Immunologic Checkpoint Blockade in Lung Cancer

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Immunologic Checkpoint Blockade in Lung Cancer Martin Reck MD, PhD, Luis Paz-Ares MD, PhD

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S0093-7754(15)00029-9 http://dx.doi.org/10.1053/j.seminoncol.2015.02.013 YSONC51816

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Semin Oncol

Cite this article as: Martin Reck MD, PhD, Luis Paz-Ares MD, PhD, Immunologic Checkpoint Blockade in Lung Cancer, Semin Oncol, http://dx.doi.org/10.1053/j. seminoncol.2015.02.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Immunologic Checkpoint Blockade in Lung Cancer Martin Reck, MD, PhDa and Luis Paz-Ares, MD, PhDb

a

Department of Thoracic Oncology, LungenClinic Grosshansdorf, Airway Research Center

North (ARCN), member of the German center for Lung research (DZL), Grosshansdorf, Germany; bDepartment of Medical Oncology, Instituto de Biomedicina de Sevilla – IBIS (Hospital Universitario Virgen del Rocio, Universidad de Sevilla and CSIC), Seville, Spain.

Corresponding author: Martin Reck Department of Thoracic Oncology, LungenClinic Grosshansdorf Wöhrendamm 80, 22927 Grosshansdorf, Germany Email: [email protected] Tel.: +49 4102 601 2101; fax: +49 4102 601 7101

Acknowledgments: The authors take full responsibility for the content of this publication, and confirm that it reflects their collective viewpoint and medical expertise. StemScientific, funded by Bristol-Myers Squibb, provided writing and editing support. Bristol-Myers Squibb did not influence the content of the manuscript, nor did the authors receive financial compensation for authoring the manuscript.

Conflicts of interest: Martin Reck has received honoraria for the participation in advisory boards and for lectures from Hoffmann-La Roche, Lilly, Boehringer-Ingelheim, Pfizer,

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AstraZeneca, Bristol-Myers Squibb, and Novartis. Luis Paz-Ares has received honoraria for the participation in advisory boards and for lectures from Hoffmann-La Roche, Lilly, Boehringer-Ingelheim, Pfizer, Bristol-Myers Squibb, and MSD.

Journal: Seminars in Oncology

Keywords: immunotherapy, lung cancer, NSCLC, immune checkpoint blockade, CTLA-4, PD-1, PD-L1

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ABSTRACT Despite the availability of radiotherapy, cytotoxic agents, and targeted agents, a high unmet medical need remains for novel therapies that improve treatment outcomes in patients with lung cancer who are ineligible for surgical resection. Building upon the early promise shown with general immuno-stimulatory agents, immuno-oncology is at the forefront of research in this field, with several novel agents currently under investigation. In particular, agents targeting immune checkpoints, such as the cytotoxic T-lymphocyte antigen-4 receptor and programmed death-1 receptor, have shown in early clinical trials potential for improving tumor responses and survival in patients with non-small cell lung cancer. Here, we examine the rationale for targeting immune checkpoints in lung cancer and review the clinical data from studies with immune checkpoint inhibitors currently in development. The challenges associated with optimizing treatment with these agents in lung cancer are also discussed.

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INTRODUCTION Current treatment approaches for patients with lung cancer offer limited clinical benefit except in a subpopulation of patients with treatable oncogenic driver mutations. Thus, despite ongoing research and drug development programs, there is still a high unmet need in this setting for novel, alternative therapies that improve treatment outcomes. At the forefront of clinical research in this field are several immuno-oncology agents, of which immune checkpoint targeted agents are the most advanced in clinical development. The aim of the present review is to provide an overview of the unmet clinical need in this setting, a brief summary of the rationale for targeting immune checkpoints in lung cancer, and an update on immune checkpoint inhibitors currently in development. The potential role of these agents in the future treatment of lung cancer will also be discussed.

THE MAGNITUDE OF THE UNMET NEED IN LUNG CANCER Lung cancer remains the leading cause of cancer-related deaths worldwide, with 1.8 million new cases diagnosed every year (approximately 13% of all new cancer cases).1,2 Due to the absence of suitable screening methods and a lack of characteristic clinical symptoms, diagnosis of lung cancer often only occurs at an advanced or metastatic stage, leading to a poor prognosis. Indeed, the overall 5-year survival rate for patients with nonsmall cell lung cancer (NSCLC) is 13% in Europe and 16% in the US.3,4 Worldwide statistics indicate that 5-year survival is 50–73% for patients with Stage IA NSCLC, 43– 58% for Stage IB, 36–46% for Stage IIA, 25–36% for Stage IIB, 19–24% for Stage IIIA, 7– 9% for Stage IIIB, and 2–13% for Stage IV NSCLC.5 Successful treatment options for lung cancer are limited. Surgical resection is usually only indicated for early-stage (Stage I–II and resectable Stage III) NSCLC, curing around 20–40% of these cases.6 Other options include radiotherapy and cytotoxic agents. In recent years, substantial progress has been

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made by the identification of treatable oncogenic alterations and the development of agents specifically targeting these alterations. However, the number of patients with mutations that can be treated and who have access to targeted therapies still remains approximately 20%.7 Thus, novel treatment approaches urgently need to be investigated.

THE IMMUNOGENICITY OF LUNG CANCER Until recently, therapeutic modalities in lung cancer directly targeted the tumor. However, immuno-oncology, which acts to harness the host immune system to recognize nascent tumors as non-self, thereby restoring an effective anti-cancer response, is currently being investigated as an alternative strategy.8 Indeed, there is a growing body of evidence that identifies lung cancer as a suitable candidate for immuno-oncology based treatment strategies.9 Increased tumor-infiltrating lymphocytes (TILs), such as cytotoxic T lymphocytes (CTLs), T helper cells, natural killer (NK) cells, and dendritic cells (DCs), are associated with improved survival in NSCLC.10–15 Furthermore, a high effector Tcell:regulatory T cell (T-reg) ratio is associated with improved long-term survival,16 while increased immunosuppressive T-regs as a proportion of total TILs, is associated with poorer survival in lung cancer.10,17,18 Lung tumors also increase recruitment of immunosuppressive cells, such as T-regs and myeloid-derived suppressor cells,19,20 and induce aberrant expansion of T-regs, which inhibit CTL and NK cell activity.21 Additionally, lung tumors may release inhibitory cytokines, interleukins, prostaglandins, and growth factors that down-regulate T-cell response.17,22–26 Finally, there is evidence that lung cancer can evade an immune response. For instance, major histocompatibility complex (MHC) class I expression is reduced in NSCLC,27 and lung tumors may also be able to escape routine antigen processing.28

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General immuno-stimulatory agents, such as interleukin 2 (which stimulates CTL proliferation), and early vaccines have failed to demonstrate activity in lung cancer, achieving a durable benefit in only a limited subset of patients, delivering a modest survival benefit, and being associated with a considerable toxicity burden.29–31 This led to the belief that lung cancer is a poorly immunogenic disease. However, novel and improved immunotherapeutic agents, including a range of vaccines and immuno-oncology agents, are currently in development, and although results are not yet available from Phase 3 clinical trial programs, data from early studies suggest that improved efficacy might be possible. Antigen-specific (such as anti-MAGE-A3), tumor cell-specific (such as belagenpumatucelL), or dendritic cell-based vaccines have shown some clinical benefits in subsets of patients,32–34 although they have generally failed to live up to their potential in patients with lung cancer, as is evident in a recent Phase 3 trial with the anti-MAGE-A3 vaccine.35 Advanced immuno-oncology agents include the immune checkpoint inhibitors, which are currently a promising class of immunotherapies in development for lung cancer. This evolving treatment modality targets and harnesses the potential of the patient’s own immune system to generate an effective immune response against cancer. This approach differs from ‘traditional’ treatment approaches, which target the tumor or tumor blood supply, and earlier immunotherapies, which were broadly immuno-stimulatory and were often associated with difficult to manage safety profiles. The clinical utility of this approach has been demonstrated with several checkpoint inhibitors in other tumor types. For example, ipilimumab was the first immune checkpoint inhibitor to be approved for the treatment of patients with advanced malignant melanoma in 2011.

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WHICH IMMUNE CHECKPOINTS ARE TARGETS IN LUNG CANCER? Immune checkpoints refer to a range of inhibitory pathways in the immune system that are crucial for maintaining self-tolerance and preventing excessive, prolonged, and potentially deleterious T-cell activity in peripheral tissues.36 There is growing evidence that lung cancer can utilize these immune checkpoints to circumvent the anti-tumor immune response. Examples of immune checkpoints known to be involved in lung cancer are: the cytotoxic T-lymphocyte antigen-4 (CTLA-4) receptor, the programmed death-1 (PD-1) receptor, the killer-cell immunoglobulin-like receptor (KIR), and lymphocyte-activation gene-3 (LAG-3) (Figure 1).

The CTLA-4 receptor CTLA-4 is a critical negative checkpoint molecule that controls the activation and proliferation of T cells. Binding of the CTLA-4 receptor to CD80/86 expressed on antigen presenting cells (APCs) has a coinhibitory effect on T cells. By competing with the CD28 co-stimulatory receptor for the same ligands, albeit with a higher binding affinity than CD28, CTLA-4 inhibits T-cell activation. As a CD28 CD80/86 interaction is required for optimal T-lymphocyte activation and proliferation, expression of CTLA-4 on T cells leads to an anergic phenotype.37 T-regs can also upregulate CTLA-4 expression, influencing Tcell activity further.38 In NSCLC, expression of surface and intracellular CTLA-4 is increased in T cells.39

The PD-1 signaling pathway Like CTLA-4, the PD-1 receptor is also expressed by activated T cells. However, it is additionally expressed on activated T-regs, B cells, and NK cells. The PD-1 receptor engages with the ligands PD-L1 and PD-L2, which are shared by the co-inhibitory receptor

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CD80.40 Docking of CD80 causes downregulation of downstream pathways, leading to Tcell activation and cytokine release. Consequently, by inhibiting this signaling pathway, the PD-1/PD-L1 interaction induces T-cell tolerance.40–42 It is assumed that docking with PD-L2 has the same effect, although this process is poorly understood.43 A wide variety of solid tumors overexpress PD-L1. In particular, around 50% of NSCLC tissue samples overexpress PD-L1, which is associated with poorer prognosis44–46 and reduced immune cell infiltration into tumors.47 Although less well investigated, PD-L2 is also expressed by various tumor cells, including lymphomas, and there is contradictory evidence that expression of PD-L2 in lung adenocarcinomas is a predictor of overall survival (OS).48–50

KIR and LAG-3 Although agents targeting the CTLA-4 and PD-1/PD-L1 pathways are approved or clinically advanced for some tumor types, other immune checkpoints remain potential therapeutic targets. Normally, KIR is expressed on the surface of NK cells, and is involved in the downregulation of NK cell immunological activity, thereby ensuring tolerance to selftissues that express MHC class I. Inhibitory KIR variants are selectively expressed by tumors.51 In NSCLC, expression of KIR variants has been shown to be associated with treatment response and survival.52 LAG-3 is another receptor involved in immune checkpoint regulation. It is coexpressed with PD-1 on tolerant TILs, and is also expressed on T-regs, acting to suppress APC activation by binding with MHC class II.53 Initial investigations suggest that LAG-3 not only inhibits T-cell activity, but also modulates T-cell activation.54,55 In preclinical studies, inhibition of LAG-3 slowed the growth of established tumors.56

TARGETING IMMUNE CHECKPOINTS IN LUNG CANCER

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Immuno-oncology agents targeting the CTLA-4 receptor Two antibodies that target the CTLA-4 receptor are currently being investigated in patients with lung cancer: ipilimumab and tremelimumab.

Ipilimumab Ipilimumab is a human IgG1 monoclonal antibody targeting CTLA-4 that is currently approved for the treatment of advanced (unresectable or metastatic) melanoma. Treatment with ipilimumab consistently improves OS in patients with advanced melanoma.57–62 Indeed, 3-year OS was 22% in a recent pooled analysis of 1,861 patients with advanced melanoma treated with ipilimumab across 12 different trials, with a durable survival plateau after 2–3 years. The durability of this long-term survival was supported by follow-up of up to 10 years in some patients.57 Ipilimumab has also shown promise in NSCLC. In a multicenter, randomized, placebo-controlled Phase 2 trial, ipilimumab (10 mg/kg) plus carboplatin (area under the curve, 6)/paclitaxel (175 mg/m2) was compared with carboplatin/paclitaxel alone as firstline treatment for advanced NSCLC and small cell lung cancer (SCLC).63 Ipilimumab was given alongside chemotherapy in either a phased schedule (2 cycles of chemotherapy plus placebo, then 4 cycles of chemotherapy plus ipilimumab) or a concurrent schedule (4 cycles of chemotherapy plus ipilimumab, then 2 cycles of chemotherapy plus placebo), and then given every 12 weeks until disease progression. Amongst 204 patients with advanced NSCLC, the primary endpoint, ‘immune-related’ progression-free survival (irPFS), was improved in patients receiving the phased schedule, with a median irPFS of 5.7 months, versus 4.6 months for chemotherapy alone (hazard ratio [HR] = 0.72, p=0.05). There was also a non-significant increase in OS with the phased schedule (12.2 months versus 8.3 months for chemotherapy alone), while the best overall response rate (ORR) was 32%

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compared with 14% for chemotherapy alone. The concurrent schedule did not significantly improve irPFS, OS, or best ORR versus chemotherapy alone. When contemplating combination therapy, ideally, the combination should meet the following criteria: each component should have single-agent activity with no crossresistance; there should be preclinical evidence of synergy between the components; and the components should not have overlapping safety profiles. However, in practice, all three criteria are rarely met and many combinations have failed to show significant improvements in outcomes compared with sequential administration of single agents. When combined with carboplatin/paclitaxel, ipilimumab appeared to be effective following the phased schedule, but not the concurrent. While there might be a synergistic effect between conventional chemotherapy and immune checkpoint inhibitors, it is also possible that chemotherapy itself is immunosuppressive and optimal phasing of regimens has to be defined. Furthermore, the stromal disruption and inflammation caused by chemotherapy might be first required for an effective reactivated immune response.64 A Phase 1 trial in Japan, testing a phased schedule of ipilimumab plus platinum-based chemotherapy, has demonstrated a 60% radiological response rate in the 10 evaluable patients with NSCLC.65 Further studies are required to elucidate the optimum dosing schedule of ipilimumab and chemotherapy in combination. Although data from small sample sizes should be interpreted with caution, subgroup analysis from the Phase 2 study of ipilimumab plus carboplatin/paclitaxel appeared to show greater efficacy with phased-ipilimumab in squamous cell than with non-squamous cell patients (irPFS HR: 0.55 [95% confidence interval [CI], 0.27 to 1.12] vs 0.82 [95% CI, 0.52 to 1.28]). The trial also included 130 patients with extensive-disease SCLC. The phased schedule improved irPFS (median 6.4 months) compared with chemotherapy alone (median 5.3 months), with a HR of 0.64 (p=0.03).66 The WHO best ORR was 57% for the phased schedule, versus 49% for chemotherapy alone. OS was not improved, and the concurrent

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schedule again did not improve irPFS or OS. Phase 3 trials are now ongoing to explore the phased regimen of ipilimumab with chemotherapy in patients with squamous cell NSCLC (NCT01285609) and extensive-stage SCLC (NCT01450761) (Table 1). Tumors can utilize more than one mechanism to avoid immuno-detection and removal, which provides a strong rationale for combining immuno-oncology agents that act on different targets, for example, anti-CTLA-4 and anti-PD-1 agents. Indeed, in the first trials combining ipilimumab with nivolumab in melanoma, the efficacy of dual checkpoint blockade exceeded that of either single agent.67 Preliminary results from an ongoing Phase 1 trial of first-line therapy with ipilimumab and nivolumab (an anti-PD-1 monoclonal antibody; see below) in 46 patients with advanced NSCLC showed an ORR of 22% (median duration of response not reached), with 33% having stable disease.68 These interim data suggest that the combination of ipilimumab and nivolumab is feasible and shows anti-tumor activity in patients with NSCLC. Overall, 88% of patients experienced treatment-related adverse events (AEs), with Grade 3/4 treatment-related AEs reported in 49% of patients. Treatment-related serious AEs (SAEs) were reported in 36% of patients, with Grade 3/4 SAEs experienced by 28% of patients, the most common of which were pneumonitis (6%), diarrhea (8%), and colitis (8%). In relation to the optimal safety profile of ipilimumab plus nivolumab in patients with NSCLC, the recommended combination dose for Phase 2/3 evaluation has yet to be determined.

Tremelimumab Tremelimumab is a fully human IgG2 monoclonal antibody that has a high affinity for CTLA-4. In an open-label Phase 2 trial in 87 patients with lung cancer who had stable or responding disease after first-line platinum-based chemotherapy, PFS was not significantly

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improved by tremelimumab (15 mg/kg) plus supportive care, versus supportive care only.69 However, 5% of patients had objective radiological responses. While tremelimumab is no longer in development as a single immunotherapeutic agent due to low response rates, evaluation is continuing into the combination of tremelimumab with the anti-PD-L1 antibody MEDI-4736 in advanced solid tumors, including lung cancer (NCT01975831) (Table 1). Tremelimumab is also currently under investigation for the treatment of mesothelioma. A Phase 2b, randomized, double-blind, placebo-controlled study of second- or third-line tremelimumab in patients with unresectable pleural or peritoneal mesothelioma who progressed after 1–2 lines of treatment is currently recruiting patients following promising findings in a single-arm Phase 2 study.70–72

Immuno-oncology agents targeting PD-1 Several agents that target the PD-1 receptor, thereby preventing its interaction with PD-L1 and PD-L2, are currently in development in lung cancer, including two fully human IgG4 antibodies (nivolumab and pembrolizumab) and a fusion protein (AMP-224).

Nivolumab In the NSCLC cohort (n=129) of a Phase 1 dose-ranging study in heavily pretreated (5 prior lines of therapy; 54% received 3 lines) patients with advanced tumors, nivolumab (1, 3, and 10 mg/kg) demonstrated encouraging survival data and a manageable safety profile.73 An objective response was observed in 17% of patients and median duration of response was 17 months. Median OS was 9.9 months, with median 1- and 2-year OS rates of 42% and 24%, respectively, for all patients. Median OS was 14.9 months for patients receiving 3 mg/kg nivolumab, with 1- and 2-year OS rates of 56% and 45%, respectively. In

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patients with PD-L1 positive tumors (defined as tumor membrane staining at any intensity in archived tumor samples using the Dako immunohistochemistry assay), median OS was 7.8 months versus 10.5 months in those with PD-L1 negative tumors; similar median OS rates were reported for squamous and non-squamous subtypes (9.2 months and 10.1 months, respectively), and clinical activity (ORR) was observed across patients who had received <3 (12%) and 3 (21%) prior therapies and those with or without epidermal growth factor receptor (EGFR) (17% and 20%, respectively) or Kirsten rat sarcoma viral oncogene homolog (KRAS) (14% and 25%, respectively) mutations. Nivolumab is currently being evaluated in several other ongoing clinical trials in lung cancer (Table 1). A Phase 2 trial testing nivolumab as a single agent in third-line and beyond therapy for squamous cell NSCLC is ongoing (NCT01721759). Two corresponding Phase 3 trials are testing nivolumab versus docetaxel as second-line treatment in both squamous (NCT01642004) and non-squamous (NCT01673867) NSCLC. An ongoing Phase 1 trial (NCT01454102) is investigating a variety of combinations, including nivolumab monotherapy and a variety of nivolumab combinations with chemotherapy, targeted therapy and ipilimumab, as first-line treatments in chemotherapynaïve NSCLC (Table 2). Preliminary data from the first 20 patients receiving nivolumab monotherapy in this study were recently presented.74 First-line nivolumab (3 mg/kg) had a manageable safety profile and an ORR of 30%. ORR was 50% in patients with PD-L1 positive tumors versus 0% for those with PD-L1 negative tumors. However, 1-year OS (71%) and median PFS (36.1 weeks) were unexpectedly high in PD-L1 negative patients; therefore, further exploration of an ideal predictive marker has to be performed and the assessment of PD-L1 expression requires further validation. These data support further studies of first-line nivolumab in NSCLC. To this end, a Phase 3 study of first-line therapy

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with nivolumab versus investigator’s choice chemotherapy in patients with Stage IIIb/IV or recurrent PD-L1 positive NSCLC is currently recruiting patients (NCT02041533).75 Interim results from the cohort with EGFR mutated advanced NSCLC receiving nivolumab (3 mg/kg) and erlotinib in NCT01454102 (n=21) suggest that this combination may provide durable clinical benefit coupled with an acceptable safety profile.76 ORR was 19% and 24-week PFS was 51%. Of the 20 patients with acquired erlotinib resistance, 15% showed a partial response, 45% had stable disease, and 5% had an unconventional immune response. Responses were also observed in T790M mutation-positive patients; however, these results will need to be confirmed in a larger cohort of patients. The effect of nivolumab in combination with erlotinib in EGFR mutation–positive, T790M mutation– negative patients who are refractory to an EGFR tyrosine kinase inhibitor will be of considerable interest. These data will also have to be viewed in the context of the thirdgeneration tyrosine kinase inhibitors, which are more efficacious in the T790M-mutant population. There is also a strong rationale to test the efficacy of immuno-oncology agents early in the disease continuum; therefore, studies with nivolumab after chemoradiation in patients with Stage 3 NSCLC or after surgery in patients with Stage 1–2 NSCLC may be warranted. Preliminary data from the cohort (n=43) of NCT01454102 treated with nivolumab and a platinum therapy doublet (gemcitabine, cisplatin, pemetrexed, carboplatin or paclitaxel) suggest that this combination has an acceptable safety profile.77 Total/confirmed ORRs were 43/33%, 40/33%, and 31/31% for patients receiving nivolumab and gemcitabine/cisplatin, nivolumab and pemetrexed/cisplatin, and nivolumab combined with carboplatin/paclitaxel, respectively. Trials are also ongoing to test nivolumab in combination with other immunotherapies. As described above, a Phase 1/2 trial is testing nivolumab alone or in combination with

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ipilimumab for advanced tumors, including advanced NSCLC (NCT01928394).68 A further Phase 1/2 trial is assessing nivolumab alone or in combination with ipilimumab in patients with advanced or metastatic solid tumors, including patients with SCLC (NCT01928394).

Pembrolizumab (MK-3475; formerly lambrolizumab) In an initial early phase trial, pembrolizumab showed an acceptable toxicity profile and preliminary efficacy in advanced renal cell carcinoma, melanoma, and NSCLC (1 patient, who had an unconfirmed partial response).78 A further Phase 1 study of 38 previously treated patients with advanced NSCLC treated with pembrolizumab 10 mg/kg reported a 21% ORR using ‘response evaluation criteria in solid tumors’ (RECIST), and a 24% ORR using immune-related response criteria. The median PFS reported for pembrolizumab-treated patients was 9.7 weeks, with a median OS of 51 weeks.79 A slightly higher response was reported in patients with a squamous histology, although numbers were too small to assess statistical significance. Preliminary data from an ongoing Phase 1 study of first-line pembrolizumab (2 or 10 mg/kg) in patients with locally advanced or metastatic NSCLC whose tumors expressed PD-L1 showed an ORR of 36% by irRC and an acceptable tolerability profile. In total, 25 patients remain on treatment.80 Pembrolizumab is currently being assessed at two different doses versus docetaxel in patients with NSCLC whose disease has relapsed after platinum-based chemotherapy (NCT01905657) (Table 1). It is also being evaluated in combination with cisplatin/pemetrexed or carboplatin/paclitaxel in advanced solid tumors including NSCLC (NCT01840579).

AMP-224

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AMP-224 is a B7-DC-Fc fusion protein designed to block the interaction between PD1 and PD-L1. Some patients with NSCLC were included in the first in-human Phase 1 trial, and a dose-dependent reduction in PD-1-high TILs was observed 4 hours after administration of the drug, which was still evident after 2 weeks. There was also an increase in the T-cell chemo-attractant CXCL9 reported in peripheral blood.81 However, this agent is not currently being assessed in ongoing trials in lung cancer.

Immuno-oncology agents targeting the PD-L1 ligand MPDL3280A, MEDI-4736, and BMS-936559 are all human IgG4 monoclonal antibodies that target PD-L1.

MPDL3280A (RG7446) In an initial Phase 1 expansion trial of MPDL3280A (1–20 mg/kg) that included 85 patients with NSCLC, a best ORR using RECIST, was reported in 23% of patients, with a stable disease rate at 6 months of 17%.82–85 The responses were durable, with a 6 month PFS of 45%, and almost all responders were progression-free after 1 year. Some potential predictors of response were also observed, with the caveat that this was a Phase 1 trial. Of 53 patients with NSCLC both evaluable for efficacy and with archived tissue samples, PDL1 expression was found to be predictive of response, with an ORR of 46% for those tumors with intermediate PD-L1 expression, rising to 83% in those with high PD-L1 expression. The ORR also differed slightly between patients stratified by NSCLC subtype (21% for non-squamous versus 27% for squamous NSCLC), smoking status (ORR of 26% for former or current smokers versus 10% in never-smokers), and KRAS mutation status (30% for patients whose tumors harbor wild type KRAS, versus 10% for those with mutated KRAS).

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Interestingly, activated CTLs increased in the bloodstream 2 weeks following treatment, but no correlation was found with radiological response. Three Phase 2 trials of MPDL3280A are currently ongoing in NSCLC (Table 1). The first is assessing MPDL3280A versus docetaxel as second-line treatment for advanced or metastatic NSCLC after platinum-based chemotherapy has failed (NCT01903993). Following the results of the Phase 1 trial, the second ongoing study is recruiting only those patients with advanced or metastatic NSCLC whose tumors are PD-L1-positive (NCT01846416). The third is a single-arm study, investigating MPDL3280A in three cohorts of patients with PD-L1-positive NSCLC: chemotherapy-naïve, single prior platinum-based regimen, and 2 prior therapies.86 A global multicenter, randomized, openlabel, Phase 3 trial is also ongoing to assess the efficacy and safety of MPDL3280A compared with docetaxel after failure of a platinum-containing chemotherapy in patients with metastatic or locally advanced NSCLC.86

MEDI-4736 Interim results are available from a Phase 2 trial in which MEDI-4736 was given every 2 weeks in 6 week cycles to patients with solid tumors, including lung cancers.87 Several durable remissions were achieved in patients with NSCLC (n=13) and MEDI-4736 had an acceptable safety profile.88 Expansion cohorts are currently being enrolled for this trial to further test the safety and efficacy of MEDI-4736 in advanced solid tumors, including lung cancer (NCT01693562) (Table 1). MEDI-4736 is also being tested in combination with the CTLA-4 inhibitor tremelimumab, as discussed above (NCT01975831). Furthermore, MEDI-4736 will be part of an innovative biomarker-driven, multi-arm, multi-drug Phase 2/3 registration trial in lung cancer that is expected to commence in 2014, in which five different drugs will be assessed

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(NCT02154490). The trial will enroll patients with recurrent Stage IIIB-IV squamous cell lung cancer, who will subsequently be screened for specific tumor mutations using a nextgeneration sequencing platform. Patients will then be randomized, based upon their tumor markers, to MEDI-4736 (MedImmune), rilotumumab (Amgen: Hepatocyte growth factor receptor c-MET inhibitor), AZD4547 (AstraZeneca: FGFR tyrosine kinase inhibitor), GDC0032 (Genentech; PI3 kinase inhibitor), or palbociclib (Pfizer: CDK4/6 kinase inhibitor). Docetaxel will be the active comparator to all experimental arms, with the exception of the riltumumab arm, which will be administered in combination with erlotinib and compared with erlotinib alone.

BMS-936559 An expansion cohort of the Phase 1 trial of BMS-936559 included 75 patients with NSCLC who had been previously treated in various different ways (95% had received platinum-based chemotherapy, 41% kinase inhibitors, 32% radiation).89 BMS-936559 was given every 2 weeks at three different doses (0.3–10 mg/kg). Of the 49 evaluable patients with NSCLC, the ORR was 10%, with a tumor response of 8% and 11% in patients with squamous and non-squamous NSCLC, respectively. Additionally, 12% of patients had stable disease at 6 months. This disease stabilization was prolonged; 3 of the 5 responders had responses lasting more than 6 months. The 6-month PFS was 31%. BMS-936559 is no longer being investigated in oncology but remains in development in virological indications.

OTHER IMMUNE CHECKPOINT INHIBITORS Several other agents that target other signaling pathways within the immune checkpoint system are currently in development, including KIR and LAG-3.

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Lirilumab (anti-KIR) Lirilumab (BMS-986015) is a fully human monoclonal antibody targeting KIR that has demonstrated activity in preclinical trials when combined with nivolumab.90 As a result of these preclinical findings, an early phase clinical trial is currently ongoing in which the safety of lirilumab plus nivolumab is being investigated in patients with advanced, metastatic, or unresectable solid tumors, including 32 patients with NSCLC (NCT01714739). A similar trial, including up to 20 patients with NSCLC, is testing the combination of lirilumab and ipilimumab (NCT01750580). BMS-986016 (anti-LAG-3) Mouse models have demonstrated that LAG-3 inhibition by monoclonal antibodies slows tumor growth and leads to synergistic regression in combination with anti-PD-1 antibodies.56 Hence, an early phase investigation of a human anti-LAG-3 antibody (BMS986016) both alone and in combination with nivolumab for treatment of solid tumors is ongoing (NCT01968109).

Others Other agents currently being investigated in cancer, either alone or in combination with other immune checkpoint inhibitors, include the anti-LAG-3 agent IMPA321 and an anti-T-cell membrane protein 3 agent.36,91–93 Costimulatory molecules, which act in the opposite way to inhibitory checkpoints, represent a second type of target currently under investigation.94 Drug candidates with co-stimulatory targets include: urelumab (BMS663513), an anti-4-1BB monoclonal antibody (CD137 activation); CP-870893 (Pfizer), an anti-CD40 monoclonal antibody;95–96 Chi Lob 7/4 (Southampton Experimental Cancer Medicine Centre, UK), a chimeric anti-CD30 monoclonal antibody;97 and TRX518 (Tolerx), an anti-GITR monoclonal antibody.94

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ASSESSING THE EFFICACY OF IMMUNE CHECKPOINT INHIBITORS As new therapies are developed, the methods employed to assess their efficacy are likely to evolve. For instance, the efficacy of immuno-oncology agents may be better assessed with immune-specific criteria, rather than traditional methods for assessing treatment responses in solid tumors, such as RECIST, due to the different kinetics in response caused by the immune-related mechanism of action. In particular, the phenomenon of progression and pseudo-progression (whereby a tumor can initially enlarge following treatment, then show a sustained regression) can be observed with immune checkpoint inhibitors. These may be attributable to a delayed immune responses while immune cells become active and/or infiltration of T cells.98 Thus, conventional measures such as ORR and PFS may not fully capture the clinical benefit of immuno-oncology agents. Based on their specific mode of action, it is expected that immune checkpoint inhibition leads to an improvement long-term survival. Indeed, this has already been observed with ipilimumab in melanoma.57 Therefore, time dependent endpoints (1-year/2-year OS rates), which adequately assess this specific efficacy, might be more suitable for the evaluation of the efficacy of such agents.

OPTIMIZING THE USE OF IMMUNE CHECKPOINT INHIBITORS It is clear that immune checkpoint inhibitors have considerable clinical potential, and that they potentially represent an exciting clinical advance for patients with lung cancer. However, if these agents are approved, there are still challenges that need to be overcome in order to ensure that their use will be fully optimized in clinical practice, including the management of toxicity, reliable predictors of responses, and potential resistance.

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Management of toxicity Immune checkpoint inhibitors have a generally acceptable safety profile, particularly compared with conventional cytotoxics. They are associated with irAEs that tend to be reversible and inflammatory in nature. The most notable AEs with immune checkpoint inhibitors are diarrhea/colitis and pneumonitis. The latter event may be of interest since patients with NSCLC often already have compromised respiratory function. Although appropriate and immediate management with steroids and other immune suppressors can minimize complications, development of AE management guidelines to ensure early diagnosis is of critical importance. Such guidelines should include potentially unspecific symptoms of pneumonitis such as dyspnea and cough. Furthermore, adequate pulmonological diagnostic workup with computed tomography, x-ray, bronchoscopy, lavage, and microbiological asservation should be recommended wherever possible. In addition, early treatment with sufficient doses of steroids should be highly encouraged in the absence of any bacterial infection. As further safety data are collected from ongoing clinical trials with immune checkpoint inhibitors, development of guidance for the early identification and management of AEs will be key for the clinical establishment of these drugs. Agents targeting the PD-1 pathway appear to have lower toxicity than those targeting CTLA-4. This may be because the CTLA-4 interaction takes place centrally, whereas the PD-1/PD-L1 interaction occurs in the peripheral tissues. Early data indicate that immunooncology agents can be safety combined with other agents (e.g. chemotherapy, tyrosine kinase inhibitors, and other immuno-oncology agents). However, safety and efficacy need to be further characterized in larger trials.

Predictors of efficacy

21

As with all new agents developed recently, the identification of predictive factors and biomarkers is a key issue, as establishing patient populations in which a larger effect is seen raises the benefit:risk ratio and avoids unnecessary toxicity. Several preliminary biomarker candidates for immuno-oncology agents have emerged. The most intuitive biomarker, PDL1 expression, has been reported to be a biomarker for agents targeting the PD-1 pathway in several but not all studies.74,79,82,99,100 However, such studies have been complicated by employing retrospective analysis of archived tissue, which may not accurately reflect fresh tumor tissues in standard immunohistochemistry tests.101 Added complications include the plasticity of expression patterns, and a non-standardized test design. Moreover, for immunebased therapies, ORR may not be the optimal endpoint to assess the predictive role of biomarkers. Consequently, many ongoing trials are prospectively investigating the ability of PD-L1 expression to predict or influence the activity of anti-PD1/PD-L1 pathway therapies (NCT01846416). Other potential biomarkers may include lung cancer subtype (i.e. squamous versus non-squamous histology), smoking status, and presence of somatic mutations such as EGFR.79,83 It is noteworthy that testing for somatic mutations is based on DNA sequencing, which is usually a sensitive, rapid, and robust analysis, especially when compared with immunohistochemistry, which is used to investigate the presence PD-L1. Findings from ongoing Phase 3 trials should provide further information on biomarkers of response with these agents. However, it is noteworthy that current biomarker analysis is based on several different assessment methods. Thus, harmonization of the techniques used is of critical importance for the further development of immuno-oncology agents.

Resistance

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Although there is no theoretical rationale to suspect that, unlike conventional targeted therapies, immuno-oncology agents will be associated with resistance, this requires confirmation in ongoing and future studies. There is evidence from other oncology settings that, if progression occurs after treatment has stopped, simply restarting treatment can reestablish T-cell function and achieve tumor remission.102,103 Conversely, there is the potential for CTLA-4 or PD-1/PD-L1 epitopes to become mutated in tumors in such a way that they can still bind their partners, but are no longer susceptible to blockade by the targeting antibody.47 Further investigations are required to understand whether resistance is a problem with immuno-oncology agents.

CONCLUSIONS There is currently a major unmet need for novel therapies that improve treatment outcomes in patients with lung cancer. Early trials of various immuno-oncology agents targeting immune checkpoint pathways have shown considerable potential for improving tumor responses and survival in such patients. Potential benefits of therapies that target immune checkpoints in lung cancer are a manageable safety profile, the potential for a sustained response by immunological memory, and efficacy across a broad spectrum of patients. Phase 3 trials of several immune checkpoint agents are underway, and the results are eagerly anticipated. Given the evidence so far, the expectations for immuno-oncology in lung cancer are high. Our hope is that these expectations are met and that the agents currently in development go on to offer new and improved treatment options for lung cancer.

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REFERENCES 1. Ferlay J, Shin H-R, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127:2893-917. 2. http://www.wcrf.org/cancer_statistics/world_cancer_statistics.php. 2012; Available at: http://www.wcrf.org/cancer_statistics/world_cancer_statistics.php. Accessed March 2014. 3. Surveillance, Epidemiology, and End Results [SEER] incidence and National Center for Health Statistics [NHCS] mortality statistics. Available at: http://www.cancer.gov/cancertopics/types/lung/cancer-survival-prognosis. Last accessed 4 August 2014. 4. De Angelis R, Sant M, Coleman MP, et al. Cancer survival in Europe 1999-2007 by country and age: results of EUROCARE--5-a population-based study. Lancet Oncol. 2014;15(1):23-34. 5. Goldstraw P, Crowley J, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol. 2007;2(8):706-14. 6. Carney DN. Lung Cancer — Time to Move on from Chemotherapy. N Engl J Med. 2002;346:126-8. 7. Cardarella S, Ortiz TM, Joshi VA, et al. The introduction of systematic genomic testing for patients with non-small-cell lung cancer. J Thorac Oncol. 2012;7:1767-74. 8. Pardoll DM. Does the immune system see tumors as foreign or self? Annu Rev Immunol. 2003;21:807-39. 9. Holt GE, Podack ER, Raez LE. Immunotherapy as a strategy for the treatment of nonsmall-cell lung cancer. Therapy. 2011;8:43-54.

24

10. Al-Shibli KI, Donnem T, Al-Saad S, et al. Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer. Clin Cancer Res. 2008;14:52207. 11. Hiraoka K, Miyamoto M, Cho Y, et al. Concurrent infiltration by CD8+ T cells and CD4+ T cells is a favourable prognostic factor in non-small-cell lung carcinoma. Br J Cancer. 2006;94:275-80. 12. Dieu-Nosjean M-C, Antoine M, Danel C, et al. Long-Term Survival for Patients With Non–Small-Cell Lung Cancer With Intratumoral Lymphoid Structures. J Clin Oncol. 2008;26:4410-7. 13. Kawai O, Ishii G, Kubota K, et al. Predominant infiltration of macrophages and CD8+ T Cells in cancer nests is a significant predictor of survival in stage IV nonsmall cell lung cancer. Cancer. 2008;113:1387-95. 14. Miotto D, Cascio NL, Stendardo M, et al. CD8+ T cells expressing IL-10 are associated with a favourable prognosis in lung cancer. Lung Cancer. 2010;69:355-60. 15. Zhuang X, Xia X, Wang C, et al. A High Number of CD8+ T Cells Infiltrated in NSCLC Tissues is Associated With a Favorable Prognosis. Appl Immunohistochem Mol Morphol. 2010;18:24-8. 16. Koyama K, Kagamu H, Miura S, et al. Reciprocal CD4+ T-cell balance of effector CD62Llow CD4+ and CD62LhighCD25+ CD4+ regulatory T cells in small cell lung cancer reflects disease stage. Clin Cancer Res. 2008;14:6770-9. 17. Shimizu KNM, Hirami Y, Yukawa T, Maeda A, Tanemoto K. Tumor-infiltrating Foxp3+ regulatory T cells are correlated with cyclooxygenase-2 expression and are associated with recurrence in resected non-small cell lung cancer. J Thorac Oncol. 2010;5:585-90.

25

18. Kadota K, Nitadori JI, Adusumilli PS. Prognostic value of the immune microenvironment in lung adenocarcinoma. Oncoimmunology. 2013;2:e24036. 19. Lesokhin AM, Hohl TM, Kitano S, et al. Monocytic CCR2+ Myeloid-Derived Suppressor Cells Promote Immune Escape by Limiting Activated CD8 T-cell Infiltration into the Tumor Microenvironment. Cancer Res. 2012;72(4):876-86. 20. Nagaraj S, Youn J-I, Gabrilovich DI. Reciprocal Relationship between MyeloidDerived Suppressor Cells and T Cells. J Immunol. 2013;191:17-23. 21. Woo EY, Yeh H, Chu CS, et al. Cutting Edge: Regulatory T Cells from Lung Cancer Patients Directly Inhibit Autologous T Cell Proliferation. J Immunol. 2002;168:42726. 22. Jadus MR, Natividad J, Mai A, et al. Lung cancer: a classic example of tumor escape and progression while providing opportunities for immunological intervention. Clin Dev Immunol. 2012;2012:160724. 23. Marrogi AJ, Travis WD, Welsh JA, et al. Nitric Oxide Synthase, Cyclooxygenase 2, and Vascular Endothelial Growth Factor in the Angiogenesis of Non-Small Cell Lung Carcinoma. Clin Cancer Res. 2000;6:4739-44. 24. Yao Z, Fenoglio S, Gao DC, et al. TGF- IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer. Proc Natl Acad Sci U.S.A. 2010;107:15535-40. 25. Wang Y-C, Sung W-W, Wu T-C, et al. Interleukin-10 Haplotype May Predict Survival and Relapse in Resected Non-Small Cell Lung Cancer. PLoS One. 2012;7:e39525.

26

26. Baratelli F, Lee JM, Hazra S, et al. PGE(2) contributes to TGF-beta induced T regulatory cell function in human non-small cell lung cancer. Am J Transl Res. 2010;2:356-67. 27. Ramnath N, Tan D, Li Q, et al. Is downregulation of MHC class I antigen expression in human non-small cell lung cancer associated with prolonged survival? Cancer Immunol Immunother. 2006;55:891-9. 28. Derniame S, Vignaud JM, Faure GC, Béné MC. Alteration of the immunological synapse in lung cancer: a microenvironmental approach. Clin Exp Immunol. 2008;154(1):48-55. 29. Jansen RL, Slingerland R, Goey SH, et al. Interleukin-2 and interferon-alpha in the treatment of patients with advanced non-small-cell lung cancer. J Immunother. 1992;12:70-3. 30. Murala S, Alli V, Kreisel D, et al. Current status of immunotherapy for the treatment of lung cancer. J Thorac Dis. 2011;2:237-44. 31. Kelly RJ, Giaccone G. Lung cancer vaccines. Cancer J. 2011;17:302-8. 32. Butts C, Socinski M, Mitchell P, et al. START: A phase III study of L-BLP25 cancer immunotherapy for unresectable stage III non-small cell lung cancer [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31:7500. 33. Giaccone GBL, Neumaitis J, et al. A phase III study of belagenpumatucel-L therapeutic tumour cell vaccine for non-small cell lung cancer (NSCLC), in ECC 2013. 2013. 34. Nemunaitis J, Dillman RO, Schwarzenberger PO, et al. Phase II study of belagenpumatucel-l, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non–small-cell lung cancer. J Clin Oncol. 2006;24:4721-30.

27

35. Vansteenkiste E, Zielinski M, Linder A, et al. Adjuvant MAGE-A3 immunotherapy in resected non–small-cell lung Cancer: phase II randomized study results [abstract]. J Clin Oncol (Meeting Abstracts). 2013;43:7103. 36. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12:252-64. 37. Li L, Chao QG, Ping LZ, et al. The prevalence of FOXP3+ regulatory T-cells in peripheral blood of patients with NSCLC. Cancer Biother Radiopharm. 2009;24:35767. 38. Gabriel EM, Lattime EC. Anti–CTL-Associated Antigen 4: Are Regulatory T Cells a Target? Clin Cancer Res. 2007;13:785-8. 39. Erfani N, Mehrabadi SM, Ghayumi MA, et al. Increase of regulatory T cells in metastatic stage and CTLA-4 over expression in lymphocytes of patients with nonsmall cell lung cancer (NSCLC). Lung Cancer. 2012;77:306-11. 40. Butte MJ, Keir ME, Phamduy TB, et al. Programmed Death-1 Ligand 1 Interacts Specifically with the B7-1 Costimulatory Molecule to Inhibit T Cell Responses. Immunity. 2007;27:111-22. 41. Liang SC, Latchman YE, Buhlmann JE, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol. 2003;33:270616. 42. Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol. 2008;8:467-77. 43. Pfistershammer K, Klauser C, Pickl WF, et al. No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation. Eur J Immunol. 2006;36:1104-13.

28

44. Mu C-Y, Huang J-A, Chen Y, et al. High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation. Med Oncol. 2011;28:682-8. 45. Chen YB, Mu CY, Huang JA. Clinical significance of programmed death-1 ligand-1 expression in patients with non-small cell lung cancer: a 5-year-follow-up study. Tumori. 2012;98:751-5. 46. Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793-800. 47. Konishi J, Yamazaki K, Azuma M, et al. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res. 2004;10:5094-100. 48. Rosenwald A, Wright G, Leroy K, et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med. 2003;198:851-62. 49. Zhang Y, Wang L, Li Y, et al. Protein expression of programmed death 1 ligand 1 and ligand 2 independently predict poor prognosis in surgically resected lung adenocarcinoma. Onco Targets Ther. 2014;7:567-73. 50. Hino R, Kabashima K, Kato Y, et al. Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer. 2010;116:1757-66. 51. Carrega P, Morandi B, Costa R, et al. Natural killer cells infiltrating human nonsmallcell lung cancer are enriched in CD56brightCD16 cells and display an impaired capability to kill tumor cells. Cancer. 2008;112:863-75. 52. Winiewski A, Jankowska R, Passowicz-Muszyska E, et al. KIR2DL2/S2 and HLAC C1C1 genotype is associated with better response to treatment and prolonged

29

survival of patients with non-small cell lung cancer in a Polish Caucasian population. Human Immunol. 2012;73:927-31. 53. Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P, et al. Tumor-infiltrating NY-ESO-1– specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U.S.A. 2010;107:7875-80. 54. Grosso JF, Kelleher CC, Harris TJ, et al. LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems. J Clin Invest. 2007;117:3383-92. 55. Sierro S, Romero P, Speiser DE. The CD4-like molecule LAG-3, biology and therapeutic applications. Expert Opin Ther Targets. 2011;15:91-101. 56. Woo S-R, Turnis ME, Goldberg MV, et al. Immune Inhibitory Molecules LAG-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape. Cancer Res. 2012;72:917-27. 57. Schadendorf D, Hodi FS, Robert C, et al. Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in metastatic or locally advanced, unresectable melanoma [abstract]. Eur J Cancer (Meeting Abstracts). 2013;49(Suppl. 2):24LBA. 58. Hodi FS, O'Day SJ, McDermott DF, et al. Improved Survival with Ipilimumab in Patients with Metastatic Melanoma. N Engl J Med. 2010;363:711-23. 59. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517-26. 60. Maio M, Bondarenko I, Robert C, et al. Survival analysis with 5 years of follow-up in a phase III study of ipilimumab and dacarbazine in metastatic melanoma [abstract]. Eur J Cancer (Meeting Abstracts). 2013;49(Suppl. 2):3704.

30

61. Lebbe C, Weber JS, Maio M, et al. Long-term survival in patients with metastatic melanoma who received ipilimumab in four phase II trials [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31(Suppl. 1):9053. 62. Di Giacomo AM, Calabrò L, Danielli R, et al. Long-term survival and immunological parameters in metastatic melanoma patients who responded to ipilimumab 10 mg/kg within an expanded access programme. Cancer Immunol Immunother. 2013;62:10218. 63. Lynch TJ, Bondarenko I, Luft A, et al. Ipilimumab in Combination With Paclitaxel and Carboplatin As First-Line Treatment in Stage IIIB/IV Non–Small-Cell Lung Cancer: Results From a Randomized, Double-Blind, Multicenter Phase II Study. J Clin Oncol. 2012;30:2046-54. 64. Liu H, Zhang T, Ye J, et al. Tumor-infiltrating lymphocytes predict response to chemotherapy in patients with advance non-small cell lung cancer. Cancer Immunol Immunother. 2012;61:1849-56. 65. Nokihara H. Phase 1 study of ipilimumab in combination with paclitaxel/carboplatin in patients with non-small cell lung cancer. In IASLC 15th World Conference on Lung Cancer. 2013, P2. 11-040: Sydney, Australia. 66. Reck M, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line therapy in extensive-disease-small-cell lung cancer: results from a randomized, double-blind, multicenter phase 2 trial. Ann Oncol. 2013;24:7583. 67. Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus Ipilimumab in Advanced Melanoma. New Engl J Med. 2013;369:122-33.

31

68. Antonia SJ, Gettinger SN, Chow LQM, et al. Nivolumab (anti-PD-1; BMS-936558, ONO-4538) and ipilimumab in first-line NSCLC: Interim phase I results [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):8023. 69. Zatloukal P, Heo DS, Park K, et al. Randomized phase II clinical trial comparing tremelimumab (CP-675,206) with best supportive care (BSC) following first-line platinum-based therapy in patients (pts) with advanced non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol (Meeting Abstracts). 2009;27(Suppl.):8071. 70. Calabrò L, Morra A, Fonsatti E, et al. Tremelimumab for patients with chemotherapyresistant advanced malignant mesothelioma: an open-label, single-arm, phase 2 trial. Lancet Oncol. 2013;14:1104-11. 71. Calabrò L, Morra A, Fonsatti E, et al. A phase 2 single-arm study with tremelimumab at an optimized dosing schedule in second-line mesothelioma patients [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):7531. 72. Maio M, Scherpereel A, Di Pietro A, et al. Randomized, double-blind, placebocontrolled study of tremelimumab for second- and third-line treatment of unresectable pleural or peritoneal mesothelioma [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):TPS7609^. 73. Brahmer JR, Horn L, Gandhi L, et al. Nivolumab (anti-PD-1, BMS-936558, ONO4538) in patients (pts) with advanced non-small-cell lung cancer (NSCLC): Survival and clinical activity by subgroup analysis [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):8112^. 74. Gettinger SN, Shepherd FA, Antonia SJ, et al. First-line nivolumab (anti-PD-1; BMS936558, ONO-4538) monotherapy in advanced NSCLC: Safety, efficacy, and correlation of outcomes with PD-L1 status [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):8024.

32

75. Carbone DP, Socinski MA, Chen AC, Bhagavatheeswaran P, Reck M, Paz-Ares L. A phase III, randomized, open-label trial of nivolumab (anti-PD-1; BMS-936558, ONO4538) versus investigator's choice chemotherapy (ICC) as first-line therapy for stage IV or recurrent PD-L1+ non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):TPS8128. 76. Rizvi NA, Chow LQM, Borghaei H, et al. Safety and response with nivolumab (antiPD-1; BMS-936558, ONO-4538) plus erlotinib in patients (pts) with epidermal growth factor receptor mutant (EGFR MT) advanced NSCLC [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):8022. 77. Rizvi N, Antonia SJ, Chow LQM, et al. A phase I study of nivolumab (anti-PD-1; BMS-936558, ONO-4538) plus platinum-based doublet chemotherapy (PT-doublet) in chemotherapy-naive non-small cell lung cancer (NSCLC) patients (pts) [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31(Suppl.):8072. 78. Patnaik A, Kang SP, Tolcher AW, et al. Phase I study of MK-3475 (anti-PD-1 monoclonal antibody) in patients with advanced solid tumors [abstract]. J Clin Oncol (Meeting Abstracts). 2012;30(Suppl.):2512. 79. Garon EB, Balmanoukian A, Hamid O, et al. MK-3475 monotherapy for previously treated non-small cell lung cancer (NSCLC): Preliminary safety and clinical activity [abstract]. Clin Cancer Res (Meeting Abstracts). 2014;20:A20. 80. Rizvi NA, Garon EB, Patnaik A, et al. Safety and clinical activity of MK-3475 as initial therapy in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):8007. 81. Smothers F, Hoos A, Langermann S, et al. AMP-224, a fusion protein that targets PD1. Ann Oncol. 2013;24:i7.

33

82. Spigel D, Scott NG, Horn L, et al. Clinical activity, safety, and biomarkers of MPDL3280A, an engineered PD-L1 antibody in patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31(Suppl.):8008. 83. Horn L, Herbst R, Spiegel D. An analysis of the relationship of clinical activity to baseline EGFR status, PD-L1 expression and prior treatment history in patients with non-small cell lung acners (NSCLC) following PD-L1 blockade with MPD-L3280A (anti-PDL1). In IASLC 14th World Conference on Lung Cancer. 2012, MO18.01: Amsterdam, the Netherlands. 84. Herbst RS, Gordon MS, Fine GD, et al. A study of MPDL3280A, an engineered PDL1 antibody in patients with locally advanced or metastatic tumors [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31(Suppl.):3000. 85. Soria J. Clinical activity, safety and biomarkers of PD-L1 blockade in non-small cell lung cancer (NSCLC): additional analyses from a clinical study of the engineered antibody MPDL3280A (anti-PDL1). In European Cancer Congress 2013. 2013: Amsterdam. 86. Rizvi NA, Chow LQM, Dirix LY, et al. Clinical trials of MPDL3280A (anti-PDL1) in patients (pts) with non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):TPS8123. 87. Khlief SL, Segal NH, et al. MEDI4736, an anti-PD-L1 antibody with engineered Fc domain preclinical evaluation and early clinical results from a Phase I study in patients with advanced solid tumors. In European Cancer Congress, 2013. 2013: Amsterdam. 88. Brahmer JR, Rizvi NA, Lutzky J, et al. Clinical activity and biomarkers of MEDI4736, an anti-PD-L1 antibody, in patients with NSCLC [abstract]. J Clin Oncol (Meeting Abstracts). 2014;32(Suppl. 5s):8021^.

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89. Brahmer JR, Tykodi SS, Chow LQM, et al. Safety and Activity of Anti–PD-L1 Antibody in Patients with Advanced Cancer. N Engl J Med. 2012;366:2455-65. 90. Sanborn RE, Sharfman WH, Segal NH, et al. A phase I dose-escalation and cohort expansion study of lirilumab (anti-KIR; BMS-986015) administered in combination with nivolumab (anti-PD-1; BMS-936558; ONO-4538) in patients (Pts) with advanced refractory solid tumors [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31(Suppl.):TPS3110. 91. Wang-Gillam A, Plambeck-Suess S, Goedegebuure P, et al. A phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma. Invest New Drugs. 2013;31:707-13. 92. Brignone C, Escudier B, Grygar C, et al. A phase I pharmacokinetic and biological correlative study of IMP321, a novel MHC class II agonist, in patients with advanced renal cell carcinoma. Clin Cancer Res. 2009;15:6225-31. 93. Brignone C, Gutierrez M, Mefti F, et al. Fist-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG3Ig) enahnaces immune responses and antitumour activity. J Transl Med. 2010;8:71. 94. Garber K. Beyond ipilimumab: new approaches target the immunological synapse. J Natl Cancer Inst. 2011;103:1079-82. 95. Vonderheide RH, Flaherty KT, Khalil M, et al. Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clin Oncol. 2007;25:876-83. 96. Vonderheide RH, Burg JM, Mick R, et al. Phase I study of the CD40 agonist antibody CP-870,893 combined with carboplatin and paclitaxel in patients with advanced solid tumors. OncoImmunology. 2013;2:1,e23033.

35

97. Johnson PW, Steven NM, Chowdhury, et al. A Cancer Research UK phase I study evaluating safety, tolerability, and biological effects of chimeric anti-CD40 monoclonal antibody (MAb), Chi Lob 7/4 [abstract]. J Clin Oncol (Meeting Abstracts). 2010;28(Suppl.):2507. 98. Wolchok JD, Hoos A, O'Day S, et al. Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria. Clin Cancer Res. 2009;15:7412-20. 99. Grosso JF, Horak CE, Inzunza HD, et al. Association of tumor PD-L1 expression and immune biomarkers with clinical activity in patients (pts) with advanced solid tumors treated with nivolumab (anti-PD-1; BMS-936558; ONO-4538) [abstract]. J Clin Oncol (Meeting Abstracts). 2013;31(Suppl.):3016. 100.Topalian SL, Hodi FS, Brahmer JR, et al. Safety, Activity, and Immune Correlates of Anti–PD-1 Antibody in Cancer. N Engl J Med. 2012;366:2443-54. 101.Freidin MB, Bhudia N, Lim E, et al. Impact of collection and storage of lung tumor tissue on whole genome expression profiling. J Mol Diagn. 2012;14(2):140-8. 102.Lipson EJ, Sharfman WH, Drake CG, et al. Durable Cancer Regression Off-Treatment and Effective Reinduction Therapy with an Anti-PD-1 Antibody. Clin Cancer Res. 2013;19:462-8. 103.Robert C, Schadendorf D, Messina M, et al. Efficacy and safety of retreatment with ipilimumab in patients with pretreated advanced melanoma who progressed after initially achieving disease control. Clin Cancer Res. 2013;19:2232-9.

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FIGURE LEGEND

Figure 1. Overview of immune checkpoint pathways. To be adapted from Creelan BC. Update on immune checkpoint inhibitors in lung cancer. Cancer Control. 2014 Jan;21(1):80-9. APC, antigen presenting cell; AR, activating receptor; DC, dendritic cell; HLA-C, human leukocyte antigen; HLAKIR, killer-cell immunoglobulin-like receptor; KIR, killer immunoglobulin receptors; MHC, major histocompatibility complex; NK, natural killer; PD-1, programmed death-1; PD-L1, programmed death ligand-1; TAA, tumor-associated antigen; TCR, antigen-specific T-cell receptors

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Table 1. Ongoing clinical trials with immune checkpoint inhibitors in lung cancer

Drug

Tar

Ph

g

a

ary

numb

etio

e

s

out

er

n

t

e

co

Population

Design

Prim

NCT

Compl

date

me me asu re Ipilimum ab

CT

1

NSCLC

L

Ipilimumab + neoadjuvant chemotherapy vs

%

neoadjuvant chemotherapy

sub

A

ject

-

s

4

wit

NCT018

May

20754

2018

NCT019

Decem

h det ecta ble tum orspe cifi c circ ulat ing T cell s 1

NSCLC

Ipilimumab + erlotinib or crizotinib

Safet y

98126

ber 2015

38

2

3

SCLC

Metastatic/E D SCLC

3

NSCLC SCC

Ipilimumab + carboplatin/etoposide

Ipilimumab plus etoposide/platinum vs

PFS

OS

etoposide/platinum

Ipilimumab + paclitaxel/carboplatin vs

OS

paclitaxel/carboplatin

NCT013

May

31525

2015

NCT014

March

50761

2017

NCT012

Decem

85609

ber 2016

Tremelilu mab

CT

1

Solid tumors

MEDI-4736 + tremelilumab

L

Safet y

NCT019 75831

A -

Decem ber 2016

2

NSCLC

4

Gefitinib, AZD9291, tremelimumab or

Comp

NCT021

selumetinib + docetaxel with a sequential switch

lete

79671

to MEDI4736

res

May 2016

pon se rate Nivolum

PD-1

1

Solid tumors

Nivolumab + interleukin-21

ab

Safet y

1

NSCLC

Safety of nivolumab + various standard chemotherapies

1/2

Advanced/m

Nivolumab or nivolumab + ipilimumab

Safet y

ORR

etastatic

NCT016

April

29758

2015

NCT014

August

54102

2015

NCT019

April

28394

2015

NCT017

Februa

solid tumors including NSCLC 2

Advanced/m etastatic SCC

Nivolumab as 3rd-line onwards therapy

ORR

21759

ry 2015

39

NSCLC who have received >2 previous treatments 3

SCC

Nivolumab vs docetaxel as 2nd-line therapy

OS

NSCLC

NCT016

August

42004

2015

after failure of platinum 3

NSCC

Nivolumab vs docetaxel as 2nd-line therapy

OS

NSCLC

NCT016

Nove

73867

mbe

after

r

failure of

2015

platinum 3

NSCLC

Nivolumab vs investigator’s choice of

PFS

chemotherapy

NCT020 41533

Januar y 2018

3

NSCLC

Nivolumab 2nd-line onwards

Safet y

Pembroli

PD-1

1

Advanced

zumab

solid

(MK34

tumors

75)

including

MK3475 + cisplatin/pemetrexed or carboplatin/paclitaxel

Safet y

NCT020

March

66636

2019

NCT018 40579

April 2015

NSCLC 1/2

2/3

NSCLC

Pembrolizumab +

ORR,

NCT020

paclitaxel/carboplatin/bevacizumab/pemetrexed/ip

PF

39674

2019

ilimumab/erlotinib/gefitinib

S OS,

NCT019

Januar

progresse

PF

05657

d after

S,

platinum

safe

NSCLC

Low or high dose of MK3475 vs docetaxel

June

y 2020

ty

40

3

NSCLC

Pembrolizumab vs

PFS

paclitaxel/carboplatin/pemetrexed/cisplatin/gemcit

NCT021 42738

abine MPDL32 80A

PD-

1

L1

Advanced solid

MPDL3280A + bevacuzimab + various other chemotherapies

Decem ber 2017

Safet y

NCT016

July

33970

2015

NCT020

August

y

13219

2016

OS

NCT019

March

03993

2017

tumors 1

2

NSCLC

Advanced/m

MPDL3280A + erlotinib

MPDL3280A vs docetaxel as 2nd-line

Safet

etastatic NSCLC after platinum failed 2

Advanced/m

MPDL3280A

ORR

etastatic

NCT018

May

46416

2015

NCT020

March

31458

2018

PD-1+ NSCLC 2

Advanced/m

MPDL3280A

ORR

etastatic PD-1+ NSCLC 3

MEDI-

PD-

4736

L1

1

NSCLC

NSCLC

MPDL3280A vs docetaxel

MEDI-4736 + tremelimumab

OS

Safet y

NCT020

June

08227

2018

NCT020

Januar

00947

y 2017

1

NSCLC

MEDI-4736 + gefitinib

Safet y

NCT020 88112

Octobe r 2017

2

NSCLC

MEDI-4736 3rd-line

ORR

NCT020

August

87423

2016

41

3

NSCLC

MEDI-4736 following concurrent chemoradiation

OS,

NCT021

Nove

PF

25461

mbe

S

r 2020

Liriluma

KIR

1

b

Advanced/m

Lirilumab + nivolumab

etastatic

1

Safet y

NCT017

Septe

14739

mbe

solid

r

tumors

2015

Advanced/m

Lirilumab + ipilimumab

etastatic

Safet y

NCT017

July

50580

2015

NCT019

Octobe

solid tumors BMS98601

LAG

1

-3

BMS-986016 alone or + nivolumab

solid

6 Bavituxi

Advanced

Safet y

68109

tumors PS

1

mab

Previously untreated

r 2020

Bavituximab + pemetrexed/carboplatin in 1st-line therapy

Safet

NCT013

May

y

23062

2018

OS

NCT019

Decem

stage IV NSCLC 3

Nonsquamous NSCLC

Bavituximab + docetaxel vs docetaxel

99673

ber 2016

42

AE, adverse effect; CTLA-4, cytotoxic T-lymphocyte antigen-4; DLT, dose limiting toxicity; ED, extensive disease; KIR, killer-cell immunoglobulin-like receptor; LAG-3, lymphocyte-activation gene-3; NSCC, non-squamous cell carcinoma; NSCLC, non-small cell lung cancer; ORR, objective response rate; OS, overall survival; PD-1, programmed death-1; PFS, progression free survival; PS, phosphatidylserine; SCC, squamous cell carcinoma; SCLC, small cell lung cancer.

Table 2. Overview of the treatment arms in a Phase 1 study of nivolumab monotherapy or in combination with a range of cytotoxic, targeted and immuno-oncology agents (NCT01454102)

Treatment arm

Patients

Nivolumab +

Dosing schedule Nivolumab: IV every 3 weeks

NSCLC gemcitabine +

Gemcitabine: IV on Day 1 and Day 8 of every cycle for 4 cycles (any histology)

cisplatin

Cisplatin: IV on Day 1 of each cycle for 4 cycles

Nivolumab +

Nivolumab: IV every 3 weeks NSCLC

pemetrexed +

Pemetrexed: IV on Day 1 of every cycle for 4 cycles (any histology)

cisplatin

Cisplatin: IV on Day 1 of each cycle for 4 cycles

Nivolumab +

Nivolumab: IV every 3 weeks NSCLC

paclitaxel +

Paclitaxel: IV on Day 1 of every cycle for 4 cycles (any histology)

carboplatin

Carboplatin: IV on Day 1 of each cycle for 4 cycles

Nivolumab +

Nivolumab: IV every 3 weeks NSCLC

bevacizumab

Bevacizumab: prior to IV infusion on Cycle 1 Day 1 followed by IV (any histology)

maintenance Nivolumab + erlotinib Nivolumab

infusion every 3 weeks on Cycle 2 onwards NSCLC

Nivolumab: IV every 2 weeks

(any histology)

Erlotinib: oral daily

NSCLC

Nivolumab: IV every 2 weeks

43

(any histology) Ipilimumab: IV on Day 1 of each cycle, for 4 cycles Nivolumab +

NSCLC Nivolumab: IV prior to ipilimumab on Day 1 of each cycle, then every 2

ipilimumab

(squamous) weeks Ipilimumab: IV on Day 1 of each cycle, for 4 cycles

Nivolumab +

NSCLC Nivolumab: IV prior to ipilimumab on Day 1 of each cycle, then every 2

ipilimumab

(non-squamous) weeks Ipilimumab: IV on Day 1 of each cycle, for 4 cycles

Nivolumab +

NSCLC Nivolumab: IV prior to ipilimumab on Day 1 of each cycle, then every 2

Ipilimumab

(any histology) weeks NSCLC

Nivolumab

Nivolumab: IV every 2 weeks, switch maintenance therapy (squamous) NSCLC

Nivolumab

Nivolumab: IV every 2 weeks, switch maintenance therapy (non-squamous) NSCLC (patients with

Nivolumab

untreated,

Nivolumab: IV every 2 weeks

asymptomatic brain metastases) IV, intravenous; NSCLC, non-small cell lung cancer.

44