LAG-3, TIM-3, and TIGIT: distinct functions in immune regulation

Summary

LAG-3, TIM-3, and TIGIT comprise the next generation of immune checkpoint receptors being harnessed in the clinic. Although initially studied for their roles in restraining T cell responses, intense investigation over the last several years has started to pinpoint the unique functions of these molecules in other immune cell types. Understanding the distinct processes that these receptors regulate across immune cells and tissues will inform the clinical development and application of therapies that either antagonize or agonize these receptors as well as the profile of potential tissue toxicity associated with their targeting. Here, we discuss the distinct functions of LAG-3, TIM-3, and TIGIT, including their contributions to the regualtion of immune cells beyond T cells, roles in disease, and the implications for their targeting in the clinic.

Etoc blurb:

LAG-3, TIM-3, and TIGIT comprise the next generation of immune checkpoint receptors being harnessed in the clinic. Kuchroo, Anderson, and Joller review advances in our understanding of the specialized functions of these receptors, focusing on their roles in immune cells beyond T cells and how this knowledge can be harnessed for rational development of therapies that maximize efficacy and mitigate immune toxicity.

In this review, Joller et al. highlight the advancement in understanding the specialized functions of LAG-3, TIM-3, and TIGIT, focusing on their roles in immune cells beyond T cells and how this knowledge can be harnessed for rational development of therapies that maximize efficacy and mitigate immune toxicity.

Introduction

Co-inhibitory receptors, also known as immune checkpoints, are well-established regulators of T cell responses that are induced with varying kinetics upon T cell activation and have an important function in contracting effector T cell responses. In settings of chronic antigen stimulation, such as in cancer or persistent infections, immune checkpoints are highly and constitutively expressed on T cells, thereby limiting excessive activation that would lead to host tissue damage and activation of auto-aggressive T cell responses. Hence, checkpoint receptors are essential for maintaining homeostasis and limiting autoimmunity, but also can limit pathogen clearance and anti-tumor immunity. Among checkpoint receptors, CTLA-4 and PD-1 are the most well-studied and successfully targeted therapeutically. They act as potent on/off immune switches, but high therapeutic impact can correlate with high toxicity, particularly when targeted in combination, that manifests in the form of immune-related adverse events (irAEs). The next generation of immune checkpoints includes LAG-3, TIM-3, and TIGIT. In our 2016 review, we highlighted the potential specialized functions of these receptors¹. Indeed, it is now appreciated that these receptors may act as checkpoints on other immune cell types in addition to T cells. Here we provide an update, expanding on recent findings that establish the unique functions of these receptors and how that may guide their application in the clinic.

LAG-3

LAG-3, a structural homolog of CD4, is induced upon T cell stimulation and its expression is maintained in settings of sustained antigenic stimulation, such as in chronic infections or cancer ^2–4. While LAG-3 is also expressed on other immune cells including NK cells, B cells⁵, and plasmacytoid dendritic cells (DCs), most studies investigating LAG-3 have focused on its function in T cells, where it was shown to act as an inhibitory receptor, downregulate T cell responses (reviewed in ⁶) and contribute to T cell exhaustion⁷. Furthermore, LAG-3 is expressed on a number of regulatory T cell populations including IL-10 producing Type I regulatory (Tr1) cells and FOXP3⁺ regulatory T cells (Tregs), where it contributes to the immunosuppressive properties of these cells ^8,9. However, this seems to be context dependent as LAG-3 expression is associated with enhanced suppression at steady state and during infections ^8,10, but limits Treg function in autoimmune diabetes ¹¹.

In line with its structural homology with CD4, the canonical ligand for LAG-3 is MHC II ¹². However, a number of additional ligands have been identified including LSECtin (expressed by hepatocytes), Galectin-3, FGL1 (produced by hepatocytes and tumor cells), the TCR-CD3 complex itself, and misfolded preformed fibrils of α-synuclein in the brain ^1,13–15 (Figure 1). While this broad array of ligands potentially allows for LAG-3 to modulate the immune response in different ways, it is still largely unclear, whether different LAG-3-ligand interactions translate into distinct functional outcomes. While, FGL1 binding seems to be dispensable for suppressing autoimmune diabetes, LAG-3 mutations that disrupt binding to MHC II (but not FGL1) have a more profound effect and resemble the complete loss of LAG-3 in models of autoimmunity and cancer¹⁶. However, whether this mutation also affects LAG-3 binding to additional ligands, particularly the TCR-CD3 complex, has not been determined. Hence, future studies will need to address the relative importance of the different LAG-3 ligands depending on the disease and anatomical context.

Figure 1. The LAG-3, TIM-3, and TIGIT pathways.

A)LAG-3: LAG-3 can bind to MHC II, LSECtin, GALECTIN-3 (GAL-3), FGL1, and in cis to the CD3-TCR complex within the immunological synapse. EP repeats in the cytoplasmic tail of LAG-3 result in local acidification of the cytosol triggering the dissociation of phospho-Lck from either the CD4 or CD8 co-receptors, thereby limiting proximal TCR signaling. In addition, LAG-3 contains a membrane proximal FXXL motif followed by a KIEELE motif in its cytoplasmic tail, both of which have been implicated in inhibitory function. The extracellular domain of LAG-3 can be shed as soluble LAG-3 (sLAG-3) upon cleavage by the metalloproteases ADAM10 and ADAM17 as a means to rapidly control LAG-3 activity.

(B)TIM-3: GALECTIN-9 (GAL-9), HMGB1, CEACAM1, and Phosphatidyl Serine (PtdSer) bind to TIM-3. T cell (Left): In the absence of TIM-3 ligand, the adaptor BAT3 is bound to the region containing Y256 and Y263 in the TIM-3 cytoplasmic tail. BAT3 recruits phospho-LCK to promote TCR signaling. Upon ligand-binding, Y256 and Y263 become phosphorylated. This triggers dissociation of BAT3, thereby allowing FYN to bind and TIM-3-mediated inhibition to ensue. Additionally, TIM-3 may recruit the phosphatases CD45 and CD148 into the synapse to inhibit TCR signaling. DC (Right): TIM-3 binding to HMGB1 prevents HMGB1-DNA complex binding to RAGE and HMGB1 binding to TLR2,4, and IL1R, thereby limiting DC activation via these pathways. TIM-3 further inhibits activation of the inflammasome and cGAS-STING by as yet unknown mechanisms. BAT3 is expressed in DCs and restrains tolerogenic programs but little else is known regarding BAT3 and its effect on TIM-3 signaling in DCs.

(C)TIGIT: TIGIT and CD226 share the ligands CD112 and CD155 but bind them with different affinities (indicated by arrow thickness). TIGIT additionally binds the FAP2 protein expressed by F. nucleatum. CD112 and CD155 also serve as ligands for CD112R and CD96, respectively, completing a complex receptor-ligand network. TIGIT inhibits positive signaling via CD226 through competition for its ligands and by disrupting CD226 homodimerization, thereby disabling ligand binding and phosphorylation of the ITT motif in the CD226 tail. TIGIT further contains an ITT-like and an ITIM motif, which become phosphorylated upon ligand binding, recruiting the adapters GRB2 and β-arrestin and, subsequently, SHIP1 into the immunological synapse to inhibit TCR signaling. Lastly, TIGIT binding to CD155 results in phosphorylation of the ITIM motif in the cytoplasmic tail of CD155, delivering an inhibitory signal into the CD155-expressing cell.

Signaling and inhibitory function of LAG-3

For years, the fact that MHC II is the canonical ligand for LAG-3, yet LAG-3 could suppress both CD4⁺ and CD8⁺ T cell responses posed a conundrum. However, the identification of additional LAG-3 ligands suggested that these can compensate for the lack of LAG-3-MHC II interactions in cells other than T helper (Th) cells. Furthermore, stable MHC-II-peptide complexes expressed on APCs can also bind LAG-3 CD8⁺ T cells to deliver a weak inhibitory signal during priming¹⁷. Notwithstanding different receptor-ligand interactions, it is well-established that the cytoplasmic tail of LAG-3 is essential for its inhibitory function. The LAG-3 tail contains a number of unconventional but evolutionarily conserved signaling motifs, including a membrane proximal FXXL motif followed by a KIEELE motif and glutamic acid-proline (EP)-rich repeats ^18,19. Early studies showed that signal transduction through the KIEELE motif is required for the inhibition of IL-2 production by T cells ¹⁸. However, more recent work has shown that deletion of the KIEELE motif did not abrogate LAG-3 inhibitory function ¹⁹. Rather, LAG-3 binding to stable MHC II-peptide complexes transduces two distinct inhibitory signals, one via the FXXL motif and one via the EP-rich repeats, both of which inhibit T cell activation and can compensate for each other.

Importantly, that LAG-3 can interacts in cis with the TCR-CD3 complex itself provides an explanation for its ability to limit activation of both CD4⁺ and CD8⁺ T cells ¹⁵. Upon TCR stimulation, LAG-3 associates and tracks with the TCR-CD3 complex into the immunological synapse. Within the synapse, the accumulation of EP-rich repeats results in local reduction of the pH, which causes the dissociation of phospho-Lck from the CD4 or CD8 co-receptors, thereby limiting phosphorylation of ZAP70 and consequently TCR signaling ¹⁵ (Figure 1A). Therefore, in T cells, the TCR-CD3 complex serves as a cis-LAG-3 ligand and hence low expression of MHC II or other ligands may not be limiting for LAG-3-mediated inhibition.

LAG-3 signaling itself is also regulated via the cleavage of the extracellular LAG-3 domain by the metalloproteases ADAM10 and ADAM17 ²⁰ (Figure 1A). LAG-3 shedding is an important mechanism for control of LAG-3 activity in cancer as mutant mice expressing cleavage-resistant LAG-3 have impaired anti-tumor immunity ²¹. Interestingly, abrogation of LAG-3 cleavage most strongly affected activation of CD4⁺ Th cells, thereby limiting their capacity to provide help to CD8⁺ T cells. Importantly, uncleavable LAG-3 also reduced responsiveness to PD-1 blockade ²¹, suggesting that recovery of anti-tumor CD8⁺ T cell responses post-therapy not only relies on the reduction of inhibitory signals into the cytotoxic T cells themselves but also on CD4⁺ T cells that provide crucial help. Notably, in cancer patients, low ADAM10 expression is inversely correlated with high LAG-3 surface expression on circulating CD4⁺ T cells in the blood and positively correlated with poor prognosis ²¹. Whether soluble LAG-3 (sLAG-3), the product of LAG-3 shedding, itself has a biological function is still unclear but in light of its short half-life (<4hrs), its biological impact is likely limited ²⁰. Nevertheless, several studies have explored the utility of sLAG-3 as a potential prognostic marker and shown that high levels of sLAG-3 correlate with higher immune responses and less cancer progression, likely due to less inhibition as LAG-3 is shed ²². These studies highlight the potential of sLAG-3 as a biomarker for patient selection in the future. However, further studies are needed to confirm this link and test its applicability across a wide variety of therapy combinations.

Despite recent progress in our understanding of LAG-3 biology, many open questions relating to the inhibitory signal delivered through LAG-3 remain. It will be important to determine how and to what extent different LAG-3 ligands trigger downstream signaling modalities in potentially different physiological settings and disease contexts. In particular, it will be important to determine which interactions and signal transduction modalities are targeted by current anti-LAG-3 immunotherapies. This will require structural insights that may build upon those obtained from the recently solved structure of the LAG-3 ectodomain ²³. Combining this with the recent advances in our understanding of LAG-3 signaling and binding partners ¹⁵ may uncover whether alteration in binding patterns can alter the mode of action and thus provide means to improve the efficacy or broaden the scope for current LAG-3 directed therapies.

Moving LAG-3 from bench to bedside

Suboptimal response rates and frequent irAEs represent the biggest challenge for checkpoint blockade therapy. Consequently, therapies targeting the next generation of co-inhibitory receptors are aimed at improving response rates without concomitantly increasing irAEs. Despite the improvements that checkpoint therapy has brought about, compensatory mechanisms come into play when a single co-inhibitory receptor is inactivated, particularly as many co-inhibitory receptors are co-expressed and co-regulated as a module on the surface of CD4⁺ and CD8⁺ T cells ^24,25. LAG-3 is co-expressed with other co-inhibitory receptors on both CD4⁺ and CD8⁺ tumor infiltrating lymphocytes (TILs) in numerous pre-clinical cancer models as well as in a broad range of human tumors including melanoma, HCC, NSCLC, HCSCC and ovarian cancer, where LAG-3 expression correlates with poor prognosis ²⁶. While PD-1 and CTLA-4 primarily inhibit CD28-mediated co-stimulation, LAG-3 inhibits the TCR signal itself, providing a rationale for combined targeting with these co-inhibitory receptors. The distinct inhibitory mechanisms of LAG-3 and PD-1 provide a solid rationale for their therapeutic co-blockade. Indeed, although LAG-3 blockade alone showed limited benefit in preclinical cancer models, its combination with PD-1 blockade led to a dramatic improvement of tumor control when compared to anti-PD-1 alone in both solid and hematologic cancer models ^2,27. This fueled the development of numerous anti-LAG-3 antibodies for evaluation in clinical trials across a broad spectrum of cancers (see ²⁸ for a recent summary of current activities).

The RELATIVITY-047 trial showed that the promising results from preclinical cancer models indeed translated to improved anti-tumor responses in melanoma patients and led to the FDA approval of the combination of anti-LAG-3 and anti-PD-1 therapy for melanoma (Opdualag) in early 2022. This global, randomized, double-blinded phase II/III study of patients with unresectable or metastatic melanoma found progression-free survival (PFS) to be more than double in Opdualag-treated patients compared to patients treated with anti-PD-1 (Nivolumab) alone (10.1 months versus 4.6 months, respectively) ²⁹. Although a highly promising trend toward improved overall survival (OS) was also observed (OS not reached with Opdualag vs. 33.2 months with Nivolumab alone), this did not reach significance, likely due to the study being insufficiently powered to detect these differences ³⁰. Thus, patient data obtained over the next years will determine whether the trend towards increased OS holds true. Importantly, the improved responses observed with the combination therapy did not come at the expense of higher toxicity as tolerability was comparable with anti-PD-1 alone and no new safety signals were observed ²⁹. The most common irAEs were comparable following combination therapy or anti-PD-1 alone (thyroiditis 18% vs 13.9%, rash 9.3% vs 6.7%). Differences in less frequent irAEs did not reach significance as the number of patients was still limited, but it will be important to determine whether small differences in hepatitis (5.6% vs. 2.4%) or pneumonitis (3.7% vs. 1.7%) are further confirmed in larger cohorts ²⁹. This is important as unique or localized irAEs could serve as indications for tissue contexts that may be particularly responsive to LAG-3 therapy, similar to what has been observed for anti-PD-1 therapy ³¹. Clinical trials targeting LAG-3 alone or in combination with other therapies are ongoing and are expected to expand their application to additional tumor types and modalities. Current studies include treatment of both solid and hematologic tumors with combination therapy of approved checkpoint inhibitors with different anti-LAG-3 antibodies as well as bispecific antibodies targeting LAG-3+PD-1, LAG-3+PD-L1 or LAG-3+CTLA-4 ²⁸. Future studies may expand to include further co-inhibitory receptors such as TIM-3 and TIGIT, which could have a more favorable safety profile compared to PD-1-, PD-L1-, or CTLA-4-targeting agents, but will also include combinations with classical cancer therapies such as chemo- or radiotherapy. Research in the next years should help guide the selection of potential combination partners and aim to gain a more complete understanding of what drives synergistic effects of LAG-3 blockade with other therapies to allow for further improvement of efficacy while limiting adverse effects.

TIM-3

T cell Immunoglobulin and mucin domain containing–3 (TIM-3) was identified as a co-inhibitory receptor that regulates Type I immunity due to its expression on differentiated IFN-γ-secreting CD4⁺ and CD8⁺ T cells in both mice and humans ³². Subsequent studies expanded its expression profile to natural killer (NK) cells, myeloid cells (macrophages and DC), mast cells, and, most recently, B cells ^33,34. As its name implies, TIM-3 has an immunoglobulin variable (IgV) domain, and mucin domain, followed by a transmembrane region and a cytoplasmic tail that lacks known inhibitory signaling motifs but has 6 tyrosines of which Y256 and Y263 become phosphorylated upon ligand binding (Figure 1B). Four ligands - GALECTIN-9, phosphatidylserine, HMGB1, and CEACAM1 - have been identified for TIM-3, each binding to different regions on the TIM-3 IgV domain. GALECTIN-9, a C-type lectin, binds to N-linked carbohydrates on the IgV whereas phosphatidylserine binds in a pocket shared by all members of the TIM family of proteins that is framed by the FG and CC” loops of the IgV domain and requires coordinated calcium binding ^35,36. HMGB1 binding, although not precisely mapped, also depends on this pocket ³⁷. CEACAM1 binds to the CC’ and FG loops of the TIM-3 IgV. Notably, examination of the ligand blocking properties of anti-TIM-3 antibodies that have shown functional efficacy shows interference of TIM-3 binding to phosphatidylserine and CEACAM-1 ³⁸.

TIM-3 Signaling

The lack of canonical inhibitory signaling motifs in the TIM-3 tail has posed a challenge to understanding its mechanism of action. Although much remains to be learned, it has been shown that TIM-3 can be recruited to the immunological synapse³⁹ in both human and murine T cells ^39,40. In human CD8⁺ T cells, TIM-3 has been shown to recruit the phosphatases CD45 and CD148, to disable TCR signaling. Further, both GALECTIN-9 and CEACAM-1 binding has been shown to trigger phosphorylation of Y256 and Y263 in murine T cells ^41,42. The region containing Y256 and Y263 mediates binding to HLA-B-associated transcript 3 (BAT3), an adaptor protein that recruits the catalytically active form of LCK. Phosphorylation of Y256 and Y263 triggers release of BAT3 from the TIM-3 cytoplasmic tail, allowing for TIM-3-mediated inhibition ⁴¹. Interestingly, the src-kinase Fyn, which has been shown to be involved in anergy induction ^43,44, can also bind in this region ⁴⁵. Given these observations, it is possible that a BAT3-FYN switch determines whether TIM-3 is permissive versus inhibitory for TCR signaling. Moreover, a proteomics analysis of proteins that bind the TIM-3 tail in CD4⁺ T cells using a tagged version of TIM-3 identified several additional intracellular binding partners involved in TCR signaling, such as VAV1, LCK, GRB2, SHP1, CBLB, PI3KR1, UBASH3A ⁴⁶. It is important to note that this study confirmed the association of TIM-3 with BAT3, LCK, and CD45. The above studies examined primary human and mouse CD4⁺ and CD8⁺ T cells, T cell lymphomas, and transfectant cell lines, yet TIM-3 has also been shown to function as a checkpoint receptor in DCs ⁴⁷ (Figure 1B; discussed below). Of note, BAT3 is expressed in DCs where it has an important role in restraining tolerogenic programs ⁴⁸, although whether this is due to inhibition of TIM-3 signaling requires investigation. Thus, to solidify the TIM-3 mechanism of action in T cells and DCs across species, more investigation into how TIM-3 signals in different cell types is needed.

Inhibitory role of TIM-3 in disease

TIM-3 was initially studied in the context of autoimmunity and self-tolerance due to its expression and regulation of Type I immunity ³². Interestingly, some TIM-3 expression has also been noted on IL17-secreting CD4⁺ (Th17) cells, which are also key mediators of autoimmune tissue inflammation ⁴⁹. Accordingly, TIM-3 blockade was shown to worsen disease in multiple preclinical models of autoimmune inflammation including Experimental Autoimmune Encephalomyelitis (EAE) ³², inflammatory bowel disease (Li et al., 2010), and diabetes ⁵¹. Indeed, TIM-3 expression has been found to be lowly expressed on T cells in patients with different autoimmune diseases, including multiple sclerosis (MS) ^52,53, ulcerative colitis ⁵⁴, rheumatoid arthritis ⁵⁵, and psoriasis ⁵⁶, compared to T cells from healthy controls. Moreover, therapies that ameliorate autoimmunity have been associated with restoration of TIM-3 expression on patient’s T cells. These include Type I Interferon treatment in MS patients ⁵³ and methotrexate or anti-IL-6 (Tocilizumab) in RA patients ⁵⁷. Interestingly, Type I Interferon has been shown to induce a regulatory network that drives a co-inhibitory gene module that includes not only TIM-3 but also other checkpoint receptors in human T cells ⁵⁸. In murine T cells, the co-inhibitory gene module, including TIM-3, is induced by IL-27 ²⁴, which is downstream of Type I interferon ⁵⁹.

The importance of TIM-3 for self-tolerance is further exemplified by the demonstration that TIM-3-deficient mice and mice treated with a TIM3-Ig fusion protein exhibit defects in the induction of antigen-specific tolerance after administration of high-dose soluble antigen ⁶⁰. Similarly, the induction of allogeneic transplant tolerance by transfusion of allogeneic donor splenocytes together with CD154 (CD40L) blockade is abrogated in TIM-3-deficient mice and in mice treated with TIM-3-Ig ⁵¹. Although these effects may reflect the function of TIM-3 in conventional CD4⁺ T cells. TIM-3 may also help maintain tolerance via its action in CD4⁺ Treg as TIM-3 is highly expressed on Treg that are found at sites of tissue inflammation but not in Treg present in the circulation or in lymphoid tissues. TIM-3⁺ Treg are highly suppressive ^61–64 and forced expression of TIM-3 on Treg is sufficient to confer an effector-like phenotype accompanied by a shift towards glycolytic metabolism ⁶⁴. Thus, TIM-3 may have a specialized role in preserving homeostasis by resolving inflammation at tissue sites via its action in Treg.

Although the inhibitory role of TIM-3 was initially demonstrated in the context of autoimmunity, TIM-3 gained prominence with the discovery that it marked the most terminally dysfunctional subset of CD8⁺ T cells in chronic viral infections and cancer in both pre-clinical models and in humans ^65–71 and that combined blockade of TIM-3 and PD-1 could improve viral and tumor clearance over that observed with PD-1 pathway blockade alone ^65,68,72,73. The observations drove the development of agents that bind to TIM-3 for clinical translation. Currently, there are multiple clinical trials investigating anti-TIM-3 antibodies in cancer (discussed further below).

Although the inhibitory function of TIM-3 has been disputed, germline loss-of-function mutations in HAVCR2 have been associated with two inflammatory diseases, subcutaneous-panniculitis-like T cell lymphoma (SPTCL) and hemophagocytic lymphohistiocytosis (HLH), that are characterized by uncontrolled activation of myeloid cells and CD8⁺ T cells with high serum levels of IL-1β, IL-18, TNF, CXCL10, and soluble CD25 ^74,75. The mutations in TIM-3 that are associated with these diseases are located in the GALECTIN-9 binding pocket of the IgV domain and lead to misfolded TIM-3 that aggregates intracellularly. Although SPTCL is classified as a T cell lymphoma, treatments that suppress inflammation such as corticosteroids, IL-1R antagonist (Anakinra) ⁷⁴, methotrexate, or cyclosporin A have been used successfully to ameliorate disease. The association of TIM-3 loss-of-function mutations with CD8⁺ T cell and myeloid cell dysregulation leading to inflammatory disease solidifies the inhibitory function of TIM-3.

Antagonism of TIM-3 with stemness

That TIM-3 is a more reliable marker than PD-1 for terminally dysfunctional CD8⁺ T cells is now well-established. Indeed, a distinguishing feature of TIM-3⁺ cells is their low expression of TCF-1, a transcription factor that maintains stemness and restrains effector differentiation ^76,77. Thus, TIM-3 and TCF-1 expression mark opposing ends of the spectrum of T cell functionality. The anti-correlation of TCF-1 and TIM-3 raises the important issue of whether TCF-1 can either directly or indirectly regulate TIM-3. TCF-1 can function as either a repressor or trans-activator of gene expression ⁷⁶. Examination of differentially expressed genes in P14 CD8⁺ T cells that either overexpressed TCF-1 or were TCF1-deficient compared to WT cells in LCMV clone 13-infected mice, identified Bcl6 as a candidate TCF-1-induced target, and Havcr2 (encodes TIM-3), Prdm1, and Cish as candidate TCF-1-repressed targets ⁷⁸. Chromatin immunoprecipitation assays confirmed TCF-1 binding to a known regulatory region located in the third intron of Prdm1 (encodes BLIMP-1) ⁷⁹ that has been shown to be important for TCF-1 repression of BLIMP-1 in CD4⁺ T follicular helper (Tfh) cells ⁸⁰. Notably, BLIMP-1 has been shown to induce TIM-3 expression ²⁴. TCF-1 also bound to a region upstream of the TIM-3 transcription start site ⁷⁸. Together these data indicate potential direct and indirect regulation of TIM-3 by TCF-1 via direct repression of TIM-3 itself and indirect repression of BLIMP-1-driven transactivation. Whether these regulatory nodes are part of a larger TCF-1-driven regulatory network that underlies the stemness to dysfunction differentiation trajectory of CD8⁺ T cells remains to be elucidated.

Broadening the scope: TIM-3 as a checkpoint in myeloid cells

Although originally identified and studied as a T cell checkpoint, that TIM-3 also acts as a checkpoint in myeloid cells is now appreciated. TIM-3 has been shown to regulate DC activation by at least two mechanisms. First, TIM-3 can interfere with nucleic acid sensing by sequestering HMGB1 and preventing its binding to RAGE and preventing activation of cGAS-STING ^37,81 (Figure 1B). TIM-3 can also interfere with HMGB1 binding to Toll-like receptors (TLRs) 2, 4, and IL-1R. The inhibition of nucleic acid sensing can suppress response to chemotherapies that induce cell death and HMGB1 release. Indeed, blockade of TIM-3 can improve the response to placitaxel chemotherapy in breast cancer ⁸¹. Notably, this effect depended on DCs, HMGB1, and GALECTIN-9. That TIM-3 may interfere with sensing via TLRs has also been demonstrated in monocytes and macrophages. TIM-3 blockade or siRNA-mediated TIM-3 knock-down in cell lines improves response to various TLR agonists and TIM-3 blockade in vivo exacerbates sepsis in a TLR4-dependent manner ^82,83. TLR activation is the first step in activation of the inflammasome. Accordingly, TIM-3 has been shown to dampen inflammasome activation in DCs ⁴⁷. Notably, this effect of TIM-3 in regulating inflammasome activation may be a critical component of the effect of therapies involving TIM-3 blockade in improving anti-tumor immunity. The mechanism by which TIM-3 achieves these effects in monocytes, macrophages, and DC is not known. Elucidation of the TIM-3 signaling pathway in myeloid cells will help in this regard.

The effects of TIM-3 on myeloid cells may further be clinically relevant in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia (AML). TIM-3 has been reported to be expressed on hematopoietic stem cells (HSCs) in MDS ⁸⁴ and on leukemic stem cells (LSCs) in AML ^85–87. In these diseases, high TIM-3 expression is associated with more aggressive disease. It has been proposed that TIM-3⁺ AML cells secrete GALECTIN-9, which feeds back to promote LSC self-renewal ⁸⁶. Currently, anti-TIM-3 is being investigated in clinical trials in both MDS and AML with promising results (discussed further below). Whether TIM-3 blockade acts on HSCs or LSCs in these diseases and/or on T cells and myeloid cells remains to be determined. It will be particularly interesting to study how TIM-3 regulation of the inflammasome impacts on MDS and AML as IL-1 is needed for MDS and AML cell survival and growth, but IL-1 is produced by pyroptotic death. Thus, TIM-3 may have been potentially co-opted by MDS/AML cells to prevent their death by pyroptosis and, concomitantly, prevent activation of DCs that can stimulate anti-tumor immunity. In this scenario, anti-TIM-3 would promote induction of pyroptotic tumor cell death and, concomitantly, induce activation of DCs to unleash immune responses against malignant cells.

TIM-3 Clinical Trials

The ample data in pre-clinical cancer models and in in vitro cultures of T cells from patients with advanced cancer showing the effects of TIM-3 blockade, particularly in conjunction with PD-1 blockade, led to the development of agents to target TIM-3 in cancer. Currently there are multiple clinical trials ongoing with anti-TIM-3 in patients with MDS, AML, and solid tumors across various indications (Table 1). The two most developed agents are Sabatolimab and Cobolimab. Sabatolimab is being investigated in MDS and AML either alone or in combination with hypomethylating agents (HMAs; Azacitidine or Decitabine) and/or BCL-2 inhibitor (Venetoclax). Notably, Sabatolimab was granted Fast-Track designation by the FDA and orphan drug status by the European Commission for the treatment of MDS in combination with HMAs based on promising phase 1 trial data showing 50% and 84.6% objective response rate (ORR) in high-risk and very high-risk MDS, respectively ⁸⁸. Importantly, the comination of Sabatolimab with HMAs appears to be well-tolerated with Sabatolimab + Decitabine showing 4/69 patients (~6%) with irAEs > grade 3, Sabatolimab + Azacitidine having no irAEs > grade 3, and neither combo having irAEs > grade 4 ⁸⁹ Cobolimab in combination with Dostarlimab (anti-PD-1) achieved a 42.9% ORR in a phase 1 study in patients with non-small cell lung cancer (NSCLC) and was well tolerated with no serious treatment-related adverse events ⁹⁰. Cobolimab is currently being investigated in phase 2 and 3 trials in lung, liver, melanoma, and a few other solid cancers in combination with anti-PD-1 and/or chemotherapy (Docetaxel) (Table 1).

Table 1.

Clinical activity targeting TIM-3.

Trial name/ identifier	Phase	Indication	Format	⍺TIM-3 regimen	Comparison	Results Expected	Sponsor
Sabatolimab and Combinations
STIMULUS-MDS3 (NCT04812548)	2	High/very high risk MDS	IgG4 (S228P)	Sabatolimab + Azacitidine + Venetoclax		2023	Novartis
NCT04623216	1b/2	AML	IgG4 (S228P)	Sabatolimab		2027
NCT04878432	2	MDS	IgG4 (S228P)	Sabatolimab + Azaciticidine/Decitabine		2025
NCT05367401	1b/2	AML/RR-AML/high risk MDS	IgG4 (S228P)	Sabatolimab		2029
NCT05201066	2	MDS, CMML	IgG4 (S228P)	Sabatolimab + Magrolimab +/− Azacitidine		2028, roll over study	Novartis
STIMULUS-MDS1 (NCT03946670)	2	High/very high risk MDS	IgG4 (S228P)	Sabatolimab + Azaciticidine/Decitabine		2024	Novartis
SIMULUS-AML1 (NCT04150029)	2	AML	IgG4 (S228P)	Sabatolimab + Azacitidine + Venetoclax		2026	Novartis
STIMULUS-MDS2 (NCT04266301)	3	MDS, CMML	IgG4 (S228P)	Sabatolimab + Azacitidine		2027	Novartis
Cobolimab and Combinations
COSTAR Lung (NCT04655976)	2/3	NSCLC	IgG4 (S228P)	Cobolimab + Dostarlimab + Docetaxel	Dostarlimab + Docetaxel, Docetaxel	2026	Glaxo-Smith Kline
NCT03680508	2	Liver cancer	IgG4 (S228P)	Cobolimab + Dostarlimab		2025	Glaxo-Smith Kline
NCT04139902	2	Melanoma	IgG4 (S228P)	Cobolimab + Dostarlimab	Dostarlimab + Docetaxel, Docetaxel	2027	Glaxo-Smith Kline

As TIM-3 trials are underway, a key question that arises is discerning the mechanism of action underlying observed clinical effects given that anti-TIM-3 has the potential to modulate T cells, myeloid cells, and tumor cells themselves, as in the case of AML. Another consideration is whether selective targeting to specific cell types may be beneficial in certain contexts. Approaches employing dual binding modalities such as in bi-specific antibodies can be envisioned to target TIM-3 blockade to distinct cell populations. Such approaches require a deeper understanding of how anti-TIM-3 achieves it effects in vivo. Similarly, whether anti-TIM-3 antibodies should have a silent of functional Fc region is debated. Notably, both Sabatolimab and Cobolimab have an IgG4 Fc that is mostly silent but is engineered with an S228P mutation to prevent Fab arm exchange (Table 1).

TIGIT

In 2009, several groups identified TIGIT, also called Vsig9, Vstm3, and WUCAM, as a new coinhibitory receptor ^91–93. TIGIT expression is transiently induced on T cells upon TCR stimulation and is stably expressed on a subset of NK cells and several T cell populations, including Tregs, Tr1 cells, T follicular helper (Tfh) cells, and dysfunctional CD8⁺ T cells ¹. More recently, TIGIT expression was also reported on B cells, particularly in B cells with regulatory functions ^94,95. Emerging data further suggest that in disease settings, TIGIT can also be expressed on innate immune cells including ILCs and macrophages ^96,97, indicating that in these contexts it may have functions beyond its classical role in T and NK cells.

TIGIT has two ligands, CD155 and CD112, which are expressed on APCs and a variety of non-hematopoietic cell types including tumor cells. Both ligands are shared with the co-stimulatory receptor CD226 (DNAM-1) and make up the core of receptor-ligand interactions of this pathway. Two additional molecules, CD96 and CD112R, share ligands with TIGIT and thus contribute to a complex co-stimulatory/co-inhibitory network that is outlined in Figure 1C. Additionally, the FAP2 protein derived from Fusobacterium nucleatum, a microbe found in colon, breast, and head and neck tumors, has also been reported to bind to TIGIT ⁹⁸. Recently, CD114 (Nectin-4) was reported as an additional ligand for human, but not murine TIGIT or CD226 ⁹⁹; however, its physiological relevance still needs to be substantiated given it’s low affinity ¹⁰⁰.

Signaling and inhibitory function of TIGIT

By binding to its ligands, TIGIT can inhibit both T cell activation and NK function in a cell-intrinsic manner ^93,101. The intracellular domain of TIGIT contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tail tyrosine (ITT)-like motif that are both highly conserved between mice and humans ^91,93,102 However, their respective contribution to TIGIT downstream signaling pathway(s) remain unclear. Initial studies performed in NK cell lines suggested that Fyn and Lck-mediated phosphorylation of tyrosine residues in both motifs was followed by binding of the adaptor proteins Grb2 and β-arrestin 2, which in turn, recruited SHIP1 and thus interfered with PI3K, MAPK, and NF-κB pathway activation ^103,104 (Figure 1C). In mouse NK cells, the two motifs seem redundant and only mutation of both phosphorylation sites abolishes the inhibitory function of TIGIT ¹⁰⁵. In human NK cells this is less clear as contradicting reports suggesting an essential role for either the ITIM or the ITT motif exist ^93,103,104.

A recent study found ligand engagement to induce phosphorylation in both the ITIM and the ITT motif in a human T cell line but could not detect direct binding of common adapter and downstream signaling molecules to the TIGIT phosphodomains ¹⁰⁶. However, some adaptor proteins, including Grb2, could be pulled down from cell lysates via the phosphorylated ITIM or ITT motifs ¹⁰⁶, suggesting that these and likely other adaptor proteins may contribute to TIGIT signaling. The absence of classical signaling adaptors bound at the TIGIT tail raises the possibility that the inhibitory signal delivered through TIGIT is propagated via a non-classical signaling pathway which may further differ depending on the cell type. Furthermore, additional context dependent signals, such as the inflammatory or immune suppressive environment present in tissues with autoimmune inflammation or tumors, respectively, may further modulate TIGIT signaling. Hence, it will be important to verify the mode and players of TIGIT signaling in primary cells under physiological conditions and in different disease contexts.

TIGIT also actively competes with CD226 for binding to their ligands and due to its higher affinity limits CD226-mediated co-stimulation ^{91,102,105,107} (Figure 1C). Furthermore, TIGIT can directly bind to CD226 in cis, thereby disrupting the CD226 homodimer and its ability to deliver costimulatory signals ¹⁰⁸. Interestingly, it seems that presence of either TIGIT or PD-1 can limit CD226 phosphorylation and hence its co-stimulatory activity ¹⁰⁶, suggesting a convergence of signals from the two pathways in limiting CD226 co-stimulation. In contrast to PD-1, TIGIT does not require its intracellular signaling domain to limit CD226 phosphorylation, suggesting that it mainly acts through competition in this context ¹⁰⁶. While this likely represents an important mechanism of TIGIT-mediated inhibition in dysfunctional CD8⁺ T cells and NK cells that co-express both CD226 and TIGIT, its impact in cells that express no or limited amounts of CD226, such as Tregs, is likely limited. Furthermore, it is clear that several downstream effects of TIGIT contribute to its overall inhibitory function as TIGIT blockade is effective even in the absence of CD226 ^106,108. Finally, the inhibitory effects of TIGIT are not limited to cell-intrinsic functions but also include the inhibition of APCs through the TIGIT-CD155 interaction and indirect immune suppression by enhancing Treg function ^91,109. TIGIT is a direct target of Foxp3 and is now well-recognized as an indicator for Treg functionality ^109–111. In Tregs, TIGIT induces the suppressive mediator Fgl2, which not only confers TIGIT⁺ Treg cells with superior suppressive function but also enables selectivity towards suppressing Th1 and Th17 but not Th2 responses ¹⁰⁹. This allows TIGIT to shift the cytokine balance away from a proinflammatory Th1- and Th17-cell-dominated response towards a Th2-like response to restore homeostasis. This consideration may be particularly important depending on the disease and tissue context. Due to its inhibitory function, TIGIT plays a protective role in autoimmunity and TIGIT engagement by agonistic antibodies as well as recombinant TIGIT have been shown to ameliorate autoimmune diseases in mouse models ^91,102,112. Conversely, blockade of TIGIT has successfully been applied in numerous preclinical cancer models ^108,112 and is currently being evaluated in a large number of clinical trials in cancer patients (discussed below). ¹⁰¹

Broadening the scope – a role for TIGIT on B cells

Recent studies addressing the role of TIGIT in autoimmunity have revealed that in this context, TIGIT is not only expressed on T cells and NK cells as originally described, but also on additional cell types. B cells, particularly B cells that share multiple characteristics with Tregs, including production of IL-10, express TIGIT ^34,94,95,113. TIGIT expression on B cells is TIM-1-dependent and co-regulated with several co-inhibitory receptors, including TIM-3 and LAG-3 ⁹⁴. Interestingly, the function of TIGIT on B cells with regulatory activity shows remarkable parallels to what was observed in Tregs in that it enhances suppressive function cell intrinsically but also modulates APC function through interaction with its ligand CD155. As in Tregs, IL-10 production in B cells is TIGIT-dependent and thus TIGIT directly promotes the regulatory activity of B cells^94,109. However, the B cell-specific loss of TIGIT but not IL-10 results in spontaneous organ inflammation over time as the mice age, indicating that TIGIT promotes regulatory functions in B cells that go beyond the induction of IL-10 ⁹⁴. Indeed, TIGIT⁺ B cells suppress maturation of DCs, their production of proinflammatory cytokines, and their ability to prime T cells in vitro ¹¹³. ⁸⁸

TIGIT⁺ B cells seem to play a particularly important role in two settings – control of Tfh cells and limitation of CNS inflammation. In human B cells, TIGIT expression is induced upon B cell activation, particularly by co-stimulation through CD40L as it would occur in germinal center responses ⁹⁵. In vitro, TIGIT expression by B cells contributes to their ability to suppress Tfh cells ⁹⁵. Furthermore, DCs co-cultured with TIGIT⁺ B cells are less efficient at inducing CXCR5⁺ICOS⁺ Tfh-like cells in vitro and these cells also produce less IL-21 but more IL-10 ¹¹³. In line with these findings, abundance of TIGIT⁺ B cells and CCR6⁺ Tfh cells in humans are inversely correlated ⁹⁵. This process seems to be particularly relevant for controlling CNS inflammation as mice lacking TIGIT specifically in B cells develop spontaneous CNS inflammation as they age, while inflammation in other organs is limited ⁹⁴. This regulatory mechanism seems to be defective in MS patients as patient-derived memory B cells display decreased TIGIT expression, but a concomitant increase of CCR6⁺ Tfh cells ⁹⁵. TIGIT thus not only limits autoimmune inflammation of the CNS by limiting T cell responses directly ¹⁰¹, but also by promoting immune regulation through B cells ^94,95,113. Notably, the spontaneous development of CNS tissue inflammation has not been observed with deletion of any other checkpoint receptor including CTLA-4 and PD-1, either on T cells or B cells, underscoring the importance of TIGIT-mediated B cell regulation in this setting.

Role of TIGIT in chronic diseases

Over the last years, TIGIT has received considerable attention as a potential target in cancer therapy due to its ability to negatively regulate anti-tumor responses ^114,115. Tumor infiltrating lymphocytes (TILs), particularly Tregs and dysfunctional CD8⁺ T cells, express high levels of TIGIT while its ligands are present on tumor cells and some tumor-associated bacteria, namely Fusobacterium nucleatum ^{98,108,115,116}. Interestingly, high TIGIT expression also serves as a marker for CAR T cells with a dysfunctional phenotype present in non-responders to CAR T cell therapy ¹¹⁷. In contrast to TIM-3, which is only expressed on terminally dysfunctional CD8⁺ T cells, TIGIT is also expressed together with PD-1 on TCF-1⁺ stem-like CD8⁺ T cells that and are known to seed the CD8⁺ T cell response upon checkpoint blockade and eventually give rise to dysfunctional T cells ^118–120. Interestingly, upon chronic LCMV infection, which provides a very strong TCR stimulus, blockade of TIGIT resulted in reduction of both TIM-3⁺ terminally dysfunctional CD8⁺ T cells, as well as partial reduction of TCF-1⁺ stem-like CD8⁺ T cells, likely due to activation induced cell death as the inhibitory TIGIT signal is blocked ¹²¹. Furthermore, co-blockade of TIGIT with PD-1, PD-L1, or TIM-3 was shown to be very potent at restoring effector functions in CD8⁺ T cells both in patient-derived CD8⁺ TILs in vitro and in preclinical tumor models in vivo ^{108,112,115,116}. Hence, TIGIT synergizes with other co-inhibitory receptors to dampen effector T cell responses and promote their dysfunction.

TIGIT not only limits immune responses through its T cell intrinsic function but also by delivering an inhibitory signal into the APCs and by enhancing Treg function and stability ^{91,109,122,123}. Whether TIGIT binding to CD155 on tumor cells also delivers an inhibitory signal via CD155 as it does in DCs or Tfh ^91,95 remains to be investigated. However, it is clear that immune regulation through TIGIT⁺ Tregs plays a central role in anti-tumor immunity as loss of TIGIT on Tregs, but not other T cells restores effector T cell functions and slows tumor growth in pre-clinical cancer models ¹¹⁵. Compared to Tregs in peripheral lymphoid organs, Tregs in tumor tissue express much higher levels of TIGIT, exhibit a highly active and suppressive phenotype ^115,122, and hence can potently limit anti-tumor immunity. Overall, TIGIT may suppress cancer immunity via multiple mechanisms that include direct suppression of effector CD8⁺ T cells and enhanced suppression through Tregs. Therapeutically, TIGIT blockade removes the inhibitory signal on effector cells and can free up CD155 to engage with CD226 to deliver a costimulatory signal. Furthermore, TIGIT blockade can dampen immune suppression by Tregs. Finally, depending on the Fc portion of the TIGIT blocking antibody, TIGIT blockade may also deplete TIGIT-expressing effector as well as regulatory T cells. Whether this contributes to the therapeutic effects observed upon anti-TIGIT treatment remains to be elucidated and will have to be monitored closely in ongoing and future clinical trials as both Fc-competent and Fc-silent anti-TIGIT antibodies are currently being tested in clinical trials for cancer.

TIGIT Clinical Trials

Clinical trials targeting TIGIT in cancer gave promising initial results, demonstrating therapeutic activity in a phase 2 clinical trial comparing non-small cell lung cancer (NSCLC) patients treated with anti-PD-L1 plus anti-TIGIT or anti-PD-L1 alone (CITYSCAPE-02 trial, see Table 2). In the cohort receiving TIGIT blockade, the ORR doubled compared to the controls and there was a clear, significant increase in both progression free survival (PFS) and overall survival (OS) ¹²⁴. The therapeutic benefit was even clearer in patients with high PD-L1 expression in tumors (>50% of tumor cells from a biopsy express PD-L1), suggesting an impressive therapeutic benefit. However, in this study, the control arm underperformed compared to historic data, likely leading to an overestimation of the therapeutic gain of TIGIT blockade in this context ¹²⁴. The follow-up phase 3 trial (SKYSCRAPER-01) is still ongoing but a communication from Roche in 2022 stating that the co-primary endpoint of increased PFS will not be met ¹²⁵, clearly dampened the enthusiasm for TIGIT-directed therapies. While the study is still ongoing, it seems that it will not be able to corroborate the results from the phase 2 CITYSCAPE-02 trial. Similarly, the KeyVibe-002 trial testing anti-TIGIT and anti-PD-1 in combination with docetaxel in patients with metastatic NSCLC showed no therapeutic benefit¹²⁶. Nevertheless, the ARC-7 trial, another phase 2 trial comparing anti-TIGIT plus anti-PD-1 co-blockade with PD-1 blockade alone in PD-L1^hi NSCLC patients, showed similar results as the initial CITYSCAPE-02 trial, a clear increase in ORR and PFS that almost doubled with additional TIGIT blockade (Table 2). Despite the discouraging interim results from the phase 3 CITYSCAPE-02 trial, it is clear that several independent studies have found a therapeutic benefit when adding TIGIT blockade to inhibition of PD-1 or PD-L1 in NSCLC patients. As results become available from over 10 ongoing clinical trials investigating the benefit of TIGIT blockade in NSCLC patients (Table 2) a clearer picture should emerge in the next few years.

Table 2.

Clinical activity targeting TIGIT.

Trial name/ identifier	Phase	Indication	⍺TIGIT Fc	⍺TIGIT group	Control Group	ORR		PFS (months)		OS (months)		> grade 3 AE		sponsor
						⍺TIGIT	control	⍺TIGIT	control	⍺TIGIT	control	⍺TIGIT	control
Tiragolumab Combinations
CITYSCAPE-02 (NCT03563716)	2	NSCLC (PD-L1 pos., 1%)	active Fc	tiragolumab (⍺TIGIT) + atezolizumab (⍺PD-L1)	atezolizumab (⍺PD-L1)	31.30%	16.20%	5.4	3.6	23.2	14.5	21%	18%	Genentech/ Roche
SKYSCRAPER-01 (NCT04294810)	3	NSCLC (PD-L1 high)	active Fc	tiragolumab (⍺TIGIT) + atezolizumab (⍺PD-L1)	atezolizumab (⍺PD-L1)	2° endpoint		1° endpoint, not met		1° endpoint		64.4%a	64.2%a
SKYSCRAPER-02 (NCT04256421)	3	SCLC	active Fc	tiragolumab (⍺TIGIT) + atezolizumab (⍺PD-L1)	atezolizumab (⍺PD-L1)	similar		5.4a	5.6a	13.6a	13.6a	33.3%a	27.5%a
MORPHEUS-liver (NCT04524871)	1b/2	Hepatocellular Carcinoma	active Fc	tiragolumab (⍺TIGIT) + atezolizumab (⍺PD-L1) + bevacizumab (⍺VGEF-A)	atezolizumab (⍺PD-L1) + bevacizumab (⍺VGEF-A)	42.5%a	11.1%a	11.1a	4.2a	immature		33.3%a	27.5%a
Summary of other ongoing trials	2–3	Hepatocellular, renal and esophageal squamous cell carcinoma; gastric, rectal, and urothelial cancer; HNSCC	active Fc	tiragolumab (⍺TIGIT) +/− atezolizumab (⍺PD-L1) or ⍺Lag-3/PD-1 bispecific in combinationn with ⍺VGEF-A or chemo	single arm or the same combination without tiragolumab (⍺TIGIT)
Domvanalimab Combinations
ARC-7 (NCT04262856)	2	NSCLC (PD-L1 high)	Fc silent	domvanalimab (⍺TIGIT) + zimberelimab (⍺PD-1) +/− etrumadenant (A2R Ant)	zimberelimab (⍺PD-1)	40%/44%a	30%a	9.3/9.9a	5.4a	immature		47%/52%a	58%a	Gilead/ Arcus
Summary of other ongoing trials	2–3	NSCLC, melanoma, gastrointestinal tract adenocarcinoma	Fc silent	domvanalimab (⍺TIGIT) + zimberelimab (⍺PD-1) +/− chemo	zimberelimab (⍺PD-1) +/− chemo or single arm
Vibostolimab Combinations
KEYVIBE-002 (NCT04725188)	2	NSCLC (metastatic)	active Fc	vibostolimab (⍺TIGIT) + pembrolizumab (⍺PD-1) +/− docetaxel (DXL)	docetaxel	29.9% (6.0% w/o DXL)	15.3%	5.6 (2.7 w/o DXL)	3.2	10.2 (7.5 w/o DXL)	8.8	29.4% (20.5% w/o DXL)	12%	Merck
Summary of other ongoing trials	2–3	NSCLC, SCLC, melanoma, hematological and solid tumors	active Fc	vibostolimab (⍺TIGIT) + pembrolizumab (⍺PD-1) alone or in combinationn with chemo, radio or other cancer therapies	single arm or the same combination without vibostolimab (⍺TIGIT)

Table includes detailed information on trials targeting TIGIT that have reported results. Additional trials targeting TIGIT that are recruiting patients or ongoing but have not reported results are summarized for each antibody. Additional trials in phase 2 or higher by iTeos/GSK, BMS, Astra Zeneca, and BeiGene are listed on ClinicalTrails.gov.

^ainterim results

Apart from NSCLC, other cancer types have been investigated as potential targets for TIGIT blockade. Two trials in particular have received much attention over the last year. Although, the phase 3 SKYSCRAPER-02 trial targeting TIGIT in small cell lung cancer (SCLC) failed as it showed no clinical benefit (¹²⁷, Table 2), very positive results were released from a recent phase 1/2 study in first-line liver cancer, where ORR and PFS more than doubled with addition of anti-TIGIT to the treatment regimen ¹²⁸. While results of this study are again confounded by underperformance of the control arm, in this case even cross-trial comparison against the Imbrave-150 trial ¹²⁹ suggests a clear therapeutic benefit, supporting a planned phase 3 study expected to begin soon. It should be noted that in SCLC, anti-CTLA-4 has proven to provide no benefit and anti-PD-1, although an approved treatment, has not consistently shown activity throughout different trials ¹³⁰. In addition, anti-TIGIT/anti-PD-1/PD-L1 treatment in combination with chemotherapy is planned or currently being investigated in phase 2 and 3 trials in lung, liver, renal, and gastrointestinal cancers (Table 1). Overall, results obtained from clinical trials targeting TIGIT have been mixed, but the fact that multiple trials observed an impressive improvement with the addition of anti-TIGIT treatment points towards an overall benefit at least in selected indications even if the improvement may not be as dramatic as initial results had suggested.

One important and consistent result obtained from the ongoing trials was the high tolerability as irAEs were comparable to the control arms and no new safety concerns emerged with the addition of TIGIT blockade (^{124,128,130,131}; Table 2). Although the frequency of the different irAEs was generally similar across groups, both trials that have already published a detailed list of irAEs (CITISCAPE 2 and Skyscraper 2) reported a slight increase in skin-related symptoms (pruritus, rash) with addition of TIGIT blockade (^124,131, Table 2). While this will have to be confirmed by future trials, it may provide clues toward which cancer types may be most responsive to anti-TIGIT therapy. Furthermore, clear biomarkers that can be used for patient selection and a better understanding of which cell types are targeted in vivo will help to narrow down the indication in which TIGIT blockade can provide a therapeutic benefit. Overall, despite some clinical disappointments, TIGIT remains an important target for anti-cancer therapy that is currently intensely studied and shows promise in several cancer types.

A look ahead

As discussed above, LAG-3, TIM-3, and TIGIT have established roles in the preservation of self-tolerance. The role of these receptors in maintaining self-tolerance raises two important considerations regarding clinical translation. First is the possibility of harnessing them clinically to treat autoimmunity. Second is the development of autoimmune-like toxicities or irAEs that ensue upon their blockade in cancer patients. Regarding the former, clinical development for autoimmune indications is far behind that for cancer indications. This is in spite of evidence in several autoimmune diseases that the expression and/or function of some of these receptors is defective and the demonstration that the expression of a T cell dysfunction signature that contains multiple checkpoint receptors positively correlates with reduced relapse rate and better response to therapy and prognosis in human autoimmune diseases ¹³². Thus, strategies aimed at increasing receptor expression such as administration of Type I Interferon, which promotes the co-inhibitory module ⁵⁸ may be beneficial. Indeed, restoration of TIM-3 expression has been associated with positive response to Type I Interferon in MS patients ⁵³. An additional strategy may be to combine therapies that induce receptor expression with receptor agonists to augment signaling. In this regard, a clinical trial investigating an agonistic anti-PD-1 antibody has shown beneficial effects in Rheumatoid Arthritis ¹³³. A potential challenge for the development of LAG-3 agonists is receptor shedding, which could limit their applicability ²¹.

Regarding therapy-induced toxicities, we had proposed that targeting LAG-3, TIM-3, and TIGIT would be associated with fewer and less severe irAEs, reflecting their cellular specification, i.e. the expression profile on T lymphocyte subsets ¹. This cellular specification is now extended beyond T cells, to include B cells and myeloid cells (Figure 2A). This specialized expression goes hand-in-hand with stage specification, i.e. expression during different stages of the immune response. Currently, most of our knowledge of stage specification for LAG-3, TIM-3, and TIGIT is in T cells (Figure 2B). These receptors have been widely studied in acute and chronic T cell activation settings. In acute activation settings, LAG-3 and TIGIT are expressed together with PD-1 soon after T cell activation in both CD4⁺ and CD8⁺ T cells, but are then downregulated in effector cells. In contrast, TIM-3 is expressed later when CD4⁺ T cells become fully differentiated and commited to the Th1 lineage or when CD8⁺ T cells become fully cytotoxic. The expression of these receptors in chronically activated T cells differs as does the trajectory of T cell differentiation. In such settings, PD-1 and TIGIT are found on stem-like CD8⁺ T cells that give rise to effector and dysfunctional T cell progeny. Notably LAG-3 and TIM-3 are not expressed on stem-like CD8⁺ T cells rather LAG-3 is expressed as cells differentiate to the effector lineage and TIM-3 expression follows as cells fully differentiate towards terminally dysfunctional cells.

Figure 2. Cellular and Stage specification.

(A)Cellular specification: Expression profile of LAG-3, TIM-3, and TIGIT on different immune cell subsets. The relevant references are included in the respective sections in the text.

(B) Stage specification: Checkpoint receptor expression at different stages of immune activation can tune T cell activation and differentiation. During acute T cell activation, LAG-3 and TIGIT are co-expressed with PD-1 whereas TIM-3 expression comes later when cells are fully differentiated and commited to the CD4⁺ Th1 lineage or to fully cytotoxic CD8⁺ T cells. During chronic activation, TIGIT is expressed together with PD-1 in stem-like T cells, highlighting unique roles for these checkpoint receptors in regulating this important cell population. LAG-3 expression follows on effector-like cells and finally TIM-3 is expressed in terminally differentiated dysfunctional T cells.

Another layer of specification is functional specification (Figure 3A). The elucidation of the mechanism by which LAG-3 affects T cell signaling positions LAG-3 as a modulator that can affect early T cell activation and priming. TIM-3 may dominate in settings where DC activation and Type 1 immune responses are induced. TIGIT has a dominant effect in promoting suppressive function in Tregs and B cells and in promoting a tolerogenic DC phenotype, selectively inhibiting proinflammatory Type 1 and Type 17 immunity and thereby restoring homeostasis. The final layer of specification is anatomic specification, which reflects the dominant functions of the different receptors in distinct tissues due to ligand expressions or the activity of receptor-expressing cell types. LAG-3 may be particularly important in the pancreas and the heart as its deficiency accelerates diabetes onset and enhances myocarditis ¹³⁴. TIGIT may be particularly important in maintaining gut homeostasis given its function in Treg. In the lung and skin where the balance between Type 1 and 17 versus type 2 immunity can influence disease manifestations, TIGIT may also be very important. Lastly, TIGIT’s role in regulating B cells appears to be critical for preventing CNS inflammation and blockade or KO of TIGIT exacerbates EAE ^94,101,112. TIM-3 may particularly be important in the CNS as TIM-3 expression has been shown to be defective in patients with Multiple sclerosis ⁵² and blockade of TIM-3 exacerbates EAE ³². TIM-3 may also be important in the gut where the TIM-3 ligands GALECTIN-9¹³⁵ and CEACAM-1¹³⁶ are highly expressed (Figure 3B). Building upon this knowledge base should be a focus, not only for refining future clinical development, but also for understanding the mechanism of action and propensity for treatment-induced toxicities of global targeting agents currently in use and in clinical development. Understanding the cellular types and processes where receptor biology is active will inform predictions of the types of irAEs that may ensue from global targeting and the development of more refined targeting approaches and their application to specific disease contexts.

Figure 3. Functional and Anatomic specification.

A) Functional specification: Cellular and stage specification of checkpoint receptors determines specialized functions in immune regulation. LAG-3 limits T cell priming due to its expression early during T cell activation ¹⁵. TIM-3 has a specialized role in determining DC function through regulation of DNA-sensing and inflammasome activation and in regulating Type 1 Immunity due to its expression in terminally differentiated IFN-γ-secreting CD4⁺ and CD8⁺ T cells⁴⁷. TIGIT has a specialized role in promoting Treg and regulatory B cell responses as well as dampening inflammation by selectively inhibiting Type 1/ Type 17 immunity and thereby shifting the balance towards Type 2 immune responses ^94,109,137.

B)Anatomic specification: LAG-3-, TIM-3-, and TIGIT-driven regulation may dominate in different tissues reflecting ligand expressions or the activity of receptor-expressing cell types. LAG-3 may be particularly active in the heart and pancreas; loss of LAG-3 has been associated with myocarditis and LAG-3 expression with poor prognosis in pancreatic cancer ¹³⁴. TIM-3 may be critically important for controlling inflammation in the CNS and in the gut; TIM-3 expression has been shown to be defective in patients with Multiple sclerosiss ^52,53 and the TIM-3 ligands GALECTIN-9 and CEACAM-1 are highly expressed in the gut ^135,136. TIGIT may be active in the lung and skin, where the balance of Type 1/Type 17 vs Type 2 immunity is important ^109,137. Further, TIGIT function in regulatory T cells and B cells seem critical for controlling inflammation in the gut and CNS ^94,109.

Concluding remarks

The immune system has evolved to contain multiple checkpoint receptors that serve to regulate active immune responses and restore homeostasis. As we learn more about each of the known checkpoints, it is becoming clear that these molecules do not simply serve as fail-safes for each other, but rather provide sophistication to immune regulation by tempering different aspects of immunity. The prevailing dogma holds that CTLA-4 serves to regulate T cell priming and PD-1 serves to regulate expansion of activated T cells. Investigation of the next wave of checkpoints (LAG-3, TIM-3, TIGIT) has identified specialized roles in regulating TCR signaling, myeloid cells, B cells, and the balance of T helper responses. As therapies blocking this next wave of checkpoints are applied in the clinic and tested in trials, it will be important to bear in mind their specialized functions. As we look ahead, it will be crucial to leverage and build upon the information we have gleaned thus far to inform: 1) disease context-specific targeting, e.g. therapies targeting TIGIT in diseases where modulation of B cell-driven suppression is desirable or TIM-3 where activation of DCs is desirable; 2) cell type-specific targeting to focus effects on desired cell types, thereby limiting irAES due to global targeting; 3) rational design of therapeutic combinations to hit different arms of the immune response for appropriate disease indications. Although much remains to be learned, it is high time to build upon the established success of checkpoint-directed therapies to create a new generation of therapies that work smarter, with increased efficacy but limited toxicity.

Highlights.

• Second generation checkpoint receptors have specialized functions

• LAG-3 regulates T cell receptor signaling via cis interactions that regulate pH

• TIM-3 regulates myeloid cell activation via the inflammasome and STING pathways

• TIGIT regulates B cell responses