Therapeutic Targeting of Checkpoint Receptors Within the DNAM1 Axis

Published OnlineFirst March 9, 2021; DOI: 10.1158/2159-8290.CD-20-1248

Review

Therapeutic Targeting of Checkpoint Receptors within the DNAM1 Axis

Zoya Alteber1, Maya F. Kotturi2, Sarah Whelan2, Sudipto Ganguly3, Emmanuel Weyl1, Drew M. Pardoll3, John Hunter2, and Eran Ophir1

abstract Therapeutic antibodies targeting the CTLA4/PD-1 pathways have revolutionized cancer immunotherapy by eliciting durable remission in patients with cancer. How- ever, relapse following early response, attributable to primary and adaptive resistance, is frequently observed. Additional immunomodulatory pathways are being studied in patients with primary or acquired resistance to CTLA4 or PD-1 blockade. The DNAM1 axis is a potent coregulator of innate and adaptive immunity whose other components include the immunoglobulin receptors TIGIT, PVRIG, and CD96, and their nectin and nectin-like ligands. We review the basic biology and therapeutic relevance of this family, which has begun to show promise in cancer clinical trials.

Significance: Recent studies have outlined the immuno-oncologic ascendancy of coinhibitory receptors in the DNAM1 axis such as TIGIT and PVRIG and, to a lesser extent, CD96. Biological elucidation backed by ongoing clinical trials of single-agent therapy directed against TIGIT or PVRIG is beginning to provide the rationale for testing combination regimens of DNAM1 axis blockers in conjunction with anti–PD-1/PD-L1 agents.

Introduction similarly inducible inhibitory checkpoint receptor whose discovery postdates that of CTLA4 is PD-1, which upon Immunostimulatory monoclonal antibodies (mAb) that binding to one of its two defined ligands, PD-L1 or PD-L2, block inhibitory checkpoint proteins such as CTLA4 or restricts T-cell activation (2). In the clinic, several mAbs PD-1 and PD-L1 have made a paradigm shift in the develop- targeting either PD-1 or PD-L1 in solid tumors were tested ment of anticancer therapies. Since the early studies target- before the approval of the first PD-1 blockers, nivolumab ing these checkpoints in the 2000s, roughly 10 additional and pembrolizumab (3, 4). A mAb targeting CTLA4, three coinhibitory molecules have been targeted clinically. The mAbs targeting PD-1, and three targeting PD-L1 have been biology of these molecules is complex, in part because most approved by the FDA for treating 18 different malignan- T-cell regulatory pathways are multicomponent. CTLA4 (or cies (5, 6). Exploratory research is ongoing to discover CD152) is a homolog of the costimulatory receptor CD28, novel checkpoints and eventually generate mAbs that could with both capable of binding the ligands CD80 and CD86. potentially augment the clinical results observed with PD-1 Reflecting its status as a key regulator of autoimmunity, or CTLA4 blockade. With that in mind, additional T cell– CTLA4 displays an affinity for CD80 and CD86 that is specific inhibitory receptors have been studied extensively in several orders of magnitude greater than that of CD28 (1). preclinical cancer models and are being tested in the clinic CTLA4 outcompetes CD28 for access to their shared ligands either as single agents or in combination with other check- and delivers inhibitory signals that dampen T-cell recep- point blockers or chemotherapy. Among these, lymphocyte- tor (TCR; signal 1) and costimulatory signals (signal 2). A activation gene 3 (LAG3), T-cell Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), T-cell Ig and mucin domain 3 (TIM3), and B and T lympho- 1Compugen Ltd, Holon, Israel. 2Compugen USA, Inc., South San Francisco, California. 3Bloomberg Kimmel Institute for Cancer Immunotherapy, John cyte attenuator (BTLA4) have all been shown to overcome Hopkins University, Baltimore, Maryland. T-cell exhaustion in the tumor microenvironment (TME) Z. Alteber, M.F. Kotturi, S. Whelan, and S. Ganguly are co–first authors of and are considered to be promising druggable targets in this review. the immuno-oncology space (7–10). Although blockers of Corresponding Author: Eran Ophir, Compugen Ltd, Azrieli Center, 26 each of these inhibitory checkpoints are in varying stages Harokmim Street, Building D, Holon 5885849, Israel. Phone: 972-37658504; of clinical testing, none have secured FDA approval for use E-mail: [email protected]. in cancer. Antibodies targeting TIM3 have shown encourag- Cancer Discov 2021;11:1–13 ing preliminary antitumor effects in patients with hema- doi: 10.1158/2159-8290.CD-20-1248 tologic malignancies, including myelodysplastic syndrome ©2021 American Association for Cancer Research. (MDS) and acute myeloid leukemia (AML; ref. 11). The

may 2021 CANCER DISCOVERY | OF1

REVIEW Alteber et al.

antigen 1 (LFA-1) has been demonstrated in both NK and activated T cells. This physical interaction was shown to Tumor cell promote the formation of stable target cell conjugates, and or APC NK cells lacking LFA-1 expression are deficient in DNAM1- driven cytotoxicity, suggesting a functional dependency between the two molecules (16). Further supporting the func- PVR PVRL2 tional interaction between the two molecules, cross-linking of LFA-1 results in FYN protein tyrosine kinase phosphorylation of DNAM1 (16). LFA-1 and DNAM1 demonstrate DNAM1 LFA-1 coordinated expression and colocalization in the immune PVRIG TIGIT synapse of activated NK cells in the presence of target cells + CD96 (17), and DNAM1-deficient CD8 T cells have a decreased ability to recruit LFA-1 and lipid rafts to the immune synapse, T or NK cell + – – while also showing reduced conjugation with target tumor + ? – cells (18). PVR and PVRL2, belonging to the superfamily of cell adhesion molecules, have been identified as the ligands for Figure 1. TIGIT and PVRIG are parallel inhibitory pathways in the DNAM1 (Fig. 1). PVR and PVRL2 mediate cell–cell adhe- DNAM1 axis. Interactions between DNAM1 axis molecules and their sion by generating heterotypic and homotypic (only PVLR2) cognate ligands on tumor or antigen-presenting cells (APC) induces interactions (19). High expression of these molecules on activation (+) or inhibition (−) of T- and NK-cell function. Arrow thickness tumor cells renders them susceptible to T and NK cell– represents the relative affinity between the interacting molecules. mediated cytotoxicity, which can be abrogated by an anti- DNAM1 antagonist antibody (20, 21). Overexpression of PVR promising candidates directed against LAG3 and TIGIT or PVRL2 in the RMA mouse tumor cell line increased tumor are currently in phase III clinical trials in combination with rejection and host survival via T- and NK-mediated mecha- antibodies blocking the PD-1 pathway. For LAG3 alone, nisms, suggesting that each can act as costimulatory ligands 10 agents are being tested in clinical trials for a variety of in the TME, presumably via their activation of DNAM1. cancers. Multiple anti-TIGIT antibodies are being evalu- However, identification and characterization of the immune- ated in different clinical trials as single agents or in com- checkpoint proteins TIGIT, CD96, and PVRIG have generated bination with PD-1/PD-L1 blockers, as will be discussed in a more complex picture of their function, because the interac- this review (12). Moreover, we will discuss the biology and tion of PVR with TIGIT and CD96, and that of PVRL2 with immunotherapeutic significance of all constituents of the PVRIG, can also mediate suppressive signaling through the DNAM1 (DNAX accessory molecule 1, CD226) axis, includ- coinhibitory arms of the DNAM1 signaling axis, suggesting ing the coinhibitory receptors TIGIT, poliovirus receptor– that they act to counterbalance DNAM1 costimulation. At related Ig domain–containing protein (PVRIG), and CD96, the 30,000-foot level, this is analogous to CD28 and CTLA4 their respective primary ligands poliovirus receptor (PVR, as counterbalancing costimulatory and coinhibitory recep- CD155) and PVR-related 2 (PVRL2, Nectin-2, CD112), and tors, respectively, for CD80 and CD86; however, at a closer the eponymous costimulatory receptor DNAM1 (Fig. 1). glance, there are significant differences that give the DNAM1 family its unique character. Although CD28 is expressed DNAM1: The Central Costimulatory exclusively on T cells, DNAM1 axis members are expressed on Molecule in an Interacting Immune- both T and NK cells. In addition, CD28 is downregulated on T cells upon activation and differentiation, whereas DNAM1 Regulatory Network in the TME maintains its expression also on differentiated effector cells DNAM1 is the central node that links together four recep- (22, 23). Furthermore, CD80 and CD86 are restricted to tors (DNAM1 itself, TIGIT, PVRIG, and CD96) and two immune cells, mostly antigen-presenting cells (APC), whereas ligands (PVR and PVRL2) into one family. It is a 65 kilo- PVR and PVRL2 are expressed on multiple cell types, includ- dalton (kD) member of the Ig superfamily that contains ing APC and tumor and endothelial cells. These differences two extracellular Ig-like domains, an intracellular immu- in expression pattern suggest that whereas CD28 and CTLA4 noreceptor tyrosine–based activation motif, and is constitu- probably play a dominant role in T-cell priming, DNAM1 tively expressed on the surface of natural killer (NK) cells, axis members presumably regulate both priming and effec- T lymphocytes, platelets, monocytes, and a subset of B cells. tor phase of T cells, resembling the PD-1 pathway. Never- DNAM1 was identified in a screen for cell-surface molecules theless, similar to the higher-affinity CD80/86 interactions that mediate T-cell cytotoxicity and was subsequently shown with CTLA4 versus CD28, TIGIT, CD96, and PVRIG have to act as a costimulatory molecule on NK cells as well as T higher affinities to their cognate ligands than DNAM1 and cells (13). Consistent with a costimulatory function on T thus can inhibit T-cell and NK-cell activity through ligand and NK cells, it was shown to mediate antitumor immune competition (24–28). This was supported by several studies responses as demonstrated by increased tumor growth in showing that the costimulatory effects driven by targeting DNAM1 knockout mice (14, 15). the TIGIT/PVR interaction are dependent on the engagement In addition to DNAM1’s direct costimulatory activity, of DNAM1 by PVR (27, 29). A recent study suggested that the a physical association with lymphocyte function-associated infiltration of CD8+DNAM1hi T cells into TME may predict

OF2 | CANCER DISCOVERY may 2021 AACRJournals.org

Targeting of Checkpoint Receptors within the DNAM1 Axis REVIEW

+ + Dendritic cells – Suppressive activity + + – –

Tumor cells Tregs – – –

+ + ? KEY – – + PVR PVRL2 CD96

TIGIT PVRIG DNAM1 Tumor cells T/NK cells

Figure 2. The effects of DNAM1 axis modulation on immune cells. DNAM1 axis molecules and nectin and nectin-like ligands are expressed on various cells, and their interactions modulate immune function. A, TIGIT and DNAM1 exert opposite effects in Tregs upon PVR binding. TIGIT further stimulates Treg immunomodulatory activity, whereas DNAM1 disrupts Treg suppression. B, Although DNAM1 stimulates effector T- and NK-cell function, TIGIT and PVRIG induce their suppression. PVR engagement on dendritic cells by TIGIT induces a tolerogenic phenotype and reduces proinflammatory cytokine secretion. the efficacy of TIGIT blockade, as the presence of these cells Potential drawbacks of anti-PVR or anti-PVRL2 mAbs are is essential for the anti-TIGIT immunomodulatory activity that direct targeting may result in interference of DNAM1 (30). Additional complexity arises from the direct interrup- costimulatory signaling, as well as in the induction of ADCC tion of DNAM1 homodimerization by TIGIT, which results against APCs. Finally, PVR and PVRL2 are broadly expressed in its impaired functionality (29). Furthermore, DNAM1 in peripheral normal tissues, creating a large target sink and expression on lymphocytes is modulated by membrane PVR increasing the risk of developing on-target, off-tumor tox- that upon binding to DNAM1 induces its internalization and icities. Indeed, thrombocytopenia was identified in monkeys degradation, resulting in decreased antitumor function (31). administered an anti-PVRL2 mAb (36). Similarly, it was shown that PVRIG outcompetes DNAM1 for Altogether, costimulatory DNAM1 activity in T and NK PVRL2 binding (ref. 32; Fig. 1). cells is tightly regulated at the expression level and by its One approach to boost antitumor immunity by modulating interaction with other axis members; understanding the biol- the DNAM1 axis could arise from agonizing DNAM1, which ogy of these interactions is crucial for the success of immuno- has the potential to show therapeutic benefit in patients with therapies targeting this axis. cancer due to its potent T- and NK-cell coactivatory function. However, a caveat for this approach is the broad expression TIGIT Blockade: A Promising New of DNAM1 on different cell types, including on platelets, Clinical Modality for Treating Solid where it was shown to stimulate activation and cell adhesion (33). Indeed, an agonist antibody directed against DNAM1 and Hematologic Malignancies (LY3435151) was clinically evaluated in solid malignancies; TIGIT was initially identified as an inhibitory receptor however, the study (NCT04099277) was recently terminated on T and NK cells based on shared gene and protein struc- for undisclosed reasons. ture with known immune checkpoints (8, 24). TIGIT is also Another interesting approach could be the direct targeting expressed on regulatory T cells (Treg) where it enhances their of PVR or PVRL2 with antagonist antibodies. This may result activation and suppressive activity (Fig. 2A). Similar to other in T- and NK-cell activation by interrupting the dominant immune checkpoints, TIGIT contains an extracellular Ig vari- interactions with TIGIT and PVRIG, respectively. When using able domain, a transmembrane domain, and an ITIM in the effector function antibodies, an antibody-dependent cellular intracellular signaling domain (8, 24). The dominant ligand cytotoxicity (ADCC) against PVR/PVRL2-expressing tumor for TIGIT is PVR with a binding affinity (KD) reported as 1–3 cells could be applied, while avoiding the risk of poten- nmol/L (Fig. 1; ref. 24). A recent study described Nectin4 tial ADCC against T cells. Moreover, in addition to direct (PVRL4) as a functional ligand of TIGIT with an affinity simi- effects on immune cells, interfering with nonimmune tumor- lar to that of TIGIT/PVR (37). PVRL2 and PVRL3 (CD113) promoting effects of PVR or PVRL2 could provide additional have also been reported to bind TIGIT albeit at much weaker benefit for this approach (34). Accordingly, an anti-PVRL2 affinities (8, 19, 24). PVR expression was shown to positively antagonizing mAb developed by Takeda exhibited antitumor correlate with an unfavorable prognosis in multiple malig- effects attributed mainly to ADCC against tumor cells (35). nancies such as lung adenocarcinoma (38) and breast (39),

may 2021 CANCER DISCOVERY | OF3

REVIEW Alteber et al. pancreatic (25), and bladder (40) cancers. In patients with CD8+ T cells and wild-type CD4+ T cells into B16F10 mela- metastatic melanoma, PVR expression on tumor cells was noma tumor–bearing RAG-deficient mice did not alter tumor associated with resistance to anti–PD-1 immunotherapy (41). growth compared with transfer of wild-type TIGIT+ CD8+ T As mentioned above, TIGIT’s higher binding affinity for PVR cells (49). In contrast, the transfer of TIGIT-deficient Tregs compared with DNAM1 allows it to outcompete DNAM1 and wild-type CD4+FOXP3− and CD8+ T cells significantly for ligand binding, similar to CTLA4 versus CD28 (21, 24, delayed B16F10 tumor growth compared with mice that 27, 28). Although the expression of both CTLA4 and TIGIT received wild-type Tregs. These data suggest that in at least is induced upon activation, CD28 is mainly expressed on some tumor models, TIGIT+ Tregs play a more dominant role naïve T cells, whereas DNAM1 has broad expression on mul- in suppressing antitumor immune responses compared with tiple T-cell subsets (23). Moreover, unlike CTLA4, TIGIT has direct signaling of TIGIT on CD8+ T cells. Other studies have been shown to directly interact with DNAM1, reducing the examined the mechanisms of TIGIT+ Treg-mediated immune ability of DNAM1 to homodimerize and enhance immune suppression. Joller and colleagues showed that increased activation (27, 29, 42). Recently, it was demonstrated that amounts of IL10 produced by TIGIT+ Tregs contributed to the TIGIT/PVR interaction induces dephosphorylation of the generation of tolerogenic dendritic cells, thereby inhib- DNAM1 at tyrosine 322 and interferes with its costimulatory iting the generation of effector T-cell responses (42, 51). responses on T cells (30). In addition to counteracting the Moreover, TIGIT blocking mAbs were shown, in addition to stimulatory function of DNAM1 via direct binding and com- enhancing conventional T-cell activity, to reduce Treg secre- petition for their common PVR ligand, TIGIT can directly tion of IL10 (52). Finally, a recent study showed that Tregs inhibit T- and NK-cell effector function by signaling through in melanoma further upregulate TIGIT and downregulate an ITIM domain (8, 43). TIGIT also indirectly regulates CD8+ DNAM1 expression, resulting in a higher TIGIT/DNAM1 T-cell priming and proliferation by engaging PVR on den- ratio (53). This high TIGIT/DNAM1 ratio in Tregs regulates dritic cells, which reportedly induces a tolerogenic phenotype their suppressive function and stability and correlates with a and reduces their proinflammatory cytokine production via poor clinical outcome following PD-1 and/or CTLA4 path- “reverse signaling” (Fig. 2B; ref. 24). way blockade in patients with melanoma (44, 49, 53). Several studies have shown that TIGIT is a marker of T-cell In addition to TIGIT’s role in T-cell biology, numerous dysfunction and is upregulated on human viral-specific CD8+ groups have shown higher TIGIT expression on viral and T cells, and human CD8+ and CD4+ T cells infiltrating a tumor-infiltrating NK cells and that increased TIGIT levels are variety of solid and hematologic tumors. In the LCMV Clone negatively correlated with the ability of NK cells to secrete pro- 13 model of chronic viral infection, Johnston and colleagues inflammatory cytokines and kill PVR-expressing tumor cells, demonstrated that TIGIT is highly expressed on CD8+ T cells suggesting that TIGIT abrogates NK-cell activation (Fig. 2B; that also express high levels of PD-1 (29), and that inhibition refs. 8, 43, 54). In mouse models, lack of TIGIT+ NK cells was of these pathways increased viral clearance and T-cell effector sufficient to slow B16 melanoma tumor growth, while antibody function. Similarly, in murine models of colon and breast car- blockade of the TIGIT pathway reinvigorated the antitumor cinomas, coblockade of TIGIT and PD-L1 resulted in tumor NK-cell response in multiple solid tumors and hematologic rejection and restoration of CD8+ T cells within the TME models (43, 55–57). Overall, survival was increased in these (29). Johnston and colleagues further demonstrated that tumor models, indicating that NK cells may be critical for the mice treated with anti-TIGIT and anti–PD-L1 were unable therapeutic effects of TIGIT pathway–based immunotherapies to reject CT26 tumors when depleted of CD8+ T cells (29). (56, 57). Collectively, these studies demonstrate important Chauvin and colleagues also demonstrated that the TIGIT roles for TIGIT-expressing CD8+ T cells, Tregs, and NK cells in pathway blockade either alone or in combination with anti– regulating antitumor responses in mouse tumor models and PD-1 synergistically increased effector function of human human in vitro functional systems. NY-ESO-1–specific CD8+ tumor–infiltrating lymphocytes An open question in the TIGIT therapeutic antibody field (TIL) from patients with melanoma (44). Collectively, these is whether Fc receptor (FcR) coengagement is required to data demonstrate that TIGIT and PD-1/PD-L1 coblockade exploit the full clinical potential from TIGIT blockade. Mul- acts through CD8+ T cells to generate an effective antitumor tiple antibodies targeting TIGIT are currently in clinical immune response. Interestingly, TIGIT was shown to be development, some with FcR binding (typically human IgG1) uniquely expressed by T memory stem (Tscm) cells, an emerg- and others with reduced binding to FcR (Table 1). Murine ing population of T cells, shown to be important for response studies with anti-TIGIT mouse IgG2a antibodies (increased to immunotherapy. Tscm cells display a functional state FcR-binding capacity) demonstrate monotherapy activity associated with enhanced self-renewal, have multipotency to in vivo and increased potency compared with mouse IgG1 Abs generate memory and effector T-cell subsets, and were found (reduced FcR-binding capacity; refs. 58, 59). Recently, Yang to be associated with increased response to immunotherapy and colleagues demonstrated that TIGIT blockade induced and to correlate significantly with PD-1 blockade therapeutic Fc-mediated depletion of Tregs that activate antitumor CD8+ activity (45–47). Importantly, TIGIT and PD-1 are the domi- T-cell responses, targeting tumor-shared antigens that are nant inhibitory T-cell checkpoints expressed on this memory normally cryptic or suppressed by Tregs (60). In contrast, a cell subset (48). mouse IgG1 anti-TIGIT antibody (low FcR-binding capacity) Other studies reported that TIGIT predominantly sup- has been shown to significantly enhance the antitumor activ- presses antitumor CD8+ T-cell responses indirectly by ity, particularly when combined with a blocking anti–PD-L1 enhancing Treg function in the TME (49–51). Kurtulus and antibody (53). Ultimately, the ability of mouse models to colleagues found that adoptively transferred TIGIT-deficient predict the importance of FcR binding by a therapeutic

OF4 | CANCER DISCOVERY may 2021 AACRJournals.org

Targeting of Checkpoint Receptors within the DNAM1 Axis REVIEW

Table 1. List of antagonistic antibodies targeting DNAM-1 axis family members that are under preclinical and clinical development

Antibody Development Combination Patient ClinicalTrials.gov Company Compound Target isotype stage therapy population identifier Roche MTIG7192A TIGIT IgG1 Phase III Atezolizumab, NSCLC, cervical NCT04300647, tiragolumab carboplatin + cancer, multiple NCT04294810, etoposide, myeloma, NCT04256421, rituximab, non-Hodgkin NCT04045028, daratumumab, lymphoma, NCT03563716, carboplatin + B-cell lym- NCT02794571, paclitaxel, phoma, gastric NCT03281369, cisplatin adenocarcinoma, NCT03869190, urothelial carci- NCT03193190 noma, pancreatic adenocarcinoma Merck MK-7684 vib- TIGIT IgG1 Phase II Pembrolizumab, Advanced solid NCT02964013, ostolimab carboplatin + tumors, NSCLC, NCT04165070, paclitaxel, melanoma NCT04305054, pemetrexed, NCT04305041, MK-1308 (anti- NCT04303169 CTLA4 mAb) Arcus Bio- AB154 TIGIT Inactive IgG1 Phase III AB122 (anti–PD-1 Advanced solid NCT03628677, sciences mAb), AB928 tumors, NSCLC NCT04262856 (adenosine receptor antagonist) Phase III Durvalumab NSCLC To be announced BMS BMS-986207 TIGIT Inactive IgG1 Phase I/IIa Nivolumab, Advanced solid NCT02913313, pomalidimide + tumors, multiple NCT04150965 dexamethasone myeloma Mereo OMP-313M32 TIGIT IgG1 Phase I Nivolumab Advanced solid NCT03119428 etigilimab tumors Compugen COM902 TIGIT IgG4 Phase I N/A Advanced malig- NCT04354246 nancies iTeos EOS884448 TIGIT IgG1 Phase I N/A Advanced malig- NCT04335253 nancies Innovent IBI939 TIGIT N/A Phase I Sintilimab (anti– Advanced malig- NCT04353830 PD-1 mAb) nancies Beigene BGB-A1217 TIGIT IgG1 Phase I Tislelizumab (anti– Metastatic solid NCT04047862 PD-1 mAb) tumors Seattle SGN-TGT TIGIT Effector- Phase I Pembrolizumab Advanced malig- NCT04254107 Genetics enhanced IgG1 nancies EMD Serono M6223 TIGIT N/A Phase I Bintrafusp alfa Metastatic solid NCT04457778 (anti–PD-L1/ tumors TGFß Trap) Compugen COM701 PVRIG IgG4 Phase I Nivolumab Advanced solid NCT03667716 tumors GSK GSK6097608 CD96 N/A Phase I Dostarlimab Neoplasms NCT04446351

Abbreviation: N/A, not applicable.

antibody is limited, because murine FcRs do not mirror a TIGIT antibody with potent effector function carries the the structural diversity, expression, and Fc binding pattern risk of depleting the effector CD8+ TILs in addition to tumor of the human FcR (61). Moreover, given the overlap in Tregs. Studies on mechanisms of action of CTLA4 blockade TIGIT expression between CD8+ effector T cells and Tregs, emphasize caution in the direct extrapolation of FcR roles

may 2021 CANCER DISCOVERY | OF5

REVIEW Alteber et al. between mouse and human. Although previous murine stud- CD96: A Second PVR-Binding Receptor ies showed that antitumor effects of a FcR-binding mouse with Unclear Function in Human and IgG2a anti-CTLA4 were dependent on FcR expression and Mouse were associated with selective intratumoral depletion of Treg, ipilimumab, a human IgG1 that binds human FcR, failed Originally described as T-cell activation, increased late to decrease intratumoral Treg density (62). Likewise, PD-L1 expression (TACTILE), CD96 was discovered two decades ago blockade was shown to require FcR engagement for medi- as a member of the Ig superfamily that is highly upregulated ating activity in vivo in mice (17), whereas in the human on activated T and NK cells and mediates cell adhesion via settings, anti–PD-L1 antibodies with silent Fc backbones interaction with its dominant ligand PVR (20, 69, 70). CD96 exhibited clinical efficacy and are approved by the FDA. has an expression pattern similar but not identical to TIGIT, Therefore, it remains to be determined in patients which cell including on hematopoietic stem cells, αβ and γδ T cells, NK type plays a dominant role in generating antitumor responses cells, and a subpopulation of B cells in humans (23, 71–75) during anti-TIGIT therapy and whether FcR engagement is a and mice (23, 75, 76). It is highly expressed on human tumor– required feature in the clinic. infiltrating CD8+ T cells, compared with normal tissues (23, Another unanswered question being investigated in clinical 77). Moreover, its expression on T cells is increased upon trials is which treatments should be paired with anti-TIGIT stimulation, resembling the expression profile kinetics of therapy to generate robust and durable antitumor responses, DNAM1 (23). Like TIGIT, the high-affinity binding partner especially in anti–PD-1/PD-L1-resistant patients. Although for CD96 is PVR. Although the binding affinity of CD96 to TIGIT is highly coexpressed with PD-1 on human TILs from PVR is higher than that of DNAM1, it is lower than that of different solid tumors and lymphomas, its expression also TIGIT (Fig. 1; ref. 24). CD96 also binds PVRL1 (CD111), but highly correlates with the expression of other checkpoint the functional relevance of the CD96/PVRL1 interaction has receptors, including LAG3, TIM3, and BTLA4 (63–65). One not been fully explored (75, 78). study showed that TIM3 pathway blockade in TIGIT-defi- The functional role of CD96 has been mainly investigated in cient mice acts synergistically to reduce B16F10 melanoma murine NK cells. CD96 appears to act as an inhibitory recep- tumor growth (49). Recently, we demonstrated that PVRIG/ tor in murine NK cells, decreasing NK-cell activation. Chan PVRL2 blockade induces TIGIT expression (26), and combin- and colleagues demonstrated that in CD96-deficient mice, NK ing TIGIT with PVRIG coblockade synergistically activates cells produced greater IFNγ in response to LPS, IL12, or IL18 T cells in human functional assays which are translated into stimulation (76). A role for CD96 in resistance to experimental in vivo antitumor effects (66). Furthermore, TIGIT-blocking melanoma lung metastases, MCA-induced fibrosarcomas, and mAbs are being tested clinically in combination with other prostate carcinomas was also demonstrated in CD96-deficient therapeutic modalities such as chemotherapy, IL15 cytokines, mice or following anti-CD96 antibody treatment in wild-type and myeloma-targeting antibodies (31). mice. Combined blockade of CD96 and PD-1 or CTLA4 path- Although the majority of anti-TIGIT antibodies are cur- ways increased cytokine secretion and survival in tumor-bear- rently in early clinical investigation, several have progressed ing mice in comparison with anti–PD-1 or anti-CTLA4 alone beyond phase I studies. These include tiragolumab (Roche- (76, 79). Furthermore, an additional report found that in mice Genentech), which is currently recruiting patients in phase that lack both PD-1 and CD96, or both TIGIT and CD96, III studies in non–small cell lung cancer (NSCLC) and small there is enhanced tumor growth inhibition and/or complete cell lung cancer (SCLC); domvanalimab (Arcus), initiating response in dual receptor–deficient mice compared with mice a phase III studies in NSCLC; and vibostolimab (Merck). lacking PD-1, TIGIT, or CD96 alone (80). Although CD96 was Initial results have been presented from three clinical stud- originally identified as an Ig receptor expressed on activated ies evaluating anti-TIGIT antibodies as a single agent and T cells, there are very few studies examining the functional have demonstrated limited clinical activity, with overall role of CD96+ T cells. In addition to its inhibitory role in NK response rates ranging from 0% to 3% (67, 68). Recently, data cells, recently, Mittal and colleagues showed that CD96 block- were reported from a randomized, phase II double-blind, ade suppressed tumor growth of several murine models in a placebo-controlled study of 135 patients with previously CD8+ T cell–dependent manner (77). The antitumor efficacy untreated NSCLC who were randomized 1:1 to tiragolumab of CD96 was mediated by increasing IFNγ secretion and was plus atezolizumab versus placebo plus atezolizumab (67). dependent on DNAM1, as CD96/DNAM1 coblockade abol- The study met its coprimary endpoints of objective response ished CD96 effect (77). rate (ORR) and progression-free survival for the combina- There are limited studies characterizing the functional role tion versus atezolizumab alone in the intent-to-treat (ITT) of CD96 in human NK cells, and the results are conflicting. population, demonstrating an improvement in ORR of 31.3% Transcriptomic analysis comparing human hepatic CD96+ compared with 16.2%, and in median progression-free sur- and CD96− NK-cell subsets suggests that CD96+ NK cells are vival of 5.4 months compared with 3.6 months. Thus, this functionally exhausted compared with CD96− NK cells and study provided the first proof of concept for TIGIT inhibi- that CD96+ cells are enriched with published gene signatures tion in a larger, randomized clinical trial. The clinical benefit such as inhibition- and exhaustion-related genes that are cor- derived from the combination was particularly effective in the related with the regulation of NK-mediated immunity (81). PD-L1–positive ≥ 50% population, demonstrating a 66% ORR Fuchs and colleagues showed that the cytotoxic activity of and a 70% reduction in the risk of disease worsening or death. human polyclonal NK cell lines was enhanced in the presence This combination is currently being evaluated in a phase III of a CD96 binding antibody (70), suggesting that human study in PD-L1 ≥ 50% first-line NSCLC. CD96 promotes NK-cell activation rather than inhibition. In

OF6 | CANCER DISCOVERY may 2021 AACRJournals.org

Targeting of Checkpoint Receptors within the DNAM1 Axis REVIEW contrast to this report, Carlsten and colleagues demonstrated CD8+ T cells upregulate PVRIG expression, although at a that although DNAM1 blockade significantly reduced NK much slower rate compared with related coinhibitory check- degranulation and cytotoxicity, an anti-CD96 mAb did not points. The relative expression of mouse PVRIG on TILs is affect tumor cell killing when NK cells were cocultured with lower than that seen in humans, and mouse PVRL2 in the fresh ovarian carcinoma cells (82). Additionally, we have TME and mouse tumor cell lines is lower than the expression demonstrated that inhibition of the CD96/PVR interaction detected in human TME, further supporting the idea that with two distinct, antagonistic anti-CD96 antibodies had no PVRIG plays a diminished role in mice compared with its role effect on IFNγ secretion from primary human T cells (26). in humans (85). These findings, including the acquisition of The cytoplasmic tail of both human and mouse CD96 has an an ITIM in human PVRIG and higher levels of human PVRIG ITIM-like motif for inhibitory signaling, but human CD96 and PVRL2 in the TME, suggest that greater effects of PVRIG also contains a YXXM motif, which is found within other inhibition may be seen in a human tumor setting relative to known activating receptors including CD28 and NKG2D mouse preclinical models. (72). The presence of a potential activating motif might Nevertheless, although mouse PVRIG lacks an ITIM motif, explain why CD96 has been reported as either an inhibitory PVRIG-deficient T cells do have increased function compared or costimulatory receptor in different human in vitro assay with wild-type T cells following antigen challenge. Moreover, systems. Accordingly, it was recently reported that CD96 has in vivo syngeneic models utilizing MC38 colon carcinoma costimulatory functions in human and mouse CD8+ T cells. and B16 melanoma demonstrated that tumors grew slower It was shown in vitro and in vivo that depletion or disruption in PVRIG-deficient compared with wild-type mice, andex vivo of CD96/PVR interaction results in impaired T-cell prolifera- analysis pointed to functional differences in T-cell responses. tion and inflammatory cytokine secretion (83). In the CT26 colon carcinoma model, reduced tumor growth Given these conflicting results, the role of CD96 in human and increased survival were observed after PVRIG and PD-1 T and NK cells is not fully understood, and collectively these coblockade (85). This was further supported by increased studies indicate that CD96 pathway blockade may either antitumor responses in PVRIG/TIGIT double-knockout enhance or inhibit lymphocyte killing of PVR+ tumor cells mice (66). (Fig. 1). This will soon be tested clinically, as GlaxoSmith- In the human setting, as in the mouse setting, results indi- Kline recently reported initiation of a phase I clinical trial cate that inhibition of this pathway may result in enhanced evaluating a CD96-blocking antibody in patients with solid antitumor immunity (26). The activating effect of PVRIG tumors, and that data should further inform the role and blockade on CD8+ T cells has been demonstrated in vitro relevance of CD96 in the DNAM1 axis. with antibodies that block the PVRIG/PVRL2 interaction. The ability of anti-PVRIG mAbs to promote T-cell responses, PVRIG: The Recently Discovered either individually or in combination with other immune checkpoints, was assessed using multiple human T cell–based Inhibitory Receptor in the DNAM1 Axis assays with both TILs and T cells from healthy donors. In PVRIG was identified usingin silico analyses designed to all assay systems, PVRIG blockade increased T-cell prolifera- discover novel immunoreceptors that are involved in regulat- tion, cytokine secretion, and cytotoxicity (26, 32). Although ing lymphocyte function in the context of cancer (32, 84). clinical monotherapy effects are rarely observed with inhibi- Human and cynomolgus PVRIG share high sequence identity, tory receptor antagonists other than anti–PD-1 (60, 86–89), and both proteins have a conserved ITIM in the intracellular numerous pathways modulate immune responses, suggesting signaling domain, which is lacking in the mouse homolog. that combinatorial approaches may increase rates of response PVRL2 is the cognate ligand for PVRIG, with a binding (89–92). Compared with blockade of PVRIG, TIGIT, or PD-1 affinity of human PVRIG to human PVRL2 reported as alone, the dual and triple combination of anti-PVRIG with 88 nmol/L (32). Studies investigating whether DNAM1 and anti–PD-1 or with anti-TIGIT further increased cytokine pro- TIGIT compete with PVRIG for binding to PVRL2 showed duction and T cell–mediated killing of PVRL2+PVR+ tumor that TIGIT had little effect on interrupting the PVRIG/ target cells (26). PVRL2 interaction, whereas DNAM1 was able to compete In addition to PVRIG’s role in regulating T-cell responses, for this interaction, despite its reduced affinity to PVRL2 we have recently shown that the PVRIG blockade significantly compared with PVRIG (26, 32). Although TIGIT is reported enhances NK cell–mediated killing of PVRL2+ cancer cells (93), to have a very low-affinity interaction with PVRL2, its physi- in line with other emerging data supporting the inhibitory ologic relevance is unclear, and our recent data suggest that functional role of PVRIG in these cells (94). Accordingly, PVRIG/PVRL2 serves as a distinct inhibitory signaling path- Xu and colleagues demonstrated that blockade of PVRIG and way in the DNAM1 axis and has a nonoverlapping function TIGIT alone or in combination enhances trastuzumab-triggered with the TIGIT/PVR pair (Fig. 1; ref. 26). antitumor response by human NK cells (95). Collectively, these Mouse PVRIG has a 59% protein identity with human data suggest that PVRIG and TIGIT receptors regulate NK-cell PVRIG and a truncated intracellular signaling domain that functions and that NK activation may be a determinant of contains phosphorylated tyrosine but lacks an ITIM, suggest- clinical efficacy for inhibitors targeting each receptor (Fig. 2B). ing that mouse PVRIG may have a reduced role as a DNAM1 In assessing tumor expression, hormonally regulated pathway checkpoint receptor compared with human PVRIG tumors, such as ovarian, endometrial, and breast cancers, (85). At steady state, expression of murine PVRIG is detected along with kidney and lung tumors, demonstrated the high- on both T and NK cells, whereas in peripheral immune tis- est PVRIG expression on T and NK cells. PVRIG expression sues, NK but not T cells express PVRIG. Upon activation, in tumors was significantly increased on TILs compared with

may 2021 CANCER DISCOVERY | OF7

REVIEW Alteber et al.

A Tumor cell or APC T cell Tumor cell or APC

PVRL2 PVRIG TCR MHC-I

– +

DNAM1 – –

PD-1 PD-L1 PVR TIGIT

B Tumor cell or APC T cell Tumor cell or APC Anti-PVRIG Ab

PVRIG TCR MHC-I PVRL2 + + DNAM1 PVR

PD-1 PD-L1 TIGIT Anti-TIGIT Ab Anti–PD-1 Ab

Figure 3. Functional interplay between the PD-1 pathway and the DNAM1 axis. A, T-cell inhibition is induced by the interaction of PVRIG and/or TIGIT with cognate ligands expressed on tumor cells or APCs. PD-1 stimulation by PD-L1 further suppresses T-cell function by dephosphorylating the DNAM1 signaling motif and reducing its expression. B, Full restoration of T-cell activation by coinhibition of PVRIG, TIGIT, and PD-1.

T cells infiltrating normal adjacent tissue, further highlight- and its expression is relatively low on Tregs (26), an IgG4 ing its potential as a checkpoint receptor on lymphocytes. backbone was selected to avoid potential depletion of effec- Moreover, PVRIG was coexpressed with PD-1 and TIGIT on tor cells. COM701 inhibits the binding of PVRL2 to PVRIG TILs, peripheral memory T cells, and activated T cells. These in a dose-dependent manner with complete inhibition of data indicate that all three inhibitory molecules play a role in PVRIG/PVRL2 interaction observed at saturating levels of regulating the immune response and provide a rationale for COM701 (26). It is currently in phase I clinical testing, both the dual and triple blockade of these checkpoint receptors, as a monotherapy and in combination with the anti–PD-1 depending on the ligand expression pattern in the TME (26). drug nivolumab (NCT03667716). In initial results presented PVRL2 is frequently expressed in various malignancies, and from the dose-escalation arms of a phase I study, COM701 specifically PVRL2/PVR mRNA ratios are generally highest was shown to be well tolerated as a monotherapy and in in hormonally regulated cancers, which was further validated combination with nivolumab, with no dose-limiting toxici- by both IHC and flow cytometry. This, together with PVRIG ties reported. Additionally, encouraging disease control rates levels in those tumors, suggests that these cancers are promis- of 69% (11/16) for the monotherapy and 75% (9/12) for ing indications for PVRIG-blocking antibodies. In addition, the combination arm were observed in a heavily pretreated increased RNA and protein PVRL2 expression levels were dem- patient population. Moreover, two patients were reported onstrated in cancer relative to normal tissues, with expression with confirmed partial responses, one from the monotherapy seen in both PD-L1+ and PD-L1− patient samples across tumor arm (microsatellite-stable primary peritoneal cancer) and one types (26, 96). These analyses highlighted cancers in which from the combination arm (microsatellite-stable colorectal PVRL2 might primarily regulate the immune response, and cancer, which has an extremely low response rate to anti– how this pathway may be relevant in patients who are PD-L1− PD-1 alone). Future clinical trials involving triple blockade of or those who develop PD-1 resistance (96–98). PVRIG, TIGIT, and PD-1 are planned. Based on the supportive preclinical data, an anti-PVRIG antibody was developed for clinical testing. COM701 is a humanized anti-PVRIG hinge stabilized IgG4 mAb that binds PD-1: A Player in the DNAM1 Axis? specifically to human and cynomolgus monkey PVRIG and Although several studies have demonstrated additive or disrupts the binding of PVRIG to PVRL2 (Table 1). Because synergistic effects of PD-1 pathway blockade with inhibi- PVRIG is predominantly expressed on CD8+ T and NK cells, tion of TIGIT or PVRIG (26, 29, 32, 44, 56), the mechanism

OF8 | CANCER DISCOVERY may 2021 AACRJournals.org

Targeting of Checkpoint Receptors within the DNAM1 Axis REVIEW

PVRIG TIGIT

Junshi JS006

Innovent IBI-939 Biothera BAT6005

SGEN-SGN-TGT

Imab-TJT6 BeiGene BGB-A1217

Compugen Merck COM902 MK-7684 Mogam MG1131 Surface Onc SRF813 Iteos Compugen Roche EOS-448 COM701 Tiragolumab Yuhan YH29143

BMS MARKETED Arcus/Gilead BMS-986207 AB154 Agenus AGEN1327

PHASE 3 Mereo OMP313M32 Y Biologics PHASE 2 Merck KgAA YBL-012 M6223

PHASE 1 Henlius HLX-53

PRE-CLINICAL

GSK GSK6097608

CD96

Figure 4. Clinical landscape of the DNAM1 axis. was poorly understood. Wang and colleagues have provided in vivo tumor model responsive to anti–PD-1 in combina- some insight into the intersection of the DNAM1 and PD-1 tion with a glucocorticoid-induced tumor necrosis factor pathways (58). Using a cell-free system, the authors dem- receptor (GITR) agonist antibody. The survival of tumor- onstrated that DNAM1 is dephosphorylated by the PD-1/ bearing mice was dramatically increased by the combina- SHP2 complex, suggesting that it is a downstream target of tion treatment and could be reversed either by the addition PD-1 signaling (Fig. 3A). This was further reinforced by the of an antagonist DNAM1 antibody or upon treatment observation that antigen-specific intratumoral CD8+ T cells of tumors engrafted into DNAM1 knockout mice. The isolated from MC38-OVA tumor–bearing mice treated with authors also demonstrated an effect of PD-1 inhibition an anti–PD-1 antibody showed a high level of DNAM1 phos- on DNAM1 expression; increased expression was seen on phorylation, whereas DNAM1 on TILs isolated from isotype antigen-reactive clones following anti–PD-1 treatment, and control-treated mice showed a lack of phosphorylation. further increased with GITR agonism. Taken together, the Extending the above results, Wang and colleagues results implicate derepression of DNAM1 costimulatory assessed the impact of DNAM1 blockade or ablation in an signaling in response to PD-1 inhibition, similar to the role

may 2021 CANCER DISCOVERY | OF9

REVIEW Alteber et al. that has been proposed for CD28 involvement in an anti– the submitted work. J. Hunter reports personal fees from Compu- PD-1 response (99). Another study has similarly shown that gen Ltd outside the submitted work; in addition, J. Hunter has a DNAM1 is required for PD-1 blockade antitumor activity patent for anti-PVRIG antibodies formulations and uses thereof in preclinical mouse models (100). In accordance with these pending. E. Ophir reports personal fees from Compugen Ltd out- results, DNAM expression on TILs was shown to correlate side the submitted work; in addition, E. Ophir has a patent for triple-combination antibody therapies pending and anti-PVRIG positively with the response to PD-1 blockade in patients antibody–related provisional patent applications pending; and with melanoma (101). These data provide a molecular Compugen Ltd is developing anti-PVRIG and anti-TIGIT mono- rationale for the combination effects observed with PD-1 clonal antibodies being evaluated in phase I clinical trials. No inhibition when combined with blockade of coinhibitory disclosures were reported by the other authors. molecules in the DNAM1 axis (Fig. 3B). Received August 27, 2020; revised November 3, 2020; accepted December 1, 2020; published first March 9, 2021. Summary Given recent advances in our understanding of DNAM1 regulation, including a possible connection with the PD-1 References pathway and the broad and upregulated expression of the . 1 Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of axis ligands PVR and PVRL2 in the TME, the coinhibitory CTLA-4 function. Annu Rev Immunol 2006;24:65–97. 2. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of receptors in the axis are increasingly attractive as targets for PD-1, a novel member of the immunoglobulin gene superfamily, cancer immunotherapy. Preclinical and early clinical data in upon programmed cell death. EMBO J 1992;11:3887–95. solid tumors have begun to emerge from clinical trials with 3. Brahmer JR, Tykodi SS, Chow LQM, Hwu W-J, Topalian SL, Hwu P, TIGIT inhibitors, and additional clinical data are expected by et al. Safety and activity of anti–PD-L1 antibody in patients with the end of 2020 (Fig. 4). Interestingly, although initial studies advanced cancer. N Engl J Med 2012;366:2455–65. in NSCLC indicate greater efficacy for TIGIT blockade in PD- 4. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, L1hi patients, early clinical data for PVRIG blockade highlight McDermott DF, et al. Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. N Engl J Med 2012;366:2443–54. activity in indications that are generally unresponsive to PD-1 5. Rotte A. Combination of CTLA-4 and PD-1 blockers for treatment therapy and that typically demonstrate low PD-L1 expression. of cancer. J Exp Clin Cancer Res 2019;38:1–12. Emerging clinical data for the PVRIG inhibitor COM701 and 6. Riley RS, June CH, Langer R, Mitchell MJ. Delivery technologies for multiple TIGIT inhibitors should yield additional insight cancer immunotherapy. Nat Rev Drug Discov 2019;18:175–96. into functional interactions between the immune-checkpoint 7. Ruffo E, Wu RC, Bruno TC, Workman CJ, Vignali DAA. Lympho- inhibitors in the DNAM1 axis, both with each other and in cyte-activation gene 3 (LAG3): the next immune checkpoint receptor. Semin Immunol 2019;42:101305. conjunction with modulation of the PD-1 signaling pathway. 8. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A, et al. Furthermore, defining the patient population most likely The interaction of TIGIT with PVR and PVRL2 inhibits human NK to respond to each therapeutic intervention is of great inter- cell cytotoxicity. Proc Natl Acad Sci U S A 2009;106:17858–63. est. As mentioned above, early data suggest that PD-L1 might 9. Pasero C, Olive D. Interfering with coinhibitory molecules: BTLA/ be a potential biomarker of response to TIGIT blockade (67), HVEM as new targets to enhance anti-tumor immunity. Immunol at least when combined with PD-L1 blockers. However, the Lett 2013;151:71–5. 10. Das M, Zhu C, Kuchroo VK. Tim-3 and its role in regulating anti- expression of other pathway members is also important. tumor immunity. Immunol Rev 2017;276:97–111. PVRIG and/or PVRL2 high-expressing tumors might support 11. Borate U, Esteve J, Porkka K, Knapper S, Vey N, Scholl S, et al. Phase dominance of the PVRIG pathway and thus preferentially Ib study of the anti-TIM-3 antibody MBG453 in combination with respond to PVRIG blockade (26). Likewise, patients with decitabine in patients with high-risk myelodysplastic syndrome TIGIT and/or PVR high-expressing tumors could potentially (MDS) and acute myeloid leukemia (AML). Blood 2019;134:570. better benefit from TIGIT blockade. Finally, the expression 12. Harjunpää H, Guillerey C. TIGIT as an emerging immune check- levels of DNAM1, which is in the center of the axis, could also point. Clin Exp Immunol 2020;200:108–19. 13. Shibuya A, Campbell D, Hannum C, Yssel H, Franz-Bacon K, serve as a biomarker of response to combination blockade of McClanashan T, et al. DNAM-1, a novel adhesion molecule involved the axis members. in the cytolytic function of T lymphocytes. Immunity 1996;4:573–81. 14. Iguchi-Manaka A, Kai H, Yamashita Y, Shibata K, Tahara-Hanaoka Authors’ Disclosures S, Honda SI, et al. Accelerated tumor growth in mice deficient in Z. Alteber reports personal fees from Compugen Ltd outside DNAM-1 receptor. J Exp Med 2008;205:2959–64. the submitted work; in addition, Z. Alteber has a patent for 15. Gilfillan S, Chan CJ, Cella M, Haynes NM, Rapaport AS, Boles KS, triple-combination antibody therapies pending and anti-PVRIG et al. DNAM-1 promotes activation of cytotoxic lymphocytes by antibody–related provisional patent applications pending; and nonprofessional antigen-presenting cells and tumors. J Exp Med Compugen Ltd is developing anti-PVRIG and anti-TIGIT antibod- 2008;205:2965–73. ies being evaluated in phase I clinical studies. M.F. Kotturi reports 16. Shibuya K, Lanier LL, Phillips JH, Ochs HD, Shimizu K, Nakayama E, et al. Physical and functional association of LFA-1 with DNAM-1 a patent for US10124061 issued and a patent for US20190010246 adhesion molecule. Immunity 1999;11:615–23. pending; and is a former employee of Compugen Inc. with stock 17. Enqvist M, Ask EH, Forslund E, Carlsten M, Abrahamsen G, Béziat options. S. Whelan reports being a former employee of Compugen V, et al. Coordinated expression of DNAM-1 and LFA-1 in educated Inc. and received stock options. E. Weyl reports personal fees from NK cells. J Immunol 2015;194:4518–27. Compugen Ltd outside the submitted work; in addition, E. Weyl 18. Ramsbottom KM, Hawkins ED, Shimoni R, McGrath M, Chan CJ, has Anti-PVRIG antibody–related provisional patent applications Russell SM, et al. Cutting edge: DNAX accessory molecule 1–defi- pending. D.M. Pardoll reports grants from Compugen Ltd during cient CD8 + T cells display immunological synapse defects that the conduct of the study and grants from Compugen Ltd outside impair antitumor immunity. J Immunol 2014;192:553–7.

OF10 | CANCER DISCOVERY may 2021 AACRJournals.org

Targeting of Checkpoint Receptors within the DNAM1 Axis REVIEW

19. Mandai K, Rikitake Y, Mori M, Takai Y. Nectins and nectin-like 40. Zhang J, Zhu Y, Wang Q, Kong Y, Sheng H, Guo J, et al. Poliovirus recep- molecules in development and disease. Curr Top Dev Biol 2015;112: tor CD155 is up-regulated in muscle-invasive bladder cancer and predicts 197–231. poor prognosis. Urol Oncol Semin Orig Investig 2020;38:41.e11–41. 20. Bottino C, Castriconi R, Pende D, Rivera P, Nanni M, Carnemolla 41. Lepletier A, Madore J, O’Donnell JS, Johnston RL, Li X-Y, McDonald B, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as E, et al. Tumor CD155 expression is associated with resistance to cell surface ligands for the human DNAM-1 (CD226) activating anti-PD1 immunotherapy in metastatic melanoma. Clin Cancer Res molecule. J Exp Med 2003;198:557–67. 2020;26:3671–81. 21. Tahara-Hanaoka S, Shibuya K, Onoda Y, Zhang H, Yamazaki S, Miy- 42. Joller N, Hafler JP, Brynedal B, Kassam N, Spoerl S, Levin SD, amoto A, et al. Functional characterization of DNAM-1 (CD226) et al. Cutting edge: TIGIT has T cell-intrinsic inhibitory functions. interaction with its ligands PVR (CD155) and nectin-2 (PRR-2/ J Immunol 2011;186:1338–42. CD112). Int Immunol 2004;16:533–8. 43. Li M, Xia P, Du Y, Liu S, Huang G, Chen J, et al. T-cell immunoglob- 22. Dardalhon V, Schubart AS, Reddy J, Meyers JH, Monney L, Sabatos ulin and ITIM domain (TIGIT) receptor/poliovirus receptor (PVR) CA, et al. CD226 is specifically expressed on the surface of Th1 cells ligand engagement suppresses interferon-γ production of natural and regulates their expansion and effector functions. J Immunol killer cells via β-arrestin 2-mediated negative signaling. J Biol Chem 2005;175:1558–65. 2014;289:17647–57. 23. Lepletier A, Lutzky VP, Mittal D, Stannard K, Watkins TS, Ratna- 44. Chauvin J, Pagliano O, Fourcade J, Sun Z, Wang H, Sander C, et tunga CN, et al. The immune checkpoint CD96 defines a distinct al. TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in lymphocyte phenotype and is highly expressed on tumor-infiltrat- melanoma patients. J Clin Invest 2015;125:2046–58. ing T cells. Immunol Cell Biol 2019;97:152–64. 45. Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley MF, et al. A 24. Yu X, Harden K, Gonzalez LC, Francesco M, Chiang E, Irving B, et al. human memory T cell subset with stem cell–like properties. Nat The surface protein TIGIT suppresses T cell activation by promot- Med 2011;17:1290–7. ing the generation of mature immunoregulatory dendritic cells. Nat 46. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Immunol 2009;10:48–57. Lundh S, et al. Determinants of response and resistance to CD19 25. Nishiwada S, Sho M, Yasuda S, Shimada K, Yamato I, Akahori T, chimeric antigen receptor (CAR) T cell therapy of chronic lympho- et al. Clinical significance of CD155 expression in human pancreatic cytic leukemia. Nat Med 2018;24:563–71. cancer. Anticancer Res 2015;35:2287–97. 47. Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, 26. Whelan S, Ophir E, Kotturi MF, Levy O, Ganguly S, Leung L, et al. Jenkins RW, et al. Defining T cell states associated with response to PVRIG and PVRL2 are induced in cancer and inhibit CD8 + T-cell checkpoint immunotherapy in melanoma. Cell 2018;175:998–1013. function. Cancer Immunol Res 2019;7:257–68. 48. Brummelman J, Mazza EMC, Alvisi G, Colombo FS, Grilli A, Mikulak 27. Lozano E, Dominguez-Villar M, Kuchroo V, Hafler DA. The J, et al. High-dimensional single cell analysis identifies stem-like TIGIT/CD226 axis regulates human T cell function. J Immunol cytotoxic CD8+ T cells infiltrating human tumors. J Exp Med 2012;188:3869–75. 2018;215:2520–35. 28. Pauken KE, Wherry EJ. TIGIT and CD226: tipping the balance 49. Kurtulus S, Sakuishi K, Ngiow S, Joller N, Tan DJ, Teng MWL, et al. between costimulatory and coinhibitory molecules to augment the TIGIT predominantly regulates the immune response via regulatory cancer immunotherapy toolkit. Cancer Cell 2014;26:785–7. T cells. J Clin Invest 2015;1:1–10. 29. Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y, 50. Levin SD, Taft DW, Brandt CS, Bucher C, Howard ED, Chadwick et al. The immunoreceptor TIGIT regulates antitumor and antiviral EM, et al. Vstm3 is a member of the CD28 family and an important CD8+ T cell effector function. Cancer Cell 2014;26:923–37. modulator of T-cell function. Eur J Immunol 2011;41:902–15. 30. Jin H, Ko M, Choi D, Kim JH, Lee D, Kang S-H, et al. CD226 hi CD8 + 51. Joller N, Lozano E, Burkett PR, Patel B, Xiao S, Zhu C, et al. T cells are a prerequisite for anti-TIGIT immunotherapy. Cancer Treg cells expressing the coinhibitory molecule TIGIT selectively Immunol Res 2020;8:912–25. inhibit proinflammatory Th1 and Th17 cell responses. Immunity 31. Chauvin J-M, Ka M, Pagliano O, Menna C, Ding Q, DeBlasio R, 2014;40:569–81. et al. IL15 Stimulation with TIGIT blockade reverses CD155-mediated 52. Dixon KO, Schorer M, Nevin J, Etminan Y, Amoozgar Z, Kondo T, et al. NK-cell dysfunction in melanoma. Clin Cancer Res 2020;26:5520–33. Functional anti-TIGIT antibodies regulate development of autoim- 32. Zhu Y, Paniccia A, Schulick AC, Chen W, Koenig MR, Byers JT, et al. munity and antitumor immunity. J Immunol 2018;200:3000–7. Identification of CD112R as a novel checkpoint for human T cells. 53. Fourcade J, Sun Z, Chauvin JM, Ka M, Davar D, Pagliano O, et al. J Exp Med 2016;213:167–76. CD226 opposes TIGIT to disrupt Tregs in melanoma. JCI insight 33. Kojima H, Kanada H, Shimizu S, Kasama E, Shibuya K, Nakauchi 2018;3:1–13. H, et al. CD226 mediates platelet and megakaryocytic cell adhesion 54. Yin X, Liu T, Wang Z, Ma M, Lei J, Zhang Z, et al. Expression of to vascular endothelial cells. J Biol Chem 2003;278:36748–53. the inhibitory receptor tigit is up-regulated specifically on NK cells 34. Li M, Qiao D, Pu J, Wang W, Zhu W, Liu H. Elevated Nectin-2 with cd226 activating receptor from HIV-infected individuals. Front expression is involved in esophageal squamous cell carcinoma by Immunol 2018;9:1–12. promoting cell migration and invasion. Oncol Lett 2018;15:4731–6. 55. Liu S, Zhang H, Li M, Hu D, Li C, Ge B, et al. Recruitment of 35. Oshima T, Sato S, Kato J, Ito Y, Watanabe T, Tsuji I, et al. Nectin-2 Grb2 and SHIP1 by the ITT-like motif of TIGIT suppresses gran- is a potential target for antibody therapy of breast and ovarian can- ule polarization and cytotoxicity of NK cells. Cell Death Differ cers. Mol Cancer 2013;12:1–13. 2013;20:456–64. 36. Oshima T, Miyashita H, Ishimura Y, Ito Y, Tanaka Y, Hori A, et al. Fc 56. Zhang Q, Bi J, Zheng X, Chen Y, Wang H, Wu W, et al. Blockade of engineering of anti-Nectin-2 antibody improved thrombocytopenic the checkpoint receptor TIGIT prevents NK cell exhaustion and adverse event in monkey. PLoS One 2018;13:1–17. elicits potent anti-tumor immunity. Nat Immunol 2018;19:723–32. 37. Reches A, Ophir Y, Stein N, Kol I, Isaacson B, Charpak Amikam Y, 57. Stamm H, Klingler F, Grossjohann EM, Muschhammer J, Vet- et al. Nectin4 is a novel TIGIT ligand which combines checkpoint torazzi E, Heuser M, et al. Immune checkpoints PVR and PVRL2 inhibition and tumor specificity. J Immunother Cancer 2020;8:1–9. are prognostic markers in AML and their blockade represents a new 38. Nakai R, Maniwa Y, Tanaka Y, Nishio W, Yoshimura M, Okita Y, therapeutic option. Oncogene 2018;37:5269–80. et al. Overexpression of Necl-5 correlates with unfavorable prog- 58. Wang B, Zhang W, Jankovic V, Golubov J, Poon P, Oswald EM, et al. nosis in patients with lung adenocarcinoma. Cancer Sci 2010;101: Combination cancer immunotherapy targeting PD-1 and GITR can 1326–30. rescue CD8+ T cell dysfunction and maintain memory phenotype. 39. Triki H, Charfi S, Bouzidi L, Ben Kridis W, Daoud J, Chaabane K, et al. Sci Immunol 2018;3:1–14. CD155 expression in human breast cancer: clinical significance and 59. Schachter J, Ribas A, Long GV, Arance A, Grob JJ, Mortier L, et al. relevance to natural killer cell infiltration. Life Sci 2019;231:116543. Pembrolizumab versus ipilimumab for advanced melanoma: final

may 2021 CANCER DISCOVERY | OF11

REVIEW Alteber et al.

overall survival results of a multicentre, randomised, open-label 78. Holmes VM, De Motes CM, Richards PT, Roldan J, Bhargava AK, phase 3 study (KEYNOTE-006). Lancet 2017;390:1853–62. Orange JS, et al. Interaction between nectin-1 and the human natu- 60. Yang F, Zhao L, Wei Z, Yang Y, Liu J, Li Y, et al. A Cross-species ral killer cell receptor CD96. PLoS One 2019;14:1–19. reactive TIGIT-blocking antibody Fc dependently confers potent 79. Blake SJ, Stannard K, Liu J, Allen S, Yong MCR, Mittal D, et al. Sup- antitumor effects. J Immunol 2020;205:2156–68. pression of metastases using a new lymphocyte checkpoint target 61. Nimmerjahn F, Ravetch J V. Fcγ receptors as regulators of immune for cancer immunotherapy. Cancer Discov 2016;6:446–59. responses. Nat Rev Immunol 2008;8:34–47. 80. Harjunpää H, Blake SJ, Ahern E, Allen S, Liu J, Yan J, et al. Deficiency 62. Sharma A, Subudhi SK, Blando J, Scutti J, Vence L, Wargo J, et al. of host CD96 and PD-1 or TIGIT enhances tumor immunity with- Anti-CTLA-4 immunotherapy does not deplete Foxp3 þ regulatory out significantly compromising immune homeostasis. Oncoimmu- T cells (Tregs) in human cancers. Clin Cancer Res 2019;25:1233–8. nology 2018;7:1–11. 63. Lee WJ, Lee YJ, Choi ME, Yun KA, Won CH, Lee MW, et al. Expression 81. Sun H, Huang Q, Huang M, Wen H, Lin R, Zheng M, et al. Human of lymphocyte-activating gene 3 and T-cell immunoreceptor with CD96 correlates to natural killer cell exhaustion and predicts immunoglobulin and ITIM domains in cutaneous melanoma and the prognosis of human hepatocellular carcinoma. Hepatology their correlation with programmed cell death 1 expression in tumor- 2019;70:168–83. infiltrating lymphocytes. J Am Acad Dermatol 2019;81:219–27. 82. Carlsten M, Björkström NK, Norell H, Bryceson Y, Van Hall T, 64. Anzengruber F, Ignatova D, Schlaepfer T, Chang YT, French LE, Baumann BC, et al. DNAX accessory molecule-1 mediated recogni- Pascolo S, et al. Divergent LAG-3 versus BTLA, TIGIT, and FCRL3 tion of freshly isolated ovarian carcinoma by resting natural killer expression in Sézary syndrome. Leuk Lymphoma 2019;60:1899–907. cells. Cancer Res 2007;67:1317–25. 65. Yang Z-Z, Kim HJ, Wu H, Jalali S, Tang X, Krull JE, et al. TIGIT 83. Chiang EY, Almeida PE, Almeida Nagata DE, Bowles KH, Du X, Chitre expression is associated with T-cell suppression and exhaustion AS, et al. CD96 functions as a co-stimulatory receptor to enhance and predicts clinical outcome and anti–PD-1 response in follicular CD8 + T cell activation and effector responses. Eur J Immunol lymphoma. Clin Cancer Res 2020;26:5217–31. 2020;50:891–902. 66. Logronio K, Qurashi S, Whelan S, Hansen K, Kumar S, Leung L, 84. Levy O, Chan C, Cojocaru G, Liang S, Ophir E, Ganguly S, et al. Com- et al. COM902, a novel therapeutic antibody targeting TIGIT aug- putational identification, functional characterization, and antibody ments t cell function and the activity of PVRIG pathway blockade blockade of a new immune checkpoint in the TIGIT family of inter- in vitro and in vivo. 34th Annual Meeting and Pre-Conference acting molecules. 31st annual meeting and pre-conference programs Programs of the Society for Immunotherapy of Cancer (SITC 2019): of the Society for Immunotherapy of Cancer (SITC 2016): late break- part 1. J Immunother Cancer 2019;7:282. Abstract P322. ing abstracts. J Immunother Cancer 2016;4:91. Abstract O4. 67. Rodriguez-Abreu D, Johnson ML, Hussein MA, Cobo M, Patel AJ, 85. Murter B, Pan X, Ophir E, Alteber Z, Azulay M, Sen R, et al. Mouse Secen NM, et al. Primary analysis of a randomized, double-blind, PVRIg has CD8+ T cell-specific coinhibitory functions and dampens phase II study of the anti-TIGIT antibody tiragolumab (tira) plus antitumor immunity. Cancer Immunol Res 2019;7:244–56. atezolizumab (atezo) versus placebo plus atezo as first-line (1L) 86. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman treatment in patients with PD-L1-selected NSCLC (CITYSCAPE). WH, et al. Phase I study of single-agent anti-programmed death-1 J Clin Oncol 2020;38:9503–3. (MDX-1106) in refractory solid tumors: Safety, clinical activity, 68. Golan T, Bauer TM, Jimeno A, Perets R, Niu J, Lee JJ, et al. Phase 1 dose- pharmacodynamics, and immunologic correlates. J Clin Oncol finding study of the anti–TIGIT antibody MK-7684 as monotherapy 2010;28:3167–75. and in combination with pembrolizumab in patients with advanced 87. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen solid. 33rd Annual Meeting and Pre-Conference Programs of the JB, et al. Improved survival with ipilimumab in patients with meta- Society for Immunotherapy of Cancer (SITC 2018). J Immunother static melanoma. N Engl J Med 2010;336:711–23. Cancer 2018;6:115. Abstract O25. 88. Sharma P, Callahan MK, Bono P, Kim J, Spiliopoulou P, Calvo E, 69. Wang PL, O’Farrell S, Clayberger C, Krensky AM. Identification and et al. Nivolumab monotherapy in recurrent metastatic urothelial molecular cloning of tactile: a novel human T cell activation antigen that carcinoma (CheckMate 032): a multicentre, open-label, two-stage, is a member of the Ig gene superfamily. J Immunol. 1992;148:2600–8. multi-arm, phase 1/2 trial. Lancet Oncol 2016;17:1590–8. 70. Fuchs A, Cella M, Giurisato E, Shaw AS, Colonna M. Cutting edge: 89. Brignone C, Escudier B, Grygar C, Marcu M, Triebel F. A phase I CD96 (tactile) promotes NK cell-target cell adhesion by interacting pharmacokinetic and biological correlative study of IMP321, a novel with the poliovirus receptor (CD155). J Immunol 2004;172:3994–8. MHC class II agonist, in patients with advanced renal cell carci- 71. Toutirais O, Cabillic F, Le Friec G, Salot S, Loyer P, Le Gallo M, et al. noma. Clin Cancer Res 2009;15:6225–31. DNAX accessory molecule-1 (CD226) promotes human hepatocellular 90. Brahmer JR, Schneider BJ, Gardner JM. Treated with immune check- carcinoma cell lysis by Vγ9Vδ2 T cells. Eur J Immunol 2009;39:1361–8. point inhibitor therapy: American Society of Clinical Oncology 72. Meyer D, Seth S, Albrecht J, Maier MK, Du Pasquier L, Ravens I, Clinical Practice Guideline. J Clin Oncol 2018;36:1714–68. et al. CD96 interaction with CD155 via its first Ig-like domain is 91. Postow MA, Chesney J, Pavlick AC, Robert C, Grossmann K, modulated by alternative splicing or mutations in distal Ig-like McDermott D, et al. Nivolumab and ipilimumab versus ipilimumab domains. J Biol Chem 2009;284:2235–44. in untreated melanoma. N Engl J Med 2015;372:2006–17. 73. Garg S, Madkaikar M, Ghosh K. Investigating cell surface markers 92. Page DB, Postow MA, Callahan MK, Allison JP, Wolchok JD. on normal hematopoietic stem cells in three different niche condi- Immune modulation in cancer with antibodies. Annu Rev Med 2014; tions. Int J Stem Cells 2013;6:129–33. 65:185–202. 74. Hosen N, Park CY, Tatsumi N, Oji Y, Sugiyama H, Gramatzki M, 93. Li J, Whelan S, Kotturi MF, Meyran D, D’Souza C, Hansen K, et al. et al. CD96 is a leukemic stem cell-specific marker in human acute PVRIG is a novel NK cell immune checkpoint receptor in acute mye- myeloid leukemia. Proc Natl Acad Sci U S A 2007;104:11008–13. loid leukemia. Haematologica 2020 Nov 5 [Epub ahead of print]. 75. Seth S, Maier MK, Qiu Q, Ravens I, Kremmer E, Förster R, et al. The 94. Panduro M, Dornbrook RM, Doshi KA, Hua J, Strand J, Palombella murine pan T cell marker CD96 is an adhesion receptor for CD155 VJ, et al. SRF813, a fully human monoclonal antibody targeting the and nectin-1. Biochem Biophys Res Commun 2007;364:959–65. inhibitory receptor CD112R, enhances immune cell activation and 76. Chan CJ, Martinet L, Gilfillan S, Souza-Fonseca-Guimaraes F, Chow demonstrates preclinical in vivo anti-tumor activity. Cancer Res MT, Town L, et al. The receptors CD96 and CD226 oppose each 2020; abstract nr4548. other in the regulation of natural killer cell functions. Nat Immunol 95. Xu F, Sunderland A, Zhou Y, Schulick RD, Edil BH, Zhu Y. Blockade 2014;15:431–8. of CD112R and TIGIT signaling sensitizes human natural killer cell 77. Mittal D, Lepletier A, Madore J, Aguilera AR, Stannard K, Blake SJ, functions. Cancer Immunol Immunother 2017;66:1367–75. et al. CD96 is an immune checkpoint that regulates CD8þ T-cell 96. Cerboni C, Fionda C, Soriani A, Zingoni A, Doria M, Cippitelli M, antitumor function. Cancer Immunol Res 2019;7:559–71. et al. The DNA damage response: a common pathway in the regulation

OF12 | CANCER DISCOVERY may 2021 AACRJournals.org

Targeting of Checkpoint Receptors within the DNAM1 Axis REVIEW

of NKG2D and DNAM-1 ligand expression in normal, infected, and 100. Weulersse M, Asrir A, Pichler AC, Lemaitre L, Braun M, Carrié N, cancer cells. Front Immunol 2014;4:1–7. et al. Eomes-dependent loss of the co-activating receptor CD226 97. O’Donnell JS, Smyth MJ, Teng MWL. Acquired resistance to anti-PD1 restrains CD8+ T cell anti-tumor functions and limits the efficacy of therapy: checkmate to checkpoint blockade? Genome Med 2016;8:1–3. cancer immunotherapy. Immunity 2020;53:824–39. 98. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and 101. Braun M, Aguilera AR, Sundarrajan A, Corvino D, Stannard K, acquired resistance to cancer immunotherapy. Cell 2017;168:707–23. Krumeich S, et al. CD155 on tumor cells drives resistance to immu- 99. Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, et al. T cell notherapy by inducing the degradation of the activating receptor costimulatory receptor CD28 is a primary target for PD-1–mediated CD226 in CD8+ T cells. Immunity 2020;53:805–23. inhibition. Science 2017;355:1428–33.

may 2021 CANCER DISCOVERY | OF13

Therapeutic Targeting of Checkpoint Receptors within the DNAM1 Axis

Zoya Alteber, Maya F. Kotturi, Sarah Whelan, et al.

Cancer Discov Published OnlineFirst March 9, 2021.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-20-1248

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/early/2021/04/13/2159-8290.CD-20-1248. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.