MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 2, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0003

Development of Novel Mouse Monoclonal Antibodies
Against Human CD19

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Shinji Yamada,1 Mika K. Kaneko,1 Yusuke Sayama,1 Teizo Asano,1 Masato Sano,1 Miyuki Yanaka,1Takuro Nakamura,1 Saki Okamoto,1 Saori Handa,1 Yu Komatsu,1 Yoshimi Nakamura,1
Yoshikazu Furusawa,1 Junko Takei,1 and Yukinari Kato1,2

CD19 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is expressed in normal
and neoplastic B cells, and it modulates the threshold of B cell activation for amplifying B cell receptor signaling.
Blinatumomab (a CD3–CD19-bispecific T cell-engaging antibody) and tisagenlecleucel (genetically modified T cells
that express a CD19 chimeric antigen receptor [CART-19]) provide significant benefits for patients with CD19-positive
relapsed or refractory B cell malignancies. In this study, we first employed the Cell-Based Immunization and Screening(CBIS) method to produce anti-CD19 monoclonal antibodies using CD19-overexpressing cells for both immunizationand screening. One established clone—C19Mab-1—proved to be useful in flow cytometry assays against lymphoma
cell lines, such as BALL-1, P30/OHK, and Raji. Second, the extracellular domain of CD19 was immunized into mice,and enzyme-linked immunosorbent assays were performed for the first screening. One established clone—C19Mab3—was determined to be useful for Western blotting and immunohistochemical analysis. Due to their complementary
utility, a combination of C19Mab-1 (established using CBIS) and C19Mab-3 (established using conventional method)
could be useful for the pathological analysis of CD19.
Keywords: CD19, hybridoma production, monoclonal antibody
restricted expression on most stages of B cells (except forplasma cells).(7) Blinatumomab (Blincyto) and tisagenlecleucel (Kymriah) represent recent breakthroughs in CD19targeted therapy. Blinatumomab is a CD3–CD19-bispecific T
cell-engaging antibody; it comprises two single-chain variable fragments (scFv) and binds to both CD19 on B cells andCD3 on T cells.(8) Blinatumomab is approved for treating
relapsed and refractory pediatric B cell acute lymphoblastic
leukemia (B-ALL). Similarly, considerable effects have beenachieved by administration of tisagenlecleucel, such as genetically modified T cells that express a CD19-specific antigen receptor (CART-19). Tisagenlecleucel is approved forthe treatment of children and young adults with B-ALL that isrefractory or at a second or later relapse.(9) Treatment withthe anti-CD19 antibody–drug conjugate coltuximab ravtansine (SAR3419) resulted in moderate clinical responses in
pretreated patients with relapsed/refractory diffuse large B
cell lymphoma.(10) Fc-engineered anti-CD19 monoclonal
antibodies that enhance the killing function of effector cells
have also shown potential as candidate therapies.(11,12)
Recently, we developed a Cell-Based Immunization andScreening (CBIS) method to produce useful monoclonal antibodies (mAbs) against several membrane proteins.(13–16) In thisstudy, we generated two novel anti-CD19 mAbs—C19Mab-1

IntroductionC

D19 (B-lymphocyte surface antigen B4), a type I
transmembrane glycoprotein of 95 kDa, belongs to theimmunoglobulin (Ig) superfamily and comprises two extracellular C2-set Ig-like domains, a transmembrane domain, anda cytoplasmic tail.(1) CD19 is expressed in normal and neoplastic B cells as well as in follicular dendritic cells; it functions coordinately with several cell surface receptors, such ascomplement receptor CD21, tetraspanin membrane protein
CD81, and CD225.(2) These receptors form a cell surface
complex with the B cell receptor (BCR) that modulates thethreshold of B cell activation for amplifying BCR signaling.(3,4) A previous report has clarified that B cells from CD19KO mice are hyporesponsive to BCR stimulation in vitro andshow relatively modest immune responses in vivo.(5) In addition, another study has revealed that deletion of the cytoplasmic domain of CD19 in transgenic mice resulted in abnormal
B cell development, modest mitogen responses, minimal serum Ig levels, and attenuated humoral immunity, indicatingthat specific signals through the cytoplasmic domain of CD19
are prerequisites for B lymphocyte function.(6)CD19 is a biomarker for lymphoma and an immunotherapy
target for B cell lymphoproliferative diseases because of its
1
2

Department 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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and C19Mab-3—using CBIS and a conventional method(a soluble protein-based method), respectively. We further
compared their utility in flow cytometry, Western blotting, andimmunohistochemical analyses.
Materials and Methods

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Cell lines

CHO-K1, LN229, and P3U1 cell lines were obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA).
Lymphoma-derived cell lines P30/OHK, BALL-1, and Raji wereobtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer at Tohoku
University (Miyagi, Japan). CD19-overexpressed CHO-K1 cells(CHO/CD19) and LN229 cells (LN229/CD19) were producedby transfecting pCAG/CD19-RAP-MAP into CHO-K1 and
LN229 cells using a Gene Pulser Xcell electroporation system(Bio-Rad Laboratories, Inc., Berkeley, CA) and a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA),
respectively. CD19 extracellular domain-producing LN229 cells(LN229/CD19ec) were prepared by transfecting pCAG/PACD19ec-RAP-MAP into LN229 cells using the Neon transfection system. The PA tag system (PA12: GVAMPGAEDDVV vs.
clone: NZ-1), RAP tag system (RAP12: DMVNPGLEDRIE vs.
clone: PMab-2), and MAP tag system (MAP12: GDGMVPPGIEDK vs. clone: PMab-1) were previously established at ourlaboratory.(17–19) The stable transfectants of CHO/CD19 were
established using a cell sorter (SH800; Sony Corp., Tokyo, Japan). The stable cell lines of LN229/CD19 and LN229/CD19ec
were established by limiting dilution.
LN229, LN229/CD19, and LN229/CD19ec were cultured in
Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Inc.,
Kyoto, Japan), and CHO-K1, CHO/CD19, P3U1, P30/OHK,
BALL-1, and Raji cells were cultured in RPMI 1640 medium(Nacalai Tesque, Inc.) at 37C in a humidified atmosphere
containing 5% CO2 and 95% air. Media were supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific, Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin, and 25 lg/mL amphotericin B (Nacalai Tesque, Inc.).
Animals and human tissues

Female BALB/c mice (6 weeks old) were purchased from
CLEA Japan (Tokyo, Japan) and kept under specific pathogenfree conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments described inthis study. We used tonsil tissues from a patient with oropharyngeal squamous cell carcinoma who had undergone surgery
at the Sendai Medical Center. Informed consent for sample
procurement and subsequent data analyses was obtained fromthe patient or the patient’s guardian.
Production of mouse monoclonal antibodies
against human CD19

Hybridomas were produced as follows. LN229/CD19 cells(1 · 108 cells/dose), or soluble CD19 (100 lg/dose), were intraperitoneally administered into the BALB/c mice. Imject
Alum (Thermo Fisher Scientific, Inc.) was mixed with an
immunogen at initial immunization as an adjuvant. After threebooster immunizations at 1-week intervals, the final injection
was intraperitoneally administered 2 days before harvesting
the spleen cells. These spleen cells were then fused with P3U1
cells using PEG1500 (Roche Diagnostics, Indianapolis, IN).

YAMADA ET AL.

The resulting hybridomas were grown in RPMI medium supplemented with 10% FBS, hypoxanthine, aminopterin, thymidine selection medium supplement (Thermo Fisher Scientific,Inc.), and 5% BriClone hybridoma cloning medium (QED
Bioscience, Inc., San Diego, CA). Subsequently, 100 U/mL
penicillin, 100 lg/mL streptomycin, and 25 lg/mL amphotericin B (Nacalai Tesque, Inc.) were added to the medium. Hybridomas were selected on the basis of the reactivity of culture
supernatants against cell-bound or soluble CD19 by flow cytometry or enzyme-linked immunosorbent assay (ELISA), respectively. mAbs were purified from the supernatants ofhybridomas and cultured in hybridoma-SFM medium (Thermo
Fisher Scientific, Inc.) using protein G Sepharose 4 Fast Flow
(GE Healthcare UK Ltd., Buckinghamshire, England).
Flow cytometry

Cells were harvested by briefly exposing them to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin/
phosphate-buffered saline (PBS), the cells were treated withthe hybridoma supernatant or 10 lg/mL of purified antibodies
for 30 minutes at 4C and subsequently with Alexa Fluor 488conjugated anti-mouse immunoglobulin G (IgG, 1:2000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescence data
were collected using SA3800 Cell Analyzers (Sony Corp.).
Enzyme-linked immunosorbent assay

Soluble CD19 protein, purified from the supernatant of
LN229/CD19ec, was immobilized on Nunc MaxiSorp 96-wellimmunoplates (Thermo Fisher Scientific, Inc.) at 1 lg/mL for
30 minutes at 37C. After blocking with the SuperBlock T20(PBS) blocking buffer (Thermo Fisher Scientific, Inc.), theplates were incubated with the hybridoma culture supernatant,followed by a 1:2000 dilution of peroxidase-conjugated antimouse IgG (Agilent Technologies, Inc., Santa Clara, CA). The
enzymatic reaction was conducted using 1-Step Ultra TMBELISA (Thermo Fisher Scientific, Inc.). Optical density (OD)
was measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA).
Western blotting

Cell lysates (10 lg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.), and the proteins were
electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan) and transferred onto polyvinylidene fluoride membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), the membranes were incubated with
10 lg/mL anti-CD19 (clone C19Mab-1 and C19Mab-3) or
1 lg/mL anti-RAP tag (clone PMab-2). Anti-b-actin mAb(clone AC-15; Sigma-Aldrich Corp., St. Louis, MO) was usedwith 1 lg/mL as an internal control. The membranes were thenreacted with peroxidase-conjugated anti-mouse IgG (Agilent
Technologies Inc.; 1:1000 dilution) and developed using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation)using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Immunohistochemistry

Cell blocks were produced using iPGell (Genostaff Co., Ltd.,Tokyo, Japan) and processed into 4-lm paraffin-embedded cell

ANTI-CD19 MAB

47

sections, which were directly autoclaved in citrate buffer(pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20
minutes. Tissue sections of 4-lm thickness were directly
autoclaved in EnVision FLEX Target Retrieval Solution,
High pH (Agilent Technologies, Inc.), for 20 minutes. Thesesections were blocked using the SuperBlock T20 (PBS)
blocking buffer (Thermo Fisher Scientific, Inc.) and incubatedwith 1 lg/mL (cell sections) or 10 lg/mL (tissue sections) of
anti-CD19 mAbs (clone C19Mab-1 and C19Mab-3) for 1 hourat room temperature. Peroxidase-conjugated anti-mouse IgG
was then applied using an Envision+ Kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3, 3¢diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.) for 2 minutes and counterstaining was performed using
hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

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Determination of binding affinity

CHO/CD19 and P30/OHK were suspended in 100 lL ofserially diluted anti-CD19, followed by addition of Alexa
Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling
Technology, Inc.). Fluorescence data were collected using
the EC800 Cell Analyzer (Sony Corp.). The dissociation
constant (KD) was obtained by fitting the binding isotherms tobuilt-in, one-site binding models in GraphPad PRISM 6(GraphPad Software, Inc., La Jolla, CA).
Results
Production of anti-CD19 mAbs using CBISIn the first trial, we immunized two mice with LN229/CD19
cells and performed flow cytometry. Hybridoma supernatantsof 1056 wells were screened against CHO/CD19 cells and
CHO-K1 cells, and three wells (3/1056; 0.3%) that were positive against CHO/CD19 cells and negative against CHO-K1
cells were selected. Although we performed Western blotting
and immunohistochemical analyses using CHO/CD19 cells
and CHO-K1 cells, none of these three wells produced specific signals against CD19. After performing limiting dilutionof the three wells that showed specific signals in flow cytometric analyses, one clone—C19Mab-1 (IgG1, kappa)—was
established (Supplementary Table S1).
Production of anti-CD19 mAbs using a soluble
protein-based method

In the next trial, we immunized two mice with soluble
CD19 (LN229/CD19ec) and performed ELISA. The culture
supernatants of 958 wells were screened against LN229/
CD19ec, and 18 wells (18/958; 1.9%) that showed a positive
reaction (OD >0.1) were selected. We then performed immunohistochemical analyses using CHO/CD19 cells and CHOK1 cells. Of these 18 wells, 7 (38.9%) were positive against
CHO/CD19 cells and negative against CHO-K1 cells. We
further performed flow cytometry using CHO/CD19 cells and
CHO-K1 cells and found that two wells were positive against
CHO/CD19 cells and negative against CHO-K1 cells. After
performing limiting dilution, one clone—C19Mab-3 (IgG1,
kappa)—was established (Supplementary Table S1).
Flow cytometric analysis against lymphoma cell lines

C19Mab-1 reacted with CHO/CD19 cells, but did not reactwith CHO-K1 cells, suggesting that C19Mab-1 is specific

FIG. 1. Flow cytometric analysis of C19Mabs. Cells weretreated with 10 mg/mL C19Mab-1 (red line) or C19Mab-3 (blueline), followed by treatment with Alexa Fluor 488-conjugatedanti-mouse IgG. Black line, fluorescence-activated cell sorting
buffer; dashed line, mouse IgG1 isotype control (10 mg/mL).
IgG, immunoglobulin G.

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48

YAMADA ET AL.

FIG. 2. Western blotting analysis using C19Mabs. Cell lysates (10 mg) were electrophoresed under reducing conditions on
5%–20% polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. After blocking, the membrane wasincubated with C19Mab-1 (10 mg/mL), C19Mab-3 (10 mg/mL), anti-RAP tag (1 mg/mL), and anti-actin (1 mg/mL) and thenwith peroxidase-conjugated secondary antibodies.
for CD19 (Fig. 1). C19Mab-1 also detected endogenous
CD19 on human lymphoma cell lines BALL-1, P30/OHK,and Raji, indicating that C19Mab-1 is useful for detecting
endogenous CD19 by flow cytometry. Although C19Mab-3
reacted with CHO/CD19 cells weakly, it could not detect
the endogenous CD19 of human lymphoma cell lines inflow cytometry (Fig. 1).
Subsequently, we performed a kinetic analysis of the interaction of C19Mab-1 with CHO/CD19 or P30/OHK cells using
flow cytometry. The dissociation constants KD of C19Mab-1
were determined to be 4.0 · 10-9 M against CHO/CD19 and
1.5 · 10-8 M against P30/OHK, indicating that C19Mab-1 possesses moderate affinity for these cells (Supplementary Fig. S1).
Western blotting analyses

C19Mab-3 detected bands of *75 and 100 kDa in lysates of
CHO/CD19 cells, although C19Mab-1 did not detect any specific signal at the expected molecular size (Fig. 2). The difference between the 75- and 100-kDa bands might be becauseof post-translational modification(s). In Raji cells, signals between 75 and 100 kDa were detected using C19Mab-3, indicating that C19Mab-3 specifically detects endogenous CD19 inWestern blotting.
Immunohistochemical analyses

Immunohistochemical analyses revealed that C19Mab-1 did
not react with CHO-K1 (Fig. 3A) or CHO/CD19 cells (Fig. 3B).
Conversely, C19Mab-3 stained CHO/CD19 cells strongly(Fig. 3D), whereas it did not react with CHO-K1 cells (Fig. 3C),
indicating that C19Mab-3 is specific for CD19. No signal wasobserved for the negative control (Fig. 3E, F).
We further performed immunohistochemical analysis in
human tonsil tissues using C19Mab-1 and C19Mab-3. As shownin Figure 4A and B, C19Mab-1 did not stain the lymphocytes inthe tonsil tissue, whereas C19Mab-3 stained the lymphocytes
in the cortex and lymphoid follicles and in the region of theparacortex (Fig. 4C, D). No signal was observed for thenegative control in a serial section (Fig. 4E, F).

FIG. 3. Immunohistochemical analysis of C19Mabs against
CD19-expressing CHO-K1 cells. After antigen retrieval,sections were incubated with 1 mg/mL of C19Mab-1 (A, B),
C19Mab-3 (C, D), and blocking buffer (E, F), followed bytreatment with peroxidase-conjugated secondary antibodies.
Sections were counterstained with hematoxylin. (G, H) HE
staining. Scale bar = 100 mm. HE, hematoxylin and eosin.

ANTI-CD19 MAB

49

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amino acid residues 97–107, 155–166, and 216–224.(22) Furthermore, a previous report has revealed that several antiCD19 mAbs (clones AB1, B4, HD37, BU12, F974A2, andSJ25-C1) have exhibited competitive inhibition against
B43,(23) indicating that the epitope of B43 is a hotspot for
production of useful anti-CD19 mAbs.
As mentioned before, CART-19 (tisagenlecleucel) therapy
has shown remarkable potential for treatment of highly refractory B cell malignancies; however, some patients do notrespond to CART-19 immunotherapy for several weeks. The
acquired resistance of B-ALL to CART-19 is partly based onskipping of CD19 exon2, in which the epitope of CD19 scFv
might exist.(24) Therefore, the development of novel antiCD19 mAbs, which recognize epitopes different fromexisting Abs, would be valuable for gaining a better understanding of CD19-targeting immunotherapy. In addition,
future studies should investigate the epitopes recognized by
C19Mab-1 and C19Mab-3.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED), Grant Numbers: JP19am0401013 (Y.K.), JP19am0101078 (Y.K.), andJP19ae0101028 (Y.K.), and by Japan Society for the Promotion of Science (JSPS) KAKENHI, Grant Numbers:
17K07299 (M.K.K.) and 19K07705 (Y.K.).

FIG. 4. Immunohistochemical analysis of C19Mabs
against human tonsil tissue. After antigen retrieval, sections
were incubated with 10 mg/mL of C19Mab-1 (A, B),
C19Mab-3 (C, D), and blocking buffer (E, F), followed bytreatment with peroxidase-conjugated secondary antibodies.
(G, H) HE staining. Scale bar = 100 mm.

Discussion

In our first trial of producing anti-CD19 mAbs, we used
CBIS to develop specific mAbs against CD19. This method isvery convenient because it does not require the purification of
membrane proteins in all steps of mAb production.(13–16)
Using CBIS, we developed a novel anti-CD19 mAb—
C19Mab-1—which is very useful for detection of CD19 inflow cytometric analyses. Unfortunately, C19Mab-1 could notrecognize the denatured CD19 protein in Western blotting
and immunohistochemistry (IHC) analyses, suggesting that
C19Mab-1 binds to a conformational epitope of CD19. In our
second trial of producing anti-CD19 mAbs, we developed
C19Mab-3 using the purified CD19 ectodomain as an immunogen. Although C19Mab-3 is not suitable for flow cytometry, it is very sensitive to Western blotting and IHC
analyses. Taken together, the findings suggest that it is important to select a suitable immunogen and screening method
when developing optimal anti-CD19 mAbs.
The CD19 recognition arm of blinatumomab is derived
from anti-CD19 mAb B43, which was obtained from a mousehybridoma.(20,21) A recent study has revealed that B43 binds toCD19 in the middle of the ectodomain and recognizes a conformational epitope, which comprises three loops spanning

Supplementary Material

Supplementary Figure S1
Supplementary Table S1References

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 26, 2020
Accepted: February 27, 2020

Supplementary Data

SUPPLEMENTARY FIG. S1. Determination of thebinding affinity of C19Mab-1 using flow cytometry.
CHO/CD19 (A) and P30/OHK (B) were suspended in
100 mL of serially diluted C19Mab-1 (0.6–10 mg/mL), followed by addition of secondary anti-mouse immunoglobulin
G. Fluorescence data were collected using a cell analyzer.
GeoMean, geometric mean value.

Supplementary Table S1. Summary of Hybridoma Production
Trial
1
2

ImmunogenFirst screening

Second screening Third screening

LN229/CD19 (cell-bound CD19) 3/1056 (FCM)a 0/3 (IHC)
LN229/CD19ec (soluble CD19) 18/958 (ELISA) 7/18 (IHC)

0/3 (WB)
2/18 (FCM)

Hybridoma clone
used in this study
C19Mab-1 (IgG1, kappa)
C19Mab-3 (IgG1, kappa)

Hybridoma supernatants were assayed against CHO-K1 cells and CHO/CD19 cells in FCM, WB, and IHC screening. For ELISA
screening, soluble CD19 was used as a solid-phase antigen.
aNo. of positive wells/no. of tested wells (screening method).
ELISA, enzyme-linked immunosorbent assay; FCM, flow cytometry; WB, Western blotting; IHC, immunohistochemistry; IgG,
immunoglobulin G.