MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 2, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0006

Development of Anti-human T Cell Immunoreceptor
with Ig and ITIM Domains (TIGIT)
Monoclonal Antibodies for Flow Cytometry

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

Junko Takei,1,2 Teizo Asano,1 Ren Nanamiya,1 Takuro Nakamura,1 Miyuki Yanaka,1 Hideki Hosono,1Tomohiro Tanaka,1 Masato Sano,1 Mika K. Kaneko,1 Hiroyuki Harada,2 and Yukinari Kato1,3,*

Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell death-1(PD-1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) recently made a significant survival rate
improvement in cancer treatment. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed in T
and NK cells related to their activities. It has a single extracellular immunoglobulin domain, a type 1 transmembrane domain, and a single intracellular ITIM. TIGIT binds with poliovirus receptor (PVR) or PVR2,
resulting in suppressing T and NK cell activities. Some studies showed that the combined use of a TIGIT
inhibitor with another immune checkpoint inhibitor enhanced antitumor activities more strongly than theirsingle use. Therefore, TIGIT should be a new target for immunotherapy. In this study, we developed new antihuman TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening(CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells(CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG1, kappa),
specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (KD) of
TgMab-2 for CHO/hTIGIT cells were determined to be 3.5 · 10-9 M. These results suggest that TgMab-2,
which was developed by CBIS method, is useful for analyzing the function of hTIGIT by flow cytometry.
Keywords: TIGIT, monoclonal antibody, hybridoma, flow cytometry, TgMab-2

PVRL2 is expressed in various cells, especially in epithelial
cells, neurons, and fibroblasts.(6) PVR and PVRL2 are overexpressed in cancer cells, including colorectal,(7) pancreatic,(8)breast or ovarian(9) cancers, and melanoma.(10)Recently, therapies targeting immune checkpoint molecules,
such as programmed cell death-ligand 1 (PD-L1), programmed
cell death-1 (PD-1), and cytotoxic T lymphocyte-associatedantigen 4 (CTLA-4), have significantly improved the prognosisof cancer patients.(11) Johnston et al. reported that the coblockade of TIGIT and PD-L1 was more effective in enhancing CD8+ T cell effector function than that of PD-L1
blockade alone, resulting in a remarkable tumor reduction in mouse cancer models.(12) Another study has shownthat both TIGIT and PD-1 blockade increased the functionof CD8+ T cell function in melanoma patients,(13) suggesting that TIGIT could be expected as a new target for
immunotherapy.

IntroductionTcell immunoreceptor with Ig and ITIM domains(TIGIT) is an immune checkpoint molecule expressedon T and NK cells. TIGIT consists of a single extracellularimmunoglobulin domain, a type 1 transmembrane domain, anda single intracellular immunoreceptor tyrosine-based inhibitory
motif (ITIM).(1) ITIM motifs are commonly used by inhibitory
receptors to recruit SH2 domain-containing tyrosine phosphatases SHP1 and SHP2.(2) TIGIT binds to poliovirus receptor(PVR, also known as CD155) with high affinity and PVR-likeprotein 2 (PVRL2, also known as CD112) with low affinity.(1)These interactions cause suppression of T cell activation andNK cell cytotoxicity.(1,3,4) DNAM-1 (CD226) competes with
TIGIT to bind with PVR, although the affinity of PVR-DNAM1 is much lower than PVR-TIGIT.(1,5) PVR is expressed at low
levels in many cell types of epithelial origin. In contrast,
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
*ORCID ID (https://orcid.org/0000-0001-5385-8201).
2

71

72

In this study, we developed a novel anti-human TIGIT(hTIGIT) monoclonal antibodies (mAbs), useful for flow
cytometry using the Cell-Based Immunization and Screening(CBIS) method. We further characterized the establishedclone (TgMab-2) by flow cytometry.
Materials and Methods

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Synthesized DNA encoding
hTIGIT (Accession No. NM_173799) was purchased from
OriGene Technologies, Inc. (Rockville, MD, catalog No:
RC221447). hTIGIT open reading frame without signal sequence and with anN-terminal MAP16 tag were subcloned intoa pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The MAP16 tag comprises 16 aminoacids (aa) (PGTGDGMVPPGIEDKI) and is recognized by
PMab-1.(14,15) A hTIGIT plasmid was transfected using a Neon
Transfection System (Thermo Fisher Scientific, Inc., Waltham,MA). Stable transfectants were sorted using a cell sorter(SH800; Sony Corp., Tokyo, Japan) and cultivated in a medium
containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA).
CHO-K1, P3U1, and CHO/hTIGIT were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan), supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific,Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). Cells weregrown in a humidified incubator at 37C, 5% CO2, and 95% air.
NK cells (Donor Lot. 4022602 NK1602) for flow cytometry were purchased from Takara Bio, Inc. (Shiga, Japan).

TAKEI ET AL.

The procedure included three additional immunization followed by a final booster intraperitoneal injection administered 2 days before spleen cells were harvested. Harvested
spleen cells were subsequently fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI medium supplemented with
10% heat-inactivated FBS (Thermo Fisher Scientific, Inc.), 100
U/mL penicillin, 100 lg/mL streptomycin, 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.), and hypoxanthine, aminopterin, thymidine for selection (Thermo Fisher Scientific,Inc.). Culture supernatants were screened by flow cytometry.
Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin
and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). They were washed with 0.1% bovine serum albumin in
phosphate-buffered saline and treated with primary mAbs for
30 minutes at 4C. Then, the cells were treated with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc.). Fluorescence data were collected using the EC800 Cell Analyzer (Sony Corp.) and analyzed
using FlowJo (BD Biosciences, Franklin Lakes, NJ).
Determination of binding affinity by flow cytometry

CHO/hTIGIT was suspended in 100 lL of serially diluted
anti-hTIGIT mAbs, and Alexa Fluor 488-conjugated antimouse IgG (1:200; Cell Signaling Technology, Inc.) was
added. Fluorescence data were collected using the BD FACSLyric (BD Biosciences). The dissociation constant (KD) was
calculated by fitting binding isotherms to built-in one-sitebinding models in GraphPad PRISM 8 (GraphPad Software,Inc., La Jolla, CA).

Antibodies

An anti-hTIGIT mAb (clone A15153G; mouse IgG2a, kappa)
was purchased from BioLegend (San Diego, CA). Secondary
Alexa Fluor 488-conjugated anti-mouse IgG was purchasedfrom Cell Signaling Technology, Inc. (Danvers, MA).
Animals

All animal experiments were performed according torelevant guidelines and regulations to minimize animal
suffering and distress in the laboratory. Animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit No.: 2019NiA-001).
Mice were monitored daily for health during the full 4week duration of the experiment. A reduction of >25% of
total body weight was defined as a humane endpoint. Mice
were euthanized by cervical dislocation, and death was
verified by respiratory and cardiac arrest. Female BALB/cmice (6 weeks old) were purchased from CLEA Japan(Tokyo, Japan). The animals were housed under specificpathogen-free conditions.
Hybridoma production

To develop mAbs against hTIGIT, we employed the CBIS
method.(16) In brief, two BALB/c mice were immunized with
CHO/hTIGIT cells (1 · 108) by the intraperitoneal route together with Imject Alum (Thermo Fisher Scientific, Inc.).

Results
Establishment of the anti-hTIGIT mAbs

To establish anti-hTIGIT mAbs, we employed the CBIS
method (Fig. 1). First, we immunized two mice with CHO/
hTIGIT cells and produced hybridomas by fusing splenocyteswith P3U1 myeloma cells. Next, we selected hybridomas
whose supernatants were positive for CHO/hTIGIT cells andnegative for CHO-K1 cells by flow cytometry. The firstscreening approach identified strong signals from CHO/hTIGIT cells and weak or no signals from CHO-K1 cells in 11 of958 wells (1.1%). The second screening approach identified
strong signals from NK cells using 2 of the 11 hybridoma
supernatants determined in the previous step (18%). After
limiting dilution and several additional screenings, TgMab-2(IgG1, kappa), which reacted most strongly with CHO/hTIGIT,
was finally established.
Flow cytometric analyses

TgMab-2 recognized CHO/hTIGIT but not CHO-K1(Fig. 2A). Also, TgMab-2 reacted with endogenous hTIGIT
in NK cells (Fig. 2A). The concentration of 0.01 and
0.1 lg/mL were enough for recognition with CHO/hTIGIT
and NK cells, respectively (Fig. 2A). As a positive control,clone A15153G from BioLegend reacted similarly withboth cells (Fig. 2B).

ANTIHUMAN TIGIT

73

by one-site binding models. The KD of TgMab-2 for
CHO/hTIGIT was determined to be 3.5 · 10-9 M (Fig. 3),
indicating that TgMab-2 possesses a high affinity for
CHO/hTIGIT cells.

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

Discussion

FIG. 1. Production of anti-hTIGIT mAbs. The CBIS
method. CHO/hTIGIT cells were immunized into miceusing intraperitoneal injection. The screening was performed byflow cytometry. CBIS, Cell-Based Immunization and Screening; CHO, Chinese hamster ovary; hTIGIT, human T cell
immunoreceptor with Ig and ITIM domains; mAbs, monoclonal antibodies.
Determination of binding affinity using TgMab-2
on hTIGIT-expressed cells by flow cytometry

To assess the apparent binding affinity of TgMab-2 with
CHO/hTIGIT, we conducted a kinetic analysis of the interaction of TgMab-2 with CHO/hTIGIT by flow cytometry. The geometric mean of fluorescence intensity wasplotted versus the concentration of TgMab-2 and fitted

In this study, we successfully developed a novel antihTIGIT mAb, TgMab-2, using the CBIS method. In the paststudies, anti-hTIGIT mAbs were acquired by hybridoma
technology or phage display method. Yu et al. generated
mAbs by immunizing hamsters with fusion proteins ofmouse TIGIT and selected mAbs, which cross-reacted tohTIGIT.(1) Stanietsky et al. and Chauvin et al. producedmAbs by immunizing mice with TIGIT-Ig fusion protein(4)and TIGIT-Fc fusion protein,(13) respectively. Han et al. acquired mAbs with a high-throughput strategy using a phagedisplayed human single-chain antibody library and CHO/
TIGIT cells.(17) Compared with these methods, the CBIS
method can produce mAbs more quickly and simply because it
does not require purification of proteins for immunization andscreening of phage libraries.
Supplementary Table S1 shows anti-hTIGIT mAbs available on the market. Their immunogens are peptides or proteins, so TgMab-2 is the first mAb produced by immunization
with hTIGIT-expressing cells. There are 28 out of 31 clones
that can be used for flow cytometry, and most of them areavailable for one or two applications. Only three clones, suchas 4A10, 4A11, and 4A12, are useful for all applications, flow
cytometry, Western blotting, and immunohistochemistry.
Previously, we succeeded in establishing mAbs against various membrane proteins, such as CD44,(18) CD133,(16) andCD20,(19) using the CBIS method. In each protein, we got
some mAbs, useful for flow cytometry, Western blotting,and immunohistochemistry with the same clone. Therefore,

FIG. 2. Flow cytometry using anti-hTIGIT mAbs. (A) CHO/hTIGIT cells and NK cells were treated with 0.01–1 mg/mL of
TgMab-2, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control.
(B) CHO/hTIGIT and NK cells were treated with 0.01–1 mg/mL of A15153G (BioLegend), followed by treatment with Alexa
Fluor 488-conjugated anti-mouse IgG; black line, negative control.

74

TAKEI ET AL.

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

FIG. 3. Determination of the binding affinity of TgMab-2.
CHO/hTIGIT was suspended in 100 mL of serially diluted
TgMab-2 (0.006–100 mg/mL). Then, Alexa Fluor 488conjugated anti-mouse IgG was added. Fluorescence data
were collected using the BD FACSLyric.
in addition to investigating further characterizations of
TgMab-2, such as the epitope and blockade of hTIGIT, wewill produce another anti-hTIGIT mAb that can be used for
all applications in the future.
Authors’ Contributions

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

No competing financial interests exist.
Funding Information

This research was supported in part by AMED underGrant Numbers JP20am0401013 (Y.K.) and JP20am
0101078 (Y.K.), and by JSPS KAKENHI Grant Numbers
21K15523 (to T.A.), 21K07168 (to M.K.K.), 20K16322 (toM.S.), and 19K07705 (to Y.K.).
Supplementary Material

Supplementary Table S1References

1. Yu X, Harden K, C Gonzalez L, Francesco M, Chiang E,
Irving B, Tom I, Ivelja S, Refino CJ, Clark H, Eaton D,
and Grogan JL: The surface protein TIGIT suppresses T
cell activation by promoting the generation of mature
immunoregulatory dendritic cells. Nat Immunol 2009;10:
48–57.
2. Tourdot BE, Brenner MK, Keough KC, Holyst T, Newman
PJ, and Newman DK: Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated inhibitory signaling isregulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: A case study involving
PECAM-1. Biochemistry 2013;52:2597–2608.

3. Kurtulus S, Sakuishi K, Ngiow S-F, Joller N, Tan DJ, Teng
MWL, Smyth MJ, Kuchroo VK, and Anderson AC: TIGIT
predominantly regulates the immune response via regulatory T cells. J Clin Investig 2015;125:4053–4062.
4. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O,
Novik A, Levine Z, Beiman M, Dassa L, Achdout H, SternGinossar N, Tsukerman P, Jonjic S, and Mandelboim O:
The interaction of TIGIT with PVR and PVRL2 inhibits
human NK cell cytotoxicity. Proc Natl Acad Sci U S A
2009;106:17858–17863.
5. Bottino C, Castriconi R, Pende D, Rivera P, Nanni M,
Carnemolla B, Cantoni C, Grassi J, Marcenaro S, Reymond
N, Vitale M, Moretta L, Lopez M, and Moretta A: Identification of PVR (CD155) and Nectin-2 (CD112) as cellsurface ligands for the human DNAM-1 (CD226) activating
molecule. J Exp Med 2003;198:557–567.
6. Takai Y, Irie K, Shimizu K, Sakisaka T, and Ikeda W:
Nectins and nectin-like molecules: Roles in cell adhesion, migration, and polarization. Cancer Sci 2003;94:
655–667.
7. Masson D: Overexpression of the CD155 gene in humancolorectal carcinoma. Gut 2001;49:236–240.
8. Nishiwada S, Sho M, Yasuda S, Shimada K, Yamato I,
Akahori T, Kinoshita S, Nagai M, Konishi N, and Nakajima Y: Clinical significance of CD155 expression inhuman pancreatic cancer. Anticancer Res 2015;35:2287–
2297.
9. Oshima T, Sato S, Kato J, Ito Y, Watanabe T, Tsuji I, HoriA, Kurokawa T, and Kokubo T: Nectin-2 is a potential
target for antibody therapy of breast and ovarian cancers.
Mol Cancer 2013;12:60.
10. Bevelacqua V, Bevelacqua Y, Candido S, Skarmoutsou E,
Amoroso A, Guarneri C, Strazzanti A, Gangemi P, Mazzarino MC, D’Amico F, McCubrey JA, Libra M, and Malaponte G: Nectin like-5 overexpression correlates with themalignant phenotype in cutaneous melanoma. Oncotarget
2012;3:882–892.
11. Ribas A, and Wolchok JD: Cancer immunotherapy using
checkpoint blockade. Science 2018;359:1350–1355.
12. Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M,
Yang Y, Park S, Javinal V, Chiu H, Irving B, and Dan J:
The immunoreceptor TIGIT regulates antitumor and antiviral CD8 + T cell effector function. Cancer Cell 2014;26:
923–937.
13. Chauvin J-M, Pagliano O, Fourcade J, Sun Z, Wang H,
Sander C, Kirkwood JM, Chen T-HT, Maurer M, Korman
AJ, and Zarour HM: TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in melanoma patients. J Clin
Investig 2015;125:2046–2058.
14. Fujii Y, Kaneko MK, and Kato Y: MAP Tag: A novel
tagging system for protein purification and detection.
Monoclon Antib Immunodiagn Immunother 2016;35:293–299.
15. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
16. Kato Y, Ohishi T, Yamada S, Itai S, Furusawa Y, Sano M,
Nakamura T, Kawada M, and Kaneko MK: Anti-CD133
monoclonal antibody CMab-43 exerts antitumor activity ina mouse xenograft model of colon cancer. Monoclon AntibImmunodiagn Immunother 2019;38:75–78.

ANTIHUMAN TIGIT

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

17. Han D, Xu Y, Zhao X, Mao Y, Kang Q, Wen W, Yu X, Xu L,
Liu F, Zhang M, Cui J, Wang Z, Yang Z, Du P, and Qin W: A
novel human anti-TIGIT monoclonal antibody with excellent
function in eliciting NK cell-mediated antitumor immunity.
Biochem Biophys Res Commun 2021;534:134–140.
18. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C(44)Mab-5.
Biochem Biophys Rep 2018;14:64–68.
19. Furusawa Y, Kaneko M, and Kato Y: Establishment of
C20Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967.

75

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2–1, Seiryo-machi, Aoba-ku
Sendai 980–8575
Japan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: February 7, 2021
Accepted: February 24, 2021