JPWO2008041594A1 - Antibody drug sensitivity test method - Google Patents

Abstract

The present invention relates to a cell surface by reacting a cell with a label added to an antibody drug to be administered to a test subject after determining whether the cell derived from the test subject and a cell serving as a positive control as appropriate or not. Regarding the provision of a simple test method for antibody drug sensitivity (presence / absence of resistance) including a step of detecting a label bound to the antibody, complement can be added to the cells after detection of the label.

Description

The present invention relates to an antibody drug sensitivity test method.
This application claims priority based on Japanese Patent Application No. 2006-261207 filed in Japan on September 26, 2006 and Japanese Patent Application No. 2006-333331 filed in Japan on December 11, 2006, The contents are incorporated here.

  Antibodies have high binding activity, binding specificity, and stability in blood. Therefore, in recent years, attempts have been made to apply these characteristics to diagnosis, prevention, and treatment of various human diseases. Among them, some chimeric antibodies and humanized antibodies using gene recombination techniques have a remarkable therapeutic effect. In recent years, research and development of antibody drugs using these antibodies have attracted attention.

  Currently, clinically used antibody drugs include trastuzumab (generic name) (Herceptin (trade name: Roche); Her2 humanized monoclonal antibody) for breast cancer, rituximab (rituximab; CD20 chimeric monoclonal antibody) for B lymphoma, etc. It has been known.

Examples of the action mechanism of the antibody drug include growth inhibition by signaling, apoptosis induction, complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC) and the like. Among them, CDC is considered to exert an effect in a relatively short time after administration of a drug because complements abundantly present in blood directly recognize antibodies and attack target cells (Non-patent Document 1). . However, for example, in some patients with B lymphoma, it is known that some people are resistant to rituximab (Non-patent Document 2).
Oliver Manches et al., Blood, Vol. 101, No. 3, pp.949-954, 2003 James M. Foran et al., British J. Hematology, 114, 881, 2001 Miltenyi Biotech Co., Ltd., “Isolation of CD19 + cells by auto MACS”, [online], [searched on September 21, 2006], Internet <URL: http://www.miltenyibiotec.co.jp/tech_info /prot/pdf_2_human/130-050-301prot-a.pdf>

As seen in the example of rituximab, antibody drugs have a potential problem of resistance (sensitivity). In order to cope with this problem, in a conventional method, a pathological specimen is first prepared using tissues and cells collected from a patient, and antibody staining is performed on the pathological specimen.
In this way, by performing antibody staining on the pathological specimen, it is possible to identify whether the antigen recognized by the antibody drug used is expressed on the target, and how to treat it according to the test result. The doctor needs to decide.

  However, since it is necessary to fix tissues and cells in the preparation of pathological specimens, there is a problem that it is not possible to clearly distinguish between antigens expressed on the surface of target cells and antigens present inside the cells. is there. In addition, when there is a possibility that the antigen recognition site of the antibody drug has a mutation, it is difficult to predict whether the antibody drug will exert an action in actual treatment. Furthermore, when CD46, CD55, CD59, etc. are expressed, there is also a problem of acting as a complement defense system. Therefore, even if it is found by using a pathological specimen that the antigen corresponding to the antibody drug to be used is present in the target, whether CDC is effective in patients who actually administer the antibody drug. There is a problem that it is unknown.

In addition to the method using a pathological specimen, there is a method for detecting an antigen on the cell surface by a flow cytometer. However, since this method requires a large amount of target cells of about 1 × 10 ⁶ , this method cannot be adopted in many cases. For example, in the case of an early cancer patient, there may be cases where a sufficient amount of tumor cells necessary for analysis cannot be obtained because the tumor is small. Even when a relatively large tissue can be collected, the ratio of tumor cells in the tissue may be low, and analysis may not be possible.

  Another problem is that antibody drugs such as rituximab are generally expensive. If it is known in advance that the therapeutic effect cannot be expected due to the resistance, it is possible to avoid the use of the antibody drug and switch to another treatment method. Therefore, a request for a simple test method for antibody drug sensitivity (presence or absence of resistance) exists in both patients and medical workers.

In order to solve the above problems, the present invention provides the following: ~ 13. The structure is taken.
1. The following steps:
(1) A step of culturing a cell derived from a test subject and a cell as a positive control as appropriate in a cell culture container;
(2) A step of determining whether the cells cultured in the cell container are alive or dead;
(3) In the step (2), when it is determined that there are a sufficient number of viable cells for testing with respect to the cells derived from the test subject and, if appropriate, the cells serving as a positive control, scheduled to be administered to the test subject And (4) a step of detecting the label bound to the cell surface; and a method of detecting the sensitivity to the antibody drug.

2. After the step (4),
(5) adding complement to the cell culture vessel; and (6) detecting the label bound to the cell surface, and further determining whether the cell is alive or not after addition of complement. Above 1. A method for testing antibody drug sensitivity in the method of the invention.

3. After the step (6),
(7) The step of calculating | requiring the ratio of the life and death of a cell; A method for testing antibody drug sensitivity in the method of the invention.

4). The following steps:
(A) a step of culturing a cell derived from a test subject and a cell as a positive control as appropriate in a cell culture container;
(B) a step of adding and culturing a reagent for determining the viability of a cell and a label added to an antibody drug to be administered to the subject to be examined in the cell culture vessel; and (c) Determining the viability of the cells in the cell culture container, and further detecting the label bound to the cell surface.

5). After the step (c)
(D) adding a complement to the cell culture vessel; and (e) detecting the label bound to the cell surface, and further determining whether the cell is alive or not after addition of the complement. 4. above. A method for testing antibody drug sensitivity in the method of the invention.

6). After step (e)
(F) The step of calculating | requiring the ratio of the life and death of a cell; A method for testing antibody drug sensitivity in the method of the invention.

7). After the step (7) or (f),
(X) the step of comparing the survival rate of cells derived from the subject to be tested and the survival rate of cells serving as a positive control; Or 6. A method for testing antibody drug sensitivity in the method of the invention.

8). The step of determining the viability of the cells in the steps (2), (6), (c), and (e) includes an operation of adding a reagent for staining cell nuclei to each compartment in the cell culture container. 1. 2. 4. Or 5. A method for testing antibody drug sensitivity in the method of the invention.

9. After step (1), before or after step (5), after step (a), before or after step (d), at least one of:
(Y) adding oil to the respective compartments in the cell culture vessel; Or 5. A method for testing antibody drug sensitivity in the method of the invention.

10. Each of the cell culture vessel compartments has a diameter of 1 to 4 mm, a depth of 2 to 5 mm, and a volume of 1 to 15 μl. Or 4. A method for testing antibody drug sensitivity in the method of the invention.

11. The label is optically detectable, and the label is detected with a microscope. Or 4. A method for testing antibody drug sensitivity in the method of the invention.

12 1. The test subject is a patient with B-cell lymphoma, and the cells derived from the test subject are cells derived from a lymphoma tissue collected from the patient. Or 4. A method for testing antibody drug sensitivity in the method of the invention.

13. Before the step (1) or before the step (a), the method includes a step of subjecting cells derived from the test subject to screening for CD19, and only positive cells are included in the step (1) or the step (a). Above, 12. A method for testing antibody drug sensitivity in the method of the invention.

  By using the antibody drug sensitivity test method of the present invention, it is possible to easily evaluate the sensitivity of treatment with an antibody drug with a small amount of sample.

This figure is an evaluation of CDC sensitivity for a plurality of B lymphomas by the test method of the present invention. In the figure, DLBCL is diffuse large B-cell lymphoma, FL is follicular lymphoma, MCL is mantle cell lymphoma, MALT is maltolymphoma, lymphoplasmacytic lymphoma is lymphoplasmacytoma, small lymphocytic lymphoma is Small lymphocytic lymphoma, Burkit represents Burkitt lymphoma, and Hodgkin represents Hodgkin lymphoma.

In the first embodiment of the present invention, the antibody drug sensitivity test method comprises:
(1) A step of culturing a cell derived from a test subject and a cell as a positive control as appropriate in a cell culture container;
(2) A step of determining whether the cells cultured in the cell container are alive or dead;
(3) In the step (2), when it is determined that there are a sufficient number of viable cells for testing with respect to the cells derived from the test subject and, if appropriate, the cells serving as a positive control, scheduled to be administered to the test subject And (4) a step of detecting the label bound to the cell surface; and a step of adding the antibody drug added with the label to the cell culture vessel and culturing.

  Before performing the above step (1), first, cells derived from the subject to be examined and cells that are positive targets are obtained as appropriate. Obtaining cells derived from the test subject can be obtained by collecting tissue and cells from the affected area of the test subject by a surgical method or by collecting peripheral blood if leukemia has occurred. On the other hand, the positive control cell is a cell expressing an antigen recognized by the antibody drug whose sensitivity is evaluated by the method of the present invention on the cell surface, and has been established as a cell line Is obtained as appropriate. The cells to be positive are specifically CD20 positive cells such as Daudi cells and Raji cells when the antibody drug is rituximab, and Her2 positive cells such as MCF7 when trastuzumab is used.

  If the tissue obtained from the test subject is a tissue, cut the tissue finely using a scalpel or scissors, suspend it in a buffer solution (eg Hanks-Buffer), and pass the suspension through a strainer. Collect only floating cells. Alternatively, the tissue collected from the person to be examined is rubbed against frosted glass, and the released cells are collected with a buffer solution, and only those passed through a strainer are collected. Through such operations, cells in a state suitable for examination are obtained. Next, the following steps (1) to (4) are sequentially performed.

Step (1):
The cells are added to a cell culture vessel together with a culture solution suitable for examination (uncolored RPMI1640 (Roswell Park Memorial Institute) medium, PBS (phosphate buffered saline), etc.), and cultured. Examples of the cell culture container to be used include normal cell culture containers, but those having dimensions and capacity suitable for culturing a small amount of cells of about 1 × 10 ² to 1 × 10 ⁴ are preferable. Specifically, it is possible to use a 384-well plate, a container in which a silicon resin having a thickness of 4 mm with a hole of 2 mm (diameter) is pressed on a glass bottom container, and the like.

  At this stage, it is possible to shift to the evaluation of antibody drug sensitivity, but when the cells derived from the test subject are not uniform, that is, when many types of cells are mixed, a specific cell type It is also possible to carry out the step of concentrating the liquid as an option. For example, when rituximab (anti-CD20 antibody) is used as an antibody drug, cells expressing CD19 on the cell surface may be concentrated in advance using an anti-CD19 antibody. This is because the CD19 antigen, which is a surface antigen of B cells, has almost the same expression time as the CD20 antigen, and B cells expressing CD19 are considered to express CD20. The antigen used in this concentration step is particularly effective when there is clear information about the expression time of the antigen on the cell surface derived from the test subject, and there are antibodies that can be obtained at a lower price than antibody drugs. It is.

Step (2):
Next, the viability of each cell cultured in the cell incubator is determined. For this determination, it is possible to use various known cell viability test reagents. For example, when PI (Propidium Iodide) dye, which is a nuclear staining reagent, is added to a cell culture compartment and the cells are observed with a microscope after culturing for a certain period of time, living cells do not stain nuclei, whereas dead cells stain nuclei. Therefore, the viability of cells can be determined by the presence or absence of staining.

  More specifically, the step of determining whether or not the patient is alive or dead is a specification in which a plurality of cells are included in a single visual field of a microscope and the viability is at least 50% or more, more preferably 80% or more. It corresponds to searching for the field of view. More preferably, after determining a visual field suitable for evaluation, photographing of the visual field is performed. By giving an address to each cell in the field of view after photographing, it becomes easier to collate with the observation / measurement results of the subsequent process.

Step (3):
Next, in the life and death determination step of the above step (2), when a visual field in which a sufficient number of living cells are present for evaluation is obtained, a label added to the antibody drug to be administered to the test subject, Add to the culture vessel and continue culturing. When an antigen recognized by an antibody drug is expressed on the surface of a living cell derived from a test subject, the antibody to which the label is added binds to the antigen. Since some resistant patients may not express the CD20 antigen on the B cell surface, the antibody to which the label is added does not bind to the cell surface. Therefore, it is possible to determine the presence or absence of antigen expression on the surface of living cells by photographing the visual field containing living cells in a state suitable for detection of the label when a certain time has elapsed after the addition of the antibody. It is.
The greater the number of cells to be analyzed, the more reliable the analysis results. However, it takes time to acquire and analyze data. Therefore, considering the reliability of the analysis results and the analysis time, the number of cells to be analyzed is preferably 20 to 20000, more preferably 100 to 10,000, more preferably 200 to 3000, and most preferably Is 500 to 2000.

  As the antibody drug, a chimeric antibody, a humanized antibody, a human antibody, a mouse antibody, or the like can be used. From the viewpoint of action, a chimeric antibody, a humanized antibody, and a human antibody are particularly preferable.

Step (4):
Next, the presence or absence of antigen expression is determined in a state suitable for detection of a label added to the antibody drug. For example, when the label is a fluorescent dye, it is performed by irradiating light suitable for detection of the dye. An optical apparatus used for cell observation, label detection, etc. in these steps can be performed by, for example, Olympus laser scanning confocal microscope FV-1000.

  By passing through the above steps, whether the antigen recognized by the antibody drug to be administered is expressed on the cell surface derived from the test subject, that is, when the antibody drug is clinically administered to a patient, it is effective. It is possible to evaluate whether the patient is promising.

In the inspection method of the present invention, after performing the steps (1) to (4),
(5) adding a complement to the cell culture vessel; and (6) detecting the label bound to the cell surface, and further determining whether the cell is alive or not after adding the complement. Is possible.

Step (5):
In step (5), complement is added. As a complement, the same type as the antibody drug used, that is, when the antibody drug is a humanized antibody, it is preferable to use a human-derived complement, particularly preferably the test subject's own. . More specifically, serum derived from a healthy person or a test subject is added to the incubator so that, for example, the proportion of the whole culture solution after addition is about 10% (v / v), and cultured for a certain period of time. . As a complement source, serum, plasma, whole blood, purified complement fluid and the like can be used. Complements are fragile and must be handled while fresh. Therefore, serum or plasma is preferable from the viewpoint of easy availability and handling.

Step (6):
Next, the step (6) of detecting the presence of the antigen on the cell surface is performed in the same manner as in the above step (4). When the antibody drug is bound to the cell surface after the above step (3), CDC is generated by addition of complement in step (5), and the cell membrane is destroyed. Therefore, the cells labeled in the above step (4) are detected by staining the nucleus, for example, with the staining reagent added in the above step (2). On the other hand, the cells determined as dead cells in step (2) are also determined as dead cells in this step, and are determined as live cells when the cells derived from the test subject are antibody drug resistant.

Step (7):
In the inspection method of the present invention, after the step (6),
(7) The step of obtaining the ratio of cell survival can be further included. In this step, it is possible to determine whether or not complement has acted effectively by determining the ratio of life and death of cells that are positive targets. That is, when the amount of complement added was small, when complement activity was low, when stirring was insufficient, when the temperature was not appropriate, or when the incubation time after complement addition was too short The CDC is not sufficiently generated in the positive target cells, and the ratio of living cells is increased. On the other hand, when the positive target cells die at a predetermined rate (for example, 80% or more), it is defined that CDC has sufficiently acted. Therefore, when CDC by complement is fully functioning, the positive target cells become dead cells at a considerable rate. Moreover, the effectiveness of an antibody drug is quantitatively shown by calculating | requiring the survival rate of the cell derived from a test object. Therefore, by performing this step (7), it is possible to confirm whether the complement has sufficiently acted and the degree of effectiveness of the antibody drug in the treatment.

In the inspection method according to the second aspect of the present invention,
(A) a step of culturing a cell derived from a test subject and a cell as a positive control as appropriate in a cell culture container;
(B) a step of adding and culturing a reagent for determining the viability of a cell and a label added to an antibody drug to be administered to the subject to be examined in the cell culture vessel; and (c) Determining whether the cells in the cell culture container are viable or not, and detecting the label bound to the cell surface.

  In the test method of the second aspect of the present invention, a cell culture container is prepared by adding a reagent to the cell biocontainer in the step (b) and an antibody drug to be administered to the test subject. The same operation as the operation of the said aspect containing process (1)-(4) is performed except adding in the inside. This test method including the steps (a) to (c) can be applied particularly when the survival rate of cells derived from the test subject is high.

In the inspection method according to the second aspect of the present invention, after the step (c),
(D) adding a complement to the cell culture vessel; and (e) detecting the label bound to the cell surface, and further determining whether the cell is alive or not after adding the complement. Is possible.
As a complement source for the complement added in the step (d), serum, plasma, whole blood, purified complement fluid, and the like can be used. Complements are fragile and must be handled while fresh. Therefore, serum or plasma is preferable from the viewpoint of easy availability and handling.
In addition, after the step (e),
(F) determining the viability ratio of the cells.

  When complement is added in the step (d), CDC occurs when the antibody drug is bound to the antigen on the cell surface by the step (c). Then, detection of the label on the cell surface after addition of complement, determination of viability of the cell, and ratio of viability of the cell are obtained to determine whether the test subject has resistance to the antibody drug and whether or not It can be confirmed whether the body has worked effectively.

In the inspection method of the present invention, after step (7) of the first form or (f) of the second form,
(X) a step of comparing the survival rate of cells derived from the subject to be tested and the survival rate of cells serving as a positive control. By including this step, it is possible to estimate how effectively CDC acts when the antibody drug is actually administered to the subject. Furthermore, it can be determined whether or not the sensitivity evaluation has been performed correctly. In particular, when the test cells and positive control cells are processed in the same container, the comparison can be performed under exactly the same conditions, so whether the test was performed correctly (whether the labeled antibody drug is working). Such as whether complement activity was sufficient or whether the experimental conditions were appropriate).

The inspection method of the present invention may further include the step (Y) of adding oil to each compartment in the cell culture container. In the first embodiment, this addition time is after step (1) or before or after step (5), more preferably immediately after step (1) or immediately after step (5). . In the second mode, it is after step (a) or before or after step (d), more preferably immediately after step (a) or immediately after step (d).
The oil defined here is a liquid that covers cells to be examined, a cell culture solution, various drugs, and the like and has an action of preventing evaporation. Any liquid having a specific gravity smaller than water, immiscible with water, and low vapor pressure may be used. Particularly preferred are mineral oil and silicone oil.
The reason for including the oil addition step is that when the number of cells derived from the test subject is small in the test method of the present invention, the culture is performed in a culture compartment having a small volume (range of 1 μl to 1 ml). This is because medium temperature may cause a problem of evaporation of the medium. By adding oil in an amount sufficient to cover the surface of the medium in each compartment, it is possible to prevent the medium from evaporating. In addition, since the specific gravity of oil is low with respect to the medium and complement sources (serum, plasma, whole blood, purified complement fluid), the complement is not added even if the oil is added before the step (5). If dripped onto the oil, the complement automatically passes through the layer of oil and mixes with the underlying medium. Oil also does not affect optical observation. Thus, the oil can be added at any stage of the process of the present invention. However, from the viewpoint of preventing the evaporation of the medium, it is preferable to add it in an amount sufficient to cover the surface of the medium before adding the complement. Moreover, when adding oil after complement addition, it is desirable to carry out immediately after complement addition from the same viewpoint.

In the inspection method of the present invention, for example,
The cell culture vessel may have compartments, each compartment having a diameter of 1-4 mm, a depth of 2-5 mm, and a volume of 1-15 μl. By using a culture container having a compartment having a size in this range, the cells floating in the container do not cause convection and can be examined with a small number of cells. In addition, this volume is preferable because the antibody drug or complement to be added spreads within the compartment in a relatively short time after the addition.

  In the inspection method of the present invention, the label can be detected optically, and the label can be detected with a microscope. More specifically, the label is a fluorescent label such as Alexa488, Cy3, Cy5, TRITC (tetramethylrhodamine isothiocyanate), FITC (fluorescein isothiocyanate) or the like.

  The test method of the present invention can be carried out for patients with B cell lymphoma, for example. In this case, it is possible to use cells derived from lymphoma tissue collected from the patient as cells derived from the test subject, and anti-CD20 antibody (rituximab) as the antibody drug. B-cell lymphoma is a disease that accounts for about 70% of non-Hodgkin's lymphoma, and CD20 antigen is usually expressed in B cells. Therefore, it is possible to generate CDC by a combination of anti-CD20 antibody and complement, and it can be a test object of the test method of the present invention.

  Specific examples of B cell lymphoma to which the test method of the present invention can be applied include precursor B lymphoblastic leukemia / lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoid trait Cellular lymphoma, splenic marginal zone B cell lymphoma, hairy cell leukemia, plasma cell tumor, extranodal follicular marginal zone B cell lymphoma, nodal follicular marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse Large cell type B cell lymphoma, mediastinal large cell type B cell lymphoma, intravascular large cell type B cell lymphoma, primary effusion lymphoma, and the like. Examples of other antibody drugs include ibritumomab for B cell lymphoma, trastuzumab (anti-Her2 antibody drug) for breast cancer, and the like.

(experimental method)
Lymphoma tissue collected from various lymphoma patients was finely cut with scissors or a scalpel and suspended in hanks-buffer, and this suspension was further passed through a strainer to collect floating cells. Daudi cells were prepared as a positive control for cells derived from lymphoma tissue. Regarding lymphoma-derived cells, only CD19-positive cells were isolated using magnetic beads (Non-patent Document 3).

Rituximab was labeled with Alexa488 according to the method of Alexa488 Monoclonal antibody labeling kit (A20181) manufactured by Invitrogen. Labeled rituximab was used at a concentration of 10 ng / ml. As a nuclear staining dye, PI was used at a concentration of 5 μg / ml.
Labeled rituximab and PI were added to the medium, and after culturing for 5 minutes, the cells were transferred to a glass bottom container or 384 well plate for cell observation. Cells in the container were observed with an inverted microscope so that about 1000 to 10000 cells were contained per culture section. Thereafter, the container was placed on a microscope stage maintained at 37 ° C. Cells were observed from the bottom of the container, photographed, fluorescent labels were detected, and the presence or absence of nuclear staining was detected.
In this first observation, a fluorescent dye labeled with rituximab is detected to confirm the expression state of CD20 antigen and the state of nuclear staining. A living cell expressing the CD20 antigen does not stain the nucleus but only the cell membrane. In addition, dead cells are stained with the nuclear staining dye PI. Therefore, a portion where the cell density of living cells expressing CD20 antigen is appropriate is selected as the observation target region.
Next, human serum (complement source) was added at an arbitrary ratio of 1-20% of the entire culture compartment volume, and cultured at 37 ° C. for 5-30 minutes. Again, cells were observed from the bottom of the vessel, photographed, fluorescent labels were detected, and the presence or absence of nuclear staining was detected.
In this second observation, cells that live expressing CD20 antigen in the first observation are targeted, and by observing whether or not the nucleus is stained, The number and ratio of dead cells were measured. This judged CDC sensitivity.

(result)
The viability of the cells was compared from the expression state of the CD20 antigen before and after complement addition. From the number of living cells and the number of dead cells, the ratio of CDC reaction (CDC sensitivity) was determined. The result is shown in FIG. It is a figure which shows the ratio (CDC sensitivity) which the disease name and CDC reaction which were determined by pathological examination etc. produced. Thus, it was found that there is a big difference in the sensitivity of CDC even in the same clinical name. Therefore, it became clear that this invention is very effective for selection of an antibody pharmaceutical.

  The antibody drug susceptibility testing method of the present invention makes it possible to easily test the sensitivity of an antibody drug using a small amount of cells.

Claims (13)

(1) A step of culturing a cell derived from a test subject and a cell as a positive control as appropriate in a cell culture container;
(2) A step of determining whether the cells cultured in the cell container are alive or dead;
(3) In the step (2), when it is determined that there are a sufficient number of viable cells for testing with respect to the cells derived from the test subject and, if appropriate, the cells serving as a positive control, scheduled to be administered to the test subject And (4) a step of detecting the label bound to the cell surface; and a method of detecting the sensitivity to the antibody drug. (5) a step of adding complement to the cell culture vessel after the step (4); and (6) detecting the label bound to the cell surface, and further determining whether the cell is viable after the addition of complement. The antibody drug sensitivity test method according to claim 1, further comprising a step of determining.   (7) The antibody drug susceptibility testing method according to claim 2, further comprising a step of obtaining a cell death ratio after the step (6). (A) a step of culturing a cell derived from a test subject and a cell as a positive control as appropriate in a cell culture container;
(B) a step of adding and culturing a reagent for determining the viability of a cell and a label added to an antibody drug to be administered to the subject to be examined in the cell culture vessel; and (c) Determining the viability of the cells in the cell culture container, and further detecting the label bound to the cell surface. (D) After the step (c), adding complement to the cell culture vessel; and (e) detecting the label bound to the cell surface, The antibody drug sensitivity test method according to claim 4, further comprising a step of determining.   (F) After the said process (e), the process of calculating | requiring the ratio of the life and death of a cell; After the step (7) or (f),
The method for testing antibody drug sensitivity according to claim 3 or 6, further comprising: (X) comparing the survival rate of cells derived from the test subject and the survival rate of cells serving as a positive control.   The step of determining the viability of cells in the steps (2), (6), (c), and (e) includes an operation of adding a reagent for staining cell nuclei into a cell culture container. 4. The antibody drug sensitivity test method according to 4 or 5. After the step (1), before or after the step (5), after the step (a), before or after the step (d),
The method for testing antibody drug sensitivity according to claim 2 or 5, further comprising (Y) adding oil to the cell culture container.   5. The cell culture vessel has compartments, each compartment having a diameter of 1 to 4 mm, a depth of 2 to 5 mm, and a volume of 1 to 15 [mu] l. Antibody drug sensitivity test method.   The antibody drug sensitivity test method according to claim 1 or 4, wherein the label is optically detectable, and the label is detected by an optical device.   The antibody drug susceptibility testing method according to claim 1 or 4, wherein the test subject is a patient with B-cell lymphoma, and the cell derived from the test subject is a cell derived from a lymphoma tissue collected from the patient.   Before the step (1) or before the step (a), the method includes a step of subjecting cells derived from the test subject to screening for CD19, and only positive cells are included in the step (1) or the step (a). The antibody drug sensitivity test method according to claim 12, which is applied.

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