Immune checkpoint modulators in cancer immunotherapy: recent advances and emerging concepts

VISTA is a type I transmembrane protein with 279 amino acids (AAs). It contains an extracellular domain with 162 AAs, a transmembrane domain with 21 AAs, and a cytoplasmic domain containing 96 AAs [59] (Fig. 1). The extracellular region could be divided into two parts, one is an immunoglobulin (Ig) V domain with a single N-terminal, and the other is about 30 AAs stalk. An analysis showed that among the B7 family members, VISTA had been proved to have its highest homology with PD-L1, and the sequence identity could reach 22% [60]. Genetically, it is chromosome 10(10q22.1) where we found VISTS expression, and no neighboring Ig superfamily members could be found. All of this may partly explain the relatively low similarity. Among B7 members, VISTA is the most conservative, besides 76% identity was observed when considering mice and humans, and it is more likely that the translocation springs up along with evolution [45, 61]. Between the assumed B and F strands, the canonical disulfide bond could be found by the IgV domain of VISTA. Besides, VISTA has four additional unique invariant cysteines. VISTA shows its homology with CD28 and CTLA-4. Although lacking a classic ITIM/ITAM motif inside the conserved cytoplasmic tail, VISTA still plays a ligand and a receptor in regulating immune responses. The function was achieved by three C-terminal Src homology domain 3 (SH3) binding motifs, an Src homology domain 2 (SH2) binding motif detected in the medial cytoplasmic tail, multiple casein kinase 2, and phosphokinase C phosphorylation that was in the cytoplasmic domain [60, 62]. The hematopoietic compartment, especially the myeloid cells, is where VISTA mostly be found. To be detailed, VISTA can be found in microglia, monocytes, neutrophils, macrophages, and dendritic cells (DCs). PSGL-1 and VSIG3 have been identified as VISTA’s confirmed ligands. VSIG8-VISTA binding is relatively weak. It is a physiological condition where VISTA can interact with VSIG3, and PSGL-1 is bound to VISTA on T cells in an acidic environment [63]. The VSIG3-VISTA complex will downregulate the level of multiple cytokines, such as interleukin (IL)-2, IL-17, interferon γ (IFN)-γ, and chemokine (C–C motif) ligand 5 (CCL5). Glycosylation and tyrosine sulfation regulate the function of the compound consisting of PSGL-1, selectins, and VISTA, while the modification of the compound is measured by lymphocyte activation. PSGL-1 is highly linked with T cell exhaustion [59, 60, 62].

Since multiple preclinical models have proved the high therapeutic effectiveness of VISTA in restricting tumor proliferation, VISTA-targeted antagonists have stepped forward into the clinical trial [64, 65].

Intercellular signaling relies on specific molecules, among which adenosine has been identified as one of the regulators of multiple physiological and pathological processes [66]. Adenosine is a nucleoside derived from the extracellular hydrolysis of adenine nucleotides. Since adenine nucleotides play a core role in the biosynthesis of ATP, rapidly developed adenosine would indicate ischemia and anoxia, which is most likely to be found in the inflammation region. Adenosine was primarily thought to be a potent vasodilator in 1927 [66]. At the same time, advanced studies showed its capacity to suppress the immune response within the TME by several specific receptors’ engagement, for example, A1, A2a, A2b, and A3 [66]. To summarize, adenosine acts as a “rheostat” of the immune response, mediating the transition from inflammation to healing.

However, Otto Warburg pointed out that the proliferation of cancer cells was linked to energy generation mainly through the anaerobic breakdown of glucose instead of the oxidative breakdown of pyruvate in normal tissue cells [67]. Therefore, the tumor microenvironment might be a perfect place for adenosine formation adenosine probably engages in the immune escape, which has been confirmed by a recent study [68].

Adenosine is generated in the TME through the coordinated activity of the ectonucleotidases CD39 (ENTPD-1) and CD73 (5’-NT or NT5E), in which the extracellular adenosine triphosphate (ATP) [69], an inflammation-inducing factor, is converted to adenosine. Given that, CD39 and CD73 became essential targets for cancer therapy. Besides the CD39-CD73 chain, the CD38-NPP1-CD73 chain utilizes nicotinamide adenine dinucleotide (NAD) as the precursor via intermediates ADP-ribose (ADPR) and cADPR to generate AMP. Several studies have identified the functional role CD38 played in the reaction chain and targeted CD38 for therapy [70].

CD39, also known as ENTPD-1, is the rate-limiting ectoenzyme in extracellular ATP hydrolysis [71]. Moreover, CD73 utilizes the product, 5′-adenosine monophosphate (5′-AMP), to generate extracellular adenosine [72]. The expression level of CD39 is upregulated under the stimulus from hypoxia-induced factor (HIF)-1, transforming growth factor (TGF)-β, and epithelial-to-mesenchymal-transition (EMT). CD39 expression could also be increased by T cell exhaustion and chronic inflammatory factors, such as IL-6 and TNF-α. CD39 is found to be broadly produced on B cells, NK cells, DCs, monocytes, macrophages, Tregs [73], also been seen on fibroblasts, myeloid cells, vascular endothelial cells, Tregs, and tumor-specific T effector cells in TME [71]. The wide distribution of CD39 indicates its abundant biological functions. (1) NLRP3 inflammasome is activated by extracellular ATP through the P2X7 receptor to induce pyroptosis [71], and CD39 can suppress the reaction by downregulating the ATP level. (2) The release of ATP by some dying tumor cells, as well as calreticulin exposure and high-mobility group protein 1 (HMGB1) secretion, are thought to be the hallmarks of immunogenic cell death, and it has been verified that the death of transformed cells often elicits highly effective anti-tumor immune responses [74]. CD39 could deplete ATP, thus cutting off the activation of macrophages, which is critical for dying tumor cells’ immunogenicity. (3) It was shown that the chemotaxis of macrophages lies on the gradient of extracellular ATP concentrations [75]. Thus, the gradient will be blocked if adding excessive exogenous ATP or using soluble CD39-like apyrase. Since increasing extracellular ATP levels also activated P2X4 and P2X7 receptors, the activation would trigger calcium signaling among neighboring macrophages [76] and eventually promote macrophage phagocytosis. It was thought that CD39 also plays a role in suppressing phagocytosis.

CD73, an ecto-5′-nucleotidase, is a cell surface glycosylphosphatidylinositol-anchored glycoprotein. CD73 has widely been the surface marker of several cell types, such as endothelial cells, subtypes of lymphocytes [77], stromal cells, and tumor cells. In tumor-infiltrating NK cells, CD73 is transported from intracellular vesicles to the cell surface and the extracellular matrix via actin polymerization–dependent exocytosis with the engagement of 4-1BBL on tumor cells [78]. Following the focal radiotherapy, inflammation and tissue damage induce the increased level of adenosine and CD73, and CD73 blockade promotes DC infiltration of tumors and tumor rejection which facilitates IFN-I inducing the infiltration of tumor [79]. Metformin has long been a potent drug for type 2 diabetes, and it has also been recently reported to modulate CD73/CD39 expression on MDSCs through activation of the AMPKα pathway and inhibition of the HIF-1α pathway, enhancing the immune response [69].

CD38 belongs to the ribosyl cyclase family and is widely expressed on the surface of non-hematopoietic cells and several immune cells. In addition to the extracellular adenosine, NAD+ is another critical factor for immune escape. It has been confirmed to participate in a series of reactions, like cell proliferation, leucocyte differentiation, as well as function [80‐82]. However, CD38 is an ectoenzyme and transmits NAD+ to ADP-ribose (ADPR) and cADPR. It has been proven to significantly affect intracellular Ca2+, cell adhesion, and signal transduction [83]. Th1 and Th17 cells were potent anti-tumor T cells for their heightened effector function and prolonged persistence. They need a functional NAD+-Sirt1 Axis to exert an anti-tumor response, which research proved that the anti-CD38 antibody could enhance the inhibition of tumor [80].

Overall, the extracellular ATP activated by inflammation is an essential inducer of the immune response, and the adenosine suppresses the reaction. Till now, we knew CD39 and CD73 worked together to mediate the transformation of extracellular ATP to adenosine, which took up most of the synthesis of extracellular adenosine. CD38 is a main bypass of the adenosine synthesis, and the precursor used, NAD+, also plays a vital role in immunoregulation. All in all, a deeper understanding of the three enzymes is needed to promote immunotherapy.

There are 425 clinical trials of CD38 ongoing or completed on Clinicaltrials.gov. Multiple myeloma (MM) is the second most common hematologic tumor, until anti-CD38 antibodies Daratumumab and Isatuximab have been approved for treating multiple myeloma. Daratumumab induces cell death through complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), induction of apoptosis, and modulation of CD38 enzyme activities. Daratumumab may also exhaust CD38+ immune regulatory cells and promote T cell expansion. Thus, it accelerates the exhaustion of tumor cells. It was approved by FDA in 2015 and applied in treating multiple myeloma [77].

MAIA and ALCYONE put Daratumumab into the first-line treatment regimen for MM, and D-VMP and DRd became the preferred treatment regimen for patients unsuitable for NDMM transplantation.

Isatuximab is another CD38 monoclonal antibody that also targets MM. It has an entirely different antigen-binding epitope compared to Daratumumab, which can almost completely inhibit the enzymatic activity of CD38 and directly cause the apoptosis of tumor cells through FcγR-mediated cross-linking. It has a better scavenging effect on CD38-positive NK cells and Treg cells. Recent studies have stepped into phase III trials (NCT02990338), showing that Isatuximab extended patients’ PFS [87].

CD38 antibody has also been applied to lymphoma.

Till now, all clinical trials on lymphoma have reached few results. Some even terminated due to unsatisfactory outcomes [90, 91] (NCT02999633, NCT02413489). All in all, studies on lymphoma are not recommended until breakthroughs occur.

ES014 is an anti-CD39/TGF-β bispecific antibody; a relevant study (NCT05381935) has been conducted on patients with advanced solid tumors, but the result has not come out yet.

LAG-3 is identified as a type I transmembrane protein. It is mainly found on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells [95]. It comprises four extracellular immunoglobulin (Ig)-like domains (D1–D4) that share 20% amino acid homology with CD4, and the similarity lies in the proximity of LAG-3 to CD4 on the human chromosome 12. Opposite to the semblable in the extracellular regions, intracellular parts of LAG-3 and CD4 show no noticeable similarity. For example, LAG-3 lacks the cysteine motif required to link with lymphocyte-specific protein tyrosine kinase (Lck) and the palmitoylation site observed in CD4 [96, 97]. The construction of the genomic parts of CD4 and LAG-3 obtaining exons encoding their extracellular regions is similar. Still, the genomic building receiving exons encoding their intracellular regions varies, indicating an early divergence of the two genes during evolution [98]. Now, we found that the LAG-3 cytoplasmic tail contains three conserved motifs. And it mediates intracellular negative signal transduction: (1) a potentially phosphorylatable serine (S484), (2) a KIEELE motif, and (3) a glutamate-proline dipeptide multiple repeat motif (EP motif). S484 has been found its correlate with IL-2 production [99]. Lysine residue (K468) found in KIEELE was required for LAG-3 downstream signaling. The EP motif was vital in promoting colocalization of LAG-3 with CD3, CD4, and CD8 within lipid rafts through interacting with LAP protein (LAG-3 associated protein) [100].

Since LAG-3 is structurally like the CD4 co-receptor, it links to the MHC class II complex with a stronger affinity than CD4. LAG-3 also affects the activity of CD8+ T cells and NK cells, which interact with the MHC class II complex. The fact has pointed to alternate ligands for Lag-3 [101]. The DC-SIGN family member LSECtin and the liver-secreted protein FGL1 have now been identified as ligands of LAG-3. Besides, studies showed that the silence of LAG-3 and PD-1 independently has little effect on autoimmunity [102]. At the same time, the double-knockout of LAG-3 and PD-1 in the mice model resulted in multi-organ lymphocytic infiltration, indicating a closer relationship between LAG-3 and PD-1 in regulating T cell viability [98].

CD4+ T cells-expressed LAG-3 binds to the MHC class II complex on antigen-presenting cells, while CD8+ T cells-expressed and NK cells-expressed LAG-3 links to the LSECtin on tumor cells or liver cells. The unique KIEELE motif on the cytoplasmic tail of LAG-3 is critical for its immune suppressive function [101]. Studies showed that the deletion of KIEELE completely abrogates LAG-3 function on CD4 T cells [103]. Since LAG-3 has a significantly higher affinity with MHC-II than CD4, it is no surprise that LAG-3-Ig fusion proteins act as competitors in CD4/MHC class II-dependent cellular adhesion assays downregulate T cell activation, cytotoxicity, and cytokine production [103]. The cytoplasmic domain of LAG-3 would transmit inhibitory signals and inhibit CD4 T cell activation once LAG-3 is tied to MHC-II [104, 105]. Besides, the bination of LAG-3 to MHC class II complex also dampens Th cell response. Overall, the high-affinity LAG-3: MHC class II bination was believed to be the primary mechanism of the high inhibitory competence of LAG-3. It works through the competition with CD4: MHC-II binding in early studies, while along with the novel identification of additional ligands, a consensus is under controversy now.

Recent studies have focused on developing antagonistic mAbs, IMP321 (Eftilagimod Alpha), a soluble dimeric recombinant protein composed of four LAG-3 extracellular domains fused to the Fc portion of human IgG1 (LAG-3-Ig) [106].

Besides, the development of antagonistic monoclonal antibodies targeting LAG-3 drew much attention. BMS-986016 (Relatlimab) is a human IgG4 anti-LAG-3 blocking mAb, and multiple studies have proven the synergistic impact of anti-LAG-3 with PD-1/PD-L1-targeted therapies.

As a critical and a pivotal trial, the up-to-date study RELATIVITY-047 (NCT03470922) suggested that inhibiting both LAG-3 and PD-1 with Relatlimab and Nivolumab (Opdualag, BMS-986213, fixed-dose combination Relatlimab and Nivolumab at a 1:3 ratio) provides a better prognosis than those who only received Nivolumab in patients with previously untreated metastatic or unresectable melanoma, which led to FDA approval on the first anti-LAG-3 antibody Relatlimab for the treatment of melanoma.

LAG-3 is an immune checkpoint of great potential. Relatlimab, an anti-LAG-3 blocking mAb, has been approved for melanoma treatment. In the latest studies, Relatlimab can also be seen in treating NSCLC or gastric cancer. Maybe some inspiring news will come from treatments of other types of tumors soon. Although IMP321 is also expected, it lacks approval for treating melanoma, and more studies must be done.

IDO-1 is essential in regulating immune escape. It catalyzes the oxidation of Trp (l-tryptophan) to form N-formyl L-kynurenine, rapidly converted by formamidases to Kyn (kynurenine) [109]. Rocketed levels of Kyn and higher plasma Kyn/Trp ratios are often found in cancer patients at an advanced stage and correlate with poor prognosis [110]. The importance of Trp starvation responses against Kyn accumulation in the TME as the driving force for immunosuppression has been debated for over 20 years [110].

Generally, Trp is a critical amino acid for mammals. The tryptophan pool in tumor microenvironments will be restricted in response to the abnormal activation of IDO-1, leading to decreased T cells [111]. Besides, the reduced concentration of local Trp triggered the activation of the general control nonderepressible 2 kinases (GCN2) [110]. Kyn is an AhR (Aryl hydrocarbon Receptor) ligand. The interaction leads to the suppressed secretion of IFN-I and activation of NF-κB signaling [112]. Kyn also converted naive CD4+ T cells into Foxp3+ Treg cells and the Trp starvation [109]. Besides its immediate effect on AhR, in vitro Kyn is slowly transmitted from non-enzymatically to byproducts that serve as high-affinity (sub-nM) AhR agonists. In addition, the downstream metabolites of the Trp catabolism, 3′OH-kynurenine, and 3′OH-anthranilic acid, also act as AhR ligands [110]. As the intermediate product of the KP (kynurenine pathway), Kyn is further metabolized through the KP to quinolinic acid. And the latter is switched to NaMN through the enzyme quinolate phosphoribosyltransferase (QPRT). Then, it ultimately switched to NAD+ via the Preiss-Handler pathway, thus joining the NAD+ immunoregulation pathway [113].

So far, studies on IDO-1 have been extensive, and many drugs have reached phase III of clinical trials. However, there is the unexpected failure of Epacadostat (INCB024360). Epacadostat is a potent and highly selective IDO-1 enzyme inhibitor that decreases tryptophan metabolism, resulting in the enhanced viability of effector T cells, NK cells, CD86^high DCs, decreased apoptosis, and the reduced expansion of Tregs [114].

Recently, a series of researches were restarted to increase the efficacy of Epacadostat by combining Epacadostat with other drugs, vaccines, and radiation in glioblastoma (NCT03532295), metastatic pancreatic adenocarcinoma (NCT03006302), or breast cancer in females (NCT03328026), but no results have been reported yet.

Besides Epacadostat, other antibodies targeting IDO were developed to explore IDO target therapy in solid tumors. The study of PF-06840003 that was developed by Pfizer (NCT02764151) was terminated, and anti-IDO-1 agent LY3381916 was also abandoned. Encouraging news came from BMS-986205, another IDO-1 inhibitor.

Among all studies concerning IDO-1, Epacadostat is of great potential. Though its trial on melanoma was not satisfying, its efficacy for urothelial cancer was promising.

CD27 is a member of the TNF receptor superfamily (TNFRSF). Besides, it is a type I transmembrane, disulfide-linked homodimer [115] (Fig. 2). CD27 is widely expressed in human lymphocytes, including naive and central memory T (TCM) cells, germinal center and memory B cells, plasma cells, and NK cells [116]. As the unique ligand of CD27, CD70 is mainly expressed in hematologic malignancies, for example, diffuse large B cell, follicular lymphoma, Hodgkin lymphoma, Waldenström macroglobulinemia, multiple myeloma, human T-lymphotropic virus type 1- and EBV-associated malignancies [116]. The costimulatory signals induced by CD70-CD27 interaction increase T cell proliferation and activity [117]. Unlike CD27, CD70 expression is transitory and limited to a subset of strongly activated T cells, B cells, and DCs, but is undetectable in homeostasis [118]. And CD70 expression is also being detected in nonhematologic malignancies, for example, renal cell carcinoma and glioblastoma [119].

After binding to CD70, the TRAF2 and TRAF5 adaptor proteins are recruited to the cytoplasmic tail of CD27, which activates either the JNK or NF-kb signaling pathways [115, 120] and finally induces the effective primary and memory cell-mediated responses. There was evidence that anti-CD70 treatment could alleviate Th17-cell-mediated inflammatory disease and experimental autoimmune encephalomyelitis (EAE) on CD4+ T cells. Besides, CD70-transgenic mice were detected with increased interferon-γ-producing CD4+ and CD8+ T cells [117].

Apart from the typical costimulatory pathway, other mechanisms account for the CD27 expression on B cells and NK cells. In human peripheral B cells cultivated with IL-2 and IL-10, the binding of CD27 with CD70 upregulates the secretion of IgG and IgM. Plasmacytoid dendritic cells (pDCs) are believed to be another important source of CD70 [121] that plays a vital role in regulating the differentiation of B cell [122]. CD27 has also been proved to stimulate Akt signaling in NK cells and upregulate IFN-γ secretion [115].

So far, the only fully human immunoglobulin G1 kappa anti-CD27 monoclonal antibody [123] is Varlilumab (CDX-1127), which interacts with the CD70-binding site and serves as an agonist of CD27.

At present, clinic trials on CD70 mainly focus on ADC (antibody–drug conjugate), mAb (monoclonal antibody), and CAR (chimeric antigen receptor)-T therapy. Though its overexpression fails to rouse immune response, CD70 can still be used to indicate tumor cells.

Besides ADC, mAb Cusatuzumab was developed by Argenx, and studies showed that it could both induce cytotoxicity against CD70+ tumor cells through enhanced ADCC, complement‐dependent cytotoxicity, or Ab‐dependent cellular phagocytosis) and improve the anti-tumor immune response by interrupting the CD70‐CD27 signaling with Tregs [137]. In recent studies, researchers reported that combination therapy of Cusatuzumab plus Azacitidine has a higher ORR than monotherapy of Azacitidine in patients with phase I or 2 AML.

The third CD70-related treatment is CAR-T, which consists of diverse T‐lymphocytes and CAR transmembrane molecules encoded by artificial fusion genes. It redirects T cell cytotoxicity against cells expressing a specific target antigen. CAR binding to target antigens is independent of the MHC receptor, resulting in abundant T cell activation and robust anti-tumor responses [138]. Therefore, CAR-T therapy was born for tumor immunotherapy. In one study, researchers constructed seven anti-human CD70 CARs. They cured NSG mice bearing established human tumors that secreted CD70 and human lymphocytes transduced with the CAR, which side effect includes transient weight loss and hematopoietic suppression [139]. A similar trial reported favorable outcomes that CD70-specific CAR-T cells could recognize and kill CD70-positive head and neck squamous cell carcinoma cells efficiently [140]. ALLO-316 is an allogeneic CAR-T cell therapy targeting CD70, and the phase I study has not been completed yet.

Research on ADCs or mAbs of CD70 is relatively few, but it has nothing to do with its efficacy on AML, especially in combination with Azacitidine. CAR-T therapy is vital in immune therapies targeting CD70, and further studies are promising.

T cell immunoglobulin and mucin domain-containing protein 3 is a member of the TIM family of immunoregulatory proteins [50]. TIM-3 was initially classified as a receptor expressed on IFN-γ-producing CD4+ Th1 and CD8+ T cytotoxic 1 (Tc1) T cells [101]. Recent studies have also shown its capacity for immune evasions like PD-1 and CTLA-4. TIM-3 is encoded by HAVCR2 and located on chromosome band 5q33.2 in humans. Human TIM-3 protein comprises an amino-terminal immunoglobulin variable domain (V domain) with five noncanonical cysteines, a mucin stalk, a transmembrane domain, and a cytoplasmic tail. Unlike other immune checkpoint molecules, for example, PD-1 and TIGIT. The TIM-3 cytoplasmic tail has no classical inhibitory signaling motifs, like immune receptor tyrosine-based inhibitory motif (ITIM) or immune receptor tyrosine-based switch motif (ITSM). Instead, TIM-3 obtains five conserved tyrosines. Among the five tyrosines, Tyr256 and Tyr263 allow interactions with HLA-B-associated transcript 3 (BAT3) and the tyrosine kinase FYN. TIM-3 was firstly found on IFN-γ-producing CD4+ and CD8+ T cells. Gradually, Tregs, myeloid cells, NK cells, and mast cells are also revealed to express TIM-3 abundantly [141, 142].

TIM-3 is also reported with four distinct ligands, including galectin-9, HMGB1, carcinoembryonic antigen cell adhesion molecule 1 (Ceacam-1), and phosphatidyl serine (PtdSer) [50]. Galectin-9 is a C-type lectin widely expressed and secreted by many hematopoietic cells. Galectin-9 binds to carbohydrate moieties on cell surface proteins, which induces intracellular calcium influx and cell death of TIM-3+ T cells. The discovery of PtdSer could be contributed to elucidating the crystal structures of the TIM family. PtdSer binds to the pocket framed by the FG and CC′ loops in the TIM-3 immunoglobulin V domain [143], acts as a surface marker for apoptotic cells, and coordinates calcium-binding. The binding site of TIM-3 and HMGB1 remains uncertain, and their interaction was proposed to impact innate immune activation. Ceacam-1 works in the regulation of antiviral responses [102].

There are over ten TIM-3 antagonistic mAbs being registered on ClinicalTrials.gov. Sym023 is a recombinant anti-TIM-3 monoclonal human antibody.

To date, there are 34 records on CD47 antagonistic mAbs registered on ClinicalTrials.gov. Magrolimab (Hu5F9-G4) is an anti-CD47 antibody and firstly entered the phase I clinical trial in 2014 [164].

AK117 is another anti-CD47 antibody with high expectations, nine clinical trials are registered on Clinicaltrials.gov, but no results are reported.

Since the receptor of B7-H4 remains unknown, only three studies are currently registered on ClinicalTrials.gov.

Exciting news came from Enoblituzumab (MGA271), an engineered Fc humanized IgG1 mAb against B7-H3, where Enoblituzumab was used for participants with localized intermediate and high-risk prostate cancer before performing radical prostatectomy (NCT02923180). Thirty-two participants received treatment, among which most patients only experienced grade 1 (n = 31) or grade 2 (n = 12) treatment-related adverse events, and only four underwent grade 3 adverse events. By the way, we could see cardiac disorders as major serious adverse events in the study: 1 case with myocarditis and 1 with pericardial effusion). The result showed that 31% of patients had a more than 10% decline in PSA before post-prostatectomy, and an altered Gleason score was observed, indicating the efficacy of Enoblituzumab for prostate cancer patients.

An agent MGD009 (NCT02628535) is a humanized B7-H3/CD3 dual-affinity re-Targeting (DART) protein. For unknown reasons, the project has been terminated. The same team turned to MGD009 administered in combination with MGA012 (Anti-PD-1Antibody) (NCT03406949). This trial ended without an exact explanation.

Several clinical trials on TIGIT are ongoing or completed on Clinicaltrials.gov, including Tiragolumab, Etigilimab (OMP-313M32), etc.

A phase II study recently released its result (NCT03563716, CITYSCAPE). Totally, 155 patients with NSCLC were enrolled. They were given Atezolizumab monotherapy (with placebo, 68 patients) or combination therapy of Tiragolumab plus Atezolizumab (67 patients). Objective response occurred in 21 patients (31.3% [95% CI 19.5–43.2]) in the Tiragolumab plus Atezolizumab group versus 11 patients (16.2% [6.7–25.7]) in the placebo plus Atezolizumab group (p = 0.031), and median PFS was 5.4 months and 3.6 months (HR = 0.57), respectively. Fourteen (21%) patients receiving combination therapy and 12 (18%) patients receiving monotherapy had serious adverse effects. In patients with high PD-L1 expression (TPS ≥50%), the improvement effect of Tiragolumab combined with Atezolizumab was more obvious, ORR: 55.2% vs 17.2%, HR = 0.33. The most frequently reported grade 3 or worse treatment-related adverse event was lipase increase (in six [9%] patients with combination therapy against two [3%] with monotherapy). Two treatment-related deaths (including pyrexia and infection) occurred in the combination therapy group [179]. This trial showed that Tiragolumab plus Atezolizumab improved ORR and PFS compared with Atezolizumab monotherapy in PD-L1 positive NSCLC.

Several phase III studies on Tiragolumab have been conducted (SKYSCRAPER-01, NCT04294810; SKYSCRAPER-02, NCT04256421; SKYSCRAPER-02C, NCT04665856), targeting NSCLC and small-cell lung carcinoma relatively. However, the results did not meet our expectations.

On May 11, Genentech announced that the study of SKYSCRAPER-01 did not meet its co-primary endpoint of progression-free survival. At this first analysis, the other co-primary endpoint of overall survival (OS) was immature, and the trial phase of TIGIT immunotherapy in combination with first-line PD-L1 non-small-cell therapy failed.

ASCO reported the result of the study NCT04256421. In this study, 490 eligible patients with untreated extensive-stage small-cell lung cancer (ES-SCLC) were randomized 1:1 to receive Atezolizumab, carboplatin, and etoposide with or without Tiragolumab (n = 243, n = 247). The result showed that the mPFS of the experimental group is 5.4m (95% CI 4.7–5.5), and that is 5.6m (95% CI 5.4–5.9) of the placebo group; mOS is 13.6m (95%CI 10.8–14.9) vs 13.6m (95%CI 12.3–15.2), and grade 3/4 TRAEs occurred in 52.3% of the experimental group and 55.7% of the placebo group and Grade 5 TRAEs occurred in 0.4% of experimental group and 2.0% placebo group [180]. The result showed that Tiragolumab combined with Atezolizumab plus chemotherapy (carboplatin/etoposide) did not improve the efficacy of ES-SCLC.

Despite the failure of Tiragolumab phase III studies, the search for TIGIT targets continues. In another phase I study (NCT03119428), 33 patients with locally advanced or metastatic cancer were enrolled (Phase Ia, n = 23. Phase Ib, n = 10). In phase Ia study, patients underwent 14-day treatment cycles with anti-TIGIT antibody Etigilimab alone (0.3, 1.0, 3.0, 10.0, 20.0 mg/kg IV) while in the phase Ib study, patients received a combination therapy of Nivolumab and Etigilimab (3.0, 10.0, 20.0 mg/kg Etigilimab and 240 mg Nivolumab). The study was terminated for a sponsor decision but still released part of the result. It reported no DLT detected. The maximum administered dose was 20 mg/kg. At the same time, MTD for monotherapy and combination therapy was not determined. The median PFS was 56.0 days and 57.5 days for phase Ia and phase Ib, respectively. Besides, in the phase Ia study, seven patients (30.0%) had SD, and no PR was reported; in the phase Ib study, one patient had a partial response. One patient had prolonged SD of nearly eight months. The most reported AEs were rash (43.5%), nausea (34.8%), and fatigue (30.4%) in phase Ia and decreased appetite (50.0%), nausea (50.0%), and rash (40%) in Phase Ib. Six patients experienced Grade ≥3 treatment-related AEs [181].

At least 50 clinical trials are registered on Clinicaltrials.gov, though only two studies have yielded results. Etigilimab has not reached phase III study, but recent outcomes indicated its potential for cancer therapy. NSCLC is a good target, and more combination therapies on Tiragolumab are appreciated.