MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0046
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Epitope Mapping of an Anti-CD10 Monoclonal Antibody(MME/1870) Using Enzyme-Linked Immunosorbent Assay

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Hiroki Kawabata,1 Hiroyuki Suzuki,2 Junko Takei,1 Mika K. Kaneko,1 and Yukinari Kato1,2CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. CD10 is expressedon predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Antibodies against CD10 areused for the diagnosis of follicular lymphoma. Anti-human CD10 monoclonal antibody (clone MME/1870) canbe used for Western blotting and immunohistochemical analyses. This study examined the critical epitope ofMME/1870 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed
ELISA with deletion mutants, and MME/1870 reacted to the 501–520 amino acid sequence of CD10. Next, weanalyzed the reaction to 20 point mutants, and MME/1870 did not recognize the alanine-substituted peptides ofY507A, I511A, I512A, and L515A. These results indicate that the binding epitope of MME/1870 includes
Tyr507, Ile511, Ile512, and Leu515 of CD10.
Keywords: CD10, clone MME/1870, epitope mapping, monoclonal antibody, enzyme-linked immunosorbent
assay

inactivating the physiological action of the peptides bylowering the extracellular concentration available for theirreceptors. Furthermore, CD10 cleaves growth factors, such as
fibroblast growth factor 2, which induces Akt signaling inendothelial cells and stimulates angiogenesis.(12)In addition to its enzymatic activity, CD10 regulates intracellular signaling pathways. For example, CD10 interacts
with p85, a PI3K subunit, and Lyn kinase, which indirectly
prevents Focal Adhesion Kinase (FAK) activation byPI3K.(13) Moreover, the association between CD10 and thetumor suppressor Phosphatase and Tensin Homolog Deleted
from Chromosome 10 (PTEN) leads to decreased PIP3
phosphorylation, which activates the Akt pathway.(14)Sensitive and specific monoclonal antibodies (mAbs)
against CD10 are needed for the diagnosis of malignancies.
Among them, anti-human CD10 mAb (clone MME/1870) is
useful for Western blotting and immunohistochemical analyses but not for flow cytometry. The understanding ofantibody–antigen binding by revealing the epitope is essential for future application to antibody technology. To
clarify further characteristics of MME/1870, we performed
epitope mapping using enzyme-linked immunosorbent assay (ELISA).

IntroductionCD10 is a type II transmembrane glycoprotein witha molecular weight of 100,000 and is known as acommon acute lymphoblastic leukemia antigen.(1–3) CD10
was identified as membrane endopeptidase-24.11, an important role in the hydrolysis of biologically active peptides.(4) CD10 is expressed on predifferentiated lymphocyte
progenitor cells, and the expression disappears when cells
differentiate into T cells. In the case of B cell lineage, it
disappears at the time of cell surface immunoglobulin expression. CD10 is detected on activated and proliferating B
cells in the germinal center(5) and on bone marrow stromalcells and neutrophils.(6) CD10 is also expressed in human B
cell malignancies, including follicular,(7) Burkitt, and lymphoblastic lymphomas.(8) Moreover, CD10 is expressed innormal epithelial cells of the small intestine, kidney, andlung, and its decreased or increased expression is observed inmelanoma, thyroid cancer, and several carcinomas.(9)CD10 is a neutral endopeptidase that cleaves at the amino
side of hydrophobic residues of several hormones, including
glucagon, enkephalins, substance P, neurotensin, oxytocin,
and bradykinin.(10,11) This function plays crucial roles in

Departments of 1Antibody Drug Development, and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai,
Miyagi, Japan.

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KAWABATA ET AL.

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Table 1. Identification of Anti-CD10 Monoclonal
Antibody Epitope Using Synthesized Peptides
by Enzyme-Linked Immunosorbent Assay
PeptidesSequence

Anti-CD10mAb

51–70
61–80
71–90
81–100
91–110
101–120
111–130
121–140
131–150
141–160
151–170
161–180
171–190
181–200
191–210201–220
211–230
221–240231–250
241–260
251–270
261–280
271–290281–300
291–310
301–320
311–330
321–340
331–350
341–360
351–370
361–380
371–390
381–400
391–410401–420
411–430
421–440
431–450
441–460
451–470
461–480
471–490481–500
491–510501–520511–530
521–540
531–550
541–560
551–570
561–580571–590581–600
591–610
601–620
611–630
621–640
631–650TYDDGISKSSDSIKSAARLIDSIKSAARLIQNMDATTEPSQNMDATTEPSTDFFKYASGGTDFFKYASGGWLKRNVIPETWLKRNVIPETSSRYGNFDILSSRYGNFDILRDELEVVLKDRDELEVVLKDVLQEPKTEDIVLQEPKTEDIVAVQKAKALYVAVQKAKALYRSSINESAIDRSSINESAIDSRGGEPLLKLSRGGEPLLKLLPDIYGWPVALPDIYGWPVATENWEQKYGATENWEQKYGASWTAEKAIAQSWTAEKAIAQLNSKYGKKVLLNSKYGKKVLINLFVGTDDKINLFVGTDDKNSVNHVIHIDNSVNHVIHIDQPRLGLPSRDQPRLGLPSRDYYESTGIYKEYYESTGIYKEASTAYVDFMIASTAYVDFMISVARLIRQEESVARLIRQEERLPIDENQLARLPIDENQLALEMNKVMELELEMNKVMELEKEIANATAKPKEIANATAKPEDRNDPMLLYEDRNDPMLLYNKMTLAQIQNNKMTLAQIQNNFSLEINGKPNFSLEINGKPFSWLNFTNEIFSWLNFTNEIMSTVNISITNMSTVNISITNEEDVVVYAPEEEDVVVYAPEYLTKLKPILTYLTKLKPILTKYSARDLQNLKYSARDLQNLMSWRFIMDLVMSWRFIMDLVSSLSRTYKESSSLSRTYKESRNAFRKALYGRNAFRKALYGTTSETATWRRTTSETATWRRSANYVNGNMESANYVNGNMENAVGRLYVEANAVGRLYVEAAFAGESKHVVAFAGESKHVVEDLIAQIREVEDLIAQIREVFIQTLDDLTWFIQTLDDLTWMDAETKKRAEMDAETKKRAEEKALAIKERIEKALAIKERIGYPDDIVSNDGYPDDIVSNDNKLNNEYLELNKLNNEYLELNYKEDEYFENNYKEDEYFENIIQNLKFSQSIIQNLKFSQSKQLKKLREKVKQLKKLREKVDKDEWISGAADKDEWISGAAVVNAFYSSGRVVNAFYSSGRNQIVFPAGIL
NQIVFPAGILQPPFFSAQQS
QPPFFSAQQSNSLNYGGIGMNSLNYGGIGMVIGHEITHGF
VIGHEITHGFDDNGRNFNKDDDNGRNFNKDGDLVDWWTQQ
GDLVDWWTQQSASNFKEQSQSASNFKEQSQSMVYQYGNFSSMVYQYGNFSWDLAGGQHLN
WDLAGGQHLNGINTLGENIA+++(continued)

Table 1. (Continued)
PeptidesSequence

Anti-CD10mAb

641–660
651–670
661–680
671–690681–700
691–710
701–720711–730
721–740731–750

GINTLGENIADNGGLGQAYRDNGGLGQAYRAYQNYIKKNG
AYQNYIKKNGEEKLLPGLDLEEKLLPGLDLNHKQLFFLNFNHKQLFFLNFAQVWSGTYRPAQVWSGTYRPEYAVNSIKTD
EYAVNSIKTDVHSPGNFRIIVHSPGNFRIIGTLQNSAEFSGTLQNSAEFSEAFHSRKNSYEAFHSRKNSYMNPEKKSRVW-+++, OD655 S0.5; ++, 0.3 &OD655 < 0.5; +, 0.1 &OD655 <0.3; -, OD655 < 0.1.
mAb, monoclonal antibody.

Materials and Methods
Antibodies

Anti-CD10 mAb, clone MME/1870 (cat. no. ab238021),
was purchased from Abcam (Cambridge, UK).
CD10 peptidesCD10 peptides (accession no. NM_000902.3), including
69 deletion mutants (Table 1) and 20 point mutants (Table 2),
were synthesized by utilizing PEPScreen (Sigma-AldrichCorp., St. Louis, MO).
Enzyme-linked immunosorbent assay

Synthesized CD10 peptides (Tables 1 and 2) were immobilized on Nunc Maxisorp 96-well immunoplates (ThermoFisher Scientific Inc., Waltham, MA) at a concentration of10 lg/mL for 30 minutes at 37C. After washing with

Table 2. Identification of Anti-CD10 Monoclonal
Antibody Epitope Using Alanine-SubstitutedPeptides by Enzyme-Linked Immunosorbent Assay
PeptidesSequence

Anti-CD10 mAb

N501A
Y502A
K503A
E504A
D505A
E506A
Y507A
F508A
E509A
N510A
I511A
I512A
Q513A
N514A
L515A
K516A
F517A
S518A
Q519A
S520A

AYKEDEYFENIIQNLKFSQS
NAKEDEYFENIIQNLKFSQSNYAEDEYFENIIQNLKFSQSNYKADEYFENIIQNLKFSQS
NYKEAEYFENIIQNLKFSQS
NYKEDAYFENIIQNLKFSQS
NYKEDEAFENIIQNLKFSQS
NYKEDEYAENIIQNLKFSQS
NYKEDEYFANIIQNLKFSQS
NYKEDEYFEAIIQNLKFSQS
NYKEDEYFENAIQNLKFSQS
NYKEDEYFENIAQNLKFSQSNYKEDEYFENIIANLKFSQS
NYKEDEYFENIIQALKFSQS
NYKEDEYFENIIQNAKFSQS
NYKEDEYFENIIQNLAFSQS
NYKEDEYFENIIQNLKASQS
NYKEDEYFENIIQNLKFAQS
NYKEDEYFENIIQNLKFSAS
NYKEDEYFENIIQNLKFSQA++++++
+++
++
++++
++++
+++
+++
++++
+++
+++
+++
+++

+++, OD655 S0.5; ++, 0.3 &OD655 < 0.5; +, 0.1 &OD655 <0.3; -, OD655 < 0.1.

EPITOPE MAPPING OF ANTI-CD10 MAB

phosphate-buffered saline containing 0.05% Tween20 (PBST;Nacalai Tesque, Inc., Kyoto, Japan), wells were blocked with1% bovine serum albumin-containing PBST for 30 min at37C. The plates were incubated with 1 lg/m of MME/1870,
followed by a peroxidase-conjugated anti-mouse immunoglobulins (1:2000 diluted; Agilent Technologies Inc., SantaClara, CA). Enzymatic reactions were performed using theELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Results

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Epitope mapping of an anti-CD10 mAb using
deletion mutants

To determine the binding epitope of MME/1870, we synthesized 69 CD10 peptides with their length of 20 aminoacids (aa). The peptides are summarized in Table 1. The
results showed that MME/1870 reacted to the 501–520 aa
sequence (501-NYKEDEYFENIIQNLKFSQS-520) of CD10.
We summarized the results in Figure 1.
Epitope mapping of an anti-CD10 mAb using
point mutants

Then, we synthesized 20 alanine-substituted peptides of501–520 aa of CD10 (Table 2). MME/1870 exhibited reaction to N501A, Y502A, K503A, E504A, D505A, E506A,

17F508A, E509A, N510A, Q513A, N514A, K516A, F517A,
S518A, Q519A, S520A, and wild-type 501–520 aa, whereas
it showed no reaction to Y507A, I511A, I512A, and L515A(Fig. 2A). The results indicate that Tyr507, Ile511, Ile512,
and Leu515 are the critical binding epitope of MME/1870.
We summarized the results in Figure 2B.
Discussion

In this study, we investigated the binding epitope of MME/1870 using synthesized peptides and determined Tyr507,Ile511, Ile512, and Leu515 as minimum epitope (Fig. 2B).
We could not rule out the possibility that Glu504, Asp505,
Phe508, and Asn514 are involved in the recognition since thereactivities were reduced to a lesser extent (Fig. 2A). Peptide
scanning is a simple and useful technique for determining alinear epitope. However, the peptide structure is unfolded anddifferent from native proteins. According to the X-ray crystal
structure analysis, the epitope region forms a helical structure.(15) In a previous study, we developed two novel epitope
mapping methods, such as the RIEDL insertion for epitope
mapping (REMAP) method to insert the RIEDL tag, and Histag insertion for epitope mapping (HisMAP) method to insert
five His tags into targeted proteins.
These methods are useful for determining both linear andconformational epitopes. We have already determined theepitope of anti-EGFR mAbs (EMab-51 and EMab-134),(16,17)
an anti-CD44 mAb (C44Mab-46),(18) and an anti-CD20 mAb

FIG. 1. Determination of the anti-CD10 mAb (clone MME/1870) epitope by ELISA using deletion mutants. Schematic
illustration of CD10 and synthesized peptides. Synthesized peptides of CD10 were immobilized on immunoplates. The
plates were incubated with anti-CD10 mAb (1 mg/mL), followed by incubation with peroxidase-conjugated anti-mouse
immunoglobulins. The peptides sequence and the result of ELISA are summarized in Table 1. A black bar (501–520 aa)
indicates the anti-CD10 mAb epitope. aa, amino acids; ELISA, enzyme-linked immunosorbent assay; IC, intracellulardomain; mAb, monoclonal antibody; TM, transmembrane domain.

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KAWABATA ET AL.

FIG. 2. Determination of the anti-CD10 mAb (clone MME/1870) epitope by ELISA using point mutants. (A) Synthesizedpeptides of CD10 were immobilized on immunoplates. The plates were incubated with anti-CD10 mAb (1 mg/mL), followedby peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of CD10 and the anti-CD10 mAbepitope. The anti-CD10 mAb epitope of CD10 involves Tyr507, Ile511, Ile512, and Leu515.
(C20Mab-60).(19) Application of these strategies will be important for further understanding of the epitope of MME/1870.
Furthermore, this epitope is not conserved among the family
member of mammalian neutral endopeptidases, including
endothelin-converting enzymes (ECE1/2), Kell metalloendopeptidase, and phosphate-regulating endopeptidase homolog
X-linked. Therefore, immunization of the epitope is thought tobe important for the further development of mAbs.
CD10 belongs to the M13 family of mammalian neutral
endopeptidases and has an N-terminal cytoplasmic domain,
followed by a single transmembrane domain and C-terminal
extracellular domain that contains the active site.(6,10,11) The
X-ray crystal structure of the extracellular domain (52–749aa) of CD10 was solved with a metalloproteinase inhibitor
phosphoramidon.(15) The structure reveals two consensus
sequences HExxH and ExxxD, multiplying connected folding domains that embrace a large central cavity containing
the active site. The inhibitor is bound to this cavity, which
provides a detailed ligand recognition. Furthermore, 543NAFY-546, closely localized to the identified epitope, forms ab-strand, which also participates in ligand recognition. Thereis no information on whether MME/1870 affects the enzymatic activity of CD10. Further studies are warranted for theinhibitory effect of MME/1870 on CD10-mediated ligand
recognition and endopeptidase activity.
Recently, the functional anti-CD10 antibody (JMAM-1) has
been reported.(20) JMAM-1 has a cytostatic effect on mesothelioma MSTO-211H cells in vitro and exhibits prolonged survivalof NCI-H226 tumor-bearing mice. These results expect the useof anti-CD10 antibody for pathological diagnosis and tumor
therapy. However, the detailed mechanism of the antitumor effect has not been investigated. Therefore, in cancer treatment, thefunctional elucidation of anti-CD10 mAbs, including JMAM-1and MME/1870, is required for future application.
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) under grant nos.

EPITOPE MAPPING OF ANTI-CD10 MAB

JP21am0401013 (to Y.K.) and JP21am0101078 (to Y.K.), andby the Japan Society for the Promotion of Science ( JSPS)
Grants-in-Aid for Scientific Research (KAKENHI) grant nos.
21K07168 (to M.K.K.) and 19K07705 (to Y.K.).

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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 17, 2021
Accepted: January 11, 2021