WO2008041594A1

WO2008041594A1 - Method for testing on sensitivity to antibody drug

Info

Publication number: WO2008041594A1
Application number: PCT/JP2007/068723
Authority: WO
Inventors: Natsuhiko Sugimura; Yuji Mishima; Yasuhito Terui; Kiyohiko Hatake
Original assignee: Olympus Corporation; Japanese Foundation For Cancer Research
Priority date: 2006-09-26
Filing date: 2007-09-26
Publication date: 2008-04-10
Also published as: JPWO2008041594A1

Abstract

Disclosed is a simple method for testing on the sensitivity to (the presence or absence of resistance against) an antibody drug, which comprises the steps of: determining the life or death of a cell derived from a subject to be tested and optionally a positive control cell, reacting the cell with an antibody drug which is intended to be administered to the subject and has a label, and detecting the label bound to the surface of the cell. In the method, after the label is detected, a complement may be added to the cell.

Description

Specification

Antibody drug sensitivity test method

Technical field

[0001] The present invention relates to an antibody drug sensitivity test method.

This application (September 26, 2006, Japanese Patent Application No. 2006-261207, filed with Takimoto Ryoko, and December 2006, November 11, 2006, Japanese Patent Application No. 2006-333331, filed with Tachimoto ί. This claim is based on the priority and the contents are incorporated herein.

Background art

[0002] 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 technology have a remarkable therapeutic effect. In recent years, research and development of antibody medicines using these antibodies have attracted attention.

[0003] Currently, clinically used antibody drugs include trastuzumab (generic name) for breast cancer (Herceptin (trade name: Roche); Her2 humanized monoclonal antibody) and rituximab (CD20 chimera) for B lymphoma. Monoclonal antibodies) are known.

[0004] The action mechanism of an antibody drug includes growth inhibition by signaling, induction of apoptosis, complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and the like. Among them, CDC is considered to be effective in a relatively short time after drug administration because complements abundant in blood directly recognize antibodies and attack target cells (non-patent literature). 1). However, for example, in some patients with B lymphoma, it is known that some people are resistant to rituximab! /, (Non-patent document 2).

Non-Patent Document 1: Oliver Manches et al., Blood, Vol. 01, No. 3, pp.949-954, 2003

Non-Patent Document 2: James M. Foran et al., British J. Hematology, Vol. 114, p. 881, 2001 Year

Non-Patent Document 3: Miltue Biotech Co., Ltd., “Isolation of CD 19+ 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.pdr ^

Disclosure of the invention

Problems to be solved by the invention

[0005] As seen in the example of rituximab, antibody drugs have a potential problem of resistance (sensitivity). In order to cope with this problem, in the conventional method, a pathological specimen is first prepared using tissues and cells collected from patient force, and antibody staining is performed on the pathological specimen.

By conducting antibody staining on the pathological specimen in this way, it is possible to identify whether the antigen recognized by the antibody drug used is expressed on the target, and how to treat it based on the test results! / The doctor needs to decide.

[0006] In preparation of pathological specimens, it is necessary to fix tissues and cells, so the antigen expressed on the surface of the target cell and the antigen present inside the cell are clearly distinguished. There is a problem that it cannot be done. 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 effect in actual treatment. Furthermore, when CD46, CD55, CD59, etc. are expressed, there is also a problem of acting as a complement protection system. Therefore, even if it is found by using pathological specimens that the antigen corresponding to the antibody drug used is present in the target, whether CDC will work effectively in patients who actually receive the antibody drug. problems force ^s that it is unknown.

[0007] In addition to the method using a pathological specimen, there is a method of detecting an antigen on the cell surface using a flow cytometer. In the practice of the method and power, because a large amount of target cells of about IX 10 ⁶ is required, in many cases it is not possible to adopt this method. For example, in the case of an early stage cancer patient, the tumor is small, so it is impossible to obtain a sufficient amount of tumor cells for analysis! /. Even if a relatively large tissue can be collected, the ratio of tumor cells in the tissue may be low, and analysis may not be possible. [0008] 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 resistance, it is possible to avoid the use of antibody drugs and switch to other treatment methods. Therefore, there is a demand for a simple test method for antibody drug sensitivity (presence / absence of resistance) in both patients and healthcare professionals.

Means for solving the problem

In order to solve the above problems, the present invention has the following configurations 1. to 13.

1. The following steps:

(1) a step of culturing cells derived from a test subject and, if appropriate, cells serving as a positive control in a cell culture container;

(2) a step of determining the viability of the cells cultured in the cell container;

(3) When it is determined in the step (2) that there are sufficient number of living cells for the test regarding the cells derived from the test subject and, if appropriate, the cells serving as a positive control, the test subject is administered to the test subject. Adding a labeled antibody drug to the cell culture vessel and culturing; and

(4) A method for testing antibody drug sensitivity, comprising a step of detecting the label bound to the cell surface.

[0010] 2. After step (4) above,

(5) adding complement to the cell culture vessel; and

(6) A method for examining antibody drug sensitivity in the method of the invention of the above (1), further comprising the step of detecting the label bound to the cell surface and further determining the viability of the cell after addition of complement.

[0011] 3. After the step (6),

(7) The method for testing antibody drug susceptibility in the method of the invention of the above (2), further comprising the step of obtaining a cell viability ratio.

[0012] 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 a reagent for determining the viability of a cell and an antibody drug to be administered to the subject to be examined to which a label is added to the cell culture vessel and culturing; and

(c) determining the viability of the cells in the cell culture container, and further detecting the label bound to the cell surface;

[0013] 5. After step (c) above,

(d) adding complement to the cell culture vessel; and

(e) a method for testing antibody drug sensitivity in the method of the invention of (4), further comprising the step of detecting the label bound to the cell surface and further determining the viability of the cell after addition of complement.

[0014] 6. After the step (e),

(f) A method for testing antibody drug sensitivity in the method of the invention of the above (5), further comprising the step of obtaining a cell viability ratio.

[0015] 7. After the step (7) or (f),

(X) comparing the survival rate of cells derived from the subject to be tested and the survival rate of cells serving as a positive control; Method.

[0016] 8. Process power for determining cell viability in the above steps (2), (6), (c), and (e). A reagent that stains cell nuclei is added to each compartment in the cell culture vessel. A method for testing antibody drug susceptibility in the method of the invention of the above-mentioned 1., 2., 4., or 5.

[0017] 9. After at least one of step (1), before or after step (5), after step (a), before or after step (d),

(Y) The method for examining antibody drug sensitivity in the method of the invention of the above item 2 or 5, further comprising the step of adding oil to each compartment in the cell culture container.

[0018] 10. Each force of the compartment of the cell culture vessel; having a diameter of! ~ 4mm, having a depth of 2-5mm; and having a volume of! ~ 151; An antibody drug sensitivity test method according to the method of the invention.

[0019] 11. The antibody drug susceptibility testing method according to the method of the above invention 1 or 4, wherein the label is optically detectable, and the label is detected by a microscope. [0020] 12. In the method of the invention of the above 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. Antibody drug sensitivity test method.

[0021] 13. Before the step (1) or before the step (a), the method comprises screening cells derived from a test subject for CD19, wherein only positive cells are obtained in the step (1) or The method for testing antibody drug susceptibility in the method of the invention described in 12. above, which is applied to step (a).

The invention's effect

[0022] 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.

Brief Description of Drawings

[0023] [FIG. 1] This figure is an evaluation of CDC sensitivity of 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, and lymphopla smacytic lymphoma is lymphoid $ 10 cells. Small lymphocytic lymphoma represents small lymphocytic lymphoma, Burkit represents Burkitt lymphoma, and Hodgkin represents Hodgkin lymphoma.

BEST MODE FOR CARRYING OUT THE INVENTION

[0024] In the first embodiment of the present invention, an antibody drug sensitivity test method comprises:

(4) detecting the label that binds to the cell surface! /,

[0025] Before performing the above step (1), a cell derived from a test subject and, optionally, a positive target cell, Get first. Obtaining cells derived from the test subject can be performed by collecting tissue cells from the affected area of the test subject by a surgical method or by collecting peripheral blood in the case of leukemia. 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. Get things as appropriate. Specifically, the cells to be positive are 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.

[0026] If the tissue obtained from the subject is a tissue, cut the tissue finely using a scalpel or scissors as appropriate, suspend it in a buffer solution (for example, Hanks-Buffer), and then add the suspension to the strainer. Collect only floating cells that have passed through. Alternatively, the tissue collected from the test subject is rubbed against frosted glass, and the released cells are collected with a buffer solution, and only those that have passed through the strainer are collected. Through these operations, cells in a state suitable for testing are obtained. Next, the following steps (1) to (4) are sequentially performed.

[0027] Step (1):

Add the cells to a cell culture vessel together with a culture medium suitable for the test (such as unpigmented RPMI1640 (Roswell Park Memorial Institute) medium or PBS (phosphate buffered saline)). As a cell culture vessel to be used, a normal cell culture vessel can be mentioned. A cell culture vessel having a size and capacity suitable for culturing a small amount of cells of about 1 × 10 ² to 1 × 10 ⁴ is 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 having a diameter of 2 mm is bonded to a glass bottom container, and the like.

[0028] At this stage, it is possible to shift to the evaluation of antibody drug susceptibility, but if the cells derived from the test subject are not uniform, that is, if many types of cells are mixed, specify The step of concentrating the cell types can optionally be performed. 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 CD19 antigen, which is a surface antigen of B cells, has almost the same expression time as CD20 antigen, and B cells expressing CD19 are considered to express CD20. For the antigen used in this concentration step, information on the expression time of the antigen on the cell surface derived from the test subject is clarified. Therefore, it is particularly effective when there is an antibody that can be obtained at a lower price than an antibody drug.

[0029] Step (2):

Next, the viability of each cell cultured in the cell incubator is determined. For this determination, various known reagents for cell viability can be used. For example, if a dye for nuclear staining (P Propidium Iodide) is added to the cell culture compartment and the cells are observed with a microscope after incubation for a certain period of time, the nuclei of living cells will not be stained, whereas the cells of dead cells will be stained. Therefore, cell viability can be determined by the presence or absence of staining.

[0030] More specifically, the step of determining whether or not the patient is alive or dead includes a plurality of cells within a single visual field of a microscope, and the viability thereof is at least 50% or more, more preferably 80%. This corresponds to searching for a specific field of view. More preferably, after determining a visual field suitable for evaluation, photographing of the visual field is performed. By assigning an address to each cell in the field of view after imaging, it becomes easier to collate with the observation and measurement results of the subsequent process.

[0031] Step (3):

Next, in the viability determination step of the above step (2), when a field of view with a sufficient number of viable cells for evaluation is obtained, the antibody drug to be administered to the subject to be examined is added with a label. Add to the culture vessel and continue culturing. When the antigen recognized by the antibody is expressed on the surface of the living cell derived from the test subject, the antibody to which the label is attached binds to the antigen. Since some resistant patients may not express the CD20 antigen on the B cell surface, the labeled antibody does not bind to the cell surface. Therefore, when a certain period of time has elapsed after the addition of the antibody, the presence or absence of antigen expression on the surface of the living cell can be determined by photographing the visual field containing the living cell in a state suitable for detection of the label. Is possible.

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 10000, even more preferably 200, and 3000 , Most preferably 500 pcs, 2,000 pcs [0032] The antibody drug is particularly preferably a chimeric antibody, a humanized antibody, or a human antibody from the viewpoint of the ability to use a chimeric antibody, humanized antibody, human antibody, mouse antibody, or the like.

[0033] 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.

[0034] 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, the antibody drug is administered to the patient in the clinic. In some cases, it is possible to evaluate whether or not a patient can be effective.

[0035] In the inspection method of the present invention, after performing the steps (1) to (4),

(5) adding complement to the cell culture vessel; and

(6) a step of detecting the label bound to the cell surface and further determining the viability of the cell after addition of complement.

[0036] Step (5):

In step (5), complement is added. As the complement, the same type of antibody drug as used, that is, when the antibody drug is a humanized antibody, it is preferable to use human-derived complement, particularly preferably the subject of the subject. is there. More specifically, serum derived from healthy subjects or test subjects is added to the incubator, for example, so that the ratio to the total culture broth after addition is about 10% (v / v), and cultured for a certain period of time. To do. 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.

[0037] 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). If the antibody drug is bound to the cell surface after the step (3), the step (5 ) Complementation causes CDC and destroys the cell membrane. Therefore, the cells labeled in the above step (4) are detected by staining the nucleus with the staining reagent added in the above step (2), for example. On the other hand, the cells determined as dead cells in the step (2) are also determined as dead cells in this step, and when the cells derived from the test subject are antibody drug resistant, they are determined as live cells.

[0038] Step (7):

In the inspection method of the present invention, after the step (6),

(7) The step of determining the ratio of cell viability can be further included. In this process, it is possible to determine whether complement has worked effectively by determining the ratio of life and death of cells to be positive. That is, when the amount of complement added is small, when complement activity is low, when agitation is insufficient, when the temperature is appropriate, and when the incubation time after addition of complement is too short In this case, CDC does not occur sufficiently in the positive target cells, and the proportion of living cells increases. On the other hand, when positive target cells die at a predetermined rate (for example, 80% or more), it is defined that CDC is fully activated. Therefore, if complement CDC works sufficiently, positive target cells become dead cells in a significant proportion. In addition, the effectiveness of the antibody drug can be quantitatively shown by determining the survival rate of cells derived from the test subject. Therefore, by performing this step (7), it is possible to confirm whether or not the complement has sufficiently acted and the degree of effectiveness of the antibody drug in the treatment.

[0039] 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 a reagent for determining the viability of a cell and an antibody drug to be administered to the subject to be examined to which a label is added to the cell culture vessel and culturing; and

(c) determining the viability of the cells in the cell culture container, and further detecting the label that is bound to the cell surface.

[0040] In the test method of the second aspect of the present invention, a reagent for determining the viability of cells in the step (b) and an antibody drug to be administered to the test subject are labeled. The operation according to the above aspect including the steps (1) to (4), except that it is added to the cell culture vessel Do the same operation. This test method including the steps (a) to (c) can be applied particularly when the survival rate of the cells derived from the test subject is high.

[0041] In the inspection method of the second aspect of the present invention, after the step (c),

(d) adding complement to the cell culture vessel; and

(e) detecting the label bound to the cell surface, and further determining the viability of the cell after addition of complement.

As a complement source for complement added in the above 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.

[0042] By the complement addition in the above step (d), CDC occurs when the antibody drug is bound to the cell surface antigen 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 determined to determine whether the test subject has resistance to the antibody drug and complement. Whether or not the body worked effectively.

[0043] In the inspection method of the present invention, after step (7) of the first form or (f) of the second form,

(X) comparing the viability ratio of cells derived from the subject to be tested and the viability ratio of cells serving as a positive control. By including this step, it is possible to estimate how effectively the CDC acts when the antibody drug is actually administered to the test subject. In addition, it is possible to judge whether or not the sensitivity has been correctly evaluated. 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 (the labeled antibody drug is Whether the complement activity was sufficient, whether the experimental conditions were appropriate, etc.).

[0044] The inspection method of the present invention may further include the step of (Y) adding a foil 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). After or just after step (5). In the second embodiment, 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 the cells to be examined, cell culture fluid, various drugs, etc., and has an action of preventing evaporation. Any liquid that is immiscible with water with a specific gravity lower than water and has a low vapor pressure is acceptable. 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 from 11 1 to 1 ml). This is because the temperature of the culture environment may cause a problem of medium evaporation. 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 light relative to the medium and complement sources (serum, plasma, whole blood, purified complement fluid), complement may be added even if oil is added before step (5). Drops on the oil, the complement automatically passes through the oil layer and mixes with the underlying medium. Oil also does not affect optical observations. Thus, the oil can be added at any stage of the process of the present invention. However, from the viewpoint of preventing the medium from evaporating, it is preferable to add it in an amount sufficient to cover the medium surface before adding the complement. In addition, when oil is added after complement is added, it is desirable to carry out immediately after complement addition from the same viewpoint.

[0045] In the inspection method of the present invention, for example,

The cell culture vessel may have compartments, each compartment having a diameter of 1 to 4 mm, a depth of 2 to 5 mm, and a volume of! . By using a culture vessel having a compartment in this range, the cells floating in the vessel 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, y5, TRITC (tetramethylrhodamine isothiocyanate), or riTC (fluorescein isothiocyanate). [0047] The examination method of the present invention can be carried out on, for example, a patient with B-cell lymphoma. In this case, it is possible to use a cell derived from a lymphoma tissue collected from the patient as a cell derived from the test subject and an anti-CD20 antibody (rituximab) as an antibody drug. B-cell lymphoma is a disease that accounts for about 70% of non-Hodgkin 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.

[0048] 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 prolymphocyte Leukemia, lymphoid plasma cell lymphoma, splenic marginal zone B cell lymphoma, hairy cell leukemia, plasma cell tumor, extranodal follicular marginal B cell lymphoma, nodal follicular marginal zone B cell lymphoma, follicular lymphoma Mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal large cell B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma. Other antibody drugs include ibritumomab for B-cell lymphoma, trastuzumab for breast cancer (anti-Her2 antibody drug), and the like.

Example

[0049] (Experimental method)

Lymphoma tissues collected from various lymphoma patients were chopped 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. For lymphoma-derived cells, only CD19-positive cells were isolated using magnetic beads (Non-patent Document 3).

).

[0050] Rituximab was labeled with Alexa488 according to the method of Alexa488 Monoclonal antibody labeling kit (A20 181) manufactured by Invitrogen. Labeled rituximab was used at a concentration of 10 ng / ml. PI was used as a nuclear staining dye at a concentration of S ^ g / 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 a 384 well plate for cell observation. Observe the cells in the container with an inverted microscope, and 1000 to 10,000 cells per culture compartment I did it. The container was then placed on a microscope stage held at 37 ° C. From the bottom of the container, cells were observed, 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 the CD20 antigen and the state of nuclear staining. Cells that express CD20 antigen live without staining their nuclei but only their cell membranes. Dead cells are stained with the nuclear dye PI. Therefore, a region where the cell density of living cells expressing CD20 antigen is appropriate is selected as the observation region.

Next, human serum (complement source) was added at an arbitrary rate of 1-20% of the total culture compartment volume and incubated at 37 ° C for 5-30 minutes. Again, cells were observed and photographed from the bottom of the container, fluorescent labels were detected, and the presence or absence of nuclear staining was detected.

In this second observation, the CD20 antigen was expressed and lived in the first observation! /, And the cells were lived after complementation by observing whether the nucleus was stained. V, the number of cells and the number of dead cells were measured. This judged CDC sensitivity.

[0051] (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 results are shown in Fig. 1. It is a figure which shows the ratio (CDC sensitivity) which the disease name and CDC reaction which were determined by the pathological examination etc. produced. Thus, it was found that there is a big difference in the sensitivity of CDC even with the same clinical name. Therefore, it became clear that the present invention is very effective for the selection of antibody drugs.

Industrial applicability

[0052] The antibody drug sensitivity test method of the present invention makes it possible to easily test the sensitivity of an antibody drug using a small amount of cells.

Claims

The scope of the claims

[1] (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 vessel;

[2] (5) Step of adding complement to the cell culture vessel after the step (4); and

(6) The method for testing antibody drug susceptibility according to claim 1, further comprising the step of detecting the label bound to the cell surface and further determining the viability of the cell after addition of complement.

[3] The antibody drug susceptibility testing method according to claim 2, further comprising: (7) after the step (6), a step of obtaining a cell death rate.

[4] (a) a step of culturing cells derived from the test subject and cells as appropriate positive controls in a cell culture vessel;

[5] (d) After the step (c), a step of adding complement to the cell culture vessel; and

5. The antibody drug susceptibility testing method according to claim 4, further comprising: (e) detecting the label bound to the cell surface, and further determining the viability of the cell after addition of complement.

6. The method for testing antibody drug sensitivity according to claim 5, further comprising: (f) after the step (e), a step of obtaining a cell death rate.

[7] After the step (7) or (f),

(X) Life / death ratio of cells derived from the test subject and life / death of cells serving as a positive control The antibody drug sensitivity test method according to claim 3 or 6, further comprising a step of comparing the ratios of

[8] 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 vessel. 2. The method for testing antibody drug sensitivity according to 2, 4 or 5.

[9] After step (1), before or after step (5), after step ω, after step (d)

) At least before or after!

6. The antibody drug susceptibility testing method according to claim 2, further comprising the step of (Y) adding oil to the cell culture container.

[10] 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! To 15 ^ 1 Item 6. The method for testing antibody drug susceptibility according to Item 1 or 4.

[11] The antibody drug susceptibility testing method according to [1] or [4], wherein the label is optically detectable, and the label is detected by an optical device.

[12] The antibody drug sensitivity test 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. .

[13] Before the step (1) or before the step (a), the method includes a step of subjecting a test subject-derived cell to screening for CD19, wherein only positive cells are subjected to the step (1) or the step 13. The antibody drug sensitivity test method according to claim 12, which is subjected to (a).