Cancer immunotherapy through checkpoint blockade: the future of cancer treatment

Johns Hopkins University School of Medicine
Baltimore, MD, USA

Cancer immunotherapy through checkpoint blockade: the future of cancer treatment

by D. Pardoll, USA

After decades of research and clinical trials aimed at harnessing a cancer patient’s immune system to attack their cancer, clinical efficacy with both vaccines and inhibitors of immune checkpoints has been demonstrated. The past few years have, therefore, become a turning point establishing active immunotherapy as a viable approach to cancer therapy. These advances have been fueled by basic molecular and cellular discoveries related to immune system activation, as well as study of the tumor microenvironment to identify resistance mechanisms that can be directly targeted. Future work will concentrate on targeting multiple pathways of immune regulation and developing rationally designed combinatorial approaches.

Medicographia. 2014;36:274-284 (see French abstract on page 284)

Without question, the major molecules to be successfully targeted in clinical cancer immunotherapy are the growing class of ligand-receptor pairs, commonly referred to as immune checkpoints. The notion of immune checkpoint blockade stems from many years of analysis of the immune microenvironment of cancer. These studies demonstrated upregulation within the tumor microenvironment of many inhibitory cytokines (eg, interleukin 10 [IL-10], transforming growth factor β [TGF-b]), ligands for inhibitory receptors on T cells (eg, programmed cell death 1 ligand 1 [PD-L1] and PD-L2) and metabolic enzymes (eg, indoleamine- pyrrole 2,3-dioxygenase [IDO] and inducible nitric oxide synthase [iNOS]) that consume amino acids essential for immune function or produce immune-inhibitory metabolites (Figure 1). A number of these inhibitory signals derive from inhibitory cell populations that accumulate in the tumor microenvironment, such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). In considering the mechanism(s) of action of inhibitors of various checkpoints, it is critical to appreciate the diversity of immune functions that they regulate. For example, the two immune checkpoint receptors that have been most actively studied in the context of clinical cancer immunotherapy, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4, also known as cluster of differentiation 152 [CD152]) and programmed cell death protein 1 (PD-1, also known as CD279), regulate immune responses at very different levels and by very different mechanisms (Figure 2). The clinical activity of blocking antibodies for each of these receptors implies that antitumor immunity can be enhanced at multiple levels, and that combinatorial strategies can be intelligently designed, guided by mechanistic considerations and preclinical models. This review will focus particular attention upon the CTLA-4 and PD-1 pathways, since they were the two checkpoints whose inhibition has revolutionized clinical cancer im- munotherapy. However, it is important to emphasize that multiple additional checkpoints represent promising targets for therapeutic blockade based on preclinical experiments, and inhibitors of many of these are under active development.

Figure 1
Figure 1. The immune microenvironment of a tumor
expresses multiple molecules that inhibit immune

Immune inhibition in the tumor microenvironment is mediated
by upregulation of ligands that bind inhibitory receptors on
helper T cells (TH) and cytotoxic T cells (CTC). Multiple immune
inhibitory cells, such as myeloid-derived suppressor cells
(MDSC) and regulatory T cells (Treg), as well as the tumor, express
these inhibitory ligands. Additionally, metabolic enzymes,
such as indoleamine-pyrrole 2,3-dioxygenase (IDO), consume
nutrients critical for effector T-cell function and produce products
inhibitory to T-cell activation. Expression of the molecules
is driven by signaling pathways, such as signal transducer and
activator of transcription 3 (STAT3), that are upregulated in
many tumor types. Most of these inhibitory molecules are
“druggable” with either small molecules or antibodies.
Abbreviations: A2aR, A2A adenosine receptor; CD4, cluster of
differentiation 4; CTLA-4, cytotoxic T lymphocyte–associated
antigen 4; DC, dendritic cells; IL, interleukin; LAG-3, lymphocyte-
activation gene 3; MΦ, macrophages; MEK, mitogen-activated
protein kinase (MAPK)/extracellular signal-related kinase
(ESK) kinase; Raf, rapidly accelerated fibrosarcoma; NK, natural
killer cells; PD-1, programmed cell death protein 1; TGF-β,
transforming growth factorβ ; VEGF, vascular endothelial
growth factor.

The CTLA-4 checkpoint—a global regulator of T-cell activation

CTLA-4, the first immune checkpoint receptor to be clinically targeted, is expressed exclusively on T cells, where it primarily regulates the amplitude of the early stages of T-cell activation. CTLA-4 knockout (KO) mice die within three weeks from immune destruction of multiple organs, which attests to its critical role as an inhibitory regulator of T cell–dependent immune responses. Primarily, CTLA-4 counteracts the activity of the T-cell costimulatory receptor CD28.1-3 CD28 does not affect T-cell activation unless the T-cell receptor (TCR) is first engaged by a cognate antigen. Once antigen recognition occurs, CD28 signaling strongly amplifies the TCR signal to activate T cells. CD28 and CTLA-4 share identical ligands, CD80 (B7.1) and CD86 (B7.2).4-8 Because CTLA-4 has a much higher overall affinity for both ligands, its expression on the surface of T cells dampens the activation of T cells by both outcompeting CD28 in binding CD80 and CD86, as well as actively delivering inhibitory signals to the T cell.9-14 The specific signaling pathways by which CTLA-4 blocks T-cell activation are still under investigation, although a number of studies suggest that activation of the phosphatases, Src homology region 2 (SHP2) and protein phosphatase 2A (PP2A) are important in counteracting kinase signals induced by TCR and CD28.2 However, CTLA-4 also confers “signaling-independent” T-cell inhibition through sequestrationof CD80 and CD86 from CD28 engagement, as well as active removal from the antigen presenting cell (APC) surface.15 The central role of CTLA-4 in maintaining T-cell activation in check is dramatically demonstrated by the systemic immune hyperactivation phenotype of CTLA-4 KO mice.16,17

Figure 2
Figure 2. Cytotoxic T lymphocyte–associated antigen
4 (CTLA-4) and programmed cell death protein 1
(PD-1) checkpoints act to regulate different elements
of the T-cell response to tumors.

Naïve T cells and resting T cells express little CTLA-4 or PD-1
on their surface. Upon initial T-cell activation via triggering of the
T-cell receptor (TCR) by cognate peptide/major histocompatibility
complex (MHC) complexes together with engagement of
cluster of differentiation 28 (CD28) by B7-1 and/or B7-2, CTLA-4
becomes expressed on the cell surface while the T cell is still
engaged with its antigen-presenting cell (APC; generally a
dendritic cell), usually in the secondary lymphoid tissue. The
greater the TCR stimulus, the more CTLA-4 is expressed on the
surface. Inhibitory signals from CTLA-4 interaction of CTLA-4
with B7-1 and B7-2 results in a counter-regulatory signal that
downmodulates the ultimate amplitude of T-cell activation.
Activated T cells then traffic into the tumor where the PD-1
pathway becomes important. The PD-1 checkpoint primarily
operates within the tumor where PD-1 ligands (PD-L1 and
sometimes PD-L2) are upregulated.
Abbreviation: Ag, antigen.

Even though CTLA-4 is expressed by activated CD8 killer T cells, the major physiologic role of CTLA-4 appears to be through distinct effects on the two major subsets of CD4 T cells; downmodulation of helper T cell (TH) activity and enhancement of Treg suppressive activity.1,18,19 CTLA-4 blockade results in a broad enhancement of immune responses dependent on TH and conversely, CTLA-4 engagement on Tregs enhances their suppressive function. CTLA-4 is a target gene of the transcription factor Foxp3,20,21 the expression of which determines the Treg lineage22,23 and Tregs therefore express CTLA-4 constitutively. While the mechanism by which CTLA-4 enhances the inhibitory function of Tregs is not known, Tregspecific CTLA-4 KO or blockade significantly inhibits their ability to regulate both autoimmunity and antitumor immunity.18,19 Thus, in considering the mechanism of action for CTLA-4 blockade, both enhancement of effector CD4 T-cell activity and inhibition of Treg-dependent immune suppression are likely important factors.

Clinical application of CTLA-4 blocking
Blockade of CTLA-4 as a general strategy was initially questioned because there is no tumor specificity to expression of the CTLA-4 ligands (other than certain myeloid and lymphoid tumors) and also because the dramatic lethal autoimmune/ hyperimmune phenotype of CTLA-4 KO mice predicted a high degree of immune toxicity associated with blockade of this receptor. However, Allison and colleagues used preclinical models to demonstrate that a therapeutic window was indeed achieved when CTLA-4 was partially blocked with antibodies.24 The initial studies demonstrated significant antitumor responses without overt immune toxicities when mice bearing partially immunogenic tumors, particularly melanomas, were treated with anti–CTLA-4 antibodies as single agents. Poorly immunogenic tumors did not respond to anti–CTLA-4 as a single agent, but did respond when anti–CTLA-4 was combined with a granulocyte-macrophage colony-stimulating factor (GMCSF)– transduced cellular vaccine.25 These findings suggested that, if there was an endogenous antitumor response present in the animals after tumor implantation, CTLA-4 blockade could enhance that endogenous response, which ultimately induced tumor regression. In the case of poorly immunogenic tumors, which do not induce significant endogenous responses, the combination of a vaccine and an anti–CTLA-4 antibody could induce a strong enough immune response to slow tumor growth, and in some cases, eliminate established tumors. These preclinical findings encouraged the production and testing of two fully human anti–CTLA-4 antibodies, ipilimumab and tremilimumab, which began clinical testing in 2000. As with virtually all anticancer agents, initial testing was as a single agent in patients with advanced disease, not responding to conventional therapy.26 Both antibodies produced objective clinical responses in roughly 10% of melanoma patients, but also immune-related toxicities involving various tissue sites in 25% to 30% of patients, with colitis being a particularly common event.27-29 The first randomized phase 3 clinical trial to be completed was for tremilimumab in patients with advanced melanoma. In the trial, 15mg/kg tremilimumab was given every three months as a single agent and compared with dacarbazine (DTIC), a standard melanoma chemotherapy treatment. The trial showed no survival benefit with this dose and schedule relative to DTIC.30

However, ipilimumab faired better. Even though the two antibodies appear to have similar intrinsic activity, response rates in phase 2 trials, and immune toxicity profiles, ipilimumab was more carefully evaluated at different doses and schedules. Additionally, more careful definition of algorithms for improved clinical management of the immune toxicities (using steroids and tumour necrosis factor (TNF)–blockers) mitigated the overall morbidity and mortality associated with immunologic toxicities.

The toxicity rate for ipilimumab is quite significant (14% to 30% grades 3-5 in various studies) and is generally immunologic in nature, implying that it is “on target”. This was predicted from the dramatic lethal hyperimmune/autoimmune phenotype of the CTLA-4 KO mice. The most common toxicities with both ipilimumab and tremilimumab are cutaneous (rash) and colitis. However, hepatitis, pneumonitis, hypophysitis, and thyroiditis are also observed. Interestingly, while there is evidence that clinical responses might be associated with immune-related adverse events, this correlation is modest.31

Finally, in a randomized three-arm clinical trial of patients with advanced melanoma that received either a melanoma-specific glycoprotein 100 (gp100) peptide vaccine alone, the gp100 vaccine plus ipilimumab, or ipilimumab alone, there was a 3.5 month survival benefit for patients in both groups receiving ipilimumab (ie, with or without the gp100 peptide vaccine), compared with the group receiving peptide vaccine alone.32 As the first therapy ever to demonstrate a survival benefit for patients with metastatic melanoma (DTICwas approved based on response rate and has never been shown to provide a survival benefit in melanoma) ipilimumab was Food and Drug Administration (FDA) approved for treatment of advanced melanoma in 2010.

More impressive than the mean survival benefit was the effect on long-term survival: 20% of the ipilimumab treated patients survived beyond two years (compared with 5% of patients receiving the peptide vaccine alone).32 In this and other studies, the proportion of long-term survivors is higher than the proportion of objective responders. The finding of ongoing responses and survival long after completion of a relatively short course of therapy (4 doses of 10mg/kg over 3 months) support the concept that immune-based therapies might reeducate the immune system to maintain tumors in check after completion of the therapeutic intervention.

As with all oncology agents that benefit a limited proportion of treated patients, there has been much effort in defining biomarkers predictive of clinical response to anti–CTLA-4 treatment. To date, no such pretreatment biomarker has been validated to the point where it could be applied as part of standard-of-care therapeutic decision-making, though insights have emerged from identification of certain posttreatment immune responses that seem to correlate with clinical outcome.33-35

An important feature of the anti–CTLA-4 clinical responses that distinguishes them from conventional chemotherapeutic agents and oncogene-targeted small molecule drugs is their kinetics. While chemotherapy and tyrosine kinase inhibitor (TKI) responses commonly occur within weeks of initial administration, the response to immune checkpoint blockers is slower and, in a number of patients, delayed (up to 6 months after treatment initiation). In some cases, metastatic lesions actually increase on computed tomography (CT) or magnetic resonance imaging (MRI) scans prior to regressing. These findings demand a reevaluation of response criteria for immunotherapeutics that does not use conventional time-toprogression or objective response evaluation criteria in solid tumors (RECIST), which were developed based on the experience with chemotherapy agents and as the primary measure of drug efficacy.36

Biology of the PD-1 checkpoint—a pathway that functions within the tumor microenvironment

Another immune checkpoint receptor, PD-1, is emerging as a promising target, emphasizing the diversity of potential molecularly defined immune manipulations capable of inducing antitumor responses by the patient’s own immune system. In contrast to CTLA-4, the major role of PD-1 is to limit the activity of T cells in the peripheral tissues at the time of an inflammatory response to infection, and to limit autoimmunity.37-43 This translates to a major immune resistance mechanism within the tumor microenvironment.44-46 PD-1 expression is induced when T cells become activated.38 When engaged by one of its ligands, PD-1 inhibits kinases involved in T-cell activation via the phosphatase SHP2,37 although additional signaling pathways are also likely induced, and because PD-1 engagement inhibits the TCR stop signal, this pathway could modify the duration of T cell/APC or T cell/target cell contact.47 Similar to CTLA-4, PD-1 is highly expressed on Tregs, where it may enhance their proliferation in the presence of ligand.48 Because many tumors are highly infiltrated with Tregs that likely further suppress effector responses, PD-1 pathway blockade may also enhance antitumor responses by diminishing the number and/or suppressive activity of intratumoral Tregs.

The two ligands for PD-1 are PD-L1 (B7-H1, CD274) and PDL2 (B7-DC, CD273).37,49-51 These B7 family members share 37% sequence homology and arose via gene duplication, positioning them within 100kB of each other in the genome.51 Recently, an unexpected molecular interaction between PD-L1 and CD80 was discovered,52 whereby CD80 expressed on T cells (and possibly APCs) can potentially behave as a receptor rather than a ligand, delivering inhibitory signals when engaged by B7-H1,53,54 the relevance of this interaction in tumor immune resistance has not yet been determined. Finally, genetic evidence from PD-1–deficient T cells suggests that both PD-L1 and CD80 may bind to a costimulatory receptor expressed on T cells.53 These complex binding interactions are reminiscent of the CD80/CD86 ligand pair, which binds the costimulatory CD28 expressed on resting T cells and the inhibitory CTLA-4 expressed on activated T cells, though, as stated above, PD-1 predominantly regulates effector T-cell activity within tissue and tumors while CTLA-4 predominantly regulates T-cell activation. Understanding the role of these various interactions in given cancer settings is highly relevant for selection of both antibodies and recombinant ligands for use in the clinic.

PD-1 is more broadly expressed than CTLA-4; it is induced on other activated non–T cell subsets, including B cells and NK cells,55,56 limiting their lytic activity. Thus, while PD-1 blockade is typically viewed as enhancing the activity of effector T cells in tissues and in the tumor microenvironment, it likely also enhances NK activity in tumors and tissues and may also enhance antibody production, either indirectly or through direct effects on PD-1 positive B cells.57

In addition, chronic antigen exposure, such as occurs with chronic viral infection and cancer, can lead to high levels of persistent PD-1 expression, which induces a state of exhaustion or anergy among cognate antigen-specific T cells. This state, which has been demonstrated in multiple murine and human chronic viral infections, appears to be partially reversible by PD-1 pathway blockade.58 Finally, while the PD-1 pathway plays its major role in limiting immune effector responses in tissues (and tumors), it can also shift the balance from T-cell activation to tolerance at early stages in T-cell responses to antigen within secondary lymphoid tissues (ie, at a similar point as CTLA-4). Taken together, these findings imply a complex set of mechanisms of action for PD-1 pathway blockade.

PD-1 is expressed on a large proportion of tumor-infiltrating lymphocytes (TILs) from many different tumor types.59,60 Some of the enhanced PD-1 expression among CD4 TILs reflects a generally high level of PD-1 on Tregs, which, as noted above, can represent a large fraction of intratumoral CD4 T cells. Increased PD-1 expression on CD8 TILs may either reflect an anergic/exhausted state, as has been suggested by decreased cytokine production by PD-1 positive vs PD-1 negative TILs from melanomas.59

Just as PD-1 is highly expressed on TILs from many cancers, the PD-1 ligands are commonly upregulated on many different human tumors.45,61 On solid tumors, the major PD-1 ligand to be expressed is PD-L1. Forced expression of PD-L1 on murine tumors inhibits local antitumor T-cell responses.45,62 Indeed, this combination of findings provides the basis for PD-1 pathway blockade to enhance antitumor effector function in the tumor microenvironment. As immunohistochemistry techniques and flow cytometry analysis of surface expression has been employed, it has become clear that the selective upregulation of PD-1 ligands in various human tumor types is heterogeneous at a number of levels.46 Expression patterns of PD-1 ligands may very well be critical in choosing suitability for therapeutic blockade of this pathway, since its primary role in cancer is thought to be immune inhibition within the tumor microenvironment, and PD-1 only inhibits lymphocyte function when it is engaged by cognate ligand.

Initially, the majority of melanoma, ovarian, and lung cancer samples were reported to have high expression of PD-L145,62,63 and subsequently, many other human cancers were reported to upregulate PD-L1. In addition to tumor cells, PD-L1 is commonly expressed on myeloid cells in the tumor microenvironment.64-66 An initial report in renal cancerdemonstrated that expression of PD-L1 on either tumor cells or infiltrating leukocytes in primary tumors predicted a worse prognosis, ie, decreased overall survival relative to PD-L1 negative tumors.67 Since that report, analyses of various tumors have suggested that PD-L1 status can either correlate with poor prognosis, better prognosis, or show no correlation with prognosis.46,68-72 Variability in immunohistochemistry technique, cancer type, stage of cancer analyzed (most analyses are of primary, not metastatic lesions), and treatment history in the analyzed cohort all likely contribute to the wide range of reported outcomes. While most of the analyses of PD-1 ligand expression has focused on PD-L1, PD-1 has also been reported to be upregulated on a number of tumors. It is highly upregulated on certain B-cell lymphomas such as primary mediastinal, follicular cell B-cell lymphoma, and Hodgkin’s disease.70 Upregulation in these lymphomas is commonly associated with gene amplification, or rearrangement to the class II transactivator (CIITA) locus, which is highly transcriptionally active in B-cell lymphomas.73

Given the heterogeneity of expression and potential relevance as a biomarker for blockade of the PD-1 pathway, it is important to understand the signals that induce expression of PD-1 ligands on tumor cells, and also hematopoietic cells, within the tumor microenvironment. Two general mechanisms for regulation of PD-L1 have emerged: innate and adaptive. For some tumors, such as glioblastoma, it has been demonstrated that PD-L1 is driven by constitutive oncogenic signaling pathways in the tumor cell. Expression on glioblastomas is enhanced upon deletion or silencing of phosphatase and tensin homolog (PTEN), implicating the phosphatidylinositol- 3-kinase (PI3K)–AKT pathway.74 Similarly, constitutive anaplastic lymphoma kinase (ALK) signaling, observed in certain lymphomas and occasionally in lung cancer, has been reported to drive PD-L1 expression via signal transducer and activator of transcription 3 (STAT3) signaling.75

The alternative mechanism for PD-L1 upregulation on tumors that has emerged from both clinical and preclinical studies reflects their adaptation to endogenous tumor-specific immune responses, a process termed adaptive resistance (Figure 3).46 In adaptive resistance, the tumor utilizes the natural physiology of the PD-1 ligand induction for tissue protection in the face of an immune response to infection in order to protect itself from an antitumor response. Expression of PD-L1 as an adaptive response to endogenous antitumor immunity can occur because it is induced on most cancers in response to interferons, predominantly ϒ-interferon, similar to what is observed in epithelial and stromal cells in normal tissues.76-78 This mechanism represents an alternative to the conventional drug resistance mechanisms that involve mutation of drug targets. It also contrasts with mechanisms of viral immune escape that involve mutation of immunodominant epitopes. The mechanism of adaptive resistance intrinsically implies that immune surveillance does exist even in some advanced cancers, but the tumor ultimately resists immune elimination by upregulating ligands for inhibitory receptors on tumor-specific lymphocytes that turn off antitumor responses within the tumor microenvironment.

A number of preclinical and clinical studies support the adaptive resistance hypothesis. Gajewski and colleagues have demonstrated that melanomas can be roughly divided into “inflammatory” and “noninflammatory” categories defined by expression of multiple inflammatory genes, including those involved in the interferon pathway.79 A recent study in melanoma demonstrated a very high correlation between cell surface PD-L1 expression on tumor cells and both lymphocytic infiltration and intratumoral g-interferon expression. This correlation was not only seen among tumors, but within individual PD-L1+ tumors at the regional level, in which regions of lymphocyte infiltration were exactly the regions where PD-L1 was expressed, on both tumor cells and infiltrating leukocytes.46

Evidence of clinical activity for PD-1 blockade
Taken together, the general findings of increased PD-1 expression by TIL and increased PD-1 ligand expression by tumor cells created an important rationale for the capacity of antibody blockade of this pathway to enhance intratumoral immune responses. This was validated through many murine tumor studies demonstrating enhanced antitumor immunity through antibody blockade of PD-1 or its ligands (see above). Furthermore, the relatively mild phenotypes of PD-1, PD-L1, and PD-L2 KO mice suggest that blockade of this pathway would result in less collateral immune toxicity than CTLA-4 blockade, a finding that appears to be the case in clinical trials.

Figure 3
Figure 3. Two mechanisms for programmed cell death 1 ligand 1 (PD-L1) induction
on tumors: innate and adaptive.

PD-L1 can be constitutively expressed on tumors as a consequence of oncogene driven transcriptional
activation. Alternatively, PD-L1 can be induced in an adaptive fashion when there are the right
inflammatory cytokines in the tumor microenvironment consequent to an ongoing immune response
to the tumor. This mechanism of tumor resistance to immune attack is co-opted from physiologic
PD-L1 expression for tissue protection in the setting of antimicrobial immune responses. Innate and
adaptive mechanisms of PD-L1 expression on tumors can coexist. The adaptive resistance mechanism
implies that PD-L1 expression is a “marker” of endogenous antitumor immunity.
Abbreviation: TCR, T-cell receptor.

While the clinical experience with anti–PD-1 antibodies is less extensive than with anti-CTLA antibodies at this time, results look extremely promising. In the first phase 1 clinical trial with a fully human immunoglobulin G4 (IgG4) anti–PD-1 antibody, there were a number of cases of tumor regression, including mixed responses, partial responses, as well as a complete response.80 Tumor regressions were observed in four of the five histologies examined (melanoma and colon, renal, and lung cancer) and were associated with significant increases in lymphocyte infiltration into metastatic tumor deposits. Results from a second, larger clinical trial, sponsored by Bristol-Myers Squibb (BMS) and extending the treatment with anti–PD-1 (named nivolumab) to 2 years, demonstrated objective responses observed in 31% of patients with advanced melanoma, with an additional 7% achieving disease stabilization for >6 months. Similar response rates were observed in renal cancer, with an additional 27% with disease stabilization for >6 months. Most surprisingly, there was an 18% response rate in non–small cell lung cancer (NSCLC), with additional 7% disease stabilization >6 months. Efficacy against melanoma, renal cancer, and lung cancer was also observed with an anti–PD-L1 antibody.81

Among 270 nivolumab-treated patients with lung, melanoma, or kidney cancer, one-/two-year landmark survival rates were 42%/14% for lung cancer, 62%/43% for melanoma, and 70%/ 50% for kidney cancer. Median overall survival in these heavily- pretreated patients (47% with 3 to 5 prior systemic therapies) was 9.6, 16.8, and >22 months, respectively. Among all responders, median response duration was 74, 104, and 56 weeks, respectively. Among responders who discontinued therapy for reasons other than disease and followed for at least 4 months (range 4-14 months), 70% retained their response.82

As predicted by the distinct phenotypes of the PD-1 KO vs CTLA-4 KO mice, the frequency of immune-related toxicities from anti–PD-1 treatment appears to be less than with anti–CTLA-4. Grade 3/4 drug-related toxicity was <15% and was also largely immune related. In contrast to anti–CTLA-4, the most significant toxicity was pneumonitis, which produced a 1% mortality rate. Recently instituted protocols to manage pneumonitis with steroids and, when necessary, anti-TNF–blocking antibodies appear to mitigate lung toxicity. It is logical to imagine that the enhancement of antitumor immune responses upon blockade of this pathway would depend, in significant part, on expression of a ligand for PD-1 within the tumor. Analysis of 42 patients treated with anti–PD-1 in the trial described above demonstrated a strong correlation between PD-L1 expression and response. None of the 17 patients with no membrane PD-L1 expression on pretreatment biopsies responded to anti–PD-1, whereas 44% patients with >5% of tumor cells expressing membrane PD-L1 displayed either an objective or mixed response.83 The lack of response in patients whose tumors exclusively expressed cytosolic PD-L1 was also notable, as cytosolic PD-L1 would fail to activate the PD-1 pathway.

If validated in a larger series, this finding sets the stage for a broader assessment of immune checkpoint ligands and receptors as targets for antibody blockade, as well as assessment of ligand expression in the tumor as a biomarker for success in blockade of a specific checkpoint pathway.

Figure 4
Figure 4. Multiple costimulatory and coinhibitory
ligand-receptor interactions ultimately
determine the amplitude of T-cell activation
and the potency of effector T-cell responses
in tissue and tumor.

B7 family ligands and CD28 family receptors are shown
in purple, and tumor necrosis factor (TNF)/TNF receptor
(TNFR) family ligand-receptor pairs are shown in
blue. There are additional inhibitory ligand-receptor pairs
that do not fit into either of these families. Some of the
receptors for B7 family members are not yet discovered.
While TNF/TNFR interactions are usually one-onone
pairs, B7 family ligands often interact with multiple
receptors. Herpes virus entry mediator (HVEM) is a
TNFR family member; in addition to its interaction with
the TNF member LIGHT, it also interacts with the inhibitory
receptor B and T lymphocyte attenuator (BTLA),
which is a member of the cluster of differentiation 28
(CD28) family. Additional signals of activation or inhibition
are contributed by cytokines.
Abbreviations: A2aR, A2A adenosine receptor; APC,
antigen-presenting cell; CTLA-4, cytotoxic T lymphocyte–
associated antigen 4; ICOS, inducible costimulator;
IL, interleukin; KIR, killer inhibitory receptors; LAG-3,
lymphocyte-activation gene 3; MHC, major histocompatibility
complex; PD-1, programmed cell death
protein 1; TCR, T-cell receptor; TGF-β, transforming
growth factor β; Tim-3, T-cell immunoglobulin domain
and mucin domain 3.

There are a number of companies developing and testing antibodies that block the PD-1 pathway; a recent study with a different anti–PD-1 antibody produced by Merck (named lambrolizumab) demonstrated a 38% response rate in melanoma84 and an anti–PD-L1 antibody produced by Genentech gave similar response rates in melanoma and NSCLC (but a somewhat lower response rate in kidney cancer) to the BMS anti– PD-1 antibody.85 These results validate the PD-1 pathway as an important target for immunotherapeutic targeting. Based on the known interactions between the PD-1 ligands, it is theoretically possible that a PD-1 antibody would have distinct biologic activity from an anti–PD-L1 antibody; an anti–PD-1 antibody would block PD-1 interaction with both PD-L1 and PD-L2, but not the interaction between PD-L1 and CD80. Most anti–PD-L1 antibodies block the interaction between PD-L1 and CD80 and between PD-L1 and PD-1, but would not block PD-1 interaction with PD-L2. Thus, it is possible that, depending on which interactions dominate in a particular cancer, PD-1 and PD-L1 antibodies might not have redundant activity.

Based on the distinct roles of CTLA-4 and PD-1 in regulating distinct components of the immune response, it was postulated that combined blockade of these pathways might provide an additive or synergistic antitumor effect. Indeed, a recent study demonstrated a 41% response rate in melanoma patients treated concurrently with ipilimumab and nivolumab. A larger proportion of the responses were “deep” (>80%) with ipilimumab and nivolumab than observed with nivolumab alone, and there were ≈20% additional mixed responses and stable disease >6 months. However, toxicity was also greater than with nivolumab alone, with a 53% grade 3/4 toxicity rate.86

While the ultimate long-term clinical benefit of this combination remains to be determined, the study emphases the potential for combinatorial blockade of multiple checkpoints.

Additional checkpoints participate in tumor immune resistance and tolerance

Successful clinical outcomes of CTLA-4 and PD-1 pathway targeting have garnered great interest in a number of additional checkpoints. Basic immunologic studies have demonstrated that a number of checkpoint receptors (Figure 4) are expressed coordinately under circumstances of tolerance to self-antigens and chronic infections, as well as in inflammatory settings.

In addition to defined lymphocyte inhibitory receptors, a number of B7-family inhibitory ligands—in particular B7-H3 (CD276) and B7-H4—do not yet have defined receptors, but murine KO experiments support an inhibitory role for both these molecules.87 In addition, they are upregulated on tumor cells or tumor-infiltrating cells.88 B7-H3 appears to be upregulated on endothelial cells of the tumor vasculature and B7-H4 has been reported to be expressed on tumor-associated macrophages.87 Preclinical tumor models have been used to demonstrate that blockade of many of these individual immune checkpoint ligands or receptors can enhance antitumor immunity and dual blockade of coordinately expressed receptors can produce additive or synergistic antitumor activity. Inhibitors for a number of these immune checkpoint targets are either entering the clinic or are under active development. Those described below are targets with currently available blocking antibodies or small molecule inhibitors, but do not represent a comprehensive list.

Lymphocyte-activation gene 3 (LAG-3orCD223), 2B4 (CD244), B and T lymphocyte attenuator (BTLA or CD272), T-cell immunoglobulin domain and mucin domain 3 (Tim-3), A2A adenosine receptor (A2aR), and the family of killer inhibitory receptors have each been associated with inhibition of lymphocyte activity and in some cases induction of lymphocyte anergy. Antibody targeting of these receptors, either alone or in combination with a second immune checkpoint blocker has been shown to enhance antitumor immunity in animal models of cancer. Because many tumors express multiple inhibitory ligands, and TIL express multiple inhibitory receptors, there are many opportunities to enhance antitumor immunity via dual or triple blockade of immune checkpoints. While human blocking antibodies specific for a number of these “second generation” inhibitory receptors are under development, none have entered the clinic at this time. Most of these receptors are induced upon T-cell activation, in keeping with the biologic theme that they play roles in feedback inhibition of T-cell responses when their cognate ligands are present. In addition to providing inhibitory signals to activated effector T cells, some of these receptors, such as LAG-3, are highly expressed on Tregs, where they are important to amplify their inhibitory activity.89 This implies that, as with CTLA-4 and PD-1, these receptors play a dual role in ultimately inhibiting effector immune responses and blocking antibodies, therefore have multiple potential mechanisms of action.

LAG-3 was cloned over 20 years ago as a CD4 homologue,90 but its function in the immune checkpoint was only defined in 2005, when it was shown to play a role in enhancing Treg function.89,91 LAG-3 also inhibits CD8 effector function independently of its role on Tregs.92 The only known ligand for LAG-3 is major histocompatibility complex class II molecule (MHCII), which is upregulated on some epithelial cancers (generally in response to g-interferon), but is also expressed on tumor-infiltrating macrophages and dendritic cells. The role of the LAG-3/ MHCII interaction in LAG-3 mediated inhibition of T-cell responses is unclear, since anti-LAG-3 antibodies that do not block the LAG-3/MHCII interaction nonetheless enhance T-cell proliferation and effector function in vitro and in vivo. The MHCII interaction of LAG-3 may be most important for its role in enhancing Treg function. LAG-3 is one of a number of immune checkpoint receptors coordinately upregulated on both Tregs and anergic T cells, and simultaneous blockade can result in enhanced reversal of this anergic state relative to blockade of either receptor. In particular, PD-1 and LAG-3 are commonly coexpressed on anergic or exhausted T cells.93,94 Dual blockade of LAG-3 and PD-1 provide synergy in reversing anergy among tumor-specific CD8Tcells, as well as virus-specificCD8 T cells, in the setting of chronic infection. Dramatic evidence of the effects of coordinate T-cell inhibition by PD-1 and LAG-3 comes from PD-1/LAG-3 double KO mice, which completely reject even poorly immunogenic tumors in a T-cell–dependent fashion, but also develop autoimmune syndromes much more quickly than PD-1 or LAG-3 single knockouts, which are ultimately fatal (though not as quickly as CTLA-4 KOs).95 These findings emphasize the balance between antitumor effects and autoimmune side effects that must be taken into consideration in all of the immune checkpoint blockade strategies.

Tim-3, the ligand of which is galectin-9 (a galectin reported to be upregulated in a number of cancer types, such as breast cancer) inhibits type 1 TH–cell (TH1) responses96 and anti– Tim-3 antibodies enhance antitumor immunity.97 Tim-3 has also been reported to be coexpressed with PD-1 on tumor specific CD8 T cells and dual blockade of both molecules significantly enhances the in vitro proliferation and cytokine production of human T cells when stimulated by the NY-ESO-1 cancer-testis antigen. In animal models, coordinate blockade of PD-1 and Tim-3 was reported to enhance antitumor responses and tumor rejection under circumstances where only modest effects from blockade of each individual molecule were observed.98-100

BTLA was first identified as an inhibitory receptor on T cells based on enhanced T-cell responses observed in the BTLA KO mice.101 Subsequently, herpes virus entry mediator (HVEM), which is expressed on certain tumor cell types (ie, melanoma), as well as on tumor-associated endothelial cells, was demonstrated to be the BTLA ligand.102 This is a rare case in which a TNF family member interacts with an immunoglobulin supergene family member. BTLA expression on activated virusspecific CD8 T cells is relatively low, but it has been demonstrated to be much more highly expressed on tumor-infiltrating lymphocytes from melanoma patients. BTLAhi T cells are inhibited in the presence of its ligand, HVEM. Thus, BTLA may also be a relevant inhibitory receptor for T cells in the tumor microenvironment.103 The system of HVEM interacting molecules is complex; two additional interacting molecules, CD160 (an immunoglobulin superfamily member) and LIGHT (a TNF family member) appear to mediate inhibitory and costimulatory activity respectively. It also appears that signaling can be bidirectional, depending on the specific combination of interactions. The complexity of this system makes therapeutic inhibition strategies less straightforward than other inhibitory receptors or ligands, though dual blockade of BTLA and PD-1 clearly enhances antitumor immunity.104

A2aR inhibits T-cell responses, in part by driving CD4 T cells to express Foxp3 and develop into Tregs.105 KO of this receptor results in enhanced and sometimes pathologic inflammatory responses to infection. This receptor is particularly relevant in tumor immunity, because the rate of cell death in tumors from cell turnover is high and dying cells release adenosine. In addition, Tregs express high levels of the exoenzymes CD39, which converts extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP), and CD73, which converts AMP to adenosine.106 Given that A2aR engagement by adenosine drives T cells to become Tregs, this can produce a self-amplifying loop within the tumor. Indeed, tumors grow more slowly in A2aR KO mice, and tumor vaccines are much more effective against established tumors in these mice.107 A2aR can be inhibited either by antibodies that block adenosine binding or by adenosine analogues, some of which are fairly specific for A2aR. While these drugs have been used in clinical trials for Parkinson’s disease, they have not yet been tested clinically in cancer patients.

Killer inhibitory receptors are a broad category of inhibitory receptors that can be divided into two classes based on structure: killer immunoglobulin receptors (KIR) and C-type lectin receptors, which are type II membrane receptors.108-110 These receptors were originally described as critical regulators of the killing activity of NK cells, though many are expressed on T cells and APCs.111 The importance of their inhibitory role on T cells and APCs (ie, dendritic cells) is less well studied, but the resulting activation of NK cells can provide potent antitumor activity. Many of the killer inhibitory receptors are specific for subsets of human leukocyte antigen (HLA) molecules and possess allele-specificity. However, other receptors recognize broadly expressed molecules, for example, the C-type killer cell lectin-like receptor G1 (KLRG1) recognizes e-cadherin. The potential value of NK cells in antitumor responses when their inhibitory receptors are not appropriately engaged is best exemplified by the significantly enhanced graft-vs-tumor effects in allogeneic bone marrow transplants, elicited by mismatches between donor NK inhibitory receptors and recipient HLA alleles. The big question in therapeutic blockade of NK inhibitory receptors is this: among >20 receptors, which should be targeted? ■

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Keywords: BTLA; cancer; checkpoint; CTLA-4; immunotherapy; interleukin; LAG-3; PD-1; Tim-3