Immunotherapy for Lung Cancer

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					Immunotherapy for Lung Cancer
Edward A. Hirschowitz1,2 and John R. Yannelli3
1
 Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Kentucky, Chandler Medical Center,
Lexington, Kentucky; 2Lexington Veterans Administration Medical Center, Lexington, Kentucky; and 3Department of Microbiology,
Immunology, and Human Genetics, University of Kentucky, Lexington, Kentucky


Immunotherapy is a conceptually attractive approach, because it is                   trastuzumab (Herceptin), which target epidermal growth factor
highly specific and can deal with disseminated disease with minimal                   (EGF) receptors, HER-1 and HER-2, respectively, and bev-
impact on normal tissues. Ability to induce antigen-specific immune                   acizumab (Avastin), which interferes with tumor angiogenesis
responses in patients with lung cancer is now well established in                    by binding to vascular endothelial growth factor (1–4). Adop-
early-phase clinical trials using a variety of immunotherapeutic                     tive transfer is another example of passive immunotherapy that
approaches. Although no immunotherapy is likely to be a panacea,                     typically involves ex vivo amplification and infusion of autolo-
randomized phase IIB studies offer promise of therapeutic activity in                gous tumor-infiltrating T cell or lymphokine-activated killer cell
both early- and late-stage lung cancer. This review will cover basic
                                                                                     therapy (5). By contrast, active immunotherapy uses the host’s
concepts of immunotherapy, provide perspective on vaccine devel-
                                                                                     immune cells and requires an intact immune system to function.
opment, and update the status of ongoing clinical trials in lung
                                                                                     Active immunotherapy is derived from the knowledge that the
cancer.
                                                                                     immune system can discriminate cancer cells from normal cells
Keywords: immunotherapy; vaccines; lung cancer; clinical trials                      based on tumor antigen recognition (6–9). Approaches that
                                                                                     directly incorporate tumor antigen are conventionally referred
                                                                                     to as vaccines. Although some active immunotherapies are
INTRODUCTION                                                                         designed to induce antibodies as the primary effector mecha-
                                                                                     nism, and may be rationally applied with certain logical in-
A number of nonconventional therapeutic modalities are being                         tention, cancer-specific antibody responses are widely viewed as
developed to improve unacceptably poor outcomes in lung                              having limited direct cytotoxic capability against cancer. With
cancer. Immunotherapy is an attractive approach that, by                             few exceptions, induction of antigen-specific T-cell responses is
design, is cancer specific and can target disseminated disease                        the primary objective of active immunotherapy (6–10). Natural
with minimal impact on normal tissues. Active immunotherapy                          killer (NK) cells are an antigen-independent arm of immune
describes those approaches that use host immune machinery to                         defense that targets major histocompatibility complex (MHC)-
generate antitumor effects. This review presents basic concepts                      mismatched (allogeneic) cells or those cells lacking surface
of active immunotherapy, provides perspective on therapeutic                         MHC molecules. Because cancer cells often downregulate
development, and updates the status of ongoing clinical immu-                        surface MHC molecules, induction of NK is a logical, but not
notherapy trials in lung cancer.                                                     common, primary objective of immunotherapy (11).

IMMUNOTHERAPY AND IMMUNOBIOLOGY                                                      Immune Recognition
Immunotherapy                                                                        Identification of tumor-associated antibodies and antigen-
                                                                                     specific cytotoxic T lymphocytes (CTL) in patients with lung
Novel therapies should improve outcomes, but have a side-
                                                                                     cancer indicates that the immune system can distinguish lung
effect profile that is more favorable than conventional chemo-
                                                                                     cancer cells from normal cells (6, 7). Antigen-specific immune
therapy (1). A number of therapeutic approaches are being
                                                                                     responses are dependent on antigen presentation in the patient’s
developed that selectively target malignant cells or their
                                                                                     lymphoid tissues. When antigen is contacted, antigen-presenting
microenvironment, but leave normal cells intact. An ideal agent
                                                                                     cells (APCs) digest whole proteins into smaller peptides that are
should also be easy to administer to address both quality-of-life
                                                                                     then presented on HLA class I or class II molecules on the APC
issues and compliance concerns. Although the term ‘‘targeted
                                                                                     surface. Coordinated expression of costimulatory molecules
therapy’’ has been most widely applied to pharmacologic
                                                                                     (CB80/CD86) on the APC surface prompts antigen-specific
agents, including small molecule inhibitors and antisense oligo-
                                                                                     lymphoid precursors to activate at appropriate times. Depending
nucleotide, immunotherapy closely fits the definition.
                                                                                     on the type or source of antigen and the existing cytokine milieu,
    Immunotherapy is categorized as either passive or active.
                                                                                     class II peptides may stimulate IL-2 and IFN-g release by antigen-
Passive immunotherapy includes any immunologically active
                                                                                     specific CD41 T cells (Th1). Antigen-specific CTL are generated
agent that is made outside the body and does not rely on host
                                                                                     when CD81 T cells bound to class I antigens on APCs are
machinery to function. The most widely applied passive immu-
                                                                                     stimulated by Th1 cytokines. Alternatively, class II antigen
notherapies are monoclonal antibodies that disrupt tumorigenic
                                                                                     binding may induce a different T-cell phenotype (Th2) that
cascades by blocking the binding of hormones or growth factors
                                                                                     releases IL-4 and IL-10, and interacts with B cells to promote
to their receptors; examples include cetuximab (Erbitux) and
                                                                                     antigen specific antibody production. While Th1 and Th2 are not
                                                                                     necessarily mutually exclusive in the immune response, Th2-
                                                                                     related cytokines tend to suppress Th1-mediated responses.
(Received in original form June 16, 2008; accepted in final form July 21, 2008)       Notably, activated Th cells have longevity compared with
Correspondence and requests for reprints should be addressed to Edward A.            other activated immune cells (APCs and CTL), and are conven-
Hirschowitz, M.D., Division of Pulmonary and Critical Care Medicine, University of   tionally viewed as being responsible for ‘‘immune memory,’’
Kentucky, Chandler Medical Center, 740 S. Limestone, Room K528, Lexington,
                                                                                     capable of ‘‘revitalizing’’ the immune response if a specific
KY. E-mail: eahirs2@uky.edu
                                                                                     antigen is reencountered; memory subsets of CD81 T cells and
Proc Am Thorac Soc Vol 6. pp 224–232, 2009
DOI: 10.1513/pats.200806-048LC                                                       B cells provide other mechanisms for rapid response to known
Internet address: www.atsjournals.org                                                antigens.
Hirschowitz and Yannelli: Immunotherapy for Lung Cancer                                                                              225

Immune Regulation and Tumor Evasion                                   and confounding comparability within the study population,
Physiologic checks and balances, that work throughout the             both of which can hinder rational therapeutic development in
immune cascade to control antigen hyperresponsiveness, create         early clinical testing. Nonetheless, autologous vaccines have
a permissive environment for tumor growth (9, 11, 12). Immu-          been shown to have immunologic activity in a number of studies
nosuppressive cytokines, APC senescence, and regulatory T             (10, 11). Allogeneic tumor is a rational alternative that can
cells (T-regs) each present an important challenge to successful      provide a renewable source of antigen and offers greater
development of immunotherapy (9, 11–13). Most established             potential for ‘‘off-the-shelf’’ application. Using allogeneic anti-
tumors also have mechanisms of immune evasion (9, 11, 12).            gen also generates a uniform preparation, which facilitates
The production and induction of immunosuppressive cytokines           immune assessment and comparability not afforded by use of
by tumor cells, such as IL-10 and transforming growth factor-b,       autologous tumor antigen. Availability of these tangible mile-
has been well characterized (12). Some tumor cells may avoid          stones makes this, and other allogeneic approaches, attractive
immune destruction by down-regulating HLA class I molecules           during therapeutic development and clinical testing.
on which antigens are presented for recognition, or by over-             Two additional allogeneic sources of antigen are synthetic
expressing B7-H1, a ligand for the T-cell receptor, PD-1, which       peptide and recombinant protein (including gene therapy–
is known to negatively regulate T-cell activation. Defined             derived antigen). In contrast to allogeneic tumor, peptides and
mechanisms of CTL resistance and counter defense include              proteins are characteristically used in monovalent formulations.
tumor expression of the apoptosis-inducing molecule, Fas              Peptides are an attractive monovalent antigen source, because
ligand, and downregulation of surface Fas receptors (12).             they are small and easily synthesized, are uniform, and provide
                                                                      the simplest and most reproducible immunologic measures of
                                                                      biological efficacy (11). A disadvantage is that peptides are
CANCER ANTIGENS
                                                                      exclusive to specific HLA types and require patient selection
There are multiple incarnations of antigen used in active             based on HLA tissue typing. Furthermore, peptides have de-
immunotherapy, each with independent advantages. A major              signated restriction to class I or class II pathways, selectively
division in antigen targeting is the use of a multivalent or          stimulating either CD81 cytotoxic T-cell effectors or CD41 Th
monovalent strategy. By targeting several antigens, multivalent       cells, responsible for immune memory (11). By contrast, whole
approaches lend themselves to many of the clinical and bio-           recombinant proteins are processed into multiple peptides and
logical realities of lung cancer. Specifically, phenotypic hetero-     presented by APCs via class I and class II pathways to CD41 and
geneity characteristic of non–small cell lung cancer (NSCLC)          CD81 T cells, respectively, and have the potential for generating
and small cell lung cancer (SCLC) dictates high variability of        a response that includes immune effectors and immune memory.
antigen expression, and precludes the ideal circumstance of one          Xenogeneic antiidiotype antibodies are somewhat unique
antigen being uniformly expressed by all lung tumors. Addi-           antigen-mimic preparations, generated as antibodies to tumor
tionally, because a vast majority of patients with lung cancer        antigen–binding sites on other antibodies (that generates a tem-
are nonsurgical, most lung cancer diagnoses are made with             plate of the antigen). The foreign (xenogeneic) nature of these
minimal tissue sampling, and the material is often too limited        preparations makes them inherently immunogenic, and the
for adequate antigen characterization; in context, multivalent        similarity of the antiidiotype antibody to the tumor antigen
approaches circumvent the need to know which antigens are             allows cross recognition of the parent/native protein (14). In
expressed by a specific tumor. By contrast, monovalent applica-        contrast to the vast majority of the other active immunothera-
tions all have the common feature of being selectively delivered to   pies under development, antiidiotypic vaccines are used to elicit
individuals with corresponding tumor antigen expression, which        tumor-specific antibodies as the dominant effectors for thera-
distinctly requires individual tumor antigen characterization.        peutic activity; these have been the most widely tested immu-
Notably, monovalent approaches are more readily incorporated          notherapy approaches in SCLC.
into proprietary products, and lend themselves to commercial
development; not surprisingly, a majority of therapies advancing      PROMOTING ANTIGEN RECOGNITION: IMMUNE
to phase III investigation are monovalent approaches.                 ADJUVANTS AND DELIVERY
   The most commonly used multivalent formulations employ
autologous or allogeneic tumor cells, although multiprotein/-         Success of any active immunotherapy ultimately depends on
peptide mixtures or fusion constructs can achieve similar             tumor antigen capture and presentation by APCs (11, 15).
multivalent objectives. Tumor-derived antigen mixtures include        Tumor antigens, however, are not, in themselves, immunogenic.
multiple dominant and minor antigenic determinants within             In order to initiate or promote antigen-specific responses, all
whole proteins, allowing the host to select, process, and present     strategies incorporate adjuvants; these are essentially activating
on MHC, the most immunogenic epitopes relevant to that                agents or stimulants for various arms of the immune cascade,
individual (11). Autologous tumor can be utilized either              intended to augment antigen recognition, uptake, presentation,
in vivo or ex vivo. The former relies on a locoregional or            and/or antigen-specific cellular reactivity (11, 15). Effects of
systemic intervention to promote antigen recognition in situ.         various adjuvants are not mutually exclusive, and mechanisms
One example of this is the systemic administration of cytokine;       often overlap and intersect. There are myriad choices that fall
alternatively, if the tumor bed can be directly accessed, gene        into one of several categories: (1) biologic and chemical
therapy strategies can be used to modify tumor cell antigenicity      adjuvants; (2) recombinant cytokines and chemokines; (3) auto-
or alter the tumor microenvironment. Ex vivo approaches most          logous dendritic cells (DCs); (4) immune modifiers; (5) gene
commonly combine surgically obtained tumor with an immune             therapy/gene transfer vectors.
adjuvant to produce a cancer vaccine. A disadvantage of using
autologous tumor in vaccines is that adequate amounts of fresh        Chemical and Biologic and Adjuvants
tumor must be available for vaccine production, generally             These adjuvants have been used for decades as a key compo-
restricting this approach to the individual whose tumor is            nent in antigen-specific immunotherapy to induce inflamma-
surgically resected with the forethought and intent of making         tion, either at the site of tumor, or used in conjunction with
a vaccine. An additional limitation is that antigens differ for       exogenously supplied antigen. Biologic adjuvants take advantage
each subject, thereby complicating immunological assessment           of the fact that they are immunogenic compounds, naturally
226                                                                     PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 6              2009


recognized as foreign and known to induce migration of APCs to           to cytotoxic therapy, where other effects may be unique to
the site of delivery. APCs responding to adjuvant stimulation are        specific chemotherapeutic classes or agents. It has been postu-
thus able to coincidentally capture and process tumor antigens           lated that apoptosis of tumor cells induced by most commonly
present in the inflammatory milieu. The most commonly used                used cytotoxic therapies somewhat paradoxically stimulates the
                                                     ´
biologic adjuvants include bacillus Calmette-Guerin (BCG),               immune system (25–27). Therapy may also mitigate inhibitory
diphtheria toxoid, and tetanus toxoid (11, 15). Chemical adju-           cytokine production in the local milieu, or induce proinflamma-
vants function similarly to biologic adjuvants as irritants that         tory signals that augment APC numbers and function or promote
induce an inflammatory response at the site of delivery, and some         generation of antigen-specific CTL (25, 26, 28). The literature
may also provide a matrix that sequesters antigen at a specific           also describes several drug-related phenotypic alterations that
location, allowing a timed release of antigen to APCs. Examples          lead to increased susceptibility to CTL lysis, including the in-
include aluminum hydroxide, montanide ISA 51, and incomplete             duction of tumor antigens and chaperone molecules (heat shock
Freund’s adjuvant (11, 15, 16).                                          proteins), upregulation of MHC, intercellular adhesion molecule,
                                                                         tumor necrosis factor–related apoptosis-inducing ligand, and Fas
Recombinant Cytokines and Chemokines                                     receptors, each of which could facilitate immune-mediated
Systemically administered cytokines have also been explored              recognition and destruction of tumor cells (22, 26, 29–33).
for decades as stand-alone agents to nonspecifically activate                ‘‘Antisuppressive agents’’ are a subcategory of immune mod-
CTL (and/or NK cells), to skew the immune response toward                ulators with capacity to reduce regulatory elements in the host
a CTL response through effects on Th cells and APCs, to induce           environment—most specifically, T-regs. Abrogation of T-regs
APC differentiation and function, or to promote HLA class I              has been most extensively described with cyclophosphamide and,
molecule expression on tumor cells. Cytokines have also been             to a lesser extent, with the antimetabolite, fludarabine (22–26,
combined with vaccines, most notably by using cytokine–gene              34). Ongoing development and testing of selective inhibitors of
transfer to generate high local concentrations of stimulatory            T-regs is likely to yield multiple agents with therapeutic potential
cytokines at the site of antigen delivery (11, 17). Granulocyte-         that could synergize with cancer vaccines and other active
macrophage colony–stimulating factor (GM-CSF) and IL-2 have              immunotherapy strategies (11, 25).
been the two most widely investigated cytokines in clinical trials.
The list of cytokines is far too extensive to be detail here, and the    Gene Therapy and Gene Transfer Vectors
reader is directed to References 11 and 17 for a further discussion      Gene therapy is not a uniquely independent category, but,
of therapeutic cytokine uses in cancer. Similar to cytokines,            rather, describes a strategy that employs a transfer vector for
chemokines may be used at the site of tumor or combined with             direct expression of gene-derived proteins that modify cells for
exogenous antigen to attract APCs to a site of vaccination.              alternate function. Because the designation of gene therapy
                                                                         as a ‘‘therapy’’ can be misleading, an appropriate distinction
Autologous DCs                                                           should be made between gene transfer vectors and gene-based
DCs are professional APCs that possess all necessary elements            therapeutic proteins (oncogenic viruses are a notable exception,
to initiate and potentiate an antigen-specific immune response.           as the innate cytolytic properties of these viruses are being
Most commonly, DC precursors are harvested by the leukaphe-              employed therapeutically). A variety of gene transfer vectors,
resis procedure from a patient with disease, cultured in vitro,          each with differing rates of efficiency, can be coupled with
and supplied antigen ex vivo. When delivered back to the                 innumerable gene-derived proteins with adjuvant properties.
individual, it is expected that the programmed DC migrates to               Gene transfer of cytokines or costimulatory molecules di-
lymph node in vivo and orchestrates the desired antigen-specific          rectly to tumor cells ex vivo and in vivo is an attractive way of
immune response (18).                                                    making nonimmunogenic cells more immune stimulatory (11).
                                                                         In vivo cytokine gene transfer can also target normal cells in the
Immune Modulators                                                        tumor milieu, thereby achieving high local concentrations of
                                                                         cytokine that avoid toxicities associated with systemic adminis-
Most immune modulators are pharmacologic agents that have                tration. Other gene-based vaccine strategies modify normal
independent therapeutic effects, but also have immunologic               cells in vivo to express and present tumor antigens; dual
properties that promote a favorable immune environment.                  expression of antigen and gene-based cytokine or costimulatory
Examples include cyclooxygenase-2 inhibitors and thalidomide-            molecule expression has also been explored as a means of
like agents (called ‘‘immunomodulatory drugs’’ or IMiDs), such           enhancing biological efficacy. Secondary advantages of viral
as lenalidomide (Revlimid). These and other similar agents have          gene-transfer vectors used in vivo or ex vivo is an adjuvant
the significant potential for synergy with cancer vaccines (19, 20).      stimulation of the immune system responding to viral proteins.
    Small molecules that stimulate Toll-like receptors, a class of       This may be an important factor in experimental success with
molecular mediators involved in the initiation of the innate and         gene-based antigen vaccines (35).
adaptive immune responses, have also been shown to activate
DCs and induce a favorable cytokine environment. CpG
oligodeoxynucleotides are Toll-like receptor-9 agonists being            CLINICAL APPLICATION
evaluated for monotherapy in clinical trials, but also hold
promise as adjuvants for cancer vaccines (21).                           Clinical Population
    Interestingly, there is an emerging body of literature that          Based on poor treatment outcomes in advanced-stage lung
suggests that chemotherapy, conventionally viewed as antago-             cancer, investigating therapies that may consolidate clinical
nistic with immunotherapy, can have adjuvant properties that             responses to chemotherapy and radiation is warranted. Simi-
somewhat counter-intuitively promote immunological objec-                larly, adjuvant therapies that could destroy limited residual
tives (22–26). Beyond the relative clinical and biological impor-        disease and small metastatic deposits after surgical resection are
tance of tumor debulking, cytotoxic therapies have numerous              highly attractive. The low risk and potential benefit of tumor
systemic and local effects that might lead to synergy of immuno-         vaccines is appealing in both populations, but is most pro-
and chemotherapy; it can be hypothesized that multiple mech-             nounced for patients having surgically resected NSCLC; mini-
anisms act in concert. Some described mechanisms are generic             mal tumor burden is generally perceived as the most amenable
Hirschowitz and Yannelli: Immunotherapy for Lung Cancer                                                                                227


clinical target for immunotherapy, and the nominal risk of the         during therapeutic development. To date, there is not a standard
intervention is highly relevant, because we cannot yet predict         assay nor consensus on what constitutes a positive immunologic
recurrence beyond statistical probability. In contrast, patients       response (36). Considerations when choosing assays for analysis
with advanced-stage lung cancer, especially those individuals          include the information desired (induction of antibody, T cell,
with bulky disease, have not been widely viewed as likely              or T-cell subsets), the number of samples to be assayed, and the
to benefit from immunotherapy. Beyond mechanical factors                volume of sample obtainable from each subject on serial blood
related to tumor burden, there are numerous biological ele-            draws. Serum antibody measurement is well established, stan-
ments that could negate potential therapeutic effects of immune        dardized, simple, and accurate; nonetheless, few vaccines are
intervention. Notably, larger and metastatic tumors are known          designed to generate antibodies as a primary effector mecha-
to acquire enhanced resistance capability during progression;          nism, and the therapeutics relevance of antibody is uncertain.
immunosuppressive elements are also more prominent in                  Conversely, measurement of antigen-specific T cells in samples
advanced-stage cancer, including a purported corresponding             from immunized patients is less-well standardized, laborious,
increase in T-regs with increasing tumor burden. Nonetheless,          but more highly relevant to desired effects of most immuno-
multiple studies indicate that vaccines can induce immune              therapies.
responses in a percentage of patients with advanced-stage                  Functional T-cell assays include cytotoxicty, proliferation,
disease, and data from completed phase IIB clinical trials             and cytokine production assays that all measure T-cell reactivity
suggest that lung cancer vaccines may slow disease progression         upon antigen stimulation. Each is employed to demonstrate higher
and/or improve quality of life in this population. When assessing      frequency of antigen-specific T cells in circulating peripheral blood
efficacy of immunotherapy for advanced-stage NSCLC, it is               mononuclear cells after vaccination compared with baseline
important to keep in mind that an added therapy that may only          (prevaccine). Interpretation is based on the notable assumption
provide marginal improvements in survival or quality of life, but      that the T-cell responses of peripheral blood mononuclear cells
has minimal risk, is commensurate with the definition of benefi-         reflect responses in draining nodes or site of tumor, and analysis
cial treatment currently applied to conventional chemotherapy.         may over- or underrepresent compartmental activity. Delayed-
    Immunotherapy for SCLC has not been as widely investi-             type hypersensitivity to antigen challenge is a qualitative func-
gated. The disease tends to be very aggressive, and immuno-            tional assay performed in vivo. Similar to the Mantoux skin test for
therapy has not been widely regarded as having a significant            tuberculosis, a mononuclear cell response is mounted at the site
potential to impact outcomes. Because SCLC is an exclusively           of antigen challenge if the patient has preexisting T-cell immunity.
nonsurgical disease, tumor is not routinely available for autol-       This is a widely used assay that can be practically applied to most
ogous vaccine production. The lack of pathologic specimens has         vaccine approaches; however, sensitivity is limited and results in
also slowed preclinical investigation, as SCLC is not available        clinical trials have been variable. Preexisting immunity, which may
for in vitro study or xenogeneic tumor modeling, nor does there        be induced by a patient’s own tumor, and biological differences
exist an appropriate SCLC animal model. Regardless, the poor           in immune competence can cloud the readout. Tetramer staining
prognosis associated with SCLC makes it a rational choice for          is a nonfunctional, quantitative measure of antigen-specific T-cell
further investigation, and several groups are auspiciously pur-        frequency in peripheral blood. Florescent-labeled tetramers are
suing vaccines for SCLC.                                               constructed to bind a unique MHC/peptide-specific T-cell re-
                                                                       ceptor, and tetramer-tagged T cells are quantified by flow cytom-
Clinical Testing                                                       etry. MHC class I–peptide tetramers used to measure CD81 CTL
                                                                       specific for select antigens are the most commonly available,
The fundamental objective of clinical immunotherapy testing is
                                                                       although some MHC class II–peptide tetramers have also been
to gain relevant biological and/or therapeutic information that
                                                                       developed for the assessment of CD41 T-cell responses. Because
promotes rational development of therapeutic strategies. The
                                                                       tetramers are specific for a single, specific HLA-matched epitope,
complexity of the immune system and reliance on an incom-
                                                                       this assay is most practical for use with HLA-restricted peptide
pletely characterized series of events makes this a challenging
                                                                       vaccines. Tetramers are, however, not available for all antigens or
prospect. Animal studies do provide insight into immunobiology,
                                                                       all HLA types (the reader is directed to References 36 and 37 for
and have been critical in rational design of immunotherapy
                                                                       comprehensive discussion of tools for immunologic monitoring of
strategies; the parallels to human disease are, however, imperfect,
                                                                       cancer vaccine trials).
and clinical trials are built on only partial understanding of human
                                                                           There are also a number of ancillary measurements that may
cancer immunobiology. Although clinical study design is based
                                                                       reflect the receptiveness of the host to immune induction.
on a fair amount of speculation, extrapolation from measurable
                                                                       Serum and cellular cytokine characterization has been used
immunological parameters in early-phase clinical trials provides
                                                                       to gauge immune hyporesponsiveness, and to measure the effec-
valuable information. A number of in vitro assays that measure
                                                                       tiveness of immune modulators. Similarly, immunological charac-
a variety of parameters in peripheral blood after intervention
                                                                       terization of the tumor compartment has indicated presence of
allow comparative appraisal of immunological activity. Impor-
                                                                       multiple elements that inhibit antigen presentation and neutral-
tantly, observed correlation in some clinical trials between
                                                                       ize effector cells; these measurements are, however, neither
immunologic activity (measured by a variety of assays) and
                                                                       readily available nor routinely sought. In parallel with evolving
clinical response criteria strongly supports the hypothesis that
                                                                       knowledge of T-regs, measurement of percent circulating T-regs
immunological response to relevant antigens translates into
                                                                       by cell surface cytometric analysis appears to offer an additional
clinical benefit. Definitive evidence of therapeutic effect in phase
                                                                       gauge of host immune responsiveness. CD41CD251FoxP31
III trials and an established link between immunological assays
                                                                       T-regs are most well characterized, although several different
and clinical outcomes will both facilitate and promote logical
                                                                       T-reg populations have been identified. Notably, the literature
development of cancer immunotherapy.
                                                                       reports variably elevated levels of CD41CD251FoxP31 T-regs in
                                                                       peripheral blood and in tumor beds of patients with lung cancer,
Immunologic Monitoring                                                 a finding that may correlate with stage and/or prognosis (38–43).
Immunologic endpoints are critical for determining biologic            T-reg measurements may prove to be a relative predictor of
activity of active immunotherapies in phase I and phase II             resistance to immune induction and become a directed endpoint
studies, and serve as reasonable measures of vaccine potency           for immune modulation.
228                                                                   PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 6              2009

Clinical Endpoints                                                     strongly support a correlation between clinical and immunolog-
Clinical endpoints are the foundation of phase IIB and phase III       ical response.
trials. Rate of recurrence or risk of recurrence is the primary
clinical endpoint in studies with surgically resected patients with    ADVANCED STUDIES USING SELECTED AGENTS
lung cancer; advanced-stage disease offers several measures of
efficacy, including objective tumor response, duration of re-           A handful of immunotherapies for lung cancer have been
sponse, time to progression (TTP), disease-free survival, and          investigated in advanced-phase studies. Nine vaccines tested
overall survival. Objective tumor response may be measured             in independent trials are discussed here (an overview is
by Response Evaluation Criteria in Solid Tumors (RECIST) or            presented in Table 1). All but one study has been conducted
World Health Organization criteria, but are reserved for patients      in NSCLC; the only SCLC vaccine of this group is also the only
with measurable disease at baseline; this typically includes serial    one to have been completely evaluated in a phase III trial. One
measurement of one or more target lesions by computed tomog-           other study in NSCLC, designed as a phase III, had a high
raphy. Increasingly sensitive radiographic techniques that have        dropout rate and has statistical power that more closely resem-
yet to be validated for clinical investigation may provide newer       bles a randomized phase IIB study. Four of the other seven
clinical response criteria that could be used as intermediate          agents discussed here have been tested in phase IIB, random-
endpoints in phase II studies (44). Tumor markers may be used          ized, controlled trials; the remaining three represent a series of
as a supplementary measurement, but, alone, are not accepted as        vaccines from a single group, which were each tested in larger,
a measure of response.                                                 nonrandomized phase II studies. With few exceptions, adverse
    Although targeting early-stage disease is appealing, an            events reported in all studies were limited to local site reactions,
important consideration for therapeutic development is that            and less frequently, flu-like symptoms and fatigue.
relatively low rates of recurrence in surgically resected patients
dictates a large study sample size to show clinical benefit in phase    BEC2 and BCG
III trials. By contrast, radiographically measurable disease, rapid    BEC2 is an antiidiotypic vaccine for SCLC and is the only lung
progression, and terminal prognosis of advanced-stage lung             cancer vaccine to have been completely evaluated in phase III
cancer provides timely endpoint analysis for therapeutic clinical      studies. In the initial phase I/II trial, patients with limited-stage
trials. The comparatively small study numbers required to see          SCLC achieving a partial or complete response from chemo-
statistical differences between study groups also makes this an        therapy were vaccinated with an antiidiotype GD3 monoclonal
attractive population for therapeutic investigation. Of note,          antibody (BEC2) and BCG. Those who developed anti-GD3
novel molecular analysis may soon be able to determine which           antibodies had prolonged survival compared with historical
individuals are likely to recur after surgical resection of NSCLC,     control subjects. These encouraging results led to a phase III
making immunotherapy studies more rational and efficiently              trial sponsored by Merck KGaA and ImClone. A total of 515
conducted in a refined population of patients having had surgical       patients were randomized 1:1 to receive five immunizations of
resection.                                                             BEC2 plus BCG over a 10-week period or best supportive care
                                                                       (BSC); the phase III trial disappointingly did not confirm
                                                                       clinical benefit suggested by the early-phase study (45). Notably,
NONRANDOMIZED CLINICAL TRIALS IN                                       vaccine induced humoral response in only one-third of the 213
LUNG CANCER                                                            patients who were able to be evaluated immunologically (negative
Myriad approaches with multiple antigens have been explored            in 142 cases and positive in 71 cases). Stratified analysis suggested
in a variety of malignancies. The published literature describes       a trend toward improved survival in patients that showed immu-
fewer than 600 patients with lung cancer that have been treated        nological response, although differences were not statistically
in 25 pilot, phase I, or early phase II studies using 17 different     significant. The authors concluded that the study was essentially
vaccines. Categorization of these trials roughly parallels the         negative; however, they acknowledged that the agent has limited
classification of immune adjuvants, including: (1) antigen plus         ability to induce antibody response, and that the reduced number
chemical/biologic adjuvant; (2) antigen plus cytokine; (3) anti-       of patients in stratified groups likely limited the power of their
idiotype plus biologic adjuvant; (4) antigen-loaded DCs; and (5)       analysis. Furthermore, the authors conclude that a monovalent
gene therapy. The objectives of each of these trials included          approach may not have been ideal, indicating that multivalent
safety, tolerability, and biological activity (10).                    approaches may be better (46).
    Comparison of these small studies is difficult if not impos-
sible (10, 36, 37). Each involves a different study population,        Anti-EGF Vaccine
vaccine protocols, doses, frequency, and even routes of admin-         The anti-EGF vaccine is unique among other lung cancer
istration. A number of different antigens have been incorpo-           vaccines in that it does not directly target tumor, but, rather,
rated into a variety of vaccine preparations, and responses were       has the primary objective of inducing anti-EGF antibodies that
assessed by variable means of immune assessment and non-               neutralize endogenous EGF and deprive tumor of this important
rigorous clinical response criteria. Nonetheless, the literature       growth factor (47, 48). The vaccine is comprised of recombinant
collectively indicates that immunotherapy for lung cancer is           EGF, chemically conjugated to a recombinant P64K bacterial
feasible and rational. Specifically, it is apparent that toxicity is    protein as carrier protein, and emulsified with the adjuvant
limited, and that multiple agents can induce measurable immu-          Montanide ISA51. The randomized phase IIB study enrolled 80
nologic response. A consistent observation in all studies is that      patients with stage IIIB/IV NSCLC who had completed first-
immune induction is not uniform, even within homogenous                line chemotherapy. Patients were randomized 1:1 to BSC or
study populations, indicating a biological variability that regu-      EGF vaccinations. The treatment group received four induction
lates individual responses, which is independent of vaccine            immunizations and monthly boosters until disease progression.
potency. Furthermore, although early-phase trials are not              Vaccine induced a desirable anti-EGF antibody response in
powered to define therapeutic efficacy, several trials offer             roughly 50% of immunized patients, which corresponded to a
anecdotal evidence of clinical benefit. An important and highly         significant decrease in serum EGF concentrations. There was a
relevant corollary to these observations is that several studies       significant correlation between anti-EGF antibody titers, serum
Hirschowitz and Yannelli: Immunotherapy for Lung Cancer                                                                                                 229

TABLE 1. RANDOMIZED CLINICAL VACCINE TRIALS IN LUNG CANCER: PHASE IIB AND PHASE III RANDOMIZED STUDIES
                                            Tumor/                         Randomization/            Endpoint
Vaccine              Description             Stage         Patient n         Trial Design          (Significance)          Secondary Analysis         Ref. No.

BLP25           MUC-1 peptide 1          NSCLC IIIB/IV       171       1:1, vaccine vs. BSC      Survival (NS)        Trend toward increased         (54)
  (Stimuvax)     liposome                                                                                               survival in subgroup
                                                                                                                        with stage IIIB disease.
MAGE-A3         MAGE protein 6           NSCLC I/II          182       2:1, vaccine vs. BSC      Recurrence (NS)      Trend toward delay in          (55, 56)
 (ASCI)          AS02B                    (MAGE-A31)                                                                    time to recurrence/
                                                                                                                        nonstatistical reduction
                                                                                                                        in relative risk of cancer
                                                                                                                        recurrence.
TG4010          Dual gene therapy        NSCLC IIIB/IV       148       1:1, vaccine 1 chemo      Survival (pending)   Interim results indicate a     (57)
                 vaccinia/MUC1/IL-2                                      vs. chemo alone                                nonstatistical increase in
                                                                                                                        6-mo survival, with
                                                                                                                        statistical differences in
                                                                                                                        clinical response (WHO
                                                                                                                        criteria).
EGF             EGF protein 1 P64        NSCLC IIIB/IV        80       1:1. vaccine vs. BSC      Survival (NS)        Significant survival            (47, 48)
                  carrier protein 1                                                                                     advantage in subgroup
                  montanide ISA51                                                                                       with vaccine-induced
                                                                                                                        anti-EGF Ab response
                                                                                                                        who were ,60 yr old;
                                                                                                                        significant correlation
                                                                                                                        between anti-EGF
                                                                                                                        antibody titers, serum
                                                                                                                        EGF levels, and clinical
                                                                                                                        outcomes (Ab response
                                                                                                                        in only 50%).
*SRL172         Heat-killed M. vaccae    NSCLC IIIB/IV       419*      1:1, vaccine 1 chemo      Survival (NS)        Significant survival            (49, 50)
                  1 chemotherapy                                         vs. chemo alone                                advantage in subgroup
                                                                                                                        with adenocarcinoma;
                                                                                                                        improved quality of life
                                                                                                                        overall.
†BEC2           Antiidiotype GD3 1       Small cell          515       1:1, vaccine vs. BSC      Survival (NS)        Low rate of immune             (45, 46)
                  BCG                      (limited)                                                                    induction (1/3); trend
                                                                                                                        toward improved
                                                                                                                        survival in subgroup
                                                                                                                        with vaccine-induced
                                                                                                                        anti-GD3 Ab response.

                                                                                                                    ´
  Definition of abbreviations: Ab 5 antibody; ASCI 5 antigen-specific cancer immunotherapy; BCG 5 bacillus Calmette-Guerin; BSC 5 best supportive care; EGF 5
epidermal growth factor; M. vaccae 5 Mycobacterium vaccae; MUC1 5 mucin-1; NS 5 not significant; NSCLC 5 non–small cell lung cancer; WHO 5 World Health
Organization.
  * SRL172: designed as phase III trial; fewer than 50% completed the prescribed number of immunizations.
  †
    BEC2: the only completed phase III trial in lung cancer.



EGF levels, and clinical outcomes. The authors report an                        223 vs. 225 days; P 5 0.65). Secondary analysis showed that
overall trend toward increased survival in the treatment group                  patients with adenocarcinoma who completed the vaccine pro-
compared with control subjects that reached statistical signifi-                 tocol (n 5 45) versus matched control subjects receiving
cance in a subgroup of patients less than 60 years old (47, 48).                chemotherapy alone did have a survival advantage (median
                                                                                overall survival, 302 vs. 177 days; P , 0.01) that was not ob-
SRL172                                                                          served in patients with squamous cell cancer (n 5 61) (49, 50).
The SRL172 approach relies on the immunogenicity of a heat-
killed Mycobacterium vaccae (SRL172) to induce a favorable,                     GVAX, Allo-GVAX, and Lucanix
albeit nonspecific, systemic immune response that promotes                       GVAX has been highly publicized through promotion by in-
autologous antigen recognition. SR Pharma plc (UK) sponsored                    dustry sponsors, and the high-profile nature alone makes GVAX
a randomized trial using SRL172 concurrently with platinum-                     and two related studies worthy of discussion. Although none of
based chemotherapy with the intention of inducing systemic                      the agents have undergone randomized, controlled investigation,
reactivity during endogenous tumor antigen release. Although                    the considerable logic of phased development behind this series
designed as a phase III, non–placebo controlled, randomized                     of gene therapy approaches is highly instructive.
study, the trial was subject to a high dropout rate, and the                        GVAX. The initial study evaluated an autologous tumor cell
reduced number of evaluable patients limited the statistical                    vaccine (GVAX) in 33 individuals with advanced- and 10 with
power. A total of 419 patients with stage III/IV NSCLC were                     early-stage NSCLC. Autologous tumor obtained from surgery
randomized 1:1 to receive serial injections of 109 bacilli (five                 was transduced ex vivo with an adenoviral vector delivering
monthly injections followed by monthly maintenance) admin-                      GM-CSF cDNA to processed tumor cells. Of 33 patients with
istered concurrently with six cycles of chemotherapy delivered                  advanced NSCLC, three (two of which were bronchoalveolar cell
on a 21-day schedule. Fewer than 50% of the subjects com-                       carcinoma) achieved complete response and prolonged remission.
pleted the prescribed series of vaccines. Including all subjects,               Eight of the 10 patients with early-stage lung cancer remained free
the authors concluded that SLR172 significantly improved                         of disease with a median follow-up time of 12 months. The authors
patient quality of life without affecting overall survival (median,             reported a positive correlation between GM-CSF production by
230                                                                   PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 6            2009


the vaccine and clinical response. An important aspect of this         vaccine vs. BSC). A total of 182 patients with resected stage IB/
study was that, of 83 tumors harvested, vaccines could not be          II, MAGE-A3–positive NSCLC were serially immunized and
successfully generated in 16 patients, and 11 other died before        evaluated for time to recurrence. Investigators observed
vaccine could be delivered. The median production time was             a delayed time to recurrence at 28 months (31.6 [vaccine] vs.
31 days, and median time from tumor harvest to immunization was        43.3% [control]), which translated into a 27% reduction in
49 days. A total of 43 patients were immunized (51).                   relative risk of cancer recurrence (55, 56). Although differences
    Allo-GVAX (bystander GVAX). Based on encouraging                   failed to meet statistical significance, results were suggestive
anecdotal response that correlated with higher levels of GM-           enough to pursue phase III investigation. The international,
CSF production, and in response to the limitations imposed by          multicenter MAGRIT study (MAGE-A3 Adjuvant Non–Small
protracted vaccine production, the group modified their ap-             Cell Lung Cancer Immunotherapy), opened to enrollment in
proach. An updated vaccine mixed autologous tumor with an              2007, plans to accrue 2,270 MAGE-A3–positive patients with
allogeneic, non–lung cancer cell line (K562 erythroleukemia            completely resected stage IB, II, or IIIA NSCLC.
cells) engineered to express GM-CSF, and thereby removed the
requirement for gene modification of individual tumors. The             TG4010
vaccine was tested in a nonrandomized study of 86 patients with        The TG4010 vaccine uses a dual-gene–based approach to
advanced-stage NSCLC, with the objectives of safety, feasibil-         coexpress MUC1 antigen and IL-2 (MVA–MUC1–IL-2) with
ity, pharmacokinetics, and efficacy. Cell processing was success-       a recombinant vaccinia virus. Two serial studies, sponsored by
ful in 76 patients, and 49 proceeded to vaccination. Although          Transgene (Strasbourg, France) evaluated clinical endpoints in
the GM-CSF secretion was 25-fold higher than autologous                patients with stage IIIB/IV NSCLC administered TG4010
GVAX, the investigators disappointingly did not observe an             concurrently with conventional chemotherapy. The initial phase
objective tumor response similar to that seen in the GVAX              IIA, nonrandomized study evaluated 44 patients, and observed
study. A higher frequency of injection site reactions than with        tumor response rate of 37%; 13 partial responses (RECIST),
the parent GVAX was also observed (52).                                71% with partial response (PR) or SD for over 12 weeks;
    Lucanix. The same group then evaluated an entirely alloge-         estimated median TTP was 6.4 months; estimated overall
neic antigen approach that standardized vaccine production and         survival was 13 months. In the follow-up phase IIB study, 148
eliminated the need for tumor cell harvest. Lucanix is comprised       patients were randomized to TG4010 plus chemotherapy or
of four allogeneic NSCLC cell lines transduced with transform-         chemotherapy alone (gem/cis). Patients in the vaccine arm were
ing growth factor-b antisense plasmid. The study was conducted         immunized weekly with TG4010 for 6 weeks, then received
as a phase II nonrandomized trial with dose escalation in 75           maintenance injections every 3 weeks until progression. Interim
patients with NSCLC (14 early stage, 61 late stage). Endpoints         results reported at the American Society of Clinical Oncology
included safety, feasibility, pharmacokinetics, and efficacy.           in June 2008 indicate statistical differences in clinical response
Results reported in 2006 observed 15% response by RECIST,              criteria, and a non–statistically significant increase in progres-
with an increase in estimated survival compared with historical        sion-free survival at 6 months. Data on overall survival are not
control subjects, and a correlation of survival with higher dose       yet mature, but show an encouraging trend favoring survival in
levels. In 2008, the group initiated a phase III placebo-controlled    the treatment group compared with matched control subjects;
trial of Lucanix in stage III and IV NSCLC (53).                       final study results are expected in late 2008 (57).
BLP25 (Stimuvax)                                                       Phase II Studies: What Have We Learned?
BLP25 liposome vaccine (L-BLP25) carries the mucin-1                   Supplementing information from early-phase trials, these stud-
(MUC1) protein admixed with monophosphoryl lipid A as an               ies indicate that immunotherapy carries limited toxicity. Con-
immune adjuvant. The randomized, phase IIB trial (1:1 BLP25            sistent with phase II objectives and study design, there is
vs. BSC), sponsored by Merck KGaA, enrolled 171 patients               suggested benefit in early- and late-stage NSCLC. Data indicate
with stage IIIB/IV NSCLC with response or stable disease (SD)          that phase III investigation is warranted and necessary. In
after first-line therapy. Patients, stratified by stage (IIIB vs.        addition to the need for definitively addressing therapeutic
‘‘wet’’ IIIB/IV), received eight weekly subcutaneous injections        efficacy, a number of relevant questions are afield. These
of BLP25 SQ; patients also received additional treatment with          include relative potency and comparative efficacy of different
300 mg/m2 cyclophosphamide 3 days before immunization. The             formulations, and practical information about necessary dura-
primary endpoint was clinical outcomes. Data analysis showed           tion of response, ideal dosing schedule, and appropriate timing
no statistical difference in overall survival (17.4 vs. 13 mo; P 5     relative to administration of conventional therapies.
0.1), but a strong trend in median survival (30.6 vs; 13.3 mo) in
a subgroup of patients with stage IIIB locoregional disease (n 5       FUTURE DIRECTIONS AND OBJECTIVES
35) compared with matched control subjects (n 5 30). Based on
the promising results in this subgroup of patients, Merck KGaA         The lack of proven clinical benefit continues to encumber
sponsored the multicenter (international) phase III, random-           development of immunotherapy for lung cancer. This fact
ized, double-blind, placebo-controlled trial. The START study          makes proven clinical efficacy in phase III trials an overarching
(Stimulating Targeted Antigenic Responses to NSCLC) opened             objective. Assuming one or more agents are shown to have
to enrollment in 2007. Targeted accrual is 1,300 patients with         clinical benefit in lung cancer, however small, attention can
unresectable stage III NSCLC that have responded to first-line,         be judiciously turned to optimizing efficacy. There are several
platinum-based chemoradiotherapy (54).                                 key points to consider in that pursuit. It is probable that
                                                                       measurable immunological response to relevant tumor antigens
MAGE-A3 Antigen-Specific Cancer Immunotherapy                           will positively correlate with clinical benefit. In the context of
GlaxoSmithKline produced a recombinant MAGE-A3 fusion                  measurable activity already shown by multiple agents and
protein (His-tagged/full-length MAGE-A3 protein/influenza               formulations, it is to be expected that a number of different
protein D) plus immune adjuvant AS02B (monophosphoryl                  agents can yield similar results. Well standardized and validated
lipid A and QS21) that was tested in a double-blind, random-           immunological assays used as a comparative measure of po-
ized, placebo-controlled, phase IIB trial (2:1 randomization,          tency may show some differences in biological activity, but it is
Hirschowitz and Yannelli: Immunotherapy for Lung Cancer                                                                                                     231


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