneous pedunculated soft tumors or anal tumors .

10. How to administer canine IL12 to dogs The dose will be determined depending on the size of the individual, the method of administration, the type of disease, symptoms, etc., but it is sufficient to show therapeutic and prophylactic effects. For example, administration of 0.1 pg to 100 μg / kg of canine IL12 per day, a sufficient effect can be obtained.

  In adoptive immunotherapy, sufficient effects can be obtained by stimulating lymphocytes isolated from 1 to 100 ml of dog blood with 0.001 pg to 1 μg of canine IL12 for 12 hours to 6 days and then returning them to the body again. .

11. Preparation of white blood cells from dog blood In preparation of white blood cells from dog blood, an anticoagulant is first added immediately after blood collection to prevent blood coagulation. As the anticoagulant, heparin and ethylenediaminetetraacetate are used. In order to prepare a cell group from the obtained blood, specific gravity centrifugation is mainly used. That is, this is a method in which blood is layered on a solution smaller than the specific gravity of erythrocytes and granulocytes and larger than that of lymphocytes and monocytes, and a cell group is fractionated by centrifugation. Ficoll-isopac solution is used as the separation solution. After centrifugation, a fraction of lymphocytes and monocytes, which are target white blood cells, is observed between the diluted plasma fraction and the separated solution, and this fraction is fractionated. Further, in order to separate only lymphocytes, the Petri dish attachment method is mainly used. The lymphocyte fraction obtained thereafter can separate and concentrate only T cells by using a nylon wool column. This method has an advantage that a relatively pure T cell group can be recovered from a diverse cell population without subjecting the cell group to intense manipulation.

12 Gene preparation from leukocytes Preparation of genes from leukocytes can be carried out, for example, as follows. After extracting poly (A) RNA from the T cell group prepared in 11 above, cDNA was synthesized and polymerase chain reaction (hereinafter abbreviated as PCR) using primers based on the gene sequences encoding each subunit. Is possible by doing Examples of methods for obtaining RNA from canine T cells include conventional methods such as polysome separation, sucrose density gradient centrifugation, and methods utilizing electrophoresis. As a method for extracting RNA from the above canine cells, a guanidine thiocyanate-cesium chloride method in which CsCl density gradient centrifugation is performed after guanidinium thiocyanate treatment (Reference 13) is treated with a surfactant in the presence of a ribonuclease inhibitor using a vanadium complex. Then, phenol extraction, guanidine / thiocyanate-hot phenol method, guanidine / thiocyanate / guanidine hydrochloride method, guanidine / thiocyanate / phenol / chloroform method, guanidine / thiocyanate and then treatment with lithium chloride to precipitate RNA An appropriate method can be selected from among the methods to be performed. Furthermore, mRNA is isolated by a conventional method, for example, lithium chloride / urea method, guanidine isothiocyanate method, oligo dT cellulose method, etc., and a normal method, for example, the method of Gubler et al. (Reference 14), H . CDNA is synthesized by the method of Okayama et al. (Reference 15). In order to synthesize cDNA from the obtained mRNA, basically a reverse transcriptase such as avian osteoblast virus (AMV) may be used, or a method using a DNA polymerase or the like using a one-part primer may be combined. It is convenient to use a commercially available synthesis or cloning kit. DNA encoding interferon-γ or interleukin 10 can be obtained by performing PCR using this cDNA as a template and a primer based on the base sequence of the gene encoding interferon-γ or interleukin 10.

13. Canine anti-tumor effect evaluation method As a method for evaluating canine anti-tumor effect by canine interleukin 12, it is a method of measuring changes in expression of cytokine genes in leukocytes in dog blood, and as cytokines, canine interferon γ and canine interleukin 10. Judgment is that when the expression of canine interferon γ is seen, it is judged that there is an antitumor effect by canine interleukin 12, and when the expression of canine interleukin 10 is seen, it is judged that there is no antitumor effect by canine interleukin 12 Can be performed.

  Canine interleukin 12 is administered to tumor affected dogs, blood is collected, white blood cells are separated, genes are further extracted, and expression variations of canine interferon-γ and canine interleukin 10 are examined by gene agarose gel electrophoresis・ Evaluate. Immediately after the start of administration, expression of canine interferon-γ, increased expression within the administration period, non-expression or low expression of interleukin 10 at the start of administration, and decreased expression within the administration period, canine anti-interleukin 12 On the contrary, when the expression of canine interferon-γ is decreased and the expression of canine interleukin 10 is increased at the start of administration and within the administration period, the antitumor effect of canine interleukin 12 is evaluated to be low. In addition, regarding tumors of dogs that are expressed in many cases, such as breast tumors, changes in tumor size over time can be recorded and evaluated together with changes in the expression of cytokine genes.

14 Evaluation Kit for Evaluating Canine Anti-Tumor Effect A kit used for measuring changes in expression of canine interferon-γ gene and canine interleukin 10 gene including the above reaction can be prepared by a method known per se. The reagents used in these kits include an agarose gel electrophoresis solution, an electrophoresis device, and a UV irradiation gel photographing device in addition to the dog interferon-γ gene and the dog interleukin 10 gene as the sample to be measured obtained by the above method. It is. Also included as optional elements are a molecular weight marker, a loading buffer that imparts a charge to the DNA solution, and the like.

  Hereinafter, the present invention will be described more specifically with reference to reference examples and examples.

Reference Example 1 Cloning of Canine IL12P40 and P35 Genes (1) Preparation of Canine cDNA 7 hours with canine peripheral blood mononuclear cells and chicken Newcastle disease virus stimulated with canine liver, lipopolysaccharide (LPS) (50 μg / ml) for 48 hours Total RNA was prepared from the treated (10 ⁷ pfu / ml) canine spleen-derived lymphocytes using isogen (Nippon Gene). The obtained RNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (hereinafter abbreviated as TE) containing 1 mM EDTA, treated at 70 ° C. for 5 minutes, and then the same amount of TE containing 1 M LiCl was added. . The RNA solution was applied to an oligo dT cellulose column equilibrated with TE containing 0.5 M LiCl and washed with the same buffer. Further, after washing with TE containing 0.3 M LiCl, poly (A) RNA adsorbed with 2 mM ethylenediaminetetraacetate (EDTA) (pH 7.0) containing 0.01% sodium dodecyl sulfate (SDS) was eluted. Single-stranded cDNA was synthesized using the poly (A) RNA thus obtained. Specifically, 5 μg of poly (A) RNA and 0.5 μg of oligo dT primer (12-18mer) were placed in a sterilized 0.5 ml microcentrifuge tube, and diethylpyrocarbonate-treated sterilized water was added to make 12 μl. Incubated for 10 minutes and then placed on ice for 1 minute. To this was added 2 μl of 200 mM Tris-HCl (pH 8.4), 500 mM KCl solution, 2 μl of 25 mM MgCl ₂ , 1 μl of 10 mM dNTP and 2 μl of 0.1 M DTT, and incubated at 42 ° C. for 5 minutes, and then 200 units of Gibco (GibcoBRL) Co. Superscript II RT (1 μl) was added and incubated at 42 ° C. for an additional 50 minutes to carry out a cDNA synthesis reaction. The reaction was stopped by further incubation at 70 ° C. for 15 minutes and placed on ice for 5 minutes. 1 μl of RNase H (2 units / ml) was added to the reaction solution and incubated at 37 ° C. for 20 minutes.

(2) Preparation of Canine cDNA Phage Library Using 1 μg of the poly (A) RNA obtained in (1) above, two copies using oligo dT primers according to the attached manual with Amersham Timesaver cDNA Synthesis Kit Strand cDNA was synthesized and an EcoRI / NotI adapter was further ligated. Using this, a recombinant λgt10 vector was prepared in accordance with the attached manual with Amersham cDNA Rapid Cloning Module-λgt10, and a recombinant phage was prepared with an in vitro packaging module of Amersham according to the attached manual. .

(3) Cloning of canine IL12P40 gene Based on the nucleotide sequences of N-terminal and C-terminal of human IL12P40 (Reference 1), two primers of SEQ ID NOs: 3 and 4 were synthesized with a DNA synthesizer. 2 μl of cDNA obtained from dog liver and LPS-stimulated canine peripheral blood (1) above was taken in separate 0.5 ml microcentrifuge tubes, and each primer was 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1 Add 5 mM MgCl ₂ , 25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP, 4 units Taq DNA polymerase to add each reagent to a total volume of 100 μl. DNA thermal of Perkin-Elmer Cetus Co. under the conditions of denaturation of DNA at 94 ° C. for 1 minute, primer annealing at 55 ° C. for 2 minutes, primer extension at 72 ° C. for 3 minutes The cycler was reacted for 35 cycles. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 990 bp was prepared according to a conventional method (Reference 16).

  This DNA fragment was transferred to T-vector (Vector) (Novagen) from Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 2 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, it was confirmed by PCR under the same conditions as described above that the PCR fragment had been inserted into this plasmid, and the dideoxy method (Reference) using Genesis 2000 DNA analysis system (DuPont). In 17), the base sequence of one P40 subunit DNA out of the two subunits that seem to exhibit canine IL12 activity was determined. This sequence is shown in SEQ ID NO: 1.

Further, a 990 bp DNA fragment containing this sequence was labeled with ³² P using a random primer DNA labeling kit manufactured by Takara Shuzo Co., Ltd. to prepare a probe. The recombinant phage library prepared from the canine liver cDNA obtained in (2) above was formed as a plaque on E. coli NM514, and transcribed into Abondham's Hybond-N + according to a conventional method.

Hybond-N + is 5 × SSPE (ethylenediaminetetraacetate-sodium phosphate-saline buffer) (0.9 M NaCl, 50 mM NaH ₂ PO ₄ , 5 mM ethylenediaminetetraacetate (EDTA) pH 7.4), 5 × Denhardt Incubate at 65 ° C. for 2 hours in solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.1% sodium dodecyl sulfate (SDS), 100 μg / ml salmon sperm DNA Then, it was hybridized with the labeled probe 1 × 10 ⁶ cpm / ml prepared as described above in the same solution. After overnight incubation at 65 ° C., Hybond-N + was washed 3 times for 15 minutes in 0.2 × SSC (sodium citrate-saline buffer) (30 mM NaCl, 3 mM sodium citrate), 0.1% SDS. The sample was exposed to the Fuji Imaging Plate of Fuji Photo Film Co., Ltd. for 12 hours and analyzed with a bioimaging analyzer of Fuji Photo Film Co., Ltd. Plaques with a positive signal were rescreened according to conventional methods. As a result of screening three times, one recombinant phage having a positive signal was obtained. Phage DNA was extracted from this recombinant phage according to a conventional method, cleaved with the restriction enzyme ECOAR I, and then a DNA fragment of about 1.5 kb obtained by 1% agarose gel electrophoresis was prepared according to the conventional method. DNA Ligation Kit Ver. 2 was used to ligate with pUC118BAP-treated DNA (Eco-R I / BAP) from Takara Shuzo Co., Ltd. Using this, a plasmid DNA was prepared by a conventional method, and obtained using a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer Co.) and using a dye terminator cycle sequencing kit manufactured by PerkinElmer Co. according to the attached protocol. The base sequence of the DNA fragment was determined. Among these, the sequence encoding canine IL12P40 is shown in SEQ ID NO: 11.

(4) Cloning of canine IL12P35 gene Based on the nucleotide sequences of the N-terminus of human IL12P35 (Reference 1) and the C-terminus of bovine IL12P35, two primers of SEQ ID NOs: 5 and 6 were synthesized using a DNA synthesizer. Using the cDNA obtained from canine spleen-derived lymphocytes treated with chicken Newcastle disease virus in (1) above as a template, a DNA fragment of about 670 bp was obtained in the same manner as in (3) above, inserted into T-Vector, and canonical IL12 The base sequence of one P35 subunit DNA of the two subunits that seem to exhibit activity was determined. This sequence is shown in SEQ ID NO: 2.

  A labeled probe was prepared using a 670 bp DNA fragment containing this sequence. A recombinant phage library prepared from cDNA obtained from canine spleen-derived lymphocytes treated with chicken Newcastle disease virus obtained in (2) above was hybridized with a labeled probe in the same manner as in (3) above, Screening was performed. As a result, DNA was extracted from one recombinant phage having a positive signal, cleaved with restriction enzyme knot I, and then a DNA fragment of about 1.2 kb obtained by 1% agarose gel electrophoresis was obtained. It was ligated to a Not I site of pBluescript II (Stratagene) according to a conventional method. Plasmid DNA was prepared using this, and the base sequence of the obtained DNA fragment was determined using a fluorescent DNA sequencer. Among these, the sequence encoding canine IL12P35 is shown in SEQ ID NO: 12.

Reference Example 2 Production of Canine IL12 (1) Preparation of Canine IL12 Expression Vector Expression vector pCDL-SRα296 (Reference 18) was cleaved with restriction enzyme EcoRI, and the end was dephosphorylated with bacterial alkaline phosphatase. This was prepared by 1% agarose gel electrophoresis and a DNA fragment of about 3.6 kb was prepared according to a conventional method. On the other hand, the CaIL12P40 DNA fragment was prepared by adding a primer to which two types of EcoRI cleavage sites of SEQ ID NOs: 7 and 8 were added, and appears to exhibit canine IL12 activity inserted into the T-Vector prepared in Reference Example 1 (2). Using one P40 subunit DNA as a template, DNA denaturation conditions are 94 ° C for 1 minute, primer annealing conditions are 55 ° C for 2 minutes, primer extension conditions are 72 ° C for 3 minutes, number of cycles PCR was performed at 30, and after ethanol precipitation, the DNA was cleaved with restriction enzyme EcoRI and a DNA fragment of about 990 bp was prepared by 1% agarose gel electrophoresis. In addition, primers were prepared by adding two types of EcoRI cleavage sites of SEQ ID NOs: 13 and 14, PCR was performed using canine IL12P40 DNA inserted into pUC118 prepared in Reference Example 1 (2) as a template, and digested with EcoRI. An approximately 990 bp DNA fragment was prepared. Each obtained canine IL12P40 DNA fragment was ligated to pCDL-SRα296 prepared as described above using T4 DNA ligase. Using this, Escherichia coli was transformed, and plasmid DNA was prepared from the obtained transformant to obtain FOCaIL12P40 and FOCaIL12P40FL expressing canine IL12P40.

  Further, pCDL-SRα296 was cleaved with the restriction enzyme PstI, dephosphorylated, and a DNA fragment of about 3.6 kb was prepared by electrophoresis. The canine IL12P35 DNA fragment was prepared by adding two types of Pst I cleavage sites of SEQ ID NOs: 9 and 10 and showing canine IL12 activity inserted in the T-Vector prepared in Reference Example 1 (3). PCR was performed at 94 ° C, 1 minute, 55 ° C, 2 minutes, 72 ° C, 3 minutes, 30 cycles using one P35 subunit DNA of the two possible subunits as a template, and after ethanol precipitation, the restriction enzyme PstI Disconnected with About 670 bp DNA fragment was prepared from this by 1% agarose gel electrophoresis. In addition, primers were prepared by adding two types of PstI cleavage sites of SEQ ID NOS: 15 and 16, PCR was performed using the canine IL12P35 DNA inserted into pUC118 prepared in Reference Example 1 (2) as a template, and cleaved with PstI. An approximately 670 bp DNA fragment was prepared. Each of the obtained canine IL12P35 DNA fragments was ligated to pCDL-SRα296 prepared by cutting with PstI using T4 DNA ligase as described above, E. coli transformation, plasmid DNA preparation was performed, and FOCaIL12P35 and FOCaIL12P35FL expressing canine IL12P35 were obtained. Obtained.

  Furthermore, the base sequences of canine IL12P40 DNA and canine IL12P35 DNA in these four expression plasmids were confirmed.

(2) Production of canine IL12 in monkey COS cells 5 μg of FOCaIL12P40 and FOCaIL12P35 obtained in (1) above were added to 50 mM Tris-HCl buffer (pH 7.5), 400 μg / ml DEAE dextran (manufactured by Amersham) and It is added to 4 ml of ERDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 100 μM chloroquine (manufactured by Sigma). On the other hand, COS-1 cells (ATCC CRL-1650) grown to 10% fetal bovine serum (Gibco, hereinafter abbreviated as FBS) using a 10 cm diameter dish until 50% confluent were once with PBS. After washing, 4 ml of the DNA mixture obtained above was added and cultured under conditions of 5% CO ₂ and 37 ° C. After 4 hours, the cells were washed with PBS and then cultured in 20 ml of ERDF medium under conditions of 5% CO ₂ and 37 ° C. for 4 days to obtain a culture supernatant in which canine IL12 was produced.

(3) Preparation of canine IL12-producing recombinant baculovirus P40 and P35 subunit cDNAs were respectively ligated to the restriction enzyme XbaI and SmaI cleavage sites downstream of the promoter of baculovirus transfer vector pAcAB3 (manufactured by Pharmingen) according to a conventional method. A recombinant transfer vector was obtained. Furthermore, a recombinant baculovirus was prepared using a Pharmingen baculovirus transfection kit according to the attached manual.

(4) Production of canine IL12 in insect cells Sf21 cells obtained by planarizing the recombinant baculovirus obtained in (3) above to confluence in a 75 cm ² flask with Pharmingen's baculogold protein-free insect medium ( Sponoptera Flugiperda origin, obtained from Farmingen) and cultured for 4 days to obtain a culture supernatant in which canine IL12 was produced.

Reference Example 3 Antitumor Effect of Canine IL12 The activity of canine IL12 produced in Reference Examples 2 (2) and (4) was measured as follows. For assaying canine IFNγ-inducing activity from canine lymphocytes, antiviral activity and canine cell class II MHC expression enhancing activity were measured. Lymphocytes were isolated from the dog spleen and suspended in 10% FBS-ERDF at a cell density of 10 ⁶ cells / ml, 2.5 ml of which was added to a 250 U, 6 cm dish of Genzyme. To this, 2.5 ml of the culture supernatant obtained in (2) above and 250 U of human IL2 (Zenzyme) were added, and cultured for 2 days under the conditions of 5% CO ₂ and 37 ° C. The antiviral activity of this culture solution was measured using MDCK (ATCC CCL-34) as a sensitive cell and MDCK (ATCC CCL-34) as a sensitive cell. As a result, an antiviral activity of 2 × 10 ⁵ dilution units / ml or more was confirmed. Also, the (4) result of measurement in the same manner as the antiviral activity of the resulting culture supernatant, 10 ⁷ dilution units / ml or more antiviral activity was detected. On the other hand, no antiviral activity was observed in the control cell culture medium in which 10 μg of pCDL-SRα296 was introduced into COS-1 cells in the same manner as in (2) and the culture medium in which Sf21 cells were cultured for 3 days.

In addition, using a canine mammary tumor tissue-derived cell line FCBR1 expressing class II MHC, the class II MHC expression enhancing activity in each of the above culture solutions was measured. 10 ⁵ FCBR1s were adhered to a 6 cm dish, and ⁵ ml of each culture solution was added thereto and cultured overnight under 5% CO ₂ and 37 ° C. conditions. After culture, the cells were detached with trypsin and centrifuged in a 1.5 ml microcentrifuge tube. To this, 10 μl of rat anti-dog class II MHC monoclonal antibody from Stratagene was added, suspended in 50 μl of 10% FBS-ERDF, and allowed to stand on ice for 1 hour. After washing with phosphate buffer (PBS), the suspension was suspended in 5 μl of FITC-labeled rabbit anti-rat monoclonal antibody (manufactured by Serotech) and 50 μl of 10% FBS-ERDF, and allowed to stand on ice for 1 hour. After washing with PBS, analysis was performed with FACScan from Becton Dickinson Co., Ltd. As a result, canine IL12 produced with COS1 and Sf21 increased the expression level of class II MHC on FCBR1 by about 20% and 60%, respectively. From these results, it was found that canine IL12 has an activity of inducing canine IFNγ by acting on canine lymphocytes.

In addition, the proliferation promoting activity of blasted canine lymphocytes was measured. Lymphocytes were isolated from canine peripheral blood, suspended in 10% FBS-ERDF at a cell density of 10 ⁶ cells / ml, and 5 ml of this was added to a 6 cm dish. To this was added PHA at a concentration of 5 μg / ml, and the cells were cultured under conditions of 5% CO ₂ and 37 ° C. for 3 days to allow lymphocytes to blast. The blasted lymphocytes were suspended in 10% FBS-ERDF at a cell density of 10 ⁶ cells / ml, and 50 μl was added per well of a 96-well microplate. To this, 50 μl of the culture supernatant obtained in (2) above was added per well. As a control, 50 μl of 10% FBS-ERDF alone was added per well. These were further cultured under conditions of 5% CO ₂ and 37 ° C. for 3 days, and then the proliferation promoting activity of blastogenic lymphocytes of canine IL12 was measured by the MTT assay method of Reference 14. That is, 10 μl of a 5 mg / ml MTT (Sigma) solution was added per well and further cultured for 6 hours. After adding 150 μl of a 0.01 N hydrochloric acid isopropanol solution, the cells were disrupted with ultrasonic waves, and the absorbance at a wavelength of 595 nm was measured with a microplate reader (Bio Rat Model 13550). As a result, while the absorbance of the control was 0.69 on average, the canine IL12 produced by COS-1 had an average of 1.52, and the proliferation promoting activity of blastogenic lymphocytes was confirmed to be about twice or more. It was.

Furthermore, the antitumor effect of canine IL12 on canine tumors was examined. Lymphocytes were isolated from canine peripheral blood, suspended in 10% FBS-ERDF at a cell density of 5 × 10 ⁶ cells / ml, 5 ml of which was added to a 6 cm dish. To this, 500 U of recombinant human IL2 from Boehringer Mannheim Co., Ltd. was added and cultured for 3 days under conditions of 5% CO ₂ and 37 ° C. On the other hand, canine tumor cells FCBR1 and A72 (ATCC CRL-1542) were suspended in 10% FBS-ERDF to a cell density of 10 ⁵ cells / ml, and 50 μl per well of 96-well plate was added and allowed to adhere to the plate. . To this was added 50 μl of canine lymphocytes stimulated with human IL2, and further 100 μl of the culture supernatant obtained in (2) above expressing canine IL12 or 100 μl of 10% FBS-ERDF as a control, 5% CO ₂ , The cells were cultured for 2 days at 37 ° C. After incubation, the supernatant was completely removed and MTT assay was performed. % Cytotoxicity was calculated by the following formula.
% Cytotoxicity = (1-OD2 / OD1) × 100

  Here, OD1 = absorbance at a wavelength of 595 nm of canine tumor cells cultured only in a medium OD2 = absorbance at a wavelength of 595 nm of canine tumor cells cultured with canine lymphocytes.

  As a result, the control was 34% in FCBR1, whereas the canine IL12 produced by COS-1 showed about 75% cytotoxicity. In addition, control was 22% for A72, whereas canine IL12 showed about 83% cytotoxicity. It was found that canine IL12 activates canine lymphocytes and exerts an antitumor effect on canine tumor cells.

Reference Example 4 Purification of Canine IL12 Using the cell culture supernatant obtained in Reference Example 2 (4), fractionation of canine IL12 and canine P40 homodimer with a sulfopropyl carrier was examined. Purification was performed at pH: 7. The culture supernatant was applied to a column packed with a sulfopropyl carrier, and then the column was washed with a sufficient amount of 20 mM phosphate buffer (pH: 7). Elution was performed in increments of 5 mM until the NaCl concentration reached 1M. As a result, canine IL12 was detected at 350 to 550 mM, and canine P40 homodimer was detected in elution fractions at respective NaCl concentrations of 600 to 750 mM (Western blotting). Dog IL12 and dog P40 homodimer were not detected in the other eluted fractions and alkali-washed fractions.

  Each fraction from which canine IL12 and canine P40 homodimer was eluted was further applied to a column packed with blue sepharose carrier, and the column was washed with a sufficient amount of 20 mM phosphate buffer (pH: 7), and then the NaCl concentration was adjusted. Elution was performed in increments of 5 mM from 500 mM to 2M. As a result, the canine IL12 was 1.1 to 1.2 M, and the canine P40 homodimer was detected in the elution fractions having respective NaCl concentrations of 1.5 to 2 M (Western blotting). According to SDS-PAGE analysis, the purity of canine IL12 in each fraction with the blue sepharose carrier from which canine IL12 was eluted was 98% or more.

Reference Example 5 Detection and biological activity of purified canine IL12 In vitro detection and activity measurement of purified canine IL12 obtained in Reference Example 4 were performed as follows. First, for detection, canine IL12 in the culture supernatant of insect cells obtained in Reference Example 2 (4) was detected by Western blotting. The culture supernatant was subjected to SDS-PAGE in a pagel of Ato Corporation. Then, after blotting according to a conventional method on a clear blot membrane of Ato Co., Ltd., the membrane is blocked ace containing rabbit serum containing anti-canine P40 polyclonal antibody obtained in (1) above (Dainippon Pharmaceutical Co., Ltd.) The solution was reacted for 6 hours, washed three times with PBS containing 0.02% Tween 20, and further reacted with a block ace solution containing peroxidase-labeled goat anti-rabbit IgG (manufactured by Biorat) for 6 hours. After washing, color was developed with Konica Immunostain HRP1000 manufactured by Konica. As a result, a band of about 75 kD was detected. Furthermore, as a result of analyzing the culture supernatant of insect cells obtained in Reference Example 2 in the same manner using rabbit serum containing anti-canine P35 polyclonal antibody, only a band of about 75 kD was detected. From this, it was found that the band of about 75 kD was canine IL12 composed of the heterodimers of canine P40 and P35. Antiviral activity was measured for assay of canine IFNγ induction activity from canine lymphocytes of purified canine IL12. Lymphocytes were isolated from canine peripheral blood, and 100 μl per well at a cell density of 10 ⁶ cells / ml was added to 10% FBS-ERDF in a 96-well microplate, and recombinant human IL2 from Genzyme was added to each well. Was added thereto, and purified canine IL12 (1 to 10 ³ pg / ml) was added thereto, followed by culturing for 72 hours under conditions of 5% CO ₂ and 37 ° C. Antiviral activity in 100 μl of each culture supernatant after culture was measured. The antiviral activity of this culture solution was measured according to the CPE method of Reference 16, using vesicular Stomatis Virus as the virus and FCBR1 as the sensitive cell. As a result, 2 × 10 ³ to 10 ⁵ units / ml of antiviral activity was confirmed in the culture supernatant added with purified canine IL12 (1 to 10 ³ pg / ml) alone.

Reference Example 6 Production of Canine IL12 Formulation Each solution of purified canine IL12 obtained in Reference Example 4 (containing 0.01% by weight of polyoxyethylene hydrogenated castor oil 60) was desalted by dialysis, and physiological for injection. Saline, low molecular weight gelatin for injection (manufactured by Nitta Gelatin Co., Ltd.), and sorbitol were added to prepare a final gelatin concentration of 0.5% and a final concentration of sorbitol of 30%. Further, the pyrogen was removed by treatment with positivedyne (manufactured by Paul Co., Ltd.) and then dispensed in 1 ml portions into glass vials sterilized by dry heat at 250 ° C. for 2 hours. Thereafter, it was aseptically freeze-dried to obtain a canine IL12 preparation containing 1 pg to 5 μg of canine IL12 in one vial. This formulation was stable at room temperature and dissolved well in distilled water or saline.

Reference Example 7 Examination of Antitumor Effect of Canine IL12 Formulation The activity measurement of the canine IL12 formulation of Reference Example 6 was performed in the same manner as in Reference Example 3 as follows. For assaying canine IFNγ-inducing activity from canine lymphocytes, antiviral activity and canine cell class II MHC expression enhancing activity were measured. Lymphocytes were isolated from the dog spleen and suspended in 10% FBS-ERDF at a cell density of 10 ⁶ cells / ml, 2.5 ml of which was added to a 250 U, 6 cm dish of Genzyme. To this, 1 ml of the preparation obtained in Reference Example 6 and 250 U of human IL2 (Zenzyme) were added, and cultured for 2 days under the conditions of 5% CO ₂ and 37 ° C., as a virus, and as a sensitive cell MDCK (ATCC CCL-34) was used to measure the antiviral activity of this culture solution according to the CPE method of Reference 19. As a result, an antiviral activity of 2 × 10 ⁵ dilution units / ml or more was confirmed. As a result of measurement in the same manner as the anti-viral activity, 10 ⁷ dilution units / ml or more antiviral activity was detected. On the other hand, no antiviral activity was observed in the preparation containing no canine IL12 (control).

Example 1 Administration of Canine IL12 to Dogs with Tumors (1) Method of Administration of Canine IL12 Dogs with tumors were administered to 19 cases including mammary tumors, fibrosarcomas, cutaneous lymphomas, and melanomas. Prior to administration of canine IL12, blood collection and measurement of body weight, body temperature, etc. were also performed for general hematological examinations. Canine IL12 is then administered according to the schedule shown in Table 1. That is, 0.1 μg / kg was administered twice weekly by intravenous injection in the first week, and 4 μg / kg was administered twice weekly by intravenous and local injections in the second to fourth weeks. On week 5, 4 μg / kg was administered twice a week by intravenous injection and local injection. In the case of local injection, even if there were multiple tumors, it was administered only to one tumor and only to the same tumor throughout the period. The topical injection was administered to the tumor parenchyma and its vicinity. Other drugs were basically not used in combination. However, administration of drugs unrelated to the antitumor effect was not limited.

(2) Items, timing, and results of observation test Table 2 shows the observation test of clinical symptoms on the day of canine IL12 administration. Moreover, it implemented before dog IL12 administration once a week. In other words, the observation test items are clinical symptoms (dehydration, breathing mode, energy, appetite, appetite for drinking, diarrhea, vomiting), body temperature, weight, hematology (blood cell system (white blood cell count, hematocrit, platelet count, blood image) , Electrolyte (Na, K, Cl), biochemical examination (BUN, Crea., GOT, GPT, CPK, Glucose, TP, Alb, Glob, ALP). There were no serious symptoms and no abnormalities were observed in the laboratory values.

(3) Preparation of canine leukocytes from canine blood The canine whole blood obtained by blood collection is layered so as not to be mixed in a centrifuge tube containing Histopaq-1077 in advance. Centrifugation was performed at 400 g for 30 minutes, and a leukocyte fraction was obtained using a Pasteur pipette. Next, ERDF-10% FBS medium was added and centrifuged again, and the precipitate fraction was again added with ERDF-10% FBS medium, placed in a 10 cm petri dish, and cultured at 37 ° C. in a CO ₂ incubator. After 1 hour, the culture supernatant of the petri dish was collected, and after centrifugation, the precipitated fraction was suspended in ERDF-10% FBS medium to obtain dog leukocytes. The prepared white blood cells 1 × 10 ⁷ cells were treated in nylon wool packed in a 10 mL syringe (manufactured by Terumo Corporation) and allowed to stand at 37 ° C. for 15 minutes. Next, the T cell fraction was prepared by extrusion with phosphate buffer (PBS). MRNA was prepared from the T cell fraction using an mRNA microprep quick pulley kit (Amersham). cDNA synthesis was performed with Oligo dT primer using Rever Tra-ACE- (TOYOBO). PCR was performed using premix taq (Takara Shuzo Co., Ltd.). PCR conditions were heat denaturation: 94 ° C., 1 minute, annealing: 56 ° C., 30 seconds, extension reaction: 72 ° C., 1 minute, and 30 cycles. The primer sequences are SEQ ID NOs: 17, 18 for β-actin, SEQ ID NOs: 19, 20 for IFN-γ, and SEQ ID NOs: 21, 22 for IL10.

  The obtained PCR product was electrophoresed on a 1% agarose gel. In addition, the electrophoresed sample was extracted from the gel using Geneclean II kit (manufactured by Funakoshi) and ligated to T-vector (Novagen) using DNA Ligation kit Ver.2. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, it was confirmed by PCR under the same conditions as described above that the PCR fragment had been inserted into this plasmid, and ABI PRISM 377XL (Applied Biosystems) was used with the BigDye Terminator Cycle Sequencing method. When the nucleotide sequence of each cytokine DNA was confirmed, it was confirmed that it was canine IFN-γ or canine IL10.

Example 2 Correlation Study between Cytokine Expression in Dog Blood and Tumor Regression Effect Of 19 cases administered with canine IL12 in Example 1, tumor regression was observed in 8 cases. When IFN-γ and IL10 gene analysis was performed in dog blood of all cases, as shown in Table 3, it was confirmed that all 8 effective cases showed increased IFN-γ expression and decreased IL10 expression. . In addition, regarding the expression level of each gene, in the period of 8 weeks in which the expression was examined, when the period during which expression is observed is observed over 8-6 weeks, +++, when it is observed over 5-3 weeks, +
+, In the case of 2 to 1 week, +, and in the case where no detection was possible at all.

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Claims (4)

A method for evaluating canine anti-tumor effect by canine interleukin 12, which comprises measuring changes in expression of cytokine gene of leukocytes in dog blood. When the cytokine is canine interferon γ and / or canine interleukin 10, and when the expression of canine interferon γ gene is observed, it is judged that canine interleukin 12 has an antitumor effect, and the expression of canine interleukin 10 gene is observed. The method for evaluating canine anti-tumor effect by canine interleukin according to claim 1, wherein it is determined that there is no anti-tumor effect by canine interleukin. An evaluation kit for evaluating canine anti-tumor effects of canine interleukin 12 by measuring changes in cytokine gene expression in canine leukocytes collected from dogs administered canine interleukin 12. The evaluation kit for evaluating the canine anti-tumor effect of canine interleukin 12 according to claim 3, wherein the cytokine is canine interferon γ and canine interleukin 10.