Immune Checkpoints: The Rising Branch in Cancer Immunotherapy

Abstract

In the cancer therapy realm, concepts of immunotherapy rose as a response to emerging adverse effects caused by conventional therapies, which to some cases even more quality-of-life-reducing than the cancer itself. Immunotherapy is aimed to systematically enhance immunity to eradicate cancerous cells without harming healthy neighbor cells. In this platform, immune checkpoint molecules are under massive explorations and have been thought to be bringing excellent outlook clinically. These molecules hinder anticancer immunity. As a result, cancer growth is favored. Therefore, inactivation of immune checkpoint by blocking engagement of checkpoint receptors and their cognate ligands will restore the anticancer functions of immune system elements; hence, they can reclaim their power to eradicate cancers. Each checkpoint possesses specific downstream mechanism for which the inhibitors are formulated. In this chapter, we discuss four major checkpoints in the context of general characteristics, structures, and their roles in some cancers. Relevant recent progress in respective checkpoint molecules is also discussed to broaden our horizon on how cancers and immune checkpoint molecules are at interplay.

1. Introduction

Cancer immunotherapy is a course of treatments by which the anticancer immunity is restored. This has transformed plethora in curing cancers [1] and rapidly evolving field of oncology. There are two primary therapeutic strategies employed in cancer immunotherapy. Immune checkpoint inhibitors, cytokines and vaccines, are principally aimed at enhancing the patient’s own antitumor immunity. The other approach is administration of tumor-reactive immune cells which can be as chimeric antigen receptor (CAR) T cells, or T-cell receptor-engineered T cells. Excellent results have already been achieved in cancers including melanoma, leukemias, and lymphoma for which immunotherapy is now employed as a standard care [2]. Of these, immune checkpoint inhibitor approach has received much growing attention, especially after ipilimumab was first approved by FDA to treat melanoma [3]. Several immune checkpoints have been investigated for various types of cancers in the past decades, including but not limited to CTLA-4, PD-1, LAG-3, and TIGIT. They are named after “immune checkpoints” to indicate their function as gatekeepers of immune responses in physiological condition [4]. In order to deliver inhibitory signals, the receptors use mono-tyrosine signaling motifs such as immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). As surface molecule, their activity can be inhibited by blocking antibodies to prevent ligation of ligand receptor. This is the big idea of developing immune checkpoint blockade [5]. Application of anti-PD-1/PD-L1 as checkpoint inhibitors has achieved success therapeutically as well as commercially [6]. This encourages much more exploration on other identified checkpoints, and they also show potential in animal models. Highlights in this chapter are the four major checkpoint molecules: CTLA-4, LAG-3, PD-1, and TIGIT, in regard with their structures, signaling pathways, and progress reports on their respective implementation clinically.

2. CTLA-4: the god father of immune checkpoint molecules

2.1 General description

Although immune responses are needed to assure protection against any harmful agents, excessive response potentiates damage. Therefore, an effective control must be warranted. Cytotoxic T-lymphocyte antigen-4 (CTLA-4), known as CD152, is constitutively expressed on regulatory T cells (Tregs) and conventional T cells after activation [7]. Often, this particular subset is overexpressed on exhausted T cells [8]; hence, it is used as one of prominent markers for T cell exhaustion. Mostly, CTLA-4 is situated within intracellular vesicles and expressed only transiently following activation of the immunological synapse prior to being endocytosed [9]. CTLA-4 is a pivotal brake system in immune responses. Its genetic ablation, which causes fatal lymphoproliferative diseases, renders it unique among other checkpoints [10, 11].

CTLA-4 is a member of the CD28 family receptors. It has a significantly higher affinity to ligands B7.1 or B7.2 than that seen for CD28. Consequently, CTLA-4 abrogates co-stimulatory signal which is elicited by CD28 [12, 13], In the context of recognition of tumor antigens, CTLA-4 is highly expressed in FOXP3-expressing regulatory T cells (Tregs) which leads toco-stimulatory prevention, braking the T cells response and facilitation of cancer cells immune escape [11]. Therefore, prohibiting negative regulation via binding of CTLA-4 is seen to be a plausible way to repromote stimulation and potentiation of T cell activation. CTLA-4 blocking antibodies have been reported to regress tumor growth and improve disease-free survival in various murine malignancy models [14].

There have been two antibodies which have been developed to inhibit the binding of CTLA-4, namely ipilimumab (known previously as MDX-010) and tremelimumab (known previously as ticilimumab). While ipilimumab is a fully IgG1 ƙ mAb, tremelimumab is an IgG2 mAb. Compared to ipilimumab, tremelimumab’s half-life is twofold longer. However, it was ipilimumab that received approval from FDA to be harnessed as a checkpoint-based anticancer therapy [15].

CTLA-4is used for the treatment of metastatic melanoma and is undergoing clinical trials for lung, colorectal, gastric, kidney, pancreatic,ovarian, and prostate which commenced Phase III thereof [7]. Initially, the overall strategy of blocking CTLA-4 appeared to invite doubts, since there is no tumor specificity on which CTLA-4 ligands can bind. Moreover, lethal autoimmune and hyperimmune phenotype in CTLA-4-knockout mice seems to be positively correlated with immune toxicity caused by the blockade of this receptor [16]. This was not until Allison et al. showed a therapeutic window of this inhibitor. Harnessing mouse model, the team demonstrated that anti-CTLA-4 negatively affect the growth of colon carcinoma as well as fibrosarcoma. Intriguingly, anti-CTLA is able to exert its robust after effect palpable tumors are stably established [17].

2.2 Signaling pathway of CTLA-4

The first mechanism is coupling CD28 to its ligands CD80 (B7-1) and CD86 expressed on the surface of APCs. TCR ligation induces conformational changes in the CD28 molecule by which bivalent enhanced avidity binding to CD80 is mediated. Although these conformational changes are yet to be clearly addressed, the higher affinity of CTLA-4 as monovalent compared to that seen for CD28 is widely thought to be the reason. With no TCR stimulation, CD28 might still be able to bind to its ligands yet at low affinity. However, CTLA-4, which structurally is close to CD28, may initiate its bivalent binding before CD28. Accordingly, it is assumed that the high avidity of CTLA-4 for the shared ligands contributes to CTLA-4-mediated inhibition that overrides CD28-induced co-stimulation [18].

Following binding to either CD80 (B7-1) or CD86, CTLA-4 turns off APCs and then increases its activity upon TCR engagement. This culminates after 2–3 days of activation of conventional CD4+ and CD8+ T cells. As CD80 (B7-1) and CD86 elicit a co-stimulatory signal via CD28, a competitive role showed by CTLA-4 is vital for T cell attenuation to fine-tune the immune response. Rapid binding kinetics of CTLA-4 and CD28 to CD80 has been seen approximately at k_off ≥1.6 and ≥0.43 s⁻¹ which allows their instant competition [7]. CTLA-4 is upregulated on the surface of Treg cells with which the level of CD80/CD86 co-stimulatory molecules, including their cytoplasmic domains, on APCs is reduced in a trans-endocytosis manner [8]. Subsequently, this dampens proliferation of non-Treg T cells and the cytokine productions [19] to modulate immune suppression on bystander cells [8].

2.3 The interplay of CTLA-4 in cancer

As for dissecting more on CTLA-4 role in cancers, first we need to understand the architecture of the one particular T cell subset referred to as T_reg cells. These are particular compartment in CD4+ T subset which co-express CD25, the α-subunit of the interleukin-2 (IL-2) receptor that is canonical marker for T_reg cells and has been implicated in immune suppression in cancer [20]. These cells were identified to carry mutations of FOXP3, the master transcription factor that regulates Treg phenotypes and function as immunosuppressant, years later. Consequently, CD25 and forkhead box P3 (FOXP3) are used to probe if CD4+ T cells are T_reg cells instead of conventional T helper (T_H) cells [19].

The role of Treg in cancers is well seen in inflammatory site, where they infiltrate in to inactivate different types of CD4⁺ T helper (T_H) cells and CD8⁺ cytotoxic T cells (CTLs). This is why reversing Tregs’ activities could revive the immune system and help in combating cancer [19]. Antihuman CTLA-4 monoclonal antibodies (mAbs) can effectively exert agonist not antagonist effect for it is not capable of binding more than 50% of CTLA-4 molecules. This statement was firmly supported by findings that in homozygous human CTLA knock-in mice (ctla^h/h) anti-CTLA-4 mAbs induce B7 upregulation, but this is not observed in hetereozygous mice (ctla^h/m). Moreover, this demonstrates that functional blocking would be required to block more than 50% CTLA-4, probably due to trans-endocytosis, could be facilitated by leaving 50% of CTLA-4 unoccupied. Therefore, upregulation of B7 on dendritic cells (DCs) is physiologically connected in the blockade of B7-CTLA-4 counteraction [21].

2.4 Potential use of CTLA-4 in cancer immunotherapy

Ipilimumab was the first FDA-approved anti-CTLA-4 blocking antibody [8]. It response is markedly different to that of traditional chemotherapy. While patients receiving conventional chemotherapy exhibit a quick reduction of baseline tumor without evidence of new lesions, patients receiving ipilimumab may see first increase in their tumor burden followed by a reduction or total eradication of all lesions. This is attributable to late activation of the immune system as infiltrating T cells may take some time to destroy the tumor [22].

As the advantage from ipilimumab takes place often after what previously has been defined as “progression” by World Health Organization (WHO) or “Response Evaluation Criteria in Solid Tumors (RECIST)” criteria, new immune response criteria have been proposed. Therapeutic response toward ipilimumab culminates between 12 and 24 weeks with slow response persists even beyond 12 months. The adverse effect, which was observed in 10–15% grade 3 or higher, is immune-related and consists of colitis, hypophysitis, thyroiditis, rash, and hepatitis [22]. The treatment with immunosuppressive agents such as corticosteroids, which are aimed at alleviating immune-related side effects, does not seem to weaken antitumor response [23, 24]. Taken all these together, ipilimumab is safe to administer if monitoring and management of the side effects are conducted properly [22].

A new design of anti-CTLA4-NF mAb referred to BMS986218 has commenced its Phase I/II clinical trial to evaluate its side effect either as monotherapy or combination therapy with nivolumab (PD-1 inhibiting antibody). This particular trial is still recruiting patients with solid cancers at advanced stages [25]. Although it is still under initial phase of clinical trials, it bulks up body of evidence of promising and safe use of checkpoint-based cancer immunotherapy.

3. Lymphocyte activation Gene-3 (LAG-3)

3.1 General description

Lymphocyte activation gene-3 (LAG-3), also known as CD233, is expressed on the various hematopoetic lineage ranges from natural killer (NK) cells, B cells, γδ T cells, and activated and regulatory CD4 and CD8 T cells. In addition, this is expressed on tumor-infiltrating lymphocytes (TILs) [26]. In humans, LAG-3 is situated in chromosome 12 (12p13.32), while in mice it lies in chromosome 6 encoding a 498-amino acid protein [27]. LAG-3 locus and CD-4 co-receptor-encoding gene are adjacent to each other with similar exon/intron architecture which indicate strongly that LAG-3 and CD4 genes have evolved from a preexisting common evolutionary ancestor IgSF domain encoding gene [27]. It is surprising that, unlike other checkpoint molecules that become a hindrance for activated T cell proliferation, T cells lacking LAG-3 precisely show defect expansion. This was observed in vitro. Given that LAG-3 has relatively a higher affinity for MHC class II than that of CD4, hence if LAG-3 is present, theoretically CD4:MHC class II complex is perturbed. However, this was not observed by an experiment by Workman and Vignali where a set of transgenic mice carrying a knock-out mutation of LAG-3 were pre-crossed with OT-II-TCR mice [28]. The OT-II-TCR is defined as MHC class II-restricted TCR that responds to residues 323–339 of chicken ovalbumin [29]. Workman and Vignali figured out that LAG-3 did not interfere CD4:MHC class II interaction. This seems to oppose previous finding by Huard and colleagues a decade earlier where human LAG-3:Ig fusion proteins were shown to be disrupting CD4:MHC class II interaction although not in a CD4:MHC class II-dependent manner [30]. Discrepancy of these results probably caused by spatial separation between LAG-3 and CD4 in the immunological synapse which might not restrict the function of the soluble LAG-3:Ig fusion protein. Non-overlapping binding sites on MHC class II molecules by LAG-3 and CD4 might as well contributed to the limitedly disturbed CD4:MHC class II interplay in the presence of LAG-3. Or alternatively, it was due to subtle binding and function mode differences in murine and human [28].

As a homolog of CD4, LAG-3 binds non polymorphic MHC class II [26] that leads to the negative regulation of T lymphocytes activation and homeostasis. This checkpoint molecule has a direct role in maintaining the tolerogenic state of CD8+ T cells in vivo [31]. In the genetic level, LAG-3 and CD4 share similarity in less than 20%. However, both demonstrate a striking similarity structurally [32]. CD4 and LAG-3 belong to a distinct class of immunoglobulin super family- (IgSF-) related protein with four extracellular Ig-like domains and tryptophan (W) x cystein (C) signature motif in domain 2 and domain 4 [26]. Differ to that of CD4, the interaction of LAG-3 and MHC class II is initiated is mediated via proline-rich, 30 amino acid loop in D1 (motif domain). Other than this, LAG-3 has a longer connecting peptide spanning the fourth Ig domain and the transmembrane region because of which LAG-3 is susceptible to cell surface shedding by disintegrin and metalloproteinase domain containing protein (ADAM) [27]. LAG-3 function is activated through a conserved KIEELE motif in its cytoplasmic domain. Hence lacking of this motif leads to negative regulatory function and reverse negative modulation on the T cells [28].

3.2 Signaling pathway of LAG-3

LAG-3 expression is induced by activation of either TCR or various cytokines particularly interleukin-12 (IL-12). On the other hand, its transcription is regulated by interplay of inducing and regulating elements including transcription factor binding sites. There have been several transcription factors, reported to have inductive effects to LAG-3 expression, such as thymocyte selection-associated high mobility group box protein (TOX), nuclear factor of activated T cells (NFAT) [33], and nuclear receptor subfamily 4 group A (NR4A) [34]. In contrast, T-box transcription factor (T-bet) is inversely correlated with LAG-3 and leads to cytotoxic T cell differentiation. Thereby, it is a critical transcription factor in regulating immune exhaustion. However, the correlation between T-bet and LAG-3 is bidirectional. Deletion of either T-bet or LAG-3 enhances the expression of the other which suggests that LAG either promotes T cell exhaustion or vice versa [34]. LAG-3 interferes TCR:CD3 complex on the T cell surface; hence, TCR signal transduction is perturbed. As a result, cell proliferation as well as CD3-induced cytokine production are terminated. LAG-3 and CD3 engagement in the immunological synapse down-modulates TCR signal transduction which results in inhibition of TCR:CD3-dependent intracellular calcium fluxes to ultimately halt T cell responses [35].

3.3 LAG-3 inhibitor cancer therapy

Britstol Myers Squibb [36]. Bristol Myers Squibb has formulated opdualag which consists of anti-LAG-3 mAb relatlimab with Opdivo (nivolumab) for the first-line treatment for metastatic or unresectable melanoma [37]. It was previously reported that combination therapy of relatlimab and nivolumab increased PFS to 10.1 months compared with nivolumab monotherapy that shows PFS of 4.6 months only. Furthermore, 36% of patients treated with nivolumab monotherapy showed PFS of 12 months while in relatlimab combined with nivolumab, PFS was 47.7% [38]. In addition to this, Novartis has formulated a combination therapy targeting PD-1 and LAG-3, known as spartalizumab +LAG-525. It is now in a Phase II clinical trials to evaluate its role in unresectable or metastatic disease [39].

Tebolimab, manufactured by Macrogenic, is one of the first in class bispecific antibodies which is now undergoing Phase I clinical trials. This is composed of antigen binding fragments (Fab) targeting LAG-3 and PD-1. Pieris formulated PRS-332 which had two Fab regions targeting PD-1 and engineered lipocalins (anticalins) which was designed to target LAG-3. Similarly, F-star Therapeutics produced antibody FS118 that harbored a Fab targeting LAG-3 in its constant region and withPD-L1 targeting domains [34].

Although not all of these are approved yet, but various platforms that are offered to put more volumes in LAG-3-based cancer immunotherapy indicates that the pertinent checkpoint is clinically pivotal in cancers and hence an excellent target for therapy.

4. Programmed Death-1 (PD-1)

4.1 General description

PD-1 gene was first identified in two different types of lymphoid cell lines be that 2B4.11 (a murine T cell hybridoma) and LyD9 (a murine hematopoietic progenitor cell line) following manipulations using ionomycin/phorbol 12-myristate 13-acetate (PMA) and IL-3-deprivation, respectively. Given the two cell lines shared the same feature in common that was programmed cell death, it was plausible that PD-1 was a player in the death-inducing process in the two manipulated cell lines. In addition, in mRNA level PD-1 was deemed to be one of the molecules whose de novo synthesis induced death in the two cell lines. Owing to its patterns in death-increasing manipulation-induced augmentation as well as in thymus-restricted expression, PD-1 is enforced as a cell-death-associated gene [40].

PD-1 as a checkpoint molecule is a 55 kDa transmembrane protein consisting of 288 amino acids with a membrane-permeating domain, an extracellular N-terminal domain (IgV-like) and a cytoplasmic tail at N and C ends [41] containing two tyrosine-based signaling motifs, tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine-based switch motif (ITSM) [42]. This is expressed on B cells, T cells, natural killer T (NKT) cells, activated dendritic cells (DCs), and monocytes. In resting T cells, PD-1 is not expressed, but this can be induced. In normal human lymphoid tissue, PD-1 is expressed on germinal center-associated T cells [43]. PD-1 can be both beneficial and harmful. In the context of physiological condition, PD-1 reduces ineffective immune responses and maintains immune tolerance, to prevent autoimmune reactions [44]. As oppose, the expression of this checkpoint molecule in a tumor microenvironment (TME) mediates dilation of malignant cells and silence of the immune surveillance. Therefore, blocking interaction of PD-1 and its ligands either PD-L1or PD-L2 was deemed to potentially augment endogenous antitumor responses [45].

4.2 Signaling pathway of PD-1 in cancers

The primary ligand of PD-1 is PD-L1 that is also known as CD279 or B7-H. This ligand is a 290 amino acid-containing 33 kDa type I transmembrane glycoprotein with Ig and IgC domains in its extracellular region [46]. PD-L1 is not only a ligand, but it also carries receptor functions. PD-1 could act as a ligand to mediate transmission of antiapoptotic signal to tumor cells via PD-L1. The second known counter receptor of PD-1 is PD-L2 or B7-DC which binds with RGMb (repulsive guidance molecule b). This interaction leads to induction of pulmonary tolerance [47].

PD-L1 is expressed by tumor cells as an immune escape strategy [48]. It is associated with production of Th1 cytokines and interferons. It has been demonstrated that IFN-γ causes PD-L1 upregulation in ovarian cancer cells, whereas the inhibition of this interferon leads to reduction of PD-L1 expression in acute myeloid leukemia in mouse models. Both have been reported to take mitogen-activated protein kinase kinase (MEK/extracellular signal-regulated kinase (ERK)) and MYD88/TRAF6 pathways [49].

The PD-L1 secreted-IFN-γ subsequently induces protein kinase D isoform 2 (PKD2); thus, inhibition of this PKD2 activity inhibits the expression PD-L1. NK and T cells produce IFN-γ via Janus Kinase (JAK)1, JAK2, and signal transducer and activator of transcription (STAT)1 pathways, to ultimately upregulate PD-L1 expression on the tumor cells’ membranes [50]. PD-L1, therefore, acts as a pro-tumorigenic factor in cancer cells via binding to its receptors and activating proliferative and survival signaling pathways. This finding further indicates that PD-L1 is implicated in subsequent tumor progression. In addition, PD-L1 has been shown to exert nonimmune proliferative effects on a variety of tumor cell types [41]. These are the basis of PD-1/PD-L1 blocking antibody development, which is intended for downregulating these expressions, to allow functional immune cells to perform robust tumor surveillance [51].

4.3 PD-1/PD-L1 in cancer immunotherapy

When a T cell recognizes antigen:MHC complex, expressed on the surface of the target cell, inflammatory process begins which is marked by the secretion of inflammatory cytokines that subsequently induces the expression of PD Ligand-1 (PD-L1) in the affected tissue. This PD-L1 activates secretion of PD-1 protein on the T cells which causes immune tolerance, an event where the immune system is no longer capable of mounting an inflammatory response even in the presence of antigen [52].

In the tumor microenvironment (TME), PD-1 and its ligand PD-L1 play a fundamental role in tumor progression and survival by escaping immune surveillance. As aforementioned, while PD-1 is expressed on various subsets of immune cells, PD-L1 is expressed on tumor cells and APCs. Upon their engagement, T cells become dysfunctional and exhausted. Moreover, interleukin-10 (IL-10) is produced largely in the tumor [53]. This is known as the cytokine synthesis inhibitory factor which inhibits the productions of diverse pro-inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8, IL-12, IL-18, tumor necrosis factor-α (TNF-α), and granulocyte macrophage-colony-stimulating factor (GMSF) in T cells as well as in macrophages. Moreover, IL-10 diminishes the expression of interferon-γ (IFN- γ) in T helper (Th) cells and peripheral blood mononuclear cells (PBMCs). On the other hand, cytokines stimulate proliferation of mast cells [54], a particular subset of tissue-resident myeloid cells that contain coarse granules of inflammatory mediators like histamine that contribute to shaping of tumor cells and tumor microenvironment (TME) [55]. Foxp3+ CD4+ Tregs have recently been observed to maintain PD-1 expression on their surfaces in order to create a highly immunosuppressive TME [44]. The FDA-approved PD-1/PD-L1-based immune checkpoint inhibitor therapy apparently outnumbers CTLA-4. Like CTLA-4, PD-1/PD-L1 checkpoint can be administered as monotherapy or combination with CTLA-4 [56].

Nivolumab and pembrolizumab are antibodies designed to block binding of PD-1 to its ligands. First approval for nivolumab with brand name Opdivo was in December 2014 when nivolumab was evidently great for treating unresectable metastatic melanoma. Few months later, again nivolumab was approved to be harnessed to treat NSCLC which resulted in 23.7% of objective response rate (ORR) and 91 days of progression-free survival (PFS) [57]. It was also used for treating Hodgkin’s lymphoma patients for which overall survival (OS) was observed at 3 years for 80% patients with median PFS between 12 and 18 months [58]. As nivolumab, pembrolizumab with brand name Keytruda was initially approved in 2014 for metastatic melanoma. The ORR obtained in this treatment was at 18%. In May 2017, it received its second approval for its use in locally advanced or metastatic urothelial carcinoma. In non-Hodgkin’s lymphoma and head and neck squamous cell carcinoma (HNSCC), treatment using pembrolizumab resulted in ORR of 53% and 19%, respectively [51]. The other FDA-approved anti-PD-1 antibody is Cemiplimab, also known as Libtayo, which is the first checkpoint designed for advanced cutaneous squamous cell carcinoma (CSCC). It was shown that the effects of Cemiplimab was durable, and no recurrence was observed even after more than 16 months [59].

As anti-PD-L1 antibodies, Atezolimumab (Tecentriq), Avelumab (Bavencio), and Durvulumab (Imfinzi) were also approved for cancer type targets. Atezolimumab is widely used for urothelial carcinoma [60, 61] and differs to the other two as it is a phage-derived human IgG1, whereas Avelumab and Durvulumab are fully human anti-PD-L1-IgG1 [51]. Avelumab is used to treat patients with metastatic Merkel cell carcinoma and NSCLC with ORR values of 62.1% and 12%, respectively [62, 63]. Durvulumab is designed to directly target PD-L1, to prevent tumor immune escape and enhance immune responses. In head and neck squamous cell carcinoma (HNSCC) ORR of 9.2% was observed. Moreover, 6-month progression-free in HNSCC patients rose to 25% for those patients who were PD-L1+ [64]. For NSCLC patient cohort, the ORR was observed at 66.3% [65].

5. T Cell Immunoreceptor with Immunoglobulin and ITIM Domain (TIGIT)

5.1 General description

TIGIT, also known as V-set and transmembrane domain containing (Vstm), Washington University cell adhesion molecule (wucam) or V-set and immunoglobulin domain-containing protein 9 (VSIG9), is a receptor of the Ig superfamily. It is an important player in adaptive as well as innate immunity regulation [66]. TIGIT is expressed on activated T cells, both in T_H and CTL subsets, NK cells, Tregs, and follicular T_H [67]. In cancer, TIGIT is often co-expressed with PD-1 on tumor-antigen-specific CD8+ T cells and C8+ tumor-infiltrating lymphocytes (TILs) in human and mice [68]. In addition, it is co-expressed with Tim-3 and LAG-3 on exhausted CTLs in tumors [66]. When homozygously knocked out, TIGIT -/- mice do not develop autoimmunity. But when compared with wild-type mice, TIGIT -/- mice suffer severe autoimmune encephalitis, after immunized with myelin oligodendrocyte glycoprotein. This supports the role of TIGIT as a negative regulator of T cell functions [69].

Structurally, TIGIT is composed of an extracellular immunoglobulin variable domain, a type I transmembrane domain, a short intracellular domain encompassing one immunoreceptor tyrosine-based inhibitory motif (ITIM) and one immunoglobulin tyrosine tail (ITT)-like motif. The immunoglobulin variable domain and members of poliovirus receptor (PVR)-like family such as DNAM-1, CD96, CD155, CD111, CD112 (PVR-related 2 (PVRL2), nectin-2, CD113 (poliovirus receptor-related 3 (PVRL3), nectin-3), and PVRL4 share sequence homology in common [70].

5.2 TIGIT signaling pathways

TIGIT has three ligands namely CD155, CD112, and CD113. These ligands belong to nectin and nectin-like (NECL) molecules that mediate cell adhesion, cell polarization, and tissue organization [71]. Among these, CD155 is the main receptor for TIGIT. Both homodimerize and upon engagement form heterotetramers. CD155 is mainly expressed on dendritic cells (DCs), T cells, B cells, and macrophages but also in non-haematopoietic tissues [72]. Following the ligation of TIGIT:CD155, inhibitory signaling is initiated via ITT-like motif. Amino acid tyrosine at position of 225 (tyr225) of the ITT-like motif is phosphorylated and coupled onto cytosolic adapter of growth factor receptor-bound protein 2 (Grb2) and β-arrestin 2 to facilitate recruitment of Src homology-2 (SH2)-containing inositol phosphatase-1 (SHP-1). This recruitment thwarts phosphoinositide 3 kinase and mitogen-activated protein kinase signaling, thus reducing killing capacity in NK cells [73]. SHIP-1 also impairs TRAF6 and nuclear factor ƙ light-chain-enhancer of activated B cells (NF-ƙB) which impedes production of IFN-γ by NK cells [74].

Other than CD155 as the highest binding magnitude ligand, CD112 and CD113 are also reported to bind well to TIGIT, but at lesser magnitude . While CD112 shows a broad range of expression in both hematopoietic and non-hematopoietic tissues, CD113 is restricted to non-hematopoietic tissues. Their overexpression is observed in in several malignancies [70]. CD112R has recently been found as a receptor for CD112 other than TIGIT. It was reported that the combined CD112R and TIGIT blockade allowed diffusion of CD4+ T cells and increased the productions of cytokines such as IFN-γ, IL-2, IL-5, IL-10, and IL-13. Moreover, blockade to these two checkpoints led to restored cytotoxicity and expansion of CD8+ T cells [75].

5.3 TIGIT’s bright future in cancer immunotherapy

TIGIT is relatively new discovered checkpoint molecule that seems to be a promising target in immune checkpoint-based cancer immunotherapy. This checkpoint was evidently shown to prevent tumor antigen release by CD8+ T cells and impair T cell priming by DCs or obstruct cancer cells killing by CD8+ T cells. And it is now under investigation in various clinical trials [72].

Recently, six human-anti-TIGIT monoclonal antibodies (mAbs) of the IgG1 isotype are being investigated in clinical trials. OncoMed Pharmaceuticals (US) produced Etigilimab (OMP-313M32). The mouse version of this 313R12 mAb was reported to function similar to etigilimab, i.e. suppression of syngeneic colon and kidney tumors in immune competent mice and improved T_H1-type response. Now, etigilimab commences its pharmacokinetics assessment in a Phase I trial. Furthermore, Arcus Bioscience’s AB154 and Merck’s MK-7684 drugs, which are designed as monotherapy for advanced solid cancers and solid cancers, respectively, are in Phase 1 trials. Merck’s MK-7684 is also being tested in combination with pembrolizumab (anti PD-1 mAb). Tiragolumab by Genentech/Roche is also in Phase II trial designed as combination therapy with atezolumab (anti-PD-1 mAb) for advanced or metastatic non-small cell lung cancer (NSCLC). Bristol-Myers Squibb bulks up the trials for TIGIT by producing BMS-986207 which is IgG1 mAb (FcγR-null) anti-TIGIT to target advanced or metastatic solid cancers. The Fc portion on the IgG of this mAb has been mutated to avoid ligating to Fcγ receptor (FcγR) because Fcγ-dependent mechanisms were found to inhibit antitumor activity of anti-PD-1 mAbs. With similar strategy (IgG1 mAb and FcyR-null), Astellas Pharma (Potenza Therapeutics) has also designed ASP 8374 [72].

6. Conclusions

Immune checkpoint molecules are promising for cancer immunotherapy. Each has specific structure and specific ligands that can be engineered in such a way that immune-suppressing activity is redirected into immune-activating activity either as monotherapy or in combination with other checkpoints to help in tumor growth reduction. PD-1 and CTLA-4 are the two most common checkpoints with FDA approvals for several cancers, years ahead of other checkpoints. TIGIT and LAG-3 are also known to show promising potential in cancer immunotherapy. As in the context of cancer immunity, immune checkpoint molecules favor cancers by facilitating mechanisms that may support cancer growth or help to negate the effects of checkpoint molecules via inhibitory mechanisms. Combination of checkpoint molecules has also been shown to outperform single checkpoint therapy. However, immune checkpoint-based immunotherapy requires proper monitoring and management for adverse effects and exudes minimum risk to patients.