JP5115921B2 - Diagnostic and therapeutic agents for renal cancer - Google Patents

Description

  The present invention relates to a diagnostic agent for renal cancer using an anti-TLR3 antibody and a therapeutic agent for renal cancer using a TLR3 agonist.

  Renal cell carcinoma (RCC) accounts for 2-3% of adult malignant tumors, and both morbidity and mortality tend to increase in Japan and the United States (Non-Patent Documents 1 and 2). The main histological types of renal cell carcinoma are (1) Clear cell RCC (about 80%), (2) Papillary RCC (10-15%), (3) Chromophobe RCC (about 5%), (4) Collecting There is a duct carcinoma (about 1%) (Non-patent Document 3). In addition, sarcomatoid change in which differentiation becomes poor and cancer cells change like sarcoma can be associated with all tissue types.

  For kidney cancer, the number of early detection cases has increased due to advances in imaging techniques, but the number of cases with distant metastasis has not yet decreased. In other words, for renal cancer, it means that there was no delay in discovery or there was no appropriate treatment in the past. Therefore, development of a simple and accurate early detection technique and an effective treatment method for renal cancer is desired. In recent years, research on diagnostic methods and therapeutic methods targeting proteins that are specifically expressed in many cancer types has been actively conducted, but most of them have only been analyzed at the gene level and are not progressing at present. It is.

  Early surgical resection is considered effective as a current treatment for renal cell carcinoma, but there is no effective treatment for advanced renal cell carcinoma such as patients who cannot be resectable or relapsed. That is, as treatment for these advanced renal cell carcinomas, chemotherapy and radiation therapy usually performed in other carcinomas are said to have little effect (Non-patent Documents 4 and 5). On the other hand, immunotherapy such as interferon α (IFNα) and interleukin 2 is effective in some cases. Renal cell carcinoma is a characteristic, and IFNα is widely used for the treatment of advanced renal cell carcinoma in Japan. It has been. However, the success rate of IFNα for advanced renal cell carcinoma cases is only about 10-20% (Non-patent Documents 6 and 7). Therefore, development of an effective treatment for advanced renal cell carcinoma is desired.

  Toll-like receptor (TLR) is a molecule that acts on defense against infection by activating innate immunocytes in response to stimuli from pathogens. To date, 13 types of TLRs are known (10 types in humans), and most of them have been identified as ligands (Non-patent Document 8). These include LPS, lipoteichoic acid, and peptidoglycan, which are bacterial cell wall components. And bacterial flagellar component flagellin, bacterial or virus-derived CpG DNA, virus-derived single-stranded RNA, virus-derived double-stranded RNA, and virus-derived protein. Many TLRs exhibit the same inflammatory action as cytokines TNFIα and IL-6 by transferring the transcription factor NF-κB to the nucleus via the MyD88-dependent pathway of the adapter molecule after ligand binding. It has traditionally been thought to have its main role in defending against infection by pathogens. However, some TLRs are also expressed in cancer cells, and it has recently been considered that TLRs create an environment that is advantageous for the growth of cancer cells (Non-patent Document 9). ). On the other hand, there is a possibility that TLR agonists may be used for cancer treatment (Non-patent Document 10). In other words, TLR can be said to be a “double-edged sword” that can work both favorably and disadvantageously for cancer (Non-patent Document 11).

  TLR3 was developed in 1998 by Rock, F. et al. L. After the discovery by (Non-Patent Document 12), many documents were reported and revealed to be involved in innate immunity. That is, unlike other TLRs, TLR3 is a double-stranded RNA (dsRNA) produced by a virus, and is an antiviral action by inducing IFN production in the MyD88-independent TRIF pathway after ligand binding. And activation of NFκB are known (Non-patent Document 13).

  Since TLR3 is involved in innate immunity, many literatures relating to medical care have been reported based on the inference that it can be applied to inflammation and cancer (Patent Documents 1 to 4). They are patent documents showing an antagonistic or agonistic action assuming an influence on IFN-β as a physiological function of TLR3, an IFN-inducible gene group, and an inflammatory cytokine.

  TLR3 expression is expressed in dendritic cells, fibroblasts, intestinal epithelial cells, glial cells, umbilical vein endothelial cells as cells, endometrium in normal tissues, kidney, colon cancer in cancer tissues (Patent Document 5) and It has been shown that TLR3 is highly expressed in kidney cancer (Non-Patent Document 14) tissue. However, Patent Literature 5 and Non-Patent Literature 14 are only gene expression analysis by microarray, and protein expression is not clarified.

Patent Literature 5 discloses the diagnosis and treatment of TLR3 and cancer using antibodies for breast cancer, colon cancer, lung cancer, gastric cancer, prostate cancer, uterine cancer, cervical cancer, endometrial cancer, skin cancer, and leukemia. Treatment is described in the claims, but only shows results in colorectal cancer and there is no example for renal cancer. Furthermore, in Patent Document 4, although the diagnosis and treatment of various types of cancers including renal cancer using anti-TLR3 antibodies are described in the claims, Examples relating to renal cancer are only described for breast cancer. There is no.
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  Although early surgical resection is effective for the treatment of renal cancer, the treatment by surgery is not only difficult to treat metastases, but often involves both invasion and complications. For advanced cancers such as radically unresectable and recurrent cases, there is no effective treatment, so early renal cancer needs to be diagnosed early. Therefore, development of an early diagnosis method and treatment method for renal cancer is desired.

  An object of the present invention is to provide a method for accurately diagnosing early renal cancer and an effective treatment method.

As a result of conducting a search for cell surface molecules specifically expressed in renal cancer by microarray analysis, it has been clarified that in Clear cell RCC, a gene group involved in innate immune response is characteristically highly expressed, It was considered that the innate immune response could lead to the pathogenesis of Clear cell RCC. In addition, it was confirmed that TLR3, which is reported to be involved in innate immune response, was highly expressed specifically at Clear cell RCC at the gene level, and at the same time, high expression specifically at Clear cell RCC was observed by RT-PCR. It could be confirmed.
Next, as a result of examining tissue immunostaining with an anti-TLR3 antibody for Clear cell RCC tissue, TLR3 protein was stained in 184 cases out of 189 cases as fine granules in the cytoplasm RCC tissue. Of these, 139 cases were confirmed to be intensely stained, whereas Chromophobe RCC was not stained at all. Furthermore, TLR3 positive images could be confirmed in 6 out of 8 cases of lung metastasis of renal cancer.
On the other hand, by adding a ligand or siRNA to a plurality of renal cancer cell lines, a growth inhibitory effect proportional to the TLR3 expression level was observed. Furthermore, the expression of IFNβ, which can be expected to have an antitumor effect, was induced by the addition of a ligand to the renal cancer cell line. This result is a method for directly treating TLR3-expressing renal cancer cells, and is found to be different from conventional cancer treatment through activation by ligands such as tree cells that are innate immunity responsible cells. The invention has been completed.

That is, the present invention provides a diagnostic agent for renal cancer containing an anti-TLR3 antibody.
The present invention also provides a method for diagnosing renal cancer, wherein a TLR3 protein is detected by reacting an anti-TLR3 antibody with a sample collected from a subject or administering an anti-TLR3 antibody to a subject. It is.
Furthermore, the present invention provides a method for treating renal cancer, characterized by administering a TLR3 agonist, and a therapeutic agent for renal cancer containing a TLR3 agonist.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to perform early diagnosis (biopsy) of renal cancer using a method and blood diagnosis performed by molecular imaging (PET) that can be specifically detected, and tissue collected by biopsy, and new renal cancer can be quickly developed. A treatment plan can be formulated. In addition, non-invasive treatment of renal cancer that does not involve surgery and places no burden on the patient can be performed.

  The disease diagnosed and treated according to the present invention is renal cancer. The animal to be diagnosed and treated is preferably a human, but may be a mammal other than a human such as a dog, cat, rabbit, mouse, rat, or guinea pig.

  When diagnosing renal cancer in the present invention, if TLR3 protein is detected in the blood and organ tissue of the subject, it is determined that the subject is likely to have renal cancer. Further, by measuring the TLR3 protein concentration in the blood or tissue of a patient diagnosed with renal cancer, it is possible to determine whether or not the patient is a treatment target patient (selection of the treatment target patient). Furthermore, after the treatment of renal cancer, in the TLR3 protein measurement, if the amount of protein is decreased from before surgery, it is determined that the course of treatment is good. On the other hand, when the amount of TLR3 protein after treatment does not decrease or increases, it is determined that there is recurrence and metastasis. Diagnosis of renal cancer can be performed by image diagnosis, biopsy and blood diagnosis.

Image diagnosis can be performed by detecting TLR3 protein by image diagnosis after administration of a labeled anti-TLR3 antibody. More specifically, a probe labeled with a radioisotope as a labeling substance is administered to a subject to an anti-TLR3 antibody, and renal cancer tissue can be detected by PET or SPECT. The radioactive isotope used may be a substance known to those skilled in the art, but is preferably a positron emitting radioactive isotope, more preferably ¹¹ C, ¹³ N, ¹⁸ F, ¹⁵ O, ^{94 m} Tc, ¹²⁴ I. It is. The anti-TLR3 antibody can be labeled with a radioisotope by a method known to those skilled in the art.

  As a method for diagnosing renal cancer by biopsy, an immunoassay can be performed using an organ tissue obtained from a subject as a sample. For example, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method and the like can be mentioned, and immunostaining is preferred. The above-described immunological methods such as immunostaining can be performed by methods known to those skilled in the art.

  As another mode of diagnosing renal cancer by biopsy, an immunohistological staining method using an anti-TLR3 antibody as a primary antibody can be performed. Specifically, a specimen obtained from a subject is fixed by paraffin or freezing by a known method to prepare a section. Next, the section is treated with an anti-TLR3 antibody as a primary antibody and a biotin-labeled antibody that recognizes IgG as a secondary antibody. As the secondary antibody, a known antibody that recognizes IgG can be used, and examples thereof include a rabbit anti-IgG antibody. A labeling substance is bound to the secondary antibody, and the presence or absence of a protein in the section is detected by a known method suitable for each labeling substance. Further, without using a secondary antibody, a labeling substance can be bound to an anti-TLR3 antibody and an immunohistological staining method can be performed. As the labeling substance, a substance known to those skilled in the art can be used, and examples thereof include peroxidase and FITC. The binding between the antibody and the labeling substance can be performed by a method known to those skilled in the art, and specific examples include a binding method using streptavidin and biotin.

  As another mode of diagnosing renal cancer, an immunological assay can be performed using blood, serum, or plasma obtained from a subject as a sample. For example, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunodiffusion method, etc. are preferable, but enzyme immunoassay is preferable, and enzyme binding is particularly preferable. It is an enzyme-linked immunosorbent assay (ELISA) (for example, sandwich ELISA). The above-described immunological methods such as ELISA can be performed by methods known to those skilled in the art.

  As a method for diagnosing renal cancer using blood, serum, or plasma as a specimen, for example, an anti-TLR3 antibody is immobilized on a support, a test sample is added thereto, and incubation is performed to bind the anti-TLR3 antibody and protein. A method of washing and detecting the TLR3 protein bound to the support via the anti-TLR3 antibody can be mentioned.

  Examples of the support used for immobilizing the anti-TLR3 antibody in the present invention include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resin, polystyrene resin, polyacrylamide resin, nylon resin, and polycarbonate resin, And an insoluble support such as glass and ferrite. These supports can be used in the form of beads or plates. In the case of beads, a column packed with these can be used. In the case of a plate, a multiwell plate (96-well multiwell plate or the like), a biosensor chip, or the like can be used. The anti-TLR3 antibody and the support can be bound by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be commercially available.

  The binding between the anti-TLR3 antibody and the TLR3 protein in the sample is usually performed in a buffer. As the buffer solution, for example, a phosphate buffer solution, a Tris buffer solution, a citrate buffer solution, a borate buffer solution, a carbonate buffer solution, or the like is used, and it may be in a pH range that is usually used. Incubation is performed under conditions that are already well used, for example, incubation at 4 ° C. to 37 ° C. for 1 hour to 24 hours. The washing after the incubation may be anything as long as it does not interfere with the binding between the anti-TLR3 antibody and the TLR3 protein. For example, a buffer containing a surfactant such as Tween-20 is used.

  In the method for detecting a TLR3 protein according to the present invention, a control sample may be installed in addition to a test sample for which the TLR3 protein is desired to be detected. Examples of the control sample include a negative control sample not containing TLR3 protein and a positive control sample containing TLR3 protein. In this case, it is possible to detect the TLR3 protein in the test sample by comparing the result obtained with the negative control sample containing no TLR3 protein with the result obtained with the positive control sample containing the TLR3 protein. is there. In addition, a series of control samples with varying concentrations are prepared, the detection results for each control sample are obtained as numerical values, a standard curve is created, and a test curve is created based on the standard curve from the test sample values. It is also possible to quantitatively detect the TLR3 protein contained in the sample.

  As a preferred embodiment of the detection of the TLR3 protein bound to the support through the anti-TLR3 antibody, a method using an anti-TLR3 antibody labeled with a labeling substance can be mentioned. For example, the test sample is brought into contact with the anti-TLR3 antibody immobilized on the support, and after washing, detection is performed using a labeled antibody that specifically recognizes the TLR3 protein.

The anti-TLR3 antibody can be labeled by a generally known method. As the labeling substance, a labeling substance known to those skilled in the art, such as a fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance, and a radioactive substance, can be used. Specific examples include radioisotopes ( ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide Examples include oxidase, microperoxidase, biotin, and ruthenium. When biotin is used as the labeling substance, it is preferable to further add streptavidin to which an enzyme such as peroxidase is bound after adding the biotin-labeled antibody. For binding of the labeling substance and the anti-TLR3 antibody, a known method such as a glutaraldehyde method, a maleimide method, a pyridyl disulfide method, or a periodic acid method can be used.

  Specifically, a solution containing an anti-TLR3 antibody is added to a support such as a plate or a bead, and the anti-TLR3 antibody is fixed to the support. After washing the plate or beads, blocking is performed with, for example, BSA, gelatin, albumin or the like in order to prevent non-specific binding of proteins. Wash again and add test sample to plate or beads. After incubation, wash and add labeled anti-TLR3 antibody. After moderate incubation, the plate or beads are washed and the labeled anti-TLR3 antibody remaining on the support is detected. Detection can be performed by methods known to those skilled in the art. For example, in the case of labeling with a radioactive substance, it can be detected by liquid scintillation or RIA. In the case of labeling with an enzyme, a substrate is added, and an enzymatic change of the substrate, for example, color development can be detected with an absorptiometer. Specific examples of the substrate include 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylenediamine (ortho-phenylenediamine), 3,3 ′ , 5,5′-tetramethylbenzidine (TMB) and the like. In the case of a fluorescent substance or a chemiluminescent substance, it can be detected by a luminometer.

  As a particularly preferred embodiment of the TLR3 protein detection method of the present invention, a method using an anti-TLR3 antibody labeled with biotin and streptavidin can be mentioned.

  Specifically, a solution containing an anti-TLR3 antibody is added to a support such as a plate to immobilize the anti-TLR3 antibody. After washing the plate, it is blocked with, for example, BSA in order to prevent non-specific protein binding. Wash again and add the test sample to the plate. After incubation, wash and add biotin-labeled anti-TLR3 antibody. After moderate incubation, the plate is washed and avidin conjugated with an enzyme such as alkaline phosphatase or peroxidase is added. After the incubation, the plate is washed, a substrate corresponding to the enzyme bound to avidin is added, and TLR3 protein is detected using the enzymatic change of the substrate as an index.

  Other embodiments of the TLR3 protein detection method of the present invention include a method using one or more primary antibodies specifically recognizing TLR3 protein and one or more secondary antibodies specifically recognizing the primary antibody. Can do.

  For example, the test sample is contacted with one or more types of anti-TLR3 antibodies immobilized on a support, incubated, washed, and the TLR3 protein bound after washing is treated with the primary anti-TLR3 antibody and the primary antibody. It is detected by one or more secondary antibodies that are specifically recognized. In this case, the secondary antibody is preferably labeled with a labeling substance.

As another embodiment of the method for detecting a TLR3 protein of the present invention, a detection method using an agglutination reaction can be mentioned. In this method, TLR3 protein can be detected using a carrier sensitized with an anti-TLR3 antibody. As the carrier for sensitizing the antibody, any carrier may be used as long as it is insoluble, does not cause a non-specific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles and the like can be used, but it is preferable to use polystyrene latex particles. The sensitized particles are mixed with the sample and stirred for a certain time. The higher the concentration of TLR3 protein in the sample, the greater the degree of particle aggregation. Therefore, the TLR3 protein can be detected by observing the aggregation with the naked eye.
It is also possible to detect turbidity due to aggregation by measuring with a spectrophotometer or the like.

  As another embodiment of the protein detection method of the present invention, for example, a method using a biosensor utilizing the surface plasmon resonance phenomenon can be mentioned. A biosensor using the surface plasmon resonance phenomenon can observe a protein-protein interaction in real time as a surface plasmon resonance signal without labeling with a minute amount of protein. For example, by using a biosensor such as BIAcore (manufactured by Biacore International AB), it is possible to detect the binding between the anti-TLR3 antibody and the TLR3 protein. Specifically, a test sample is brought into contact with a sensor chip on which an anti-TLR3 antibody is immobilized, and a TLR3 protein that binds to the anti-TLR3 antibody can be detected as a change in resonance signal.

  The detection method of the present invention can be automated using various automatic inspection apparatuses, and a large number of samples can be inspected at a time.

  The diagnostic agent for renal cancer of the present invention may be in the form of a kit. The diagnostic agent for renal cancer of the present invention contains at least an anti-TLR3 antibody. When the diagnostic agent is based on an EIA method such as an ELISA method, a carrier for immobilizing the antibody may be included, and the antibody may be bound to the carrier in advance. When the diagnostic agent is based on an agglutination method using a carrier such as latex, it may contain a carrier to which an antibody is adsorbed. In addition, the diagnostic agent may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.

  The diagnostic anti-TLR3 antibody using a sample such as a biopsy tissue and blood of the present invention may be specifically bound to the TLR3 protein, regardless of its origin, type (monoclonal, polyclonal) and shape. Specifically, known antibodies such as a mouse antibody, a rat antibody, an avian antibody, a human antibody, a chimeric antibody, and a humanized antibody can be used. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody, and a commercially available antibody may be used as long as it allows high sensitivity and specific measurement.

  The anti-TLR3 antibody immobilized on the support and the anti-TLR3 antibody labeled with a labeling substance may recognize the same epitope of the TLR3 protein, but preferably recognize different epitopes, and the site is not particularly limited. .

  The antibody used for diagnostic imaging of the present invention may be a chimeric antibody, a humanized (CDR grafted) antibody, or a human antibody as long as it is a monoclonal antibody as long as it specifically binds to the TLR3 protein. Moreover, as for these antibodies, as long as the binding with respect to cancer is recognized, you may use the antibody marketed.

  The anti-TLR3 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using a known means. As the anti-TLR3 antibody used in the present invention, a mammal-derived or avian-derived monoclonal antibody is preferable. In particular, a monoclonal antibody derived from a mammal is preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.

A monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, using TLR3 protein as a sensitizing antigen, this is immunized according to a normal immunization method, the resulting immune cells are fused with a known parent cell by a normal cell fusion method, and a monoclonal antibody is obtained by a normal screening method. It is possible to prepare by screening for an antibody-producing cell.
Specifically, the monoclonal antibody can be produced as follows.

  First, a TLR3 protein used as a sensitizing antigen for obtaining an antibody is obtained by expressing the gene / amino acid sequence disclosed in GenBank number (NM_003265). That is, after inserting each gene sequence encoding TLR3 protein into a known expression vector system to transform an appropriate host cell, the target human TLR3 protein is known from the host cell or culture supernatant. Purify by method. Natural TLR3 protein can also be purified and used.

  Next, this purified TLR3 protein is used as a sensitizing antigen. Alternatively, a partial peptide of TLR3 protein can be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human TLR3 protein, or can be obtained by incorporating a part of human TLR3 gene into an expression vector, and further, natural human TLR3 protein can be obtained by proteolytic enzyme. It can also be obtained by decomposing. The portion and size of the human TLR3 protein used as the partial peptide are not limited.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, birds, rabbits, monkeys and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline or the like, and mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. The mammal is dosed several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is desirable to immunize by binding to a carrier protein such as albumin or keyhole limpet hemocyanin.

  Thus, after immunizing a mammal and confirming that the desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Cell.

  Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. This myeloma cell is known in various known cell lines such as P3 (P3x63Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SF et al., J. Immunol. Methods (1980) 35 , 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are preferably used. Is done.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Kohler and Milstein, C., Methods Enzymol. (1981) 73, 3- 46) etc.

  More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of a cell fusion promoter, for example. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency if desired.

  The use ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used. Serum replacement fluid such as fetal calf serum (FCS) can be used in combination.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60% The target fusion cell (hybridoma) is formed by adding at a concentration of w / v) and mixing. Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.

  Screening and single cloning of the target antibody may be performed by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as beads made of polystyrene or a commercially available 96-well microtiter plate, reacted with the culture supernatant of the hybridoma, the carrier is washed, and then the enzyme-labeled secondary antibody is reacted. Thus, it can be determined whether or not the target antibody reacting with the sensitizing antigen is contained in the culture supernatant. A hybridoma producing the target antibody can be cloned by limiting dilution or the like. In this case, the antigen used for immunization may be used.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes are sensitized to TLR3 protein in vitro, and the sensitized lymphocytes are fused with human-derived myeloma cells having permanent mitotic potential. Thus, a desired human antibody having binding activity to TLR3 protein can also be obtained (see Japanese Patent Publication No. 1-59878). Furthermore, TLR3 protein as an antigen is administered to a transgenic animal having all repertoires of human antibody genes to obtain anti-TLR3 antibody-producing cells, and human antibodies against TLR3 protein are obtained from the immortalized cells. (See International Patent Application Publication Number WO 94/25585, WO 93/12227, WO 92/03918, WO 94/02602).

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith to proliferate, and as ascites is obtained. The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

In the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host, and a recombinant type produced using gene recombination technology can be used. (See, for example, Vandamme, AM et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
Specifically, mRNA encoding the variable (V) region of the anti-TLR3 antibody is isolated from the hybridoma producing the anti-TLR3 antibody. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), APGC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) and the like to prepare total RNA, and mRNA of interest is prepared using mRNA Purification Kit (manufactured by Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

  Antibody V region cDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis is performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation). In order to synthesize and amplify cDNA, 5′-Ampli FINDER RACE Kit (manufactured by Clontech) and 5′-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA) (1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) and the like can be used.

  The target DNA fragment is purified from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method such as the dideoxynucleotide chain termination method.

  After obtaining the DNA encoding the V region of the desired anti-TLR3 antibody, this is incorporated into an expression vector containing DNA encoding the desired antibody constant region (C region).

  In order to produce an anti-TLR3 antibody used in the present invention, an antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody.

  The expression of the antibody gene may be carried out by co-transforming the host cell by separately incorporating DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) into an expression vector, or H chain and L chain. Alternatively, a host cell may be transformed by incorporating a DNA encoding s into a single expression vector (see WO 94/11523).

  In addition to the above host cells, transgenic animals can be used for the production of recombinant antibodies. For example, an antibody gene is inserted in the middle of a gene encoding a protein (such as goat β casein) inherently produced in milk to prepare a fusion gene. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its offspring. In addition, hormones may be used in transgenic goats as appropriate to increase the amount of milk containing the desired antibody produced from the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).

  The antibody used in the present invention is not limited to the whole antibody molecule, and may be a fragment of an antibody or a modified product thereof as long as it binds to TLR3 protein, and includes both a bivalent antibody and a monovalent antibody. For example, antibody fragments include Fab, F (ab ′) 2, Fv, Fab / c having one Fab and a complete Fc, or a single chain obtained by linking Hv or L chain Fv with an appropriate linker. Examples include chain Fv (scFv). Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, followed by appropriate host cells. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc. Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669, Bird, RE et al., TIBTECH (1991) 9, 132-137).

  scFv is obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879. -5883). The H chain V region and the L chain V region in scFv may be derived from any of those described as antibodies herein. As the peptide linker that links the V regions, for example, any single chain peptide consisting of amino acid residues 12 to 19 is used.

  The DNA encoding scFv is the DNA encoding the H chain or H chain V region of the antibody, and the DNA encoding the L chain or L chain V region. Amplification by PCR using a DNA pair encoding as a template and a primer pair defining both ends thereof, and then further encoding DNA encoding a peptide linker part and both ends thereof linked to H chain and L chain, respectively. Obtained by combining and amplifying primer pairs defined in 1.

  In addition, once a DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, ScFv can be obtained according to the method.

  These antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above. The “antibody” in the present invention includes fragments of these antibodies.

  As a modified antibody, an anti-TLR3 antibody conjugated with various molecules such as a labeling substance can be used. The “antibody” in the present invention includes these modified antibodies. Such a modified antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field.

  Furthermore, the antibody used in the present invention may be a bispecific antibody. The bispecific antibody may be a bispecific antibody having an antigen binding site that recognizes a different epitope on the molecule, or one antigen binding site recognizes the TLR3 protein and the other antigen binding site is labeled. Substances etc. may be recognized. Bispecific antibodies can be prepared by binding HL pairs of two types of antibodies, or by producing hybrid cells producing bispecific antibodies by fusing hybridomas producing different monoclonal antibodies. it can. Furthermore, bispecific antibodies can be produced by genetic engineering techniques.

  The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of mammalian cells, it can be expressed by functionally binding a useful promoter commonly used, an antibody gene to be expressed, and a poly A signal downstream of the 3 ′ side thereof. For example, examples of the promoter / enhancer include human cytomegalovirus immediate early promoter / enhancer.

  In addition, other promoters / enhancers that can be used for expression of the antibodies used in the present invention include viral promoters / enhancers such as retrovirus, polyomavirus, adenovirus, simian virus 40 (SV40), or human elongation factor 1α ( And a promoter / enhancer derived from mammalian cells such as HEF1α).

  When using the SV40 promoter / enhancer, the method of Mulligan et al. (Nature (1979) 277, 108), and when using the HEF1α promoter / enhancer, the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322). ) Allows easy gene expression.

  In the case of E. coli, the gene can be expressed by functionally combining a useful promoter commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed. Examples of the promoter include lacZ promoter and araB promoter. When using the lacZ promoter, the method of Ward et al. (Nature (1098) 341, 544-546; FASEBJ. (1992) 6, 2422-2427) or when using the araB promoter, the method of Better et al. (Science ( 1988) 240, 1041-1043).

  As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the periplasm of E. coli is produced. Then, after the antibody produced in the periplasm is separated, the structure of the antibody is appropriately reassembled and used.

  As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. Furthermore, the expression vector is a selectable marker for amplification of gene copy number in the host cell system. The aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene and the like can be included.

  Any expression system can be used for the production of the antibodies used in the present invention, for example, eukaryotic or prokaryotic cell systems. Examples of eukaryotic cells include cells such as established mammalian cell lines, insect cell lines, filamentous fungal cells, and yeast cells. Examples of prokaryotic cells include bacterial cells such as E. coli cells.

  Preferably, the antibodies used in the present invention are expressed in mammalian cells such as CHO, COS, myeloma, BHK, Vero, HeLa cells.

  Next, the transformed host cell is cultured in vitro or in vivo to produce the desired antibody. Host cells are cultured according to a known method. For example, DMEM, MEM, RPMI 1640, and IMDM can be used as a culture solution, and a serum supplement such as fetal calf serum (FCS) can be used in combination.

  The antibody expressed and produced as described above can be isolated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed using an affinity column. For example, HyperD, POROS, Sepharose FF (manufactured by Pharmacia) and the like can be mentioned as a column using a protein A column. In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns other than the affinity column, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies: A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

  Moreover, since it became clear that TLR3 protein is specifically expressed in Clear Cell RCC according to the present invention, renal cell carcinoma (RCC) to be diagnosed and treated in the present invention includes Clear Cell. RCC and Clear Cell RCC metastatic cancer are particularly preferred.

  Examples of the TLR3 agonist used in the therapeutic agent for renal cancer of the present invention include double-stranded RNA and an antibody exhibiting an agonistic action on TLR3.

  The therapeutic agent for renal cancer of the present invention is formulated by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, lyophilizing, etc. with a TLR3 agonist together with a pharmaceutically acceptable carrier well known in the art. can do.

  For oral administration, the TLR3 agonist is combined with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., as well as tablets, pills, dragees, soft capsules, hard capsules, solutions, suspensions. It can be formulated into dosage forms such as suspension, emulsion, gel, syrup, and slurry.

  For parenteral administration, the TLR3 agonist is combined with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., and solutions for injection, suspensions, emulsions, creams, ointments And can be formulated into dosage forms such as inhalants and suppositories. In injectable formulations, the anti-TLR3 antibody can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. In addition, the composition can take the form of a suspension, solution, emulsion or the like in an oily or aqueous vehicle. Alternatively, the therapeutic agent may be produced in the form of a powder, and an aqueous solution or suspension may be prepared using sterile water or the like before use. For administration by inhalation, the TLR3 agonist can be powdered and made into a powder mixture with a suitable base such as lactose or starch. Suppository formulations can be made by mixing the TLR3 agonist with a conventional suppository base such as cocoa butter. Furthermore, the therapeutic agent for renal cancer of the present invention can be encapsulated in a polymer matrix or the like and formulated as a sustained release preparation.

  The dosage and frequency of administration vary depending on the dosage form and administration route, and the patient's symptoms, age, and body weight, but in general, the TLR3 agonist is in the range of about 0.001 mg to 1000 mg per kg body weight per day, preferably about It can be administered once to several times a day so as to be in the range of 0.01 mg to 10 mg.

The therapeutic agent for renal cancer of the present invention is usually administered parenterally, for example, by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, nasal, transpulmonary, etc. Is preferred.
In addition to the TLR3 agonist, the therapeutic agent for renal cancer of the present invention is particularly preferably used in combination with interferon, particularly interferon α. When a TLR3 agonist is used in combination with interferon, administration of interferon α follows the method for treating hepatitis C. That is, it is preferable to administer interferon α by 600 to 10 million IU intramuscularly for 2 consecutive weeks, further 22 weeks and 3 times per week.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Comprehensive Gene Expression Analysis of Renal Cell Carcinoma (1-1) Target Cases Thirty clinical cases of renal cell carcinoma excised surgically at the University of Tokyo Hospital from 2000 to 2004 and normal of the same case An exhaustive gene expression analysis was performed using GeneChip (Affymetrix), an oligonucleotide microarray, for 9 kidney tissues. All cases were given informed consent and written consent, and all experimental protocols with clinical specimens in this study were approved by the Ethics Committee. After excision by surgery, a portion of the cancerous part or normal kidney tissue was cut with a scalpel and snap frozen in liquid nitrogen. The frozen tissue was stored in a liquid nitrogen tank or -80 ° C deep freezer until RNA extraction. A summary of clinicopathological factors in the subject case group is listed in Table 1. Regarding the TNM classification, the standard of American Joint Committee on Cancer was followed. Regarding Nuclear Grade, the classification according to Furhmann was followed.

(1-2) Extraction of Total RNA The frozen specimen was ground with a pestle in a mortar filled with liquid nitrogen, and 1 mL of Trizol (Invitrogen) was added and homogenized. Total RNA was extracted according to Trizol protocol.

(1-3) Oligonucleotide microarray analysis According to the instructions of GeneChip, from SuperRNA 3 μg of each sample, using Oligo-dT (24mer) primer with T7 promoter, SuperScript ^TM II reverse transcriptase (Inverse transcription reaction) Double-stranded cDNA synthesis was performed by Subsequently, biotin-labeled cDNA was synthesized by an in vitro transcription reaction using T7 RNA polymerase (ENZO). Fragmentation was performed and hybridization was performed for 16 hours with GeneChippHG-U133 plus 2.0 having 54675 probe using 5 μg of cRNA per GeneChip. After washing, the signal was enhanced by Strrepyoavidin Phycoerythrin staining, biotinylated antibody treatment, and Strepyoavidin Phycoerythrin re-staining, then read with a scanner, and Affymetrix Microarray Analysis 5.0 image (MAS analysis 5.0). Thereafter, for each gene on each GeneChip, the signal value displayed by MAS 5.0 was used as the expression level, and the data was standardized so that the average gene expression level in each GeneChip was 100.

(1-4) Gene expression analysis (1-4-1) Cluster analysis Based on the 54675 probe signal value obtained from GeneChip, GeneSpring (Agilent) is used to perform hierarchical cluster analysis by probe and case, Thirty-nine specimens (27 Clear cell RCC, 2 Chromphobe RCC, 1 Sarcomotoid RCC and 9 non-cancerous normal kidney) were classified. First, for each probe, at least one sample out of 39 samples having a signal value exceeding 200 was selected and used as a probe set. Next, using the expression level of the probe set, a Pearson correlation coefficient was determined between genes, and cluster analysis was performed. The Pearson correlation coefficient was similarly obtained between cases, and cluster analysis was performed. Thus, cluster analysis was performed in two directions between genes and between cases, and the similarity of cases was compared. First, out of all probes of GeneChip (54675 probes), a set of 17512 probes was selected by excluding probes with signal values of 200 or less in all cases. When hierarchical cluster analysis was performed with this probe set, it was roughly divided into two groups: a group of Clear cell RCC (including Sarcomatoid RCC) and a group including normal kidney and Chromophobe RCC (FIG. 1). Nine normal kidneys and two Chromophore RCCs each formed a cluster.

(1-4-2) Selection of genes that are highly expressed specifically in tissue type Genes that are specifically highly expressed in Clear cell RCC or Chromophobe RCC using the expression values of the probe set used for cluster analysis Was elected. The selection conditions are as follows.

i) Clear cell RCC-specific high expression gene Clear cell RCC Median of 27 cases / median of 9 normal kidneys> 5 and
Clear cell RCC 27 medians / Chromophore RCC 2 medians> 5
ii) Chromphobe RCC specific high expression gene Chromphobe RCC 2 minimum value / median value of 9 normal kidney> 5 and
Chromophobe RCC 2 cases minimum / Clear cell RCC 27 cases median> 5

315 and 196 probes were selected as genes that are specifically highly expressed in Clear cell RCC or Chromophobe RCC, respectively. In addition, there was no common gene that was more than 5 times more expressed in Clear cell RCC than in normal kidney and a gene that was more than 5 times more expressed in Chromophore RCC than in normal kidney. . That is, the expression of completely different gene groups was enhanced in these two tissue types.
Using the selected probe set, analysis by the software EXPRESSION ANALYSIS SYSTEM EXPLORER provided by NIH and what types of genes are included in the gene group whose expression is specifically enhanced in each tissue type I investigated. As shown in Tables 2 and 3, Clear cell RCC was characteristically highly expressed in gene groups related to immune response, biological defense and immune response. In addition, it has been clarified that a group of genes involved in ion transport, homeostasis and the like are characteristically highly expressed in Chromphobe RCC. The genes that were specifically highly expressed in each tissue type and the top 50 probes are shown in Tables 4 and 5 on the following pages.

(1-4-3) Gene ontology analysis of genes that are highly expressed in a tissue type specific manner Regarding what functions of genes are included in the gene group selected in (1-4-2) above, Analysis was performed by software EXPRESSION ANALYSIS SYSTEM EXPLORER provided by NIH.
This is to test how much each gene ontology (gene function) attached to each probe is concentrated in the target gene group with respect to the frequency in all probes by modified Fisher's exact test. It is. The test value is expressed as Ease Score, and it is evaluated that the lower the value, the more concentrated the target gene group, that is, the more the gene having the function is contained in the target gene group.

(1-4-4) Selection of target gene for renal cell carcinoma The expression data of renal cell carcinoma and normal kidney analyzed this time, and analysis using GeneChip HG-U133 plus2.0 at the Department of Genomic Sciences, University of Tokyo. Expression data of 15 normal organs (Brain, Muscle, Heart, Skin, Lung, Liver, Stormach, Colon, Pancreas, Kidney, Blader, Bone marrow, Peripheral blood, Overy, Testis) The genes having low expression and clear cell RCC-specific expression were selected. The selection criteria are as follows.

Clear cell RCC median of 27 cases / median of 9 normal kidneys> 5 and
Clear cell RCC 27 median> 500 (signal value) and
Maximum value of 15 normal organs <500 (signal value)

As a result, as shown in Table 6, 36 probes (23 as the number of known genes) were selected.
Of these 36 probes, further investigation was conducted focusing on TLR3. The gene expression value of TLR3 (GeneChip signal value) is shown in FIG.
Oligonucleotide microarray analysis revealed that the expression of the TLR3 gene was increased in Clear cell RCC compared to normal kidney and various normal tissues.

Example 2 Increased expression of TLR3 gene by quantitative RT-PCR in Clear cell RCC The expression of TLR3 gene in 7 pairs of Clear cell RCC and normal kidney tissue of the same patient was measured by quantitative RT-PCR. For cDNA synthesis, DNase I Amp grade (Invitrogen) was added to 1 μg of total RNA and allowed to stand at room temperature for 15 minutes to decompose genomic DNA. Subsequently, a reverse transcription reaction was performed at 50 ° C. for 60 minutes using an Oligo (dT) primer and SuperScript ^™ III reverse transcriptase (Invitrogen). Thereafter, RNase H was added to decompose the template RNA strand. The synthesized cDNA solution was diluted with nuclease free water and used for PCR reaction. PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, ₂ mM MgCl ₂ , 0.01% gelatin), 200 μM dNTPs (TaKaRa), 0.2 μM primer, Taq Polymerase, SYBR Green using the synthesized cDNA as a template Quantitative RT-PCR was performed using (BMA). The TLR3 Forward primer uses SEQ ID NO: 1 and the reverse primer uses SEQ ID NO: 2, PCR conditions: 94 ° C. for 3 minutes (94 ° C. for 15 seconds, 60 ° C. for 15 seconds, 72 ° C. for 30 seconds) × 45 cycles Carried out. For ACTB as an internal standard gene, SEQ ID NO: 3 for Forward primer and SEQ ID NO: 4 for Reverse primer, PCR conditions: 94 ° C. for 3 minutes (94 ° C. for 15 seconds, 62 ° C. for 15 seconds, 72 ° C. 30 seconds) × 35 cycles, and the amount of initial template cDNA was determined by measuring the amount of fluorescence emitted by SYBR Green. Furthermore, the amount of the initial template cDNA of ACTB was determined, and the amount of the initial template cDNA of the target gene was corrected by the value, and the obtained value was used as the relative amount of mRNA of the target gene. Three 50 μL systems were prepared for the same sample, and the average value was used as a result. As shown in FIG. 3, in all cases, increased expression of the TLR3 gene was observed in Clear cell RCC compared to normal kidney.

Example 3 Cloning of TLR3 cDNA, Preparation of Full Length Forced Expression Cell Extract Primers were designed for the coding region of TLR3, and PCR was performed using the cDNA obtained in Example 2 as a template. As DNA polymerase, KOD-plus-ver. 2 (TOYOBO) was used. The PCR conditions are as follows. SEQ ID NO: 5 was used as a Forward primer and SEQ ID NO: 6 was used as a reverse primer, and PCR conditions were 94 ° C. for 2 minutes (94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 3 minutes) × 25 cycles. The obtained PCR product was excised by electrophoresis on an agarose gel and recovered using a QIAquick Gel Extraction Kit (QIAGEN). The collected DNA and Zero Blunt TOPO PCR Cloning Reagents (Invitrogen) were used for integration into a vector. Cloning was performed using TOPO10 (Invitrogen) as competent cells. A construct that was confirmed to contain the correct TLR3 cDNA sequence by colony PCR and sequencing was subjected to restriction enzyme treatment with EcoRI and SmaI (TaKaRa Bio), and the coding region of TLR3 was excised from the plasmid, and QIAquick Gel was used. DNA fragments were recovered using an Extraction Kit. Similarly, the coding region of the obtained TLR3 was incorporated into phCMV vector (Genlantis), an animal cell expression vector treated with restriction enzymes. T4 DNA Ligase (Promega) was used as a ligation reagent. The resulting construct was transformed into TOP10. Plasmid DNA was purified and recovered using QIAGEN's MidPrep kit.

Next, the purified plasmid DNA was introduced into CHO cells (Dainippon Sumitomo Pharma Co., Ltd.) using FuGENE Transfection Reagent (Roche). Cells were seeded 8 × 10 ⁵ cells in a 10 cm dish the day before transfection. At the time of transfection, 400 μL of OptiMEM medium (Invitrogen), 16 μL of FuGENE reagent, and 8 μg of plasmid DNA were mixed and allowed to stand at room temperature for 15 minutes. Thereafter, the mixture was added to a 10 cm dish. After 48 hours, limiting dilution was performed and cloning was started. G418 (Invitrogen) was used as a selection drug at a concentration of 1 mg / mL. The cloned cells were cultured in a 10 cm dish, and when they became confluent, proteins were recovered using a RIPA buffer to which a protease inhibitor was added.

Example 4 Preparation of antigen for TLR3 immunization A primer with a histidine tag added to the C-terminal side of the extracellular region of TLR3 (1-700aa) was designed, and the coding region of TLR3 obtained in Example 3 above was designed. PCR reaction was performed using DNA as a template. Examples of DNA polymerase include KOD-plus-ver. 2 (TOYOBO) was used. The PCR conditions are as follows. SEQ ID NO: 7 was used as a forward primer, SEQ ID NO: 8 was used as a reverse primer, and PCR conditions were 94 ° C. for 2 minutes (94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 3 minutes) × 25 cycles. The obtained PCR product was excised by electrophoresis on an agarose gel and recovered using a QIAquick Gel Extraction Kit (QIAGEN). The collected DNA and Zero Blunt TOPO PCR Cloning Reagents (Invitrogen) were used for integration into a vector. Cloning was performed using TOPO10 (Invitrogen) as competent cells. A construct confirmed to have the correct TLR3 cDNA sequence incorporated by colony PCR and sequencing was subjected to restriction enzyme treatment with NotI and XbaI (TaKaRa Bio), and the extracellular region of TLR3 was excised from the plasmid. The DNA fragment was recovered using Gel Extraction Kit. Similarly, the extracellular region of the obtained TLR3 was incorporated into a pNOW vector (Immuno Japan), which is an animal cell expression vector treated with restriction enzymes. T4 DNA Ligase (Promega) was used as a ligation reagent. The resulting construct was transformed into TOP10. Plasmid DNA was purified and recovered using QIAGEN's MidPrep kit.

Subsequently, the purified and recovered plasmid DNA was introduced into CHO cells (Dainippon Sumitomo Pharma Co., Ltd.) using FuGEN Transfection Reagent (Roche). Cells were seeded 8 × 10 ⁵ cells in a 10 cm dish the day before transfection. At the time of transfection, 400 μL of OptiMEM medium (Invitrogen), 16 μL of FuGENE reagent, and 8 μg of plasmid DNA were mixed and allowed to stand at room temperature for 15 minutes. Thereafter, the mixture was added to a 10 cm dish. After 48 hours, limiting dilution was performed and cloning was started. G418 (Invitrogen) was used as a selection drug at a concentration of 1 mg / mL. The cloned cells were cultured in a roller bottle (Falcon) using a serum-free medium CHO-SFM-II (Invitrogen), and the culture supernatant was collected. The TLR3 extracellular region (1-700aa) -His tag fusion protein was purified from the obtained culture supernatant using a His Trap HP column (manufactured by GE Healthcare Bioscience). This purified fusion protein was dialyzed against PBS and used as an antigen for immunization.

Example 5 Preparation of Anti-TLR3 Monoclonal Antibody MRL / Ipr was prepared by mixing equal amounts of 50 μg of TLR3 extracellular region (1-700aa) -His tag fusion protein and Titer-MAX (TiterMax USA, Inc.) dissolved in PBS. Initial immunization was performed by intraperitoneal injection into mice (Sankyo Lab Service). The second and subsequent immunizations were carried out by mixing the TLR3 extracellular region (1-700aa) -His tag fusion protein equivalent to the amount of 25 μg protein prepared in the same manner and Titer-MAX and intraperitoneally injecting them. Three days after the final immunization, spleen cells were aseptically prepared from the mice, and cell fusion with mouse myeloma cells NS1 was performed by the polyethylene glycol method.
Screening of the anti-TLR3 antibody in the hybridoma culture supernatant was performed by ELISA on which the extracellular region of TLR3 (1-700aa) -His tag fusion protein was immobilized.

Example 6 Immunoblotting of kidney cancer tissue extract TLR3 RIPA buffer containing frozen tissue and TLR3 forced expression CHO cells in a mortar filled with liquid nitrogen using a pestle and protease inhibitor (SIGMA) added ( 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) was added. Place on ice for 30 minutes, centrifuge at 4 ° C., 10 minutes, 10,000 rpm, collect the supernatant in a tube, add an equal volume of sample buffer (2 × SDS buffer, 10% mercaptoethanol), and add 95 ° C. For 15 minutes. After heating, the centrifuged supernatant was electrophoresed on a 12% acrylamide gel. Electrophoresis was performed at 100V for 10 minutes and 200V for 50 minutes. After blocking with 2% skim milk / TBS-T (Triton X-100) for 60 minutes, the mixture was reacted with the primary antibody at room temperature for 60 minutes. As primary antibodies, anti-TLR3 monoclonal antibody (2.5 μg / mL, IMGENEX) and anti-β-actin monoclonal antibody (0.3 μg / mL, Sigma) were used. After washing with TBS-T, the mixture was reacted with the secondary antibody at room temperature for 45 minutes. As the secondary antibody, an HRP-labeled anti-mouse IgG antibody (Amersham Biosciences) diluted to 1 / 10,000 was used. After washing with TBS-T, HRP was emitted with ECL-PLUS (Amersham Biosciences), which is a chemiluminescence detection reagent, and a band was detected with an LAS 3000 image analyzer (Fuji Film).

  As shown in FIG. 4, in the protein recovered from CHO cells in which TLR3 was forcibly expressed, a single band was obtained around 120 kDa, which is the expected size of TLR3 protein, but the protein recovered from control CHO cells. In the case of no band was recognized. On the other hand, a single band was also observed at the same position in the specimen (9T) in which the expression enhancement of TLR3 gene in the cancer site was confirmed by quantitative RT-PCR. A very weak band was observed at the same position even in the normal kidney (9N) of the same patient.

Example 7 Localization of TLR3 Protein in Renal Cell Carcinoma, and Expression Frequency in Each Tissue Type, and Correlation with Clinicopathological Data (7-1) Preparation of TMA Block From 1993 to 2004, University of Tokyo A tissue microarray (TMA) block was prepared from a paraffin block of 216 renal tumors surgically excised at the hospital. First, the slide glass of each case was microscopically examined to evaluate the tissue type, and two representative portions of the tumor were marked. Then, while collating with the marked slide glass, two places were punched out from the paraffin block of each case with a needle having a diameter of 2 mm and transplanted to the TMA block. Nine TMA blocks were prepared by transplanting 24 cases and 48 tissues per block. The TMA block was sliced to a thickness of 4 μm and attached to silane-coated glass to prepare a specimen for immunostaining. In addition, HE (hematoxylin and eosin) staining was performed on one piece of serial sections, and the tissue type was evaluated.

(7-2) Staining method First, deparaffinization and hydrophilization were performed with a xylene / ethanol series on a slice sliced to 4 μm. Next, antigen activation was performed by autoclaving (121 ° C.) for 10 minutes in a state where the sections were immersed in a pH 6.0 citrate buffer. As a primary antibody, an anti-TLR3 monoclonal antibody (IMGENEX) was diluted 10-fold with an antibody diluent (Chemmate Antibody Diluent, DakoCytomation) (concentration 2.5 μg / mL after dilution), and reacted at room temperature for 60 minutes. After washing with TBS, ENVISION + / HRP (DAKO) was reacted as a secondary antibody at room temperature for 30 minutes and washed with TBS. Color was developed with diaminebenzoline tetrahydrochloride (DAB), followed by nuclear staining with hematoxylin, dehydrated with ethanol and xylene series, and sealed. As a negative control, the same staining step was performed on all sections without the primary antibody alone. No positive signal was observed in the negative control sections.

  The intensity of staining was evaluated in four grades: 0 (negative), 1+ (weak positive), 2+ (moderate positive), 3+ (strong positive). The determination of staining intensity was performed independently by two pathologists. In the immunostaining for TMA, 189 Clear cell RCC cases, 11 Pillary RCC cases, 8 Chromophore RCC cases, and 208 cases in total were evaluated.

  As a result of staining for TMA, a weak positive image (1+) was observed in the cytoplasm of most tubules and collecting ducts in the normal kidney (FIG. 5A). Rarely, tubules showing moderately positive (2+) were also found (FIG. 5B). In rare cases, weak positive images were also observed in Bowman's sac epithelium.

In Clear cell RCC, positive images were obtained in 184 (97.4) of 189 cases. Among 189 cases, 139 cases (73.5%) showed stronger expression than normal tubules (2+ and 3+). In the cancer area, fine granular stained images were observed in the cytoplasm of the cancer cells (FIGS. 5D and 5E). There was also a case where the expression was particularly enhanced at the boundary with the stroma (FIG. 5 (F)). Cancer cells were stained specifically for cancer cells, and positive images were not observed for vascular endothelial cells and inflammatory cells other than cancer cells.
In the Pillary RCC, expression was observed as frequently as in the Clear cell RCC (FIG. 6 (A), Table 7), but in the Chromophobe RCC, all of the 8 cases were completely negative (FIG. 6 (B), Table 7). 7).

(7-3) Examination of correlation with clinicopathological data Table 8 shows the correlation between TLR3 staining intensity and clinicopathological factors for 189 cases of Clear cell RCC. The TLR3 high expression group tended to have less venous invasion than the low expression group, but there was no significant difference between the staining intensity of TLR3 and age / sex / nuclear atypia / pT / venous invasion It was.

Example 8 Expression of TLR3 Protein in Clear Cell RCC Lung Metastases Immunostaining was performed in the same manner as in Example 7 using 8 paraffin-embedded specimens of Clear cell RCC lung metastases.
In clear cell RCC lung metastases, 6 (8%) out of 8 cases (75%) showed moderate (2+) or more positive images in 50% or more cancer cells (FIG. 6 (C, D)). In addition, almost no expression of TLR3 was observed in normal alveolar epithelium or terminal bronchiolar epithelium.

Example 9 Suppression of Cell Growth by Poly I: C Single Agent Eight types of renal cancer cell lines were tested at Tohoku University Age Research Institute (Caki-1, ACHN, SW839, VMRC-RCW), RIKEN Cell Bank (OS-RC-2, TUHR10-). (TKB, TUHR14-TKB) and American Type Culture Collection (Caki-2). The cells were cultured at 37 ° C. under aeration of 5% CO ₂ using a medium containing 10% FBS and antibiotics (penicillin / streptomycin) as a culture solution. The media used were ACHN, SW839, VMRC-RCW, OS-RC-2, TUHR10-TKB, TUHR14-TKB for RPMI 1640 medium (SIGMA), Caki-1 and Caki-2 for Macoy 5A modified medium (Invitrogen) It is. Each cell was cultured at 10 cm dish, and RNA was collected when it became 70-80% confluent. CDNA synthesis was performed using 1 μg of RNA as a template, and the expression level of the target gene was measured by quantitative RT-PCR. TLR3 gene expression was observed in all cell lines (FIG. 7A). The expression level varied depending on the cell line, and TUHR10-TKB, which had the highest expression, showed an expression level 9.9 times that of Caki-2, which had the lowest expression. Poly I: C (Invitrogen) was administered to these 8 types of renal cancer cell lines, and the degree of cell proliferation was measured. Each cell was seeded on a 96-well plate so that it would be 2 × 10 ³ or 4 × 10 ³ per well (the number of seeded cells was adjusted according to the growth rate). After 24 hours, the medium was removed, medium containing various concentrations of Poly I: C was added, and further culturing was performed. After a certain time from adding Poly I: C, the number of viable cells was compared using WST-8 assay Kit (Dojin). The absorbance at 450 nm was measured with a plate reader, and the value obtained by subtracting the absorbance at 630 nm as the background was used as the result. Four wells were measured under the same conditions, and the average value was used as a result. When 10 μg / mL Poly I: C was administered and absorbance was measured after 72 hours, 5 out of 8 cell lines (TUHR10-TKB, OS-RC-2, TUHR14-TKB, SW839, VMRC-RCW) A significant (p <0.01) growth inhibitory effect was observed (FIG. 7B). And the cell line with higher expression of TLR3 gene tended to show a stronger growth inhibitory effect. In other words, administration of 10 μg / mL Poly I: C showed the greatest growth inhibitory effect on TUHR10-TKB, which has the highest TLR3 gene expression. The expression of TLR3 gene was low in 3 strains (ACHN, Caki-1, Caki-2) that did not show a significant growth inhibitory effect when 10 μg / mL Poly I: C was added (FIG. 7A).
The growth inhibitory effect of Poly I: C was dose-dependent. That is, when various concentrations of Poly I: C were administered to TUHR10-TKB with the highest expression of the TLR3 gene, the higher the concentration of Poly I: C, the stronger the growth inhibitory effect was shown (FIG. 7 (C)).

Example 10 Induction of apoptosis by addition of Poly I: C It was examined by Annexin-V staining whether apoptosis contributes to the growth inhibitory effect of Poly I: C. The renal cancer cell line TUHR10-TKB was seeded on a 4-well tissue culture culture slide (BDbiosciences) at 5 × 10 ⁴ cells per well. After 24 hours, the medium was removed, and a medium containing 0 or 50 μg / mL Poly I: C was added, followed by culturing for 5 hours. Then, according to the protocol of Annexin-V-FLUOS Staining Kit (Roche), the cells which fell into apoptosis were observed. That is, after completion of the culture, the medium was removed and washed twice with HEPES buffer. Next, Annexin-V-FLUOS labeling solution was added at 100 μL per well and allowed to react at room temperature for 15 minutes. The sample was sealed with a cover glass and observed with a fluorescence microscope at a wavelength of 488 nm. In the group added with Poly I: C, about 10% of the cells were positive, whereas in the group not added with Poly I: C, the positive cells were rare (about 1%) (FIG. 8). That is, it was found that cell apoptosis was induced by addition of Poly I: C.

Example 11 Analysis of change in expression of TLR3 downstream gene during Poly I: C administration by quantitative RT-PCR After culture of renal cancer cell lines TUHR10-TKB and Caki-1, 50 μg / mL Poly I: C was administered, After 0, 2, 6, 12, and 24 hours, the cells were washed twice with PBS and then homogenized by adding Trizol. CDNA synthesis was performed using 1 μg of the recovered RNA as a template, and the expression change of the target gene was measured by quantitative RT-PCR. cDNA synthesis and quantitative RT-PCR were performed as described above. The initial template cDNA amount of ACTB was determined as an internal standard gene, and the initial template cDNA amount of the target gene was divided by the value to make a correction. The obtained value was used as the relative amount of mRNA of the target gene. Three 50 μL systems were prepared for the same sample, and the average value was used as a result. The forward primer (F) and reverse primer (R) of each cytokine and chemokine used are shown below. Each PCR condition is the same as IFNβ except for the number of cycles.

<IFNβ>: F (SEQ ID NO: 9), R (SEQ ID NO: 10); 94 ° C. 3 minutes, (94 ° C. 15 seconds, 60 ° C. 15 seconds, 72 ° C. 30 seconds) × 38 cycles <IL6>: F (SEQ ID NO: 11), R (SEQ ID NO: 12); x 40 cycles <IL8>: F (SEQ ID NO: 13), R (SEQ ID NO: 14); x 40 cycles <IP10 (CXCL10)>: F (SEQ ID NO: 15), R ( SEQ ID NO: 16); x 35 cycles <RANTES (CCL5)>: F (SEQ ID NO: 17), R (SEQ ID NO: 18); x 35 cycles

  First, the expression of IFNβ, which plays the most important role in TLR3 signal, was examined. In both TUHR10-TKB and Caki-1, almost no expression of IFNβ was observed in the steady state, but the expression was remarkably increased by addition of poly I: C (FIG. 7D). The expression enhancement of IFNβ was more remarkable in the TUHR10-TKB strain with high TLR3 expression. Moreover, about TUHR10-TKB strain, the expression change with time by poly I: C addition of IL6, IL8, IP10 (CXCL10), and RANTES (CCL5), which are known as downstream genes of TLR3, was examined. None of the genes was expressed in a steady state, but increased 2 hours after addition of poly I: C, and further increased over time (FIG. 9 (AD). )). The expression of TLR3 also increased from 6 hours after addition of poly I: C (FIG. 9 (E)).

Example 12 Suppression of TLR3 Gene Expression by siRNA TLR3 expression suppression analysis by siRNA targeting TLR3 was performed on the renal cancer cell line OS-RC-2. The siRNA was introduced using the reverse transfection method according to the protocol of HiPerFect (QIAGEN). HiPerFect and siRNA were mixed in OptiMEM medium (Invitrogen) to a final concentration of 20 nM, and an antibiotic-free cell suspension was added. After 48 hours of culture on a 6-well plate, RNA was recovered. CDNA synthesis was performed using 1 μg of RNA as a template, and the expression change of the target gene was measured by quantitative RT-PCR. The sequence of siRNA is shown in SEQ ID NO: 19. As a negative control, Stealth RNAi Negative Control Kit with Medium GC (GC 48%) (Invitrogen) was used. Further, in the same manner as described above, siRNA having a final concentration of 20 nM was introduced into the renal cancer cell line OS-RC-2, and cultured for 72 hours at 96-well plate. Thereafter, 0 or 50 μg / mL poly I: C solution (including antibiotics) was added, and further cultured for 48 hours, and then the number of viable cells was compared using the WST-8 assay Kit as described above. . Four wells were measured under the same conditions, and the average value was used as a result. As a result, the expression of the TLR3 gene was significantly suppressed by siRNA targeting TLR3 as compared with the negative control (FIG. 10 (A)). Next, changes in cell proliferation when TLR3 gene expression was suppressed by siRNA, growth inhibitory effect by poly I: C, and changes in IFNβ gene expression were examined. First, suppressing the expression of the TLR3 gene itself did not change cell proliferation (FIG. 10B). However, by suppressing the expression of the TLR3 gene, the growth inhibitory effect by poly I: C was significantly attenuated. That is, in the control strain, the addition of poly I: C showed a 37% growth inhibitory effect (indicating the decrease rate of the absorbance of the administration group relative to the absorbance of the poly I: C non-administration group), whereas the expression of TLR3 gene was shown. In the case of the inhibition, the growth inhibition effect was halved to 17% (FIG. 10B). That is, it was shown that the growth inhibitory effect by poly I: C depends on the expression of TLR3. Further, by suppressing the expression of the TLR3 gene, the induction of the expression of IFNβ by poly I: C was suppressed (FIG. 10C). That is, it was shown that the expression induction of IFNβ by poly I: C also depends on the expression of TLR3.

Example 13 Sensitivity test by using poly I: C and IFNα in combination The change in proliferation by using poly I: C and IFNα in combination was measured for the renal cancer cell line Caki-1. First, cells were seeded on a 96-well plate so that there were 2 × 10 ³ per well. At 24 hours after seeding, the medium was removed, and a medium containing IFNα (0, 100, 1000, 10,000 U / mL) was added. 24 hours after the addition of IFNα, the medium was removed, and a medium containing poly I: C (0, 10, 50 μg / mL) was added. After further culturing for 48 hours, the number of viable cells was compared using WST-8 assay Kit. Four wells were measured under the same conditions, and the average value was used as a result.
In contrast to Caki-1, IFNα and poly I: C showed a synergistic growth inhibitory effect (FIG. 11 (A)). That is, when IFNα was not added, the growth inhibitory effect of poly I: C alone was poor (the poly I: C 50 μg / mL was 17% less than the poly I: C non-proliferative effect). ), IFNα administration significantly enhanced the growth inhibitory effect of poly I: C. Specifically, when IFNα is added at 100, 1000, and 10000 U / mL (only 11, 19 and 23%, respectively, exhibiting a growth inhibitory effect), poly I: C is added at 50 μg / mL. The growth inhibitory effect (representing the rate of decrease in absorbance of the target group relative to the absorbance of the group to which neither poly I: C nor IFNα was added; the same applies hereinafter) was enhanced to 50, 71, and 76%, respectively. Moreover, when IFNα alone was used, the growth inhibitory effect was poor (the one with IFNα 10000 U / mL was a 23% growth inhibitory effect compared to the one without IFNα), but when poly I: C of IFNα-added culture was added, The growth inhibitory effect by IFNα was remarkably enhanced. Specifically, when 10 or 50 μg / mL of poly I: C is added (only 9 or 17% of the growth inhibitory effect is obtained, respectively), the growth inhibitory effect of the addition of IFNα 10000 U / mL is respectively Increased to 57, 76%.

Example 14 Expression Change of TLR3 Gene by Addition of IFNα IFNα (0,100,1000,10000 U / mL) was added to the renal cancer cell line Caki-1 in the same manner as in Example 13, and RNA was recovered after 6 hours. CDNA synthesis was performed using 1 μg of RNA as a template, and changes in the expression of the TLR3 gene were measured by quantitative RT-PCR. Expression of TLR3 gene was increased by administration of IFNα. Compared to the steady state, the expression level of the TLR3 gene increased 3.4 times by adding IFNα 100 U / mL and 4.8 times by adding 10,000 U / mL (FIG. 11B).

Example 15 Expression Change of TLR3 Downstream Gene by Addition of IFNα and Poly I: C Similar to Example 13, 24 hours after administration of 0, 100, 1000, 10000 U / mL of IFNα to renal cancer cell line Caki-1. After culturing, 0 or 50 μg / mL poly I: C was administered, and RNA was recovered 6 hours later. CDNA synthesis was performed using 1 μg of RNA as a template, and the expression change of the target gene was measured by quantitative RT-PCR. In any of IFNβ, IL6, IL8, IP10, and RANTES, when poly I: C was not added, almost no expression was observed regardless of the preincubated IFNα concentration (IFNβ, IL6, IL8, RANTES). Is shown in FIG. 10 (D-F). Addition of poly I: C increased the expression of all genes. The degree of increase in gene expression was dependent on the concentration of IFNα. That is, when poly I: C at the same concentration (50 μg / mL) was added, the higher the preincubated IFNα concentration, the higher the expression level of the TLR3 downstream gene (FIG. 10 (CF)).

The result of the hierarchical cluster analysis by the two directions of a renal cancer case and a gene is shown. The gene expression level of TLR3 by the GeneChip analysis of renal cancer and normal tissue is shown. The expression level of the TLR3 gene by quantitative RT-PCR in renal cancer and normal tissues is shown. The immunoblotting result of the clear cell RCC, the normal renal tissue pair specimen, and the renal cancer tissue extract TLR3 in TLR3 forced expression CHO cells is shown. Figure 3 shows the localization of TLR3 protein in renal cancer. The expression frequency in each histological type and metastasis of renal cancer is shown. The cell growth inhibitory effect result by Poly I: C single agent is shown. The result of apoptosis induction by addition of Poly I: C is shown. The analysis result of the expression change of TLR3 downstream gene at the time of Poly I: C administration by quantitative RT-PCR is shown. The expression suppression result of TLR3 gene by siRNA is shown. The synergistic effect result with respect to cell proliferation by combined use of poly I: C and IFNα is shown.

Claims (8)

  A diagnostic agent for renal cancer containing an anti-TLR3 antibody.   The diagnostic agent for renal cancer according to claim 1, wherein the renal cancer is Clear cell RCC.   The diagnostic agent for renal cancer according to claim 1 or 2, wherein the renal cancer is Clear cell RCC metastatic cancer.   The diagnostic agent for renal cancer according to any one of claims 1 to 3, wherein the anti-TLR3 antibody is an antibody that binds to a TLR3 protein.   The diagnostic agent for renal cancer according to any one of claims 1 to 4, which is used by reacting an anti-TLR3 antibody with an organ tissue to detect TLR3 protein.   The diagnostic agent for renal cancer according to any one of claims 1 to 5, which is a diagnostic agent for selecting Clear cell RCC or Clear Cell RCC metastatic cancer among renal cancer patients.   A method for using as an indicator of renal cancer, comprising reacting a sample collected from a subject with an anti-TLR3 antibody and detecting TLR3 protein in the sample.   The method for using as an index of renal cancer according to claim 6, wherein the sample collected from the subject is an organ tissue.