MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 4, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0015

Original Articles

Establishment of an Anti-CD20 Monoclonal Antibody(C20Mab-60) for Immunohistochemical Analyses

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Yoshikazu Furusawa,1,2 Mika K. Kaneko,1 and Yukinari Kato1,2Sensitive and specific monoclonal antibodies (mAbs) are needed for detecting CD20. This antigen, one ofseveral B lymphocyte antigens, is a target for every application used in the diagnosis of B cell lymphoma. Many
anti-CD20 mAbs have been established, although applications of these antibodies are limited. This study aims
to establish sensitive and specific anti-CD20 mAbs suitable for broad application, such as flow cytometry,
Western blotting, and immunohistochemical analyses. Using the Cell-Based Immunization and Screening(CBIS) method, all procedures were performed by utilizing CD20-stable transfectants, and a clone, C20Mab-60(IgG2a, kappa), was developed. In flow cytometry, C20Mab-60 detected overexpression of CD20 in LN229 celland endogenous CD20 in BALL-1 (a human B cell leukemia cell line) but did not react with CD20-knockout
BALL-1 (BINDS-24), indicating specificity for CD20. In Western blotting, C20Mab-60 detected CD20overexpressing Chinese hamster ovary-K1, BALL-1, and Raji (a human Burkitt’s lymphoma cell line) displaying both sensitivity and specificity. Furthermore, B cell but not T cell lymphomas were strongly stainedwith C20Mab-60 in immunohistochemical analyses. C20Mab-60, which was developed by CBIS method, isshown to be useful for the detection of cells expressing CD20 in lymphoma tissues by flow cytometry, Western
blotting, and immunohistochemical analyses.
Keywords: CD20, monoclonal antibody, Western blotting, flow cytometry, immunohistochemistry
Introduction

C

D20 is expressed in B cells from pre- to maturephases of cell development and is also detected in many
types of non-Hodgkin lymphoma (NHL).(1) CD20 is detected
in 50% of B-lymphoblastic leukemia/lymphomas (B-ALL/LBL), originating from pre-B cells; however, it is not detected
in terminally differentiated plasma cell malignancies.(2,3)
CD20 has four membrane-spanning domains, consisting of 297 amino acids (aa), with a molecular weight of
33–37 kDa.(4,5) The two extracellular domains are located at
72–80 and 142–182 aa. Sensitive and specific monoclonal
antibodies (mAbs) are critical for the diagnosis of many typesof cancer, but the detection of extracellular loops of multipass
transmembrane proteins is quite difficult.(6) CD20 possesses
two small extracellular transmembrane loops, increasing thedifficulty of developing useful mAbs for use in multiple applications. This difficulty contrasts with the relative ease ofproduction of mAbs against single-pass transmembraneproteins, such as CD44(7) or PD-L1.(8)We have developed the Cell-Based Immunization andScreening (CBIS) method, in which cell lines are exclusively
used for both immunization and screening. The CBIS method
has previously been used for developing several mAbs
12against various proteins, including the five transmembraneprotein CD133.(6) Using the CBIS method, we have successfully produced sensitive and specific mAbs useful for not
only flow cytometry, but also Western blot and immunohistochemical analyses.(6–16) In this study, we aimed to develop
multiuse anti-CD20 mAbs that can be used for flow cytometry, Western blot, and immunohistochemical analyses.
Materials and Methods
Cell lines

P3X63Ag8U.1 (P3U1), Chinese hamster ovary (CHO)K1, and LN229 cells were obtained from the American Type
Culture Collection (Manassas, VA, USA). Raji (humanBurkitt’s lymphoma cell line) and BALL-1 (human B cellleukemia cell line) were obtained from the Cell Resource
Center for Biomedical Research, Institute of Development,
Aging and Cancer, Tohoku University, Miyagi, Japan. DNA
encoding the CD20 gene (IRAL012D02) was provided by theRIKEN BRC through the National BioResource Project ofMEXT, Japan. An open reading frame of CD20, plus an
N-terminal PA tag, was subcloned into a pCAG-Neo orpCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). CHO/CD20 was produced byDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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ESTABLISHMENT OF AN ANTI-CD20 MAB

P3U1, CHO-K1, CHO/CD20, Raji, BALL-1, and BINDS24 were cultured in Roswell Park Memorial Institute (RPMI)
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan). LN229
and LN229/CD20 were cultured using Dulbecco’s Modified
Eagle’s Medium (Nacalai Tesque, Inc.). Media were supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin,
100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B(Nacalai Tesque, Inc.). Cells were grown inside an incubator
at 37C with humidity and 5% CO2/95% air atmosphere.

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transfecting pCAG-Neo/CD20 into CHO-K1 cells using aGene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). LN229/CD20 was produced by transfecting pCAG-Ble/CD20 into LN229 cells
using a Neon transfection system (Thermo Fisher Scientific,Inc., Waltham, MA, USA). The cell line BALL-1/CD20-KO(BINDS-24) was generated by transfecting CRISPR/Cas9
plasmids for CD20 (Thermo Fisher Scientific, Inc.) using aNeon transfection system. Stable transfectants were established using SH800 (Sony Corp., Tokyo, Japan).

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FIG. 1. Detection of CD20 by C20Mab-60 using flow cytometry and Western blotting. (A) Detection of CD20 by C20Mab60. LN229, LN229/CD20, BALL-1, and BIND-24 were treated with C20Mab-60 (red line) at a concentration of 1 mg/mL or
0.1% BSA in PBS (gray) for 30 minutes, followed by incubation with secondary antibodies. (B) Fluorescence intensity wasquantified. **p < 0.01. n.s., not significant. All data are expressed as mean – SEM. (C) Detection of CD20 by C20Mab-60
using Western blotting. Cell lysates of CHO-K1, CHO/CD20, BALL-1, BINDS-24, and Raji cells were electrophoresed andtransferred onto PVDF membranes. These membranes were treated with C20Mab-60 (left panel), NZ-1 (anti-PA tag; middlepanel), or anti-b-actin (right panel), followed by incubation with secondary antibodies. BSA, bovine serum albumin; CHO,
Chinese hamster ovary; PBS, phosphate-buffered saline; SEM, standard error of the mean.

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Hybridoma production

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Two female BALB/c mice (6 weeks old) were purchasedfrom CLEA Japan (Tokyo, Japan). Animals were housed
under specific pathogen-free conditions. Animal experiments
for hybridoma production were approved by the Animal Careand Use Committee of Tohoku University (Permit No.:
2016MdA-153). In brief, LN229/CD20 cells (1 · 108 cells) wereinjected intraperitoneally into two BALB/c mice together with
Imject Alum (Thermo Fisher Scientific, Inc.). After three additional immunizations, a booster injection was administered 2days before harvesting spleen cells. Spleen cells were fused withP3U1 cells using polyethylene glycol 1500 (Roche Diagnostics,Indianapolis, IN, USA). The resulting hybridomas weregrown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine (Thermo Fisher
Scientific, Inc.). Culture supernatants were utilized forhybridoma screening by flow cytometry.
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin
with 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with phosphate-buffered saline (PBS)
containing 0.1% bovine serum albumin, cells were treatedwith 1 lg/mL of C20Mab-60 for 30 minutes at 4C, followed
by treatment with Alexa Fluor 488-conjugated anti-mouse
IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA,USA). Fluorescence intensity data were collected using
Spectral Cell Analyzer SA3800 (Sony Corp.).

FURUSAWA ET AL.

(Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3,3¢-diaminobenzidine tetrahydrochloride(DAB, Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin (FUJIFILM
Wako Pure Chemical Corporation).
Statistical analyses

Statistical analysis used Student’s t-test in GraphPad Prism
6 (GraphPad Software, Inc., La Jolla, CA, USA). p < 0.05 wasconsidered statistically significant. All data are expressed asmean – standard error of the mean.
Results
Establishment of anti-CD20 mAbs

Two mice were immunized with LN229/CD20 cells. Supernatants from cultured hybridomas, positive for CHO/CD20
and negative for CHO-K1, were selected by flow cytometry.
Further screening using Western blotting and immunohistochemistry was performed, and C20Mab-60 (IgG2a, kappa) wasestablished.

Western blotting analyses

Cells were lysed in 1% Triton X-100, and cell debris wasremoved by centrifugation. Cell lysates were boiled in sodium dodecyl sulfate sample buffer with a reducing reagent(Nacalai Tesque, Inc.). Proteins (10 lg) were separated on
5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and were then transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt,Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), the membranes were incubated with 10 lg/mL ofC20Mab-60, 1 lg/mL of NZ-1 (anti-PA tag), or 1 lg/mL ofanti-b-actin for control (clone AC-15; Sigma-Aldrich Corp.,
St. Louis, MO, USA), followed by incubation with the secondary antibody, peroxidase-conjugated anti-mouse immunoglobulins (1:1000; Agilent Technologies, Inc., Santa Clara,CA, USA), or anti-rat IgG (1:10,000; Sigma-Aldrich Corp.).
Finally, proteins were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using the SayacaImager (DRC Co. Ltd., Tokyo, Japan).
Immunohistochemical analyses

A formalin-fixed paraffin-embedded (FFPE) tissue microarray (Cat. No.: Z7020072) for lymphomas was purchasedfrom BioChain Institute Inc. (Newark, CA, USA). Tissue
sections were directly autoclaved in citrate buffer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min. After
blocking with the SuperBlock T20 (PBS) Blocking Buffer(Thermo Fisher Scientific, Inc.), tissue sections were incubated with C20Mab-60 (5 lg/mL) for 1 hour at room temperature and were then treated with a mouse Envision+ Kit

FIG. 2. Immunohistochemical analyses using C20Mab-60
for B cell lymphomas. Tissue sections of B cell (A–F) and T
cell lymphomas (G, H) were incubated with C20Mab-60,
followed by an Envision+ kit. (B, D, F, H) are enlargementsof (A, C, E, G), respectively. Counterstaining used hematoxylin. Scale bar = 100 mm.

ESTABLISHMENT OF AN ANTI-CD20 MAB
Flow cytometry

C20Mab-60 reacted with LN229/CD20 cells, but not with
LN229 cells, which allowed cells to be distinguished using
flow cytometry analysis (Fig. 1A). Furthermore, C20Mab-60
reacted with BALL-1 cells, but not with CD20-knockout
BALL-1 cells (BINDS-24), which indicated that C20Mab-60
is specific for CD20 (Fig. 1B).

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Western blot analyses

Western blotting detected CD20 using C20Mab-60 in a 45kDa protein band from CHO/CD20 cells and in a 37-kDa
band in BALL-1 and Raji cells. No detection was observed inproteins from CHO-K1 cells and CD20-knockout BALL-1(BIND-24) cells (Fig. 1C). Thus, C20Mab-60 is specific forCD20. An anti-PA tag mAb (NZ-1) detected CD20 at a
45-kDa band and did not detect this band in proteins fromCHO-K1, BALL-1, BINDS-24, and Raji cells. The difference in molecular size of CD20 obtained from CHO/CD20and BALL-1 cells might be due to the N-terminal PA-tag ofCD20 introduced into CHO-K1 cells.
Immunohistochemical analyses

C20Mab-60 was also assessed for its immunohistochemical
utility in a B cell lymphoma tissue microarray. C20Mab-60
strongly stained B cell lymphomas (Fig. 2A–F), and nostaining was observed in T cell lymphomas (Fig. 2G, H).
Thus, C20Mab-60 staining is B cell specific. C20Mab-60 isuseful for detecting B cells in immunohistochemical analyses
in FFPE-processed tissues.
Discussion

In this study, a sensitive and specific anti-CD20 mAb,
C20Mab-60, was developed using the CBIS method.(6) Thismethod is easier and more effective in acquiring mAbs
against multipass transmembrane proteins because it has norequirement for purified proteins for immunization andscreening. Rituximab (a mouse–human chimeric mAb; theoriginal mouse clone is 2B8(17)) is the first anti-CD20 mAb
approved by the US FDA and is currently used to treat B cell
NHL and B cell chronic lymphocytic leukemia.(18) Rituximab shows high sensitivity in flow cytometry and exerts highantibody-dependent cell-mediated cytotoxicity, complementdependent cytotoxicity, and antitumor activity. Rituximab
does not detect CD20 protein in Western blotting (data notshown), but C20Mab-60 does detect endogenous CD20 protein expressed in BALL-1 and Raji cells with high sensitivity(Fig. 1C). C20Mab-60 also stains B cell lymphoma cells in
immunohistochemical analyses (Fig. 2). Nonetheless, further
study validating the results in supplemental tissues that display different stages of disease progression will provide usefuladditional characterization of the mAb.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by AMED under Grant
Numbers JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),

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and JP20ae0101028 (Y.K.), and by JSPS KAKENHI Grant
Numbers 17K07299 (M.K.K.) and 19K07705 (Y.K.).
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 19, 2020
Accepted: June 17, 2020