MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 3, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0013
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TgMab-2: An Anti-human T Cell Immunoglobulin
and Immunoreceptor Tyrosine-Based Inhibitory Motif
Domain Monoclonal Antibody for Immunocytochemistry

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Masaki Saito,1 Hiroyuki Suzuki,1 Mika K. Kaneko,2 and Yukinari Kato1,2

T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is one of theimmune checkpoint molecules. TIGIT is expressed in T or natural killer (NK) cells and is upregulated in several
cancers. Because TIGIT suppresses the antitumor activity of the T or NK cells by binding to its ligand, such as
CD155, CD112, and CD113, TIGIT can be a molecular marker or a therapeutic target for cancer immunotherapy. We previously developed an anti-human TIGIT (hTIGIT) monoclonal antibody (mAb; clone TgMab-2;
mouse IgG1, kappa) by the Cell-Based Immunization and Screening method. TgMab-2 binds to hTIGIT withhigh binding affinity in flow cytometry. In this study, we investigated the availability of TgMab-2 and its
recombinant mAb (recTgMab-2) in immunocytochemistry. We found that TgMab-2 and recTgMab-2 bind to
hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells, but not parental CHO-K1 cells, indicating thatboth mAbs specifically recognize hTIGIT. Furthermore, both mAbs recognized endogenous hTIGIT expressedin human NK cells in immunocytochemistry. These results demonstrate that TgMab-2 and recTgMab-2 areapplicable for immunocytochemistry against hTIGIT.
Keywords: TIGIT, TgMab-2, monoclonal antibody, immunocytochemistry
Introduction

D4+ T, CD8+ T, and natural killer (NK) cells suppress
tumor progression by antitumor immunity.(1) Tumor
cells, in turn, escape from the immune cell recognition by
activating immune checkpoint molecules, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) andprogrammed cell death 1 (PD-1) and by suppressing thefunctions of T or NK cells. Specific monoclonal antibodies(mAbs) against CTLA-4, PD-1, and PD-1 ligand 1 (PD-L1)
could block their function, and have provided great benefits
in improving the prognosis of cancer patients.(2) However,
due to the limited number of patients who respond to thosemAbs, the development of novel mAbs against other immune
checkpoint molecules has been desired.(3)T cell immunoglobulin and immunoreceptor tyrosinebased inhibitory motif domain (TIGIT) is another immune
checkpoint molecule, which is expressed in CD4+ T, CD8+ T,
NK, and regulatory T (Treg) cells.(4,5) TIGIT is a type 1

C

transmembrane protein, which possesses an extracellular immunoglobulin domain and an intracellular immunoreceptor
tyrosine-based inhibitory motif domain.(4) CD155 (poliovirus
receptor [PVR]), CD112 (PVR-like protein 2), and CD113(PVR-like protein 3) are identified as TIGIT ligands that are
expressed in antigen-presenting cells and cancer cells.(6)TIGIT interacts with CD155, CD112, and CD113 withhigh, moderate, and low affinities, respectively.(4,5) The binding of CD155 to TIGIT suppresses the activity of NK cells
through the phosphorylation of Y225 on the immunoreceptor
tail tyrosine-like motif of TIGIT, the recruitment of a cytosolic adaptor protein Grb2 and SH2 domain-containing
tyrosine phosphatase-1, and the subsequent termination ofphosphatidylinositol 3-kinase and mitogen-activated protein
kinase signaling.(7)Elevated expression of TIGIT and its ligands is identified
in tumor-infiltrating lymphocytes.(6) For example, TIGIT
is increased in Tregs of melanoma patients(8) and CD8+ T
cells of gastric cancer patients.(9) TIGIT is also increased in

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

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tumor-infiltrating NK cells or CD8+ T cells in mouse xenograft models of colorectal cancer, breast cancer, melanoma, andfibrosarcoma.(10) In addition, CD155 is upregulated in melanoma, colorectal, gastric, and pancreatic carcinomas,(9,11–13)
whereas CD112 is upregulated in breast, gastric, and ovariancarcinomas.(9,14)
These reports suggest that TIGIT can be a novel molecularmarker or a target molecule for cancer immunotherapy.
Moreover, despite advances in research of physiological
functions of TIGIT, the regulatory mechanisms of TIGIT
in the TIGIT-overexpressing CD8+ T and NK cells haveremained unclear. Thus, the development of novel mAbs
against TIGIT has been required.
We have developed mAbs against membrane proteinsby the Cell-Based Immunization and Screening (CBIS)
method, including C–C motif chemokine receptor 3(CCR3),(15,16) CCR8,(17–19) CCR9,(20) CD10,(21,22) CD19,(23)CD20,(24,25)
CD44,(26)CD133,(27)EpCAM,(28,29)
(30)
(15)
(31)
HER3,
KLRG1,
PD-L1,
podoplanin,(32–46) and(47,48)
TROP2.
We have also established an anti-human TIGIT(hTIGIT) mAb (clone TgMab-2; mouse IgG1, kappa).(49)
TgMab-2 reacts to hTIGIT with high binding affinity in flow
cytometry.(49) In this study, we investigated whether TgMab-2
and its recombinant mAb (recTgMab-2) could recognize endogenous and exogenous hTIGIT in immunocytochemistry.
Materials and Methods

SAITO ET AL.

10 min. Then, the cells were blocked in BPA buffer (PBSc/m
supplemented with 0.5% bovine serum albumin and 0.02%
sodium azide) for 30 min, and incubated with primary antibodies (10 lg/mL in BPA buffer) for 1 h and Alexa Fluor
488-conjugated anti-mouse IgG (1:400 dilution in BPA
buffer) for 45 min.
Finally, the cells were mounted using ProLong Glass
antifade mounting medium (Thermo Fisher Scientific,Inc.). The cell nuclei were stained with 4¢,6-diamidino-2phenylindole (DAPI; Thermo Fisher Scientific, Inc.). Fluorescence images were acquired on a BZ-X800 digital
microscope (Keyence, Osaka, Japan) with a 40 · objective.
Immunocytochemistry of suspension cells

The suspension of NK cells was centrifuged at 270 · g for
5 min. The obtained cell pellet was suspended in 4% PFA in
PBS for 10 min, followed by 50 mM NH4Cl in PBSc/m for
10 min. After centrifugation, NK cells were suspended in
BPA buffer for 30 min, primary antibodies (10 mg/mL in BPA
buffer) for 2 h, and Alexa Fluor 488-conjugated antimouse IgG (1:400 dilution BPA buffer) for 45 min. Subsequently, NK cells were suspended in ProLong Glass antifade
mounting medium and mounted on a slide glass. The
cell nuclei were stained with DAPI. Fluorescence images
were acquired on a BZ-X800 digital microscope with a 40 ·objective.

Cell lines

Chinese hamster ovary (CHO)-K1 was obtained fromthe American Type Culture Collection (Manassas, VA).
hTIGIT-overexpressed CHO-K1 (CHO/hTIGIT) was established in our previous report.(49) CHO-K1 and CHO/hTIGIT
were cultured in Roswell Park Memorial Institute 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented
with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.,
Waltham, MA), 0.25 lg/mL of amphotericin B, 100 U/mL of
penicillin, and 100 lg/mL of streptomycin (Nacalai Tesque,Inc.). The cells were maintained in a humidified atmosphere
under 5% CO2 and 95% air condition at 37C. Human NK
cells (donor lot. 4022602, purity >70%) were purchased fromTakara Bio (Shiga, Japan).
Antibodies

TgMab-2 was developed in our previous report.(49)
Recombinant TgMab-2 (recTgMab-2) was generated bysubcloning VH and CH of complementary DNAs (cDNAs) of
TgMab-2 into the pCAG-Neo vector, along with VL and CL
cDNAs of TgMab-2 into the pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan), respectively. An anti-hTIGIT mAb (clone A15153G) was purchasedfrom BioLegend (San Diego, CA). Alexa Fluor 488conjugated anti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).
Immunocytochemistry of adherent cells

CHO-K1 and CHO/hTIGIT were seeded on acid-washcoverslips. They were fixed with 4% paraformaldehyde (PFA)
contained in phosphate-buffered saline (PBS) for 10 min andquenched with 50 mM NH4Cl contained in PBS supplemented with 0.2 mM Ca2+ and 2 mM Mg2+ (PBSc/m) for

Results

We previously showed that TgMab-2 specifically recognizes CHO/hTIGIT cells in flow cytometry.(49) In this study,
we investigated the availability of TgMab-2 and recTgMab-2
in immunocytochemistry. We found TgMab-2 and recTgMab-2,
but not buffer control, reacted to CHO/hTIGIT cells(Fig. 1A). In contrast, TgMab-2 and recTgMab-2 did not bindto parental CHO-K1 cells (Fig. 1B). Another anti-hTIGIT
mAb (A15153G) also reacted to CHO/hTIGIT cells, but not
to CHO-K1 cells (Fig. 1A, B). This result demonstrates that
TgMab-2 and recTgMab-2 specifically recognize exogenous
hTIGIT in immunocytochemistry.
Next, we have applied TgMab-2 and recTgMab-2 in NK
cells to investigate whether both antibodies recognize
endogenously expressing hTIGIT. We found that TgMab-2
and recTgMab-2, as well as A15153G, showed sensitive
fluorescent signals in NK cells (Fig. 1C), indicating that
TgMab-2 and recTgMab-2 reacted to endogenous hTIGIT in
immunocytochemistry.
Discussion

This study demonstrated that TgMab-2 and recTgMab-2
specifically recognize endogenous and exogenous hTIGIT
in immunocytochemistry. These mAbs would be powerfultools for the diagnosis of hTIGIT-positive cancers throughdetection of the tumor-infiltrating NK and CD8+ T cells.
Importantly, TgMab-2 and recTgMab-2 provided highcontrast fluorescent images against both exogenously andendogenously expressing hTIGIT in CHO/hTIGIT and NK
cells, respectively. We speculate that mAbs, developed by the
CBIS method,(27) are suitable in immunocytochemistry because a mammalian cell line, which stably expresses a target

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FIG. 1. Immunocytochemistry using TgMab-2 and recTgMab-2. (A, B) CHO/hTIGIT cells (A) or CHO-K1 (B) cells weretreated with buffer control, 10 mg/mL of A15153G, 10 mg/mL of TgMab-2, or 10 mg/mL of recTgMab-2 for 1 h. The cells
were further treated with Alexa 488-conjugated anti-mouse IgG and DAPI for 45 min. (C) Immunocytochemistry of NK
cells using TgMag-2 and recTgMab-2. NK cells were treated with buffer control, 10 mg/mL of A15153G, 10 mg/mL of
TgMab-2, or 10 mg/mL of recTgMab-2 for 2 h. The cells were further treated with Alexa 488-conjugated anti-mouse IgG
and DAPI for 45 min. Scale bars; 20 mm. CHO, Chinese hamster ovary; DAPI, 4’,6-diamidino-2-phenylindole; hTIGIT,
human TIGIT; IgG, immunoglobulin G; NK, natural killer; TIGIT, T cell immunoglobulin and immunoreceptor tyrosinebased inhibitory motif domain.
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membrane protein, is used for an immunogen. Furthermore,
the conformation and post-translational modification of theimmunogen would be physiological.
In fact, we have confirmed that an anti-CCR3 mAb [clone
C3Mab-2(50)], anti-CCR8 mAbs [clone C8Mab-1,(19) C8Mab2,(51) and C8Mab-3(18)], and an anti-CCR9 mAb [clone
C9Mab-1(52)] also provided high-contrast images against
both endogenously and exogenously expressing target molecules in immunocytochemistry.
We consider that the high-contrast images by TgMab-2
and recTgMab-2 would enable us to identify the cellular
distribution of hTIGIT and support elucidate the physiological functions of hTIGIT. To further clarify the function of
TIGIT using TgMab-2 and recTgMab-2, we need to investigate whether both mAbs are applicable for immunohistochemistry, immunoprecipitation, and Western blotting in
the future study.
Some studies have revealed that blockade of TIGIT by
its specific mAbs elicited antitumor responses and tumor regression, including colorectal carcinoma, breast cancer, melanoma, and fibrosarcoma.(10,53,54) Interestingly, co-blockade
of TIGIT with PD-1 and CTLA-4 more potently elicits antitumor responses.(53–55) Moreover, co-blockade of TIGIT and
PD-1 together with CD40 agonist suppressed the progressionof pancreatic cancer.(56) In the future, it is necessary to investigate the antitumor responses of TgMab-2 and recTgMab2, in addition to the antibody-dependent cellular cytotoxicity
and complement-dependent cytotoxicity activities.
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: JP22ama121008 (to Y.K.), JP22am0401013 (toY.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.),
and JP21am0101078 (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: March 4, 2022
Accepted: April 19, 2022