JP5721342B2 - Drug efficacy markers for renal cell carcinoma - Google Patents

Description

  The present invention detects susceptibility to interleukin-2 (IL-2) and interferon-α (IFN-α) combination therapy in renal cell carcinoma, particularly in patients with renal cell carcinoma with metastasis, based on patient-specific genetic information And a material (reagent and reagent kit) used for the method.

  Renal cell carcinoma is not common among Japanese people, but its incidence is increasing year by year. In particular, it is known that the prognosis of treatment for patients with metastatic renal cell carcinoma is extremely poor. For the treatment of renal cell carcinoma, surgical excision of the tumor is a common method, but for some patients, recurrence in the kidney or metastasis to other organs such as the lung in several years after excision. Is often accepted. Renal cell carcinoma, usually diagnosed with a high degree of malignancy and invasion and metastasis to the lymph nodes, is prone to cancer metastasis and recurrence, and after surgery, immunotherapeutic agents, chemotherapy, radiation therapy and hormones Therapies have been used to kill cancer cells that could not be removed by surgery.

  Cytokines such as interferon-α (hereinafter referred to as “IFN-α”) and interleukin-2 (hereinafter referred to as “IL-2”), which are immunotherapeutic agents, are effective when used alone or in combination. It is used in clinical practice as one of the therapeutic methods for renal cell carcinoma (see, for example, Non-Patent Documents 1 to 4).

  IFN-α is a therapeutic agent that has an immunopotentiating effect such as a carcinogenesis-preventing effect as well as an antiviral effect, compared to other drugs. The therapeutic effect of IFN-α has been confirmed to be dose-dependent, and it is said that the higher the dose of IFN-α, the higher the effect. For this reason, in order to improve the therapeutic effect, attempts are often made to extend the daily administration period or increase the dose to the maximum amount, but IFN-α also has the problem of strong side effects. It is a fact. Although the burden of side effects such as fever, malaise, and depression is increased for patients, the therapeutic effect has not been improved. And if a side effect is strong, the situation where the administration must be stopped sometimes occurs. The reason why the side effects of IFN-α cannot be eliminated is that the detailed mechanism of various effects including immune activity of IFN-α and the mechanism of side effect expression have not been clarified. Continued research.

  IL-2 promotes the proliferation of T cells, which are central when attacking immune system cancer cells, and enhances the function of NK (natural killer) cells to destroy cancer cells, thereby attacking cancer There is. As with IFN, the higher the dose of IL-2, the higher the anti-tumor effect.However, the side effects such as fever, chills, headache, rash, nausea / vomiting, loss of appetite, diarrhea, weight gain, and sleep disorders The number of people who can be used at high doses is limited.

  The combined therapy of IFN and IL-2 is higher than the response rate of each single agent, but has not reached a satisfactory level. In fact, in the treatment of renal cell carcinoma with IL-2 and IFN-α, the complete and partial response rates are only 10-20%, which has not changed in recent years, The present condition is that the patient died within two years (for example, refer nonpatent literature 2 and 5-6). On the other hand, as described above, since the side effects of each cytokine are not negligible for patients, the development of diagnostic means for predicting the therapeutic effect when deciding on the combination therapy with IL-2 and IFN-α It can be said that it is urgent.

  By the way, HLA, which is a human MHC (major histocompatibility complex or major histocompatibility antigen) molecule, was found in 1952 as an antigen against a leukocyte agglutination test in a blood transfusion patient serum. It is a human leukocyte antigen. HLA is a gene product controlled by a gene group encoded by an MHC region present within about 4,000 kbp of 6p21.3 of the short arm of chromosome 6. This MHC region is expressed only in the class I gene region that controls the HLA-A, B, and C antigen systems expressed on the surface of most eukaryotic cell membranes and in limited tissues or cells such as B cells and macrophages. It consists of a class II gene region that governs cell-specific HLA-DP, DQ, and DR antigen systems, and a class III gene region that governs complement components and 21-hydroxylase.

  HLA-DQA1 (Major histocompatibility complex, class II, DQ alpha 1) and HLA-DQB1 (Major histocompatibility complex, class II, DQ beta 1) belong to the side system homologues of HLA class II α chain and β chain, respectively. This class II molecule is a heterodimer consisting of an α chain (DQA) and a β chain (DQB), both of which are anchored to the membrane. It is known to play a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed on antigen presenting cells, B lymphocytes, dendritic cells, and macrophages. The α chain is approximately 33-35 kDa and its gene contains five exons (exon 1 is the leader peptide, exons 2 and 3 are the two extracellular regions, and exon 4 directs the transmembrane region and cytoplasmic tail). The β chain is approximately 26-28 kDa, and its gene is composed of six exons (exon 1 is the leader peptide, exons 2 and 3 are the two extracellular regions, exon 4 is the transmembrane region, and exon 5 is the cytoplasm. )including.

  In DQ molecules, it is known that both α and β chains contain polymorphisms that direct peptide binding specificity, resulting in up to four different molecules.

  Conventionally, the expression level of HLA-DQA1 gene or HLA-DQB1 gene and the presence of polymorphisms in these genes are likely to cause progressive type I diabetes, therapeutic effect of chronic B large cell lymphoma (DLBCL), solid It has been reported that it becomes an index for evaluating the sensitivity of cancer to tyrosinase inhibitors, side effects caused by administration of antiplatelet drugs, and the sensitivity of harmful blood reactions caused by drugs (see Patent Documents 1 to 5). However, it is not known about the relationship between these genes and renal cell carcinoma, and that the expression of these genes correlates with the sensitivity of renal cell carcinoma to immunotherapy.

WO2009 / 149297 WO2008 / 134729 WO2007 / 099852 JP 2007-117085 US20060177860

Negrier S, Escudier B, Lasset C, Douillard JY, Savary J, Chevreau C, Ravaud A, Mercatello A, Peny J, Mousseau M, Philip T, Tursz T. Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma. Groupe Francais d'Immunotherapie. N Engl J Med. 1998; 338 (18): 1272-8 Atzpodien J, Hoffmann R, Franzke M, Stief C, Wandert T, Reitz M. Thirteen-year, long-term efficacy of interferon 2alpha and interleukin 2-based home therapy in patients with advanced renal cell carcinoma.Cancer. 2002; 95 ( 5): 1045-50 Miyake H, Hara I, Sakai I, Harada K, Inoue TA, Eto H, Takechi Y, Fujisawa M. Clinical outcome of combined immunotherapy with low-dose interleukin-2 and interferon-alpha for Japanese patients with metastatic renal cell carcinoma who had undergone radical nephrectomy: a preliminary report. Int J Clin Oncol. 2005; 10 (5): 338-41 Koji Kawai et al. "Low-volume IL-2 and IFNα combination therapy is effective for lung metastases of renal cell carcinoma (renal tumor / pharmacotherapy 2, general presentation, 97th Annual Meeting of the Japanese Urological Association)" Japanese Journal of Urology 100 (2) pp.298 2009) Tourani JM, Pfister C, Tubiana N, Ouldkaci M, Prevot G, Lucas V, Oudard S, Malet M, Cottu P, Ferrero JM, Mayeur D, Rixe O, Sun XS, Bernard O, Andre T, Tournigand C, Muracciole X , Guilhot J; Subcutaneous Administration Propeukin Program Cooperative Group.Subcutaneous interleukin-2 and interferon alfa administration in patients with metastatic renal cell carcinoma: final results of SCAPP III, a large, multicenter, phase II, nonrandomized study with sequential analysis design--the Subcutaneous Administration Propeukin Program Cooperative Group. J Clin Oncol. 2003; 21 (21): 3987-94. Bukowski RM. Natural history and therapy of metastatic renal cell carcinoma: the role of interleukin-2. Cancer. 1997; 80 (7): 1198-220.

  The present invention relates to genes associated with susceptibility to IL-2 and IFN-α combination therapy in renal cell carcinoma patients, preferably metastatic renal cell carcinoma patients, particularly renal cell carcinoma patients with metastasis (hereinafter referred to as “IL-2 / IFN-α combination therapy susceptibility gene ”), and using the expression level of the gene as an index, the susceptibility of IL-2 / IFN-α combination therapy to renal cell cancer patients, preferably patients with metastatic renal cell carcinoma An object is to provide a method of detection. Another object of the present invention is to provide reagents and reagent kits useful for carrying out the method.

  In the present specification, metastatic renal cell carcinoma patients are collectively referred to as “metastatic renal cell carcinoma patients”, including those already having metastasis.

  In order to solve the above-mentioned problems, the present inventors have studied gene information of renal cell carcinoma patients, particularly metastatic renal cell carcinoma patients, and found that the expression of HLA-DQA1 gene or HLA-DQB1 gene and By finding that there is a significant statistical correlation between the sensitivity to IL-2 / IFN-α combination therapy and using the expression level of at least one of HLA-DQA1 gene and HLA-DQB1 gene as an index, It was confirmed in advance that the effectiveness of IL-2 / IFN-α combination therapy for renal cell carcinoma patients can be determined. That is, by using the expression level of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene as an index, a therapeutic method suitable for renal cell cancer patients, particularly metastatic renal cell cancer patients can be selected. It is possible to avoid ineffective treatment for patients who are not effective with IFN-α combination therapy and effective and accurate IL-2 / IFN-α combination therapy for patients who are effective It becomes possible to apply.

The present invention has been completed on the basis of such findings and has the following aspects.
(I) Method for detecting sensitivity to renal cell carcinoma patient IL-2 / IFN-α combination therapy (I-1) Characteristic of using expression level of one or both of HLA-DQA1 gene and HLA-DQB1 gene as an index A method for detecting the sensitivity of a renal cell carcinoma patient to IL-2 / IFN-α combination therapy.
(I-2) The method according to (I-1), comprising a step of measuring the expression level of one or both of the HLA-DQA1 gene and the HLA-DQB1 gene for a sample derived from a renal cell carcinoma patient.
(I-3) The expression level (E1) of one or both of the HLA-DQA1 gene and the HLA-DQB1 gene is compared with the predetermined expression level (E0) of the corresponding gene. Determining a group higher than the dose (E0) as a response group to the IL-2 / IFN-α combination therapy and a lower group as a non-response group to the IL-2 / IFN-α combination therapy (I-1 ) Or (I-2).
(I-4) The expression level of the HLA-DQA1 gene or HLA-DQB1 gene is the amount of mRNA expressed by the HLA-DQA1 gene or HLA-DQB1 gene or the amount of cDNA derived therefrom (I-1) to (I The method described in any one of I-3).
(I-5) The expression level of the HLA-DQA1 gene or HLA-DQB1 gene is the amount of HLA-DQA1 protein or HLA-DQB1 protein expressed by the HLA-DQA1 gene or HLA-DQB1 gene (I-1 ) To (I-3).

(I-6) The method according to any one of (I-1) to (I-5), wherein the renal cell carcinoma patient is a metastatic renal cell carcinoma patient.
(I-7) The method according to (I-6), wherein the metastasis is lung metastasis.

  In addition, these inventions can be paraphrased as “a method for selecting a successful group for IL-2 / IFN-α combination therapy from patients with renal cell carcinoma”.

(II) Reagent or reagent kit for detecting sensitivity to IL-2 / IFN-α combination therapy for renal cell carcinoma patients (II-1) Reagent for detecting sensitivity to IL-2 / IFN-α combination therapy for renal cell cancer patients A reagent or reagent kit comprising at least one of the primer shown in (a) below and the probe shown in (b):
(A) an oligonucleotide primer having a length of at least 15 bases having a base sequence for specifically amplifying mRNA or cDNA of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene;
(B) An oligonucleotide probe having a length of at least 15 bases having a base sequence that specifically hybridizes to DNA derived from mRNA of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene.

  (II-2) A reagent or reagent kit for detecting sensitivity to IL-2 and IFN-α combination therapy for renal cell carcinoma patients, comprising an antibody against at least one of HLA-DQA1 protein and HLA-DQB1 protein.

(II-3) The reagent or reagent kit according to (II-1) or (II-2), wherein the renal cell carcinoma patient is a metastatic renal cell carcinoma patient.
(II-4) The reagent or reagent kit according to (II-3), wherein the metastatic lesion is lung.

(III) IL-2 determined by the method described in any of (III-1), (I-3) to (I-7), a therapeutic agent for renal cell carcinoma comprising a combination of IL-2 and IFN-α And a therapeutic agent for renal cell carcinoma comprising a combination of IL-2 and IFN-α, which is used for patients with renal cell carcinoma belonging to the group susceptible to IFN-α combination therapy.
(III-2) 7 to 2.1 million units (JRU) of IL-2 are administered by intravenous infusion for 1 month every day, and IFN-α is 3 to 10 million units (IU) of each administration. The therapeutic agent for renal cell carcinoma according to (III-1), which is used by being administered 2 to 5 times a week by subcutaneous or intramuscular injection.

  According to the present invention, by measuring the expression level of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene in renal cell carcinoma patients, particularly metastatic renal cell carcinoma patients, IL-2 / The effectiveness of IFN-α combination therapy can be determined. Specifically, patients judged to have high effectiveness of IL-2 / IFN-α combination therapy (sensitivity to IL-2 / IFN-α combination therapy) (at least one of HLA-DQA1 gene and HLA-DQB1 gene) Patients with high expression levels of IL-2 / IFN-α are selected as active treatment and IL-2 / IFN-α combination therapy as a treatment policy. be able to.

  Conversely, in the present invention, patients who were judged to have low efficacy (sensitivity to IL-2 / IFN-α combination therapy) of IL-2 / IFN-α combination therapy (HLA-DQA1 gene and HLA-DQB1 gene For patients with low expression level of at least one gene), treatment options other than IL-2 / IFN-α combination therapy are treated as non-successful IL-2 / IFN-α combination therapy. Can be given. This is effective both in reducing the burden of treatment costs (health) caused by unnecessary (ineffective) treatment and reducing the side effects that can be caused by IL-2 / IFN-α combination therapy. It is.

ROC (Receiver Operating Characteristics) curve showing the relationship between the expression level of HLA-DQA1 gene in patients with renal cell carcinoma and the therapeutic response of IL-2 / IFN-α combination therapy, and the cut-off value calculated from it and kidney cells It is a figure which shows the relationship with 42 cancer patients (sensitivity group, tolerance group). ROC (Receiver Operating Characteristics) curve showing the relationship between the expression level of HLA-DQB1 gene and the therapeutic response of IL-2 / IFN-α combination therapy in renal cell carcinoma patients, and the cut-off value calculated from it and renal cells It is a figure which shows the relationship with 42 cancer patients (sensitivity group, tolerance group). Based on the cut-off values of the expression levels of HLA-DQA1 and HLA-DQB1 genes calculated in Example 3, renal cell carcinoma patients (learning case: sensitive group [-◆-], resistant group [-■-], test The case sensitivity group [-▲-] and tolerance group [-◆-]) are grouped.

1. IL-2 / IFN-α combination therapy susceptibility gene The present inventors have a renal cell carcinoma patient, particularly a metastatic renal cell carcinoma patient, in which the expression level of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene is high. (For example, renal cell carcinoma patients with lung metastases (pulmonary metastasis patients)) are more sensitive to IL-2 / IFN-α combination therapy than patients with low expression of these genes. 2 / IFN-α combination therapy enjoys high cancer treatment effect, whereas patients with renal cell carcinoma, especially those with metastatic renal cell carcinoma (eg, lung metastasis patients) with low expression of these genes It is resistant to IL-2 / IFN-α combination therapy compared to patients with high expression levels, and IL-2 / IFN-α combination therapy does not provide a significant cancer treatment effect. The above-mentioned HLA-DQA1 gene and HLA-DQB1 gene are useful for IL-2 / IFN-α combination therapy in patients with renal cell carcinoma, especially metastatic renal cell carcinoma. To susceptibility I was convinced that it is related (efficacy) and significantly.

  Based on this, the present inventors have used individual HCC-DQA1 gene and HLA-DQB1 gene expression level as an index, so that individual renal cell carcinoma patients, particularly metastatic renal cell carcinoma We were convinced that it was possible to determine whether IL-2 / IFN-α combination therapy was effective as a cancer therapy for patients.

  Based on the findings thus obtained, the present invention provides a susceptibility gene (IL-2 / IFN-α combination therapy sensitivity to IL-2 / IFN-α combination therapy in patients with renal cell carcinoma, particularly patients with metastatic renal cell carcinoma). HLA-DQA1 gene and HLA-DQB1 gene are provided as genes).

  Both the sequence information of these genes and the information (amino acid sequences) of their expression products are known. For example, information on human-derived HLA-DQA1 mRNA and protein (HLA-DQA1 protein) is registered in NCBI as NM_002122.3 and NP_002113.2, respectively. Information on human-derived HLA-DQB1 gene mRNA and protein (HLA-DQB1 protein) is registered in the NCBI as NM_002123.3 and XP_002343953.1, respectively. However, since these genes are known to have many polymorphisms, they are not particularly limited to the sequence information registered therein.

  The origin of the HLA-DQA1 gene and HLA-DQB1 gene targeted by the present invention is preferably human. However, it is not limited to humans, but may be derived from mammals such as mice, rats, guinea pigs, monkeys, rabbits, pigs, sheep and cows.

2. Medicinal marker for renal cell cancer The present invention relates to a patient who is effective in IL-2 / IFN-α combination therapy as a cancer therapy among patients with renal cell carcinoma, particularly patients with metastatic renal cell carcinoma (for example, patients with lung metastasis). HLA-DQA1 gene as a marker for screening (IL-2 / IFN-α combination therapy sensitive group) (IL-2 / IFN-α combination therapy sensitive group selection marker, also called “renal cell cancer drug efficacy marker”) And the expression product of either or both of the HLA-DQB1 gene. As such expression products, for the HLA-DQA1 gene, mRNA of the gene or cDNA derived therefrom and HLA-DQA1 protein, and for the HLA-DQB1 gene, mRNA of the gene or cDNA derived therefrom and HLA-DQB1 Mention may be made of proteins.

  By detecting the IL-2 / IFN-α combination therapy sensitive group selection marker in patients with renal cell carcinoma, especially metastatic patients, and measuring the amount thereof, IL-2 and IFN-α It can be evaluated and judged in advance whether the combination therapy is effective for treating the cancer.

3. Method for Detecting Sensitivity of IL-2 / IFN-α Combination Therapy for Renal Cell Carcinoma Patients The present invention provides a method for detecting the sensitivity of IL-2 / IFN-α combination therapy for patients with renal cell carcinoma. In this method, sensitivity to IL-2 / IFN-α combination therapy is detected in advance for each renal cell cancer patient before cancer treatment, and IL-2 / IFN-α combination therapy is effective for the patient. It is used to determine whether or not. In this method, in the IL-2 / IFN-α combination therapy, a sensitivity group (response group) to the IL-2 / IFN-α combination therapy is selected from the renal cell carcinoma patient group, and the resistance group (non-response group). It can also be said to be a method of sieving.

  Such a method is used in patients with renal cell carcinoma, particularly metastatic renal cell carcinoma patients (pulmonary metastasis patients), in which the expression level of either or both of the HLA-DQA1 gene and the HLA-DQB1 gene is a predetermined expression level ( More patients than E0) are more sensitive to IL-2 / IFN-α combination therapy, and anti-cancer such as renal cell carcinoma (primary focus) and reduction of metastases with IL-2 / IFN-α combination therapy Patients who can enjoy the action effectively, but patients with lower HLA-DQA1 and HLA-DQB1 gene expression levels (E0) than IL-2 / IFN-α combination therapy It is based on the finding that it is resistant to IL-2 / IFN-α combination therapy and cannot receive an effective anticancer effect.

  Are renal cell carcinoma patients sensitive to IL-2 / IFN-α combination therapy (whether they belong to the IL-2 / IFN-α combination therapy group) or IL-2 / IFN-α combination therapy To detect whether it is resistant to IL-2 / IFN-α combination therapy, use the expression level of one or both of HLA-DQA1 gene and HLA-DQB1 gene as an index Done in Therefore, the detection method includes a step of measuring the expression level of one or both of the HLA-DQA1 gene and the HLA-DQB1 gene for a biological sample of a renal cell carcinoma patient.

  Renal cell carcinoma patients are human. The tissue type of renal cell carcinoma of these patients is not particularly limited, and may be any of clear cell carcinoma type, papillary renal cell carcinoma type, or mixed type. Further, such patients may be those who have renal cell carcinoma as the primary focus and have metastatic focus. Preferred examples of such metastatic organs include lungs, but those having metastatic lesions in organs other than lungs may also be used. The renal cell carcinoma patient targeted by the present invention is preferably a metastatic renal cell carcinoma patient, more preferably a renal cell carcinoma patient having lung metastasis.

  The sample derived from these patients may be any sample that can contain the HLA-DQA1 gene or HLA-DQB1 gene and its expression product, and any cell (cultured) isolated from renal cell carcinoma patients and clinical specimens. Preparation of biological samples such as cells), tissues (eg, liver, etc., including cultured tissues), or body fluids (eg, blood, saliva, lymph, oral mucosa, airway mucosa, sweat, urine, etc.) as materials. Can do. As the material, leukocytes or mononuclear cells separated from peripheral blood are preferable, and leukocytes are particularly preferable. These materials can be isolated according to methods commonly used in clinical laboratory tests.

For example, when leukocytes are used as a material, leukocytes are first separated from peripheral blood isolated from a renal cell carcinoma patient according to a conventional method. Subsequently, proteinase K and sodium dodecyl sulfate are added to the obtained leukocytes to degrade and denature the protein, followed by phenol / chloroform extraction to obtain a gene (including mRNA). Incidentally, extraction of the gene (including mRNA) is not limited to the above method, methods well known in the art (e.g., Sambrook J. et al, "Molecular Cloning:.. A Laboratry Manual (2 nd Ed. ) ”Cold Spring Harbor Laboratory, NY) or a commercially available DNA extraction kit.

  In the detection method of the present invention, the HLA-DQA1 gene or the HLA-DQB1 gene (hereinafter collectively referred to as “HLA-DQA1 / B1 gene”) is used for the sample derived from the above renal cell carcinoma patient. Or it is implemented by measuring the expression level of both.

  Measurement of the expression level of these HLA-DQA1 / B1 genes can be performed according to a conventional method based on known information. For example, the expression level (E1) of the HLA-DQA1 / B1 gene is measured by measuring the expression level (mRNA amount) of the gene. In this case, the amount of mRNA is determined by, for example, Northern blotting or microarray using the nucleotide sequence of HLA-DQA1 / B1 gene or a nucleic acid containing a part thereof as a probe, or one of the nucleotide sequences of HLA-DQA1 / B1 gene. It can be measured by a PCR method or RT-PCR method using a part-containing nucleic acid as a PCR primer or a method analogous thereto. The expression level (E1) of the HLA-DQA1 gene or HLA-DQB1 gene may be measured by measuring the amount of HLA-DQA1 protein or HLA-DQB1 protein expressed and produced from these genes, respectively. At this time, the amount of the protein is determined by Western blotting or ELISA using an antibody that recognizes the amino acid sequence of HLA-DQA1 protein or HLA-DQB1 protein or a part of the protein, in a cell extract or the like. It can be measured by a method such as a method, an immunohistochemical method or the like. In the detection method of the present invention, the gene whose expression level is measured may be either one of the HLA-DQA1 gene or the HLA-DQB1 gene, or the expression level of both genes is measured. Sensitivity to IL-2 / IFN-α combination therapy may be comprehensively judged.

In the detection method of the present invention, subsequently, there is a step of comparing the expression level (E1) of the HLA-DQA1 / B1 gene measured in the above step with a predetermined expression level (E0) for each gene. Here, the “predetermined expression level (E0)” is an expression level that serves as a reference for determining the sensitivity of IL-2 / IFN-α combination therapy for renal cell carcinoma patients, and can be arbitrarily set in advance. Value. In setting the “predetermined expression level (EO)” in advance, the present inventors discovered for the first time that the expression level of HLA-DQA1 / B1 gene is IL-2 / IFN-α of renal cell carcinoma patients. The finding that “shows a positive correlation with sensitivity to combination therapy” is taken into account. That is, in the subsequent process, a cutoff value that serves as a reference when determining the presence or absence of IL-2 / IFN-α combination therapy sensitivity in renal cell carcinoma patients (if the expression level exceeds this value, IL- 2 / IFN-α combination therapy ”is determined, and if the expression level is less than this value, the“ threshold value determined to be resistant to IL-2 / IFN-α combination therapy ”) The expression level (E0) can be preset. More specifically, as shown in Example 2, with regard to renal cell carcinoma patients, particularly metastatic patients, the expression levels of HLA-DQA1 gene or HLA-DQB1 gene and IL-2 / IFN-α combination therapy in advance Using the ROC (Receiver Operating Characteristics) curve, both sensitivity and specificity are close to 1, and the expression “(1-sensitivity) ² + (1-specificity) ² ”can be determined as the smallest value, and this can be set as“ predetermined expression level (E0) ”.

  The detection method of the present invention subsequently determines whether the expression level (E1) of the HLA-DQA1 gene or HLA-DQB1 gene is significantly higher than the predetermined expression level (E0) as a result of the comparison performed above. to decide. There is no particular limitation on the method for determining the presence or absence of a significant difference, and statistical methods well known to those skilled in the art can be used. As an example, statistically “significantly high” often means that the null hypothesis H0 is rejected at a significance level of 0.05 (5%). Based on the results of this determination, the sensitivity of renal cell carcinoma patients to IL-2 / IFN-α combination therapy is evaluated. That is, if the expression level (E1) of the HLA-DQA1 / B1 gene is significantly higher than the predetermined expression level (E0), “IL-2 / IFN-α combination” “Sensitive to therapy” or “highly effective with IL-2 / IFN-α combination therapy”. On the other hand, if the expression level (E1) of the HLA-DQA1 / B1 gene is not significantly higher than the predetermined expression level (E0), the IL-2 / IFN- It can be determined that “tolerant to α combination therapy” or “low efficacy with IL-2 / IFN-α combination therapy”.

  Based on such determination, determination of a more appropriate treatment policy can be promoted. For example, for patients with renal cell carcinoma that have been determined to have “high (sensitivity)” to IL-2 / IFN-α combination therapy by the detection method of the present invention, IL-2 / IFN-α combination therapy is the first. The treatment policy selected as one option will be determined. As a result, surgical resection with physical and mental burdens can be avoided for such patients, and it is possible to provide effective treatment with less burden and risk of side effects for the patients. It becomes. On the other hand, in patients with renal cell carcinoma determined to be “not (low)” (tolerant) to IL-2 / IFN-α combination therapy by the detection method of the present invention, IL-2 / IFN-α combination Treatment with options other than therapy can avoid medications that are not only effective but can also cause side effects.

4). Reagent for detecting IL-2 / IFN-α sensitivity and kit containing the same The present invention provides a reagent and a reagent kit for detecting the sensitivity of a renal cell carcinoma patient to IL-2 / IFN-α combination therapy.

  Examples of the components of the reagent or reagent kit include probes, primers, and antibodies described below.

(1) Probe The probe is used to measure the expression level of one or both of the HLA-DQA1 gene and the HLA-DQB1 gene and the expression level (mRNA level) of the gene. Such a probe can be used when measuring the mRNA level of HLA-DQA1 gene or HLA-DQB1 gene by Northern blotting or microarray method.

  Specific examples of such probes include oligonucleotide probes having a length of at least 15 bases having a base sequence that specifically hybridizes to mRNA derived from mRNA of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene. it can. Specifically, the probe has a length of 15 to 500 bases, preferably 20 to 200 bases, more preferably 20 to 50 bases in the base sequence of at least one of the HLA-DQA1 gene or the HLA-DQB1 gene. It is designed as an oligo or polynucleotide having the above base length so as to specifically hybridize with the gene region.

  Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the second edition, conditions described in 1989), it means that cross-hybridization with other DNA does not occur significantly. Preferably, the oligo or polynucleotide preferably has a base sequence complementary to the base sequence of the HLA-DQA1 gene or HLA-DQB1 gene to be detected, but such specific hybridization is possible. For example, it need not be completely complementary.

  These oligos or polynucleotides can be prepared based on the base sequence of the HLA-DQA1 gene or HLA-DQB1 gene by, for example, a commercially available nucleotide synthesizer according to a conventional method.

  More preferably, the probe is labeled with a radioactive substance, a fluorescent substance, a chemiluminescent substance, an enzyme, or the like (described later).

  The probe (oligo or polynucleotide) can be used by immobilizing on an arbitrary solid phase. Therefore, the present invention also provides the probe (oligo or polynucleotide) as an immobilized probe (for example, a gene chip, a cDNA microarray, an oligo DNA array, a membrane filter, etc. on which the probe is immobilized). The probe can be preferably used as a DNA chip for detecting sensitivity to IL-2 / IFN-α combination therapy.

  The solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to. The immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).

(2) Primer When measuring the expression level of one or both of the HLA-DQA1 gene and HLA-DQB1 gene, the primer determines the amount of mRNA of HLA-DQA1 gene or HLA-DQB1 gene and the amount of its derivative. Can be used to specifically amplify by RT or RT-PCR.

  As such a primer, specifically, an oligonucleotide primer having a length of at least 15 bases having a base sequence that specifically hybridizes to DNA (cDNA) derived from mRNA of at least one gene of HLA-DQA1 gene or HLA-DQB1 gene Can be mentioned. Specifically, the primer is a continuous sequence having a length of 15 to 500 bases, preferably 20 to 200 bases, more preferably 20 to 50 bases in at least one base sequence of the HLA-DQA1 gene or the HLA-DQB1 gene. It is designed as an oligo or polynucleotide having the above base length so as to specifically hybridize with the gene region.

  Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the second edition, conditions described in 1989), it means that cross-hybridization with other DNA does not occur significantly. Preferably, the oligo or polynucleotide preferably has a base sequence complementary to the base sequence of the HLA-DQA1 gene or HLA-DQB1 gene to be detected, but such specific hybridization is possible. For example, it need not be completely complementary.

  These oligos or polynucleotides can be prepared based on the base sequence of the HLA-DQA1 gene or HLA-DQB1 gene by, for example, a commercially available nucleotide synthesizer according to a conventional method.

(3) Labeled substance The probe or primer of the present invention includes an appropriate labeling substance for detecting the expression level of HLA-DQA1 gene or HLA-DQB1 gene, such as fluorescent dye, enzyme, protein, radioisotope, chemical A substance to which a luminescent substance, biotin or the like is added is included.

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′ , 7'-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems) , Yellow-green fluorescent dye), rhodamine or a derivative thereof [eg, tetramethylrhodamine (TMR)], but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, Amersham Pharmacia, oligonucleotide ECL 3'-oligo labeling system, etc.).

  The above-mentioned probe (which may be labeled) or primer (which may be labeled) is an IL-2 / IFN-α combination therapy as a cancer treatment for patients with renal cell carcinoma, particularly those with metastasis. To determine whether or not is effective (IL-2 / IFN-α combination therapy sensitivity detection reagent), in other words, patients with renal cell carcinoma, especially those with metastasis, are treated with IL-2 / IFN-α It can be used as a reagent for selecting a successful group and a non-success group in combination therapy. In other words, the above-mentioned probe (which may be labeled) or primer (which may be labeled) is effective for IL-2 / IFN-α combination therapy for renal cell carcinoma patients, particularly metastatic patients, from another angle. It can be defined as a reagent for selecting whether a person is an effective person or an ineffective person.

(4) Reagent kit for detecting IL-2 / IFN-α sensitivity The present invention also provides a reagent for detecting (selecting) the above-mentioned IL-2 / IFN-α combination therapy sensitive group (response group). As provided. The kit includes at least one oligo or polynucleotide used as the probe or primer (which may be labeled or immobilized on a solid phase). In addition to the probe or primer described above, the kit of the present invention includes other reagents and instruments necessary for carrying out the method of the present invention, such as a hybridization reagent, a probe label, a label detection agent, and a buffer as necessary. Etc. may be included as appropriate. Furthermore, the reagent kit of the present invention includes a method for using reagents such as primers and probes, and a method for detecting an IL-2 / IFN-α combination therapy sensitive group (response group) using the same (determination criteria, predetermined expression level). (E0)) may be included.

5. Therapeutic agent for renal cell carcinoma comprising a combination of IL-2 and IFN-α The present invention provides a therapeutic agent for renal cell carcinoma comprising a combination of IL-2 and IFN-α. The therapeutic agent for renal cell carcinoma according to the present invention is used in advance for IL-2 and IFN-α combination therapy according to the above-described “method for detecting sensitivity to IL-2 / IFN-α combination therapy for renal cell cancer patients” described above. It is a therapeutic agent exclusively prescribed and administered to patients with renal cell carcinoma judged to be sensitive.

  Here, IL-2 and IFN-α are individually packaged and administered in parallel to the above renal cell carcinoma patients by different administration routes. Specifically, IL-2 is used in a schedule in which 7 to 2.1 million units (JRU) per administration are administered to the above renal cell carcinoma patient by intravenous infusion over a period of one month every day. On the other hand, IFN-α is used in a schedule in which 3 to 10 million units (IU) per administration are administered at a rate of 2 to 5 times per week by subcutaneous or intramuscular injection to the above renal cell carcinoma patients.

  The therapeutic agent for renal cell carcinoma of the present invention is not particularly limited as long as it is a combination used in the above-mentioned administration form for the above-mentioned specific renal cell carcinoma patient, and the liquid form (infusion solution, injection solution) is not particularly limited. For example, it may have a form of a freeze-dried preparation used by dissolving in physiological saline or water for injection at the time of use.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1 Criteria for non-invasive treatment of renal cell carcinoma in 42 patients (see Table 1) with renal cell carcinoma with selected lung metastases (Japanese Journal of Urology, Vol. 83, No. 4, 1992) Based on this, the clinical effect was determined.

  Based on the judgment results (response rate: reduction rate of lesions that can be measured in two directions), the response group (CR: Complete Response), the effective group (PR: Partial Response), the unchanged group (NC: No Change), and the progression group ( Classified as PD: Progressive Disease. Of these, cases in which the reduction rate of the bilaterally measurable lesions was 25% or more and less than 50% were regarded as a slightly effective group (MR: Minor Response) (Table 2). Of these, a total of 15 cases in the effective group and the effective group were treated as susceptibility groups (response groups) to the IL-2 / IFN-α combination therapy, and a total of 27 cases in the effective group, unchanged group, and advanced group were treated with IL- As the tolerance group (non-response group) to the 2 / IFN-α combination therapy, analysis described in Example 2 and later was performed.

Example 2 By using 38,500 gene expression information obtained from screening array analysis of target genes , IL-2 / IFN-α combination therapy sensitive group (hereinafter referred to as sensitivity group) and IL-2 / IFN-α combination Genes with significantly different expression levels were screened between therapy resistant groups (hereinafter referred to as resistance groups).

  Based on the reduction rate of lung metastases, the top 10 cases with a high reduction rate among 15 cases in the susceptibility group and the lower 18 cases (a total of 28 cases) with a low reduction rate among the 27 cases in the resistance group were designated as “learning cases” The rest was designated as a “test case”. That is, the “test case” includes 5 cases from the lower reduction rate among the 15 cases in the sensitivity group, and 9 cases (total 14 cases) from the upper case in the 27 tolerance group having the higher reduction rate.

The “learning case” was analyzed using a random permutation test (Clin Cancer Res., Vol. 11, 2005, p2565-2636) (1,000,000 times). In addition, a gene satisfying both of the following conditions: (i) the p-value is less than 5 × 10 ⁻⁴ , (ii) 60% or more of the cases having expression information in at least one of the sensitive group and the resistant group The gene to be analyzed was used. As a result, 24 genes were identified as candidate genes for predicting sensitivity to IL-2 / IFN-α combination therapy. Of these 24 genes, 14 genes were highly expressed in the sensitive group compared to the resistant group.

Example 3 Calculation of Cut-off Value Predicting Treatment Effect Among 14 genes selected in Example 2, HLA-DQA1 (random permutation test p value; 2.83E-05), HLA-DQB1 (same as 2.66E) -04) were selected and a detailed analysis was performed on the relationship between the expression level of each gene and the response of IL-2 / IFN-α combination therapy (Table 2). Specifically, a ROC (Receiver Operating Characteristic) curve is drawn for each gene, and the sensitivity (sensitivity) and specificity (specificity) are both close to 1, using such a curve, and “(1-sensitivity) ² + (1 -specificity) The value at which ² "was the smallest was taken as the best cut-off value for the expression level of each gene. As a result, the cut-off value of HLA-DQA1 was 0.315 (see FIG. 1), and HLA-DQB1 was 1.177 (see FIG. 2).

Example 4 Correlation between Expression Levels of HLA-DQA1 and HLA-DQB1 Genes and Treatment Effects Sensitivity groups of learning cases (10 cases, FIG. 3) based on the cut-off value of the expression level of each gene determined above. The middle “-♦-” and tolerance groups (18 cases, indicated by “-■-” in FIG. 3) were classified into groups higher and lower than these cut-off values. The results are shown in FIG.

  As shown in FIG. 3, for the HLA-DQA1 gene, the number of patients whose expression level is lower than the cut-off value is 1 in 10 in the sensitive group and 13 in 18 in the resistant group, which is lower than the cut-off value. Among patients with low expression (14 cases), the proportion of patients in the tolerance group was high (13 cases / 14 cases: 92.9% ineffective rate), and even when test cases were included, it was as high as 85.7% ( Of the patients (21 cases) whose expression level is lower than the cut-off value, 18 patients (18 cases / 21 cases) accounted for by the resistant group.

  On the other hand, for HLA-DQB1, the number of patients whose expression level is lower than the cut-off value is 2 out of 10 in the sensitivity group, and 14 out of 18 cases in the resistance group. Among the patients whose expression level is lower than the cut-off value The percentage of patients in the tolerance group was as high as 14 cases (14 cases / 16 cases: 87.5% ineffectiveness rate), and even when including test cases, it was as high as 82.6% (patients whose expression level was lower than the cutoff value ( Among the 23 cases), 19 patients (19 cases / 23 cases) accounted for by the resistant group.

  In this study, the effective rate of IL-2 / IFN-α combination therapy for renal cell carcinoma patients was 35.7%. From the above results, it was confirmed that HLA-DQA1 gene expression level is low in renal cell carcinoma patients. On the other hand, by avoiding IL-2 / IFN-α combination therapy, this effective rate increases to 64.3% (57.1% including the test case), and renal cell carcinoma with low HLA-DQB1 gene expression. By avoiding IL-2 / IFN-α combination therapy for patients, the above-mentioned effective rate was confirmed to increase to 66.6% (57.9% including the test case).

  Based on the above, by measuring the expression level of HLA-DQA1 gene and HLA-DQB1 gene, treatment response of IL-2 / IFN-α combination therapy for renal cell carcinoma patients, especially renal cell carcinoma patients with lung metastases Sex (response) can be predicted before treatment, and clinical application can be expected.

Claims (4)

Characterized in that the one or index both the expression level of HLA-DQAl gene and HLA-DQB1 genes, met sensitive method for detecting renal cell carcinoma patients to IL-2 and IFN-alpha combination therapy with lung metastases And
Compare the expression level (E1) of one or both of the HLA-DQA1 gene and HLA-DQB1 gene in renal cell carcinoma patients with lung metastases with the specified expression level (EO) for each of the above genes. ) Having a higher expression level (EO) than the group that is sensitive to IL2 and IFNα combination therapy, or a group that is lower than the group that is resistant to IL-2 and IFNα combination therapy . The method according to claim 1, further comprising the step of measuring the expression level of one or both of the HLA-DQA1 gene and the HLA-DQB1 gene for a sample derived from a renal cell carcinoma patient having lung metastasis . Reagent or reagent kit for detecting the sensitivity of IL-2 and IFN-α combination therapy for renal cell carcinoma patients with lung metastasis containing one or both of the primers shown in (a) and (b) below:
(A) an oligonucleotide primer having a base sequence of at least 15 bases having a base sequence for specifically amplifying mRNA or cDNA of at least one of the HLA-DQA1 gene and the HLA-DQB1 gene; and (b) above (a) of a probe used in combination with oligonucleotide primers, at least 15 bases having a nucleotide sequence that specifically hybridizes to c DNA derived from at least one of the genes of mRNA of HLA-DQAl gene and HLA-DQB1 gene Long oligonucleotide probe. A reagent or reagent kit for detecting sensitivity to IL2 and IFNα combination therapy for renal cell carcinoma patients with lung metastases, comprising an antibody against at least one of HLA-DQA1 protein and HLA-DQB1 protein.

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