Published OnlineFirst July 22, 2013; DOI: 10.1158/2326-6066.

CIR-13-0078

Cancer Immunology at the Crossroads: Thoracic Oncology

Cancer
Immunology
Research

Immune Checkpoint Inhibitors: Making Immunotherapy

a Reality for the Treatment of Lung Cancer
Julie R. Brahmer and Drew M. Pardoll

Abstract
Despite the limited success of immunotherapies in solid malignancy, two human cancers, melanoma and renal
cancer, have, for many years, responded to systemic administration of immune-targeted biologics and showed
signals of response to certain therapeutic vaccines. These ndings underpinned a long-held perception that
melanoma and renal cancer were uniquely "immunogenic" but that virtually all other human cancers were not and
thus would not respond to immune modulation. That notion has now been shattered by the signicant and
durable responses in nonsmall cell lung cancer induced by therapeutic treatment with antibodies blocking the
PD-1 checkpoint. The immunotherapy success in lung cancer thus provides a gateway to development of
treatments for multiple cancer types that were previously believed not accessible to immune-based therapies.
Cancer Immunol Res; 1(2); 8591.
2013 AACR.

Introduction

Immune Resistance Mechanisms in Lung Cancer

Even with the approval of immune system-targeted biologics

such as IFN, interleukin-2 (IL-2), and anti-CTLA-4 monoclonal
antibody (mAb) in melanoma and kidney cancer, immunotherapy has, until recently, not had a signicant impact on the
treatment of lung cancer. Despite multiple trials of lung cancer
vaccines, few objective responses were observed and none have
yet shown a clear survival benet in randomized trials (1); the
most recent being a phase III trial of the Liposomal-BLP MUC-1
peptide vaccine given after denitive chemotherapy and radiotherapy in stage III nonsmall cell lung cancer (NSCLC; ref. 2).
As more is learned about the biology of lung cancers and their
immune microenvironment, a number of specic mechanisms
of immune resistance have emerged that are particularly
relevant to T-cell responses. Taken together, these insights,
along with the clinical results from blockade of the programmed death-1 (PD-1) checkpoint (see below) suggest that
a repertoire of tumor-specic or tumor-selective T cells indeed
exists in many patients with lung cancer and this latent pool
can be mobilized therapeutically once specic resistance
mechanisms are blocked. While multiple immune effector
mechanisms, both innate and adaptive, can be brought to
bear against lung cancer, the focus of most translational efforts
is directed at T cells. However, as will be discussed in the last
section, opportunities to activate both innate and adaptive
immune effector mechanisms in concert offer particular promise for the future.

Direct T-cell recognition of tumor cells requires the presentation of antigenic peptides by MHC molecules. These peptides
are generated by proteasomal digestion and transported to the
endoplasmic reticulum, where they are rst loaded onto
nascent MHC molecules, which ultimately transport them to
the cell membrane. A signicant proportion of lung cancers
downregulates components of the antigen-presenting machinery such as the immunoproteasome subunits LMP2 and LMP7,
the antigenic peptide transporters TAP1 and TAP2, and the
MHC molecules. The downregulation is most commonly via
epigenetic mechanisms but it can also involve mutation (35).
These alterations represent fundamental "immune resistance"
mechanisms that help explain how lung and other cancers
evade detection and killing by T cells.
Suppression of the antigen-presenting machinery is likely a
particularly important immune resistance mechanism for
smoking- and pollution-associated lung cancers because these
tumors possess among the highest density of missense mutations in expressed genes of any cancer type (roughly 12 mutations per megabase of expressed exonic sequence; ref. 6). These
genetic alterations, together with activation of many genes due
to epigenetic dysregulation (including induction of cancertestes antigens that are otherwise only expressed on germ
cells), endow lung cancer cells with huge numbers of tumorspecic and tumor-selective neoantigens that are able to be
recognized by T cells. Restifo and colleagues showed that, in
the majority of lung cancer cell lines, suppressed antigenpresenting molecules could be upregulated by IFN-g (5). This
nding is highly relevant to immunotherapy because it suggests that if T cells or NK cells (the two major producers of
IFN-g) could be activated within the tumor microenvironment,
suppression of tumor antigen presentation can be reversed in
the majority of lung cancers.
Given the plethora of potential antigenic targets in lung
cancer, it has also been postulated that they can escape

Authors' Afliation: The Sidney Kimmel Comprehensive Cancer Center at

Johns Hopkins, Baltimore, Maryland
Corresponding Author: Drew M. Pardoll, The Bunting-Blaustein Cancer
Research Building, 1650 Orleans Street, Room G-94, Baltimore, MD
21287-0013. Phone: 410-502-7159; Fax: 410-614-9334
doi: 10.1158/2326-6066.CIR-13-0078

2013 American Association for Cancer Research.

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Brahmer and Pardoll

immune rejection by either "editing" out particularly immunogenic neoepitopes (7) or through the induction of antigenspecic tolerance (8, 9). These mechanisms are quite different:
editing implies that T-cell recognition of a tumor neoantigen
has resulted in selection for antigen-loss variants, whereas
tolerance induction implies that tumor-specic T cells have
been rendered incapable of attacking antigen-bearing cells.
Evidence for both processes has been produced in a murine
model of lung carcinogenesis created by pulmonary instillation
of a replication-defective lentivirus encoding cre plus a foreign
antigen into mice bearing an oncogenically mutated K-ras gene
whose promoter contains a lox-stop-lox cassette. In this model,
only infected pulmonary epithelial cells transform and express
the foreign antigen as a tumor-specic neoantigen (10). Transfer
of T cells specic for "neoantigens" into these mice early after
transformation can induce editing, whereas T cells transferred
later can slow tumor growth, but eventually the transferred T
cells are rendered tolerant and ultimately deleted from the
tumor microenvironment. The relative importance of editing
versus tolerance induction in human lung cancer remains to be
determined. A subset of small cell lung cancers does elicit CD4dependent antibody and even CD8 T-cell responses against
neuronal antigens ectopically expressed by this tumor type
(11, 12). These antineuronal antigen responses are generally
associated with paraneoplastic neurologic syndromes (PNS)
that are thought to be mediated by the induced immune
responses. The presence of PNS is frequently associated with
limited disease, better prognosis, and rarely spontaneous regressions of this normally aggressive cancer type (13).
In NSCLC, the most therapeutically relevant mechanism for
immune resistance is expression of immune inhibitory molecules in the tumor microenvironment. These molecules fall
into a number of classes based on the nature of the inhibitory
ligand: cytokines, membrane ligands, and metabolites. The two
inhibitory cytokines commonly expressed in lung cancers are
IL-10 and TGF-b. Among the membrane inhibitory ligands (socalled checkpoint ligands), PD-L1 has been the most studied in
NSCLC, though PD-L2, B7-H3, and B7-H4 have also been
reported as upregulated in lung cancer (14, 15). PD-L1 is
expressed on tumor cells in roughly half of NSCLC but is
sometimes expressed on myeloid cells in the stroma surrounding tumor nests. Grossly, increased numbers of CD4 and
CD8 tumor-inltrating T-lymphocytes (TIL) have been
reported to be a good prognostic factor in lung cancer, whereas
increased numbers of Foxp3 TIL have been reported to be a
poor prognostic factor (16, 17); these are relatively crude
analyses and much more work needs to done to assess the
biologic and therapeutic relevance of expression patterns of
multiple inhibitory ligands as well as the distribution and
expression pattern of their cognate receptors on TILs. Though
not often considered, immune inhibitory metabolites are likely
important players in local immune resistance in lung cancer.
Concentrations of adenosine, which binds to the inhibitory Gprotein-coupled A2a receptor expressed on lymphocytes, have
been shown to be extremely high in NSCLC tissue. A2aRtriggering both inhibits effector T-cell function and drives the
development of Tregs, another inhibitory component of the
tumor microenvironment (18, 19).

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Cancer Immunol Res; 1(2) August 2013

Relevant to the immune inhibitory ligand-receptor interactions in the tumor microenvironment, a major turning point
in cancer immunotherapy came with the clinical application
of antibodies that block immune checkpoints. Of the many
molecularly dened checkpoint ligands and receptors
(reviewed in refs. 14, 15), blockers of only two pathways,
CTLA-4 and PD-1, have been tested clinically to date. In the
case of the PD-1 pathway, both PD-1 and one of its ligands, PDL1, have been blocked. These two checkpoints seem to modulate very distinct components of T-cell immunity. CTLA-4
counterbalances the costimulatory signals delivered by CD28
during T-cell activationboth bind the B7 family ligands, B7.1
and B7.2 (reviewed in ref. 20; Fig. 1). PD-1 is also induced upon
T-cell activation but seems to predominantly downmodulate
T-cell responses in tissues. The PD-1 ligands, PD-L1 and PD-L2,
are induced by distinct inammatory cytokineswhile PD-L1
expression can be induced on diverse epithelial and hematopoietic cell types, PD-L2 is predominantly expressed on dendritic cells (DC) and macrophages (2124). Both CTLA-4 and
PD-1 pathway-blocking antibodies have shown activity in lung
cancer and, as mentioned above, the responses to anti-PD-1
and anti-PD-L1 monotherapy in NSCLC have garnered significant attention by the lung cancer community (reviewed in
ref. 1).

CTLA-4 Inhibitors in Lung Cancer

In contrast with melanoma (25), ipilimumab, an anti-CTLA4 blocking mAb has virtually no effect as a single agent in lung
cancer (26); however, it does seem to provide modest benet in
NSCLC as well as small cell lung cancer (SCLC) when tested in
combination with chemotherapy. A randomized phase II trial
of two different schedules of combining ipilimumab with
standard chemotherapy compared with standard combination
chemotherapy (paclitaxel and carboplatin) alone was reported
recently. Patients with advanced NSCLC and SCLC were randomized to paclitaxel and carboplatin versus a "phased"
schedule of chemotherapy alone for 2 cycles followed by
ipilimumab combined with the chemotherapy regimen for 4
additional cycles versus concurrent administration of chemotherapy with ipilimumab upfront for 4 cycles followed by 2
cycles of chemotherapy alone (27). If patients had stable or
responding disease, patients were given ipilimumab "maintenance" once every 12 weeks until progression.
Evaluation of patient responses and progression-free survival (PFS) used immune-related response criteria, which take
into account tumor regression in the face of new lesionsa
unique pattern of response that can be seen with checkpoint
blockade. The study showed that addition of ipilimumab
improved immune-related PFS (ir-PFS) in patients with
advanced NSCLC. On the basis of the immune mechanism of
action of ipilimumab, the immune-related grades 3 and 4 side
effects were increased in the two ipilimumab-containing arms;
however, the overall side effects were not signicantly different. The phased schedule signicantly improved the ir-PFS
compared with that of the control arm (median 5.7 months vs.
4.6 months respectively, HR 0.72; P 0.05), whereas the
concurrent schedule did not improve the ir-PFS or overall

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Figure 1. Distinct roles of CTLA-4 and PD-1 in the regulation of antitumor T-cell responses. CD28 is the master costimulatory receptor expressed on T cells and
enhances T-cell activation upon antigen recognition when the antigen presenting cell (APC) expresses its ligands, B7-1 and B7-2. Tumor or tumor
vaccine is the source of tumor antigens that must be processed and presented by the MHC complex to activate T cells. CTLA-4 is rapidly expressed on
T cells once antigen is recognized, and it binds the same ligands (B7.1/2) as CD28 but at higher afnity, thereby counterbalancing the costimulatory
effects of CD28 on T-cell activation. Tumor-specic T-cell activation leads to proliferations and effector function, but also the upregulation of PD-1. After
trafcking to the tumor microenvironment, PD-1 T cells might encounter PD-1 ligands, which can inhibit them from mediating their killing function. Thus, the
CTLA-4 and PD-1 pathways provide complementary mechanisms to regulate antitumor effector T cells, and blocking each one may prove to be synergistic.

survival (OS). The overall survival for the phased arm was 12.2
months compared with 8.3 months in the control arm but this
difference did not achieve statistical signicance. Interestingly,
in the phased schedule group, the ir-PFS was signicantly
improved in patients with squamous cell histology and not in
patients with nonsquamous histology (mostly adenocarcinoma). The OS was only improved in the patients with squamous
histology. Alternatively, for the nonsquamous histology group,
the phased schedule resulted in worse OS compared with
standard chemotherapy [HR 1.17; 95% condence interval
(CI), 0.741.86]. On the basis of these phase II data, patients
with squamous cell histology are being recruited for a phase III
trial of the phased schedule combining ipilimumab with
chemotherapy versus chemotherapy alone (paclitaxel and
carboplatin) for rst-line treatment.
A separate ongoing study is comparing the phased schedule
of ipilimumab combined with cisplatin and etoposide in the
rst-line treatment setting for SCLC. Ipilimumab's phase III
study in SCLC was based on the same trial above but in an
extensive stage SCLC population (28). The trial endpoint of irPFS was improved in the phased schedule arm with a median of
6.4 months compared with chemotherapy alone with a median

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of 5.3 months (HR 0.64; 95% CI, 0.401.02; P 0.03), although

OS was not improved. The immune-related side effects were
increased in the two ipilimumab arms but the discontinuation
rate based on treatment-related side effects was similar across
the arms.

Anti-PD-1 Inhibitors
While the anti-CTLA-4 antibodies have not resulted in
signicant single-agent responses, PD-1 checkpoint blockade
with blocking antibodies targeted to either the receptor or its
major ligand, PD-L1, have resulted in signicant single-agent
activity in advanced, heavily pretreated patients with NSCLC,
in terms of OR, stable disease (SD), and associated long-term
survival. Several antibodies have been developed to either
block the PD-1 receptor or its ligand PD-L1. The anti-PD-1
antibodies [Nivolumab (BMS-936558/MDX-1106/ONO-4538)
or Lambrolizumab (MK3475)] block the binding of PD-1
receptor to its 2 ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC).
The rst-in-human, single dose phase I study of Nivolumab
in refractory solid tumors revealed no maximum tolerated
dose (MTD) and initial durable activity in solid tumors (29). A

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two-week dosing schedule was explored in the next phase I trial

(30). Because of initial signs of activity in patients with NSCLC
in the dose-escalation phase of the trial, expansion cohorts of
patients with lung cancer were randomized to one of three
doses, 1, 3, 10 mg/kg once every two weeks. 129 patients with
NSCLC were enrolled in the trial of which 54% had received 3
previous therapies. The most common side effects in the lung
cancer cohort were fatigue (24%), decreased appetite (12%),
and diarrhea (10%), which were similar to the full study
population. Grade 3 or 4 treatment-related select adverse
events were seen in 14% of patients with NSCLC. Eight patients
(6%) with NSCLC developed pneumonitis, 3 of which were
grade 3 or 4. Two patients with NSCLC died from pneumonitis
on this trial. On the basis of the role of the PD-1 pathway in
downmodulating tissue inammation, it is generally believed
that organ-specic immune toxicities observed in patients
receiving blockers of this pathway reect underlying subclinical inammation that is exacerbated upon initiation of therapy. The lung is a major organ site in which immunity must be
carefully modulated, as excessive tissue destructive inammation from responses to inhaled microbes will compromise
oxygen exchange. Because NSCLC is commonly associated
with chronic (chronic obstructive pulmonary disease) and
acute (i.e., post-obstructive) pneumonia, oncologists must pay
special attention to this toxicity in this patient population.
Of the 122 patients with NSCLC evaluable for response on
this trial, 22 (17%) achieved a partial response (PR) based on
RECIST 1.0 criteria and 10% had stable disease at 24 weeks.
Unconventional immune-related responses occurred in an
additional 5% of patients. While the adenocarcinoma subset
of NSCLC shows reasonable initial responses to both chemotherapy and targeted tyrosine kinase inhibitors (TKI; when
matched to appropriate driver oncogene mutations), squamous cell carcinoma is highly refractory to virtually all chemotherapies and TKIs. In contrast, Nivolumab activity in both
NSCLC histologies is similar, with antitumor effects observed
particularly at the 3 mg/kg dose level. The response rate was
28% (5/18) for nonsquamous histology and 27% (4/15) for
squamous histology at this dose.
Beyond the signicant response and disease stabilization
rate in advanced chemotherapy-refractory patients, the durability of responses was unprecedented. Median duration of
response was 74 weeks, compared with 48 months for chemotherapy regimens and oncogene-targeted TKI. Even stable
disease was durableroughly half of the patients with stable
disease 24 weeks maintained stable disease beyond 48 weeks
at the time of most recent evaluation. These durable responses
were associated with improved survival outcomes relative to
reports of other salvage therapies applied to this population of
advanced inoperable and multiply pretreated patients: 1 year
and 2 year overall survival was 42% and 14%, respectively (31).
Taken together, the durable responses, unique to immunotherapy, support the long held concept within the cancer
immunology community that immune modulators may reset
the patient's endogenous tumor-specic T-cell immunity in a
fashion that allows it to adapt to the evolution of the tumors
genetics, something that tumor-targeted drug therapies
cannot do.

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Given that the PD-1 pathway seems to mediate tumor

immune resistance at the level of the tumor microenvironment,
expression within the tumor of the major PD-1 ligand, PD-L1 has
been explored as a potential biomarker for response to blockade
of this pathway. As part of the initial phase I trial and the
expanded trial reported in 2012, PD-L1 expression by tumor cells
was assessed in a subset of the patients. Among all histologies
tested in the second trial, 0 of 17 patients whose tumors were PDL1(-) and 11 of 25 patients whose tumors were PD-L1 (dened
as >5% of tumor cells displaying a clear membrane pattern of
expression on immunohistochemistry), showed responses to
anti-PD-1. Of these cases, 10 were NSCLC. Of the 10 NSCLC
patients evaluated, 0 of 5 patients whose tumors were PD-L1(-)
and 1 of 5 patients whose tumors were PD-L1 showed
responses to anti-PD-1 (30). Preliminary results correlating
PD-L1 expression in NSCLC with response to the anti-PD-L1
antibody MPDL3280A (see below, refs. 3234) showed that 4 of 4
PD-L1 tumors showed an objective response in contrast to
only 4 of 26 PD-L1(-) tumors. Taken together, the results of the
two studies suggest that PD-L1 expression in lung cancer may be
predictive of response to blockade of this pathway but these
numbers are clearly too small to draw any denitive conclusions.
There is ongoing analysis of both PD-L1 and PD-L2 expression in
follow-up trials to determine their suitability as biomarkers for
patient selection in NSCLC.
The encouraging results of the reported clinical trials have
led to two separate phase III trials of Nivolumab as a single
agent compared with docetaxel (35). One trial is enrolling only
patients with nonsquamous histology in the second- or thirdline treatment setting with a goal to improve OS. In the other
trial with coprimary endpoints of OS and response rate,
patients with squamous cell histology after one prior platinum
combination chemotherapy are being enrolled. In addition to
these pivotal phase III trials, a single-arm study of Nivolumab
in the third-line treatment setting and beyond in only squamous histology patients has recently nished recruitment. In
addition to single-agent trials, multiple treatment-arm phase I
trials in NSCLC combining Nivolumab with various chemotherapy regimens as well as with ipilimumab have been
launched. The combination of Nivolumab and ipilimumab
makes sense given the distinct roles of the CTLA-4 and PD1 pathways in regulating the initiation and execution of
immune responses within tumors; in additon, a recent phase
I trial testing this combination in patients with melanoma was
highly encouraging (36). A second anti-PD-1 monoclonal antibody (Lambrolizumab) was recently reported to manifest
substantial antitumor effects in patients with advanced melanoma (37); testing is underway in NSCLC and the results
should be of great interest.

Anti-PD-L1 Antibodies
Three blocking anti-PD-L1 antibodies, BMS-936559,
MPDL3280A, and MedI-4736 have been or are being evaluated
in NSCLC. There has been much debate about the relative
merits of antibodies directed at PD-1 versus PD-L1. Antibodies
directed to PD-1 block its binding to both known PD-1 ligands,
PD-L1, and PD-L2, whereas anti-PD-L1 antibodies only block

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the PD-1:PD-L1 interaction. However, another unexpected

inhibitory interaction between PD-L1 and B7.1 (with B7.1
acting as an inhibitory receptor on T cells) has been described;
this interaction is blocked by anti-PD-L1 antibodies but not
anti-PD-1 antibodies. While the PD-L1:PD-1 interaction is
considered the most important mediator of tumor immune
resistance, the importance of the PD-L2:PD-1 and PD-L1:B7.1
interactions in human cancers is not well studied and could
play a role in distinguishing the clinical activities of anti-PD-1
versus anti-PD-L1 antibodies. It is also possible that these
additional interactions may be important in organ-specic
immune modulation, which could impact relative immune
toxicities of the two classes of antibody.
The rst-in-human trial of BMS-936559 resulted in no MTD
being identied, and in general the antibody was well tolerated
(38). Only 6% of patients stopped treatment due to drug-related
side effects and there were no drug-related deaths. Initial
activity was seen in melanoma, renal cell carcinoma, ovarian
cancer, and NSCLC. Of the 49 patients with lung cancer that
were evaluable at the time of reporting, 5 patients (10%) had a
response to therapy. The duration of response was long lasting
(2.3 months up to 16.6 months and ongoing). The stable disease
rate after 6 months of treatment was 12%. Results from 37
patients with NSCLC (both squamous and non-squamous
histologies included) treated with MPDL3280A and evaluable
for response showed a 24% response rate with additional
immune-related responses (progression before regression or
new lesions in the face of overall decrease in tumor burden;
ref. 32). However, results with the two anti-PD-L1 antibodies
cannot be directly compared as the patient population in the
MPDL3280A trial was partially enriched for tumor expression
of PD-L1. Given that multiple anti-PD-1 and anti-PD-L1 antibodies are under development, an important question is
whether there are fundamental efcacy and toxicity differences between antibodies targeting the ligand versus the
receptor. While Nivolumab and BMS-936559 (no longer in
development) were never compared in a randomized trial, the
similarities in patient characteristics between the two trials
reported simultaneously in 2012 suggest that anti-PD-L1 is
somewhat less active than anti-PD-1 but may also be associated with slightly lower toxicity (30, 38). Ongoing studies with
the anti-PD-L1 antibodies MPDL3280A and MedI-4736 as well
as the anti-PD-1 antibodies Nivolumab and Lambrolizumab
will ultimately shed light on whether there are target-specic
differences in activity or toxicity in NSCLC.

The Future: Beyond Single-Agent

Immunotherapy
Now that the door for immunotherapy in lung cancer has
been opened, the future lies in application of immunotherapies
at earlier stages of lung cancer, introduction of modulatory
antibodies and drugs against additional targets (both costimulatory pathway agonists and coinhibitory pathway antagonists), and nally, development of combinatorial therapy.
Ultimately, proling of the tumor microenvironment will
provide biomarkers to guide precision immunotherapy. Multiple studies are trying to capitalize on the single-agent activity

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of checkpoint blockers, particularly PD-1 pathway blockers, by

combining these antibodies either with each other, vaccines,
tyrosine kinase inhibitors, or even standard chemotherapy
(Fig. 2). One could envision checkpoint blockade being an
option alone or in combination at any stage of the treatment
algorithm, though care in scheduling driven by preclinical
modeling and treatment science will be critical. Even though
vaccines have yet to show activity in lung cancer, new vaccine
formulations have been shown in animal models to display
increased potency and combinations of vaccines and checkpoint inhibitors show the most potent synergy of all in multiple
preclinical models. It is likely that the lack of activity of
vaccines used as single agents in lung cancer is related to the
inability of vaccine-enhanced T-cell responses to overcome
intratumoral checkpointsthis notion underpins the value of
vaccine-checkpoint inhibitor combinations.
Another highly promising opportunity in lung cancer comes
from the signicant proportion of adenocarcinomas that
possess oncogene mutations that confer susceptibility to
targeted TKI. Four mutationsEGFR, ALK, BRAF, and ROS1,
are druggable and collectively comprise 25%30% of adenocarcinomas. While the response rate to targeted TKI in patients
whose tumors bear these mutations is rapid and frequent,
resistance also develops rapidly. Given that the mutant oncogene-specic TKIs would not be expected to inhibit T-cell
responses (this has been shown for the BRAF inhibitors), it
makes sense that the initial tumor lysis driven by the TKI would
provide a large release of tumor antigen that could prime
immune responses, and that could kill residual tumor when
checkpoints are simultaneously blocked.
Opportunities to leverage immune effects of many therapeutic strategies abound. One such example in development in
lung cancer is using epigenetic therapy (i.e., DNA-demethylating agents and HDACi) before Nivolumab treatment to prime
the tumor to become more responsive to immunotherapy. This
concept is based on both preclinical ndings as well as the
recent clinical observation that patients with lung cancer that
rst received a combination of 5-Aza-CR (a DNMT inhibitor
that induces gene expression via demethylation of promoter
regions) and an HDACi followed by either anti-PD-1 or antiPD-L1 had signicant benet from the checkpoint blockade. Of
the 5 patients treated this way, 3 had durable PRs ongoing for
over a year and the other 2 patients had stable disease on
therapy for at least 6 months. Preclinical data in a set of lung
cancer lines show that the mechanism by which epigenetic
priming of immunotherapy could improve outcomes may
involve upregulation of a broad but related set of genes
associated with IFN-signaling, antigen processing, and presentation as well as genes encoding cancertestes antigens. In
addition, epigenetic treatment of these lines increased expression of PD-L1 (39). Thus, a combination of this epigenetic
therapy together with PD-1 blockade may shift the balance
toward enhanced adaptive and innate immune responses
within the tumor microenvironment, a hypothesis being tested
prospectively in a combination clinical trial.
Results from the past 3 years have not only validated the
clinical potential for immunotherapy but also the sense that we
are just scratching the surface. Lung cancer, the number one

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Figure 2. Potential mechanisms of therapeutic synergy between epigenetic modulation and PD-1 pathway blockade. Epigenetic modulation, originally tested
in lung cancer based on its capacity to induce expression of epigenetically silenced tumor suppressor genes, also has signicant immunologic activity.
Treatment of lung cancer cells with the DNA-demethylating agent 5-azacytidine (AZA) can induce the type I IFN pathway as well as multiple components of
antigen presentation, thereby enhancing intratumoral inammatory responses. However, PD-L1 is also induced on tumor cells, which could blunt immunity.
Concomitant blockade of the PD-1 pathway would shift the balance such that the immune-enhancing effects of epigenetic modulation would dominate. DNA
demethylation also induces expression of cancertestes (C-T) antigens, which are known targets for tumor-specic T cells. Finally, epigenetic modulation can
activate silenced effector cytokine genes in anergized T cells and induce PD-1 expression. Again, concomitant PD-1 pathway blockade would favor the
immune-enhancing effects of epigenetic modulators on T cells. These potential mechanisms of synergy may account for recent preliminary clinical
observations in 5 patients with NSCLC treated with either anti-PD-1 or anti-PD-L1 antibodies after receiving a combination of 5-azacytodine and entinostat
(a class 1-specic HDACi); durable objective responses were observed in 3 patients and stable disease for more than 6months in the other two patients.
This combination approach is a potential example of leveraging the immunologic effects of "nonimmunologic" therapies.

cancer killer in the United States and worldwide, has been the
frontier for immunotherapy's leap beyond the melanoma and
renal cancer and will continue to provide vistas for future
innovation in this burgeoning eld.
Disclosure of Potential Conicts of Interest
J.R. Brahmer has a commercial research grant from Bristol Myers Squibb and
is a consultant/advisory board member of Bristol Myers Squibb, Merck, and Eli
Lilly. D. Pardoll receives no monetary remuneration or equity from companies
mentioned in this article. He is coinventor of patents covering therapies
mentioned in this article.

Authors' Contributions
Conception and design: J.R. Brahmer, D. Pardoll
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J.R. Brahmer, D. Pardoll
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): J.R. Brahmer
Writing, review, and/or revision of the manuscript: D.M. Pardoll, J.R.
Brahmer

Received June 20, 2013; accepted June 20, 2013; published OnlineFirst July 22,
2013.

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Published OnlineFirst July 22, 2013; DOI: 10.1158/2326-6066.CIR-13-0078

Immune Checkpoint Inhibitors: Making Immunotherapy a Reality

for the Treatment of Lung Cancer
Julie R. Brahmer and Drew M. Pardoll
Cancer Immunol Res 2013;1:85-91. Published OnlineFirst July 22, 2013.

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