JP6882777B2 - Kits and methods for selecting cancer patients for whom administration of antibody drugs targeting the HER2 protein as a therapeutic target molecule is effective - Google Patents

Description

The present invention relates to a kit and a method for selecting a cancer patient for whom administration of an antibody drug having a HER2 protein as a therapeutic target molecule is effective.

The HER2 protein is a gene product of the human oncogene HER2 / neu (c-erbB-2) and is one of a series of epithelial growth factor receptor proteins (HER1-4) known as the HER family, on the cytoplasmic side. It is a transmembrane protein having a tyrosine kinase active region and a molecular weight of about 185,000. The HER2 protein forms a homodimer or a heterodimer with the HER family protein expressed on the cell surface, and plays a role of transmitting the proliferation signal of epithelial growth factor to the nucleus. So far, breast cancer, stomach cancer, esophageal cancer, colon cancer, bile duct cancer, bile sac cancer, non-small cell lung cancer, head and neck cancer, bladder cancer, ovarian cancer, uterine cancer, salivary adenocarcinoma, etc. HER2 overexpression has been confirmed in certain patients, and the relationship between prognosis and HER2 overexpression has been studied.

Trastuzumab (trade name "Herceptin (registered trademark)"), which is an anti-HER2 humanized monoclonal antibody, is listed in the NHI price list as a molecular-targeted drug that targets the HER2 protein, and breast cancer in which HER2 overexpression has been confirmed, and HER2 Approved for application to unresectable advanced / recurrent gastric cancer with confirmed overexpression. In recent years, while the development of molecular-targeted drugs has brought about an improvement in the cure rate, medical economics are also emphasized and use in more appropriate subjects is required. Therefore, trastuzumab is also used in the target molecule prior to administration. A test to confirm the overexpression of a certain HER2 protein and a test to confirm the amplification of the HER2 gene are indispensable. In the case of gastric cancer, immunohistochemistry (IHC) is used for HER2 protein overexpression, and in situ hybridization (ISH) method is used for HER2 gene amplification (Non-Patent Document 1).

Non-Patent Document 2 reports a concordance rate of HER2 overexpression test results between a sample obtained by endoscopic biopsy before surgery and a sample obtained by surgery in 61 cases of gastric cancer. According to this, there were 3 cases (4.9%) in which a positive result was obtained in the sample obtained by surgery after a negative result was obtained in the biopsy sample. This result indicates that false negatives occur in biopsy samples, and although these patients are originally treated with trastuzumab, trastuzumab is not obtained unless a subsequent positive result is obtained in the surgical sample. It means that you cannot receive standard treatment by. That is, it must be said that the current test method is insufficient to make an accurate judgment on a biopsy sample.

Thus, HER2 overexpression tests (IHC and ISH) using current biopsy samples produce false negatives of around a few percent, so cases that should benefit from trastuzumab treatment may be overlooked. .. In addition, both IHC and ISH have a large number of steps, the procedure is complicated, there are a plurality of factors that affect the test results, and the results may differ depending on the facility or pathologist. Furthermore, it takes several days to several weeks from the biopsy sampling to the acquisition of the test result, and there is also a problem that the speed is lacking. Therefore, it is desired to establish a test method that can quickly, easily and surely select a HER2 overexpressing patient.

Bang YJ, Van Cutsem E, Feyereislova A, et al: Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomized controlled trial . Lancet 2010 Aug 28; 376 (9742): 687-97. Pirrelli M, Caruso ML, Di Maggio M, Armentano R, Valentini AM. Are biopsy specimens predictive of HER2 status in gastric cancer patients? Digestive diseases and sciences. 2013; 58 (2): 397-404.

An object of the present invention is to provide a kit and a method capable of selecting a HER2 protein overexpressing patient quickly, easily and surely without causing false negatives even when using a biopsy sample. Another object of the present invention is to provide a kit and a method for selecting a cancer patient for which administration of an antibody drug having a HER2 protein as a therapeutic target molecule is effective. Furthermore, it is an object of the present invention to provide a kit and a method capable of selecting an antibody drug suitable for a patient.

The present invention includes the following inventions in order to solve the above problems.
[1] A kit for selecting cancer patients for whom administration of an antibody drug targeting HER2 protein as a therapeutic target molecule is effective, and a test strip for lateral flow immunoassay for detecting HER2 protein in cancer tissue. The test strip contains, in order from the upstream in the direction in which the sample flows, a sample pad to which the sample is added, a conjugate pad holding the first anti-HER2 protein antibody, and a second anti-HER2 protein antibody are immobilized. A membrane having a region in which an antibody that binds to the first anti-HER2 protein antibody is immobilized, and an absorption pad are linked to the first anti-HER2 protein antibody and the second anti-HER2 protein antibody. A kit characterized in that the antibody drug is used for any one of the HER2 protein antibodies.
[2] The kit according to the above [1], wherein the antibody drug is selected from the group consisting of trastuzumab, pertuzumab and trastuzumab-emtancin.
[3] The kit according to the above [1] or [2], wherein the first anti-HER2 protein antibody is labeled with colloidal fine particles.
[4] The kit according to any one of claims 1 to 3, wherein the antibody drug is used for the first anti-HER2 protein antibody.
[5] The kit according to any one of [1] to [3], wherein the antibody drug is used for the second anti-HER2 protein antibody.
[6] The membrane is a region in which a third anti-HER2 protein antibody is immobilized, or a region in which a third anti-HER2 protein antibody is immobilized and a fourth anti-HER2 protein antibody are immobilized. The second anti-HER2 protein antibody, the third anti-HER2 protein antibody, and the fourth anti-HER2 protein antibody are different antibody drugs. The kit according to any one of [3].
[7] The kit according to any one of [1] to [6] above, wherein a protein extract prepared from a cancer tissue collected from a subject is used as a sample.
[8] The kit according to the above [7], wherein the cancer tissue is a biopsy tissue.
[9] The kit according to the above [7] or [8], wherein the cancer tissue is a cancer tissue of the upper gastrointestinal tract.
[10] A method for selecting a cancer patient for which administration of an antibody drug using the HER2 protein as a therapeutic target molecule is effective, which comprises the following steps (1) to (3):
(1) Step of preparing a sample from cancer tissue collected from a subject,
(2) A step of detecting the HER2 protein in the sample by a lateral flow immunoassay using an antibody drug using the HER2 protein as a therapeutic target molecule, and (3) a step of selecting a subject who is positive in the lateral flow immunoassay. ..
[11] The method according to [10] above, wherein the antibody drug is selected from the group consisting of trastuzumab, pertuzumab and trastuzumab-emtancin.
[12] In the step (2), the sample pad to which the sample is added, the conjugate pad holding the first anti-HER2 protein antibody, and the second anti-HER2 protein antibody are solidified in order from the upstream in the direction in which the sample flows. Using a membrane having a phased region and a region in which an antibody that binds to the first anti-HER2 protein antibody is immobilized, and a test strip for lateral flow immunoassay having a structure in which an absorption pad is linked, the first test strip is used. The method according to the above [10] or [11], wherein the antibody drug is used for either the anti-HER2 protein antibody and the second anti-HER2 protein antibody.
[13] The method according to the above [12], wherein the first anti-HER2 protein antibody is labeled with colloidal fine particles.
[14] The method according to the above [12] or [13], wherein the antibody drug is used for the first anti-HER2 protein antibody.
[15] The method according to the above [12] or [13], wherein the antibody drug is used for the second anti-HER2 protein antibody.
[16] The membrane is immobilized with a region in which a third anti-HER2 protein antibody is immobilized, or a region in which a third anti-HER2 protein antibody is immobilized and a fourth anti-HER2 protein antibody are immobilized. The second anti-HER2 protein antibody, the third anti-HER2 protein antibody, and the fourth anti-HER2 protein antibody are different antibody drugs. The method according to [13].
[17] The method according to any one of [10] to [16], wherein the cancer tissue is a biopsy tissue.
[18] The method according to any one of [10] to [17], wherein the cancer tissue is a cancer tissue of the upper gastrointestinal tract.

According to the present invention, it is possible to provide a kit and a method capable of selecting a HER2 protein overexpressing patient quickly, easily and surely without causing false negatives even when using a biopsy sample. Further, since the present invention uses an antibody drug having the HER2 protein as a therapeutic target molecule as an antibody for detecting the HER2 protein, the effectiveness of the antibody drug on patients can be evaluated. In addition, compared to current biopsy samples, HER2 overexpression tests (IHC and ISH) are easier to operate and require less skill, which improves accuracy control and results in variability between facilities and judgment physicians. Can be improved.

It is a schematic diagram which shows the structure of an example of the test strip for lateral flow immunoassay used in this invention, (A) is a plan view, (B) is a side view. It is a figure which shows the result of having confirmed the HER2 protein in the protein extract prepared from various gastric cancer cell lines by the Western blotting method, and the result of quantifying the HER2 protein concentration in a sample by densitometry based on a Western blotting image. It is a figure which shows the result of having detected the HER2 protein in the protein extract prepared from various gastric cancer cell lines using the half strip of Example 1. It is a figure which shows the result of having detected the HER2 protein in the protein extract prepared from MKN7 cells using 5 kinds of half strips which different the immobilization amount of trastuzumab. It is a figure which shows the result of having detected the HER2 protein in the protein extract prepared from the breast cancer cell line MCF7 which has a low expression level of MKN7 cell and HER2 using the lateral flow strip of Example 1. It is a figure which shows the result of confirming the HER2 protein in the protein extract prepared from various gastric cancer cell lines by the Western blotting method, and quantifying the amount of HER2 protein per unit protein in a sample by ELISA. It is a figure which shows the result of having detected the HER2 protein contained in the protein extract prepared from various gastric cancer cell lines using the lateral flow strip of Example 5. It is a figure which shows the result of having detected the HER2 protein in the sample which diluted the protein extract prepared from GLM1 cell using the lateral flow strip of Example 5. The HER2 protein in the protein extract prepared from the GLM1 cell-derived tumor and the MKN45 cell-derived tumor formed under the skin of the nude mouse and the biopsy sample collected from the HER2-positive gastric cancer patient was subjected to the lateral flow strip of Example 5. It is a figure which shows the result of detection using.

The present invention provides a kit for selecting a cancer patient for whom administration of an antibody drug targeting a HER2 protein as a therapeutic target molecule is effective. The present invention also provides a method for selecting a cancer patient for whom administration of an antibody drug targeting a HER2 protein as a therapeutic target molecule is effective. Both the kits and methods of the present invention are characterized by detecting the HER2 protein in cancer tissue by a lateral flow immunoassay using an antibody drug targeting the HER2 protein as a therapeutic target molecule. The present invention is preferably carried out using a lateral flow immunoassay test strip configured to detect the HER2 protein in the sample.

The basic configuration of the test strip for lateral flow immunoassay that can be used in the present invention is not particularly limited, and the configuration of a known test strip used for ordinary lateral flow immunoassay can be adopted. For example, as shown in FIG. 1, it is composed of a membrane 1, an absorption pad 2, a conjugate pad 3, a sample pad 4, and a back sheet 5 in order from the upstream in the direction in which the sample flows (in the direction of the arrow in the development direction in FIG. 1). ), A test strip to which the sample pad 4, the conjugate pad 3, the membrane 1 and the absorption pad 2 are connected can be preferably used.

[Membrane]
The material of the membrane is not limited as long as it has a developing speed that allows sufficient sensitivity to be obtained in a short time as a chromatographic medium. For example, nitrocellulose membrane, cellulose membrane, acetylcellulose membrane, polysulfone membrane, polyethersulfone membrane, nylon membrane, glass fiber, non-woven fabric, filter paper and the like can be preferably used. As will be described later, a region on which the antibody is immobilized is formed on the membrane.

[Sample pad]
Examples of the material of the sample pad include, but are not limited to, those having uniform characteristics such as cellulose film, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth. The sample pad is a part to which the sample is added, but also has a function of filtering insoluble particles and the like in the sample.

[Conjugate pad]
Examples of the material of the conjugate pad include, but are not limited to, a cellulose film, glass fiber, and a non-woven fabric. The conjugate pad is prepared by uniformly impregnating the pad material with a labeled anti-HER2 protein antibody and drying it. The method for manufacturing the conjugate pad will be described later.

[Absorption pad]
The absorption pad is a site where the added sample is physically absorbed by capillarity and absorbs and removes the complex of the HER2 protein and the anti-HER2 protein antibody in the sample that did not bind to the immobilized antibody of the membrane. Is. As the material, a water-absorbent material such as a cellulosic film, a non-woven fabric, a cloth, or a cellulose acetate film can be preferably used. Depending on the material and size of the absorption pad, the developing speed of the added sample can be set to a desired speed.

[Anti-HER2 protein antibody]
The present invention presents the HER2 protein to either the first anti-HER2 protein antibody or the second anti-HER2 protein antibody in a lateral flow immunoassay using at least the first anti-HER2 protein antibody and the second anti-HER2 protein antibody. It is characterized by using an antibody drug whose therapeutic target molecule is. Examples of antibody drugs that use the HER2 protein as a therapeutic target molecule include trastuzumab (trade name: Herceptin, Chugai Pharmaceutical), pertuzumab (trade name: Perjeta, Chugai Pharmaceutical), and trastuzumab-emtancin (trade name: Kadcyla, Chugai Pharmaceutical). Be done. These antibody drugs are listed in the NHI price list and can be purchased and used.

Antibodies that use the HER2 protein as a therapeutic target molecule The anti-HER2 protein antibody other than the drug is not particularly limited as long as it is an antibody that can specifically bind to the human HER2 protein. A commercially available anti-human HER2 protein antibody, an anti-HER2 protein antibody having cross-reactivity with the human HER2 protein, and the like can be preferably used. In addition, anti-human HER2 protein antibody can be prepared and used by oneself. The anti-HER2 protein antibody may be a polyclonal antibody, a monoclonal antibody, or an antibody fragment. Preferably, it is an anti-human HER2 protein monoclonal antibody, or fragment thereof capable of binding to the HER2 protein. The first anti-HER2 protein antibody and the second anti-HER2 protein antibody are preferably different antibodies. When the first anti-HER2 protein antibody is an antibody drug, the second anti-HER2 protein antibody is preferably an anti-human HER2 protein antibody other than the antibody drug, and the first anti-HER2 protein antibody is other than the antibody drug. In the case of an anti-human HER2 protein antibody, the second anti-HER2 protein antibody is preferably an antibody drug.

[Labeling of anti-HER2 protein antibody]
The first anti-HER2 protein antibody retained on the conjugate pad is preferably labeled. The labeling substance is not particularly limited as long as it is a known labeling substance used for labeling an antibody, but it is preferable to use known colloidal fine particles that can be used for an immunoagglutination reaction. Specific examples thereof include gold colloid, silver colloid, platinum colloid, iron colloid, aluminum hydroxide colloid, latex particles, and composite colloids thereof. Preferably, it is a colored metal colloid such as gold colloid, silver colloid, platinum colloid, or a composite colloid thereof. More preferably, it is a gold colloid showing red color, a silver colloid showing yellow color, and even more preferably a gold colloid. The average particle size of the colloidal fine particles is preferably from about 1 nm to about 500 nm, more preferably from about 10 nm to about 150 nm, and even more preferably from about 20 nm to about 100 nm. When gold colloid is used as the colloidal fine particles, commercially available gold colloid particles or a gold colloid labeling kit can be preferably used. Alternatively, gold colloidal particles may be prepared by a known method. Examples of known methods for preparing gold colloidal particles include a method of reducing gold chloride acid with sodium citrate.

[Control antibody]
In the present specification, an antibody that binds to the first anti-HER2 protein antibody is referred to as a control antibody. Therefore, as the control antibody, a first anti-HER2 protein antibody, that is, an antibody capable of binding to a labeled anti-HER2 protein antibody, is used. The control antibody is not particularly limited as long as it can bind to the labeled anti-HER2 protein antibody to be used, and it should be appropriately selected from commercially available antibodies according to the animal species and antibody class of the labeled anti-HER2 protein antibody. Can be done. In addition, the control antibody can be made and used by oneself. The control antibody may be a polyclonal antibody, a monoclonal antibody, or an antibody fragment capable of binding to the first anti-HER2 protein antibody.

[Making a conjugate pad]
The method for retaining the labeled anti-HER2 protein antibody on the pad material (cellulose film, glass fiber, non-woven fabric, etc.) is not particularly limited. For example, a method can be used in which a labeled anti-HER2 protein antibody is suspended in an appropriate solvent to prepare a colloidal solution, and the colloidal solution is uniformly impregnated into the pad material by coating, spraying, dipping or the like, and then dried. .. The drying method is not particularly limited, and for example, drying means such as natural drying, vacuum drying, freeze drying, and heat drying can be used. The anti-HER2 protein antibody retained in the conjugate pad needs to be easily eluted by the sample and bind to the HER2 protein in the sample while moving with the sample. Therefore, it is preferable to add sugars such as sucrose, maltose and lactose, and sugar alcohols such as mannitol to the solvent for suspending the anti-HER2 protein antibody. Alternatively, these substances may be coated on the pad material in advance.

[Solid phase of antibody on membrane]
A region on which the second anti-HER2 protein antibody is immobilized and a region on which the control antibody is immobilized are formed on the membrane. The membrane may further have a region where a third anti-HER2 protein antibody is immobilized. The method for immobilizing the antibody on the membrane is not particularly limited, and the antibody can be immobilized by a known physical or chemical means. For example, a method of physically adsorbing the antibody to the membrane by dropping the antibody solution onto the surface of the membrane and drying it can be mentioned. The shape of the antibody-immobilized region is not particularly limited as long as the antibody is locally immobilized and the coloring can be visually confirmed. For example, a circular shape, a strip shape, a linear shape, or the like can be mentioned. The amount of antibody to be immobilized can be appropriately set in consideration of the detection sensitivity. For example, it is preferable that the amount of antibody in one region is immobilized in the range of about 0.01 μg to about 5 μg.

[Non-specific adsorption prevention treatment of sample pad]
In order to prevent the analysis target in the sample from being non-specifically adsorbed on the material of the sample pad, it is preferable to perform a non-specific adsorption prevention treatment on the sample pad. Examples of the non-specific adsorption prevention treatment include a method of uniformly impregnating a sample pad with a known blocking agent such as BSA (bovine serum albumin), skim milk, and casein and drying the sample pad. Specifically, for example, a blocking agent solution is prepared using an appropriate solvent so that the concentration is about 1% by mass to about 5% by mass, and about 60 μL of this solution is impregnated per test strip, and then dried. By doing so, non-specific adsorption prevention treatment can be performed. The non-specific adsorption prevention treatment may be performed on the membrane or both the sample pad and the membrane. The non-specific adsorption prevention treatment of the membrane can be performed in the same manner as the non-specific adsorption prevention treatment of the sample pad.

[Preparation of test strip for lateral flow immunoassay]
The lateral flow immunoassay test strip is prepared by connecting the sample pad 4, the conjugate pad 3, the membrane 1 and the absorption pad 2 from upstream to downstream in the direction in which the sample flows (the direction of the arrow in FIG. 1). be able to. In order to facilitate the occurrence of capillary action between the members, it is preferable to superimpose the adjacent members by about 1 mm to 7 mm. Further, it is preferable to attach these members on a backing sheet with an adhesive. As the backing sheet, a commercially available backing sheet for immunochromatography can be preferably used.

[Sample preparation]
Samples are prepared from cancer tissue taken from cancer patients. The target cancer patients are cancer patients who are suspected of having cancer in which HER2 is overexpressed. Cancers with confirmed overexpression of HER2 include breast cancer, stomach cancer, esophageal cancer, colon cancer, bile duct cancer, bile sac cancer, non-small cell lung cancer, head and neck cancer, bladder cancer, and ovary. Hmm, uterine cancer, salivary adenocarcinoma, etc. Therefore, the target cancer patients are preferably cancer patients suffering from these cancers. More preferably, it is a breast cancer patient, a gastric cancer patient, an esophageal cancer patient, a colon cancer patient, a bile duct cancer patient, a head and neck cancer patient, more preferably a breast cancer patient, a gastric cancer patient, and particularly preferably a gastric cancer patient. is there.

The cancer tissue may be collected from a cancer patient to be tested. It may be a biopsy tissue or a cancer tissue removed by surgery. It is preferably a biopsy tissue collected by endoscopy, and more preferably a biopsy tissue collected by upper gastrointestinal endoscopy.
It is preferable to prepare a protein extract from the above cancer tissue and use it as a sample for lateral flow immunoassay. The protein extract can be prepared using a known method. Specifically, for example, a commercially available tissue lysate (for example, RIPA (Radio-Immunoprecipitation Assay) buffer) is added to the cancer tissue for homogenization, and the obtained tissue lysate is centrifuged, and the supernatant thereof is separated. Can be used as a protein extract. The obtained protein extract can be cryopreserved at −80 ° C. The protein extract is preferably diluted appropriately so that the total amount of protein in the sample falls within a certain range when subjected to the lateral flow immunoassay. The total amount of protein in the sample can be appropriately set in consideration of the detection sensitivity. The protein concentration can be measured using a known method (eg, a commercially available protein quantification kit). The amount of the sample added to the sample pad can be appropriately set according to the size and shape of the test strip.

[Judgment method]
The determination is usually made by visually observing the coloration of the anti-HER2 protein antibody-immobilized region formed on the membrane. A commercially available immunochromatographic reader can also be used. If no coloring of the control antibody-immobilized region is observed, the test is judged to be invalid.

[First Embodiment]
The first embodiment of the present invention is carried out using a test strip for lateral flow immunoassay using an antibody drug using the HER2 protein as a therapeutic target molecule as the first anti-HER2 protein antibody. Here, the case where trastuzumab is used as the antibody drug will be described, but the same can be applied to the case where an antibody drug other than trastuzumab is used.

Trastuzumab is colloidal gold labeled and retained in a conjugate pad. As the second anti-HER2 protein antibody, a commercially available anti-HER2 protein antibody capable of specifically binding to the human HER2 protein is used. As the control antibody, an antibody capable of binding to gold colloid-labeled trastuzumab (for example, an anti-human IgG antibody) is used. The second anti-HER2 protein antibody and control antibody are immobilized in different regions on the membrane, respectively.

When the sample (protein extract) is added to the sample pad, it develops in the direction of the conjugate pad. The sample that reaches the conjugate pad develops further downstream while eluting the retained gold colloid-labeled trastuzumab. If the HER2 protein is present in the sample, the colloidal gold-labeled trastuzumab binds to the HER2 protein to form a complex. The complex is captured by a second anti-HER2 protein antibody and control antibody immobilized on the membrane, and each immobilized region is colored red. The control antibody also captures colloidal gold-labeled trastuzumab that does not form a complex. In the absence of HER2 in the sample, the colloidal gold-labeled trastuzumab does not form a complex with the HER2 protein, so the control antibody-immobilized region captures the colloidal gold-labeled trastuzumab and turns red, but the second anti-antibody. The HER2 protein antibody immobilized region is not colored. Therefore, on the premise that the control antibody-immobilized region is colored, when the second anti-HER2 protein antibody-immobilized region is colored, it is determined to be positive, and the second anti-HER2 protein antibody-immobilized region is colored. If not, it is judged as negative. Patients who are positive can be judged to be effective for treatment with trastuzumab.

In the present embodiment, when pertuzumab is used instead of trastuzumab, a patient who is determined to be positive can be determined to be a patient for whom treatment with pertuzumab is effective. Further, in the present embodiment, when trastuzumab-emtancin is used instead of trastuzumab, a patient determined to be positive can be determined to be a patient for whom treatment with trastuzumab-emtancin is effective.

[Second Embodiment]
A second embodiment of the present invention is carried out as a second anti-HER2 protein antibody using a test strip for lateral flow immunoassay using an antibody drug targeting the HER2 protein as a therapeutic target molecule. Here, the case where trastuzumab is used as the antibody drug will be described, but the same can be applied to the case where an antibody drug other than trastuzumab is used.

As the first anti-HER2 protein antibody, a commercially available anti-HER2 protein antibody capable of specifically binding to the human HER2 protein is used. The first anti-HER2 protein antibody is colloidal gold labeled and retained on a conjugate pad. As the control antibody, an antibody capable of binding to the first anti-HER2 protein antibody labeled with gold colloid is used. Trastuzumab and control antibody are immobilized in different regions on the membrane, respectively.

After adding the sample, the sample develops in the same manner as in the first embodiment. When the HER2 protein is present in the sample, both the trastuzumab-immobilized region and the control antibody-immobilized region are colored red, and when the HER2 protein is not present in the sample, only the control antibody-immobilized region is colored. Color red. Therefore, on the premise that the control antibody-immobilized region is colored, it is determined to be positive when the trastuzumab-immobilized region is colored, and it is determined to be negative when the trastuzumab-immobilized region is not colored. Patients who are positive can be judged to be effective for treatment with trastuzumab.

In the present embodiment, when pertuzumab is used instead of trastuzumab, a patient who is determined to be positive can be determined to be a patient for whom treatment with pertuzumab is effective. Further, in the present embodiment, when trastuzumab-emtancin is used instead of trastuzumab, a patient determined to be positive can be determined to be a patient for whom treatment with trastuzumab-emtancin is effective.

[Third Embodiment]
The third embodiment of the present invention is a lateral flow in which an antibody drug having a HER2 protein as a therapeutic target molecule, which is different from the second anti-HER2 protein antibody in the second embodiment, is further immobilized on a membrane. It is performed using a test strip for immunoassay. Different from the second anti-HER2 protein antibody, there may be one type or two or more types of antibody drugs using the HER2 protein as a therapeutic target molecule. When two types of antibody drugs are used as the second and third anti-HER2 protein antibodies, for example, a combination of trastuzumab and pertuzumab, a combination of trastuzumab and trastuzumab-emtancin, a combination of trastuzumab and trastuzumab-emtancin, etc. can be selected. it can. When the three antibody drugs are used as the second, third and fourth anti-HER2 protein antibodies, for example, a combination of trastuzumab, pertuzumab and trastuzumab-emtancin can be selected.

In the third embodiment, on the premise that the control antibody immobilization region is colored, when at least one of the plurality of antibody drug immobilization regions is colored, it is determined to be positive, and the plurality of antibody drug solid phases are determined to be positive. If all of the chemical regions are not colored, it is judged as negative. For example, when a test strip for lateral flow immunoassay having three types of antibody drug immobilization regions was used, the cancer tissue of the patient in which all three antibody drug immobilization regions were colored was immobilized. Since it is considered that the HER2 protein having high binding property to any antibody drug is overexpressed, it can be judged that all the immobilized antibody drugs are effective patients. The cancer tissue of a patient in which a colored antibody drug-immobilized region and an uncolored antibody drug-immobilized region coexist has high binding to the antibody drug immobilized on the colored region and is not colored. Since it is considered that the HER2 protein, which has low binding property to the antibody drug immobilized on the region, is overexpressed, treatment with the antibody drug immobilized on the colored region is effective, but it is colored. It can be determined that treatment with an antibody drug immobilized on a region that has not been present is not effective. The same judgment can be made when two types of antibody drugs are used and when four or more types of antibody drugs are used.

As described above, the third embodiment is considered to be very useful in that it can be used for selecting an antibody drug to be used for treating an individual cancer patient. When a new antibody drug is further developed, an antibody drug suitable for the patient can be selected from a plurality of available antibody drugs by increasing the number of anti-HER2 protein antibody-immobilized regions. it is conceivable that.

The kit of the present invention may include a test strip for lateral flow immunoassay for detecting the HER2 protein in cancer tissue. The test strip for the lateral flow immunoassay has already been described. The specific configuration of the kit other than these is not particularly limited, and other necessary reagents, instruments, instruction manuals, etc. may be appropriately selected to configure the kit. By using the kit of the present invention, it is possible to quickly, easily and surely select a cancer patient for which administration of an antibody drug using the HER2 protein as a therapeutic target molecule is effective.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. In the following, unless otherwise specified,% indicates mass%.

[Reference Example 1: Examination of HER2 expression in gastric cancer cell lines by Western blotting]
1. 1. Experimental materials and methods (1) Cells used As human gastric cancer cell lines, MKN1 (RIKEN BioResource Center No .: RCB1003), MKN7 (RIKEN BioResource Center No .: RCB0999), MKN45 (RIKEN BioResource Center No .: RCB1001) , MKN74 (RIKEN BioResource Center No .: RCB1002), GLM1 (Nakanishi, H., K. Yasui, Y. Ikehara, H. Yokoyama, S. Munesue, Y. Kodera and M. Tatematsu. Novel Human Gastric Cancer Cell Lines with Differentiated Intestinal Phenotype Derived from Liver Metastasis. "Clin Exp Metastasis 22, no. 2 (2005): 137-47.), GLM2 (same as above), GLM4 (Yokoyama, H., Y. Ikehara, Y. Kodera, S. Ikehara, Y. Yatabe, Y. Mochizuki, M. Koike, M. Fujiwara, A. Nakao, M. Tatematsu and H. Nakanishi. "Molecular Basis for Sensitivity and Acquired Resistance to Gefitinib in Her2-Overexpressing Human Gastric Cancer Cell Lines Derived from Liver Metastasis. "Br J Cancer 95, no. 11 (2006): 1504-13.), AGS (ATCC number: CRL-1739), KATOIII (RIKEN BioResource Center number: RCB2088) , HGC27 (RIKEN BioResource Center No .: RCB0500) was used. GLM1, GLM2 and GLM4 were donated by the Aichi Cancer Center Research Institute.
As a control, a breast cancer cell line MCF7 (RIKEN BioResource Center No .: RCB1904) was used. MCF7 is known to have low HER2 expression levels.
The cells were cultured in a 100 mm cell culture dish. As the medium, a known medium suitable for each cell was selected and used.

(2) Preparation of protein extract The medium of the dish in which the cells were cultured was discarded, and the cells were washed twice with PBS. RIPA buffer (10 mM Tris-HCl, pH7.4 150 mM NaCl, 1% NP40, 0.1% SDS, 0.1% sodium deoxycholate (DOC), 1 mM EDTA, 1 mM Na ₃ VO _{4) with protease inhibitor (Roche, Complete Mini)} , 2 mM PMSF) was added to the dish to lyse the cells, and the cell lysate was scraped off with a scraper and collected. The obtained cytolytic solution was centrifuged (14,000 rpm, 4 ° C., 30 min), and the supernatant was stored as a protein extract at −80 ° C. It was thawed at the time of use and subjected to the test.

(3) Western blotting The protein concentration of the protein extract of each cell prepared in (2) above was quantified using PIERCE BCA Protein Assay Kit (ThermoFischer Scientific). It was diluted with a sample buffer (Nacalai Tesque, Sample Buffer Solution with Reducing Reagent (6x) for SDS-PAGE) so that the protein concentration was 15 μg / lane, and subjected to SDS-PAGE. It was transferred to a PVDF membrane using a transblot Turbo transcription system (BIO-RAD). Anti-HER2 antibody (CST HER2 / ErbB2 Antibody), secondary antibody (GE healthcare ECL Rabbit IgG, HRP-linked whole Ab from donkey), anti-β-Actin antibody (CST β-Actin (13E5) Rabbit mAb HRP Conjugate ), An immunoblot was performed using each antibody. Immobilon Western (Merck Millipore) was used as the color former. The photo was taken with Chemidoc XRS + (BIO-RAD).

(4) Quantitative measurement by densitometry The captured image was analyzed by Image Lab software (BIO-RAD) to obtain the signal intensity of each band. A calibration curve was prepared using a recombinant HER2 protein (SignalChem, HER2, Active, E27-11G, molecular weight 131), and the HER2 concentration of each protein extract was determined based on this calibration curve.

2. Results The results are shown in FIG. The upper row shows the result of detecting HER2 protein by Western blotting, the middle row shows the result of detecting β-actin by Western blotting, and the lower row shows the result of quantifying HER2 protein by densitometry based on the Western blotting image of HER2 protein. From these results, it was clarified that the HER2 expression level of MKN7, GLM1, GLM2 and GLM4 was high.

[Example 1: Preparation of test strip (1)]
1-1 Strip material A nitrocellulose membrane card (Merck Millipore, HF120MC100, 60 mm × 301 mm) was used as the membrane. A glass fiber pad (Merck Millipore, GFCP103000) was used as the conjugate pad. Cellulose pads (Merck Millipore, CFSP173000) were used as sample pads and absorption pads.

1-2 Antibodies used The first anti-HER2 protein antibody (hereinafter referred to as "first antibody") is an anti-HER2 mouse monoclonal antibody (HER-2 / ErbB2 Monoclonal Antibody (N24), Thermo Scientific, MA1-12691. )It was used. Trastuzumab (Herceptin® injection 60, manufactured by Chugai Pharmaceutical Co., Ltd.) was used as the second anti-HER2 protein antibody (hereinafter referred to as “second antibody”). As a control antibody, an anti-mouse IgG antibody (Goat anti-Mouse IgG Secondary Antibody, Thermo Scientific, PA1-28555) was used. Antibody Stabilizer Based on PBS (CANDOR Bioscience, 131050, hereinafter referred to as "antibody diluent") was used for diluting the antibody.

1-3 Immobilization of the second antibody Herceptin® for injection 60 was dissolved according to the package insert to prepare a 21 mg / mL trastuzumab solution. This was diluted with an antibody diluent to prepare a trastuzumab solution having a desired concentration. The anti-mouse IgG antibody was diluted with an antibody diluent to prepare a 0.5 mg / mL control antibody solution. 1 μL of each antibody solution was attached to the membrane using a micropipette and dried at 37 ° C. for 120 minutes.

1-4 Preparation of conjugate pad 1-4-1 Gold colloid labeling of first antibody A gold colloid labeling kit (BBI GoldLink Kits, BB International, GLK 10.40) was used for the gold colloid labeling of the anti-HER2 mouse monoclonal antibody. .. Gold colloid labeling was performed according to the protocol of the kit, and a solution of the first antibody labeled with gold colloid containing 1 μg of the antibody in 50 μL of the solution was obtained.
1-4-2 Preparation of conjugate pad To 1 volume of the obtained solution of colloidal gold-labeled primary antibody, 25 volumes of application buffer (5% sucrose, 2 mM boric acid solution) was added and diluted to dilute it. The conjugate pad (Glass Fiber Conjugate Pad) was uniformly impregnated. The area of the conjugate pad and the antibody solution soaked so that the amount of the gold colloid-labeled primary antibody contained in the conjugate pad (5 mm × 10 mm) per test strip is 1 μL. The amount was adjusted as appropriate. Then, the conjugate pad was kept horizontal and dried at room temperature overnight.

Preparation of 1-5 sample pad BSA (Wako Pure Chemical Industries, Ltd., 015-21274) was dissolved in PBS (PBS tablet (Takara Bio) dissolved in ultrapure water) to prepare 5% BSA / PBS. This was uniformly impregnated into a sample pad (Cellulose Fiber Sample Pad). The area of the sample pad and the amount of liquid to be impregnated were appropriately adjusted so that 60 μL of 5% BSA / PBS was contained in the sample pad (5 mm × 17 mm) per test strip. Then, with the sample pad kept horizontal, it was completely dried at room temperature for at least one night.

Preparation of 1-6 Half Strip A half strip is a preliminary study strip composed of a membrane on which trastuzumab and a control antibody (anti-mouse IgG antibody) are immobilized and an absorption pad. One end of the membrane (the one to which the absorption pad is not connected) is immersed in a solution containing the sample and the colloidal gold-labeled primary antibody, and the solution is developed in the direction of the absorption pad for use.
The half strip was prepared by the following procedure.
(1) Separate the sample pad and conjugate pad attachment part of the membrane card (2) Attach the absorption pad to the membrane card (3) Cut to a width of 5 mm (4) Immobilize trastuzumab on the membrane (see 1-3 above)
(5) Immobilization of anti-mouse IgG antibody on the membrane (see 1-3 above)

1-7 Preparation of Lateral Flow Strip The lateral flow strip was prepared by the following procedure.
(1) Attach the absorption pad to the membrane card (2) Attach the conjugate pad holding the gold colloid-labeled primary antibody to the membrane card (3) Attach the sample pad holding the BSA to the conjugate pad (4) ) Cut to 5 mm width (5) Immobilize trastuzumab on the membrane (see 1-3 above)
(6) Immobilization of anti-mouse IgG antibody on the membrane (see 1-3 above)
In the following examples, the region on which the second antibody is immobilized is referred to as a "test line", and the region on which the control antibody is immobilized is referred to as a "control line".

[Example 2: Detection of HER2 protein by half strip]
1. 1. Experimental materials and methods (1) Sample The protein extract prepared in Reference Example 1 was used. The cells used were MKN1, MKN7, MKN45, MKN74, AGS, KATOIII, HGC27, MCF7 (negative control).
(2) Strip The half strip prepared in Example 1 was used. The amount of antibody (trastuzumab) on the test line was 5 μg.
(3) Detection method A 96-well plate was used. The amount of protein from the protein extract is 60 μg / well, the BSA concentration is 3% / well, the solution amount of the gold colloid-labeled first antibody prepared in 1-4-1 of Example 1 is 1 μL / well, and the liquid per well. Samples were prepared to a volume of 100 μL. One end of the half-strip membrane was immersed in the sample solution and developed, and coloration of the test line and control line was observed. Those whose coloration on the test line could be visually confirmed were judged to be positive.

2. Results The results are shown in FIG. The numerical values in the upper part of FIG. 3 indicate the HER2 protein concentration in the protein extract of each cell line quantified by densitometry in Reference Example 1 above. As is clear from FIG. 3, a clear signal was detected in MKN7 with a high HER2 expression level. Weak signals were also detected in MKN74, KATOIII and HGC27. From this result, it was shown that the lateral flow assay can detect HER2-positive gastric cancer.

[Example 3: Examination of test line antibody amount]
1. 1. Experimental materials and methods (1) Sample The protein extract of MKN7 prepared in Reference Example 1 was used.
(2) Strip The half strip prepared in Example 1 was used. Five types were used in which the amount of antibody (trastuzumab) on the test line was 1, 3, 5, 10 or 20 μg.
(3) Detection method The same method as in Example 2 was used.

2. Results The results are shown in FIG. As is clear from FIG. 4, the signal intensity was enhanced depending on the amount of antibody in the test line. This result indicates that the detection sensitivity can be adjusted by increasing or decreasing the amount of antibody on the test line.

[Example 4: Detection of HER2 protein by lateral flow strip]
1. 1. Experimental Materials and Methods (1) Samples The protein extracts of MKN7 and MCF7 (control) prepared in Reference Example 1 were used.
(2) Strip The lateral flow strip prepared in Example 1 was used. The amount of antibody (trastuzumab) on the test line was 5 μg.
(3) Detection Method A sample was prepared so that the amount of protein from the protein extract was 200 μg and the amount of the liquid was 100 μL, and the entire amount of the sample was added to the sample pad portion and developed. The coloration of the control line and test line was visually observed.

2. Results The results are shown in FIG. As is clear from FIG. 5, control line and test line signals were observed in MKN7, but test line signals were not observed in MCF7.

[Reference Example 2: Examination of HER2 expression in gastric cancer cell lines by Western blotting]
1. 1. Experimental materials and methods (1) Cells used (see Reference Example 1)
As human gastric cancer cell lines, MKN1, MKN7, MKN45, MKN74, GLM1, GLM2, GLM4, AGS, and HGC27 were used. A breast cancer cell line MCF7 was used as a control.
(2) Preparation of protein extract A protein extract was prepared from each cell by the same method as in Reference Example 1.
(3) Western blotting Western blotting was performed by the same method as in Reference Example 1.
(4) Quantification by ELISA Using ErbB2 / Her2 ELISA Kit (Novus Biologicals, NBP1-84823), the HER2 protein concentration in the protein extract prepared from each cell was quantified. The quantitative value was expressed not by the concentration of HER2 protein in the solution but by the amount of HER2 protein per unit amount of protein in the solution.

2. Results The results are shown in FIG. The upper row shows the result of detecting HER2 protein by Western blotting, the middle row shows the result of detecting β-actin by Western blotting, and the lower row shows the result of quantifying HER2 protein by ELISA. From these results, it was clarified that the HER2 expression level of GLM1 and GLM4 was very high, followed by the HER2 expression level of MKN7 and GLM2.

[Example 5: Preparation of test strip (2)]
5-1 Strip material A nitrocellulose membrane (Merck Millipore, HF13504) was used as the membrane. A glass fiber pad (Merck Millipore, GFDX203000) was used as the sample pad and the conjugate pad. A cellulose pad (Merck Millipore, CFSP223000) was used as the absorption pad.

5-2 Antibody used A test strip was prepared by reversing the first antibody and the second antibody of the test strip prepared in Example 1. That is, trastuzumab (Herceptin® injection 60, manufactured by Chugai Pharmaceutical Co., Ltd.) was used as the first antibody. As the second antibody, an anti-HER2 mouse monoclonal antibody (HER-2 / ErbB2 Monoclonal Antibody (N24), Thermo Scientific, MA1-12691) was used. As a control antibody, an anti-mouse IgG antibody (Goat anti-Mouse IgG's, Nippon Flour Mills) was used. The same antibody diluent as in Example 1 was used for diluting the antibody.

5-3 Immobilization of the second antibody Anti-HER2 mouse monoclonal antibody and anti-mouse IgG are each diluted with an antibody diluent to prepare a 0.1 μg / μL antibody solution, and 1 μL of the antibody solution per spot is micropipedted. Was attached to the membrane using (antibody 0.1 μg / spot). The anti-HER2 mouse monoclonal antibody was spotted at one site and the anti-mouse IgG was spotted at two sites. The membrane was dried at 50 ° C. for 30 minutes. Subsequently, the membrane was immersed in blocking buffer (0.5% casein, 50 mM boric acid, pH 8.5) and allowed to stand at room temperature for 30 minutes. The membrane was transferred to a washing / stabilizing buffer (0.5% sucrose, 0.05% sodium cholic acid, 50 mM Tris-HCl, pH 7.5) and immersed, and allowed to stand at room temperature for 30 minutes or more. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature. The membrane thus obtained can be stored in an aluminum pouch together with a desiccant.

5-4 Preparation of Conjugate Pad 5-4-1 Gold Colloid Labeling of First Antibody Gold colloid labeling of trastuzumab was carried out by the following procedure.
(1) Herceptin® 60 for injection was dissolved according to the package insert and diluted with distilled water to prepare a 70 μg / mL trastuzumab solution.
(2) 18 mL of gold colloid solution (Gold Colloid 40 nm, BBI solution) was added to 2 mL of 50 mM KH ₂ PO _{4 (pH 8.0), and 2 mL of the trastuzumab solution was added with stirring.}
(3) Further, 1.1 mL of 1% PEG20000 was added and stirred.
(4) Centrifugation was carried out at 8,000 × g for 15 minutes, and excess supernatant was removed so that about 1 mL of the supernatant remained.
(5) Using an ultrasonic cleaner, the precipitate was dispersed in the remaining supernatant.
(6) 20 mL of gold colloid storage buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG 20,000, 150 mM NaCl, 1% BSA, 0.1% NaN ₃ ) was added and stirred.
(7) (4) and (5) were repeated.
(8) 50 μL of the dispersion obtained in the second (5) was separated, 950 μL of a gold colloid storage buffer was added, and OD520 was measured.
(9) The gold colloid storage buffer was prepared so that OD520 = 6.0, and a solution of the gold colloid-labeled first antibody was obtained.

5-4-2 Preparation of Conjugate Pad In 500 μL of the above-prepared solution of gold colloid-labeled primary antibody, 500 μL of distilled water and gold colloid coating buffer (20 mM Tris-HCl (pH 8.0), 0.05% PEG 20,000, 5%) (Colloid) 1 mL was mixed and lightly stirred. 1.6 mL of the above mixed solution was evenly applied to each glass fiber pad (300 mm). The pad was placed in a desiccator and dried under reduced pressure at room temperature for at least one night. The dried conjugate pad can be stored in a light-shielded plastic bag containing silica gel.

5-5 Preparation of Lateral Flow Strip A backing sheet for immunochromatography having a length of 60 mm and a width of 300 mm was attached so as to be 15 mm from the upper end of the membrane so that the upper end of the membrane having a length of 25 mm and a width of 300 mm came. The lower edge of the membrane is 20 mm from the lower edge of the backing sheet. Next, the upper end of the absorption pad having a length of 20 mm and a width of 300 mm was aligned with the upper end of the backing sheet and attached. At this time, the absorption pad overlaps the membrane by about 5 mm. Next, a conjugate pad having a length of 8 mm and a width of 300 mm was attached so that the lower end thereof was at a position 14 mm from the lower end of the back sheet. At this time, about 2 mm on the upper end side of the conjugate pad overlaps the membrane sheet. Further, the lower end of the sample pad having a length of 18 mm and a width of 300 mm was aligned with the lower end of the back sheet and attached. At this time, 4 mm on the upper end side of the sample pad overlaps the conjugate pad. The bonded sheets were cut to a width of 5 mm to prepare a lateral flow strip. The prepared lateral flow strip can be placed in an aluminum pouch together with silica gel and stored at room temperature.

[Example 6: Detection of HER2 protein in gastric cancer cell line-derived protein extract]
1. 1. Experimental Materials and Methods (1) Samples The protein extracts derived from 9 types of human gastric cancer cell lines prepared in Reference Example 2 were used. As a control, a protein extract derived from the breast cancer cell line MCF7 prepared in Reference Example 2 was used.
(2) Strip The lateral flow strip prepared in Example 5 was used.
(3) Detection method Prepare a sample so that the amount of protein from the protein extract is 2 μg and the amount of liquid is 100 μL (protein concentration 0.02 mg / mL), and add the entire amount of the sample to the sample pad portion to develop it. It was. The coloration of the control line and test line was visually observed.

2. Results The results are shown in FIG. The numerical value in the upper part of FIG. 7 indicates the amount of HER2 protein per unit protein amount quantified by ELISA in Reference Example 2, and the numerical value in the middle part of FIG. 7 is the amount of HER2 protein per strip calculated based on the numerical value in the upper part. Is shown. It was considered from FIG. 7 that if the amount of HER2 protein per strip exceeds 10 ng, it can be detected by the lateral flow strip prepared in Example 5.

[Example 7: Examination of HER2 protein concentration in sample]
1. 1. Experimental Materials and Methods (1) Sample The GLM1 protein extract prepared in Reference Example 1 was used. Five types of samples having a total protein concentration (μg / mL) of 0.2, 0.02, 0.002, 0.0002 and 0.00002 were prepared.
(2) Strip The lateral flow strip prepared in Example 5 was used.
(3) Detection method 100 μL of each sample was added to the sample pad portion and developed, and the coloring of the control line and the test line was visually observed.

2. Results The results are shown in FIG. The numerical value in the upper part of FIG. 8 indicates the total protein concentration in the sample, and the numerical value in the middle part of FIG. 8 indicates the amount of HER2 protein per strip. It was also considered from FIG. 8 that if the amount of HER2 protein per strip exceeds 10 ng, it may be detected by the lateral flow strip prepared in Example 5.

[Example 8: Examination of HER2 protein concentration in xenograft-derived sample and human biopsy sample]
1. 1. Experimental Materials and Methods (1) Preparation of Samples Derived from Xenograft GLM1 and MKN45 were inoculated subcutaneously into nude mice, respectively, to form tumors. Samples were taken from the tumor tissue using biopsy forceps used during upper gastrointestinal endoscopy, weighed, and then stored in liquid nitrogen. T-PER Tissue Protein Extraction Reagent (Thermo Scientific) was added to the collected tissue, and homogenized with a homogenizer (Nippi, BioMasher II). The obtained tissue lysate was centrifuged (10,000 × g, 4 ° C., 5 minutes), and the supernatant was stored as a protein extract at −80 ° C. It was thawed at the time of use and subjected to the test.
(2) Preparation of human sample-derived sample A biopsy sample collected from a gastric cancer patient was used. Specimens of cases confirmed to be HER2 positive by immunostaining for HER2 were used. As a control, a healthy gastric biopsy sample of the same patient was used. In the same manner as in (1) above, T-PER Tissue Protein Extension Reagent was added, homogenized, and centrifuged to prepare a protein extract, which was stored at -80 ° C. It was thawed at the time of use and subjected to the test.
(3) Strip The lateral flow strip prepared in Example 5 was used.
(4) Detection method The HER2 concentration of each protein extract prepared in (1) and (2) above was calculated using ErbB2 / Her2 ELISA Kit (Novus Biologicals, NBP1-84823). All of the above 4 samples were diluted 200-fold with ultrapure water, and 100 μL thereof was added to the sample pad portion for development. The coloration of the control line and test line was visually observed.

The results are shown in FIG. The numbers in the upper part of FIG. 9 indicate the amount of HER2 protein per strip. As is clear from FIG. 9, HER2 could be detected in GLM1-derived xenograft and biopsy specimens of human tumors. From this result, it was shown that the HER2 protein can be detected even in the protein extract from the tumor tissue using the prepared strip.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all the academic documents and patent documents described in the present specification are incorporated herein by reference.

1 Membrane 2 Absorption pad 3 Conjugate pad 4 Sample pad 5 Backsheet 6 Anti-HER2 protein antibody solid phase region 7 Control antibody solid phase region

Claims (11)

A kit for selecting cancer patients for whom administration of an antibody drug targeting HER2 protein as a therapeutic target molecule is effective, which comprises a test strip for lateral flow immunoassay for detecting HER2 protein in cancer tissue. The test strips consist of a sample pad to which the sample is added, a conjugate pad holding the first anti-HER2 protein antibody, a region where the second anti-HER2 protein antibody is immobilized, and a region in which the sample is immobilized, in order from the upstream in the direction in which the sample flows. The first anti-HER2 protein antibody and the second anti-HER2 protein antibody and the second have a structure in which a membrane having regions on which the antibody binding to the first anti-HER2 protein antibody is immobilized is located in different regions and an absorption pad are linked. In any one of the anti-HER2 protein antibodies, the antibody drug is used, and the membrane is immobilized on the region where the third anti-HER2 protein antibody is immobilized, or the third anti-HER2 protein antibody is immobilized. The region where the second anti-HER2 protein antibody was immobilized and the region where the fourth anti-HER2 protein antibody was immobilized were divided into different regions and the region where the second anti-HER2 protein antibody was immobilized and the first anti-HER2 protein antibody. The binding antibody is further contained in a region different from any of the immobilized regions, and the second anti-HER2 protein antibody, the third anti-HER2 protein antibody and the fourth anti-HER2 protein antibody are different from each other. A kit characterized by being an antibody drug. The kit according to claim 1, wherein the antibody drug using the HER2 protein as a therapeutic target molecule is selected from the group consisting of trastuzumab, pertuzumab and trastuzumab-emtancin. The kit according to claim 1 or 2, wherein the first anti-HER2 protein antibody is labeled with colloidal microparticles. The kit according to any one of claims 1 to 3 , wherein a protein extract prepared from a cancer tissue collected from a subject is used as a sample. The kit according to claim 4 , wherein the cancer tissue is a biopsy tissue. The kit according to claim 4 or 5 , wherein the cancer tissue is a cancer tissue of the upper gastrointestinal tract. A method for selecting an effective cancer patient for administration of an antibody drug using the HER2 protein as a therapeutic target molecule, which comprises the following steps (1) to (3), and in the step (2), a sample. In order from the upstream in the direction of flow, the sample pad to which the sample is added, the conjugate pad holding the first anti-HER2 protein antibody, the region where the second anti-HER2 protein antibody is immobilized, and the first anti-HER2 Using a membrane having regions in which the antibody that binds to the protein antibody is immobilized in different regions, and a test strip for lateral flow immunoassay having a structure in which an absorption pad is linked, the first anti-HER2 protein antibody and the above-mentioned first anti-HER2 protein antibody and The antibody drug is used for either one of the second anti-HER2 protein antibodies, and the membrane is the region where the third anti-HER2 protein antibody is immobilized, or the third anti-HER2 protein antibody is immobilized. The region where the antibody was immobilized and the region where the fourth anti-HER2 protein antibody was immobilized are different regions, and the region where the second anti-HER2 protein antibody was immobilized and the region where the first anti-HER2 protein antibody was immobilized and the region where the first anti-HER2 protein was immobilized. The antibody that binds to the antibody is further contained in a region different from any of the immobilized regions, and the second anti-HER2 protein antibody, the third anti-HER2 protein antibody, and the fourth anti-HER2 protein antibody are each contained. A method characterized by being a different antibody drug:
(1) Step of preparing a sample from cancer tissue collected from a subject,
(2) A step of detecting the HER2 protein in the sample by a lateral flow immunoassay using an antibody drug using the HER2 protein as a therapeutic target molecule, and (3) selecting a subject in which the HER2 protein was detected by the lateral flow immunoassay. Process to do. The method according to claim 7 , wherein the antibody drug using the HER2 protein as a therapeutic target molecule is selected from the group consisting of trastuzumab, pertuzumab and trastuzumab-emtancin. The method according to claim 7 or 8 , wherein the first anti-HER2 protein antibody is labeled with colloidal microparticles. The method according to any one of claims 7 to 9 , wherein the cancer tissue is a biopsy tissue. The method according to any one of claims 7 to 10 , wherein the cancer tissue is a cancer tissue of the upper gastrointestinal tract.

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