**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.

**Keywords:** immunotherapy, cancer, immunity, checkpoint, inhibitors, CTLA-4, LAG-3, TIGIT, PD-1

### **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 koff ≥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 Treg 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 Treg 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 Treg cells instead of conventional T helper (TH) cells [19].

The role of Treg in cancers is well seen in inflammatory site, where they infiltrate in to inactivate different types of CD4<sup>+</sup> T helper (TH) 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 knockout mutation of LAG-3 were pre-crossed with OT-II-TCR mice [28]. The OT-II-TCR is

#### *Immune Checkpoints: The Rising Branch in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.108656*

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].
