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The Proceedings of the American Thoracic Society 6:224-232 (2009)
© 2009 The American Thoracic Society
doi: 10.1513/pats.200806-048LC
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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; 2 Lexington Veterans Administration Medical Center, Lexington, Kentucky; and 3 Department of Microbiology, Immunology, and Human Genetics, University of Kentucky, Lexington, Kentucky

Correspondence and requests for reprints should be addressed to Edward A. Hirschowitz, M.D., Division of Pulmonary and Critical Care Medicine, University of Kentucky, Chandler Medical Center, 740 S. Limestone, Room K528, Lexington, KY. E-mail: eahirs2{at}uky.edu

ABSTRACT

Immunotherapy is a conceptually attractive approach, because it is highly specific and can deal with disseminated disease with minimal impact on normal tissues. Ability to induce antigen-specific immune responses in patients with lung cancer is now well established in early-phase clinical trials using a variety of immunotherapeutic approaches. Although no immunotherapy is likely to be a panacea, randomized phase IIB studies offer promise of therapeutic activity in both early- and late-stage lung cancer. This review will cover basic concepts of immunotherapy, provide perspective on vaccine development, and update the status of ongoing clinical trials in lung cancer.

Key Words: immunotherapy • vaccines • lung cancer • clinical trials

INTRODUCTION

A number of nonconventional therapeutic modalities are being developed to improve unacceptably poor outcomes in lung cancer. Immunotherapy is an attractive approach that, by design, is cancer specific and can target disseminated disease with minimal impact on normal tissues. Active immunotherapy describes those approaches that use host immune machinery to generate antitumor effects. This review presents basic concepts of active immunotherapy, provides perspective on therapeutic development, and updates the status of ongoing clinical immunotherapy trials in lung cancer.

IMMUNOTHERAPY AND IMMUNOBIOLOGY

Immunotherapy
Novel therapies should improve outcomes, but have a side-effect profile that is more favorable than conventional chemotherapy (1). A number of therapeutic approaches are being developed that selectively target malignant cells or their microenvironment, but leave normal cells intact. An ideal agent should also be easy to administer to address both quality-of-life issues and compliance concerns. Although the term "targeted therapy" has been most widely applied to pharmacologic agents, including small molecule inhibitors and antisense oligonucleotide, immunotherapy closely fits the definition.

Immunotherapy is categorized as either passive or active. Passive immunotherapy includes any immunologically active agent that is made outside the body and does not rely on host machinery to function. The most widely applied passive immunotherapies are monoclonal antibodies that disrupt tumorigenic cascades by blocking the binding of hormones or growth factors to their receptors; examples include cetuximab (Erbitux) and trastuzumab (Herceptin), which target epidermal growth factor (EGF) receptors, HER-1 and HER-2, respectively, and bevacizumab (Avastin), which interferes with tumor angiogenesis by binding to vascular endothelial growth factor (1–4). Adoptive transfer is another example of passive immunotherapy that typically involves ex vivo amplification and infusion of autologous tumor-infiltrating T cell or lymphokine-activated killer cell therapy (5). By contrast, active immunotherapy uses the host's immune cells and requires an intact immune system to function. Active immunotherapy is derived from the knowledge that the immune system can discriminate cancer cells from normal cells based on tumor antigen recognition (6–9). Approaches that directly incorporate tumor antigen are conventionally referred to as vaccines. Although some active immunotherapies are designed to induce antibodies as the primary effector mechanism, and may be rationally applied with certain logical intention, cancer-specific antibody responses are widely viewed as having limited direct cytotoxic capability against cancer. With few exceptions, induction of antigen-specific T-cell responses is the primary objective of active immunotherapy (6–10). Natural killer (NK) cells are an antigen-independent arm of immune defense that targets major histocompatibility complex (MHC)-mismatched (allogeneic) cells or those cells lacking surface MHC molecules. Because cancer cells often downregulate surface MHC molecules, induction of NK is a logical, but not common, primary objective of immunotherapy (11).

Immune Recognition
Identification of tumor-associated antibodies and antigen-specific cytotoxic T lymphocytes (CTL) in patients with lung cancer indicates that the immune system can distinguish lung cancer cells from normal cells (6, 7). Antigen-specific immune responses are dependent on antigen presentation in the patient's lymphoid tissues. When antigen is contacted, antigen-presenting cells (APCs) digest whole proteins into smaller peptides that are then presented on HLA class I or class II molecules on the APC surface. Coordinated expression of costimulatory molecules (CB80/CD86) on the APC surface prompts antigen-specific lymphoid precursors to activate at appropriate times. Depending on the type or source of antigen and the existing cytokine milieu, class II peptides may stimulate IL-2 and IFN-{gamma} release by antigen-specific CD4+ T cells (Th1). Antigen-specific CTL are generated when CD8+ T cells bound to class I antigens on APCs are stimulated by Th1 cytokines. Alternatively, class II antigen binding may induce a different T-cell phenotype (Th2) that releases IL-4 and IL-10, and interacts with B cells to promote 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. Notably, activated Th cells have longevity compared with other activated immune cells (APCs and CTL), and are conventionally viewed as being responsible for "immune memory," capable of "revitalizing" the immune response if a specific antigen is reencountered; memory subsets of CD8+ T cells and B cells provide other mechanisms for rapid response to known antigens.

Immune Regulation and Tumor Evasion
Physiologic checks and balances, that work throughout the immune cascade to control antigen hyperresponsiveness, create a permissive environment for tumor growth (9, 11, 12). Immunosuppressive cytokines, APC senescence, and regulatory T cells (T-regs) each present an important challenge to successful development of immunotherapy (9, 11–13). Most established tumors also have mechanisms of immune evasion (9, 11, 12). The production and induction of immunosuppressive cytokines by tumor cells, such as IL-10 and transforming growth factor-β, has been well characterized (12). Some tumor cells may avoid immune destruction by down-regulating HLA class I molecules on which antigens are presented for recognition, or by over-expressing B7-H1, a ligand for the T-cell receptor, PD-1, which is known to negatively regulate T-cell activation. Defined mechanisms of CTL resistance and counter defense include tumor expression of the apoptosis-inducing molecule, Fas ligand, and downregulation of surface Fas receptors (12).

CANCER ANTIGENS

There are multiple incarnations of antigen used in active immunotherapy, each with independent advantages. A major division in antigen targeting is the use of a multivalent or monovalent strategy. By targeting several antigens, multivalent approaches lend themselves to many of the clinical and biological realities of lung cancer. Specifically, phenotypic heterogeneity characteristic of non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) dictates high variability of antigen expression, and precludes the ideal circumstance of one antigen being uniformly expressed by all lung tumors. Additionally, because a vast majority of patients with lung cancer are nonsurgical, most lung cancer diagnoses are made with minimal tissue sampling, and the material is often too limited for adequate antigen characterization; in context, multivalent approaches circumvent the need to know which antigens are expressed by a specific tumor. By contrast, monovalent applications all have the common feature of being selectively delivered to individuals with corresponding tumor antigen expression, which distinctly requires individual tumor antigen characterization. Notably, monovalent approaches are more readily incorporated into proprietary products, and lend themselves to commercial development; not surprisingly, a majority of therapies advancing to phase III investigation are monovalent approaches.

The most commonly used multivalent formulations employ autologous or allogeneic tumor cells, although multiprotein/-peptide mixtures or fusion constructs can achieve similar multivalent objectives. Tumor-derived antigen mixtures include multiple dominant and minor antigenic determinants within whole proteins, allowing the host to select, process, and present on MHC, the most immunogenic epitopes relevant to that individual (11). Autologous tumor can be utilized either in vivo or ex vivo. The former relies on a locoregional or systemic intervention to promote antigen recognition in situ. One example of this is the systemic administration of cytokine; alternatively, if the tumor bed can be directly accessed, gene therapy strategies can be used to modify tumor cell antigenicity or alter the tumor microenvironment. Ex vivo approaches most commonly combine surgically obtained tumor with an immune adjuvant to produce a cancer vaccine. A disadvantage of using autologous tumor in vaccines is that adequate amounts of fresh tumor must be available for vaccine production, generally restricting this approach to the individual whose tumor is surgically resected with the forethought and intent of making a vaccine. An additional limitation is that antigens differ for each subject, thereby complicating immunological assessment and confounding comparability within the study population, both of which can hinder rational therapeutic development in early clinical testing. Nonetheless, autologous vaccines have been shown to have immunologic activity in a number of studies (10, 11). Allogeneic tumor is a rational alternative that can provide a renewable source of antigen and offers greater potential for "off-the-shelf" application. Using allogeneic antigen also generates a uniform preparation, which facilitates immune assessment and comparability not afforded by use of autologous tumor antigen. Availability of these tangible milestones makes this, and other allogeneic approaches, attractive during therapeutic development and clinical testing.

Two additional allogeneic sources of antigen are synthetic peptide and recombinant protein (including gene therapy–derived antigen). In contrast to allogeneic tumor, peptides and proteins are characteristically used in monovalent formulations. Peptides are an attractive monovalent antigen source, because 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 exclusive to specific HLA types and require patient selection based on HLA tissue typing. Furthermore, peptides have designated restriction to class I or class II pathways, selectively stimulating either CD8+ cytotoxic T-cell effectors or CD4+ Th cells, responsible for immune memory (11). By contrast, whole recombinant proteins are processed into multiple peptides and presented by APCs via class I and class II pathways to CD4+ and CD8+ T cells, respectively, and have the potential for generating a response that includes immune effectors and immune memory.

Xenogeneic antiidiotype antibodies are somewhat unique antigen-mimic preparations, generated as antibodies to tumor antigen–binding sites on other antibodies (that generates a template of the antigen). The foreign (xenogeneic) nature of these preparations makes them inherently immunogenic, and the similarity of the antiidiotype antibody to the tumor antigen allows cross recognition of the parent/native protein (14). In contrast to the vast majority of the other active immunotherapies under development, antiidiotypic vaccines are used to elicit tumor-specific antibodies as the dominant effectors for therapeutic activity; these have been the most widely tested immunotherapy approaches in SCLC.

PROMOTING ANTIGEN RECOGNITION: IMMUNE ADJUVANTS AND DELIVERY

Success of any active immunotherapy ultimately depends on tumor antigen capture and presentation by APCs (11, 15). Tumor antigens, however, are not, in themselves, immunogenic. In order to initiate or promote antigen-specific responses, all strategies incorporate adjuvants; these are essentially activating agents or stimulants for various arms of the immune cascade, intended to augment antigen recognition, uptake, presentation, and/or antigen-specific cellular reactivity (11, 15). Effects of various adjuvants are not mutually exclusive, and mechanisms often overlap and intersect. There are myriad choices that fall into one of several categories: (1) biologic and chemical adjuvants; (2) recombinant cytokines and chemokines; (3) autologous dendritic cells (DCs); (4) immune modifiers; (5) gene therapy/gene transfer vectors.

Chemical and Biologic and Adjuvants
These adjuvants have been used for decades as a key component in antigen-specific immunotherapy to induce inflammation, either at the site of tumor, or used in conjunction with exogenously supplied antigen. Biologic adjuvants take advantage of the fact that they are immunogenic compounds, naturally recognized as foreign and known to induce migration of APCs to the site of delivery. APCs responding to adjuvant stimulation are thus able to coincidentally capture and process tumor antigens present in the inflammatory milieu. The most commonly used biologic adjuvants include bacillus Calmette-Guérin (BCG), diphtheria toxoid, and tetanus toxoid (11, 15). Chemical adjuvants function similarly to biologic adjuvants as irritants that induce an inflammatory response at the site of delivery, and some may also provide a matrix that sequesters antigen at a specific location, allowing a timed release of antigen to APCs. Examples include aluminum hydroxide, montanide ISA 51, and incomplete Freund's adjuvant (11, 15, 16).

Recombinant Cytokines and Chemokines
Systemically administered cytokines have also been explored for decades as stand-alone agents to nonspecifically activate CTL (and/or NK cells), to skew the immune response toward a CTL response through effects on Th cells and APCs, to induce APC differentiation and function, or to promote HLA class I molecule expression on tumor cells. Cytokines have also been combined with vaccines, most notably by using cytokine–gene transfer to generate high local concentrations of stimulatory cytokines at the site of antigen delivery (11, 17). Granulocyte-macrophage colony–stimulating factor (GM-CSF) and IL-2 have been the two most widely investigated cytokines in clinical trials. The list of cytokines is far too extensive to be detail here, and the reader is directed to References 11 and 17 for a further discussion of therapeutic cytokine uses in cancer. Similar to cytokines, chemokines may be used at the site of tumor or combined with exogenous antigen to attract APCs to a site of vaccination.

Autologous DCs
DCs are professional APCs that possess all necessary elements to initiate and potentiate an antigen-specific immune response. Most commonly, DC precursors are harvested by the leukapheresis procedure from a patient with disease, cultured in vitro, and supplied antigen ex vivo. When delivered back to the individual, it is expected that the programmed DC migrates to lymph node in vivo and orchestrates the desired antigen-specific immune response (18).

Immune Modulators
Most immune modulators are pharmacologic agents that have independent therapeutic effects, but also have immunologic properties that promote a favorable immune environment. Examples include cyclooxygenase-2 inhibitors and thalidomide-like agents (called "immunomodulatory drugs" or IMiDs), such as lenalidomide (Revlimid). These and other similar agents have the significant potential for synergy with cancer vaccines (19, 20).

Small molecules that stimulate Toll-like receptors, a class of molecular mediators involved in the initiation of the innate and 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 evaluated for monotherapy in clinical trials, but also hold promise as adjuvants for cancer vaccines (21).

Interestingly, there is an emerging body of literature that suggests that chemotherapy, conventionally viewed as antagonistic with immunotherapy, can have adjuvant properties that somewhat counter-intuitively promote immunological objectives (22–26). Beyond the relative clinical and biological importance of tumor debulking, cytotoxic therapies have numerous systemic and local effects that might lead to synergy of immuno- and chemotherapy; it can be hypothesized that multiple mechanisms act in concert. Some described mechanisms are generic to cytotoxic therapy, where other effects may be unique to specific chemotherapeutic classes or agents. It has been postulated that apoptosis of tumor cells induced by most commonly used cytotoxic therapies somewhat paradoxically stimulates the immune system (25–27). Therapy may also mitigate inhibitory cytokine production in the local milieu, or induce proinflammatory signals that augment APC numbers and function or promote generation of antigen-specific CTL (25, 26, 28). The literature also describes several drug-related phenotypic alterations that lead to increased susceptibility to CTL lysis, including the induction of tumor antigens and chaperone molecules (heat shock proteins), upregulation of MHC, intercellular adhesion molecule, tumor necrosis factor–related apoptosis-inducing ligand, and Fas receptors, each of which could facilitate immune-mediated recognition and destruction of tumor cells (22, 26, 29–33).

"Antisuppressive agents" are a subcategory of immune modulators with capacity to reduce regulatory elements in the host environment—most specifically, T-regs. Abrogation of T-regs has been most extensively described with cyclophosphamide and, to a lesser extent, with the antimetabolite, fludarabine (22–26, 34). Ongoing development and testing of selective inhibitors of T-regs is likely to yield multiple agents with therapeutic potential that could synergize with cancer vaccines and other active immunotherapy strategies (11, 25).

Gene Therapy and Gene Transfer Vectors
Gene therapy is not a uniquely independent category, but, rather, describes a strategy that employs a transfer vector for direct expression of gene-derived proteins that modify cells for alternate function. Because the designation of gene therapy as a "therapy" can be misleading, an appropriate distinction should be made between gene transfer vectors and gene-based therapeutic proteins (oncogenic viruses are a notable exception, as the innate cytolytic properties of these viruses are being employed therapeutically). A variety of gene transfer vectors, each with differing rates of efficiency, can be coupled with innumerable gene-derived proteins with adjuvant properties.

Gene transfer of cytokines or costimulatory molecules directly to tumor cells ex vivo and in vivo is an attractive way of making nonimmunogenic cells more immune stimulatory (11). In vivo cytokine gene transfer can also target normal cells in the tumor milieu, thereby achieving high local concentrations of cytokine that avoid toxicities associated with systemic administration. Other gene-based vaccine strategies modify normal cells in vivo to express and present tumor antigens; dual expression of antigen and gene-based cytokine or costimulatory molecule expression has also been explored as a means of enhancing biological efficacy. Secondary advantages of viral gene-transfer vectors used in vivo or ex vivo is an adjuvant stimulation of the immune system responding to viral proteins. This may be an important factor in experimental success with gene-based antigen vaccines (35).

CLINICAL APPLICATION

Clinical Population
Based on poor treatment outcomes in advanced-stage lung cancer, investigating therapies that may consolidate clinical responses to chemotherapy and radiation is warranted. Similarly, adjuvant therapies that could destroy limited residual disease and small metastatic deposits after surgical resection are highly attractive. The low risk and potential benefit of tumor vaccines is appealing in both populations, but is most pronounced for patients having surgically resected NSCLC; minimal tumor burden is generally perceived as the most amenable clinical target for immunotherapy, and the nominal risk of the intervention is highly relevant, because we cannot yet predict recurrence beyond statistical probability. In contrast, patients with advanced-stage lung cancer, especially those individuals with bulky disease, have not been widely viewed as likely to benefit from immunotherapy. Beyond mechanical factors related to tumor burden, there are numerous biological elements that could negate potential therapeutic effects of immune intervention. Notably, larger and metastatic tumors are known to acquire enhanced resistance capability during progression; immunosuppressive elements are also more prominent in advanced-stage cancer, including a purported corresponding increase in T-regs with increasing tumor burden. Nonetheless, multiple studies indicate that vaccines can induce immune responses in a percentage of patients with advanced-stage disease, and data from completed phase IIB clinical trials suggest that lung cancer vaccines may slow disease progression and/or improve quality of life in this population. When assessing efficacy of immunotherapy for advanced-stage NSCLC, it is important to keep in mind that an added therapy that may only provide marginal improvements in survival or quality of life, but has minimal risk, is commensurate with the definition of beneficial treatment currently applied to conventional chemotherapy.

Immunotherapy for SCLC has not been as widely investigated. The disease tends to be very aggressive, and immunotherapy has not been widely regarded as having a significant potential to impact outcomes. Because SCLC is an exclusively nonsurgical disease, tumor is not routinely available for autologous vaccine production. The lack of pathologic specimens has also slowed preclinical investigation, as SCLC is not available for in vitro study or xenogeneic tumor modeling, nor does there exist an appropriate SCLC animal model. Regardless, the poor prognosis associated with SCLC makes it a rational choice for further investigation, and several groups are auspiciously pursuing vaccines for SCLC.

Clinical Testing
The fundamental objective of clinical immunotherapy testing is to gain relevant biological and/or therapeutic information that promotes rational development of therapeutic strategies. The complexity of the immune system and reliance on an incompletely characterized series of events makes this a challenging prospect. Animal studies do provide insight into immunobiology, and have been critical in rational design of immunotherapy strategies; the parallels to human disease are, however, imperfect, and clinical trials are built on only partial understanding of human cancer immunobiology. Although clinical study design is based on a fair amount of speculation, extrapolation from measurable immunological parameters in early-phase clinical trials provides valuable information. A number of in vitro assays that measure a variety of parameters in peripheral blood after intervention allow comparative appraisal of immunological activity. Importantly, observed correlation in some clinical trials between immunologic activity (measured by a variety of assays) and clinical response criteria strongly supports the hypothesis that immunological response to relevant antigens translates into clinical benefit. Definitive evidence of therapeutic effect in phase III trials and an established link between immunological assays and clinical outcomes will both facilitate and promote logical development of cancer immunotherapy.

Immunologic Monitoring
Immunologic endpoints are critical for determining biologic activity of active immunotherapies in phase I and phase II studies, and serve as reasonable measures of vaccine potency during therapeutic development. To date, there is not a standard assay nor consensus on what constitutes a positive immunologic response (36). Considerations when choosing assays for analysis include the information desired (induction of antibody, T cell, or T-cell subsets), the number of samples to be assayed, and the volume of sample obtainable from each subject on serial blood draws. Serum antibody measurement is well established, standardized, simple, and accurate; nonetheless, few vaccines are designed to generate antibodies as a primary effector mechanism, and the therapeutics relevance of antibody is uncertain. Conversely, measurement of antigen-specific T cells in samples from immunized patients is less-well standardized, laborious, but more highly relevant to desired effects of most immunotherapies.

Functional T-cell assays include cytotoxicty, proliferation, and cytokine production assays that all measure T-cell reactivity upon antigen stimulation. Each is employed to demonstrate higher frequency of antigen-specific T cells in circulating peripheral blood mononuclear cells after vaccination compared with baseline (prevaccine). Interpretation is based on the notable assumption that the T-cell responses of peripheral blood mononuclear cells reflect responses in draining nodes or site of tumor, and analysis may over- or underrepresent compartmental activity. Delayed-type hypersensitivity to antigen challenge is a qualitative functional assay performed in vivo. Similar to the Mantoux skin test for tuberculosis, a mononuclear cell response is mounted at the site of antigen challenge if the patient has preexisting T-cell immunity. This is a widely used assay that can be practically applied to most vaccine approaches; however, sensitivity is limited and results in clinical trials have been variable. Preexisting immunity, which may be induced by a patient's own tumor, and biological differences in immune competence can cloud the readout. Tetramer staining is a nonfunctional, quantitative measure of antigen-specific T-cell frequency in peripheral blood. Florescent-labeled tetramers are constructed to bind a unique MHC/peptide-specific T-cell receptor, and tetramer-tagged T cells are quantified by flow cytometry. MHC class I–peptide tetramers used to measure CD8+ CTL specific for select antigens are the most commonly available, although some MHC class II–peptide tetramers have also been developed for the assessment of CD4+ T-cell responses. Because tetramers are specific for a single, specific HLA-matched epitope, this assay is most practical for use with HLA-restricted peptide vaccines. Tetramers are, however, not available for all antigens or all HLA types (the reader is directed to References 36 and 37 for comprehensive discussion of tools for immunologic monitoring of cancer vaccine trials).

There are also a number of ancillary measurements that may reflect the receptiveness of the host to immune induction. Serum and cellular cytokine characterization has been used to gauge immune hyporesponsiveness, and to measure the effectiveness of immune modulators. Similarly, immunological characterization of the tumor compartment has indicated presence of multiple elements that inhibit antigen presentation and neutralize effector cells; these measurements are, however, neither readily available nor routinely sought. In parallel with evolving knowledge of T-regs, measurement of percent circulating T-regs by cell surface cytometric analysis appears to offer an additional gauge of host immune responsiveness. CD4+CD25+FoxP3+ T-regs are most well characterized, although several different T-reg populations have been identified. Notably, the literature reports variably elevated levels of CD4+CD25+FoxP3+ T-regs in peripheral blood and in tumor beds of patients with lung cancer, a finding that may correlate with stage and/or prognosis (38–43). T-reg measurements may prove to be a relative predictor of resistance to immune induction and become a directed endpoint for immune modulation.

Clinical Endpoints
Clinical endpoints are the foundation of phase IIB and phase III trials. Rate of recurrence or risk of recurrence is the primary clinical endpoint in studies with surgically resected patients with lung cancer; advanced-stage disease offers several measures of efficacy, including objective tumor response, duration of response, time to progression (TTP), disease-free survival, and overall survival. Objective tumor response may be measured by Response Evaluation Criteria in Solid Tumors (RECIST) or World Health Organization criteria, but are reserved for patients with measurable disease at baseline; this typically includes serial measurement of one or more target lesions by computed tomography. Increasingly sensitive radiographic techniques that have yet to be validated for clinical investigation may provide newer clinical response criteria that could be used as intermediate endpoints in phase II studies (44). Tumor markers may be used as a supplementary measurement, but, alone, are not accepted as a measure of response.

Although targeting early-stage disease is appealing, an important consideration for therapeutic development is that relatively low rates of recurrence in surgically resected patients dictates a large study sample size to show clinical benefit in phase III trials. By contrast, radiographically measurable disease, rapid progression, and terminal prognosis of advanced-stage lung cancer provides timely endpoint analysis for therapeutic clinical trials. The comparatively small study numbers required to see statistical differences between study groups also makes this an attractive population for therapeutic investigation. Of note, novel molecular analysis may soon be able to determine which individuals are likely to recur after surgical resection of NSCLC, making immunotherapy studies more rational and efficiently conducted in a refined population of patients having had surgical resection.

NONRANDOMIZED CLINICAL TRIALS IN LUNG CANCER

Myriad approaches with multiple antigens have been explored in a variety of malignancies. The published literature describes fewer than 600 patients with lung cancer that have been treated in 25 pilot, phase I, or early phase II studies using 17 different vaccines. Categorization of these trials roughly parallels the classification of immune adjuvants, including: (1) antigen plus chemical/biologic adjuvant; (2) antigen plus cytokine; (3) antiidiotype plus biologic adjuvant; (4) antigen-loaded DCs; and (5) gene therapy. The objectives of each of these trials included safety, tolerability, and biological activity (10).

Comparison of these small studies is difficult if not impossible (10, 36, 37). Each involves a different study population, vaccine protocols, doses, frequency, and even routes of administration. A number of different antigens have been incorporated into a variety of vaccine preparations, and responses were assessed by variable means of immune assessment and nonrigorous clinical response criteria. Nonetheless, the literature collectively indicates that immunotherapy for lung cancer is feasible and rational. Specifically, it is apparent that toxicity is limited, and that multiple agents can induce measurable immunologic response. A consistent observation in all studies is that immune induction is not uniform, even within homogenous study populations, indicating a biological variability that regulates individual responses, which is independent of vaccine potency. Furthermore, although early-phase trials are not powered to define therapeutic efficacy, several trials offer anecdotal evidence of clinical benefit. An important and highly relevant corollary to these observations is that several studies strongly support a correlation between clinical and immunological response.

ADVANCED STUDIES USING SELECTED AGENTS

A handful of immunotherapies for lung cancer have been investigated in advanced-phase studies. Nine vaccines tested in independent trials are discussed here (an overview is presented in Table 1). All but one study has been conducted in NSCLC; the only SCLC vaccine of this group is also the only one to have been completely evaluated in a phase III trial. One other study in NSCLC, designed as a phase III, had a high dropout rate and has statistical power that more closely resembles a randomized phase IIB study. Four of the other seven agents discussed here have been tested in phase IIB, randomized, controlled trials; the remaining three represent a series of vaccines from a single group, which were each tested in larger, nonrandomized phase II studies. With few exceptions, adverse events reported in all studies were limited to local site reactions, and less frequently, flu-like symptoms and fatigue.


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  		TABLE 1. RANDOMIZED CLINICAL VACCINE TRIALS IN LUNG CANCER: PHASE IIB AND PHASE III RANDOMIZED STUDIES

 
BEC2 and BCG
BEC2 is an antiidiotypic vaccine for SCLC and is the only lung cancer vaccine to have been completely evaluated in phase III studies. In the initial phase I/II trial, patients with limited-stage SCLC achieving a partial or complete response from chemotherapy were vaccinated with an antiidiotype GD3 monoclonal antibody (BEC2) and BCG. Those who developed anti-GD3 antibodies had prolonged survival compared with historical control subjects. These encouraging results led to a phase III trial sponsored by Merck KGaA and ImClone. A total of 515 patients were randomized 1:1 to receive five immunizations of 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, vaccine induced humoral response in only one-third of the 213 patients who were able to be evaluated immunologically (negative in 142 cases and positive in 71 cases). Stratified analysis suggested a trend toward improved survival in patients that showed immunological response, although differences were not statistically significant. The authors concluded that the study was essentially negative; however, they acknowledged that the agent has limited ability to induce antibody response, and that the reduced number of patients in stratified groups likely limited the power of their analysis. Furthermore, the authors conclude that a monovalent approach may not have been ideal, indicating that multivalent approaches may be better (46).

Anti-EGF Vaccine
The anti-EGF vaccine is unique among other lung cancer vaccines in that it does not directly target tumor, but, rather, has the primary objective of inducing anti-EGF antibodies that neutralize endogenous EGF and deprive tumor of this important growth factor (47, 48). The vaccine is comprised of recombinant EGF, chemically conjugated to a recombinant P64K bacterial protein as carrier protein, and emulsified with the adjuvant Montanide ISA51. The randomized phase IIB study enrolled 80 patients with stage IIIB/IV NSCLC who had completed first-line chemotherapy. Patients were randomized 1:1 to BSC or EGF vaccinations. The treatment group received four induction immunizations and monthly boosters until disease progression. Vaccine induced a desirable anti-EGF antibody response in roughly 50% of immunized patients, which corresponded to a significant decrease in serum EGF concentrations. There was a significant correlation between anti-EGF antibody titers, serum EGF levels, and clinical outcomes. The authors report an overall trend toward increased survival in the treatment group compared with control subjects that reached statistical significance in a subgroup of patients less than 60 years old (47, 48).

SRL172
The SRL172 approach relies on the immunogenicity of a heat-killed Mycobacterium vaccae (SRL172) to induce a favorable, albeit nonspecific, systemic immune response that promotes autologous antigen recognition. SR Pharma plc (UK) sponsored a randomized trial using SRL172 concurrently with platinum-based chemotherapy with the intention of inducing systemic reactivity during endogenous tumor antigen release. Although designed as a phase III, non–placebo controlled, randomized study, the trial was subject to a high dropout rate, and the reduced number of evaluable patients limited the statistical power. A total of 419 patients with stage III/IV NSCLC were randomized 1:1 to receive serial injections of 109 bacilli (five monthly injections followed by monthly maintenance) administered concurrently with six cycles of chemotherapy delivered on a 21-day schedule. Fewer than 50% of the subjects completed the prescribed series of vaccines. Including all subjects, the authors concluded that SLR172 significantly improved patient quality of life without affecting overall survival (median, 223 vs. 225 days; P = 0.65). Secondary analysis showed that patients with adenocarcinoma who completed the vaccine protocol (n = 45) versus matched control subjects receiving chemotherapy alone did have a survival advantage (median overall survival, 302 vs. 177 days; P < 0.01) that was not observed in patients with squamous cell cancer (n = 61) (49, 50).

GVAX, Allo-GVAX, and Lucanix
GVAX has been highly publicized through promotion by industry sponsors, and the high-profile nature alone makes GVAX and two related studies worthy of discussion. Although none of the agents have undergone randomized, controlled investigation, the considerable logic of phased development behind this series of gene therapy approaches is highly instructive.

GVAX.
The initial study evaluated an autologous tumor cell vaccine (GVAX) in 33 individuals with advanced- and 10 with early-stage NSCLC. Autologous tumor obtained from surgery was transduced ex vivo with an adenoviral vector delivering GM-CSF cDNA to processed tumor cells. Of 33 patients with advanced NSCLC, three (two of which were bronchoalveolar cell carcinoma) achieved complete response and prolonged remission. Eight of the 10 patients with early-stage lung cancer remained free of disease with a median follow-up time of 12 months. The authors reported a positive correlation between GM-CSF production by the vaccine and clinical response. An important aspect of this study was that, of 83 tumors harvested, vaccines could not be successfully generated in 16 patients, and 11 other died before vaccine could be delivered. The median production time was 31 days, and median time from tumor harvest to immunization was 49 days. A total of 43 patients were immunized (51).

Allo-GVAX (bystander GVAX).
Based on encouraging anecdotal response that correlated with higher levels of GM-CSF production, and in response to the limitations imposed by protracted vaccine production, the group modified their approach. An updated vaccine mixed autologous tumor with an allogeneic, non–lung cancer cell line (K562 erythroleukemia cells) engineered to express GM-CSF, and thereby removed the requirement for gene modification of individual tumors. The vaccine was tested in a nonrandomized study of 86 patients with advanced-stage NSCLC, with the objectives of safety, feasibility, pharmacokinetics, and efficacy. Cell processing was successful in 76 patients, and 49 proceeded to vaccination. Although the GM-CSF secretion was 25-fold higher than autologous GVAX, the investigators disappointingly did not observe an objective tumor response similar to that seen in the GVAX study. A higher frequency of injection site reactions than with the parent GVAX was also observed (52).

Lucanix.
The same group then evaluated an entirely allogeneic antigen approach that standardized vaccine production and eliminated the need for tumor cell harvest. Lucanix is comprised of four allogeneic NSCLC cell lines transduced with transforming growth factor-β antisense plasmid. The study was conducted as a phase II nonrandomized trial with dose escalation in 75 patients with NSCLC (14 early stage, 61 late stage). Endpoints included safety, feasibility, pharmacokinetics, and efficacy. Results reported in 2006 observed 15% response by RECIST, with an increase in estimated survival compared with historical control subjects, and a correlation of survival with higher dose levels. In 2008, the group initiated a phase III placebo-controlled trial of Lucanix in stage III and IV NSCLC (53).

BLP25 (Stimuvax)
BLP25 liposome vaccine (L-BLP25) carries the mucin-1 (MUC1) protein admixed with monophosphoryl lipid A as an immune adjuvant. The randomized, phase IIB trial (1:1 BLP25 vs. BSC), sponsored by Merck KGaA, enrolled 171 patients with stage IIIB/IV NSCLC with response or stable disease (SD) after first-line therapy. Patients, stratified by stage (IIIB vs. "wet" IIIB/IV), received eight weekly subcutaneous injections of BLP25 SQ; patients also received additional treatment with 300 mg/m2 cyclophosphamide 3 days before immunization. The primary endpoint was clinical outcomes. Data analysis showed no statistical difference in overall survival (17.4 vs. 13 mo; P = 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 = 35) compared with matched control subjects (n = 30). Based on the promising results in this subgroup of patients, Merck KGaA sponsored the multicenter (international) phase III, randomized, double-blind, placebo-controlled trial. The START study (Stimulating Targeted Antigenic Responses to NSCLC) opened to enrollment in 2007. Targeted accrual is 1,300 patients with unresectable stage III NSCLC that have responded to first-line, platinum-based chemoradiotherapy (54).

MAGE-A3 Antigen-Specific Cancer Immunotherapy
GlaxoSmithKline produced a recombinant MAGE-A3 fusion protein (His-tagged/full-length MAGE-A3 protein/influenza protein D) plus immune adjuvant AS02B (monophosphoryl lipid A and QS21) that was tested in a double-blind, randomized, placebo-controlled, phase IIB trial (2:1 randomization, vaccine vs. BSC). A total of 182 patients with resected stage IB/II, MAGE-A3–positive NSCLC were serially immunized and evaluated for time to recurrence. Investigators observed a delayed time to recurrence at 28 months (31.6 [vaccine] vs. 43.3% [control]), which translated into a 27% reduction in relative risk of cancer recurrence (55, 56). Although differences failed to meet statistical significance, results were suggestive enough to pursue phase III investigation. The international, multicenter MAGRIT study (MAGE-A3 Adjuvant Non–Small Cell Lung Cancer Immunotherapy), opened to enrollment in 2007, plans to accrue 2,270 MAGE-A3–positive patients with completely resected stage IB, II, or IIIA NSCLC.

TG4010
The TG4010 vaccine uses a dual-gene–based approach to coexpress MUC1 antigen and IL-2 (MVA–MUC1–IL-2) with a recombinant vaccinia virus. Two serial studies, sponsored by Transgene (Strasbourg, France) evaluated clinical endpoints in patients with stage IIIB/IV NSCLC administered TG4010 concurrently with conventional chemotherapy. The initial phase IIA, nonrandomized study evaluated 44 patients, and observed tumor response rate of 37%; 13 partial responses (RECIST), 71% with partial response (PR) or SD for over 12 weeks; estimated median TTP was 6.4 months; estimated overall survival was 13 months. In the follow-up phase IIB study, 148 patients were randomized to TG4010 plus chemotherapy or chemotherapy alone (gem/cis). Patients in the vaccine arm were immunized weekly with TG4010 for 6 weeks, then received maintenance injections every 3 weeks until progression. Interim results reported at the American Society of Clinical Oncology in June 2008 indicate statistical differences in clinical response criteria, and a non–statistically significant increase in progression-free survival at 6 months. Data on overall survival are not yet mature, but show an encouraging trend favoring survival in the treatment group compared with matched control subjects; final study results are expected in late 2008 (57).

Phase II Studies: What Have We Learned?
Supplementing information from early-phase trials, these studies indicate that immunotherapy carries limited toxicity. Consistent with phase II objectives and study design, there is suggested benefit in early- and late-stage NSCLC. Data indicate that phase III investigation is warranted and necessary. In addition to the need for definitively addressing therapeutic efficacy, a number of relevant questions are afield. These include relative potency and comparative efficacy of different formulations, and practical information about necessary duration of response, ideal dosing schedule, and appropriate timing relative to administration of conventional therapies.

FUTURE DIRECTIONS AND OBJECTIVES

The lack of proven clinical benefit continues to encumber development of immunotherapy for lung cancer. This fact makes proven clinical efficacy in phase III trials an overarching objective. Assuming one or more agents are shown to have clinical benefit in lung cancer, however small, attention can be judiciously turned to optimizing efficacy. There are several key points to consider in that pursuit. It is probable that measurable immunological response to relevant tumor antigens will positively correlate with clinical benefit. In the context of measurable activity already shown by multiple agents and formulations, it is to be expected that a number of different agents can yield similar results. Well standardized and validated immunological assays used as a comparative measure of potency may show some differences in biological activity, but it is unlikely that there will emerge a single ideal approach. In a search for the highest-potency vaccine formulations, pragmatic considerations of simplicity, cost, and portability are highly relevant. Efficacy will, however, still depend on relevance of antigens. Questions remain about whether there are significant differences in immunogenicity between autologous and allogeneic tumor antigen. Similarly, future investigation should address the therapeutic equivalence of various allogeneic approaches, with practical consideration to the fact that customized monovalent approaches are currently applicable and restricted to a relatively small and selected portion of the lung cancer population. It is also becoming clear that abrogation of immune regulatory elements will be necessary before immunotherapy achieves full therapeutic potential (58). Our current knowledge of immune regulation in lung cancer already offers multiple options for immune modulation, and continued investigation promises to yield additional targets and corresponding agents that could improve efficacy. In the future, it is likely that combinations of multiple immunologically active agents, conventional treatment modalities, and novel targeted therapies, used in concert, will overcome limitations of any single approach and lead to significant improvements in therapeutic outcomes of lung cancer.

FOOTNOTES

Conflict of Interest Statement: Neither of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form June 16, 2008; accepted in final form July 21, 2008)

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