Therapeutic Targeting of Checkpoint Receptors Within the DNAM-1 Axis

Author Manuscript Published OnlineFirst on March 9, 2021; DOI: 10.1158/2159-8290.CD-20-1248 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Therapeutic Targeting of Checkpoint Receptors within the DNAM-1 Axis

3 Zoya Alteber1*, Maya F. Kotturi2*, Sarah Whelan2*, Sudipto Ganguly3*, Emmanuel Weyl1, Drew 4 M. Pardoll3, John Hunter2, and Eran Ophir1 5 1 Compugen Ltd, Holon, Israel. 6 2 Compugen USA, Inc., South San Francisco, California 7 3 Bloomberg Kimmel Institute for Cancer Immunotherapy, John Hopkins University, Baltimore, 8 MD 9 * Z. Alteber, M. F. Kotturi, S. Whelan and S. Ganguly are co–first authors of this review

10 Disclosure of Potential Conflicts of Interest

11 Z. Alteber, E. Weyl and E. Ophir are Employees of Compugen Ltd. with stock options. M. F. 12 Kotturi is Former Compugen Ltd employee with company stock options; current employee with 13 company stock options at IGM Biosciences Inc; S. Whelan is Former employee of Compugen 14 Ltd. Currently an employee and equity holder of Nurix Therapeutics S. Gungluy has no conflict 15 of interest. D.M. Pardoll reports receiving commercial research funding from Bristol-Myers 16 Squibb, Compugen, and AstraZeneca; has ownership interest in Aduro Biotech, DNAtrix, 17 Dracen, Enara, Five Prime Therapeutics, Tizona, Trieza, and WindMil; and is a consultant/ 18 advisory board member for Amgen, Bayer, FLX Bio, Immunomic, Janssen, Merck, Rock 19 Springs Capitol, and Tizona. J. Hunter is Former Compugen Ltd employee with company stock 20 options; current employee and equity holder of Keyhole Therapeutics Inc

21 Corresponding Author: 22 Eran Ophir 23 Compugen Ltd

24 Azrieli Center, 26 Harokmim St. Bldg D 25 Holon 5885849, Israel 26 Phone: 972-37658543 27 Email: [email protected]

Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 9, 2021; DOI: 10.1158/2159-8290.CD-20-1248 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

28 Abstract

29 Therapeutic antibodies targeting the CTLA-4/ PD-1 pathways have revolutionized cancer 30 immunotherapy by eliciting durable remission in cancer patients. However, relapse following 31 early response, attributable to primary and adaptive resistance, is frequently observed. Additional 32 immunomodulatory pathways are being studied in patients with primary or acquired resistance to 33 CTLA-4 or PD-1 blockade. The DNAM-1 axis is a potent co-regulator of innate and adaptive 34 immunity whose other components include the immunoglobulin receptors TIGIT, PVRIG and 35 CD96, and their nectin and nectin-like ligands. We review the basic biology and therapeutic 36 relevance of this family, which has begun to show promise in cancer clinical trials.

38 Significance

39 Recent studies have outlined the immuno-oncologic ascendancy of co-inhibitory receptors in the 40 DNAM-1 axis such as TIGIT and PVRIG, and to a lesser extent, CD96. Biologic elucidation 41 backed by ongoing clinical trials of single-agent therapy directed against TIGIT or PVRIG is 42 beginning to provide the rationale for testing combination regimens of DNAM-1 axis blockers in 43 conjunction with anti-PD-(L)1 agents.

51 2

52 Introduction

53 Immuno-stimulatory monoclonal antibodies (mAbs) that block inhibitory checkpoint proteins 54 such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) or programmed death 1 (PD-1), 55 and programmed death-ligand 1 (PD-L1) have made a paradigm shift in the development of anti- 56 cancer therapies. Since the early studies targeting these checkpoints in the 2000s, roughly ten 57 additional co-inhibitory molecules have been targeted clinically. The biology of these molecules 58 is complex, in part because most T cell regulatory pathways are multicomponent. CTLA-4 (or 59 CD152) is a homolog of the co-stimulatory receptor CD28, with both capable of binding the 60 ligands CD80 and CD86. Reflecting its status as a key regulator of autoimmunity, CTLA-4 61 displays an affinity for CD80 and CD86 that is several orders of magnitude greater than that of 62 CD28 (1). CTLA-4 outcompetes CD28 for access to their shared ligands and delivers inhibitory 63 signals that dampen TCR (signal 1) and co-stimulatory signals (signal 2). A similarly inducible 64 inhibitory checkpoint receptor whose discovery postdates that of CTLA-4 is PD-1, which upon 65 binding to one of its two defined ligands, PD-L1 or PD-L2 restricts T-cell activation (2). In the 66 clinic, several mAbs targeting either PD-1 or its PD-L1 ligand in solid tumors were tested before 67 the approval of the first PD-1 blockers – Nivolumab and Pembrolizumab (3,4). A mAb targeting 68 CTLA-4, three mAbs targeting PD-1, and three targeting PD-L1 have been approved by the U.S. 69 Food and Drug Administration (FDA) for treating 18 different malignancies (5,6). Exploratory 70 research is ongoing to discover novel checkpoints and eventually generate mAbs that could 71 potentially augment the clinical results observed with PD-1 or CTLA-4 blockade. With that in 72 mind, additional T-cell-specific inhibitory receptors have been studied extensively in preclinical 73 cancer models and are being tested in the clinic either as single-agents or in combination with 74 other checkpoint blockers or chemotherapy. Among these, lymphocyte-activation gene 3 (LAG- 75 3), T cell Ig and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) (TIGIT), T cell 76 Ig and mucin domain-3 (TIM-3), and B and T lymphocyte attenuator (BTLA4) have all been 77 shown to overcome T cell exhaustion in the tumor microenvironment (TME) and are considered 78 to be promising druggable targets in the immuno-oncology space (7–10). Although blockers of 79 each of these inhibitory checkpoints are in varying stages of clinical testing, none have secured 80 FDA approval for use in cancer. Antibodies targeting TIM-3 have shown encouraging

81 preliminary anti-tumor effects in patients with hematologic malignancies, including 82 Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) (11). The promising 83 candidates directed against LAG-3 and TIGIT are currently in Phase 3 clinical trials in 84 combination with antibodies blocking the PD-1 pathway. For LAG3 alone, ten agents are being 85 tested in clinical trials for a variety of cancers. Multiple anti‐TIGIT antibodies are being 86 evaluated in different clinical trials as a single-agent or in combination with PD-(L)1 blockers, as 87 will be discussed in this review (12). Moreover, we will discuss the biology and 88 immunotherapeutic significance of all constituents of the DNAM-1 (DNAX accessory molecule 89 1, CD226) axis including the co-inhibitory receptors, TIGIT, poliovirus receptor-related Ig

90 domain-containing protein (PVRIG), and CD96, their respective primary ligands, poliovirus 91 receptor (PVR, CD155) and PVR-related 2 (PVRL2, Nectin-2, CD112), and the eponymous co- 92 stimulatory receptor, DNAM-1 (Figure 1).

93 DNAM-1: the central co-stimulatory molecule in an interacting immune regulatory 94 network in the TME

95 DNAM-1 is the central node that links together four receptors (DNAM-1 itself, TIGIT, PVRIG, 96 and CD96) and two ligands (PVR and PVRL2) into one family. It is a 65 kilodalton (kD) 97 member of the Ig superfamily that contains two extracellular Ig-like domains, an intracellular 98 immunoreceptor tyrosine-based activation motif (ITAM) and is constitutively expressed on the 99 surface of natural killer (NK) cells, T lymphocytes, platelets, monocytes and a subset of B cells. 100 DNAM-1 was identified in a screen for cell surface molecules that mediate T cell cytotoxicity 101 and was subsequently shown to act as a co-stimulatory molecule on NK cells as well as T cells 102 (13). Consistent with a co-stimulatory function on T and NK cells, it was shown to mediate anti- 103 tumor immune responses as demonstrated by increased tumor growth in DNAM-1 knockout 104 mice (14,15). 105 In addition to DNAM-1’s direct co-stimulatory activity, a physical association with lymphocyte 106 function-associated antigen 1 (LFA-1) has been demonstrated both in NK and activated T cells. 107 This physical interaction was shown to promote the formation of stable target cell conjugates, 108 and NK cells lacking LFA-1 expression are deficient in DNAM-1 driven cytotoxicity, suggesting

109 a functional dependency between the two molecules (16). Further supporting the functional 110 interaction between the two molecules, crosslinking of LFA-1 results in Fyn protein tyrosine 111 kinase phosphorylation of DNAM-1 (16). LFA-1 and DNAM-1 demonstrate coordinated 112 expression and colocalization in the immune synapse of activated NK cells in the presence of 113 target cells (17), and DNAM-1 deficient CD8+ T cells have a decreased ability to recruit LFA-1 114 and lipid rafts to the immune synapse, while also showing reduced conjugation with target tumor 115 cells (18). 116 PVR and PVRL2, belonging to the superfamily of cell adhesion molecules, have been identified 117 as the ligands for DNAM-1 (Figure 1). PVR and PVRL2 mediate cell-cell adhesion by 118 generating heterotypic and homotypic (only PVLR2) interactions (19). High expression of these 119 molecules on tumor cells renders them susceptible to T and NK cell-mediated cytotoxicity, 120 which can be abrogated by an anti-DNAM-1 antagonist antibody (20,21). Overexpression of 121 PVR or PVRL2 in the RMA mouse tumor cell line increased tumor rejection and host survival 122 via T and NK mediated mechanisms, suggesting that each can act as co-stimulatory ligands in 123 the TME, presumably via their activation of DNAM-1. However, identification and 124 characterization of the immune checkpoint proteins TIGIT, CD96, and PVRIG has generated a 125 more complex picture of their function, since the interaction of PVR with TIGIT and CD96, and 126 that of PVRL2 with PVRIG can also mediate suppressive signaling through the co-inhibitory 127 arms of the DNAM-1 signaling axis, suggesting that they act to counterbalance DNAM-1 co- 128 stimulation. At the 30,000-foot level, this is analogous to CD28 and CTLA-4 as 129 counterbalancing co-stimulatory and co-inhibitory receptors, respectively, for CD80 and CD86; 130 however, at a closer glance, there are significant differences that give the DNAM-1 family its 131 unique character. While CD28 is expressed exclusively on T cells, DNAM-1 axis members are 132 expressed on both T and NK cells. In addition, CD28 is downregulated on T cells upon 133 activation and differentiation while DNAM-1 maintains its expression also on differentiated 134 effector cells (22,23). Furthermore, CD80 and CD86 are restricted to immune cells, mostly 135 antigen presenting cells, while PVR and PVRL2 are expressed on multiple cell types, including 136 antigen presenting cells, tumor and endothelial cells. These differences in expression pattern 137 suggest that while CD28 and CTLA-4 probably play a dominant role in T cell priming, DNAM-1

138 axis members presumably regulate both priming and effector phase of T cells, resembling PD-1 139 pathway. Nevertheless, similar to the higher affinity CD80/86 interactions with CTLA-4 versus 140 CD28, TIGIT, CD96, and PVRIG have higher affinities to their cognate ligands than DNAM-1 141 and thus can inhibit T and NK cell activity through ligand competition (24–28). This was 142 supported by several studies showing that the co-stimulatory effects driven by targeting the 143 TIGIT/PVR interaction are dependent on the engagement of DNAM-1 by PVR (27,29). A recent 144 study suggested that the infiltration of CD8+DNAM-1high T cells into TME may predict the 145 efficacy of TIGIT blockade, as the presence of these cells is essential for the anti-TIGIT 146 immunomodulatory activity (30). Additional complexity arises from the direct interruption of 147 DNAM-1 homodimerization by TIGIT which results in its impaired functionality (29). 148 Furthermore, DNAM-1 expression on lymphocytes is modulated by membrane PVR that upon 149 binding to DNAM-1 induces its internalization and degradation, resulting in decreased anti- 150 tumor function (31). Similarly, it was shown that PVRIG outcompetes DNAM-1 for PVRL2 151 binding (32) (Figure 1). 152 One approach to boost anti-tumor immunity by modulating the DNAM-1 axis could arise from 153 agonizing DNAM-1, which has the potential to show therapeutic benefit in cancer patients, due 154 to its potent T and NK cells co-activatory function. However, a caveat for this approach is the 155 broad expression of DNAM-1 on different cell types, including on platelets, where it was shown 156 to stimulate activation and cell adhesion (33). Indeed, an agonist antibody directed against 157 DNAM-1 (LY3435151) was clinically evaluated in solid malignancies, however the study 158 (NCT04099277) was recently terminated for undisclosed reasons. 159 Another interesting approach could be the direct targeting of PVR or PVRL2 with antagonist 160 antibodies. This may result in T and NK cell activation by interrupting the dominant interactions 161 with TIGIT and PVRIG, respectively. When using effector function antibodies, an antibody- 162 dependent cellular cytotoxicity (ADCC) against PVCR/PVRL2-expressing tumor cells could be 163 applied, while avoiding the risk of potential ADCC against T cells. Moreover, in addition to 164 direct effects on immune cells, interfering with non-immune tumor promoting effects of PVR or 165 PVRL2 could provide additional benefit for this approach (34). Accordingly, an anti-PVRL2 166 antagonizing mAb developed by Takeda exhibited anti-tumor effects attributed mainly to ADCC

167 against tumor cells (35). Potential drawbacks of anti-PVR or anti-PVRL2 mAbs are that direct 168 targeting may result in interference of DNAM-1 co-stimulatory signaling, as well as in the 169 induction of ADCC against antigen presenting cells. Finally, PVR and PVRL2 are broadly 170 expressed in peripheral normal tissues, creating a large target sink and increasing the risk of 171 developing on-target, off-tumor toxicities. Indeed, thrombocytopenia was identified in monkeys 172 administered an anti-PVRL2 mAb (36).

173 Altogether, co-stimulatory DNAM-1 activity in T and NK cells is tightly regulated at the 174 expression level and by its interaction with other axis members; understanding the biology of 175 these interactions is crucial for the success of immunotherapies targeting this axis.

176 TIGIT blockade - a promising new clinical modality for treating solid and hematological 177 malignancies

178 TIGIT was initially identified as an inhibitory receptor on T and NK cells based on shared gene 179 and protein structure with known immune checkpoints (8,24). TIGIT is also expressed on Treg 180 cells where it enhances their activation and suppressive activity (Figure 2A). Similar to other 181 immune checkpoints, TIGIT contains an extracellular Ig variable domain, a transmembrane 182 domain, and an ITIM in the intracellular signaling domain (8,24). The dominant ligand for

183 TIGIT is PVR with a binding affinity (KD) reported as 1-3 nM (Figure 1) (24). A recent study 184 described Nectin4 (PVRL4) as a functional ligand of TIGIT with an affinity similar to that of 185 TIGIT/PVR (37). PVRL2 and PVRL3 (CD113) have also been reported to bind TIGIT albeit at 186 much weaker affinities (8,19,24). PVR expression was shown to positively correlate with an 187 unfavorable prognosis in multiple malignancies such as lung adenocarcinoma (38), breast (39), 188 pancreatic (25), and bladder (40) cancers. In metastatic melanoma patients, PVR expression on 189 tumor cells was associated with resistance to anti-PD-1 immunotherapy (41). As mentioned 190 above, TIGIT’s higher binding affinity for PVR compared to DNAM-1 allows it to outcompete 191 DNAM-1 for ligand binding, similar to CTLA-4 versus CD28 (21,24,27,28). While the 192 expression of both CTLA-4 and TIGIT is induced upon activation, CD28 is mainly expressed on 193 naïve T cells, whereas DNAM-1 has broad expression on multiple T cell subsets (23). Moreover, 194 unlike CTLA-4, TIGIT has been shown to directly interact with DNAM-1, reducing the ability of

195 DNAM-1 to homodimerize and enhance immune activation (27,29,42). Recently, it was 196 demonstrated that the TIGIT/PVR interaction induces de-phosphorylation of DNAM-1 at 197 tyrosine 322 and interferes with its co-stimulatory responses on T cell (43). In addition to 198 counteracting the stimulatory function of DNAM-1 via direct binding and competition for their 199 common PVR ligand, TIGIT can directly inhibit T and NK cell effector function by signaling 200 through an ITIM domain (8,44). TIGIT also indirectly regulates CD8+ T cell priming and 201 proliferation by engaging PVR on dendritic cells, which reportedly induces a tolerogenic 202 phenotype and reduces their pro-inflammatory cytokine production via “reverse signaling” 203 (Figure 2B) (24).

204 Several studies have shown that TIGIT is a marker of T cell dysfunction and is upregulated on 205 human viral-specific CD8+ T cells, and human CD8+ and CD4+ T cells infiltrating a variety of 206 solid and hematological tumors. In the LCMV Clone 13 model of chronic viral infection, 207 Johnston et al. demonstrated that TIGIT is highly expressed on CD8+ T cells that also express 208 high levels of PD-1 (29), and that inhibition of these pathways increased viral clearance and 209 T cell effector function. Similarly, in murine models of colon and breast carcinomas, co- 210 blockade of TIGIT and PD-L1 resulted in tumor rejection and restoration of CD8+ T cells within 211 the TME (29). Johnston et al. further demonstrated that mice treated with anti-TIGIT and anti- 212 PD-L1 were unable to reject CT26 tumors when depleted of CD8+ T cells (29). Chauvin et al. 213 also demonstrated that the TIGIT pathway blockade either alone or in combination with anti-PD- 214 1 synergistically increased effector function of human NY-ESO-1-specific CD8+ tumor- 215 infiltrating lymphocytes (TILs) from melanoma patients (45). Collectively, these data 216 demonstrate that TIGIT and PD-1/PD-L1 co-blockade acts through CD8+ T cells to generate an 217 effective anti-tumor immune response. Interestingly, TIGIT was shown to be uniquely expressed 218 by T memory stem (Tscm) cells, an emerging population of T cells, shown to be important for 219 response to immunotherapy. Tscm cells display a functional state associated with enhanced self- 220 renewal, have multipotency to generate memory and effector T cell subsets, were found to be 221 associated with increased response to immunotherapy, and to correlate significantly with PD-1 222 blockade therapeutic activity (46–48). Importantly, TIGIT and PD-1 are the dominant inhibitory 223 T cell checkpoints expressed on this memory cell subset (49). 8

224 Other studies reported that TIGIT predominantly suppresses anti-tumor CD8+ T cell responses 225 indirectly by enhancing Treg function in the TME (50–52). Kurtulus et al. found that adoptively 226 transferred TIGIT-deficient CD8+ T cells and wild type CD4+ T cells into B16F10 melanoma 227 tumor-bearing Rag-deficient mice did not alter tumor growth compared to transfer of wild type 228 TIGIT+ CD8+ T cells (50). In contrast, the transfer of TIGIT-deficient Tregs and wild type 229 CD4+FoxP3- and CD8+ T cells, significantly delayed B16F10 tumor growth compared with mice 230 that received wild type Tregs. These data suggest that in at least some tumor models, TIGIT+ 231 Tregs play a more dominant role in suppressing anti-tumor immune responses compared to direct 232 signaling of TIGIT on CD8+ T cells. Other studies have examined the mechanisms of TIGIT+ 233 Treg-mediated immune suppression. Joller et al. showed that increased amounts of IL-10 234 produced by TIGIT+ Tregs contributed to the generation of tolerogenic dendritic cells, thereby 235 inhibiting the generation of effector T cell responses (42,52). Moreover, TIGIT blocking mAbs 236 were shown, in addition to enhancing conventional T cell activity, to reduce Tregs secretion of 237 IL-10 (53). Finally, a recent study showed that Tregs in melanoma further upregulate TIGIT and 238 downregulate DNAM-1 expression resulting in a higher TIGIT/DNAM-1 ratio (54). This high 239 TIGIT/DNAM-1 ratio in Tregs regulates their suppressive function and stability and correlates 240 with a poor clinical outcome following PD-1 and/or CTLA-4 pathway blockade in melanoma 241 patients (45,50,54).

242 In addition to TIGIT’s role in T cell biology, numerous groups have shown higher TIGIT 243 expression on viral and tumor-infiltrating NK cells and that increased TIGIT levels are 244 negatively correlated with the ability of NK cells to secrete pro-inflammatory cytokines and kill 245 PVR-expressing tumor cells, suggesting that TIGIT abrogates NK cell activation (Figure 2B) 246 (8,44,55). In mouse models, lack of TIGIT+ NK cells was sufficient to slow B16 melanoma 247 tumor growth, while antibody blockade of the TIGIT pathway reinvigorated the anti-tumor 248 NK cell response in multiple solid tumors and hematological models (44,56–58). Overall, 249 survival was increased in these tumor models, indicating that NK cells may be critical for the 250 therapeutic effects of TIGIT pathway-based immunotherapies (57,58). Collectively, these studies 251 demonstrate important roles for TIGIT-expressing CD8+ T cells, Tregs, and NK cells in 252 regulating anti-tumor responses in mouse tumor models and human in vitro functional systems. 9

253 An open question in the TIGIT therapeutic antibody field is whether Fc receptor (FcR) co- 254 engagement is required to exploit the full clinical potential from TIGIT blockade. Multiple 255 antibodies targeting TIGIT are currently in clinical development, some with FcR binding 256 (typically human IgG1) and others with reduced binding to FcR (Table 1). Murine studies with 257 anti-TIGIT mouse IgG2a antibodies (increased FcR binding capacity) demonstrate monotherapy 258 activity in-vivo and increased potency compared to mouse IgG1 Abs (reduced FcR binding 259 capacity) (59,60). Recently, Yang et al. demonstrated that TIGIT blockade induced Fc-mediated 260 depletion of Tregs that activate anti-tumor CD8+ T cell responses, targeting tumor-shared 261 antigens that are normally cryptic or suppressed by Tregs (61). In contrast, a mouse IgG1 anti- 262 TIGIT antibody (low FcR binding capacity) has been shown to significantly enhance the 263 antitumor activity, particularly when combined with a blocking anti-PD-L1 antibody (62). 264 Ultimately, the ability of mouse models to predict the importance of FcR binding by a 265 therapeutic antibody is limited, since murine FcRs do not mirror the structural diversity, 266 expression, and Fc binding pattern of the human FcR (63). Moreover, given the overlap in TIGIT 267 expression between CD8+ effector T cells and Tregs, a TIGIT antibody with potent effector 268 function carries the risk of depleting the very CD8+ TILs in addition to tumor Tregs. Studies on 269 mechanisms of action of CTLA-4 blockade emphasize that caution in the direct extrapolation of 270 FcR roles between mouse and human. While previous murine studies showed that anti-tumor 271 effects of a FcR-binding mouse IgG2a anti-CTLA-4 were dependent on FcR expression and 272 were associated with selective intra-tumoral depletion of Treg, Ipilimumab, a human IgG1 y that 273 binds human FcR, failed to decrease intra-tumoral Treg density (64). Likewise, PD-L1 blockade 274 was shown to require FcR engagement for mediating activity in-vivo in mice (17), whereas in the 275 human settings anti-PD-L1 antibodies with silent Fc backbones exhibited clinical efficacy and 276 are FDA approved. Therefore, it remains to be determined in patients which cell type plays a 277 dominant role in generating anti-tumor responses during anti-TIGIT therapy and whether FcR- 278 engagement is a required feature in the clinic.

279 Another unanswered question being investigated in clinical trials is which treatments should be 280 paired with anti-TIGIT therapy to generate robust and durable anti-tumor responses, especially in 281 anti-PD-1/PD-L1 resistant patients. While TIGIT is highly co-expressed with PD-1 on human 10

282 TILs from different solid tumors and lymphomas, its expression also highly correlates with the 283 expression of other checkpoint receptors including LAG-3, TIM-3, and BTLA4 (65–67). One 284 study showed that TIM-3 pathway blockade in TIGIT deficient mice acts synergistically to 285 reduce B16F10 melanoma tumor growth (50). Recently, we demonstrated that PVRIG/PVRL2 286 blockade induces TIGIT expression (26), and combining TIGIT with PVRIG co-blockade 287 synergistically activates T cells in human functional assays which are translated into in-vivo anti- 288 tumor effects (68). Furthermore, TIGIT blocking mAbs are being tested clinically in combination 289 with other therapeutic modalities such as chemotherapy, IL-15 cytokines, and myeloma targeting 290 antibodies (31).

291 Whilst the majority of anti-TIGIT antibodies are currently in early clinical investigation, several 292 have progressed beyond Phase 1 studies. These include tiragolumab (Roche-Genentech), which 293 is currently recruiting patients in Phase 3 studies in Non-Small Cell Lung Cancer (NSCLC) and 294 Small Cell Lung Cancer (SCLC), domvanalimab (Arcus) initiating a Phase 3 studies in NSCLC, 295 and vibostolimab (Merck). Initial results have been presented from three clinical studies 296 evaluating anti-TIGIT antibodies as a single agent and have demonstrated limited clinical 297 activity, with overall response rates ranging from 0-3% (69,70). Recently, data were reported 298 from a randomized, Phase 2 double-blind, placebo-controlled study of 135 patients with 299 previously untreated NSCLC who were randomized 1:1 to tiragolumab plus atezolizumab versus 300 placebo plus atezolizumab (69). The study met its co-primary endpoints of objective response 301 rate (ORR) and progression-free survival for the combination versus atezolizumab alone in the 302 intent to treat (ITT) population, demonstrating an improvement in objective response rate (ORR) 303 of 31.3% compared to 16.2%, and in median progression-free survival (PFS) of 5.4 months 304 compared to 3.6 months. Thus, this study provided the first proof-of-concept for TIGIT 305 inhibition in a larger, randomized clinical trial. The clinical benefit derived from the combination 306 was particularly effective in the PD-L1 positive ≥ 50% population, demonstrating a 66% ORR 307 and a 70% reduction in the risk of disease worsening or death. This combination is currently 308 being evaluated in a Phase 3 study in PD-L1 ≥ 50% first line NSCLC.

309 CD96: a second PVR-binding receptor with unclear function in human and mouse

310 Originally described as T cell activation, increased late expression (TACTILE), CD96 was 311 discovered two decades ago as a member of the Ig superfamily that is highly upregulated on 312 activated T and NK cells and mediates cell adhesion via interaction with its dominant ligand 313 PVR (20,71,72). CD96 has an expression pattern similar but not identical to TIGIT, including on 314 hematopoietic stem cells, αβ and γδ T cells, NK cells, and a subpopulation of B cells in humans 315 (23,73–77) and mice (23,77,78). It is highly expressed on human tumor-infiltrating CD8+ T 316 cells, compared to normal tissues (23,79). Moreover, its expression on T cells is increased upon 317 stimulation, resembling the expression profile kinetics of DNAM-1 (23). Like TIGIT, the high- 318 affinity binding partner for CD96 is PVR. While the binding affinity of CD96 to PVR is higher 319 than that of DNAM-1, it is lower than that of TIGIT (Figure 1) (24). CD96 also binds PVRL1 320 (CD111), but the functional relevance of the CD96/PVRL1 interaction has not been fully 321 explored (77,80).

322 The functional role of CD96 has been mainly investigated in murine NK cells. CD96 appears to 323 act as an inhibitory receptor in murine NK cells, decreasing NK cell activation. Chan et al. 324 demonstrated that in CD96-deficient mice, NK cells produced greater IFN-γ in response to LPS, 325 IL-12, or IL-18 stimulation (78). A role for CD96 in resistance to experimental melanoma lung 326 metastases, MCA-induced fibrosarcomas, and prostate carcinomas was also demonstrated in 327 CD96-deficient mice or following anti-CD96 antibody treatment in wild type mice. Combined 328 blockade of CD96 and PD-1 or CTLA-4 pathways increased cytokine secretion and survival in 329 tumor-bearing mice in comparison to anti-PD-1 or anti-CTLA-4 alone (78,81). Furthermore, an 330 additional report found that in mice that lack both PD-1 and CD96, or both TIGIT and CD96, 331 there is enhanced tumor growth inhibition and/or complete response in dual receptor-deficient 332 mice compared to mice lacking PD-1, TIGIT or CD96 alone (82). Although CD96 was originally 333 identified as an Ig receptor expressed on activated T cells, there are very few studies examining 334 the functional role of CD96+ T cells. In addition to its inhibitory role in NK cells, recently, Mittal 335 et al. showed that CD96 blockade suppressed tumor growth of several murine models in a CD8+ 336 T cell-dependent manner (79). The anti-tumor efficacy of CD96 was mediated by increasing 337 IFN-γ secretion, and was dependent on DNAM-1, as CD96/DNAM-1 co-blockade abolished 338 CD96 effect (79). 12

339 There are limited studies characterizing the functional role of CD96 in human NK cells and the 340 results are conflicting. Transcriptomic analysis comparing human hepatic CD96+ and CD96- NK 341 cell subsets suggest that CD96+ NK cells are functionally exhausted comparing to CD96- NK 342 cells and that CD96+ cells are enriched with published gene signatures such as inhibition- and 343 exhaustion-related genes that are correlated with the regulation of NK-mediated immunity (83). 344 Fuchs et al. showed that the cytotoxic activity of human polyclonal NK cell lines was enhanced 345 in the presence of a CD96 binding antibody (72), suggesting that human CD96 promotes NK cell 346 activation rather than inhibition. In contrast to this report, Carlsten et al. demonstrated that while 347 DNAM-1 blockade significantly reduced NK degranulation and cytotoxicity, an anti-CD96 mAb 348 did not affect tumor cell killing when NK cells were co-cultured with fresh ovarian carcinoma 349 cells (84). Additionally, we have demonstrated that inhibition of the CD96/PVR interaction with 350 two distinct, antagonistic anti-CD96 antibodies had no effect on IFN-γ secretion from primary 351 human T cells (26). The cytoplasmic tail of both human and mouse CD96 has an ITIM-like motif 352 for inhibitory signaling, but human CD96 also contains a YXXM motif, which is found within 353 other known activating receptors including CD28 and NKG2D (74). The presence of a potential 354 activating motif might explain why CD96 has been reported as either an inhibitory or co- 355 stimulatory receptor in different human in vitro assay systems. Accordingly, it was recently 356 reported that CD96 has co-stimulatory functions in human and mouse CD8+ T cells. It was 357 shown in-vitro and in-vivo that depletion or disruption of CD96/PVR interaction results in 358 impaired T cell proliferation and inflammatory cytokines secretion (85).

359 Given these conflicting results, the role of CD96 in human T and NK cells is not fully 360 understood, and collectively these studies indicate that CD96 pathway blockade may either 361 enhance or inhibit lymphocyte killing of PVR+ tumor cells (Figure 1). This will soon be tested 362 clinically, as GlaxoSmithKline recently reported initiation of Phase 1 clinical trial evaluating a 363 CD96 blocking antibody in patients with solid tumors and that data should further inform the 364 role and relevance of CD96 in the DNAM-1 axis.

365 PVRIG – the recently discovered inhibitory receptor in the DNAM-1 axis

366 PVRIG was identified using in silico analyses designed to discover novel immunoreceptors that 367 are involved in regulating lymphocyte function in the context of cancer (32,86). Human and 368 cynomolgus PVRIG share high sequence identity, and both proteins have a conserved ITIM in 369 the intracellular signaling domain, which is lacking in the mouse homolog. PVRL2 is the cognate 370 ligand for PVRIG with a binding affinity of human PVRIG to human PVRL2 reported as 88 nM 371 (32). Studies investigating whether DNAM-1 and TIGIT compete with PVRIG for binding to 372 PVRL2 showed that TIGIT had little effect on interrupting the PVRIG/PVRL2 interaction, 373 whereas DNAM-1 was able to compete for this interaction, despite its reduced affinity to PVRL2 374 compared with PVRIG (26,32). While TIGIT is reported to have a very low-affinity interaction 375 with PVRL2, its physiologic relevance is unclear and our recent data suggest that 376 PVRIG/PVRL2 serves as a distinct inhibitory signaling pathway in the DNAM-1 axis, and has a 377 non-overlapping function with TIGIT/PVR pair (Figure 1) (26).

378 Mouse PVRIG has a 59% protein identity with human PVRIG and a truncated intracellular 379 signaling domain that contains phosphorylated tyrosine but lacks an ITIM, suggesting that mouse 380 PVRIG may have a reduced role as a DNAM-1 pathway checkpoint receptor compared to human 381 PVRIG (87). At steady state, expression of murine PVRIG is detected on both T and NK cells, 382 whereas in peripheral immune tissues, NK but not T cells express PVRIG. Upon activation, 383 CD8+ T cells upregulate PVRIG expression, although at a much slower rate compared with 384 related co-inhibitory checkpoints. The relative expression of mouse PVRIG on TILs is lower 385 than that seen in humans, and mouse PVRL2 in the TME and mouse tumor cell lines is lower 386 than the expression detected in human TME, further supporting the idea that PVRIG plays a 387 diminished role in mice compared to its role in humans (87). These findings, including the 388 acquisition of an ITIM in human PVRIG and higher levels of human PVRIG and PVRL2 in the 389 TME, suggest that greater effects of PVRIG inhibition may be seen in a human tumor setting 390 relative to mouse preclinical models.

391 Nevertheless, although mouse PVRIG lacks an ITIM motif, PVRIG deficient T cells do have 392 increased function compared to wild type T cells following antigen challenge. Moreover, in-vivo 393 syngeneic models utilizing MC38 colon carcinoma and B16 melanoma demonstrated that tumors

394 grew slower in PVRIG-deficient compared to wild type mice and ex-vivo analysis pointed to 395 functional differences in T cell responses. In the CT26 colon carcinoma model, reduced tumor 396 growth and increased survival were observed after PVRIG and PD-1 co-blockade (87). This was 397 further supported by increased anti-tumor responses in PVRIG/TIGIT double knockout mice 398 (68).

399 In the human setting, as in the mouse setting, results indicate that inhibition of this pathway may 400 result in enhanced anti-tumor immunity (26). The activating effect of PVRIG blockade on CD8+ 401 T cells has been demonstrated in-vitro with antibodies that block the PVRIG/PVRL2 interaction. 402 The ability of anti-PVRIG mAbs to promote T cell responses, either individually or in 403 combination with other immune checkpoints, was assessed using multiple human T cell-based 404 assays with both TILs and T cells from healthy donors. In all assay systems, PVRIG blockade 405 increased T cell proliferation, cytokine secretion, and cytotoxicity (26,32). Although clinical 406 monotherapy effects are rarely observed with inhibitory receptor antagonists other than anti-PD- 407 1 (60,88–91), numerous pathways modulate immune responses, suggesting that combinatorial 408 approaches may increase rates of response (91–94). Compared to blockade of PVRIG, TIGIT, or 409 PD-1 alone, the dual and triple combination of anti-PVRIG with anti-PD-1 or with anti-TIGIT 410 further increased cytokine production and T cell-mediated killing of PVRL2+PVR+ tumor target 411 cells (26).

412 In addition to PVRIG’s role in regulating T cell responses, we have recently shown that the 413 PVRIG blockade significantly enhances NK cell-mediated killing of PVRL2+ cancer cells (95), 414 in line with other emerging data supporting the inhibitory functional role of PVRIG in these cells 415 (96). Accordingly, Xu et al., demonstrated that blockade of PVRIG and TIGIT alone or in 416 combination enhances trastuzumab-triggered anti-tumor response by human NK cells (97). 417 Collectively, these data suggests that PVRIG and TIGIT receptors regulate NK cell functions and 418 that NK activation may be a determinant of clinical efficacy for inhibitors targeting each (Figure 419 2B).

420 In assessing tumor expression, hormonally regulated tumors, such as ovarian, endometrial, breast 421 cancers, along with kidney and lung tumors demonstrated the highest PVRIG expression on T

422 and NK cells. PVRIG expression in tumors was significantly increased on TILs compared to T 423 cells infiltrating normal adjacent tissue, further highlighting its potential as a checkpoint receptor 424 on lymphocytes. Moreover, PVRIG was co-expressed with PD-1 and TIGIT on TILs, peripheral 425 memory T cells, and activated T cells. These data indicate that all three inhibitory molecules play 426 a role in regulating the immune response and provide a rationale for the dual and triple blockade 427 of these checkpoint receptors, depending on the ligand expression pattern in the TME (26). 428 PVRL2 is frequently expressed in various malignancies and specifically PVRL2/PVR mRNA 429 ratios are generally highest in hormonally regulated cancers which was further validated also by 430 both IHC and flow cytometry. This, together with PVRIG levels in those tumors, suggest that 431 these cancers are promising indications for PVRIG blocking antibodies. In addition, increased 432 RNA and protein PVRL2 expression levels were demonstrated in cancer relative to normal 433 tissues with expression seen in both PD-L1+ and PD-L1- patients samples across tumor 434 types(26,98). These analyses highlighted cancers in which PVRL2 might primarily regulate the 435 immune response, and how this pathway may be relevant in patients who are PD-L1- or those 436 who develop PD-1 resistance (98–100).

437 Based on the supportive preclinical data, an anti-PVRIG antibody was developed for clinical 438 testing. COM701 is a humanized anti-PVRIG hinge stabilized IgG4 mAb that binds specifically 439 to human and cynomolgus monkey PVRIG and disrupts the binding of PVRIG to PVRL2 (Table 440 1). Since PVRIG is predominantly expressed on CD8+ T and NK cells, and its expression is 441 relatively low on Tregs (26), an IgG4 backbone was selected to avoid potential depletion of 442 effector cells. COM701 inhibits the binding of PVRL2 to PVRIG in a dose-dependent manner 443 with complete inhibition of PVRIG/PVRL2 interaction observed at saturating levels of COM701 444 (26). It is currently in Phase I clinical testing, both as a monotherapy and in combination with 445 the anti-PD-1 drug nivolumab (NCT03667716). In initial results presented from the dose- 446 escalation arms of a Phase 1 study, COM701 was shown to be well-tolerated as a monotherapy 447 and in combination with nivolumab, with no dose-limiting toxicities reported. Additionally, 448 encouraging disease control rates of 69% (11/16) for the monotherapy and 75% (9/12) for the 449 combination arm were observed in a heavily pre-treated patient population. Moreover, two 450 patients were reported with confirmed partial responses, one from the monotherapy arm 16

451 (microsatellite stable primary peritoneal cancer) and one from the combination arm 452 (microsatellite stable colorectal cancer, which has an extremely low response rate to anti-PD-1 453 alone). Future clinical trials involving triple blockade of PVRIG, TIGIT, and PD-1 are planned.

454 PD-1: A player in the DNAM-1 axis?

455 While several studies have demonstrated additive or synergistic effects of PD-1 pathway 456 blockade with inhibition of TIGIT or PVRIG (26,29,32,45,57), the mechanism was poorly 457 understood. Wang and colleagues have provided some insight into the intersection of the 458 DNAM-1 and PD-1 pathways (59). Using a cell-free system, the authors demonstrated that 459 DNAM-1 is dephosphorylated by the PD-1/SHP2 complex, suggesting that it is a downstream 460 target of PD-1 signaling (Figure 3A). This was further reinforced by the observation that antigen- 461 specific intra-tumoral CD8+ T cells isolated from MC38-OVA tumor-bearing mice treated with 462 an anti–PD-1 antibody showed a high level of DNAM-1 phosphorylation, whereas DNAM-1 on 463 TILs isolated from isotype control-treated mice showed a lack of phosphorylation.

464 Extending the above results, Wang and colleagues assessed the impact of DNAM-1 blockade or 465 ablation in an in-vivo tumor model responsive to anti-PD-1 in combination with a glucocorticoid- 466 induced tumor necrosis factor receptor (GITR) agonist antibody. The survival of tumor-bearing 467 mice was dramatically increased by the combination treatment and could be reversed by either 468 the addition of an antagonist DNAM-1 antibody or upon treatment of tumors engrafted into 469 DNAM-1 knockout mice. The authors also demonstrated an effect of PD-1 inhibition on DNAM- 470 1 expression; increased expression was seen on antigen reactive clones following anti-PD-1 471 treatment, and further increased with GITR agonism. Taken together the results implicate de- 472 repression of DNAM-1 co-stimulatory signaling in response to PD-1 inhibition, similar to the 473 role that has been proposed for CD28 involvement in an anti-PD-1 response (101). Another 474 study has similarly shown that DNAM-1 is required for PD-1 blockade anti-tumor activity in 475 pre-clinical mouse models (102). In accordance with these results, DNAM expression on TILs 476 was shown to correlate positively with the response to PD-1 blockade in melanoma patients 477 (103). These data provide a molecular rationale for the combination effects observed with PD-1

478 inhibition when combined with blockade of coinhibitory molecules in the DNAM-1 axis (Figure 479 3B).

480 Summary

481 Given recent advances in our understanding of DNAM-1 regulation, including a possible 482 connection with the PD-1 pathway and the broad and up-regulated expression of the axis ligands 483 PVR and PVRL2 in the TME, the co-inhibitory receptors in the axis are increasingly attractive as 484 targets for cancer immunotherapy. Preclinical and early clinical data in solid tumors have begun 485 to emerge from clinical trials with TIGIT inhibitors, and additional clinical data is expected by 486 the end of 2020 (Figure 4). Interestingly, while initial studies in NSCLC indicate greater efficacy 487 for TIGIT blockade in PD-L1high patients, early clinical data for PVRIG blockade highlights 488 activity in indications that are generally unresponsive to PD-1 therapy and that typically 489 demonstrate low PD-L1 expression. Emerging clinical data for the PVRIG inhibitor COM701 490 and multiple TIGIT inhibitors should yield additional insight into functional interactions between 491 the immune checkpoint inhibitors in the DNAM-1 axis, both with each other and in conjunction 492 with modulation of the PD-1 signaling pathway.

493 Furthermore, defining the patient population most likely to respond to each therapeutic 494 intervention is of great interest. As mentioned above, early data suggests that PD-L1 might be a 495 potential biomarker of response to TIGIT blockade (69), at least when combined with PD-L1 496 blockers. However, the expression of other pathway members is also important. PVRIG and/or 497 PVRL2 high expressing tumors might support dominance of the PVRIG pathway and thus 498 preferentially respond to PVRIG blockade (26). Likewise, patients with TIGIT and/or PVR high 499 expressing tumors could potentially better benefit from TIGIT blockade. Finally, the expression 500 levels of DNAM-1, which is in the center of the axis, could also serve as a biomarker of response 501 to combination blockade of the axis members.

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511 References

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Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2021 American Association for Cancer Research. Company AuthorCompound Manuscript PublishedTarget AntibodyOnlineFirst onDevelopm March 9, 2021;Combination DOI: 10.1158/2159-8290.CD-20-1248 Patient Population ClinicalTrials.gov Author manuscripts have been peerIsotype reviewed ent and Stage accepted Therapy for publication but have not yet been edited.Identifier

Roche MTIG7192A TIGIT IgG1 Phase 3 Atezolizumab, Non-Small Cell Lung NCT04300647, Tiragolumab Carboplatin+Etopo Cancer, Cervical NCT04294810, side, Rituximab, Cancer, Multiple NCT04256421, Daratumumab, Myeloma, Non- NCT04045028, Carboplatin+Paclit Hodgkin Lymphoma, NCT03563716, axel, Cisplatin B cell Lymphoma, NCT02794571, Gastric NCT03281369, Adenocarcinoma, NCT03869190, Urothelial Carcinoma, NCT03193190 Pancreatic Adenocarcinoma Merck MK-7684 TIGIT IgG1 Phase 2 Pembrolizumab, Advanced solid NCT02964013, Vibostolimab Carboplatin+Paclit tumors, Non-Small NCT04165070, axel, Pemetrexed, Cell Lung cancer, NCT04305054, MK-1308 (anti- Melanoma NCT04305041, CTLA-4 mAb) NCT04303169 Arcus AB154 TIGIT Inactive Phase 2 AB122 (anti-PD-1 Advanced solid NCT03628677, Biosciences IgG1 mAb), AB928 tumors, Non-Small NCT04262856 (adenosine receptor Cell Lung cancer antagonist)

Phase 3 Durvalumab Non-Small Cell Lung To be announced cancer BMS BMS-986207 TIGIT Inactive Phase 1/2a Nivolumab, Advanced solid NCT02913313, IgG1 Pomalidimide + tumors, Multiple NCT04150965 Dexamethasone Myeloma

Mereo OMP- TIGIT IgG1 Phase 1 Nivolumab Advanced solid NCT03119428 313M32 tumors Etigilimab Compugen COM902 TIGIT IgG4 Phase 1 N/A Advanced NCT04354246 Malignancies iTeos EOS884448 TIGIT IgG1 Phase 1 N/A Advanced NCT04335253 Malignancies Innovent IBI939 TIGIT N/A Phase 1 Sintilimab (anti- Advanced NCT04353830 PD-1 mAb) Malignancies

Beigene BGB-A1217 TIGIT IgG1 Phase 1 Tislelizumab (anti- Metastatic Solid NCT04047862 PD-1 mAb) Tumors

Seattle SGN-TGT TIGIT Effector Phase 1 Pembrolizumab Advanced NCT04254107 Genetics Enhanced Malignancies IgG1

EMD Serono M6223 TIGIT N/A Phase 1 Bintrafusp Alfa Metastatic Solid NCT04457778 (anti-PD-L1/ TGFß Tumors Trap) Compugen COM701 PVRIG IgG4 Phase 1 Nivolumab Advanced solid NCT03667716 tumors GSK GSK6097608 CD96 N/A Phase 1 Dostarlimab Neoplasms NCT04446351

Table 1. List of antagonistic antibodies targeting DNAM-1 axis family members that are under preclinical and clinical development. N/A refers to not applicable.

Figure legends

Figure 1: TIGIT and PVRIG are parallel inhibitory pathways in the DNAM-1 Axis. Interactions between DNAM-1 axis molecules and their cognate ligands on tumor or antigen presenting cells (APCs) induces activation (+) or inhibition (-) of T and NK cells function. Arrow thickness represents the relative affinity between the interacting molecules.

Figure 2: The effects of DNAM-1 Axis modulation on immune cells. DNAM-1 axis molecules and nectin and nectin-like ligands are expressed on various cells, and their interactions modulate immune function. A) TIGIT and DNAM-1 exert opposite effects in Tregs upon PVR binding. TIGIT further stimulate Treg immuno-modulatory activity, whereas DNAM-1 disrupts Treg suppression. B) While DNAM-1 stimulates effector T and NK cell function, TIGIT and PVRIG induce their suppression. PVR engagement on DCs by TIGIT induces a tolerogenic phenotype and reduces pro- inflammatory cytokine secretion.

Figure 3: Functional interplay between the PD-1 pathway and DNAM-1 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 de-phosphorylating the DNAM-1 signaling motif and reducing its expression. B) Full restoration of T cell activation by co-inhibition of PVRIG, TIGIT, and PD-1.

Figure 4: Clinical landscape of the DNAM-1 Axis

Figure 1

Figure 2

A) B)

Figure 3

Figure 4

Therapeutic Targeting of Checkpoint Receptors within the DNAM-1 Axis

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

Cancer Discov Published OnlineFirst March 9, 2021.

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