98 - Lung Cancer: Epidemiology and Carcinogenesis
Editors: Shields, Thomas W.; LoCicero, Joseph; Ponn, Ronald B.; Rusch, Valerie W.
Title: General Thoracic Surgery, 6th Edition
Copyright 2005 Lippincott Williams & Wilkins
> Table of Contents > Volume II > Section XVI - Carcinoma of the Lung > Chapter 115 - Novel Strategies for Lung Cancer Immunotherapy
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Chapter 115
Novel Strategies for Lung Cancer Immunotherapy
David S. Schrump
Dao M. Nguyen
Steven E. Finkelstein
A growing body of literature, including that of Nagorsen and associates (2003), indicates that cancer patients can mount an immune response to their tumors, leading on occasion to complete eradication of their disease. Whereas the evidence for immune-mediated cancer regression is most firmly established for melanomas and renal cell carcinomas, a number of studies, such as those of Nakamura (2002) and Mazzoccoli (2003) and their colleagues, suggest that the proliferation and metastatic potential of lung cancers may be modulated by immunologic mechanisms as well. Lung cancers contain abundant tumor-infiltrating lymphocytes (TILs) that exhibit clonal proliferation indicative of immune recognition of tumor-associated proteins. In addition, lung cancers frequently express shared antigens that can be recognized by antibodies or cytolytic T lymphocytes (CTLs), as reported by one of us (DSS, 1988), Hirohashi (1986), Bazhin (2003), Takenoyama (1998), Yamada (2003), and Mami-Chouaib (2002) and respective coinvestigators.
In 1983, Vanky and co-workers observed that lung cancers are nearly as immunogenic as melanomas and renal cell carcinomas, and that immune recognition of autologous tumor cells enhances survival in lung cancer patients. More recently, Uchida and collaborators (1990) reported a statistically significant correlation between T cell mediated autologous antitumor responses and disease-free interval, as well as overall survival, in stage I/II lung cancer patients undergoing curative resections. However, to date, the efficacy of immunotherapy for lung cancer has not been established. This chapter summarizes recent progress concerning the identification of relevant targets for lung cancer immunotherapy, and discusses novel strategies to enhance antitumor immune responses in lung cancer patients.
MECHANISMS OF ANTITUMOR IMMUNITY
The goal of immunotherapy is the eradication of established cancers. Regression of solid tumors in humans occurs primarily, but not exclusively, by T cell mediated mechanisms, as observed by Schreiber (1999) as well as by Whitesell and Cook (1996). Experiments utilizing animals in which T-cell subsets have been depleted via gene-knockout or antibody techniques have firmly established that T cells are critical components of antitumor immune responses. As such, appreciation of how T cells recognize tumor antigens is critical for the development of efficacious lung cancer immunotherapy.
T cells can be broadly subdivided into two major classes: CD4+ and CD8+ T cells. In humans, as noted by Restifo and Wunderlich (2001), these T-cell subsets are distinguished by specific interactions with major histocompatibility complex (MHC) antigens and their effector functions. The class I human leukocyte antigen (HLA) molecule is a heterodimer consisting of a highly polymorphic chain containing three regions ( 1, 2, and 3) and 2-microglobulin; the CD8 molecule on the CTL binds to the 3 domain to stabilize interactions between the T-cell receptor and the MHC peptide complex on the target cell. The class II HLA molecule is a heterodimer consisting of an chain containing 1 and 2 subunits, and a chain consisting of 2 and 2 subunits; the CD4 molecule on the T helper cell binds to the 2 domain to stabilize interaction between the T-cell receptor and MHC peptide complex on the surface of professional antigen-presenting cells (APCs) such as macrophages, B cells, and dendritic cells. The overall architecture of the peptide-binding sites for class I and class II MHC molecules is relatively similar, as pointed out by Peace and associates (1991) as well as by Pichler and Wyss-Coray (1994). However, class I MHC molecules present peptides containing 8 to 10 amino acids, whereas class II MHC molecules usually present peptides containing 12 to 20 residues that are frequently nested within a larger sequence.
CD4+ and CD8+ T cells exhibit different functions. For instance, CD4+ cells recognize peptide antigens presented in the context of class II MHC molecules typically expressed by professional antigen-presenting cells. CD4+ T
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cells are composed of helper (TH) and T regulatory/suppressor (Treg) subsets. T helper cells are critical for initiating and perpetuating CTL reactivity to viral, tissue allograft, and autologous tumor antigens. Without CD4+ T-cell help, CD8+ T cells may become lethargic or deleted in vivo following antigen stimulation. CD4+CD25- T helper lymphocytes secrete interleukin-2 (IL-2), which enhances activation and clonal expansion of CD8 cells, as well as a variety of additional cytokines mediating immunoglobulin class switch in B cells. According to Green and colleagues (2003), CD4+CD25+ Treg cells mediate immunologic tolerance to self antigens. Diminished CD4+CD25+ Treg cell function has been implicated in the pathogenesis of autoimmune diseases such as asthma and rheumatoid arthritis by the studies of Umetsu and coinvestigators (2003). Conversely, increased CD4+CD25+ Treg cell activity may inhibit response to autologous tumor antigens in cancer patients, as noted by Wolf (2003) and Woo (2002) and their co-workers.
CD8+ T cells recognize peptide antigens in the context of class I MHC molecules, which are typically expressed on epithelial cells. CD8+ cytolytic T lymphocytes, as discussed by Hellstrom and Hellstrom (1998) and Sherman and colleagues (1998), mediate lysis of virally infected cells, tissue allografts, and cancers. Class I MHC molecules generally present peptides derived from intracellular proteins such as mutant or overexpressed oncoproteins, as well as normal tissue-differentiation antigens, as pointed out by Pichler and Wyss-Coray (1994). These peptides are generated within proteosomes composed of low-molecular-weight proteins (LMPs), including LMP2 and LMP7, and then transferred to newly synthesized MHC class I molecules in the endoplasmic reticulum by an ATP-dependent mechanism requiring several proteins including TAP (transporter associated with antigen binding) 1 and 2, as described by Restifo (1993) and Ortmann (1994) and their coinvestigators. According to studies by Restifo and associates (1996), 2-microglobulin is critical for the folding and stability of the class I MHC complex at the cell surface; deficiencies of 2-microglobulin expression result in markedly reduced MHC expression in a variety of human cancers. Expression of LMP, TAP, class I heavy chain, and 2-microglobulin can be induced by interferon- (IFN- ). In contrast, Chicz and Urban (1994) observed that class II molecules are generally involved in the presentation of soluble antigens that are endocytosed by professional APCs and subsequently bound to class II MHC molecules as they recycle to the cell surface.
T-cell activation, as noted by Restifo and Wunderlich (2001) as well as by Hellstrom and Hellstrom (1998), requires high-affinity interaction between the T-cell receptor and the appropriate MHC peptide complex on the target cell as well as the presence of a variety of adhesion molecules and costimulatory ligands such as B7.1 and B7.2. In the presence of high-affinity interaction and appropriate costimulation, T helper cells proliferate in response to antigen and secrete a variety of cytokines, particularly IL-2 and IFN- , that provide costimulation required to activate CTLs. In contrast, as shown by Seder and Mosmann (1999), interaction between T cells and their respective targets in the absence of appropriate costimulation results in antigen-specific unresponsiveness (anergy).
Recent data published by Phan and associates (2003) suggest that effective antitumor immunotherapies require induction of autoimmune reactions via breaking immune tolerance. If tolerance is dysregulated, Ryan (2001) and Espenschied (2003) and their coinvestigators have suggested that peptides corresponding to mutant as well as wild-type genes that are aberrantly expressed within cancer cells may induce an antitumor response. Nevertheless, despite the expression of self, foreign, or mutated self antigens by cancer cells, immune responses that result in complete and durable tumor regression are not consistently observed; numerous potential mechanisms exist whereby a transformed cell may escape immune surveillance (Table 115-1). Lack of antigen presentation due to insufficient binding of peptides to MHC alleles, as well as diminished expression of antigen-processing enzymes or MHC components, impairs recognition of tumor cells; this phenomenon occurs frequently in small cell lung cancers, as pointed out by Restifo and colleagues (1993). Interaction of initiator (helper) or effector (cytolytic) T cells with antigens in the tumor site or shed into the circulation
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in the absence of costimulation may induce cancer-specific anergy, as described by Seder and Mosmann (1999). CD4+CD25+ Treg cells may inhibit the proliferative and cytolytic activities of TIL and natural killer (NK) cells. Antitumor immunity also may be inhibited by soluble factors expressed by cancer cells. For instance, Sharma and coinvestigators (2003) have shown that tumor supernatants inhibit dendritic cell function via cyclooxygenase-2-dependent pathways; tumor supernatants also enhance apoptosis in activated T cells via IKB-dependent mechanisms, as noted by Batra and associates (2003). In addition, Kataki and coinvestigators (2002) reported that tumor cells secrete transforming growth factor- (TGF- ), which suppresses proliferation of T cells following antigen recognition. Tada and colleagues (2003) reported that Fas ligand on cancer cells induces apoptosis of T cells following activation by tumor antigens. Each of these complex issues must be simultaneously considered as we attempt to develop effective strategies for lung cancer immunotherapy.
Table 115-1. Mechanisms of Impaired Immune Response to Cancer | ||
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POTENTIAL TARGETS FOR LUNG CANCER IMMUNOTHERAPY
ras
The N-, R-, and K-ras genes encode 21-kilodalton proteins that are integral membrane components of mitogen signal cascades. Mutations involving ras genes have been observed by Rodenhuis and Slebos (1992) in 20% of all solid malignancies, including nearly 40% of pulmonary adenocarcinomas. Somatic mutations involving ras genes typically occur in codons 12, 13, or 61, creating tumor-specific antigens that Midgley and Kerr (2002) suggest may be exploited for lung cancer immunotherapy.
Peace and associates (1991) observed that mice immunized with mutant ras peptides developed CD4+, MHC class II restricted T cells. Subsequently, Fenton and colleagues (1993) reported that mice immunized with ras peptides containing the gly-to-arg alteration frequently observed at codon 12 (arg 12) developed CD8+ T cells that specifically lysed tumor cells expressing arg 12 ras and protected them from challenge with cells expressing mutant, but not wild-type, ras genes. Subsequently, Abrams and coinvestigators (1995) observed that mice immunized with peptide corresponding to a gly-to-val mutation at position 12 generated peptide-specific CD4+ cells.
The fact that mutant ras peptides were immunogenic in murine models prompted a number of investigators to evaluate immune response to ras proteins in cancer patients. Jung and Schluesener (1991) isolated CD4+ T cells from peripheral blood of normal individuals following in vitro exposure to (val 12) ras peptides, demonstrating the capacity of these individuals to recognize mutant ras proteins. Subsequently, Fossum and collaborators (1994, 1995) isolated CD4+ and CD8+ cells from colorectal cancer patients specific for mutant K-ras (gly 13 to asp). Collectively, these data indicate that mutant ras proteins can be recognized in normal and tumor-bearing individuals.
Normal ras peptides do not bind to HLA motifs, and therefore are not immunogenic. In contrast, mutations at codons 12 or 13 that are typically observed in cancers result in amino acid substitutions that increase interactions between ras peptides and HLA molecules, thereby enhancing peptide presentation. Nevertheless, interactions between mutant ras peptides and class I MHC molecules are relatively weak, and Bergmann-Leitner and co-workers (1998) noted that maximal killing of tumor cells by ras-specific CTLs in vitro often requires exogenous IFN- , which increases HLA and adhesion molecule expression on tumor cells. Hence, naturally occurring mutant ras epitopes are weak immunogens due to their inefficient binding to HLA molecules.
Bristol and associates (1998) reported that ras 4 12 (val 12) peptide induced a weak ras-related CTL response in mice. Substitution of valine at position 12 with leucine or isoleucine anchored the ras peptide to MHC class I and enhanced the cytotoxicity of anti-ras CTL relative to the original ras (val 12) peptide. Furthermore, mice immunized with ras 4 12 (leu 12) peptide were resistant to challenge with tumor cells expressing ras (val 12) epitopes. Yokomizo and colleagues (1997) observed that replacement of tyrosine by tryptophan at position 4 of ras 3 17 peptide enhanced the recognition of ras (val 12) by CD4+ T cells isolated from a gastric cancer patient. These studies indicate that mutant ras peptides can be processed by cancer cells for recognition by CD4 and CD8+ T cells specific for these epitopes, and that immunogenicity of mutant ras peptides can be augmented by alterations in anchor residues that enhance binding of these peptides to class I MHC molecules. Furthermore, cytotoxicity of ras-specific CTLs against tumor cells expressing mutant peptides can be augmented by cytokines such as INF- that enhance antigen processing and MHC expression. These observations are consistent with data reported by Rosenberg and coinvestigators (1998), in which experimental alterations of gp100 enhanced binding of this melanocyte differentiation antigen to HLA class I molecules. Vaccination of melanoma patients with modified gp100 peptides, particularly in the context of high-dose IL-2, enhances the response of these individuals to tumor cells expressing wild-type gp100.
Khleif and co-workers (1999) vaccinated cancer patients with peptides reflecting ras mutations in their tumors. Three of ten evaluable patients had evidence of immunization following three vaccinations with 500- g peptide in Detox adjuvant (Ribi ImmunoChem Research, Hamilton, MT, U.S.A.). Two individuals (including a 58-year-old woman with a poorly differentiated pulmonary adenocarcinoma) developed CD4+ T cell responses, and one patient developed a CD8+ T cell response to mutant ras peptides; CD8+ T cells from this latter individual specifically recognized
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HLA-matched allogeneic tumor cells expressing the appropriate ras mutation. No significant systemic toxicities were noted in this phase I study, and response to vaccination was not dose dependent. Although no clinical responses were observed in these patients (all of whom had advanced bulky disease), this clinical trial demonstrated the feasibility of generating CD4+ and CD8+ T cells specific for mutant ras epitopes in cancer patients.
In a related phase I/II study, Gjertsen and associates (2001) vaccinated 48 pancreatic cancer patients with synthetic mutant ras peptides in conjunction with granulocyte-macrophage colony-stimulating factor (GM-CSF). Peptide-specific immune responses were observed in 58% of evaluable individuals. Peptide-specific CD4+ cells were isolated from tumors containing the ras mutation, indicating that ras-specific T cells could selectively accumulate within the primary cancer. Patients demonstrating immune response to peptide vaccines experienced significantly enhanced survival times relative to individuals in whom ras-specific immune reactivity was not observed (median survival: 148 days vs. 61 days, respectively; p = 0.0002). Overall, these data indicate that ras-specific immune responses can be generated in cancer patients following peptide vaccinations in the context of a potent adjuvant cytokine, and that immunotherapy may prolong survival of patients whose tumors exhibit ras mutations.
p53
The p53 tumor suppressor gene encodes a 53-kilodalton DNA binding protein, which is critical for regulating cell cycle progression, DNA repair, and apoptosis in normal and malignant cells. Mutations involving p53 have been observed by both Mitsudomi (1992) and D'Amico (1992) and their colleagues in nearly 75% of lung cancers irrespective of histology. Hollstein and co-workers (1991) found that the majority of mutations occurred within the central, DNA binding domain of the p53 molecule. Most of these mutations alter the conformation of the p53 protein, stabilizing it against ubiquitin-mediated degradation. The prolonged half-life of mutant p53, according to Kirsch and Kastan (1998), accounts for the increased immunoreactivity observed in cancers relative to adjacent normal tissues in which p53 levels are barely detectable.
Mutant as well as wild-type p53 proteins are immunogenic in cancer patients. Winter and associates (1993) observed that 13% of lung cancer patients had circulating p53-reactive antibodies; all patients with p53 autoantibodies exhibited p53 missense mutations in their primary tumors; no p53 autoantibodies were observed in individuals whose tumors had stop, splice, or frameshift mutations involving this tumor suppressor gene. Additional studies revealed that these p53 autoantibodies exhibited cross reactivity with a variety of mutant p53 proteins. In general, as identified by Lubin and colleagues (1993), the epitopes recognized by p53 autoantibodies are wild-type sequences located in the amino or carboxy terminus of the p53 molecule; overexpression renders these wild-type peptides immunogenic in cancer patients.
Several studies have been performed to ascertain the prognostic significance of p53 autoantibodies in lung cancer patients. Iizasa and coinvestigators (1998) detected p53 autoantibodies in 13 (21%) of 62 lung cancer patients undergoing curative resections. Forty percent of patients whose tumors exhibited p53 immunoreactivity had p53 autoantibodies, compared with 3 of 37 patients whose tumors lacked detectable p53 expression. The presence of p53 autoantibodies did not correlate with clinical stage or prognosis of lung cancer patients. Mitsudomi and co-workers (1998) observed p53 autoantibodies in 38% of 188 consecutive lung cancer patients undergoing curative resections. The presence of p53 autoantibodies correlated significantly with squamous cell histology, advanced stage of disease, and p53 overexpression in primary tumors. Whereas no correlation between p53 autoantibody titers and clinical outcome was observed by Mitsudomi and coinvestigators (1998), Lai and colleagues (1998) reported that p53 autoantibodies correlated with malignant effusions and diminished survival in lung cancer patients. In contrast, Bergqvist and associates (1998) observed that p53 autoantibodies correlated significantly with prolonged survival in lung cancer patients receiving radiation therapy. Hence, the prognostic value of p53 autoantibodies in lung cancer patients remains uncertain at present.
A number of investigators have isolated HLA-restricted CTLs recognizing mutant as well as wild-type p53 epitopes in normal as well as tumor-bearing individuals. Houbiers and colleagues (1993) isolated a CTL clone specifically recognizing a mutant p53 peptide. Ropke and co-workers (1995) evaluated the precursor frequency of p53-reactive lymphocytes in peripheral blood of healthy volunteers, and observed peptide-specific responses against two wild-type peptide sequences; precursor frequencies of lymphocytes reactive with these peptides correlated with their relative binding affinities to HLA class I motifs. No differences were noted between precursor frequencies for mutant versus wild-type p53 epitopes, suggesting that mutant p53 peptides are no more immunogenic than wild-type sequences in normal individuals. Ciernik and collaborators (1996) observed that human lung cancer cells can process and present mutant p53 epitopes for recognition by HLA-restricted CTLs specific for these peptides. Ropke and associates (1996) generated a CTL clone specific for a wild-type p53 sequence from a normal individual that could lyse oropharyngeal carcinoma cells expressing mutant p53. Collectively, these data suggest that overexpression of mutant p53 in human carcinoma cells results in the presentation of wild-type p53 epitopes that can be recognized by CTLs specific for these peptide sequences, and that tolerance to endogenously processed p53 protein epitopes can be broken by peptide-specific in vitro priming.
In light of the fact that lung cancers frequently express mutant p53, Chada and colleagues (2003) suggested that
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immunization with p53 peptides or recombinant viral vectors expressing wild-type p53 might be an attractive strategy for lung cancer immunotherapy. Vierboom and co-workers (1997) reported that adoptive transfer of p53-reactive CTLs eradicated p53-expressing tumor cells without inducing autoimmunity, thus demonstrating the capacity of these CTLs to selectively target tumor cells. Zwaveling and associates (2002) reported that CD4+ T-cell response to wild-type p53 sequences is not affected by tolerance at the T helper level. Yen and coinvestigators (2000) examined immune response to p53 in 10 lung cancer patients who were receiving monthly intratumoral injections of a recombinant adenoviral vector expressing p53 (Ad-p53). No consistent alterations in posttreatment humoral or cell-mediated immunity to p53 were observed, despite evidence of disease stabilization or regression following Ad-p53 therapy in these individuals.
Additional efforts have been under way to evaluate the efficacy and toxicity of a canary pox virus expressing wild-type p53 (ALVAC-p53). Hurpin and collaborators (1998) reported that intravenous but not subcutaneous, intramuscular, or intradermal administration of ALVAC-p53 induced p53-reactive CTLs in mice; additional experiments revealed that mice receiving intravenous ALVAC-p53 were protected from challenge with tumor cells overexpressing p53. Odin and associates (2001) reported that intravenous ALVAC-p53 induced eradication of syngeneic tumors expressing mutant p53. Although ALVAC-p53 has not been evaluated in lung cancer patients, Menon and colleagues (2003) recently administered ALVAC-p53 to 15 patients with advanced colorectal carcinomas overexpressing p53. Fever was the only vaccine-related adverse event; no autoimmunity was observed. Several patients exhibited cellular and humoral immunity to p53 following vaccine therapy. Although no objective responses were noted in this study, one patient exhibited stabilization of disease. These data support further evaluation of ALVAC-p53, particularly in the context of cytokine adjuvants for lung cancer immunotherapy.
CANCER/TESTIS ANTIGENS
In addition to point mutations, allelic deletions, and gene rearrangements that irreversibly activate protooncogenes and silence tumor suppressor gene expression, two of us (DSS and DMN, 1999), as well as Jones and Baylin (2002), have pointed out that epigenetic events involving reversible alterations in chromatin structure profoundly influence gene expression during malignant transformation. In recent years, chromatin structure in normal and malignant cells has been the focus of intense research efforts; appreciation of the mechanisms that regulate chromatin structure may enable us to pharmacologically manipulate genes encoding tumor-associated antigens. Chromatin is composed of nucleosomes that contain 146 base pairs of DNA coiled around an octamer of core histone proteins. Annunziato and Hansen (2000), as well as Nakayama and Takami (2001), have described the long lysine-rich amino-terminal tails of histone proteins that protrude from the DNA, exhibiting numerous sites for phosphorylation, methylation, and acetylation. These posttranslational modifications, as noted by Jenuwein and Allis (2001), determine interactions of histone proteins with DNA and other chromatin-associated proteins, which in turn modulate gene expression.
DNA methylation and histone acetylation contribute significantly to chromatin structure in normal and malignant cells, as discussed by Jones and Laird (1999). In general, transcriptionally active chromatin contains hypomethylated DNA sequences associated with acetylated core histones, whereas transcriptionally silent regions exhibit hypermethylated DNA associated with deacetylated core histone proteins, as described by Eden and associates (1998). Recent studies by Cervoni and Szyf (2001) indicate that DNA methylation promotes deacetylation of histone proteins; conversely, histone acetylation enhances DNA demethylation. These processes are highly dynamic, as described by MacLeod and Szyf (1995), and are influenced by a variety of stimulatory as well as inhibitory signal transduction pathways during cell cycle progression.
In mammalian cells, according to the studies of Attwood and colleagues (2002), DNA methyltransferases mediate methylation of cytosines in the context of CpG dinucleotides. Clusters of CG dinucleotides (CpG islands) are frequently located in promoter and proximal coding regions of genes exhibiting dynamic expression patterns. Methylation of CpG islands within promoter regions inhibits gene expression by rendering DNA inaccessible to the transcription machinery, as pointed out in the investigations of Nguyen (2001) and Siegfried (1999) and their co-workers.
Kouzarides (1999) showed that the acetylation status of core histone proteins is governed by the opposing actions of a variety of histone acetyl transferases (HATs), such as p300/CBP and PCAF-GCN-5, and several classes of histone deacetylases (HDACs). Acetylation of lysine residues by HATs diminishes DNA histone interactions, facilitating chromatin relaxation and gene expression. In contrast, as noted by Annunziato and Hansen (2000), deacetylation of lysines by HDACs enhances DNA histone interactions, resulting in chromatin compaction and repression of transcription.
Complex alterations in chromatin structure occur early during multistep carcinogenesis, as discussed by Clark and Melki (2002) and Wade (2001). Typically, cancer cells exhibit global DNA demethylation, which facilitates expression of a variety of genes on the X chromosome that encode proteins recognized by tumor-reactive lymphocytes, as noted by Maio and associates (2003). Scanlan and colleagues (2002) record that these immunogenic proteins are referred to as cancer/testis antigens (CTAs) because they are normally expressed only in testes or ovary, yet are aberrantly expressed in cancer cells. Haas and co-workers (1988) have shown that the expression of these proteins in gonadal tissues does not result in immune response because these tissues do not express HLA class I molecules, which are necessary for antigen presentation.
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Of the CTAs defined to date, MAGE and NYESO1 are particularly relevant as potential targets for lung cancer immunotherapy. The melanoma antigen (MAGE) genes were initially identified during molecular analysis of tumor antigens recognized by CTL from a melanoma patient (MZ2) receiving an autologous tumor vaccine. Van der Bruggen and coinvestigators (1991) recognized that the MZ2E gene encoded a peptide (designated MAGE1) that could be presented in an HLA-restricted manner to autologous CTL. Similarly, Gaugler and associates (1994) noted that an additional CTL clone derived from the same patient recognized a second peptide encoded by a gene (MAGE3), exhibiting marked sequence homology to MAGE1. To date, more than 50 MAGE genes have been identified, six of which (MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, and MAGEA12) are expressed in a significant percentage of cancers, as noted by Chen and Old (1999).
MAGEA1, MAGEA2, MAGEA3, and MAGEA6 have been the most extensively analyzed in lung cancer. In general, these MAGE genes are aberrantly expressed in 30% to 50% and 60% to 80% of non small cell cancers (NSCLC) and small cell cancers (SCLC), respectively, as noted by Scanlan (2002), Yoshimatsu (1998), Bolli (2002), and Tajima (2003) and their coinvestigators, as well as by Sakata (1996). Jang and associates (2001) observed hypomethylation of MAGEA1, MAGEA3, and MAGEB2 promoter regions in 75%, 80%, and 80% of resected lung cancers respectively, and 35%, 50%, and 55% of histologically normal tissues adjacent to these neoplasms, indicating that demethylation of MAGE genes occurs early during pulmonary carcinogenesis. Whereas their precise roles remain unclear, MAGE proteins may be involved in regulating cell cycle progression, apoptosis, and chemosensitivity in cancer cells, as suggested by the studies of Ohman and Nordqvist (2001) as well as of Zendman and colleagues (2003). For instance, as shown by Barker and Salehi (2002), MAGED1 binds to the p75 neurotrophin receptor, as well as the apoptosis inhibitor protein XIAP, to induce cell cycle arrest and apoptosis in cancer cells; in contrast, Duan and associates (2003) have reported that MAGEA2 and MAGEA6 appear to mediate chemoresistance in cancer cells. MAGE3 expression appears to correlate with diminished survival in patients with pulmonary squamous cell carcinomas, according to the study of Bolli and coinvestigators (2002).
Because the production of high-titered IgG antibodies requires antigen recognition by CD4+ T helper cells, novel serologic techniques such as serologic identification of antigens by recombinant cDNA expression libraries (SEREX) may be used to define tumor antigens defined by T cells. The NYESO1 tumor antigen was initially identified by Chen and co-workers (1997) utilizing SEREX to examine autologous T-cell antitumor response in an esophageal cancer patient. Soon thereafter, Wang (1995) and Jager (1998) and their collaborators isolated HLA-A2- and HLA-A31-restricted CTL clones reactive with NYESO1 from melanoma patients exhibiting high-titered antibody reactivity to this cancer/testis antigen. Recent studies by Jager and associates (1999) suggest that NYESO1 antibodies increase with disease progression; studies by Zeng and colleagues (2001) suggest that serologic reactivity to NYESO1 coincides with class II HLA-DP4 expression in cancer patients. Available data from the investigations of Scanlan (2000, 2002) and Lee (1999) and their co-workers indicate that NYESO1 is expressed in approximately 15% to 40% of NSCLC and 70% of SCLC lines or specimens. Lee and colleagues (1999) observed a high concordance regarding NYESO1 and MAGE3 expression in cultured lung cancer cells, and demonstrated that NSCLC cells expressing NYESO1 could be recognized by CTLs specific for this CTA. Additional studies by Lee (1999) and Weiser (2001a, 2001b) and their colleagues demonstrated that MAGE3 and NYESO1 expression can be induced in lung cancer cells following exposure to the DNA demethylating agent deoxyazacytidine.
Using oligonucleotide and tissue array techniques, Sugita and associates (2002) observed that MAGE3 and NYESO1 were among the 20 most highly expressed genes in lung cancer. Nevertheless, immune response to these CTAs remains limited in lung cancer patients. Stockert and co-workers (1998) observed that 0 of 24 and 1 of 24 NSCLC patients had antibodies to MAGE3 and NYESO1, respectively. More recent data presented by Scanlan and coinvestigators (2002) confirm that antibodies to MAGE3 are exceedingly uncommon in NSCLC patients, and that NYESO1 antibodies are detectable in approximately 25% of these individuals.
Restifo and associates (1993) have observed that the inability to detect antitumor immune responses in lung cancer patients whose neoplasms express CTAs may also be due to the fact that lung cancers (particularly SCLC) frequently exhibit decreased expression of TAP1, TAP2, LMP2, and LMP7 genes that encode proteins required for processing of endogenous tumor antigens; in addition, these tumors often exhibit diminished HLA class I expression, as noted by Doyle and colleagues (1985). Traversari and coinvestigators (1997) observed that following transfection with an IFN- cDNA, SCLC cells exhibited increased TAP1, LMP2, and class I MHC expression, which enabled recognition of these cells by an HLA-restricted CTL specific for MAGE3. Collectively, these data confirm that NYESO1 and to a lesser extent MAGE3 are immunogenic in cancer patients, and suggest that strategies to augment expression and presentation of these cancer/testis antigens may prove useful for lung cancer immunotherapy.
Currently, limited information is available concerning the efficacy of MAGE3 and NYESO1 vaccines in lung cancer patients. However, recent studies indicate that peptides derived from MAGE3 and NYESO1 can induce primary immunity and mediate tumor regression in cancer patients. Marchand and associates (1999) observed tumor regressions in 7 of 25 patients receiving MAGE3 peptide without adjuvant; 3 individuals exhibited complete responses. In an additional trial conducted by Thurner and colleagues (1999), mixed responses were observed in 6 of 11 melanoma patients immunized with dendritic cells pulsed with MAGE3 peptides. In a more recent trial, Marchand and co-workers (2003)
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observed two partial responses, two mixed responses, and two disease stabilizations in 33 melanoma patients immunized with recombinant MAGE3 protein in conjunction with the adjuvant SBAS2. In these studies, immune response to peptide did not correlate with clinical response. Thurner and colleagues (1999) vaccinated 11 melanoma patients with mature, monocyte-derived dendritic cells pulsed with MAGE3 peptide, and observed significant expansion of MAGE3-specific CTL precursors in 8 of these individuals. Six of 11 patients exhibited regression of metastases. Clinical response coincided with MAGE3 expression and CTL accumulation within tumors. Reynolds and associates (2003) treated 131 melanoma patients with a polyvalent shed antigen vaccine containing MAGE3 peptide and noted a statistically significant correlation between immune response to MAGE3 and improved clinical outcome in these individuals.
Because humoral and CD8+ T-cell immune responses to NYESO1 occur in 40% to 50% of all patients with tumors that express NYESO1, as noted by Scanlan and co-workers (2002), this CTA is a highly attractive target for cancer immunotherapy. Recently, individual epitopes recognized by CD4+ and CD8+ T cells have been defined. Interestingly, several peptides derived from NYESO1 exhibit promiscuous binding to HLA alleles. For instance, Zarour and coinvestigators (2002) observed that NYESO1 peptide 119 143 binds to several different HLA-DRB4 alleles to induce CD4+ response to NYESO1. Zeng and associates (2002) reported that NYESO1 peptide 157 170 contains an HLA-DP4-restricted epitope as well as an HLA-A2-restricted epitope, suggesting that this peptide might be highly useful as a cancer vaccine given its ability to simultaneously induce CD4+ and CD8+ immune responses. Jager and colleagues (2000) examined the immunologic effects of NYESO1 vaccination in 12 patients with tumors expressing this CTA. Five patients had detectable NYESO1 antibodies prior to therapy. Primary NYESO1-specific CD8+ T-cell reactivity and delayed type hypersensitivity reactions were observed in 4 of 7 antibody-negative patients. Induction of CD8+ T-cell responses to NYESO1 coincided with disease stabilization or regression of individual metastases in 5 of 7 patients without NYESO1 antibodies. Three of 5 NYESO1 antibody-positive patients exhibited stabilization of disease. Whereas the majority of responses in this trial were mixed and transient in nature, this experience supports further evaluation of NYESO1 vaccines in cancer patients.
Gnjatic and co-workers (2002) observed CD8+ T-cell responses against a dominant cryptic epitope following immunization of melanoma patients with overlapping NYESO1 peptides. Interestingly, despite the apparent immunogenicity of NYESO1, Dutoit and associates (2002) observed that immunization of melanoma patients with overlapping NYESO1 peptides resulted in the generation of CD8+ T cells that exhibited low-avidity binding to peptide and failed to recognize NYESO1-expressing tumor targets. These data indicate the need to precisely define the immunogenic epitopes within the NYESO1 protein in order to optimize NYESO1 targeted immunotherapy in cancer patients.
Because genome-wide demethylation facilitates expression of CTAs, it is conceivable that pharmacologic inhibition of DNA methyltransferase activity could represent a novel strategy to augment immunogenicity of lung cancer cells. Recently, our group conducted a series of experiments designed to examine if deoxyazacytidine (DAC) could be utilized to induce CTA expression in lung cancer cells under exposure conditions potentially achievable in clinical settings. Jones and Taylor (1980) demonstrated that this cytidine derivative is incorporated into DNA and then covalently traps and inhibits DNA methyltransferases, resulting in DNA hypomethylation. As such, DNA replication is required for DNA demethylation by this agent, which is rapidly inactivated by cytidine deaminase.
Quantitative reverse transcriptase polymerase chain reaction experiments demonstrated a dose-dependent induction of MAGE3 as well as NYESO1 expression in cultured lung cancer cells following 72-hour exposure to low-dose DAC. Additional studies by Weiser and colleagues (2001b) revealed that the HDAC inhibitor Depsipeptide FK228 (DP) markedly enhanced DAC-mediated CTA expression in cultured tumor cells. Following exposure to DAC or sequential DAC/DP, lung cancer cells were recognized by NYESO1-specific CTLs. In contrast, cultured normal bronchial epithelial (NHBE) cells were not recognized because the treatment regimen did not induce NYESO1 in these cells. Additional experiments by Weiser and coworkers (2001b) revealed that sequential DAC/DP mediated pronounced growth inhibition and apoptosis in lung cancer cells, but not in NHBE cells.
The aforementioned data provided the preclinical rationale for several gene induction trials that have been conducted in the Thoracic Oncology Section, Surgery Branch, National Cancer Institute. The first trial involved a phase I study of DAC-mediated induction of tumor antigen expression in thoracic oncology patients. The primary end points for this study included determining the maximum tolerated dose (MTD) and pharmacokinetics of 72-hour continuous DAC infusion in cancer patients, examination of NYESO1 expression, and evaluation of immune response to NYESO1 in patients before and after DAC infusion. In this trial, 34 patients were treated, most of whom had stage IV lung cancer. No objective responses were observed; however, stabilization of disease was noted in 8 individuals, 2 of whom remained on DAC therapy for more than a year. Steady-state plasma DAC levels in representative patients corresponded to threshold concentrations for gene induction in cultured cells. The dose-limiting toxicity of DAC was myelosuppression. Immunohistochemical analysis revealed induction of NYESO1 in 60% of evaluable patients. Posttreatment antibodies to NYESO1 were observed in 2 of 13 patients, both of whom exhibited NYESO1 induction in tumor tissues.
Currently, a phase I study is under way to examine gene induction mediated by sequential DAC/DP infusion in patients with pulmonary or pleural malignancies. The primary
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end points for this trial include evaluation of the pharmacokinetics of sequential DAC/DP infusion, and analysis of CTA expression and apoptosis in pre- and posttreatment biopsies; additional end points include evaluation of immune response to NYESO1. In this study, laser capture microdissection (LCM) and cDNA array techniques are being used for comprehensive analysis of gene expression profiles in target tissues. To date, 15 patients have received sequential DAC/DP therapy, several of whom have exhibited stabilization of disease. Molecular analysis is currently in progress. Overall, these preliminary data suggest that gene induction may be a novel strategy to elicit specific and potentially efficacious immune response to lung cancer.
TUMOR CELL VACCINES
During the past 20 years, considerable research efforts have focused on the evaluation of vaccines composed of tumor cells and tumor cell lysates for cancer therapy. In an early vaccine trial, Hollinshead and associates (1987, 1988) observed that lung cancer patients vaccinated with autologous tumor cell lysates following curative resection experienced improved survival relative to control individuals; adjuvant immunotherapy was most beneficial for individuals with stage I disease. Subsequently, Kimura and Yamaguchi (1995) randomized 105 lung cancer patients undergoing noncurative resections to receive either adjuvant chemotherapy with radiation therapy, or chemotherapy, radiation therapy, and IL-2 with lymphokine activated killer (LAK) cells. Adjuvant immunotherapy significantly improved survival of patients with adenocarcinomas, and appeared beneficial for control of pulmonary or nodal metastases, but not for residual disease involving the chest wall, pleura, or diaphragm. Kimura and Yamaguchi (1996) then prospectively randomized 82 lung cancer patients to receive either chemotherapy with IL-2/LAK, chemotherapy alone, or no adjuvant therapy following curative resections. Survival rates were 58% for patients receiving chemoimmunotherapy versus 31% for those receiving either chemotherapy alone or no treatment. Chemotherapy alone had no impact on outcome in these individuals. Postsurgical survival rates for stage II and stage III patients receiving chemoimmunotherapy were 69% and 37%, respectively. In contrast, survival rates for similarly staged control patients were 46% and 15%, respectively. Multivariate analysis revealed that immunotherapy was the only significant independent variable correlating with prolonged survival in these patients.
In a subsequent study, Ratto and co-workers (1998) randomized 113 lung cancer patients undergoing resection to receive either standard therapy or adoptive immunotherapy with tumor-infiltrating lymphocytes and IL-2. Fifty-six patients received autologous TILs. Stage II patients in the control arm received no additional therapy; stage III patients in both groups received radiation therapy. Survival of patients with stage II lung cancers who received adoptive immunotherapy was not significantly improved compared with similarly staged control patients. The median survival of stage III patients who received immunotherapy was 22 months, compared with approximately 10 months for control individuals (p = 0.06). Interestingly, adoptive immunotherapy appeared to reduce local but not distant relapse rates in stage III patients.
As pointed out by Restifo and Wunderlich (2001), a variety of cytokines, including IL-2, GM-CSF, IL-12, and B7.1, are known to modulate and potentiate antitumor immunity. As recorded by Lee and associates (1997), GM-CSF is extremely potent with regard to enhancing tumor immunity due to its ability to induce differentiation of dendritic cells and macrophages from hematopoietic precursor cells; transduction of Lewis lung carcinoma cells with a recombinant adenoviral vector expressing GM-CSF inhibits the tumorigenicity of these otherwise highly malignant and poorly immunogenic cells. In a recent clinical trial, Salgia and colleagues (2003) vaccinated 34 stage IV lung cancer patients with irradiated autologous tumor cells transduced with an adenoviral vector expressing GM-CSF. No significant treatment-related toxicities were observed. Vaccination resulted in lymphoid infiltrates within tumor tissues in 18 of 25 evaluable patients. Delayed type hypersensitivity responses to nontransduced autologous tumor cells were observed in 18 of 22 patients. Five individuals exhibited disease stabilization ranging from 3 to 33 months. One mixed response was observed. Two additional patients who underwent resection of metastases for tumor cell harvest were free of disease approximately 43 months after surgery (no evidence of disease); both of these individuals manifested dramatic vaccine-related responses. In another trial, Nemunaitis (2003) treated 33 lung cancer patients with six GM-CSF gene-transduced autologous tumor cell vaccines. Three patients exhibited complete tumor regressions lasting more than 6 months.
Another strategy suggested by Engleman (2003) for the generation of autologous tumor immunity involves the use of dendritic cells to present tumor antigens in vitro and in vivo. Nair and associates (1997) noted that dendritic cells exhibit extremely high-level MHC expression, which enhances antigen presentation; these APCs also express a variety of adhesion molecules and costimulatory ligands that optimize T-cell activation. Jenne and co-workers (2000) observed that dendritic cells containing apoptotic melanoma cells can prime CD8+ cells for efficient tumor cell lysis. More recently, Zhou and colleagues (2003) reported that dendritic cells can efficiently present lung cancer antigens to induce antigen-specific CD8+ T-cell responses; irradiated tumor cells were superior to tumor cell lysates or killed tumor cells as sources of tumor antigens for loading of dendritic cells.
Although experience with dendritic cell vaccines in lung cancer patients is limited, clinical trials using dendritic cells pulsed with melanoma antigens have been undertaken, with some encouraging results. Thurner and coinvestigators (1999) reported that vaccination of melanoma patients with
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MAGE3 peptide pulsed, monocyte-derived dendritic cells led to clonal expansion of MAGE3-reactive CTLs in 8 of 11 patients, and mediated regression of metastatic lesions expressing this CTA in several individuals. Palucka and associates (2003) observed that a single injection of dendritic cells pulsed with MAGE3 induced T-cell immunity in melanoma patients. Additional studies by Schuler-Thurner and colleagues (2002) demonstrated that mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells could induce rapid expansion of type I CD4+ T cells specific for MAGE3 in melanoma patients. Toungouz and co-workers (2001) observed similar results following vaccination of melanoma patients with fresh autologous dendritic cells pulsed with MAGE3 peptides. These results are highly relevant regarding lung cancer immunotherapy because type I T helper cells secrete IFN- , which enhances antigen presentation by tumor cells and mediates angiostasis during tumor rejection by CD8+ T cells, as observed by both Travesari (1997) and Qin (2003) and their coinvestigators.
IL-12 enhances differentiation of T helper cells and activates CTLs as well as natural killer cells, in part via interferon- pathways. Guo and associates (1999) observed that inoculation of mice with Lewis lung carcinoma cells transduced with IL-12 and B7.1 protected them from challenge with parental lung carcinoma cells. Interestingly, cytokine-mediated effects observed in the murine model were achieved following viral transduction of only 1% to 2% of the tumor cells; hence, expression of appropriate cytokines from a small percentage of lung cancer cells can mediate significant tumor regression and induce systemic immunity against highly lethal lung cancer cells. Hess and colleagues (2003) observed that CD4+ T cells in the microenvironment of human lung cancers can be mobilized by sustained release of IL-12 to lyse tumor cells in situ via indirect effects of IFN- . Furthermore, Egilmez and co-workers (2002) noted that CD4+ T cells can mediate indirect IL-12-dependent and IFN- -dependent inhibition of autologous lung cancer cell growth in vivo. Hill and associates (2002) reported that intratumoral administration of IL-12- and GM-CSF-encapsulated microspheres induces antitumor immune responses resulting in eradication of disseminated disease in murine cancer models. Collectively, these data highlight the significance of IL-12, GM-CSF, and IFN- regarding efficient immune response to tumor, and suggest that future clinical trials should focus on the evaluation of local administration of these cytokines for lung cancer immunotherapy.
Heat shock proteins (HSP) play critical roles in the processing and trafficking of intracellular and membrane-bound proteins, and recent studies reported by Pockley (2001) indicate that these chaperone proteins mediate innate and adoptive immunity to a variety of microbial and tumor-associated antigens. Vaccination with tumor-derived HSP96 protects mice from tumor challenge and mediates regression of established tumors in these animals. These effects appear related to the ability of HSP96 to chaperone tumor-derived peptides to professional APCs for T-cell stimulation. In addition, HSP96 promotes activation and maturation of dendritic cells and induces secretion of a variety of proinflammatory cytokines, as observed by Hilf (2002) and Manjili (2002) and their coinvestigators.
On the basis of provocative animal data, several clinical trials have been undertaken recently to examine the potential efficacy of HSP96 vaccinations in patients with advanced malignancies. Rivoltini and associates (2003) reported that immunization with autologous tumor-derived HSP96 induced recognition of MART1 and carcinoembryonic antigen in melanoma and colon carcinoma patients, respectively. Belli and colleagues (2002) observed complete responses in 2 patients, and stabilization of disease in 3 of 28 melanoma patients receiving autologous tumor-derived HSP96 peptide complexes. Mazzafero and co-workers (2003) treated 29 patients undergoing resection of hepatic metastases with autologous tumor-derived HSP96 complexes. De novo induced responses or enhancement of preexisting anticolon cancer responses were observed in 52% of these patients. No significant treatment-related toxicities were observed. Overall and disease-free survivals were significantly prolonged in patients exhibiting immune response to HSP96 vaccines. Clinical trials evaluating HSP96 vaccines in lung cancer patients will be initiated in the near future.
Lung cancer patients exhibit increased amounts of CD4+CD25+Treg cells in peripheral blood; in addition, CD4+CD25+Treg cells constitute a major percentage of tumor-infiltrating lymphocytes in primary lung cancers. Wolf and coinvestigators (2003) reported that CD4+CD25+ Treg cells from peripheral blood of lung cancer patients can suppress proliferation of CD4+CD25- T cells and inhibit NK cell mediated cytotoxicity. Woo and associates (2002) reported that CD4+CD25+ regulatory T cells from lung cancer tissues secrete the immunosuppressive cytokine TGF- and express high levels of CTLA-4, which binds to B7.1 and B7.2 of professional APCs to antagonize T-cell activation. Recently, Espenschied and colleagues (2003) observed that an anti-CTLA-4 antibody enhanced the therapeutic effect of a recombinant viral vaccine targeting p53 in a murine tumor model. Ryan and collaborators (2001) observed regression of pulmonary metastases in mice following adoptive transfer of tumor-reactive CTL generated by immunization with sarcoma cells in the context of CTLA-4 blockade. Phan and co-workers (2003) observed two complete responses and one partial response in 14 melanoma patients receiving tumor-associated peptide vaccines in conjunction with CTLA-4 blockade. Grade III/IV autoimmune manifestations were observed in 6 individuals. Tumor regressions only occurred in patients exhibiting autoimmunity, suggesting that CTLA-4 blockade was sufficient to break tolerance to melanoma antigens. These exciting data support evaluation of CTLA-4 blockade in lung cancer patients.
CONCLUSION
Recent insights into mechanisms that regulate immune response to tumors provide new opportunities for the development
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of effective lung cancer immunotherapies. Data presented in this chapter exemplify potential shared antigens for immunotherapy resulting from oncogene or tumor suppressor gene mutations, as well as epigenetic events that frequently occur during pulmonary carcinogenesis. Currently, ras and p53 as well as MAGE3 and NYESO1 are attractive, well-defined targets for lung cancer immunotherapy. Gene induction may be an attractive strategy to augment CTA expression, and local administration of cytokines such as IFN- and IL-12 may prove useful for enhancing antigen presentation in primary tumors. Utilization of novel immunization techniques including dendritic cells, HSP96, and CTLA-4 blockade may facilitate the generation of high-avidity tumor-reactive CTLs targeting these antigens in lung cancer patients. Ideally, clinical trials pertaining to lung cancer immunotherapy should mirror those demonstrating the most promising results in melanoma patients. With continued progress, Mocellin and associates (2002) note that immunotherapy may emerge as a viable treatment modality for lung cancer, particularly when administered for low-volume or microscopic disease.
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