Clinical Insights Into Novel Immune Checkpoint Inhibitors

The success of immune checkpoint inhibitors (ICIs), notably anti-cytotoxic T lymphocyte associated antigen-4 (CTLA-4) as well as inhibitors of CTLA-4, programmed death 1 (PD-1), and programmed death ligand-1 (PD-L1), has revolutionized treatment options for solid tumors. However, the lack of response to treatment, in terms of de novo or acquired resistance, and immune related adverse events (IRAE) remain as hurdles. One mechanisms to overcome the limitations of ICIs is to target other immune checkpoints associated with tumor microenvironment. Immune checkpoints such as lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), V-domain immunoglobulin suppressor of T cell activation (VISTA), B7 homolog 3 protein (B7-H3), inducible T cell costimulatory (ICOS), and B and T lymphocyte attenuator (BTLA) are feasible and promising options for treating solid tumors, and clinical trials are currently under active investigation. This review aims to summarize the clinical aspects of the immune checkpoints and introduce novel agents targeting these checkpoints.

Despite the feasibility and anti-tumor activity of ICIs, there remain several hurdles in immunotherapy for cancer. Only a subset of patients respond to treatment, and the majority of patients who have durable responses eventually experience disease progression (Trebeschi et al., 2019). Furthermore, patients experience IRAE, some of which are highly toxic (Boutros et al., 2016;. To overcome these impediments, treatment strategies such as combination with chemotherapy, targeted agents, or radiotherapy have been implemented (Gandhi et al., 2018;Rini et al., 2019). Notably, treatment with a combination of different ICIs has resulted in increased clinical responses, as observed with the combination of nivolumab and ipilimumab in melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC) (Rizvi et al., 2016;Hellmann et al., 2018;Motzer et al., 2018).
Promising results from the combination of anti-CTLA-4 and PD-L1 mAbs have resulted in the launch of several other ICI combinations with non-overlapping mechanisms of action that may increase efficacy and minimize toxicity (Barbari et al., 2020). Currently, approximately 2/3 of all oncology trials are dedicated to T cell-targeting immunomodulators, and there are more than 3,000 ongoing clinical trials (Xin Yu et al., 2019).

Clinical Trials on LAG-3
Co-expression of LAG-3 with immune checkpoints, such as PD-1, and robust clinical data on the efficacy of LAG-3 and PD-1 dual blockade have prompted trials focusing on this combination as well as other immune checkpoint inhibitors. Currently, there are 17 agents targeting LAG-3 (Table 2), with multiple combinations of treatments across various tumors ( Table 3). Eight of these agents have interim or final clinical results, and nine of the investigational agents are ongoing clinical trials.
A phase 1 study of eftilagimod alpha (IMP321), an antigenpresenting cell (APC) activator for LAG-3, in combination with pembrolizumab was conducted in 24 patients with metastatic melanoma (NCT02676869) (Atkinson et al., 2020). The primary endpoints were the recommended phase 2 dose (RP2D), safety, and tolerability of the combined agents. The study included cohort A of dose escalation and cohort B of extension, and the patients received subcutaneous pembrolizumab and eftilagimod alpha bi-weekly at doses of 1, 6, or 30 mg for up to 6 and 12 months for Cohorts A and B, respectively. There was no dose-limiting toxicity (DLT) and the treatment was well tolerated, with the injection site as the most common adverse event (AE). The response to treatment was encouraging, with an overall response rate (ORR) of 33 and 50% for pembrolizumabrefractory cohort A and PD-1 naive cohort B patients, respectively.
Similarly, the combination of eftilagimod alpha and pembrolizumab has been investigated in NSCLC and head and neck squamous cell carcinoma (HNSCC) (NCT03625323) (Peguero et al., 2019). The AIPAC study, a placebo-controlled randomized phase IIb study on eftilagimod alpha (or placebo) with paclitaxel as the first-line treatment in patients with metastatic breast cancer (MBC), is also under investigation (NCT02614833) (Dirix and Triebel, 2019). Preliminary results show that the agent could elicit durable immune responses. Clinical data, including progression-free survival (PFS), ORR, OS, and safety, are all awaiting results.
Relatlimab (BMS-986016), an IgG4 mAb targeting LAG-3, has been investigated in various settings and agents, notably with well-established immune checkpoint inhibitors such as nivolumab and ipilimumab and other novel agents such as indoleamine 2,3-dioxygenase-1 (IDO1) inhibitors, CCR2/5 dual antagonist, and anti-TIGIT. Notably, clinical trials are ongoing for phase II/III in previously untreated metastatic melanoma, in combination with or without nivolumab (NCT03470922), phase II of nivolumab and oxaliplatin-based chemotherapy with or without relatlimab in GC or gastroesophageal junction (GEJ) cancer (NCT03662659), and phase II of relatlimab with nivolumab in mismatch repair deficient (dMMR) cancers resistant to prior PD-1/PD-L1 inhibition (Lipson et al., 2018;Feeney et al., 2019;Bever et al.,    Frontiers in Pharmacology | www.frontiersin.org May 2021 | Volume 12 | Article 681320 2020). Relatlimab is being tested in a wide range of tumor types and settings as front-or second-line treatment, in resectable status, and in stage II/III. An open label, phase 2 study including 72 patients treated with LAG-525, which is an IgG4 mAb for LAG-3, and spartalizumab (PDR001), an anti-PD-1, for advanced solid tumors and hematologic malignancies showed promising activity, especially in neuroendocrine tumors, small cell lung cancer (SCLC), and diffuse large B-cell lymphoma (DLBCL), with a clinical benefit rate at 24 weeks (CBR24) of 0.86, 0.27, and 0.804, respectively, meeting its primary endpoint (NCT03365791) (Uboha et al., 2019). In GEJ cancer, the CBR24 was 0.071, and enrollment was stopped for these subsets of patients. Other tumors such as triple-negative breast cancer (TNBC) (NCT03742349 and NCT03499899) and melanoma (NCT03484923) are ongoing trials in advanced and metastatic settings.
The preliminary results of a phase 1 study on cemiplimab (REGN3767), an mAb for LAG-3, as monotherapy (n 27), and in combination with PD-1 mAb (n 42) was conducted in advanced malignancies (NCT03005782) (Papadopoulos et al., 2019). No DLT was observed with in the monotherapy group, whereas the combination group, during treatment with R3767 3 mg/kg every 3 weeks (Q3W) + cemiplimab 3 mg/kg Q3W, experienced grade 4 elevated creatine phosphokinase levels in addition to grade 3 myasthenia gravis. Overall, both treatments were deemed tolerable; cemiplimab 20 mg/kg or 1600 mg as a fixed dose of Q3W is ongoing further evaluation as monotherapy and as a combination.
Similarly, BI 754111, an mAb for LAG-3, was also tested with BI 754091 (anti-PD-1) in treatment-refractory solid tumors, in a dose escalation phase 1 study, followed by an expansion phase in microsatellite stable (MSS) CRC and anti-PD1/PD-L1 refractory tumors including NSCLC (NCT03156114) . The primary endpoints for dose escalation and dose expansion phase were DLT and the maximum tolerated dose (MTD) and ORR, respectively. Biomarker analysis was performed in MSS CRC refractory to immunotherapy; the patients who responded to these agents with a partial response (PR) or stable disease (SD) had increased treatment-associated IFN-γ gene signature scores . Furthermore, patients with high PD-L1 gene expression in pre-treatment biopsy samples responded better to the treatment. Baseline immunohistochemistry of LAG-3 was not a predictive factor for this subset of patients.
Sym022 (anti-LAG-3) was evaluated as a single agent or in combination with sym021 (anti-PD-1) in phase 1 trials for solid tumors or lymphomas (NCT03311412, NCT03489369, and NCT03489343) (Lakhani et al., 2020). Interim analysis showed that 15 patients who were administered monotherapy and 20 patients under combination treatment, had one unconfirmed PR. Both treatment arms had tolerable safety profiles, with the combination treatment showing one grade 3-4 immune-related hypophysitis. Further assessments of the pharmacokinetic (PK) and pharmacodynamic (PD) markers and the anti-tumor activity of the monotherapy and combination are awaiting results.
MGD013 is a LAG-3 and PD-1 dual-affinity re-targeting (DART) protein; its safety, tolerability, DLT, MTD, PK/PD, and antitumor activity were analyzed in patients with unresectable and metastatic tumors in a phase 1 study (NCT03219268) (Luke et al., 2020). Fifty patients in the doseescalation phase and 157 patients in the dose-expansion phase, with 46 and 32% of patients with prior exposure to immunotherapy, respectively, were enrolled. No MTD was reached, and the most common treatment-related adverse events (TRAE), which were fatigue and nausea, were well tolerated. Despite exposure to previous immunotherapy, both cohorts included patients with objective responses. More mature clinical data are awaiting results, and biomarker analysis of LAG-3 and PD-L1 is ongoing.
May 2021 | Volume 12 | Article 681320     Recently, several studies have highlighted that TIGIT is co-expressed and associated with PD-1 expression (Johnston et al., 2014;Chauvin et al., 2015). Dual blockade of TIGIT and PD-1 resulted in the restoration of T-cell immunity in preclinical settings and provided a rationale for combination with these agents as a feasible anti-cancer therapeutic strategy (Johnston et al., 2014;Kurtulus et al., 2015;.

Clinical Trials on TIGIT
Among the 10 anti-TIGIT mAbs undergoing clinical trials, one of the most promising agents is tiragolumab (GO30103) ( Table 2). In a randomized, double-blind, phase 2 trial, 135 treatment-naïve patients with unresectable and metastatic NSCLC, positive for PD-L1 expression, were treated with tiragolumab (or placebo) in combination with atezolizumab (anti-PD-L1) (NCT03563716) (Rodriguez-Abreu et al., 2020). Primary analysis of CITYSCAPE showed that the result was significant and durable, especially in patients with a PD-L1 tumor proportion score (TPS) ≥50% in the tiragolumab and atezolizumab groups, with an ORR of 31.3 vs. 16.2% and median PFS of 5.4 and 3.6 months in the combination treatment and atezolizumab monotherapy, respectively (hazard ratio 0.57, 95% confidence interval [CI] 0.37-0.90). The combination was well tolerated and had acceptable safety profiles. The positive and robust results of this trial prompted initiation of phase III in select patients with high PD-L1 expression (SKYSCRAPER-1, NCT04294810). Furthermore, the combination was supplemented with chemotherapy in chemotherapy-naive extensive stage SCLC (SKYSCRAPER-2, NCT04256421). Phase 1 and 2 clinical trials on tiragolumab are also ongoing for esophageal and gastric cancers (NCT03281369) in metastatic settings.
Vibostolimab (MK-7684) is also an anti-TIGIT mAb. The preliminary results of a phase 1 dose-finding study of vibostolimab (200 or 210 mg) with pembrolizumab (200 mg) on day 1 of each Q3W cycle administered to patients with advanced/metastatic solid tumors without prior anti-PD-1/PD-1, showed acceptable toxicity profiles (NCT02964013) (Niu et al., 2020). The ORR and median PFS were 29% and 5.4 months for all patients, and 46% and 8.4 months for 13 patients with TPS ≥1%, respectively. The effects of vibostolimab are also being investigated in melanoma, in combination with other agents (NCT04305054, NCT04305041, and NCT04303169).

TIM-3
TIM-3, previously known as hepatitis A virus cellular receptor 2 (HAVCR2), is a member of the TIM gene family, encoding proteins such as TIM-1 and TIM-4 ( Table 1) (Monney et al., 2002). It is structured with type-1 cell surface glycoproteins, an extracellular Ig variable region (IgV)-like domain, a mucin-like and transmembrane domain, and an intracellular cytoplasmic tail composed of five tyrosine residues (Monney et al., 2002). Once the two tyrosine residues, Y265 and 272, are phosphorylated by Src kinases or interleukin inducible T cell kinase, the downstream signaling of TIM-3 is activated (van de Weyer et al., 2006;Nagahara et al., 2008).
Similar to PD-L1, TIM-3 is expressed in TILs is associated with disease progression in certain cancers (Ngiow et al., 2011). Meta-analysis of TIM-3 overexpression in solid tumors has shown that higher TIM-3 expression is associated with worse OS and may potentially be a prognostic marker . Blocking TIM-3 expression results in T cell proliferation and cytokine production, thereby eliciting immune activation (Gao et al., 2012). In addition, targeting TIM-3 with PD-1 in preclinical settings has shown a synergistic effect by reinvigorating T cell function and increasing anti-tumor immunity (Sakuishi et al., 2010;Koyama et al., 2016). Thus, the dual blockade of PD-1 and TIM-3 is a feasible and promising therapeutic option.
LY3321367 is also an anti-TIM-3 mAb; an interim analysis of a phase 1a/1b, dose-escalation and -expansion study showed that intravenous infusion of 3-1200 mg LY3321367 Q2W monotherapy (Arm A, 23 patients) or 70-1200 mg LY3321367 + 200-700 mg LY3300054 (anti-PD-L1) Q2W combination therapy (Arm B, 18 patients) was well tolerated in the treatment of refractory solid tumors; further, no DLT was observed and most TRAEs observed were grade ≤2 (NCT03099109) (Harding et al., 2019). Two patients in arm A showed >20% tumor reduction. Overall, there was no effect on the pharmacokinetics, and the antidrug antibody titers were low; thus, Eli Lily dropped the agent from its pipeline.

B7-H3
B7-H3, also called CD276, is a member of the B7 family. It was initially recognized as a co-stimulatory molecule that activates T cells and IFN-γ production ( Table 1) (Chapoval et al., 2001). B7-H3 is found in activated immune cells such as antigenpresenting cells (APCs), NK cells, T cells, and monocytes (Janakiram et al., 2017). In addition, B7-H3 is expressed in several tumors. Notably, high levels of B7-H3 expression in NSCLC, RCC, CRC, and prostate cancer are correlated with disease progression Jin et al., 2015;Benzon et al., 2017;Mao et al., 2017). In NSCLC, B7-H3 with Tregs was associated with poor prognosis, and co-expression of B7-H3 and CD14 was found to play a role in angiogenesis and tumor progression in RCC Jin et al., 2015). Patients with CRC, harboring B7-H3 and CD133 expression, have shorter survival (Castellanos et al., 2017). Similarly, high levels of B7-H3 are associated with higher Gleason grade, advanced stage, and poor outcomes in prostate cancer (Benzon et al., 2017).

Clinical Trials on B7-H3
Eleven agents targeting B7-H3 are currently under investigation in clinical trials (Table 2). Generally, patients harboring B7-H3 are enrolled in clinical trials. Enoblituzumab (MGA271), an anti-B7-H3 mAb with antibody-dependent cellular toxicity (ADCC) function, has been investigated in multiple solid tumors, including pediatric tumors. Interim analysis of enoblituzumab in refractory solid tumors revealed that it was well tolerated up to 15 mg/kg, with no DLT and MTD (Powderly et al., 2015). Although TRAEs, such as fatigue (30%) and infusion-related reactions (26%), occurred in 71% of the patients, most of these AEs were tolerated with adequate supportive care (NCT01391143). Enoblituzumab is currently being used as a monotherapy or in combination with anti-PD-1 antibody (retifanlimab or pembrolizumab), tebotelimab, a PD-1 and LAG-3 bispecific DART, or ipilimumab, as shown in Table 3.

VISTA
VISTA has several names such as differentiation of embryonic stem cells 1 (Dies1), DD1 α, Gi24, and B7H5 (  , 2013). Notably, it is also named PD-1 homologue (PD-1H), as its extracellular domain shows structural similarity to PD-1; however, it is different, as it lacks the classical ITIM or ITSM motif in the cytoplasmic domain (Flies et al., 2011). Furthermore, VISTA differs from PD-1, which functions in the effector stage, as VISTA is expressed on resting T cells, indicating its regulatory role in earlier stages (Kondo et al., 2016). Compared to that in peripheral lymph nodes, VISTA is more abundant in myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) (Le Mercier et al., 2014). High levels of VISTA are expressed by mature APCs with CD11b, whereas relatively low expression is found on Tregs, CD8 + , CD4 + , and TILs . Although the counter structures for VISTA have not been comprehensively elucidated, recent in vitro findings on V-Set and immunoglobulin domain containing 3 (VSIG-3) have shown that VISTA also acts as a coinhibitory ligand on tumor cells . VISTA promotes Treg maturation and prevents T cell activation independent of PD-1 expression (Yoon et al., 2015;Torphy et al., 2017;Popovic et al., 2018). The non-overlapping mechanisms of VISTA and PD-L1 make their combination an ideal treatment strategy to overcome immune suppression. In mouse models, dual blockade of VISTA and PD-1, using monoclonal antibodies specific for these immune checkpoints, led to synergistic activity against T-cells with anti-tumor responses .
A wide array of tumors has been studied to determine the prognostic and predictive roles of VISTA. High-grade serous ovarian cancer patients with tumor cells expressing VISTA showed longer PFS and OS (Zong et al., 2020). Furthermore, VISTA expression on TILs in pT1/2 esophageal adenocarcinoma was associated with improved OS compared to the TILs negative for VISTA (Loeser et al., 2019). Similarly, VISTA + and CD8 + TIL subtypes are associated with better OS in HCC . Contrary to these findings, VISTA + and CD8 + TIL subtypes were associated with worse prognosis in oral squamous cell carcinoma and cutaneous melanoma with VISTA expression, whereas VISTA had no correlation with survival outcome in GC expressing VISTA (Böger et al., 2017;Wu et al., 2017;Kuklinski et al., 2018).

Clinical Trials on VISTA
Ongoing clinical trials on VISTA include two anti-VISTA mAbs and one small-molecule antagonist of VISTA ( Table 2). JNJ-61610588 (NCT02671955) and CI-8993 (NCT04475523) are anti-VISTA mAbs, currently under investigation in phase 1 trials for the treatment of refractory solid tumors. CA-170 is a small molecule that targets both VISTA and PD-L1 (Musielak et al., 2019). A phase 1 study in patients with advanced solid tumors or lymphomas showed no DLT during dose escalation in 19 patients treated across six dose levels (50-800 mg) (NCT02812875) (Powderly et al., 2017). Exploratory analysis showed an increased proportion of both circulating CD8 + and CD4 + cells after oral dosing with CA-170. Further data on dose escalation, the recommended phase 2 dose, and anti-tumor responses are awaiting results.

ICOS
ICOS, also known as cluster of differentiation 278 (CD278) in T cells, is a member of the CD28 coreceptor family, which includes costimulatory CD28 and coinhibitory receptor CTLA-4 ( Table 1) (Hutloff et al., 1999). The ICOS ligand (ICOSL) is expressed in APCs such as macrophages, DCs, and B cells (Yoshinaga et al., 1999). In contrast to the expression of CD28 in both naive and memory T cells, the majority of ICOS is expressed only after the activation of memory T cells, with only small fractions expressed in resting memory T cells. Further, unlike CD28 and CTLA-4 ligands, which are expressed primarily on lymphoid tissues, ICOSL is expressed in non-lymphoid cells, such as endothelial cells, epithelial cells, mesenchymal cells, and fibroblasts, via the activation of tumor necrosis factor-α (Swallow et al., 1999;Khayyamian et al., 2002;Martin-Orozco et al., 2010). Activation of the ICOS pathway induces the production of cytokines, such as IL-4, IL-10, and IL-21, by CD4 + Th cells, CD4 + forkhead box P3 (FoxP3 + ) Tregs, and CD8 + cytotoxic T lymphocytes (CTL) (Hutloff et al., 1999;Gigoux et al., 2009;Solinas et al., 2020). ICOS interacts with its ligand (ICOSL) to increase anti-tumor effects via the regulation of memory and effector T cell development and humoral immune responses (Marinelli et al., 2018). The rationale for targeting the ICOS/ ICOSL axis with agonists and antagonists is its capacity to trigger both anti-tumor T cell responses by Th1 and other effector T cells, as well as its protumor responses via Tregs (Solinas et al., 2020).
In preclinical studies, ICOS expression on FoxP3 + Tregs and other Th subsets has been identified in multiple arrays of solid tumors, including melanoma, gastric, colorectal, and breast cancers (Strauss et al., 2008;Zhang et al., 2016;Gu-Trantien et al., 2017;Nagase et al., 2017). ICOS + Treg TILs have been found to be associated with worse survival in GC, whereas high levels of ICOS in Th1 TILs in colorectal cancer indicated better survival outcomes Nagase et al., 2017). Dual blockade of ICOS with anti-CTLA-4 has been effective in eliciting anti-tumor responses in ICOS knockout mice that were unresponsive to anti-CTLA-4 monotherapy (Fu et al., 2011;Fan et al., 2014). More importantly, the utilization of ICOStargeted agents is gaining attention in hematological malignancies owing to the enhancement of co-stimulatory receptor 4-1BB in CD4 + CAR T cells by ICOS (Guedan et al., 2018).

Clinical Trials on ICOS
Currently, both anti-ICOS agonists and anti-ICOS antagonists are under clinical investigation ( Table 2). The phase 1 trial of GSK3359609 (INDUCE-1), a humanized anti-ICOS agonist monoclonal antibody, comprised two treatment groups: part 1 patients were treated with a monotherapy of GSK3359609, and part 2 patients were Frontiers in Pharmacology | www.frontiersin.org May 2021 | Volume 12 | Article 681320 administered a combination with pembrolizumab or other immunotherapy in the treatment of advanced solid tumors. The study is ongoing, with no dose-limiting toxicities from the first three dose-limiting cohorts (Angevin et al., 2017). In head and neck cancer, the efficacy of GSK3359609 and pembrolizumab with or without platinum-based chemotherapy is currently under investigation (NCT04428333 and NCT04128696).
Another investigational anti-ICOS agonist monoclonal antibody is JTX-2011, used in combination with either anti-PD1 (pembrolizumab or nivolumab) or anti-CTLA-4 (ipilimumab) in advanced solid tumors (NCT02904226) (Yap et al., 2018). In phase 1/II of the trial, anti-tumor activity was observed with JTX-2011 monotherapy and in combination with nivolumab, in heavily treated GC and TNBC with manageable toxicity profiles. Exploratory analysis showed that the peripheral blood CD4 ICOS high T cell subsets may be a potential biomarker for the response.
Further, agonistic antibodies such as MEDI-570 alone and KY1044 with atezolizumab are under investigation in phases 1 and phase 1/II, respectively (NCT02520791 and NCT03829501).
BTLA BTLA (CD272) is also a member of the CD28 coreceptor family ( Table 1) (Ceeraz et al., 2013). It is a co-inhibitory molecule with a structure and function similar to those of PD-1 and CTLA-4 (Paulos and June, 2010). When expressed on mature lymphocytes, such as B cells and T cells, macrophages, and DCs, BTLA binds to herpes virus entry mediator (HVEM), a member of the tumor necrosis factor receptor superfamily (TNFRSF), as well as to LIGHT and lymphotoxin-α, two members of the tumor necrosis factor (TNF) superfamily (Han et al., 2004;Sedy et al., 2005;Steinberg et al., 2011). Binding of BTLA to HVEM via CD160 transmits inhibitory signals to T cells, which are necessary for proliferation and cytokine production, whereas binding to LIGHT induces co-stimulatory signals (Sedy et al., 2005;Murphy et al., 2006;Cai et al., 2008). Thus, the complexity of the BTLA receptor and ligand activity poses a challenge for BTLA blockade treatment.
Recently, the possibility of BTLA as a potential therapeutic target in cancer immunotherapy has been established in vivo, wherein human melanoma tumor antigen-specific effector CD8 + T cells expressing high levels of BTLA were downregulated with a vaccine formulated using CpG oligodeoxynucleotides, a toll-like receptor 9 (TLR9) agonist that triggers innate immunity, thereby proving that inhibition of BTLA may partially reverse the function of human CD8 + cancer-specific T cells (Derré et al., 2010;Paulos and June, 2010).

CONCLUSION
Cancer immunotherapy is one of the major pillars in the field of medical oncology, especially for the treatment of unresectable, metastatic, and recurrent cancers. The success of ICIs, such as anti-CTLA-4 and anti-PD-1/PD-L1, in combination with chemotherapy, immunotherapy, and targeted agents, has changed the paradigm of cancer treatment. Nonetheless, the limited efficacy and IRAEs of ICIs have paved way for the discovery of novel checkpoints. Among the immune checkpoint inhibitors, anti-LAG-3 and anti-TIGIT are promising targets, and their efficacy in combination with anti-PD-1/PD-L1 may help overcome the limitations seen in prior treatments. More robust data are yet to follow on agents targeting TIM-3, B7-H3, VISTA, ICOS, and BTLA.