Alternative Checkpoints as Targets for Immunotherapy.

PURPOSE OF REVIEW: Immunotherapy has shown an unprecedented response in treatment of tumors. However, challenges such as lack of cytotoxic lymphocytes to mount an immune response or development of resistance to therapy can limit efficacy. Here, we discuss alternative checkpoints that can be targeted to improve cytotoxic lymphocyte function while harnessing other components of the immune system. RECENT
FINDINGS: Blockade of alternative checkpoints has improved anti-tumor immunity in mouse models and is being tested clinically with encouraging findings. In addition to modulating T cell function directly, alternative checkpoints can also regulate activity of myeloid cells and regulatory T cells to affect anti-tumor response. Combination of immune checkpoint inhibitors can improve treatment of tumors by activating multiple arms of the immune system.

Introduction

T cell dysfunction or “exhaustion” is a phenomenon that can result from chronic antigenic stimulation in the context of a tumor [1]. Broadly defined, exhaustion refers to a progressive loss of proliferative capacity, cytotoxicity, and inflammatory cytokine production. Exhausted T cells upregulate inhibitory molecules on the cell surface which mediate loss of effector function. Although T cell exhaustion can prevent immunopathology, autoreactivity, or T cell apoptosis in chronic antigen stimulation scenarios such as chronic viral infection, in the presence of a tumor, it can lead to immune evasion and tumor progression. Therefore, targeting inhibitory molecules such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein-1 (PD-1) and their ligands has become a cornerstone of cancer immunotherapy [2].

Using immune checkpoint inhibitors (ICI) against PD-1 has generated unprecedented response in treating many solid tumors such as colon cancer, melanoma non-small cell lung cancer, and renal cell carcinoma [3]. Despite the success of ICIs in treating “hot” tumors that are inflamed and have infiltrating T cells that can be mobilized with ICIs, many “cold” tumors such as glioblastoma, prostate cancer, and pancreatic cancer are refractory to anti-PD-1 treatment due to a dearth of cytotoxic T lymphocytes (CTLs) in the tumor. Even within “hot” tumors, anti-PD-1 therapy does not work for the majority of the patients, and those who respond initially are at risk of developing resistance to the therapy. This resistance is often accompanied by a concomitant increase in alternative checkpoint molecules such as LAG-3, TIM-3, VISTA, TIGIT, and B7-H3 in the tumor. As a result, our arsenal of ICIs is constantly expanding, with alternative checkpoints being targeted to prevent resistance and to stimulate T cell infiltration of “cold” tumors.

The multitude of studies exploring the blockade of these alternative checkpoints have revealed intricate interactions between these checkpoint molecules and non-CTLs including antigen-presenting cells (APCs), immunosuppressive cells, and T regulatory cells (Tregs). In this review, we will highlight the emerging alternative checkpoints and their effect on the lymphoid and myeloid compartments in cancer.

TIM-3 (T Cell Immunoglobulin Mucin-3)

TIM-3 or hepatitis A virus cellular receptor 2 (HAVCR2) is an immune checkpoint that is present on CD4+ T cells, CD8+ T cells, NK cells, macrophages, dendritic cells (DCs), and B cells. Originally identified on the type 1 helper T cell (Th1) subset of CD4+ T cells as a suppressor of effector function in an autoimmune mouse model, it is now appreciated as a potent checkpoint molecule capable of mediating exhaustion among tumor-infiltrating CD8+ T cells [4]. Numerous animal studies have shown the efficacy of targeting TIM-3 along with PD-1 to reverse T cell exhaustion and boost anti-tumor immunity [5, 6]. Analysis of immune cells from human tumors also corroborates the association between TIM-3 expression, T cell exhaustion, and poor prognosis [7-9]. Guided by these findings, there are many ongoing phase I and phase II clinical trials exploring dual TIM-3/PD-1 blockade in recurrent glioblastoma (NCT03961971), leukemia (NCT03066648), and advanced or metastatic solid tumors (AMBER, NCT02817633) as well as other tumors [10] (see Table 1).

Table 1

Summary of clinical trials evaluating alternate immune checkpoint targets presented in this review

Checkpoint Inhibitor	Trial	Treatment arms	Phase	Number of patients	Current status	Tumor
anti-TIM-3
	NCT03961971	+ Anti-PD-1 + stereotactic radiosurgery	I	15	Active, not recruiting	Recurrent GBM
	NCT03066648	Monotherapy	I	873	Active, recruiting	Leukemia
		+ Decitabine
		+ Anti-PD-1
		+ Decitabine + anti-PD-1
		Decitabine + anti-PD-1
	NCT02817633	Monotherapy	I		Active, recruiting	Advanced or metastatic solid tumors
		+ Anti-PD-1
		+ Anti-LAG-3
LAG-3Ig fusion protein
	Brignone et al. [40]	+ Paclitaxel (for breast cancer patients)	I/II	21	Complete	Advanced renal cell cancer and metastatic breast cancer
	Romano et al. [41]	+ MART-1 analog peptide vaccine	I	12	Complete	Advanced melanoma
	Wang-Gilliam et al. [42]	+ Gemcitabine	I	18	Complete	Advanced pancreatic adenocarcinoma
Anti-LAG-3	Ascierto et al. [32••]	Monotherapy	I/IIa	43	Complete	Advanced melanoma
		+ Anti-PD-1
anti-TIGIT
	NCT04294810	+ Anti-PD-L1	III	500	Active, recruiting	Non-small cell lung cancer
		Placebo + anti-PD-L1
	NCT04256421	+ Anti-PD-L1 + carboplatin + etoposide	III	400	Active, recruiting	Small cell lung cancer
		Placebo + anti-PD-L1 + carboplatin + etoposide
	NCT04047862	+ Anti-PD-1	I/Ib	39	Active, recruiting	Metastatic solid tumors
	NCT04150965	Monotherapy	I/II	104	Active, not yet recruiting	Multiple myeloma
		+ Pomalidomide + dexamethasone
		Anti-LAG-3
		Anti-LAG-3 + pomalidomide + dexamethasone
		Elotuzumab (anti-SLAM-7) + pomalidomide + dexamethasone
anti-VISTA
	NCT02812875	None	I	300	Active, not recruiting	Advanced solid tumors or lymphomas
anti-B7-H3
	NCT02923180	None	II	33	Active, not recruiting	Prostate cancer
	NCT02475213	+ Anti-PD-1 (pembrolizumab or MGA012)	I	145	Active, not recruiting	Melanoma, head and neck cancer, non-small cell lung cancer, urothelial carcinoma
	NCT03275402	None	II/III	32	Active, recruiting	Neuroblastoma, CNS metastases, leptomeningeal metastases

What makes TIM-3 a unique candidate for immunotherapy is that it has multiple ligands including galectin-9 (Gal-9), carcinoembryonic antigen cell adhesion molecule (CEACAM-1), phosphotidylserine (PS), and high-mobility group protein B1 (HMGB-1) [11-14]. The function of TIM-3 has been shown to vary depending on the specific receptor-ligand interaction (see Table 2). For example, TIM-3 can have both pro- and anti-inflammatory effects depending on which cell type it is present on and which ligand interacts with TIM-3. TIM-3 on DCs, when ligated to galectin-9, can promote an inflammatory Th1 response. However, TIM-3 on Th1 cells can downregulate Th1 immune response when ligated with galectin-9 [15]. Furthermore, the interaction of galectin-9 with TIM3+ CD8+ T cells in an immunosuppressive context such as tumor microenvironment can promote effector function [16]; however, this same interaction in the context of an autoimmune or hyperimmune environment can promote CD8+ T cell apoptosis [17].

Table 2

Summary of ligand-checkpoint interactions

Ligand	Ligand + cell	Checkpoint + cell	Interaction context	Interaction outcome
TIM-3
Gal-9
	?	Th1 cells	Hyperimmune conditions	Apoptosis of TIM3+ cell [11]
	Secreted by tumor	CD8+ T cells	Tumor	Apoptosis of TIM3+ cells [13]
	Recombinant protein	DCs	Tumor	TIM3+ DC activates CD8+ T cells [16]
HMGB1
	Extracellular protein	DCs	Tumor	Nucleic acids released from tumor and bound to HMGB1 cannot activate TLRs. Impaired antigen presentation [19]
	Extracellular protein	CD8+ Tregs in the liver	Hepatic necrosis during infection	CD8+ Tregs suppress activation of CD8+ T effector cells against infection [14]
PS	Apoptotic cells	CD8+ DCs	Apoptosis	CD8+ DCs phagocytose apoptotic cells and cross-present antigens to CD8+ T cells [20]
CEACAM-1	CD8+ T cells	CD8+ T cells	T cell homeostasis	CEACAM-1 can form heterodimers with TIM-3 in trans or in cis, potentiating TIM-3’s inhibitory role [12]
LAG-3
MHC II	APCs	CD4+ T cells	T cell homeostasis	LAG-3 bound to stable peptide-MHC II complex, suppressed T cell activation [22]
Gal-3	T cells and non-T cells in TME	CD8+ T cells	Tumor	Activation of tumor-specific T cells suppressed [23]
LSECtin	Tumor cells	CD8+ T cells	Tumor	LSECtin induced on melanoma cells can suppress T cell activation [24]
FGL-1	Secreted by some tumors	CD8+ T cells	Tumor	Activation of antigen specific T cells suppressed [25••]
MHC II	DC	Soluble LAG-3	DC homeostasis	Suppression of monocytic differentiation to DC [38] and promotion of DC maturation [36]
TIGIT
CD155/PVR	DCs	TIGIT-Fc fusion protein	DTH	DCs exhibit tolerogenic changes, T cell priming reduced [47]
CD122/PVRL2	Target cell for cytotoxicity	NK cells	Immunosurveillance	Inhibition of NK cytotoxicity [46]
VISTA
VSIG3	Immobilized VSIG3-IgG1Fc	T cells	T cell homeostasis	Activation and proliferation of T cells inhibited [62]
PSGL-1	CD4+ T cells	VISTA-Fc fusion protein	T cell homeostasis	Activation and proliferation of T cells inhibited [63]
VISTA (homophilic binding)	Apoptotic cells, phagocytes, T cells	Apoptotic cells, phagocytes, T cells	Phagocytosis/T cell homeostasis	Apoptotic cells phagocytosed; T cell activation suppressed [64]
B7-H3
?	?	Tumor cells, APCs	Tumor	B7-H3 blockade improve anti-tumor response [81–83]

Gal-9 galectin-9

HMGB1 high mobility group box 1

PS phosphatidylserine

CEACAM-1 carcinoembryonic antigen-related cell adhesion molecule 1

Gal-3 galectin-3

TME tumor microenvironment

LSECtin liver and lymph node sinusoidal endothelial cell C-type lectin

FGL-1 fibrinogen-like protein 1

PVR poliovirus receptor

DTH delayed-type hypersensitivity

PVRL2 poliovirus receptor-related 2

VSIG3 V-set and Ig domain-containing protein 3

PSGL-1 P-selectin glycoprotein ligand-1

Another ligand, HMGB1, illustrates the unique role of TIM-3 in suppressing antigen presentation. HMGB-1 is an alarmin produced by stressed and necrotic cells and is capable of binding immunogenic nucleic acids and recruiting them to DC endosomes for interaction with TLRs (Toll-like receptors) [18]. By binding HMGB1, TIM-3 can sequester it from activating TLRs. This reduces the immunogenicity of nucleic acids from dying tumor cells and plays a role in abrogating the benefit of DNA vaccines [19]. In contrast, phosphotidylserine is a ligand of TIM-3 exposed on the surface of apoptotic cells that stimulates TIM-3+ DCs to phagocytose the apoptotic cell and cross-present their antigens to T cells [20].

By having such diverse and opposing functions depending on context, TIM-3 illustrates the necessity of considering not only the protein level of checkpoints on immune cells to determine optimal immunotherapy regimen but also the tumor microenvironment as a whole. This includes the availability of ligands, the context in which the ligand interacts with the checkpoint, and the nature of ongoing antigen presentation. Through optimization of treatment schedule, or improvements in local delivery of immunotherapy to tumor or draining lymph node, or co-blockade of select ligands along with the checkpoint molecule, we can improve cancer treatments by maximizing anti-tumor function while minimizing pro-tumor effects of checkpoint molecules.

LAG-3 (Lymphocyte Activation Gene 3)

LAG-3 is an inhibitory molecule expressed on activated T cells, NK cells, Tregs, plasmacytoid dendritic cells, and B cells. It is structurally similar to CD4 protein and is embedded in the CD4 locus. It binds to MHC II with a greater affinity than CD4, leading to early speculations that LAG-3 inhibits T cell activation by sequestering MHC II from binding to CD4 protein [21]. However, more recent studies have shown that the interaction between the extracellular region of LAG-3 and MHC II is neither universal nor sufficient to prevent T cell activation [22]. The study found that LAG-3 can only bind stable complexes of peptide-MHC II, and the intracellular regions of LAG-3 are indispensable for transducing an inhibitory signal. This provided a key piece of evidence linking LAG-3 to the maintenance of tolerance to dominant antigens, indicating the co-opting of this tolerance mechanism for cancer evasion despite the presence of immunogenic tumor antigens.

Although MHC II was long considered the canonical ligand, recent evidence shows that galectin-3, LSECtin (liver sinusoidal endothelial cell lectin), and fibrinogen-like protein 1 (FGL-1) interact with LAG-3 and negatively regulate T cell function [23, 24, 25••](see Table 2). Due to its effect on T cell regulation, blocking LAG-3 has been tested in multiple mouse models of tumor and has shown significant synergy with PD-1 blockade [26-28]. In tumor-infiltrating T cells derived from patients, co-expression of LAG-3 and PD-1 was indicative of a greater exhaustion phenotype and worse prognosis than PD-1 expression alone [29-31]. As a result, many clinical trials exploring combination LAG-3 and PD-1 blockade are currently underway. Indeed, the combination of anti-PD-1 with BMS-986016, a monoclonal antibody against LAG-3, has generated exciting results demonstrating clinical benefits in melanoma patients initially refractory to anti-PD-1 therapy [32••]. This combination is now being tested in multiple phase I and II trials for both hematological and solid malignancies.

A unique aspect of LAG-3 protein is that its transmembrane form can be cleaved by the ADAM10 and ADAM17 metalloproteases to a soluble form (s-LAG-3), which has immunomodulatory roles of its own. S-LAG-3 is released from activated T cells [33] or plasmacytoid DCs [34], and blocking cleavage of LAG-3 prevents further proliferation and activation of T cells [35]. However, the complete role of s-LAG-3 remains elusive. Findings from Triebel and colleagues show opposing effects of soluble LAG-3 on APC homeostasis. On the one hand, LAG-3-Ig (fusion of extracellular LAG-3 with IgG1 Fc portion) can mediate phenotypic maturation of DCs [36] and be used as a vaccine adjuvant to induce humoral and cellular immune response to antigens [37]. Once mature, these DCs secrete inflammatory cytokines and lose their capacity to capture further soluble antigens. On the other hand, the same group also showed that s-LAG-3 can prevent the differentiation of monocyte precursors to macrophages and dendritic cells, indicating that the positive effect of s-LAG-3 on DC maturation depends on the presence of pre-existing DCs [38]. Clinically, the use of IMP321, a soluble LAG-3 Ig, has shown robust induction of APC and CD8+ T cell function in advanced renal cell carcinoma and breast cancer [39-41], but not in pancreatic ductal adenocarcinoma (PDAC) [42]. To examine the suitability of this therapy for a wider range of tumors including those that have a heavy presence of immunosuppressive myeloid cells such as glioblastoma [43] and PDAC [44•], it will be important to deconstruct the interplay between s-LAG-3, the presence of immunosuppressive myeloid precursors, and chronic antigen persistence.

TIGIT (T Cell Immunoreceptor with Ig and ITIM Domains)

TIGIT, also known as VSTM3 or WUCAM, is an inhibitory molecule found on T cells, Tregs, and NK cells. It has been shown to mediate T cell dysfunction and is co-expressed with PD-1, LAG-3, and TIM-3 [45]. The ligands for TIGIT are CD155 (PVR or poliovirus receptor) and CD122 (PVRL2) [46]. TIGIT demonstrates multiple mechanisms of signaling. It can signal extracellularly by acting as a ligand for CD155 which is found on DCs. CD155 then promotes tolerogenic behavior in DCs by promoting secretion of anti-inflammatory IL-10 and diminishing production of the p40 subunit of IL-12p40, thereby indirectly reducing T cell activation [47]. Another mechanism of TIGIT signaling involves interaction with CD155, which outcompetes the T cell cytotoxicity-promoting DNAM-1 protein from binding to CD155 [47, 48]. In doing so, TIGIT closely mimics the role of inhibitory CTLA-4 in preventing costimulatory CD28 from binding B7 ligand. Through its immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tail tyrosine-like (ITT-like) motif, TIGIT can also signal intracellularly to directly suppress T cell activation [49].

In murine tumor models, TIGIT is upregulated on tumor-infiltrating cells compared with cells from peripheral lymphoid organs [45, 50]. Among the tumor-infiltrating CD8+ T cells, TIGIT is upregulated on severely dysfunctional cells that have the highest expression of PD-1, TIM-3, and the transcription factor Eomes, which is associated with terminally exhausted T cells [45]. Findings from Kurtulus et al. indicate that TIGIT + Tregs could promote severed dysfunction of T cells and show that TIGIT expression on Tregs drives the suppression of an anti-tumor response [45]. This highlights the potential of TIGIT blockade as a Treg-targeting therapy.

The use of TIGIT blockade alone [50] or in combination with anti-PD-1 therapy [50, 52] has shown efficacy in murine models of tumor. In human samples, TIGIT is upregulated in tumor-infiltrating lymphocytes, and co-expression with PD-1 and LAG-3 is associated with severe CD8+ T cell dysfunction and poor prognosis [50, 53–55]. In vitro assays using T cells derived from patients with multiple myeloma [51•] or advanced melanoma [54] have shown improvement in effector function following TIGIT blockade alone or with anti-PD-1 therapy, respectively. TIGIT upregulation on Tregs has also been correlated with poor clinical outcome following anti-PD-1 and/or anti-CTLA-4 therapy [56]. Currently, there are clinical trials evaluating TIGIT blockade along with anti-PD-L1 therapy for non-small cell lung cancer (SKYCRAPER-01, NCT04294810), small cell lung cancer (SKYCRAPER-02, NCT04256421), or with anti-PD-1 therapy (NCT04047862) for metastatic solid tumors. Furthermore, newer trials in 2020 are exploring novel combinations; for example, a multiple myeloma trial is studying combination of TIGIT blockade with pomalidomide (NCT04150965). The results of these trials will be eagerly anticipated and can chart the role of TIGIT blockade in the management of other tumors as well.

VISTA (V-Set Immunoglobulin Domain Suppressor of T Cell Activation)

VISTA, also known as programmed death-1 homolog (PD-1H), is a checkpoint molecule recently discovered to be involved in peripheral tolerance and T cell inactivation [57-59]. In addition to expression on T cells, VISTA is also found on myeloid cells and non-immune cells such as tumor cells. Unlike most other inhibitory molecules, VISTA does not need to be present on T cells to cause dysfunction. VISTA expression on cancer cells or APCs can also impair T cell activity [60, 61], warranting investigations of possible binding partners for VISTA used by T cells. Studies suggest VSIG3 is a ligand for VISTA [62], co-inhibitory PSGL-1 is a VISTA-binding partner [63], and VISTA can have homophilic interaction with VISTA on other cells [64].

In mouse tumor models, overexpression of VISTA on cancer cells has been associated with rapid tumor growth and impaired survival [59]. VISTA can be expressed on patient-derived tumor cells as well, and high expression is associated with tumor progression [65] and lymph node metastasis [66] as well as T cell suppression [67]. Using VISTA blockade has shown pre-clinical anti-tumor efficacy [60, 67]. VISTA engagement in vitro has been shown to drastically reduce degranulation and cytokine production of tumor-infiltrating T cells isolated from pancreatic cancer patients [44]. What makes VISTA blockade more appealing is that its upregulation is implicated as a potential mechanism of acquired resistance in melanoma patients treated with anti-PD-1 [68•] and prostate cancer patients treated with anti-CTLA-4 therapy [69•]. Currently, CA-170, an orally available small molecule inhibitor of VISTA, is being tested in combination with PD-L1/PD-L2 blockade in a phase I trial in patients with advanced tumors and lymphomas unresponsive to available therapies (NCT02812875). An anti-VISTA monoclonal antibody (JNJ-61610588) was also being examined clinically, but this study has been prematurely terminated.

B7-H3

B7-H3 (CD276) is a member of the B7 family of proteins that includes other members such as CD80, CD86, PD-L1 (B7-H1), and VISTA. It is found on the surface of tumor cells, activated dendritic cells, and macrophages and allows them to modulate T cell response [70]. The receptor for B7-H3 has not been identified, and the role of B7-H3 signaling is controversial as it is reported to have both stimulatory [70, 71] and inhibitory effect on T cells [72, 73]. A number of murine studies have shown an anti-tumor response elicited by B7-H3 [74-76], but the expression of B7-H3 in human tumors correlates with poor prognosis, advanced pathological state, and metastasis in non-small cell lung cancer, pancreatic cancer, prostate cancer, and colorectal cancer [77-80]. The identification of counter receptors for B7-H3 on T cells could shed light on a seemingly context-dependent, contradictory role of B7-H3 on T cell homeostasis.

Despite the ambivalent results from correlative studies, blockade of B7-H3 with antagonistic antibodies has consistently demonstrated an anti-tumor response in mouse models [81-83]. While these studies indicate the blockade of B7-H3 on tumor cells or tumor-infiltrating DCs and macrophages enhance T cell function, there are non-immunological reasons to be hopeful about the efficacy of anti-B7-H3 therapy. B7-H3 on cancer cells has been shown to promote epithelial-mesenchymal transition (EMT) which can maintain stem cell-like properties in tumor cells, aid in metastasis, and confer chemotherapy resistance [84, 85]. Knockdown or blockade of B7-H3 expression reduces proliferative capacity of cancer cells, increases chemotherapy sensitivity, and compromises viability of cancer-initiating cells [86, 87]. B7-H3 is also a tumor endothelial marker that is highly expressed during pathological angiogenesis and not physiological angiogenesis and can constitute tumor vasculature of colorectal cancer, esophageal cancer, non-small-cell lung cancer, breast cancer, and bladder cancer [88]. Because of its broad applicability to generating an anti-tumor response, B7-H3 blockade has entered several clinical trials. Antibodies against B7-H3 are currently being tested on patients with prostate cancer (NCT02923180), melanoma, head and neck cancer, non-small cell lung cancer and urothelial carcinoma (NCT02475213), neuroblastoma (NCT03275402), and many other tumors.

Concluding Remarks

The list of promising alternative checkpoints is not limited to the ones discussed above. The roles of other immune checkpoints like BTLA, Siglec-15, and newer B7 family members are being increasingly appreciated in dampening immune response and mediating tumor progression. While this review did not provide an exhaustive list of checkpoints or their mechanisms of action, it sought to present some common themes that the study of alternative checkpoint molecules has highlighted over recent years. While cancer immunotherapy is often considered a T cell-centric approach because of the prevalence of these markers on T cells, further examination has revealed that the regulatory roles of checkpoints extend beyond just T cells. These checkpoints can control the fate and function of other residents of the tumor microenvironment as well as of secondary lymphoid organs―from antigen-presenting cells to immunosuppressive cells to Tregs. TIM-3 and LAG-3, in addition to attenuating CD8+ T cell function, are now known to boost suppressor activity in Tregs [89, 90]. Any therapeutic success from blocking these checkpoints could be attributed to inhibition of Tregs as much as to the alleviation of T cell exhaustion. Similarly, the efficacy of anti-TIM-3 or anti-TIGIT blockade might depend not only on the reversal of T cell exhaustion but also on the restoration of APC function and T cell priming, which could enhance T cell infiltration of “cold” tumors. Our understanding of the mechanism of action of the first generation of checkpoint molecules has also evolved. Recent work has shown PD-1 ablation on myeloid cells can have a greater anti-tumor effect than PD-1 ablation on T cells, by decreasing accumulation of myeloid precursors of MDSCs [91••]. Data from clinical trials have indicated that the efficacy of anti-PD-1 treatment might rely on recruitment of newly expanded clones of T cells into the tumor and that the pre-existing TILs have limited reinvigoration capacity [92••, 93]. While it is tempting to think of the above-mentioned alternative checkpoints solely in terms of their ability to shift PD-1+ T cell along the activation-exhaustion spectrum, it is equally important to consider their impact across the different immune compartments. Such a holistic understanding can allow physicians to tailor immunotherapy regimen to the distinct immune niche of a particular tumor in individual patients.