TgMab-2: An Anti-human T Cell Immunoglobulin and Immunoreceptor Tyrosine-Based Inhibitory Motif Domain Monoclonal Antibody for Immunocytochemistry

Introduction

CD4⁺ 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) and programmed cell death 1 (PD-1) and by suppressing the functions 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 those mAbs, the development of novel mAbs against other immune checkpoint molecules has been desired.⁽³⁾

T cell immunoglobulin and immunoreceptor tyrosine-based 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 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 with high, 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 of phosphatidylinositol 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 tumor-infiltrating NK cells or CD8⁺ T cells in mouse xenograft models of colorectal cancer, breast cancer, melanoma, and fibrosarcoma.⁽¹⁰⁾ In addition, CD155 is upregulated in melanoma, colorectal, gastric, and pancreatic carcinomas,^(9,11–13) whereas CD112 is upregulated in breast, gastric, and ovarian carcinomas.^(9,14)

These reports suggest that TIGIT can be a novel molecular marker 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 have remained unclear. Thus, the development of novel mAbs against TIGIT has been required.

We have developed mAbs against membrane proteins by 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) HER3,⁽³⁰⁾ KLRG1,⁽¹⁵⁾ PD-L1,⁽³¹⁾ podoplanin,^(32–46) and TROP2.^(47,48) 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

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 bind to 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.

OPEN IN VIEWER

FIG. 1.

Immunocytochemistry using TgMab-2 and recTgMab-2. (A, B) CHO/hTIGIT cells (A) or CHO-K1 (B) cells were treated with buffer control, 10 μg/mL of A15153G, 10 μg/mL of TgMab-2, or 10 μg/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 μg/mL of A15153G, 10 μg/mL of TgMab-2, or 10 μg/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 μm. 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 tyrosine-based inhibitory motif domain.

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 powerful tools for the diagnosis of hTIGIT-positive cancers through detection of the tumor-infiltrating NK and CD8⁺ T cells.

Importantly, TgMab-2 and recTgMab-2 provided high-contrast fluorescent images against both exogenously and endogenously 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 membrane protein, is used for an immunogen. Furthermore, the conformation and post-translational modification of the immunogen would be physiological.

In fact, we have confirmed that an anti-CCR3 mAb [clone C₃Mab-2⁽⁵⁰⁾], anti-CCR8 mAbs [clone C₈Mab-1,⁽¹⁹⁾ C₈Mab-2,⁽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 progression of pancreatic cancer.⁽⁵⁶⁾ In the future, it is necessary to investigate the antitumor responses of TgMab-2 and recTgMab-2, in addition to the antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities.