MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0044
Open camera or QR reader andscan code to access this article
and other resources online.

Epitope Mapping of Rituximab Using HisMAP Method

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

Teizo Asano,1 Hiroyuki Suzuki,2 Mika K. Kaneko,1 and Yukinari Kato1,2

CD20 is expressed in the B lymphocyte, and an effective target for the detection and treatment of B cell
lymphomas. Therefore, CD20 has been studied as a therapeutic target of B cell lymphomas and autoimmune disorders. Specific anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab,
veltuzumab, and ocaratuzumab, have been developed. Revealing the recognition mechanism of antigen by
mAbs could contribute to understanding the function of mAbs and could be useful for the development ofvaccine. Rituximab is a mouse-human chimeric anti-CD20 mAb, which was developed and approved forthe treatment of the B cell malignancies. Hence, the binding epitope of rituximab for CD20 has beenstudied. Some reports show that 170-ANPS-173, especially Ala170 and Pro172 of CD20 are important for
rituximab binding. However, only phage display results showed that 182-YCYSI-186 of CD20 is also
important for rituximab binding to CD20. In this study, we tried to determine the binding epitope ofrituximab for CD20 using histidine-tag insertion for epitope mapping (HisMAP) method. The results
showed that two regions of CD20 (169-PANPSE-174 and 183-CYSIQ-187) are important for rituximabbinding for CD20.
Keywords: CD20, rituximab, epitope mapping, monoclonal antibody, His tag

lymphoma and B cell chronic lymphocytic leukemia.(13) The
binding of rituximab to CD20 causes cell destruction throughapoptosis pathway, antibody-dependent cellular cytotoxicity(ADCC), and complement-dependent cytotoxicity (CDC).(14,15)
The epitope determination could contribute to understanding function of mAbs and development of vaccine.
Therefore, the binding region of anti-CD20 mAbs, including
rituximab on CD20, has been studied using such as crystal
structure, phage display, and peptide screening.(16–23) CD20
has a small and a large extracellular loop. The small loop is
between 74th and 80th amino acids of CD20, and the large loop
is between 141st and 187th amino acids of CD20. Many antiCD20 mAbs, including rituximab, bind to the large extracellular loop.(16) Some studies showed that 170-ANPS-173, especially Ala170 and Pro172 of CD20 binds to rituximab.(18,24)
Moreover, phage display results showed that rituximab binds
to a discontinuous region, 170-ANPS-173 and 182-YCYSI-186, of
CD20.(25) However, only phage display results showed theimportance of 182-YCYSI-186 for rituximab binding to CD20,
and other studies have not indicated 182-YCYSI-186 as an
epitope. Therefore, we tried to determine the epitope region ofIntroduction

C

D20 is a transmembrane protein, which has four transmembrane domains and two extracellular loops.(1–3)
CD20 functions as a calcium-permeable cation channel in thecell membrane,(4) and is related to B cell activation, differentiation, and regulation of calcium influx.(5,6) CD20 was first
identified as a human B lymphocyte-specific antigen,(7) and isexpressed on B cells from pre-B to mature B cell, and is also
detected in many types of non-Hodgkin’s lymphoma(8,9);
therefore, CD20 is an effective target for the detection andtreatment of B cell lymphomas, and has been studied as atherapeutic target of B cell lymphomas and autoimmune
disorders.(10–12)
Specific and sensitive monoclonal antibodies (mAbs) are
critical for the diagnosis of many types of cancer. For thisreason, many anti-CD20 mAbs such as rituximab, ofatumumab, veltuzumab, and ocaratuzumab have been developed.
Rituximab is a mouse/human chimeric mAb, and is first Foodand Drug Administration approved mAb, and used for thetreatment of lymphomas, including B cell non-Hodgkin’s

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

8

EPITOPE MAPPING OF RITUXIMAB

rituximab for CD20 using a novel epitope mapping method. We
previously developed a RIEDL insertion for epitope mapping(REMAP) method, which is a simple and an efficient method
for linear and conformational epitopes.(26–28) In this study, weused a histidine tag (His-tag) insertion for epitope mapping(HisMAP) method to determine the binding region of CD20 for
rituximab.
Materials and Methods

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

Plasmid preparation

DNA encoding the CD20 gene (IRAL012D02) was provided by RIKEN BRC through the National BioResource
Project of MEXT, Japan.(29,30) The open reading frame ofCD20 was subcloned into a pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan). Insertionsof a 5xHis-tag (5xH*) in the extracellular region of CD20
were performed using the HotStar HiFidelity Polymerase Kit
with oligonucleotides containing 5xH* insertions at the selected position. Ile141_5xH*_Lys142 (I141_5xH*_K142)
was produced for instance by inserting the 5-histidine sequence between Ile141 and Lys142 of CD20. Polymerase
chain reaction fragments bearing the desired mutations wereinserted into the pCAG-Ble vector using the In-Fusion HD
Cloning Kit (Takara Bio, Inc., Shiga, Japan).
The 5xH* insertion mutants produced were the following:
I141_5xH*_K142, K142_5xH*_I143, I143_5xH*_S144,
S144_5xH*_H145, H145_5xH*_F146, F146_5xH*_L147,
L147_5xH*_K148, K148_5xH*_M149, M149_5xH*_E150,
E150_5xH*_S151, S151_5xH*_L152, L152_5xH*_N153,
N153_5xH*_F154, F154_5xH*_I155, I155_5xH*_R156,
R156_5xH*_A157, A157_5xH*_H158, H158_5xH*_T159,
T159_5xH*_P160, P160_5xH*_Y161, Y161_5xH*_I162,
I162_5xH*_N163, N163_5xH*_I164, I164_5xH*_Y165,
Y165_5xH*_N166, N166_5xH*_C167, C167_5xH*_E168,
E168_5xH*_P169, P169_5xH*_A170, A170_5xH*_N171,
N171_5xH*_P172, P172_5xH*_S173, S173_5xH*_E174,
E174_5xH*_K175, K175_5xH*_N176, N176_5xH*_S177,
S177_5xH*_P178, P178_5xH*_S179, S179_5xH*_T180,
T180_5xH*_Q181, Q181_5xH*_Y182, Y182_5xH*_C183,
C183_5xH*_Y184, Y184_5xH*_S185, S185_5xH*_I186,
and I186_5xH*_Q187.

9Flow cytometry

Cells were harvested after brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin in
phosphate-buffered saline, cells were treated with primarymAbs (1 lg/mL) for 30 minutes at 4C and subsequently with
Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA) or FITCconjugated anti-human IgG (1:1000; Sigma-Aldrich Corp.
St. Louis, MO). Fluorescence data were collected using an
SA3800 (Sony Biotechnology Corp., Tokyo, Japan) for
HisMAP analyses.
Results
Production of 5xHis-tag-inserted CD20 proteinsIn the HisMAP method, we utilized a 5xH* and an antiHis-tag mAb (HisMab-1).(31) 5xH* was inserted between two
neighboring amino acids at the extracellular region of CD20.
Rituximab can bind to CD20 when 5xH* is inserted in any
region independent of the rituximab epitope (Fig. 1A). In
contrast, rituximab lost its reactivity to CD20 when 5xH* isinserted into the rituximab epitope region while the conformation of the rituximab epitope is disrupted by 5xH* insertion (Fig. 1B).
In this study, we employed the HisMAP method toinvestigate the epitope of rituximab for CD20. We

Cell lines

CHO/CD20 was produced in our previous study.(29,30)
CD20 mutation plasmids were transfected into CHO-K1 cells
using Lipofectamine LTX with Plus Reagent (Thermo Fisher
Scientific, Inc., Waltham, MA). CHO-K1 cells and transfectants were cultured in RPMI 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,Inc.), 100 U/mL of penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in
a humidified atmosphere containing 5% CO2. The transfectants were cultivated in a medium containing 0.5 mg/mL
Zeocin (InvivoGen, San Diego, CA).
Antibodies

C20Mab-11 was developed as previously described.(30)
Rituximab was purchased from R&D Systems (Minneapolis,
MN). HisMab-1 was developed as previously described.(31)

FIG. 1. Schematic illustration of HisMAP method.
(A) The 5-histidine tag is inserted into CD20. Rituximab canbind to CD20 when 5xH* is inserted into any region, whichis independent of the rituximab epitope. (B) Rituximab will
not bind to CD20 when the conformation of the rituximab
epitope is disrupted by 5xH* tag insertion. HisMAP,
histidine-tag insertion for epitope mapping.

10

Determination of the rituximab-binding region using
HisMAP method

Mutant protein expressing CHO-K1 cells were treated with
HisMab-1 (Fig. 3A), anti-CD20 mAb (C20Mab-11, Fig. 3B),or rituximab (Fig. 4), and were analyzed using flow

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

constructed 46 mutant plasmids of CD20, in which
5xH* was inserted into the second extracellular loop
region of CD20 (from Ile141 to Gln187) (Fig. 2). Mutant plasmids were transfected into CHO-K1 cells, andmutant proteins were transiently expressed on CHO-K1
cells.

ASANO ET AL.

FIG. 2. Schematic illustration of histidine tag insertion mutants of CD20. The 5xH* was inserted into the large extracellular loop of CD20, and 46 mutants were constructed.

11

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

EPITOPE MAPPING OF RITUXIMAB

FIG. 3. Confirmation of mutant expression on CHO-K1 cells. Each 5xH* inserted mutant was expressed on CHO-K1 cells
and incubated with HisMab-1 (A) or C20Mab-11 (B) for 30 minutes at 4C, followed by treatment with secondary antibody.
Red lines: treated with HisMab-1 or C20Mab-11, black lines: without first antibody as negative control.
cytometry. Results of the flow cytometry analysis showedthat HisMab-1 used as a positive control detected 41 mutants.
Although HisMab-1 did not detect five mutants(I141_5xH*_K142, C183_5xH*_Y184, Y184_5xH*_S185,
S185_5xH*_I186, and I186_5xH*_Q187) (Fig. 3A), anti-

CD20 mAb (C20Mab-11) detected these five mutants(Fig. 3B), indicating that all CD20 mutant proteins wereexpressed on CHO-K1 cells. Since 5xH* was not inserted inwild-type CD20, HisMab-1 did not react with CHO/CD20(Fig. 3A).

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

12

ASANO ET AL.

FIG. 4. Epitope mapping of rituximab using histidine tag insertion mutants of CD20. Each 5xH* inserted mutant was
expressed on CHO-K1 cells and incubated with rituximab for 30 minutes at 4C, followed by treatment with secondary
antibody. Red lines: treated with rituximab, black lines: without first antibody as negative control.
In contrast, rituximab did not detect the nine mutants(P169_5xH*_A170, A170_5xH*_N171, N171_5xH*_P172,
P172_5xH*_S173, S173_5xH*_E174, C183_5xH*_Y184,
Y184_5xH*_S185, S185_5xH*_I186, and I186_5xH*_Q187),
and weakly detected one mutant (E174_5xH*_K175) (Fig. 4).

FIG. 5. Schematic illustration of epitope mapping of rituximab. Two regions (169-PANPSE-174 and 183-CYSIQ-187)
are important for the binding of rituximab to CD20.

These results indicate that two peptides (169-PANPSE-174, and
183-CYSIQ-187) are important for binding rituximab to CD20.
These results are summarized in Figure 5.
Discussion

The binding of rituximab to CD20 causes cell destruction
through apoptosis pathway, ADCC, and CDC. To understandthe molecular mechanism of recognition of CD20 by rituximab, the binding epitope of rituximab has been studied.
Polyak et al. conducted flow cytometric analysis using point
mutation of CD20.(32) Mutation of Ala170 and Pro172 inhuman CD20 abrogated the binding of rituximab. Although
rituximab did not recognize murine CD20, introduction of
Ala170 and Pro172 into the equivalent positions in murine
CD20 reconstituted the epitope recognized by rituximab.
These results indicated that Ala170 and Pro172 are essential
for rituximab binding to CD20. Teeling et al. also reported
that amino acids from Tyr165 to Tyr182 of CD20 contributeto binding of rituximab, especially Ala170 and Pro172 are
absolutely essential.(18)
Binder et al. demonstrated the epitope analysis of rituximab using phage display, and results showed that rituximab
binds to a discontinuous region in CD20: 170-ANPS-173 and(25)
Moreover, the crystal structure
182-YCYSI-186 of CD20.
of the rituximab Fab in complex with a synthesized peptide
were solved by Du et al.(24) The 170-ANPS-173 of CD20 isDownloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

EPITOPE MAPPING OF RITUXIMAB

embedded into the pocket on the surface of rituximab, andplays an essential role in the binding of rituximab to CD20.
These studies showed that 170-ANPS-173 of CD20 is important for rituximab binding. Especially, Ala170 and Pro172 ofCD20 are critical epitope of rituximab. In this study, we
showed that 169-PANPSE-174 and 183-CYSIQ-187 are important for binding rituximab to CD20 using HisMAP method.
The result that 169-PANPSE-174 peptide is important for rituximab binding is consistent with previous studies eventhough Pro169 and Glu174 are added.
In HisMAP method, two mutants, P169_5xH*_A170 and
S173_5xH*_E174, lost their reactivity to rituximab (Fig. 4);
therefore, we judged that Pro169 and Glu174 of CD20 are also
important for rituximab binding. Crystal structure analysis
showed that a hydrogen bonding is formed between Pro169 of
CD20 and Ser50 of rituximab heavy chain. In addition to hydrogen bonding interaction, Pro169 of CD20 makes van der
Waals contacts to Asn93 of rituximab light chain and Ser59 ofrituximab heavy chain.(24) Glu174 of CD20 also makes van der
Waals contacts to Tyr102 and Trp106 of rituximab heavy
chain. Therefore, Pro169 and Glu174 also contribute to theinteraction of CD20 with rituximab.
Moreover, our results indicated that 183-CYSIQ-187 arealso important for rituximab binding (Figs. 4 and 5). Binder
et al. showed almost the same region as a rituximab binding
motif.(25) However, crystal structure analysis showed thisregion is not binding to rituximab.(24) The large extracellularloop of CD20 has a disulfide bond between Cys167 andCys183. Reduction of CD20 caused abrogation of rituximab
binding to CD20.(3) This result indicated that Cys167-Cys183
disulfide bond is essential for rituximab binding to CD20, andCD20 recognition by rituximab is critically dependent on theconformation of CD20. Two epitope regions, 169-PANPSE-174,
and 183-CYSIQ-187, are located on adjacent site because
Cys167 and Cys183 make the disulfide bond. Insertionof 5xH* into 183-CYSIQ-187 region may affect the structure of
169-PANPSE-174 region, or may cause the disruption of
Cys167-Cys183 disulfide bond resulting in inhibition of therituximab binding.
In HisMAP method, His-tag system consists of His-tag and
HisMab-1 can be used as control for expression of mutant
proteins; therefore, HisMAP method is applicable to theepitope mapping for proteins for which there are not specificantibodies. However, five mutants (I141_5xH*_K142,
C183_5xH*_Y184, Y184_5xH*_S185, S185_5xH*_I186,
and I186_5xH*_Q187) did not react with HisMab-1. The
inserted His-tag in I141_5xH*_K142 and I186_5xH*_Q187
is located on juxtamembrane; therefore, HisMab-1 may notaccess to the His-tag. Moreover, full-length protein expressedon membrane is used in HisMAP method, whereas a partial
sequence of protein in solution is usually used in X-ray
crystallography and peptide scanning; hence, HisMAP
method can detect antigen–antibody interaction, which is
close to native state. In this report, we showed that HisMAP
method is useful for epitope mapping of rituximab; however, further studies are required for the investigation of
structure analysis of interaction between HisMab-1 andinserted His-tag.
Author Disclosure Statement

No competing financial interests exist.

13
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under grant nos.
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.
21K15523 (to T.A.), 21K07168 (to M.K.K.), and 19K07705(to Y.K.).
References

1. Tedder TF, Streuli M, Schlossman SF, and Saito H: Isolation and structure of a cDNA encoding the B1 (CD20)
cell-surface antigen of human B lymphocytes. Proc Natl
Acad Sci U S A 1988;85:208–212.
2. Einfeld DA, Brown JP, Valentine MA, Clark EA, andLedbetter JA: Molecular cloning of the human B cell CD20
receptor predicts a hydrophobic protein with multiple
transmembrane domains. EMBO J 1988;7:711–717.
3. Ernst JA, Li H, Kim HS, Nakamura GR, Yansura DG, andVandlen RL: Isolation and characterization of the B-cellmarker CD20. Biochemistry 2005;44:15150–15158.
4. Bubien JK, Zhou LJ, Bell PD, Frizzell RA, and Tedder TF:
Transfection of the CD20 cell surface molecule into ectopic
cell types generates a Ca2+ conductance found constitutively in B lymphocytes. J Cell Biol 1993;121:1121–1132.
5. Kuijpers TW, Bende RJ, Baars PA, Grummels A, Derks IA,
Dolman KM, Beaumont T, Tedder TF, van Noesel CJ,
Eldering E, and van Lier RA: CD20 deficiency in humans
results in impaired T cell-independent antibody responses.
J Clin Invest 2010;120:214–222.
6. Tedder TF, and Engel P: CD20: A regulator of cell-cycle
progression of B lymphocytes. Immunol Today 1994;15:
450–454.
7. Stashenko P, Nadler LM, Hardy R, and Schlossman SF:
Characterization of a human B lymphocyte-specific antigen. J Immunol 1980;125:1678–1685.
8. Polyak MJ, Li H, Shariat N, and Deans JP: CD20 homooligomers physically associate with the B cell antigen receptor. Dissociation upon receptor engagement and recruitmentof phosphoproteins and calmodulin-binding proteins. J Biol
Chem 2008;283:18545–18552.
9. Li H, Ayer LM, Lytton J, and Deans JP: Store-operated
cation entry mediated by CD20 in membrane rafts. J Biol
Chem 2003;278:42427–42434.
10. Press OW, Appelbaum F, Ledbetter JA, Martin PJ, Zarling
J, Kidd P, and Thomas ED: Monoclonal antibody 1F5 (antiCD20) serotherapy of human B cell lymphomas. Blood
1987;69:584–591.
11. Smith MR: Rituximab (monoclonal anti-CD20 antibody):
Mechanisms of action and resistance. Oncogene 2003;22:7359–7368.
12. Pescovitz MD: Rituximab, an anti-cd20 monoclonal antibody: History and mechanism of action. Am J Transplant
2006;6:859–866.
13. Keating GM: Rituximab: A review of its use in chronic lymphocytic leukaemia, low-grade or follicular lymphoma anddiffuse large B-cell lymphoma. Drugs 2010;70:1445–1476.
14. Glennie MJ, French RR, Cragg MS, and Taylor RP: Mechanisms of killing by anti-CD20 monoclonal antibodies.
Mol Immunol 2007;44:3823–3837.
15. Lim SH, Beers SA, French RR, Johnson PW, Glennie MJ, andCragg MS: Anti-CD20 monoclonal antibodies: Historical andfuture perspectives. Haematologica 2010;95:135–143.

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

14

16. Klein C, Lammens A, Schäfer W, Georges G, Schwaiger M,
Mössner E, Hopfner KP, Umaña P, and Niederfellner G: Epitope interactions of monoclonal antibodies targeting CD20 andtheir relationship to functional properties. MAbs 2013;5:22–33.
17. Niederfellner G, Lammens A, Mundigl O, Georges GJ,
Schaefer W, Schwaiger M, Franke A, Wiechmann K,
Jenewein S, Slootstra JW, Timmerman P, Brännström A,
Lindstrom F, Mössner E, Umana P, Hopfner KP, and Klein
C: Epitope characterization and crystal structure of GA101
provide insights into the molecular basis for type I/II distinction of CD20 antibodies. Blood 2011;118:358–367.
18. Teeling JL, Mackus WJ, Wiegman LJ, van den Brakel JH,
Beers SA, French RR, van Meerten T, Ebeling S, Vink T,
Slootstra JW, Parren PW, Glennie MJ, van and de Winkel
JG: The biological activity of human CD20 monoclonal
antibodies is linked to unique epitopes on CD20. J Immunol
2006;177:362–371.
19. Du J, Wang H, Zhong C, Peng B, Zhang M, Li B, Hou S,
Guo Y, and Ding J: Crystal structure of chimeric antibody
C2H7 Fab in complex with a CD20 peptide. Mol Immunol
2008;45:2861–2868.
20. Gordon LI, Molina A, Witzig T, Emmanouilides C, Raubtischek A, Darif M, Schilder RJ, Wiseman G, and White CA:
Durable responses after ibritumomab tiuxetan radioimmunotherapy for CD20+ B-cell lymphoma: Long-term
follow-up of a phase 1/2 study. Blood 2004;103:4429–4431.
21. Uchiyama S, Suzuki Y, Otake K, Yokoyama M, Ohta M,
Aikawa S, Komatsu M, Sawada T, Kagami Y, MorishimaY, and Fukui K: Development of novel humanized antiCD20 antibodies based on affinity constant and epitope.
Cancer Sci 2010;101:201–209.
22. Stein R, Qu Z, Chen S, Rosario A, Shi V, Hayes M, Horak
ID, Hansen HJ, and Goldenberg DM: Characterization of a
new humanized anti-CD20 monoclonal antibody, IMMU106, and Its use in combination with the humanized antiCD22 antibody, epratuzumab, for the therapy of nonHodgkin’s lymphoma. Clin Cancer Res 2004;10:2868–2878.
23. Forero-Torres A, de Vos S, Pohlman BL, Pashkevich M,
Cronier DM, Dang NH, Carpenter SP, Allan BW, Nelson
JG, Slapak CA, Smith MR, Link BK, Wooldridge JE, andGanjoo KN: Results of a phase 1 study of AME-133v(LY2469298), an Fc-engineered humanized monoclonalanti-CD20 antibody, in FcgRIIIa-genotyped patients withpreviously treated follicular lymphoma. Clin Cancer Res
2012;18:1395–1403.
24. Du J, Wang H, Zhong C, Peng B, Zhang M, Li B, Huo S,
Guo Y, and Ding J: Structural basis for recognition of

ASANO ET AL.

CD20 by therapeutic antibody Rituximab. J Biol Chem
2007;282:15073–15080.
25. Binder M, Otto F, Mertelsmann R, Veelken H, and Trepel
M: The epitope recognized by rituximab. Blood 2006;108:
1975–1978.
26. Asano T, Kaneko MK, and Kato Y: RIEDL tag: A novel
pentapeptide tagging system for transmembrane protein
purification. Biochem Biophys Rep 2020;23:100780.
27. Asano T, Kaneko MK, Takei J, Tateyama N, and Kato Y:
Epitope mapping of the anti-CD44 monoclonal antibody(C44Mab-46) using the REMAP method. Monoclon AntibImmunodiagn Immunother 2021;40:156–161.
28. Sano M, Kaneko MK, Aasano T, and Kato Y: Epitope
mapping of an antihuman EGFR monoclonal antibody(EMab-134) using the REMAP method. Monoclon AntibImmunodiagn Immunother 2021;40:191–195.
29. Furusawa Y, Kaneko MK, and Kato Y: Establishment of ananti-CD20 monoclonal antibody (C(20)Mab-60) for immunohistochemical analyses. Monoclon Antib Immunodiagn Immunother 2020;39:112–116.
30. Furusawa Y, Kaneko MK, and Kato Y: Establishment ofC(20)Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967.
31. Asano T, Takei J, Furusawa Y, Saito M, Suzuki H, Kaneko
MK, and Kato Y: Epitope mapping of an anti-CD20 monoclonal
antibody (C20Mab-60) using the HisMAP method. Monoclon
Antib Immunodiagn Immunother 2021;40:243–249.
32. Polyak MJ, and Deans JP: Alanine-170 and proline-172 are
critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed byboth amino acid sequence and quaternary structure. Blood
2002;99:3256–3262.

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: September 14, 2021
Accepted: January 11, 2022