JP2012237586A - Diagnostic reagent for selecting effective treatment method of anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means or the like capable of specifying cancer of which the treatment by an anticancer agent is effective.SOLUTION: A diagnostic reagent includes a substance having an affinity with an HER2 extracellular domain fragment and diagnoses an effect of an anticancer agent to lung cancer. A diagnostic kit includes the substance having the affinity with the HER2 extracellular domain fragment and the HER2 extracellular domain fragment and diagnoses an effect of the anticancer agent to the lung cancer. A method for testing the effect of the anticancer agent to the lung cancer includes a step (a) for measuring the concentration of HER2 extracellular domain fragments in a biological sample derived from a subject and a step (b) for comparing the measured concentration of HER2 extracellular domain fragments with a reference indicating relation between the concentration of HER2 extracellular domain fragments and the effect of the anticancer agent to the lung cancer.

Description

  The present invention relates to a diagnostic reagent for selecting an effective treatment method for an anticancer agent.

In the treatment of lung cancer, it has been reported that a specific inhibitor against EGFR exhibits an excellent therapeutic effect against non-small cell lung cancer having an active mutant EGFR. Such lung cancer has the property of proliferating depending on EGFR, and this is the basis of the therapeutic response by the EGFR inhibitor.
However, there have been reports of cases where treatment with an EGFR inhibitor is effective despite not expressing active mutant EGFR. As described above, it is considered that there are cancers other than those having active mutant EGFR as cancers for which treatment with EGFR inhibitors is effective. However, means for identifying such cancers (eg, biomarkers) have been reported. Not.

  In the treatment of lung cancer, in addition to the above-mentioned active mutant EGFR, kinase-derived genes such as gene amplification MET and fusion gene EML4-ALK have been identified as clear therapeutic targets, and these therapeutic agents can be applied to the treatment of lung cancer. Or has been shown to be effective in clinical trials. However, other kinases have not proven to be clear therapeutic targets.

In relation to the above, the following prior art has been reported.
Non-Patent Document 1 discloses that there are cases in which an EGFR inhibitor (erlotinib) is effective for squamous cell carcinoma of the lung that does not show mutated EGFR.
Non-Patent Document 2 discloses that erbB-2 (another name for HER2) was found in the serum and tumor of breast cancer patients.
Non-Patent Document 3 discloses that the extracellular domain of p185 / neu (another name for HER2) is released from the surface of a human breast cancer cell line.
Non-Patent Document 4 discloses that HER2 extracellular domain secretion and serum circulation were found in breast cancer, and that such a measurement of secretion and serum circulation is effective for treatment with anti-HER2 antibody (Herceptin). It discloses that the diagnosis of breast cancer and its therapeutic effect can be followed.

Clark GM, Cameron T, Das Gupta A et al. Clinical benefit of erlotinib in male smokers in squamous cell NSCLC. J Clin Oncol 2006; 24: 7166. Breuer, B.M. , DeVivo, I.M. Luo, J .; C. , Et al. erbB-2 and myc oncoproteins in serra and tutors of breast cancer patents. Cancer Epidemiol. Biomark. Prev. 3: 63-66, 1994. Zabrecky, J .; R. Lam, T .; McKenzie, S .; et al. The extracellular domain of p185 / neu is released from the surface of human breast carcinoma cells, SK-BR-3. J. et al. Biol. Chem. 266: 1716-1720, 1991. Kostler WJ, et al. Monitoring of serum Her-2 / neu predicts response and progression-free Survival to trastuzumab-basic treatments in patents with breast breast. Clin Cancer Res 2004; 10: 1618-24.

  As described above, there have been reported cases in which treatment with an EGFR inhibitor is effective despite not expressing active mutant EGFR, and active cancer with active mutant EGFR is an effective treatment with an EGFR inhibitor. It is thought that it exists in addition to the cancer which has this. However, no means has been reported for identifying cancers as described above for which treatment with EGFR inhibitors is effective. Moreover, it has not been reported that HER2 extracellular domain fragment is detected in a biological sample in lung cancer.

  An object of the present invention is to provide a means or the like that can identify lung cancer for which treatment with an anticancer agent such as an EGFR inhibitor is effective.

  As a result of intensive studies, the present inventors have found that HER2 extracellular domain fragments are detected in biological samples in lung cancer. The present inventors have also conducted intensive analysis on the active state of EGFR and other kinase factors in cancer cells that are sensitive to EGFR inhibitors despite having no active mutant EGFR. It was found that it proliferates depending on another HER2 kinase without depending on it. Based on these findings, the present inventors have conceived that the therapeutic effect of an anticancer drug on lung cancer can be easily determined by measuring the concentration of the HER2 extracellular domain fragment, and the present invention has been completed. It was.

That is, the present invention is as follows.
[1] A diagnostic reagent for the effect of an anticancer agent on lung cancer, comprising a substance having affinity for the HER2 extracellular domain fragment.
[2] The diagnostic reagent according to [1], wherein the substance having affinity for the HER2 extracellular domain fragment is an antibody against the HER2 extracellular domain fragment.
[3] The diagnostic reagent according to [1] or [2], wherein the lung cancer is blood HER2-positive lung cancer.
[4] The diagnostic reagent according to any one of [1] to [3], wherein the anticancer agent is an EGFR inhibitor.
[5] The diagnostic reagent according to any one of [1] to [4], wherein the lung cancer is non-small cell lung cancer.
[6] The diagnostic reagent according to any one of [1] to [5], wherein the lung cancer is lung squamous cell carcinoma.
[7] An EGFR inhibitor is represented by the following formula (1):
The diagnostic reagent of [4], which is a compound having a basic structure represented by the formula:
[8] A diagnostic kit for the effect of an anticancer agent on lung cancer, comprising a substance having affinity for the HER2 extracellular domain fragment, and the HER2 extracellular domain fragment.
[9] A method for testing the effect of an anticancer agent on lung cancer, comprising the following steps (a) and (b):
(A) measuring the concentration of the HER2 extracellular domain fragment in a biological sample from the subject; and (b) measuring the concentration of the HER2 extracellular domain fragment, the concentration of the HER2 extracellular domain fragment, and lung cancer Comparing to a criterion indicating a relationship between the effects of the anticancer agent.

  According to the present invention, the effect of an anticancer agent on lung cancer can be diagnosed by measuring soluble HER2 in a biological sample (eg, blood), and a subject suffering from lung cancer can be diagnosed by measuring soluble HER2 in the biological sample. it can.

FIG. 1 is a graph showing the sensitivity of various human lung cancer cell lines to EGFR inhibitor (Gefitinib) (n = 4). PC-9: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having an active mutant EGFR; HCC827: derived from human non-small cell lung cancer (lung adenocarcinoma) and an active mutant EGFR HCC4006: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having an active mutant EGFR; H2170: derived from human non-small cell lung cancer (lung squamous cell carcinoma) and active Cell lines without mutant EGFR; and H1703: cell lines derived from human non-small cell lung cancer (lung adenocarcinoma) and without active mutant EGFR. FIG. 2 is a diagram showing analysis of EGFR and HER2 expression and activation states. A549: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having no active mutation EGFR; PC-9: derived from human non-small cell lung cancer (lung adenocarcinoma) and active mutation Cell line with EGFR; H460: derived from human non-small cell lung cancer (Lung Large Cell Carcinoma) and without active mutant EGFR; H1650: derived from human non-small cell lung cancer (Lung Adenocarcinoma) H1703: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having no active mutant EGFR; H1975: human non-human A cell line derived from small cell lung cancer (lung adenocarcinoma) and having a secondary resistance mutation in EGFR that becomes resistant to active mutant EGFR and EGFR inhibitor; H1993: human non-small cell lung cancer (lung adenocarcinoma) Derived from activating mutant EG Cell line not having R; H2170: Cell line derived from human non-small cell lung cancer (lung squamous cell carcinoma) and having no active mutant EGFR; HCC827: Human non-small cell lung cancer (lung adenocarcinoma) A cell line derived from and having an active mutant EGFR; HCC4006: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having an active mutant EGFR. Western blotting used the following antibodies: EGFR (an antibody recognizing EGFR intracellular domain); HER2 (an antibody recognizing HER2 intracellular domain); p-Tyr (anti-phosphotyrosine antibody) and β-actin ( Control). FIG. 3 is a diagram showing HER2 activity inhibition by an EGFR inhibitor (Gefitinib). NCI-H2170: A cell line derived from human non-small cell lung cancer (lung squamous cell carcinoma) and having no active mutant EGFR. Western blotting used the following antibodies: pY (anti-phosphotyrosine antibody); HER2 (antibody recognizing HER2 intracellular domain). FIG. 4 shows EGFR or HER2 expression in human lung cancer cell lines treated with EGFR siRNA or HER2 siRNA. NCI-H2170: a cell line derived from human non-small cell lung cancer (lung squamous cell carcinoma) and having no active mutant EGFR; HCC4006: derived from human non-small cell lung cancer (lung adenocarcinoma) and active Cell line having mutant EGFR; HCC827: Cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having active mutant EGFR. Western blotting used the following antibodies: HER2 (an antibody recognizing the HER2 intracellular domain); and EGFR (an antibody recognizing the EGFR intracellular domain). FIG. 5 shows the growth of human lung cancer cell lines treated with EGFR siRNA or HER2 siRNA (n = 4). The cell line used is the same as in FIG. Four types of siRNA (si-EGFR-1, si-EGFR-2, si-HER2-1, si-HER2-2) were used. N. C. (Stealth RNAi siRNA Negative Control, manufactured by Invitrogen) FIG. 6 is a graph showing the relationship between HER2 phosphorylation and EGFR expression in human lung cancer cell lines. The details of the antibody used are the same as in FIG. FIG. 7 is a diagram showing detection by Western blot of secreted HER2 in a human lung cancer cell line. The details of the cell line used are as described above. The anti-HER2 antibody used in the Western blot recognizes secreted HER2 (ie, HER2 extracellular domain). FIG. 8 is a diagram showing detection by ELISA kit of soluble HER2 in the serum of mice in which cancer tissues are formed (n = 2). The details of the cell line used are as described above. The anti-HER2 antibody attached to the ELISA kit recognizes soluble HER2 (ie, HER2 extracellular domain). FIG. 9 shows the sensitivity of various human lung cancer cell lines to EGFR inhibitor (Erlotinib) (n = 4). PC-9: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having an active mutant EGFR; HCC827: derived from human non-small cell lung cancer (lung adenocarcinoma) and an active mutant EGFR HCC4006: a cell line derived from human non-small cell lung cancer (lung adenocarcinoma) and having an active mutant EGFR; H2170: derived from human non-small cell lung cancer (lung squamous cell carcinoma) and active Cell lines without mutant EGFR; and H1703: cell lines derived from human non-small cell lung cancer (lung adenocarcinoma) and without active mutant EGFR. FIG. 10 shows the sensitivity of various human lung cancer cell lines to EGFR inhibitor (Lapatinib) (n = 4). Details of the cells are the same as in FIG.

  The present invention provides a method for testing the effect of anticancer agents on cancer. The test method of the present invention may be a diagnostic method or a prediction method.

The test method of the present invention may include the following steps (a) and (b):
(A) measuring the concentration of the HER2 extracellular domain fragment in a biological sample derived from the subject; and (b) measuring the concentration of the HER2 extracellular domain fragment, the concentration of the HER2 extracellular domain fragment, and an anticancer agent against cancer. Comparing with criteria indicating a relationship between the effects of

  HER2 is a receptor tyrosine kinase that belongs to the same family as epidermal growth factor (EGFR). It has a structure in which an extracellular domain, a transmembrane domain, and an intracellular domain having tyrosine kinase activity are linked in a single chain (for example, Coussens et al., Science, 1985, Vol. 230, No. 4730, pp. 1132-1139. reference). For the HER2 sequence, see Genebank Accession Number: NP_001005862.1.

  As used herein, the term “HER2 extracellular domain fragment” refers to a HER2 extracellular domain polypeptide secreted from the surface of cancer cells or a degradation product of the polypeptide. Herein, the HER2 extracellular domain fragment may be referred to as secreted HER2 or soluble HER2.

  In the above (a), examples of the subject include a mammal suffering from cancer or a mammal possibly having cancer. Examples of mammals include primates, pets, and working animals. Specific examples of mammals include humans, chimpanzees, dogs, cats, cows, sheep, horses, pigs, rabbits, mice, and rats, with humans being preferred.

  Examples of cancer include lung cancer (eg, squamous cell carcinoma, non-small cell cancer such as adenocarcinoma and large cell cancer, and small cell cancer), digestive system cancer (eg, stomach cancer, small intestine cancer, colon cancer, rectal cancer). ), Pancreatic cancer, kidney cancer, liver cancer, thymic cancer, spleen cancer, thyroid cancer, adrenal cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer (eg, endometrial cancer, cervical cancer), bone cancer, Examples include skin cancer, brain tumor, sarcoma, melanoma, blastoma (eg, neuroblastoma), adenocarcinoma, squamous cell carcinoma, solid cancer, epithelial cancer, and mesothelioma.

  The cancer may be a HER2-dependent cancer. As used herein, the term “dependence” for cell growth refers to an oncogene addition or addiction state in which the cell relies on growth for a particular oncogene. Say. HER2-dependent cancer can enhance HER2 expression. Thus, whether a cancer is HER2-dependent can be determined by measuring HER2 expression. Also, the dependence on a specific oncogene (ie, whether or not growth is dependent on a specific oncogene) treats the cell with an inhibitor of the specific oncogene and then the growth of the treated cell This can be confirmed by evaluating performance. The proliferative ability can be evaluated by, for example, an MTT assay or an MTS assay. In addition, it is known that cell death due to apoptosis can be induced by treating an oncogene-dependent cell with a specific oncogene inhibitor for a specific oncogene. Therefore, the dependence on a specific oncogene may be confirmed, for example, by evaluating whether apoptosis can be induced by inhibition of the oncogene. Apoptosis induction can be evaluated by, for example, TUNEL assay, detection of active caspase, or detection of ANNEXIN V.

  In HER2-dependent cancer, since HER2 expression can be enhanced, the amount of HER2 extracellular domain fragment secreted into the biological sample (which can be correlated with the HER2 expression level) can also increase. Therefore, HER2-dependent cancer usually has a HER2 extracellular domain fragment detected in a biological sample (eg, blood HER2 positive).

  An example of a preferred subject is a cancer patient that is being treated with a particular anticancer agent. Specific anticancer agents include, for example, EGFR inhibitors, HER2 inhibitors, c-MET inhibitors, EGFR inhibitors, ALK inhibitors, PDGFR inhibitors, and c-KIT inhibitors. Examples of these inhibitors include low molecular weight organic compounds and antibodies.

  Examples of the EGFR inhibitor include gefitinib, lapatinib, erlotinib, cetuximab, ZD6474, CL-387785, HKI-272, XL647, PD15730B, E6473 2992, EKB-569, PF-299804.

  The EGFR inhibitor also has, for example, the following formula (1):

Or a salt thereof having a basic structure represented by the formula: The basic structure represented by the above formula (1) is known as a structure that can be common to EGFR inhibitors of low molecular organic compounds. The compound having a basic structure represented by the above formula (1) or a salt thereof has a predetermined structure having 1 to 5 (eg, 1, 2, 3) basic structures represented by the above formula (1). A compound substituted with a substituent or a salt thereof. The compound having the basic structure represented by the above formula (1) or a salt thereof may be a hydrate, and both a hydrate and a non-hydrate are included in the scope of the present invention. . Examples of the salt include metal salts (eg, sodium salts, potassium salts, magnesium salts, calcium salts), inorganic salts such as ammonium salts, and various salts such as organic salts (eg, alkyl ammonium).

  The structures of gefitinib, lapatinib, and erlotinib are shown below for reference of the compound or salt having the basic structure represented by the above formula (1).

  Examples of the HER2 inhibitor include trastuzumab (Herceptin), pertuzumab, lapatinib that inhibits both EGFR and HER2, and BIBW2992. HER2 inhibitors also include EGFR inhibitors that are pharmaceuticals of low molecular weight organic compounds. The present inventors have found that such an EGFR inhibitor also acts as a HER2 inhibitor (see Examples described later).

  Examples of c-MET inhibitors include PHA-666552, SU11274, XL-880, XL-184, ARQ 197, AMG208, AMG458, CE-355621, and MP470. Examples of the ALK inhibitor include WHI-P154, TAE684, and PF-234066. Examples of PDGFR inhibitors include Gleevec, Desatinib, Valatinib, and Pazopanib. Examples of c-KIT inhibitors include imatinib, sunitinib, varatinib, desatinib, masatinib, and motesanib, and zotanib.

  Another example of a preferred subject is a cancer patient whose therapeutic effect by a particular anticancer agent is being monitored. The cancer patient may also be a patient who is required to determine whether or not to switch to treatment with another anticancer agent when a decrease in the therapeutic effect with a specific anticancer agent is observed. The anticancer agent can be the same as the anticancer agent described above.

  The biological sample that can be used in the present invention can be a biological sample collected from the subject described above. The biological sample is not particularly limited as long as the HER2 extracellular domain fragment is present, and examples thereof include blood, serum, urine, saliva, ascites, tissue sample, cell sample, tissue extract, and cell extract. Of the above, blood, urine, and saliva are preferable from the viewpoint of low invasiveness. If necessary, the biological sample may be processed in advance prior to measurement. Examples of such treatment include centrifugation, extraction, concentration, fractionation, cell fixation, tissue fixation, tissue freezing, and tissue thinning.

  The concentration of the HER2 extracellular domain fragment can be measured using, for example, a substance having affinity for the HER2 extracellular domain fragment (described later). Measurement may also be performed by immunological techniques. Examples of such immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples include immunochromatography, luminescence immunoassay, spin immunoassay, western blot, and latex agglutination. Examples of methods other than the above that enable measurement of the concentration of the HER2 extracellular domain fragment include LC-MS.

  In (b) above, the measured concentration of HER2 extracellular domain fragment can be compared to a criterion indicating the relationship between the concentration of HER2 extracellular domain fragment and the effect of an anticancer agent on cancer. The criteria may indicate a relative relationship between high and low HER2 extracellular domain fragment concentrations and the strength of anticancer drug effects (sensitivity or resistance to anticancer drugs). For example, if the measured concentration of HER2 extracellular domain fragment is relatively high, the therapeutic effect of the EGFR inhibitor (or HER2 inhibitor) may be high, or other anticancer agents (eg, as described above) Such as c-MET inhibitor, ALK inhibitor, PDGFR inhibitor, c-KIT inhibitor). Also, if the measured concentration of the HER2 extracellular domain fragment is relatively low, the therapeutic effect of the EGFR inhibitor or HER2 inhibitor may be low, or other anticancer agents (eg, c -A MET inhibitor, ALK inhibitor, PDGFR inhibitor, c-KIT inhibitor) may have a high therapeutic effect. In the case of the present invention, a specific reference value may be provided as a reference. As such a reference value, patients who are sensitive to EGFR inhibitors (or HER2 inhibitors) or patients who are resistant to other anticancer agents show a high positive rate (above the reference value), and EGFR inhibitors (or The concentration of the HER2 extracellular domain fragment that may show a high negative rate (below the reference value) in patients resistant to (HER2 inhibitors) or patients sensitive to other anticancer agents may be set. An example of such a reference value is a cutoff value. The calculation method of the reference value is well known in this field.

  According to the present invention, it can be determined whether a subject is sensitive or resistant to a particular anticancer agent. Such determination is useful, for example, for determining an anticancer agent to be administered to a subject. For example, if the measured concentration of HER2 extracellular domain fragment is relatively high, the cancer patient may be sensitive to an EGFR inhibitor (or HER2 inhibitor) and the EGFR inhibitor (or There is a possibility that the therapeutic effect by the HER2 inhibitor) is high. In this case, the anticancer agent may be resistant to other anticancer agents (eg, c-MET inhibitor, ALK inhibitor, PDGFR inhibitor, c-KIT inhibitor as described above). The therapeutic effect of may be low. On the other hand, if the measured concentration of HER2 extracellular domain fragment is relatively low, the cancer patient may be resistant to an EGFR inhibitor (or HER2 inhibitor) and the EGFR inhibitor (or HER2 inhibitor) may have a low therapeutic effect. In this case, the anticancer agent may be sensitive to other anticancer agents (eg, c-MET inhibitor, ALK inhibitor, PDGFR inhibitor, c-KIT inhibitor as described above). May have a high therapeutic effect.

  In addition, according to the present invention, the therapeutic effect of a subject with a specific anticancer agent can be evaluated (eg, monitoring over time). Such evaluation is useful, for example, in determining whether to switch to treatment with another anticancer agent when a decrease in the therapeutic effect with a specific anticancer agent is observed. For example, cancer patients may still be sensitive to EGFR inhibitors (or HER2 inhibitors) if the measured concentration of HER2 extracellular domain fragments remains relatively high. Yes, there is a possibility that the therapeutic effect of the EGFR inhibitor (or HER2 inhibitor) will continue to be high. In this case, it may still be resistant to other anticancer agents (eg, c-MET inhibitor, ALK inhibitor, PDGFR inhibitor, c-KIT inhibitor as described above), The therapeutic effect of the anticancer agent may be low. On the other hand, if the measured concentration of the HER2 extracellular domain fragment remains relatively low, the cancer patient may still be resistant to an EGFR inhibitor (or HER2 inhibitor). Yes, the therapeutic effects of EGFR inhibitors (or HER2 inhibitors) may continue to be low. In this case, it may still be sensitive to other anticancer agents (eg, c-MET inhibitor, ALK inhibitor, PDGFR inhibitor, c-KIT inhibitor as described above), The therapeutic effect of the anticancer drug may continue to be high.

  The test method of the present invention may include a step of (b ') determining the effect of an anticancer agent on cancer using the measured concentration of the HER2 extracellular domain fragment as an index instead of the above (b). This step can be performed in the same manner as (b) above. The test method of the present invention is also based on such judgment, and the anticancer agent to be administered (eg, EGFR inhibitor, HER2 inhibitor, c-MET inhibitor, EGFR inhibitor, ALK inhibitor, PDGFR inhibitor as described above) , C-KIT inhibitor) and / or anticancer agent to be administered is another anticancer agent (eg, EGFR inhibitor, HER2 inhibitor, c-MET inhibitor, ALK inhibitor, PDGFR inhibitor as described above) , C-KIT inhibitor) may be included. The test method of the present invention may further include a step of administering an anticancer agent based on such determination.

  The present invention provides a diagnostic reagent for the effect of an anticancer agent on cancer. The reagent of the present invention contains a substance having affinity for the HER2 extracellular domain fragment. The diagnostic reagent of the present invention may be in the form of a kit. The diagnostic reagent of the present invention can be suitably used for performing the test method of the present invention.

  As used herein, the term “substance having affinity for a HER2 extracellular domain fragment” refers to a substance having the ability to bind to a HER2 extracellular domain fragment. Examples of the substance having affinity for the HER2 extracellular domain fragment include an antibody against the HER2 extracellular domain fragment and an aptamer against the HER2 extracellular domain fragment.

The antibody against the HER2 extracellular domain fragment is not particularly limited as long as it is an antibody that can specifically bind to the HER2 extracellular domain fragment. For example, the antibody against the HER2 extracellular domain fragment may be either a polyclonal antibody or a monoclonal antibody. The antibody may also be a fragment of an antibody (eg, Fab, F (ab ′) ₂ ), a recombinant antibody (eg, scFv). The antibody may be provided in a form immobilized on a substrate such as a plate or impregnated in a support such as a strip. An antibody can be produced by a method known per se using a HER2 extracellular domain fragment or a partial peptide thereof as an antigen. The antigen can be appropriately prepared with reference to the amino acid sequence information of HER2. Further, a HER2 extracellular domain fragment obtained from the supernatant of cultured cancer cells may be used as an antigen.

  Polyclonal antibodies can be used, for example, by using the HER2 extracellular domain fragment or a partial peptide thereof as an antigen together with a commercially available adjuvant (eg, complete or incomplete Freund's adjuvant) in the animal subcutaneously or intraperitoneally every 2 to 3 weeks. It can be obtained by administering about twice, collecting whole blood about 3 to about 10 days after the final immunization, and purifying the antiserum. The antigen may be a complex in which a HER2 extracellular domain fragment or a partial peptide thereof is cross-linked to a carrier protein (eg, bovine serum albumin, KLH). Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, cows, guinea pigs, and hamsters.

  A monoclonal antibody can be prepared, for example, by a cell fusion method. For example, a HER2 extracellular domain fragment or a partial peptide thereof is administered to a mouse 2 to 4 times subcutaneously or intraperitoneally with a commercially available adjuvant, and spleen or lymph nodes are collected about 3 days after the final administration, and leukocytes are collected. The leukocytes and myeloma cells (eg, NS-1) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. Examples of cell fusion include a PEG method and a voltage pulse method. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  The aptamer for the HER2 extracellular domain fragment is not particularly limited as long as it is an aptamer that can specifically bind to the HER2 extracellular domain fragment. The aptamer can be produced by a technique called SELEX, for example.

A substance having affinity for the HER2 extracellular domain fragment may be provided in a form labeled with a labeling substance, if necessary. Examples of the labeling substance include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, biotin, and streptavidin. And the like, and the like.

  The diagnostic reagent of the present invention may be provided in the form of a kit containing additional components in addition to a substance having affinity for the HER2 extracellular domain fragment. In this case, each component included in the kit may be provided in a form isolated from each other, for example, stored in a different container (eg, a tube). For example, when a substance having affinity for the HER2 extracellular domain fragment is not labeled with a labeling substance, such a kit may further contain a labeling substance. Such a kit may also include a HER2 extracellular domain fragment as a positive control.

  When the diagnostic reagent of the present invention is provided in the form of a kit, the kit may contain additional components depending on the type of substance having affinity for the HER2 extracellular domain fragment. For example, when the substance having affinity for the HER2 extracellular domain fragment is an antibody, the kit may further contain a secondary antibody (eg, anti-IgG antibody) and a secondary antibody detection reagent.

  When the reagent of the present invention is provided in the form of a kit, the kit may further include an instrument capable of collecting a biological sample. The device that can collect the biological sample is not particularly limited as long as the biological sample can be obtained from the subject, and examples thereof include a blood collection device such as a syringe.

  Hereinafter, the present invention will be described in detail by way of examples. In addition, this invention is not limited by these Examples.

Example 1: Sensitivity test of various human lung cancer cell lines to EGFR inhibitors Among human lung cancer cell lines, PC-9, HCC827 and HCC4006 with active mutant EGFR, and H2170 and H1703 without active mutant EGFR Are exposed to different concentrations of EGFR inhibitor (Gefitinib), and the growth inhibitory effect of the drug is reduced by MTS (tetrazolium compound [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethylphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt)]) assay.
As a result, although the effect was weaker than PC-9, HCC827, and HCC4006 having active mutant EGFR, it was shown that proliferation of H2170 was also inhibited by the EGFR inhibitor (FIG. 1).

Example 2: Analysis of EGFR and HER2 expression and activation states EGFR and HER2 expression and activation states in various human lung cancer cell lines were analyzed by Western blot.
As a result, lung cancer cell lines (PC-9, H1650, H1975, HCC827, and HCC4006) having the active mutant EGFR express the active mutant EGFR (molecular weight about 160 kDa) and increase the phosphorylation of the molecular weight about 160 kDa protein. (See FIG. 2, EGFR and p-Tyr). This suggests that phosphorylation of active mutant EGFR is enhanced in these lung cancer cell lines. On the other hand, H2170, which did not have active mutant EGFR and was sensitive to EGFR inhibitors, did not express detectable amounts of EGFR and showed expression of a phosphorylation-promoting protein with a higher molecular weight (about 200 kDa) (FIG. 2, see EGFR and p-Tyr). This indicates that phosphorylation of proteins other than EGFR is enhanced in H2170. Therefore, an attempt was made to identify a protein having a molecular weight of about 200 kDa, in which phosphorylation was enhanced by H2170. As a result, in H2170, it was found that HER2 was overexpressed as a protein having a molecular weight of about 200 kDa (see FIG. 2, HER2).
From the above, it was shown that HER2 is overexpressed specifically in H2170.

Example 3: Inhibition of HER2 activity by EGFR inhibitor H2170 was treated with different concentrations of EGFR inhibitor (Gefitinib), and the activity inhibition effect on HER2 was confirmed by analyzing phosphorylated tyrosine and HER2 by Western blot.
As a result, the EGFR inhibitor decreased the phosphorylation of HER2 at a concentration of 1-10 μM or higher (see Example 1 and FIG. 1) showing an antitumor effect against H2170 (FIG. 3).
From the above, it was shown that the EGFR inhibitor inhibits the activity of HER2 (FIG. 3).

Example 4: Sensitivity to EGFR and HER2 knockdown HCC827 and HCC4006 having active mutant EGFR and H2170 not having active mutant EGFR are small interfering RNAs (siRNA) that specifically inhibit the expression of EGFR or HER2 gene. Processed by.
As a result, the growth of HCC827 and HCC4006 was suppressed as EGFR expression was inhibited by EGFR-specific siRNA (si-EGFR-1, si-EGFR-2) (FIG. 4) (FIG. 5). . On the other hand, proliferation of H2170 was suppressed as HER2 expression was inhibited by HER2-specific siRNA (si-HER2-1, si-HER2-2) (FIG. 4) (FIG. 5).
From the above, it was shown that H2170 does not depend on EGFR but proliferates depending on HER2, and that inhibition of HER2 suppresses proliferation of H2170. Thus, it has been shown that HER2 can be a therapeutic target with EGFR inhibitors.

Example 5: Analysis of the relationship between phosphorylation of HER2 and EGFR expression HCC827 and HCC4006 with active mutant EGFR and H2170 without active mutant EGFR specifically inhibit the expression of EGFR or HER2 gene Treated with small Interfering RNA (siRNA). The phosphorylation status of HER2 in these cells was confirmed by phosphorylated tyrosine blot against HER2 immunoprecipitate.
As a result, in HCC827 and HCC4006 that depend on the survival of the activated mutant EGFR, HER2 phosphorylation decreased with inhibition of EGFR expression (FIG. 6). On the other hand, in H2170, HER2 phosphorylation was not affected by inhibition of EGFR expression (FIG. 6). Therefore, it was shown that HER2 expressed by H2170 was not cross-activated by EGFR, unlike HER2 expressed by HCC827 and HCC4006.

Example 6: Diagnosis of HER2-dependent cancer by measurement of HER2 in serum (a) Detection by immunostaining Paraffin-embedded formalin-fixed sections were prepared from lung cancer tissues derived from mice transplanted with H2170 or HCC827. Lung cancer tissue specimens prepared by slicing them and fixing on glass slides were stained with anti-HER2 antibody and anti-EGFR antibody.
As a result, cancer derived from HER2-dependent H2170 could be specifically stained with an anti-HER2 antibody. Therefore, it was shown that a tissue containing HER2-dependent cancer cells can be immunostained with an anti-HER2 antibody.

(B) Detection by Western Blot H2170 or HCC827 was cultured, and HER2 (Lysate) expressed in the cells or HER2 (Supernatant) secreted into the medium was detected by Western blot with an anti-HER2 antibody.
As a result, secreted HER2 (HER2 extracellular domain fragment) was detected specifically for HER2-dependent H2170 (FIG. 7). Therefore, measurement of secreted HER2 showed that HER2-dependent cancer cells can be identified.

(C) Detection by ELISA Blood was collected from a mouse transplanted with H2170 or HCC827, and a serum sample was prepared. Serum HER2 was quantified by an ELISA kit that specifically detects soluble HER2.
As a result, soluble HER2 (HER2 extracellular domain fragment) was detected in the blood specifically in mice in which cancer tissues derived from HER2-dependent H2170 were formed (FIG. 8). Therefore, measurement of soluble HER2 in blood indicated that an individual can be identified as having HER2-dependent cancer cells.

  From the above, by measuring soluble HER2 in a biological sample (eg, blood), the effect of an anticancer agent on cancer can be diagnosed, and by measuring soluble HER2 in a biological sample, a subject suffering from cancer It was shown that it can be diagnosed.

Example 7: Sensitivity test of various human lung cancer cell lines to EGFR inhibitors other than gefitinib (a) Sensitivity test to Erlotinib Among human lung cancer cell lines, PC-9, HCC827 and HCC4006 having active mutant EGFR, and activity H2170 and H1703 without type mutant EGFR were exposed to different concentrations of EGFR inhibitor (Erlotinib), and the growth inhibitory effect of the drug was evaluated by MTS assay.
As a result, it was shown that the growth of H2170 was inhibited by erlotinib, similar to the results of the sensitivity test for Gefitinib shown in Example 1 (FIG. 9).

(B) Sensitivity test for Lapatinib Among human lung cancer cell lines, EGFR inhibitors having different concentrations of PC-9, HCC827 and HCC4006 having active mutant EGFR, and H2170 and H1703 not having active mutant EGFR (Lapatinib) The growth inhibitory effect of the drug was evaluated by MTS assay.
As a result, it was shown that the growth of H2170 was inhibited by Lapatinib, similar to the results of the sensitivity test for Gefitinib shown in Example 1 (FIG. 10).

  The present invention is useful, for example, for diagnosing the effect of an anticancer agent on cancer and diagnosing a subject suffering from cancer.

Claims (9)

  The diagnostic reagent of the effect of the anticancer agent with respect to lung cancer containing the substance which has affinity to a HER2 extracellular domain fragment.   The diagnostic reagent according to claim 1, wherein the substance having affinity for the HER2 extracellular domain fragment is an antibody against the HER2 extracellular domain fragment.   The diagnostic reagent according to claim 1 or 2, wherein the lung cancer is blood HER2-positive lung cancer.   The diagnostic reagent according to any one of claims 1 to 3, wherein the anticancer agent is an EGFR inhibitor.   The diagnostic reagent according to any one of claims 1 to 4, wherein the lung cancer is non-small cell lung cancer.   The diagnostic reagent according to any one of claims 1 to 5, wherein the lung cancer is lung squamous cell carcinoma. The EGFR inhibitor is represented by the following formula (1):
The diagnostic reagent of Claim 4 which is a compound or its salt which has a basic structure represented by these.   The diagnostic kit of the effect of the anticancer agent with respect to lung cancer containing the substance which has affinity to a HER2 extracellular domain fragment, and a HER2 extracellular domain fragment. A method for testing the effect of an anticancer agent on lung cancer, comprising the following steps (a) and (b):
(A) measuring the concentration of the HER2 extracellular domain fragment in a biological sample from the subject; and (b) measuring the concentration of the HER2 extracellular domain fragment, the concentration of the HER2 extracellular domain fragment, and lung cancer Comparing to a criterion indicating a relationship between the effects of the anticancer agent.