TIGIT in Lung Cancer: Potential Theranostic Implications

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a co-inhibitory receptor expressed on various immune cells, including T cells, NK cells, and dendritic cells. TIGIT interacts with different ligands, such as CD155 and CD112, which are highly expressed on cancer cells, leading to the suppression of immune responses. Recent studies have highlighted the importance of TIGIT in regulating immune cell function in the tumor microenvironment and its role as a potential therapeutic target, especially in the field of lung cancer. However, the role of TIGIT in cancer development and progression remains controversial, particularly regarding the relevance of its expression both in the tumor microenvironment and on tumor cells, with prognostic and predictive implications that remain to date essentially undisclosed. Here, we provide a review of the recent advances in TIGIT-blockade in lung cancer, and also insights on TIGIT relevance as an immunohistochemical biomarker and its possible theranostic implications.


Introduction
Human neoplasms avoid immune system detection through a variety of immunological escape mechanisms [1]. Tumor cells can decrease T-cell signaling by downregulating the activity of stimulatory receptors while increasing the activity of inhibitory immunoreceptors [2]; for example, they can reduce TCR-mediated stimulatory signaling by downregulating surface MHC-I levels [3], or they may tune up PD-1-mediated inhibitory signaling by increasing PD-L1 surface expression [4]. The hypothesis that inhibiting the activation of inhibitory immunoreceptors might rejuvenate immune cell antitumor action has been shown experimentally and has been successfully applied in the clinical setting for the treatment of various forms of advanced-stage cancers [5,6]. Targeting ligands involved in those interactions with monoclonal antibodies (mAb) has proven to be effective in animal and human tumor models, and immune checkpoint blockade (ICB) with anti-PD-L1, anti-PD-1, or both mAbs is currently regarded as standard therapy for many advanced stage solid malignancies. Moreover, several additional co-inhibitory receptor-ligand interactions, aside from the PD-1/PD-L1 axis, have been described, which can either directly or indirectly suppress the anti-tumor function of CD8+ T cells in the tumor microenvironment. These co-inhibitory receptors include T cell immunoglobulin mucin domain 3 (TIM3) [7], lymphocyte-activation gene 3 (LAG3) [8], cytotoxic T-lymphocyte associated protein 4 (CTLA-4) [9], and T cell immunoreceptor with Ig and ITIM domains (TIGIT) [1]. Interestingly, many lines of evidence suggest that TIGIT is important in reducing adaptive and innate immunity towards malignancies, and anti-TIGIT mAbs have shown promising results in the field of lung cancer [1,10,11]. Specifically, the synergy between TIGIT and the PD-1/PD-L1 axis is being exploited in several clinical trials in which both mechanisms are targeted together, with promising results. towards malignancies, and anti-TIGIT mAbs have shown promising results in the field of lung cancer [1,10,11]. Specifically, the synergy between TIGIT and the PD-1/PD-L1 axis is being exploited in several clinical trials in which both mechanisms are targeted together, with promising results.
TIGIT is a T-cell receptor involved in limiting T-cell function and adaptive immune responses in the context of cancer immune evasion mechanisms. TIGIT is mostly expressed in T cells and natural killer (NK) cells and has three ligands: CD155, CD112, and CD113. When CD155 is highly expressed on tumor cells, it binds TIGIT and promotes direct and indirect downregulation of T-cell response ( Figure 1). The TIGIT/CD155 axis has been shown to play a role in the immune escape and cancer progression of pancreatic cancer [12], ovarian cancer [13], breast cancer [14], and gastric cancer [15]. The interaction of TIGIT with its ligands results in the recruitment of the SHP-1 and SHP-2 phosphatases to the immunoreceptor tyrosine-based inhibition motifs (ITIMs) present in the cytoplasmic domain of TIGIT, leading to the dephosphorylation of downstream signaling molecules and resulting in the inhibition of T-cell activation and proliferation. Additionally, TIGIT can compete with the co-stimulatory receptor CD226 (DNAM-1) in binding CD155 and CD112. CD226 is involved in the activation of T and NK cells, and its engagement with CD155 and CD112 leads to increased cytotoxicity and cytokine production. The competition between TIGIT and CD226 can therefore result in the suppression of immune responses. Moreover, TIGIT has been shown to regulate immune cell metabolism, suppressing glucose uptake and glycolysis in T cells through the inhibition of the Akt-mTOR pathway [15][16][17][18][19]. An association between TIGIT expression and poor survival was identified in multiple malignancies, although with controversial results [20]. In this review, we highlight the current knowledge about TIGIT as a molecular target for lung cancer treatment across all current clinical trials employing anti-TIGIT mAbs; furthermore, we examine the role of TIGIT as a prognostic and predictive biomarker in human cancer, with a focus on immunohistochemistry and its possible prognostic, predictive, and overall theranostic applicaptions on lung cancer. In this review, we highlight the current knowledge about TIGIT as a molecular target for lung cancer treatment across all current clinical trials employing anti-TIGIT mAbs; furthermore, we examine the role of TIGIT as a prognostic and predictive biomarker in human cancer, with a focus on immunohistochemistry and its possible prognostic, predictive, and overall theranostic applications on lung cancer.

Materials and Methods
The online database clinicaltrials.gov was accessed to retrieve the current clinical trials utilizing anti-TIGIT strategies in lung cancer. All 32 results are discussed in Section 3.1. The online database PubMed was accessed to retrieve all current published literature regarding TIGIT as an immunohistochemical biomarker with prognostic and/or predictive value in human cancer. Search keywords included a combination of "TIGIT" and the following: "immunohistochemistry", "prognostic", "predictive", and "biomarker". A total of 656 articles were identified; 624 were excluded due to lack of relevance and/or discussion about TIGIT as an immunohistochemical biomarker. A total of 32 articles remained and are discussed in Section 3.2.

Clinical Trials in Lung Cancer Utilizing TIGIT-Blockade
Several anti-TIGIT monoclonal antibodies of the IgG1 isotype are currently being evaluated in lung cancer clinical trials ( Table 1). The potential effectiveness and the safety of TIGIT inhibitors are being explored mostly in combination with other immune-checkpoint inhibitors or chemotherapies, across different development phases and clinical settings (Table 2) [18,21].
The anti-TIGIT mAb tiragolumab has progressed the furthest in clinical trials for the treatment of non-small cell lung carcinoma (NSCLC). Recently, the Phase II CITYSCAPE (NCT03563716) trial evaluated the possible efficacy of combining tiragolumab with anti-PD-1 antibody atezolizumab in the first-line treatment of NSCLC with PD-L1 expression >1%, assessed by means of the tumor proportion score (TPS). Data showed promising results in favor of the combined treatment (atezolizumab + tiragolumab vs. atezolizumab + placebo) with a longer median survival (PFS 5.6 months vs. 3.9 months; HR 0.58, 95%CI: 0.38-0.89) and an improved objective response rate (ORR 31.3 vs. 16.2%). An exploratory analysis revealed that patients with high PD-L1 expression (TPS ≥ 50%) had a 69% reduction in the risk of disease progression or death from the illness with atezolizumab + tiragolumab vs. 24% with atezolizumab + placebo (PFS 16.6 months vs. 4.11 months; HR 0.29, 95%CI: 0.15-0.53) [11,22]. These results suggest that dual inhibition of immunotherapeutic mechanisms may be effective in clinical practice, although the final results and the design of the Phase III trial in PD-L1 + TPS ≥ 50% population (NCT04294810) are still needed. The CITYSCAPE trial also evaluated the prognostic significance of TIGIT expression. Out of 105 assessable patients, 49 (46.7%) were defined as TIGIT-high (with ≥5% expression on tumor-infiltrating immune cells); no significant impact on progressionfree survival (PFS) was noted between TIGIT-high and TIGIT-low groups (HR 0.62, 95%CI: 0.30-1.32) [11,18,21].
Although the CITYSCAPE trial found that combined treatment improved ORR and PFS in NSCLC patients, the same combination did not provide any benefits in patients with small cell lung carcinoma (SCLC) in the Phase III SKYSCRAPER-02 (NCT04256421), despite being well-tolerated. However, a Phase II study (NCT04308785) is currently investigating atezolizumab ± tiragolumab as consolidation therapy in limited-stage SCLC participants who have not progressed after receiving chemoradiotherapy.
Moreover, tiragolumab is presently being assessed in a non-metastatic NSCLC setting. The ongoing Phase II SKYSCRAPER-06 trial (NCT04619797) is evaluating atezolizumab + pemetrexed and platinum-based chemotherapy with or without tiragolumab in patients with previously untreated advanced non-squamous NSCLC. Meanwhile, the Phase III SKYSCRAPER-03 trial (NCT04513925) compared atezolizumab and tiragolumab versus durvalumab among patients with locally advanced, unresectable stage III NSCLC. A Phase II study (NCT04832854) is currently underway, with the aim of comparing the effects of neoadjuvant and adjuvant tiragolumab + atezolizumab in combination with chemotherapy versus chemotherapy alone in patients with previously untreated locally advanced resectable stage II, IIIA, or select IIIB NSCLC.
Vibostolimab, another anti-TIGIT mAb, is being studied as monotherapy or in combination with pembrolizumab in NSCLC (NCT02964013). In patients with anti-PD-1/PD-L1-naive NSCLC, treatment-related adverse events (TRAEs) were observed in 85% of cases, with pruritus (38%) and hypoalbuminemia (31%) being the most common ones. The ORR was 26%, with responses occurring in both PD-L1-positive and PD-L1-negative tumors. In contrast, among patients with anti-PD-1/PD-L1-refractory NSCLC, 56% of patients receiving monotherapy and 70% of patients receiving combination therapy experienced TRAEs. The most common adverse events reported were rash and fatigue, affecting 21% of patients on monotherapy, and pruritus (36%) and fatigue (24%) in patients treated with combination therapy. The confirmed ORR was only 6% for monotherapy and 3% for combination therapy. Such results highlight that vibostolimab combined with pembrolizumab exhibited favorable tolerance and showed efficacy in the anti-PD-1/PD-L1-naive population, as well as in both patient subgroups with PD-L1 TPS >1% or <1%. However, the anti-tumor effects of vibostolimab alone or in combination with pembrolizumab were limited in the anti-PD-1/PD-L1-refractory population [23]. Moreover, an ongoing Phase III (NCT04738487) trial is assessing pembrolizumab alone and in conjunction with vibostolimab in PD-L1 positive NSCLC patients. The available data on tiragolumab and vibostolimab indicate a need for further clarification on the appropriate setting for dual anti-TIGIT+anti-PD-1/PD-L1 therapy. The results show that higher ORRs were achieved in the anti-PD-1/PD-L1-naïve population, suggesting that administering the combination therapy upfront may be optimal for preventing or delaying the development of immune checkpoint inhibitor (ICI) resistance. Conversely, the ORR was significantly lower among the anti-PD-1/PD-L1-refractory population, highlighting the limitations in treating acquired ICI resistance [24].
Other combinations of anti-TIGIT and anti-PD-L1 or anti-PD-1 have shown promising activity in NSCLC. The Phase II ARC-7 trial (NCT04262856) is currently investigating the combination of the anti-TIGIT domvanalimab and zimberelimab (an anti-PD-1 drug) on PD-L1-positive locally advanced or metastatic NSCLC patients. In addition, a phase II study (NCT 04791839) is evaluating the use of domvanalimab + zimberelimab along with etrumadenant (an adenosine receptor antagonist) in previously treated NSCLC patients [25]. A Phase III (NCT04746924) study is underway to assess the effectiveness of ociperlimab + tislelizumab, as opposed to pembrolizumab, in previously untreated patients with advanced NSCLC and PD-L1 tumor cell ≥ 50% expression [26].
In a Phase I trial (NCT03119428), the anti-TIGIT antibody etigilimab was tested alone or in combination with the anti-PD-1 antibody nivolumab in patients with locally advanced or metastatic solid tumors. The most reported adverse events in Phase Ia and Ib were rashes, nausea, fatigue, and a decreased appetite. Six patients experienced severe TRAEs, while a few patients showed stable disease or partial response. The median PFS was approximately 56 days in Phase Ia and 57 days in Phase Ib. The study also identified evidence of etiligimab's dose-dependent immune modulation through flow cytometry and PCR biomarker correlative analyses, including the activation of immune T-cell subpopulations and the decrease in peripheral Tregs [27]. Although promising results were documented in terms of safety and antitumor activity during Phase Ia, Phase Ib was not carried on due to the sponsor's decision [28,29].
Many other human anti-TIGIT mAbs are currently being tested in Phase I/II clinical trials in combination with PD-1/PD-L1 blockade or chemotherapies for the treatment of advanced lung cancer. Preliminary results show that a combination of these agents with PD-1/PD-L1 inhibition in NSCLC leads to higher response rates compared with PD-1/PD-L1 inhibition alone, possibly due to the synergistic mechanisms of action, including the increased activation of NK cells and CD8+ TILs [30][31][32]. Further studies will be necessary to determine the proper sequence of specific therapy regimens of these mAbs, and identify which patients would benefit from early chemotherapy combinations [33]. Additional research is required to comprehensively understand the approaches to enhance immune regulation in SCLC patients, possibly prioritizing the investigation of the molecular subtypes.
Anti-TIGIT strategies are being investigated in other human malignancies aside from lung cancer, with promising initial results [28]. In the future, anti-TIGIT therapies could become a standard-of-care; identifying an inexpensive and easily accessible predictive biomarker would aid greatly in patients' stratification and management, with overall improved patient care.

TIGIT as an Immunohistochemical Biomarker: Current Knowledge
Numerous studies have investigated the expression and prognostic significance of TIGIT in various human cancers, including melanoma, NSCLC, hepatocellular carcinoma, thyroid cancer, gastric cancer, and colorectal cancer. These studies have reported varying levels of TIGIT expression in different cancer types and stages, with high TIGIT expression being associated with poor prognosis in some cases and favorable prognosis in others. TIGIT immunohistochemistry (IHC) has been performed with various antibodies for various purposes, mostly in association with genomic, transcriptomic, flow cytometry, and/or fluorescence techniques, with IHC usually serving as a validation tool for TIGIT protein expression. Different scoring systems were adopted for TIGIT IHC evaluation, depending on the focus either on the tumor microenvironment or cancer cells (Table 3).
The majority of studies investigating TIGIT expression in TME across different malignancies have shown its negative impact on overall survival, progression-free survival, disease-free survival, recurrence-free survival, or its association with increased hazard for metastatic disease (Table 3). However, results are conflicting, and several studies have failed to prove a prognostic role for TIGIT expression, specifically in esophageal cancer [46], medullary thyroid carcinoma [47], NSCLC [34,48,49], and SCLC [50]. In contrast, other authors have reported a positive prognostic impact of TIGIT-enriched TME on survival, as seen in oral squamous cell carcinoma [51], breast cancer [52], and melanoma [39].   Further studies are needed to deepen our knowledge of TIGIT expression, both in the neoplastic microenvironment and in tumor cells, and its substantial correlation with the PD1/PD-L1 axis, particularly in the field of lung cancer, on which most immunotherapy trials are focused. To date, a single clinical trial evaluated the significance of TIGIT IHC as a biomarker, specifically in a prognostic way [11,22,34]. TIGIT immunohistochemical expression is currently not considered a prerequisite for the administration of TIGIT inhibitors, such as tiragolumab in non-small-cell lung cancer, for which PD-L1 positivity is deemed sufficient, and no data exist about the potential role of TIGIT expression as a predictive biomarker for response to anti-TIGIT regimens. Current clinical trials mostly utilize anti-TIGIT regimens as an addition to anti-PD-1/PD-L1 or anti-CTLA-4 inhibition, with few exceptions: a Phase I trial in which vibostolimab is administered to anti-PD-1/PD-L1-refractory NSCLC (NCT02964013), a Phase II trial in which vibostolimab is utilized in treatment naïve advanced NSCLC (NCT04165070), and a terminated Phase I trial which used etigilimab in advanced/metastatic solid malignancies, lung included (NCT03119428). These examples highlight the clinical need for a predictive biomarker of responses to anti-TIGIT therapy regimens, in which IHC could play a significant role in stratifying patients who could benefit most from the therapy and patients in which therapy could be ineffective and unnecessary, paralleling the PD-L1 experience, particularly in the lung. In this view, TIGIT IHC may reveal a theranostic utility, potentially guiding complex therapeutic approaches, and providing novel insights into the complexity of TME.

Conclusions
The importance of TIGIT as a target for immune-checkpoint inhibition in lung cancer is becoming more and more clear as clinical trials continue to progress and provide results on the therapeutic effectiveness of anti-TIGIT mAbs. As for now, the prognostic value of TIGIT expression in human malignancies, assessed with IHC, is controversial, with different results across different types of human cancer. The predictive role of TIGIT expression is understudied and largely unknown. Although solid, the current assumption that TIGIT inhibition has to rely on PD-1/PD-L1 axis inhibition, on which most clinical trials using anti-TIGIT strategies are based, may be not totally comprehensive; differences could exist between different types of human cancer in relation to the significance of TIGIT expression and its relationship with PD-1/PD-L1, and the relevance of TIGIT expression might have been overlooked, especially in anti-TIGIT monotherapy regimens trials. To investigate whether TIGIT expression in CD8+ TILs in human cancer is predictive to anti-TIGIT therapy could provide insights into a novel and inexpensive tool for patients' treatment stratification, thus potentially reducing overtreatment and collateral effects.