MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 2, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0008

Short Communication

Development of Antihuman Killer Cell Lectin-Like
Receptor Subfamily G Member 1 Monoclonal
Antibodies for Flow Cytometry

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Teizo Asano,1 Ren Nanamiya,1 Tomohiro Tanaka,1 Mika K. Kaneko,1 and Yukinari Kato1,2

Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, was identified
as an inhibitory receptor expressed on natural killer (NK) cells and certain T cells. The protein regulates effector
functions and developmental processes in these cells. In this study, we established a specific and sensitive
monoclonal antibody (mAb) for human KLRG1 (hKLRG1), which is useful for flow cytometry, using a CellBased Immunization and Screening (CBIS) method. The established anti-hKLRG1 mAb, KLMab-1 (mouse
IgG1, kappa), reacted with overexpressed hKLRG1 in Chinese hamster ovary-K1 (CHO/hKLRG1) and humanNK cells, which also expressed endogenous hKLRG1 as confirmed by flow cytometry. KLMab-1, which wasestablished by the CBIS method, could be useful for elucidating the hKLRG1-related biological response byflow cytometry.
Keywords: KLRG1, monoclonal antibody, CBIS, flow cytometry

to KLRG1 and are putative therapeutic targets for immune
checkpoints.(17–20) Anti-PD-1 monoclonal antibodies (mAbs)
and anti-CTLA-4 mAbs are reported as potential anticancer
drugs.(21,22) By contrast, mAbs against KLRG1 have not beendeveloped as potential drugs for cancer therapies.
In this study, we developed a novel anti-KLRG1 mAb using
a Cell-Based Immunization and Screening (CBIS) method(23,24) and investigated its application in flow cytometry.

Introduction

N

atural killer (NK) cells generate rapid responses tovirus-infected cells, without prior activation of the innate immunity.(1) NK cells are characterized by the expression of CD16 and CD56, and the lack of CD3. The proportionof NK cells in peripheral blood lymphocytes is *7%.(2)Several activating and inhibitory receptors are also expressedon NK cell surfaces.(3,4)
Killer cell lectin-like receptor subfamily G member 1(KLRG1) is a type II transmembrane protein and is an inhibitory receptor expressed on NK and some T cells.(5–7) The
protein is also expressed on group 2 innate lymphoid cells inmice and humans.(8,9) It is reported that the percentage of
KLRG1-expressing NK cells is increased by viral infection.(6)IL-2 receptor signaling is essential for the development of
KLRG1+ terminally differentiated T regulatory cells.(10)KLRG1 also binds to E-, N-, and R-cadherin. Of these proteins, E-cadherin is known to inhibit T cell proliferation, NK
cell cytotoxicity, and NK or T cell activation by binding to
KLRG1; therefore, KLRG1 regulates effector functions anddevelopmental processes in NK or T cells.(11–15) Moreover, theinteractive inhibition between E-cadherin and KLRG1 prevents metastasis in the 4T1 metastatic breast cancer model.(16)Programed cell death 1 (PD-1) and cytotoxic T lymphocyteassociated protein 4 (CTLA-4) are inhibitory receptors similar
1
2

Materials and Methods
Cell lines

Chinese hamster ovary-K1 (CHO-K1) and P3X63Ag8U.1(P3U1) were obtained from the American Type Culture
Collection (Manassas, VA). For flow cytometry, NK cells(donor lot 4022602 NK1602) were purchased from Takara
Bio, Inc. (Shiga, Japan). Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding human KLRG1 (hKLRG1;
accession no: NP_001316028.1) plus N-terminal PA tag(25)was subcloned into a pCAG-Ble vector (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan). The hKLRG1
plasmid was transfected into CHO-K1 cells using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific Inc.,
Waltham, MA). Stable transfectants were identified using acell sorter (SH800; Sony Corp., Tokyo, Japan) and cultivatedDepartment 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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ANTIHUMAN KLRG1

in a medium containing 0.5 mg/mL zeocin (InvivoGen,
San Diego, CA) to generate CHO/PA-hKLRG1 cells.
CHO-K1, P3U1, and CHO/PA-hKLRG1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin,
and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). Cells
were grown in a humidified incubator at 37C with 5% CO2.

77
Results
Establishment of anti-hKLRG1 mAbs

CBIS method was used to establish anti-hKLRG1 mAbs
(Fig. 1). First, two mice were immunized with CHO/PAhKLRG1 cells, and hybridomas were produced by fusing

Antibodies

An anti-hKLRG1 mAb (clone 14C2A07; mouse IgG2a,kappa) was purchased from BioLegend (San Diego, CA). Secondary Alexa Fluor 488-conjugated anti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).

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Hybridoma production

Female BALB/c mice (6 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. Animal studies were performed according to the relevant guidelines and regulations
to minimize animal suffering and distress. Studies were approved by the Animal Care and Use Committee of Tohoku
University (permit: 2019NiA-001). Mice were monitored
daily during the 4-week study. A reduction of >25% of total
body weight was defined as a humane endpoint. Mice wereeuthanized by cervical dislocation, and death was verified byrespiratory and cardiac arrest.
We used CBIS method to develop mAbs against
hKLRG1.(24) In brief, two BALB/c mice were intraperitoneallyimmunized with CHO/PA-hKLRG1 cells (1 · 108) together
with Imject Alum (Thermo Fisher Scientific Inc.). The procedure required three additional immunizations followed by afinal booster intraperitoneal injection administered 2 days before spleen cells were harvested. Once harvested, cells werefused with P3U1 cells using polyethylene glycol 1500 (Roche
Diagnostics, Indianapolis, IN). For selection, resultant hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine (Thermo Fisher ScientificInc.). Culture supernatants were screened by flow cytometry.
Flow cytometry

Cells were harvested after a brief exposure to 0.25%
trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). They were washed in 0.1% bovine serum albumin in phosphate-buffered saline, treated with primarymAbs for 30 minutes at 4C, and then treated with Alexa
Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc.). Fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity by flow cytometry

CHO/PA-hKLRG1 cells were suspended in 100 lL seriallydiluted anti-hKLRG1 mAbs, and Alexa Fluor 488-conjugatedanti-mouse IgG was added (1:200; Cell Signaling Technology,Inc.). Fluorescence data were collected using the BD FACSLyric instrument (BD Biosciences, Franklin Lakes, NJ). The
dissociation constant (KD) was calculated by fitting binding
isotherms to built-in one-site binding models in GraphPad
PRISM 8 (GraphPad Software, Inc., La Jolla, CA).

FIG. 1. Production of anti-hKLRG1 mAbs. Schematic
representation of CBIS. (1) CHO/PA-hKLRG1 cells were
immunized into BALB/c mice by intraperitoneal injection.
(2) Splenocytes were fused with myelomas for production of
hybridomas. (3) Screening of anti-hKLRG1 mAb producing
hybridomas was performed by flow cytometry using CHO/hKLRG1 and CHO-K1 cells. (4) Anti-hKLRG1 mAbproducing hybridomas were cloned using the limiting dilution
method. CBIS, Cell-Based Immunization and Screening;
CHO, Chinese hamster ovary-K1; hKLRG1, human KLRG1;
KLRG1, Killer cell lectin-like receptor subfamily G member
1; mAbs, monoclonal antibodies.

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Flow cytometric analyses

KLMab-1 recognized CHO/PA-hKLRG1 but not CHO-K1
cells (Fig. 2A). Also, KLMab-1 reacted with endogenous
KLRG1 in NK cells (Fig. 2A). A 0.01 lg/mL concentration
was adequate for KLMab-1 recognition of CHO/PA-hKLRG1,
but the KLMab-1 recognition of NK cells was weak (Fig. 2A).
As a positive control, clone 14C2A07 (BioLegend) reacted
similarly with all cell types (Fig. 2B).

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splenocytes with P3U1 myeloma cells. Next, we selectedhybridomas whose supernatants were positive for CHO/PAhKLRG1 but negative for CHO-K1 cells, using flow cytometry. The first screening identified strong signals from
CHO/PA-hKLRG1 cells and weak or no signals from CHOK1 cells in 22 of 960 wells (2.3%). After limiting dilutions
and several additional screenings, KLMab-1 (IgG1, kappa),
which reacted most strongly with CHO/PA-hKLRG1, wasestablished.

ASANO ET AL.

FIG. 2. Flow cytometry using anti-hKLRG1 mAbs. (A) CHO-K1, CHO/PA-hKLRG1, and NK cells were treated with
0.01–1 mg/mL KLMab-1, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; negative control (black).
(B) CHO-K1, CHO/PA-hKLRG1, and NK cells were treated with 0.01–1 mg/mL of clone 14C2A07, followed by treatment
with Alexa Fluor 488-conjugated anti-mouse IgG; negative control (black). (C) Determination of KLMab-1 binding affinity.
CHO/PA-hKLRG1 cells were suspended in 100 mL of serially diluted KLMab-1 (0.006–100 mg/mL). Alexa Fluor 488conjugated anti-mouse IgG was then added. Fluorescence data were collected using the BD FACSLyric instrument. NK,
natural killer.

ANTIHUMAN KLRG1
Determination of binding affinity using KLMab-1
in hKLRG1-expressing cells

We conducted a kinetic analysis of the interaction between KLMab-1 and CHO/PA-hKLRG1 cells by flow cytometry to assess the binding affinity of KLMab-1 with
CHO/PA-hKLRG1 cells. The geometric mean of fluorescence intensity was plotted against different KLMab-1
concentrations and fitted by one-site binding models. The
KD of KLMab-1 for CHO/PA-hKLRG1 cells was determined to be 2.0 · 10-8 M (Fig. 2C), suggesting that
KLMab-1 possessed a moderate binding affinity for thesecells.

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Discussion

By using CBIS method, the biological structure and modification of proteins, such as glycosylation and folding, could
be retained. In commercial products, two mAbs for flow cytometry, including clone 14C2A07 from BioLegend (Fig. 2B),
have been developed using cell-based immunization (Supplementary Table S1). It generates a more natural and functional antigen than do synthetic peptides as immunogens.
Furthermore, it is easier to acquire mAbs against multipass
transmembrane proteins, because purified proteins are notrequired as immunogens and for screening purposes. Previously, we developed mAbs against several membrane proteins, such as CD20,(26) CD44,(27) CD133,(24) EpCAM,(28) and
TROP2,(29) using CBIS method. In this study, we successfully
developed a sensitive anti-hKLRG1 mAb, KLMab-1. KLMab-1
recognized overexpressed and endogenous hKLRG1 in our celllines (Fig. 2). Because hKLRG1 is considered the next pivotal
immune checkpoint molecule,(30) KLMab-1 and in vivo mousemodels may be used to investigate molecular mechanisms behind hKLRG1-related diseases.
Authors’ Contributions

T.A., R.N., and T.N. performed experiments; M.K.K. designed the experiments; T.A. and Y.K. wrote the article.
Author Disclosure Statement

The authors have no conflict of interest.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers: JP20am0401013 (Y.K.) and JP20am0101078 (Y.K.).
Supplementary Material

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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: yukinarikato@med.tohoku.ac.jp
Received: February 15, 2021
Accepted: February 26, 2021