Fellow in the Thoracic Oncology Research Program,

Sidney Kimmel Comprehensive Cancer Center at

For many years nonsmall cell lung cancer (NSCLC), unlike melanoma and renal cell carcinoma, was thought to be a nonimmunogenic tumor unlikely to benefit from therapies aimed at augmenting endogenous antitumor immunity. Recent early-phase trial data with several agents that target immune checkpoint molecules responsible for suppression of T-cell&ndash; mediated immunity have challenged this perception. Antibodies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) are currently undergoing phase III investigation for advanced NSCLC. Vaccines targeting molecules expressed on NSCLC tumors have shown promise in earlyphase trials, and phase III studies are ongoing. In this article, we summarize early-phase trial results in NSCLC with immune checkpoint inhibitors and vaccines, and look forward to future developments in this rapidly expanding field.

Therapy for advanced nonsmall cell lung cancer (NSCLC) has shown incremental improvement over the past two decades with median overall survival (OS) in phase III trials now typically exceeding 12 months for patients with nonsquamous disease, and approaching 15 months in the most recently reported studies.

In addition, OS for the 10% to 15% of patients who have a targetable driver mutation in the epidermal growth factor receptor (EGFR) and are treated with EGFR tyrosine kinase inhibitor (TKI) therapy has ranged from 22.9 to 27.7 months in recent studies.

Results from trials enrolling patients with squamous NSCLC, which rarely has driver mutations that can be targeted with current FDA-approved agents, have been less encouraging, with typical median survival figures of 9 to 12 months.

For earlier-stage disease, approximately 50% to 75% of patients will eventually relapse and die from lung cancer despite apparently curative resection.

In this setting, novel treatments such as immunotherapy are of great interest, particularly if new biomarkers of response can be developed, allowing their use to cross the boundaries of histology and mutational status.

Vaccine-based strategies currently in phase III development in NSCLC are summarized in

Melanoma-associated antigen A3 (MAGE-A3) is a tumorspecific gene product that is recognized by cytotoxic T cells and frequently upregulated in the early development of NSCLC.

MAGE-A3 is expressed in 30% to 50% of NSCLC tumors and at a higher frequency in squamous tumors, poorly differentiated tumors, and advanced disease.

Expression has also been associated with poorer disease-free survival (DFS) for resected NSCLC.

Given the specificity of MAGE-A3 expression for tumor cells, MAGE-A3 vaccine has been created containing MAGE-A3 recombinant protein combined with a ASO2B immunologic adjuvant system, which appears to potentiate the immunologic effect. This vaccine has shown preliminary efficacy in metastatic melanoma, with five responses from 26 patients in a phase I/II study.

Initial studies in patients post-resection of early-stage NSCLC demonstrated that seven of eight vaccinated patients attained a rise in serum anti-MAGE-A3 antibodies, and repeated vaccination augmented this response.

In a double-blind phase II study, 182 patients with resected stage IB or II NSCLC, expressing MAGE-A3 by quantitative reverse transcriptasepolymerase chain reaction, were randomized 2:1 to receive adjuvant MAGE-A3 vaccine intramuscular injections or placebo.9 Analysis showed a positive trend favoring the MAGE-A3 vaccine with hazard ratios of 0.76 (95% CI, 0.48-1.21) for DFS and 0.81 (95% CI, 0.47-1.40) for OS; however, these figures did not reach statistical significance. The investigators developed a gene signature from this study, of immune-related genes that appeared to predict benefit from the vaccine, and indeed may be predictive of response to immune-based therapeutics in general.

TABLE 1. Vaccines in Phase III Clinical Development for NSCLC

Post-surgical resection of early-stage NSCLC

Trend for increased DFS vs placebo (HR = 0.74; P = .107)

MAGRIT study, stage IB-IIIA, MAGEA3- positive, vaccine or placebo after surgery alone/surgery + adjuvant chemo; completed accrual 2012

Post-CRT for unresectable stage III NSCLC

Trend for increased OS vs BSC (HR = 0.75; P = .112)

START study, stage IIIA/B, after definitive CRT, reportedly negative for OS

Allogeneic cell vaccine transfected with TGF-&beta;2 antisense gene

Response in 15%, higher dose correlated with survival

STOP study, stage III/IV, vaccine vs placebo after first-line platinum doublet chemo; completed accrual 2012

Trend for increased OS in patients developing antibody response

MUC1, recombinant viral vector coding human MUC1 and hIL-2

Chemo + TG4010 vs chemo alone, trend for improved RR for vaccine, no difference in OS

First-line MUC1-positive stage III/IV, chemo + vaccine vs chemo alone

MAGE-A3, melanoma-associated antigen-A3; DFS, disease-free survival; OS, overall survival; BSC, best supportive care; chemo, chemotherapy; CRT, chemoradiation; TGF- &beta;2, transforming growth factor-&beta;2; rHu-EGF, recombinant human epidermal growth factor; MUC1, mucin glycoprotein 1; hIL-2, recombinant human interleukin-2; RR, response rate by RECIST 1.0.

The MAGE-A3 vaccine is currently being investigated in a large phase III trial, NCT00480025, that has enrolled over 3000 patients with stage IB-IIIA resected NSCLC (MAGRIT study) and includes patients who receive adjuvant chemotherapy.

The primary endpoint for this study is DFS, and initial efficacy data are expected in mid-2013.

Mucin glycoprotein 1 (MUC 1) is a membrane-associated glycoprotein that is overexpressed in human cancers including NSCLC, and has been associated with a poor prognosis.

Liposomal BLP25 (Stimuvax) is a peptide vaccine that targets MUC 1 and has elicited T-cell responses in phase I studies.

In a phase IIb study, 171 patients with advanced NSCLC were randomized to receive either a single dose of low-dose cyclophosphamide followed by 8 weekly doses of liposomal BLP25 vaccine or best supportive care.11 Results showed a nonsignificant trend toward improved survival for patients who received liposomal BLP25 (17.2 vs 13.0 months; hazard ratio [HR] = 0&middot;745; 95% CI, 0.533-1.042). Adverse events included flu-like symptoms and injection site reaction, and were generally grade 1-2.

Two international phase III studies of liposomal BLP25 are ongoing.28,29 The START study (Stimulating Targeted Antigenic Responses to NSCLC), NCT00409188, in Europe and the United States, randomized 1300 patients with stage IIIA/B NSCLC to vaccine or placebo after definitive chemoradiation,28 while a similar study in Asia has commenced enrollment.29 Merck Serono, the pharmaceutical company responsible for liposomal BLP25, recently announced that START did not meet its primary endpoint of improving OS; however, detailed results of this study are awaited.12

Belagenpumatucel-L is an allogeneic cell vaccine consisting of four NSCLC lines (2 adenocarcinoma, 1 squamous, and 1 large cell). Allogeneic vaccines do not have the logistical concerns associated with manufacture that are seen with autologous agents such as the prostate cancer vaccine, sipuleucel-T30; however, more uncertainty exists with allogeneic vaccines regarding the degree of true antitumor activity elicited. Belagenpumatucel-L downregulates transforming growth factor-&beta;2 (TGF-&beta;2) by transfecting cells with a TGF-&beta;2 antisense gene, thus enhancing tumor antigen recognition.

TABLE 2. Immune Checkpoint Blockade in NSCLC

Phase II study, phased schedule with chemotherapy showed prolonged irPFS vs chemo alone

First-line, advanced squamous NSCLC, chemo +/- phased ipilimumab32

Phase I study included 121 evaluable, pretreated patients with advanced NSCLC; RR, 16%

Second-line, advanced NSCLC, single-agent chemo vs nivolumab

Phase I study included 49 evaluable, pretreated patients with advanced NSCLC; RR, 10%

NSCLC, nonsmall cell lung cancer; chemo, chemotherapy; CTLA-4, cytotoxic T-lymphocyte antigen-4; irPFS, progression-free-survival by immune response; PD-1, programmed death-1; RR, response rate by RECIST 1.0; PD-L1, programmed death-ligand 1.

Immunotherapy is showing unexpected promise in NSCLC, with numerous large phase III trials under way or planned.

Results from vaccine trials in both the adjuvant and advanceddisease settings are awaited. Important vaccines in development include the MAGE-A3 vaccine, liposomal BLP25 targeting MUC1, an allogeneic vaccine, belagenpumatucel-L, and a vaccine targeting epidermal growth factor.

Agents modulating immune checkpoint molecules are entering phase III investigation in NSCLC, including ipilimumab targeting CTLA-4 and nivolumab (BMS-936558) targeting PD-1.

In a phase II dose-variable study of 75 patients with stage II-IV NSCLC, a response rate of 15% was noted in 61 patients with stage III-IV disease.

OS appeared to be prolonged in patients who received higher doses of the vaccine (OS, 47% at 2 years vs 18% for lower dose), and patients who had a humoral and cellular response to the vaccine had improved OS (32.5 months vs 11.6 months;

= .011). The phase III STOP trial, NCT00676507, is comparing belagenpumatucel-L with placebo for patients with advanced NSCLC who have stable disease or a response after first-line platinum doublet chemotherapy. The primary endpoint of this study is OS.

This study completed enrollment of over 700 patients in 2012.

CIMAvax-EGF is a therapeutic cancer vaccine comprising human recombinant epidermal growth factor (EGF) conjugated to a carrier protein, P64K from Neisseria Meningitides.

After promising phase II results in advanced NSCLC, enrollment was completed on a phase III study, NCT01444118, with results expected within the next year.

To date, agents targeting two immune checkpoint molecules, CTLA-4 and PD-1, have entered phase III clinical trial investigation in NSCLC. In addition, early-phase studies of an antibody targeting the ligand of PD-1, programmed death-ligand 1 (PD-L1), have also shown promising results in NSCLC. Studies to date incorporating these agents are summarized in

CTLA-4 is a transmembrane receptor whose expression is induced by T cell activation, leading to downregulation of T cell responses and suppression of the innate immune response to tumor.

Ipilimumab is a fully human monoclonal antibody that blocks CTLA-4, thus releasing innate antitumor immunity.

In a phase II study, 204 patients with advanced NSCLC were randomized to firstline platinum doublet chemotherapy with placebo versus chemotherapy with concurrent or phased-schedule ipilimumab.

The phased schedule of two induction chemotherapy cycles followed by four cycles of chemotherapy concurrent with ipilimumab met the primary endpoint of improved progression-free survival by immune response (irPFS),

= .05), whereas the concurrent schedule did not. The benefit of ipilimumab in this study appeared to be confined to patients with squamous histology, leading to activation of an ongoing phase III study, NCT01285609, in this cohort.

PD-1 is a coinhibitory molecule, widely expressed on immune cells after antigen exposure, that acts to dampen the T-cellmediated immune response to tumors and other antigens.

In a large phase I study incorporating 296 patients with melanoma, renal cell carcinoma, and advanced NSCLC (76 patients evaluable for response at the time of report), single-agent anti-PD-1 (nivolumab [BMS-936558]) was responsible for a 16% response rate by RECIST 1.0 in patients with heavily pretreated NSCLC.

In addition, 33% of patients with NSCLC were free from tumor progression at 6 months. Responses were noted in nine of 48 patients with squamous NSCLC and 11 of 73 patients with nonsquamous NSCLC, suggesting activity in both subsets. Immunohistochemical expression of PD-L1 on tumor cells appeared to correlate with response to anti-PD-1, with no responses seen in tumors lacking PD-L1 expression; however, only 10 patients with NSCLC were included in this analysis. Pneumonitis was noted as a potential immune-related serious toxicity associated with anti-PD-1 therapy, occurring in 3% of the overall study population, with cessation of therapy and early institution of steroids leading to the resolution of several cases. Currently, a phase III study, NCT01642004, of single-agent nivolumab compared with second-line, single-agent chemotherapy in advanced NSCLC is ongoing.

Several other agents targeting PD-1 are currently either undergoing or in the planning stages of early clinical development, including MK-3475 (Merck), MEDI4736 (Medimmune), MPDL-3280A (Genentech), AMP-224 (Amplimmune/GSK), and AUR-012 (Aurigene).

In a phase I study of anti-PD-L1 (BMS-936559) that included 75 heavily pretreated patients with advanced NSCLC, anti-PD-L1 induced a 10% response rate in the 49 patients evaluable for response; an additional 12% of patients had stable disease lasting more than 6 months.

Strategies aimed at augmenting endogenous antitumor immunity have shown significant promise in earlyphase studies in NSCLC. Results are awaited from large phase III studies of vaccine-based therapies. Immune checkpoint inhibition has led to responses and prolonged remissions for some patients with advanced NSCLC, and phase III studies of anti-CTLA-4 and anti-PD-1 therapies are planned or in progress. Challenges remain, in particular validating biomarkers of response to immunotherapy in order to maximize benefit. Elucidating the optimal sequence and combinations of immune-based treatments with traditional cytotoxic chemotherapy and other targeted agents will be the next step in the development of these exciting new therapies.

Kabbinavar FF, Miller VA, Johnson BE, et al. Overall survival (OS) in ATLAS, a phase IIIb trial comparing bevacizumab (B) therapy with or without erlotinib (E) after completion of chemotherapy (chemo) with B for firstline treatment of locally advanced, recurrent, or metastatic non-small cell lung cancer (NSCLC).

Lara PN Jr, Douillard JY, Nakagawa K, et al. Randomized phase III placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (ASA404) in advanced non-smallcell lung cancer

Cancergrace.org. My Four Key Points from the PointBreak Trial. Available at: http://cancergrace.org/lung/2012/09/07/4-key-points-frompointbreak- trial/. Accessed March 22, 2013.

Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.

Inoue A, Kobayashi K, Maemondo M, et al. Updated overall survival results from a randomized phase III trial comparing gefitinib with carboplatin-paclitaxel for chemo-na&iuml;ve non-small cell lung cancer with sensitive EGFR gene mutations (NEJ002).

Pirker R, Pereira JR, Szczesna A, et al. Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX): an openlabel randomised phase III trial.

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