JP6157045B2 - CD20-recognizing monoclonal antibody, hybridoma, CD20 detection method and determination method - Google Patents

Description

  The present invention relates to a CD20-recognizing monoclonal antibody that recognizes a CD20 molecule, a hybridoma that produces the CD20-recognizing monoclonal antibody, a CD20 detection method and a diagnostic method that use the CD20-recognizing monoclonal antibody.

  The CD20 molecule expressed on the cell surface of normal B cells or B cell lymphoma cells has recently been used as a target molecule for antibody therapy. Recently, lymphoma cells that have acquired a genetic mutation in the C-terminal cytoplasmic region of the CD20 molecule may be resistant to chemotherapy combined with rituximab, one of the anti-CD20 monoclonal antibody drugs. (See Non-Patent Document 1).

  On the other hand, to date, a monoclonal antibody named L26 has been used almost exclusively for histopathochemical staining of CD20 molecule. This antibody has been reported to recognize the intracytoplasmic region of the CD20 molecule (see Non-Patent Document 2), but no report has been made on the details of its epitope.

  Various monoclonal antibodies that recognize CD20 molecules have been marketed so far, but it has been difficult to recognize CD20 molecules having a gene mutation in the C-terminal cytoplasmic region.

Clin Cancer Res., 2009 Apr 1; 15 (7): pp. 2523-30 Am J Pathol., 1990 Jun; 136 (6): pp. 1215-22

  An object of the present invention is to provide a CD20-recognizing monoclonal antibody capable of recognizing a wider range of CD20 molecules, including a CD20 molecule having a gene mutation in the C-terminal cytoplasmic region, a hybridoma producing the CD20-recognizing monoclonal antibody, and the CD20 An object of the present invention is to provide a CD20 detection method and a diagnostic method using a recognition monoclonal antibody.

The present invention relates to a CD20-recognizing monoclonal antibody that recognizes CD20 having a gene mutation in the C-terminal cytoplasmic region, and recognizes an epitope comprising amino acid residues 23 to 36 in the N-terminal cytoplasmic region of CD20. and, which is the C-terminal CD20 recognized monoclonal antibody Ru detectable der cytoplasmic region of CD20 having the mutation in immunohistochemical staining.

  The present invention also provides a hybridoma that produces the CD20-recognizing monoclonal antibody.

The present invention also relates to a method for detecting CD20, wherein an antigen-antibody reaction using the CD20-recognizing monoclonal antibody is used to detect CD20 having a gene mutation in the C-terminal cytoplasmic region by immunohistochemical staining .

Further, the present invention utilizes an antigen-antibody reaction using the CD20 recognition monoclonal antibodies, you detect CD20 with genetic mutations in the C-terminal cytoplasmic region of the measuring object in the sample by immunohistochemical staining, B cells Is a method for determining lymphoma and B-cell leukemia.

  In the present invention, a CD20-recognizing monoclonal antibody capable of recognizing the N-terminal cytoplasmic region of CD20 can recognize a wider range of CD20 molecules including a CD20 molecule having a gene mutation in the C-terminal cytoplasmic region. it can.

It is a schematic diagram which shows the structure of CD20 molecule | numerator in B cell. It is a schematic diagram which shows an example of the recognition site | part of the CD20 molecule | numerator by CD20 recognition monoclonal antibody which concerns on embodiment of this invention, and L26. It is a figure which shows the dyeing | staining result in the epitope search of the L26 antibody in the Example of this invention. It is a figure which shows the evaluation result by the western blotting method of 21 types of hybridomas in the Example of this invention. It is a figure which shows the result of the tissue immunostaining of the pathological specimen in the Example of this invention.

  Embodiments of the present invention will be described below. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.

<CD20 recognition monoclonal antibody>
FIG. 1 is a schematic diagram showing the structure of the CD20 molecule in B cells. As shown in FIG. 1, the CD20 molecule 50 has an N-terminal cytoplasmic region 54 located in the cytoplasm 72 from the N-terminal side to the cell membrane 52, and an intracellular region (TM) located in the cell membrane 52. 56, an extracellular region 58 located outside the cell membrane 74, an intracytoplasmic region 60, an intracytoplasmic region 62, an inner cell membrane region 64, an outer cell membrane region 66, an inner cell membrane region 68, and a C-terminal intracytoplasmic region 70. Has been. In the CD20 molecule 50, rituximab, which is one of the anti-CD20 monoclonal antibody drugs, usually recognizes the rituximab recognition region 76 in the extracellular region 66.

  When the present inventors experimentally specified the antibody binding position of the L26 antibody to the CD20 molecule, as shown in FIG. 2, the region from the amino acid sequence 264 to 281 in the C-terminal cytoplasmic region 70 of the CD20 molecule It became clear that the region within 78 was recognized (see Example column), and it was theoretically found that most of the CD20 molecule having a gene mutation in the C-terminal cytoplasmic region 70 could not be recognized. Therefore, the present inventors can recognize a wider range of CD20 molecules including a CD20 molecule having a gene mutation in the C-terminal cytoplasmic region 70, and recognize the N-terminal cytoplasmic region of the CD20 molecule. A monoclonal antibody was established.

  The CD20-recognizing monoclonal antibody according to an embodiment of the present invention is an antibody molecule that can recognize and bind to the N-terminal cytoplasmic region of the CD20 molecule. The CD20-recognizing monoclonal antibody according to the embodiment of the present invention is not particularly limited as long as it can recognize and bind to the N-terminal cytoplasmic region 54 of the CD20 molecule. The recognition site recognized by the CD20-recognizing monoclonal antibody is preferably as close as possible to the N-terminus of the N-terminal cytoplasmic region 54 and is preferably a site where antibodies can be easily generated.

  Here, the “N-terminal cytoplasmic region” refers to the region from the first to the 64th amino acid sequence of the CD20 molecule (MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHI). “C-terminal cytoplasmic region” refers to the region from the 210th to the 297th amino acid sequence of the CD20 molecule (GIVENEWKRTCSRPKSNIVLLSAEEKEKEQTIIEKEEVGLTETSQPKNEEDIEIIPIQEEEEEEETNFFPPPQDSSPIENDSSP).

  In the “N-terminal cytoplasmic region”, the CD20-recognizing monoclonal antibody according to the present embodiment has amino acid residues 23 to 36 in the amino acid sequence (MQSGPKPLFRRMSS) (region of FIG. 2). 80), and a CD20-recognizing monoclonal antibody that recognizes an epitope containing the 43rd to 52nd amino acid residues (sequence: SFFMRESKTTL). Among them, the 23rd to 36th amino acid residues (MQSGPKPLFRRMSS) A CD20-recognizing monoclonal antibody that recognizes the epitope to be contained is particularly preferred.

  The CD20-recognizing monoclonal antibody according to this embodiment may be a human antibody or a human antibody.

<Method for producing CD20-recognizing monoclonal antibody and hybridoma>
The CD20-recognizing monoclonal antibody according to this embodiment can be produced by various methods well known in the art. For example, a synthetic peptide in which cysteine (Cys) is arranged at the N-terminus of the amino acid sequence of a desired region in the N-terminal cytoplasmic region of the CD20 molecule is prepared, and a carrier protein such as KLH is bound to the terminal cysteine residue (conjugation) The mixture is mixed with an adjuvant, such as Freund's complete adjuvant, if necessary, and administered subcutaneously to mammals such as rats, mice, and rabbits. Administration may be performed multiple times at predetermined intervals using the same antigen. After the final sensitization, after a predetermined interval, antibody-producing cells such as spleen cells are collected from the antibody-producing mammal and cell fusion with mouse myeloma cells is performed according to a conventional method. To produce a hybridoma. Antibody production can be confirmed by collecting serum from the administered mammal and using an oxygen-linked immunosorbent assay or the like using the synthetic peptide used for immunization. The hybridoma can be cloned, the culture supernatant can be used, the oxygen-binding immunosorbent assay can be performed using the synthetic peptide used for immunization, and candidate cells can be selected to establish the hybridoma.

  The dose of the antigen per mammal is, for example, 0.5 mg to 1 mg when no adjuvant is used, and 0.05 mg to 0.1 mg when an adjuvant is used.

  Examples of the adjuvant include Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), synthetic surfactant, liposome and the like.

  Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, intramuscularly or the like. The immunization interval is not particularly limited, and for example, immunization is performed 1 to 5 times, preferably 2 to 4 times at intervals of several days to several weeks, preferably at intervals of 2 to 3 weeks. For example, antibody-producing cells are collected 1 to 7 days after the last immunization day, preferably 1 to 5 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, bone marrow cells and the like, with spleen cells being preferred.

  In order to obtain a cell fusion hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As myeloma cells to be fused with antibody-producing cells, for example, generally available cell lines of animals such as mice can be used. Examples of myeloma cells include mouse myeloma cells.

Cell fusion between antibody-producing cells and myeloma cells is performed by mixing 1-10 × 10 ⁷ cells / mL antibody-producing cells and 1-5 × 10 ⁷ cells / mL myeloma cells in a medium such as HAT medium. Then, the fusion reaction is carried out in the presence of a cell fusion promoter. The cell ratio of antibody producing cells to myeloma cells is, for example, 2: 1 to 10: 1. As the cell fusion promoter, polyethylene glycol having a weight average molecular weight of 1,000 to 6,000 or the like can be used. Alternatively, antibody-producing cells and myeloma cells may be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

  The target hybridoma is selected from the cells after cell fusion treatment. The cell suspension is cultured by a method such as limiting dilution. Cells that grow by culturing can be obtained as hybridomas. Then, it is screened whether or not the target antibody is present in the culture supernatant of the grown hybridoma. Hybridoma screening may be performed by an ordinary method, and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma may be collected and screened by oxygen-linked immunosorbent assay, flow cytometry, immunoblotting, or the like. Positive fused cells can be cloned by limiting dilution or the like, and hybridomas that are monoclonal antibody-producing cells can be established.

  As a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method, ascites formation method, or the like may be used. In the cell culture method, for example, the hybridoma is cultured in a medium such as RPMI basic medium at 30 ° C. to 38 ° C., preferably 37 ° C. for 3 to 60 days, and a monoclonal antibody is obtained from the culture supernatant. can do.

In the case of the ascites formation method, for example, 1 to 2 × 10 ⁷ hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. Then, after 1 to 2 weeks, ascites can be collected to obtain a monoclonal antibody.

  The monoclonal antibody may be purified as necessary. Examples of the purification method include ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography and the like, and these methods may be appropriately selected or purified by combining them.

<CD20 detection method>
The antigen-antibody reaction using the CD20-recognizing monoclonal antibody according to the present embodiment can be used to detect CD20, that is, to evaluate whether the sample to be measured contains CD20 (protein, DNA, RNA). it can. Samples to be measured include cells, tissues, blood, lymph fluid, bone marrow fluid, and the like. Examples of the detection method include immunohistochemical staining, Western blotting, ELISA (Enzyme-Linked Immunosorbent Assay), and RIA (Radio ImmunoAssay).

  The immunohistochemical staining method is, for example, a method in which an antigen-antibody reaction is performed on a formalin-fixed paraffin-embedded section or the like using the CD20-recognizing monoclonal antibody according to this embodiment, and visualization is performed by a color development operation. By using the CD20-recognizing monoclonal antibody according to this embodiment, it is possible to obtain a favorable stained image specific to a wider range of CD20 molecules including CD20 molecules having a gene mutation in the C-terminal cytoplasmic region.

  Western blotting is performed by, for example, separating a sample by electrophoresis such as SDS-polyacrylamide gel electrophoresis, transferring the sample to a membrane, performing an antigen-antibody reaction using the CD20-recognizing monoclonal antibody according to this embodiment, and performing a color development operation. This is a method of visualization. By using the CD20-recognizing monoclonal antibody according to this embodiment, a wider range of CD20 molecules, including CD20 molecules having a gene mutation in the C-terminal cytoplasmic region, can be detected well.

  The method for detecting CD20 using the CD20-recognizing monoclonal antibody according to this embodiment is a solid malignant such as non-Hodgkin's malignant lymphoma, which is a B-cell lymphoma such as diffuse large cell lymphoma, Burkitt lymphoma, follicular lymphoma, MALT lymphoma, etc. It can be used for clinical examination, diagnosis, etc. of tumors (phosphorformers) and B-cell leukemia (Leukemia), which is one of the blood malignant tumors.

  The CD20-recognizing monoclonal antibody according to this embodiment can be used as a therapeutic antibody for B-cell lymphoma or the like by using a human-type antibody or a human antibody, and is one of the current therapeutic anti-CD20 monoclonal antibody drugs. There is a possibility that an immunotherapeutic agent for the treatment of CD20 positive B-cell lymphoma etc., which has a higher therapeutic effect than rituximab, which is one of the above, may be obtained.

  The CD20-recognizing monoclonal antibody according to the present embodiment enables early detection of cases showing irreversible resistance to rituximab treatment, etc. in combination with a monoclonal antibody such as L26 antibody, and the continuation of treatment with rituximab etc. Therefore, it is expected to provide important knowledge for judging the change of therapeutic policy to a novel antibody drug that is expected to be effective against antigens with lower expression levels that are expected to be introduced in the future.

  Hereinafter, although an example and a comparative example are given and the present invention is explained more concretely in detail, the present invention is not limited to the following examples.

<Searching for epitope of L26 antibody>
Total RNA was extracted from the human Burkitt lymphoma cell line Daudi cell using an RNA extraction reagent (Invitrogen TRIzol). Using this RNA as a template, cDNA is prepared by reverse transcription reaction, and using an oligo DNA primer designed to sandwich the entire length of the protein coding region of the human CD20 gene, the entire length of the protein coding region of the human CD20 gene (297 amino acids) (SEQ ID NO: 3) was amplified by the PCR method. The reverse transcription reaction and PCR reaction were performed using Takara's PrimeScript RT-PCR kit. The DNA thus obtained was cloned into a retrovirus vector (Takara pDON-AI vector) using a restriction enzyme. After confirming the introduced gene sequence using a DNA sequence analyzer (Applied Biosystems Genetic Analyzer 3130), using a vector introduced with the full length of the protein coding region of this human CD20 gene as a template, a Stratagene Mutagenesis kit was used. CD20 amino acid sequence 281st glutamic acid, 263rd glutamic acid, 245th glutamic acid, 245th glutinic acid, 228th valine, gene mutation inserted so that a stop codon immediately follows the 210th glycine residue, C-terminal deletion mutation Five types of vectors were produced.

  A packaging cell line G3Thi (Takara) was used by using seven types of retovirus vectors, a full-length wild-type human CD20, a vector into which genes encoding five types of CD20 deletion mutants were introduced, and a mock vector into which no foreign gene was introduced. Was used to produce retroviruses. Using these seven types of retroviruses, human myeloma cell line KMS12PE cells that do not express CD20 protein are infected, and cell lines and mock vectors expressing six types of CD20 with different lengths in the C-terminal cytoplasmic region are used. An introduced negative control cell line was prepared.

  As these 7 types of cell lines and positive control cell lines, human Burkitt lymphoma cell line Raji cells were mounted in a cover glass (IWAKI) coated with collagen I after swelling with 75 mM potassium chloride aqueous solution. Cell specimens were fixed with 3.7% formamide / 0.5% glutaraldehyde solution, permeabilized with 0.2% Triton X-100 solution, L26 antibody (DAKO) solution was added, and antigen antibody The reaction was carried out. After the specimen was washed with 0.2% Triton X-100 solution, secondary staining was performed with an anti-mouse IgG goat antibody (Invitrogen) labeled with Alexa Fluor488. The specimen was washed with a 0.2% Triton X-100 solution and then subjected to nuclear staining with a 5 μg / mL DAPI solution. These specimens were observed using a confocal microscope (Olympus). The results are shown in FIG. Since the specific staining signal by the L26 antibody was observed only in the wild type CD20 molecule, the cell expressing the CD20 molecule having a stop codon inserted immediately after the 281st glutamic acid residue, and the positive control cell, the L26 antibody It was strongly suggested that the binding position on the CD20 molecule is between amino acid sequence 264th to 281st.

<Preparation of CD20 recognition monoclonal antibody>
A synthetic peptide homologous to CMQSGPKPLFRRMSS (SEQ ID NO: 2) in which cysteine (Cys) is arranged at the N-terminal of the amino acid sequence of the N-terminal cytoplasmic region (amino acids 23 to 36) of CD20 (SEQ ID NO: 1) Residues with KLH attached were mixed with Freund's complete adjuvant and administered subcutaneously in 3 BALB / C mice. Specifically, KLM-binding synthetic peptide prepared at 0.5 mg / mL was mixed with an equal amount of Freund's complete adjuvant to form an emulsion, and 0.05 mL each was subcutaneously administered to the back of the mouse. Mice were further sensitized four times (final sensitization intravenously) at intervals of 2 weeks with the same antigen. Four days after the final sensitization, serum was collected from each mouse, and antibody production was confirmed by an oxygen-linked immunosorbent assay using the synthetic peptide used for immunization. Spleen cells were collected from the antibody-producing mice, and cell fusion with mouse myeloma cells was performed according to a conventional method to prepare hybridomas. Specifically, 1 × 10 ⁹ spleen cells and 2 × 10 ⁸ mouse myeloma P3U1 cells were mixed and then centrifuged at 1,000 rpm for 10 minutes. The supernatant is removed by aspiration, and cells are obtained as a precipitate fraction. After adding 1 mL of 25% (w / v) polyethylene glycol 1500 (PEG1500, manufactured by Boehringer), 10 mL of RPMI medium is slowly added. The total amount was 10 mL. To this, 10 mL of RPMI medium containing 20% FCS was added and allowed to stand a little, and then centrifuged at 1,000 rpm for 5 minutes, and the resulting precipitate fraction (cell fraction) contained 20% FCS. A cell suspension prepared by adding RPMI medium to a cell concentration of 10 ⁶ cells / mL was dispensed into Corning 96-well culture plates at 200 μL / well. After culturing in a 5% CO ₂ · 37 ° C. incubator for 24 hours, a HAT solution (manufactured by Invitrogen) was added, followed by further culturing for 2 weeks. Hybridomas were cloned, and using the culture supernatant, oxygen-linked immunosorbent assay was performed again using the synthetic peptide used for immunization, and candidate cells were selected to establish 21 types of hybridomas. Subclasses included IgG1, IgG2b, and IgG3 antibodies.

<Evaluation of hybridoma clones>
Twenty-one types of hybridomas were cultured on CD-Hybridoma Medium (Invitrogen), and antibodies were evaluated by Western blotting using the culture supernatant.

  As an antigen sample used for evaluation, human Burkitt lymphoma cell line Daudi cells were collected by centrifugation, 50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol. The sample was suspended in a sample buffer solution containing 10% SDS polyacrylamide gel, an antigen sample was added to each well so that the amount of protein was 25 μg, separated by electrophoresis, and a PVDF membrane ( It was electrically transferred to BioRad (ImmmunainasuBlot PVDF). After completion of the transfer, each lane was cut after being immersed in a blocking agent (Blocking One, manufactured by Nacalai Tesque) for 30 minutes at room temperature (22-27 ° C.), and each lane was cut with Tris buffer containing 0.1% Tween-20. It was immersed in the culture supernatant diluted twice, and immersed at room temperature for 2 hours. After discarding the culture supernatant, it was washed three times with a sufficient amount of Tris buffer containing 0.1% Tween-20, and then diluted HRP-labeled anti-antibody with Tris buffer containing 0.1% Tween-20. It was immersed in mouse goat IgG (Santa Cruz) for 1 hour at room temperature. After discarding the antibody solution, it was washed three times with a sufficient amount of Tris buffer containing 0.1% Tween-20, and then treated with GE Healthcare, ECL Plus Western Blotting Detection System, and the film for chemiluminescence (GE Healthcare, Hyperfilm ECL) detected the signal. The evaluation results are shown in FIG.

<Tissue immunostaining of pathological specimen>
Immunoglobulin purified from the culture supernatant of L26 antibody (DAKO code number N1502) and hybridoma, clone 4-6H: 2C (CD20N) as a primary antibody using solid phase protein-A resin (Pierce) (Subclass IgG3) was used.

[Sample preparation]
The pathological tissue before treatment with rituximab evaluated as CD20 positive by the method using the normal L26 antibody and the pleural effusion fibrin clot after relapse evaluated as CD20 negative were immediately collected in an amount of 5 to 10 times the tissue section. Immerse in 10% neutral formalin fixative. At this time, 24 hours was not exceeded. After fixation, it was washed with water and immersed in 70% ethanol. It was dehydrated by immersing it in a series of ethanol having a high concentration successively, and further dehydrated by immersing in xylene, and then embedded in paraffin. The paraffin-embedded specimen was sliced into 3 to 5 μm with a microtome, attached to a slide glass, and sufficiently dried in a thermostatic bath.

[Deparaffinization / Hydrophilization]
The slide was immersed in the following solutions in order to perform deparaffinization and hydrophilization.
Xylene 5 minutes (twice), 100% ethanol 3 minutes (twice), 95% ethanol 3 minutes (twice), 80% ethanol 3 minutes (twice), 70% ethanol 3 minutes, 50% ethanol 3 minutes, Purified water 3 minutes (twice), Tris hydrochloric acid buffer 3 minutes

[Antigen activation treatment]
The slide subjected to the deparaffinization / hydrophilization treatment was immersed in Target Retrieval Solution, pH 9 (Dako), and heated at 97 ° C. for 40 minutes for antigen activation treatment.

[Labeling]
Antibody labeling was performed according to the protocol of Dako using an automated immunohistochemical device Autostainer plus manufactured by Dako. However, CD20N used was diluted 50 times with an antibody dilution buffer (Dako).

[Dehydration / Encapsulation]
The specimen tissue slide was immersed in each solution in the following order to perform dehydration / penetration treatment.
Purified water 1 min, 50% ethanol 3 min, 70% ethanol 3 min, 80% ethanol 3 min, 95% ethanol 3 min, 100% ethanol 3 min (3 times), xylene 3 min (3 times), then sealed Encapsulated with the agent and covered with a cover glass. The observation result by a microscope is shown in FIG. In FIG. 5, the upper row shows the results of observation of the pathological tissue before treatment with rituximab evaluated as CD20 positive by a method using a normal L26 antibody, and the lower row shows the pleural effusion cell clot after recurrence evaluated as CD20 negative. Results are shown.

  Thus, a monoclonal antibody that recognizes the N-terminal cytoplasmic region of the CD20 molecule, which recognizes a wider range of CD20 molecules including CD20 molecules having a genetic mutation in the C-terminal region, was established. This antibody provided a good stained image specific to the CD20 molecule even in immunohistochemical staining using formalin-fixed paraffin-embedded sections. Furthermore, as a result of immunohistochemical staining using the L26 antibody, a sample showing a positive result was detected by reexamination of a sample evaluated as negative for CD20. As a result of the CD20 gene sequence analysis of this case, a gene mutation was detected in the C-terminal region.

  As a result of retrospective analysis, it was found that this case had no therapeutic response to rituximab combination chemotherapy since the gene mutation was detected. In addition, as a result of analysis using the CD20-recognizing monoclonal antibody established in this example, the CD20 molecule in this case was expressed in the vicinity of the cell membrane, but the binding of rituximab was significantly attenuated compared with the normal CD20 molecule. (However, it was not completely lost).

  50 CD20 molecule, 52 cell membrane, 54 N-terminal intracytoplasmic region, 56, 60, 64, 68 intracellular membrane region, 58, 66 extra-cytoplasmic region, 62 intracytoplasmic region, 70 C-terminal intracytoplasmic region, 72 intracytoplasmic, 74 cell membrane Outside, 76 Rituximab recognition region, 78, 80 region.

Claims (4)

A CD20-recognizing monoclonal antibody that recognizes CD20 having a gene mutation in the C-terminal cytoplasmic region,
Recognizing an epitope comprising amino acid residues 23 to 36 in the N-terminal cytoplasmic region of CD20 ;
CD20 recognized monoclonal antibody, wherein the detectable der Rukoto the CD20 with genetic mutations in the C-terminal cytoplasmic region immunohistochemical staining.   A hybridoma characterized in that it produces the CD20-recognizing monoclonal antibody according to claim 1. A method for detecting CD20 comprising detecting an antigen-antibody reaction using the CD20-recognizing monoclonal antibody according to claim 1 by detecting immunohistochemical staining for CD20 having a gene mutation in the C-terminal cytoplasmic region . Using the antigen-antibody reaction using the CD20-recognizing monoclonal antibody according to claim 1, CD20 having a gene mutation in a C-terminal cytoplasmic region in a sample to be measured is detected by immunohistochemical staining. , A method for determining B-cell lymphoma and B-cell leukemia.