Abstract
In view of their potent antigen-presenting capacity and ability to induce effective immune responses, dendritic cells (DCs) have become an attractive target for therapeutic manipulation of the immune system. The application of tumor-associated antigen (TAA)-expressing DCs for cancer therapy has been the subject of intensive translational investigation. Previous clinical trials demonstrated tumor-specific immune responses without any significant toxicity. However, the clinical success has been modest, because only a limited number of immunized patients demonstrated cancer regression. Considerable progress has been made in the knowledge of DC biology, which opens new avenues for the development of optimized clinical protocols. One such promising approach that might carve its place in the future of DC-based therapy is the use of gene-modified DCs. DCs engineered to express TAAs allow multiepitope presentation by both major histocompatibility complex (MHC) class I and II molecules of full-length TAAs independent of the patient's HLA constitution, as opposed to peptide vaccination strategies. Besides transgene TAA expression, DCs can be genetically modified (1) to express a variety of immune-potentiating molecules (e.g., costimulatory molecules, cytokines, and chemokines) or (2) to downregulate negative modulators of DC functioning, all allowing an enhancement of their immunogenic potential. In the present review, gene delivery systems for DCs are discussed, as well as the various transgenes used for genetic modification of DCs. Moreover, a detailed review of the already published trials using gene-modified DCs is presented and future DC-based strategies targeting multiple layers of the complex cellular immune response are highlighted.
Introduction
One of the most evident ways to achieve cancer immunization is by means of whole tumor cell preparations. The first and most obvious types of vaccine were prepared from autologous tumor cells mixed with bacterial adjuvants in an attempt to amplify the antitumor response (Vermorken et al., 1999). More recently, genetically modified autologous tumor cell vaccines (GMTVs) expressing relevant cytokines and/or costimulatory molecules (e.g., CD80/CD86) (Pardoll, 1993; Jaffee and Pardoll, 1997; Copier and Dalgleish, 2006) have been designed to increase the immunogenicity of the tumor cell and to allow the presentation of tumor-associated antigens (TAAs) in a preferential immune-stimulatory context. However, the clinical outcome of GMTVs was rather disappointing, possibly caused by several obstacles (Nawrocki et al., 2001). Inadequate mounting of a clinical beneficial immune response might be the result of (1) an absent encounter between tumor cells and dendritic cells (DCs) or (2) because DCs become inactivated or dysfunctional due to the suppressive nature of the tumor microenvironment (Vicari et al., 2002; Pinzon-Charry et al., 2005). Therefore, methods for ex vivo generation of DCs emerged in the 1990s (Romani et al., 1994; Lardon et al., 1997) and inspired the use of DCs as nature's adjuvant to present TAAs to the patient's immune system and initiate an immune response.
DCs are a highly specialized population of white blood cells and key regulators in the induction of primary immune responses and in the maintenance of tolerance (Cools et al., 2007). They patrol the body to capture pathogens and malignant cells in order to induce efficient antimicrobial or antitumor immune responses. On encounter of a “danger” signal, DCs mature and migrate to the T cell areas of the secondary lymphoid organs, where they present captured antigens to T cells. This maturation step is believed to be a crucial event to regulate DC function and makes DCs potent inducers of T cell immunity. In addition, mature DCs are believed to be, at least in part, resistant to tumor-related suppressive factors (Kajino et al., 2007; Haenssle et al., 2008), which makes them an attractive therapeutic tool to break tolerance in cancer-bearing patients. However, until now the efficacy of DC-based cancer vaccines, and of cancer vaccines in general, has been suboptimal. Whereas activation and expansion of tumor antigen-specific T cells have frequently been demonstrated in immunized patients (Schlom et al., 2007; Dauer et al., 2008), only a limited number of immunized patients have shown biologically relevant clinical responses, that is, tumor regression and increased disease-free survival (Schadendorf et al., 2006; Small et al., 2006; Nencioni et al., 2008), in randomized controlled phase II/III trials (Finke et al., 2007). However, new concepts in antitumor immunity have emerged and provide the groundwork for improved cancer immunotherapy approaches and, perhaps more importantly, for more rational design of clinical trials and end-point analyses (extensively reviewed by Schlom et al., 2007; Finn, 2008). One way to implement these new concepts in current DC-based vaccines would be through safe and efficient gene transfer of the necessary molecules in ex vivo-generated DCs (Breckpot et al., 2004) or by in vivo targeting of DCs through gene transfer vectors (Breckpot et al., 2007; Tacken et al., 2007). Both strategies ultimately pursue improved DC functionality in order to induce effective and clinically relevant T cell immunity characterized by high-avidity cytotoxic T lymphocytes (CTLs) and long-term memory CD4+ and CD8+ T cells capable of overcoming immune suppression or tolerance mechanisms imposed by the tumor.
In the present review, we focus on current viral and nonviral vector systems for gene transfer in DCs and on the various transgenes used for genetic modification of DCs. In addition, we review the results obtained in completed clinical trials in detail and highlight future strategies in the field of DC-based cancer gene therapy.
Gene Delivery Systems for Dendritic Cells
The promising observations made in the late 1990s, in early preclinical and clinical studies using tumor peptide- or lysate-loaded DC vaccines, paved the way for further optimization of DC-based immunotherapy in order to exploit the full potential of stimulatory DCs for the treatment of cancer and infectious diseases. Capitalizing on the fact that, in the same period, the field of gene therapy had made substantial progress through rational viral vector design and Good Manufacturing Practice (GMP) production of recombinant viruses, several groups reasoned that viral transduction of DCs with full-length TAAs, whether or not combined with immune-stimulatory proteins, would render them even more immunogenic to boost the host's immune system against cancer. Therefore, the whole array of viral (including adenovirus, retrovirus, lentivirus, adeno-associated virus, poxvirus, and herpesvirus) and nonviral methods (including gene gun, lipoplexes and electroporation) was applied to ex vivo-generated dendritic cells in order to augment the stimulatory function of DCs for use as cancer vaccines (reviewed in detail by Van Tendeloo et al., 2001b; Breckpot et al., 2004). To date, the most widely used vectors for genetic engineering of DCs are adenoviral vectors (AdVs), recombinant poxviruses (rPs), and mRNA electroporation, based on their high gene transfer and expression efficiency, on the availability of clinical-grade production protocols, and on their enhanced safety profile over other (integrating) viral vectors (Ribas, 2005; Mossoba and Medin, 2006; Van Tendeloo et al., 2007).
The major disadvantage of AdV- and rP-mediated transduction of DCs, aside from their tedious and complex GMP production process as compared with nonviral strategies, is the immunogenicity of the viral particles and residual viral gene products on transduction that could hamper repeated administrations in the setting of a vaccination strategy. On the other hand, their immunogenicity may also function as a trigger for enhanced DC maturation and may provide the necessary danger signals in the vaccine, resulting in more potent antitumor immunity (Dietz et al., 2001; Timares et al., 2004; Perreau et al., 2007; Seiler et al., 2007). In preclinical murine models, adenovirally transduced DCs were shown to be more potent than their peptide-loaded counterparts for generating antitumor immunity (Nakamura et al., 2005; Oh et al., 2006). It remains to be investigated whether these observations can be translated to the human setting. Although vaccinia-based vectors have been most widely used to date, avipox viruses, including fowlpox and canarypox, have been shown to be promising vaccine candidates and are currently being tested in DC-based strategies (Tsang et al., 2005). Avipox vectors may circumvent some of the disadvantages associated with vaccinia virus and AdVs, such as preexisting immunity. Also, prime–boost strategies can be considered when using such vectors in DC-based cancer vaccines to prevent clearance of the vector as a result of humoral immunity (Arlen et al., 2005).
Interestingly, in contrast to the general paradigm of using viral gene delivery systems in the field of (cancer) gene therapy, most efforts nowadays have been put in the nonviral transfection of human DCs, more particularly using mRNA transfection (reviewed by our group in Van Tendeloo et al., 2007) because of the highly efficient cytoplasmic expression of the transgene, simplicity over viral transduction protocols, and its superior clinical safety profile. Several groups have adopted the mRNA electroporation protocol as this results in far better transfection efficiencies than lipofection, passive pulsing strategies of mRNA, or cDNA transfection (Ponsaerts et al., 2003). It is worth noting that, to date, 60% of published clinical vaccination trials with gene-modified DCs use mRNA as the transgene, be it in vitro-transcribed mRNA or total mRNA isolated from tumor cells (Table 1). It will be interesting to assess whether such RNA-modified DC vaccines exert superior clinical efficacy over virally transduced DCs in future clinical trials. Another group of emerging gene delivery vehicles consists of recombinant Sendai viruses (rSeVs), which are cytoplasmic RNA vectors that efficiently transduce DCs ex vivo and in vivo. The group of Yonemitsu has reported in preclinical rodent models the potent immune-stimulatory capacity of replication-deficient rSeVs and their antitumor activity in several tumor models (Yoneyama et al., 2007). They called this approach based on rSeV-activated DCs “immune-stimulatory virotherapy” (Yonemitsu et al., 2008).
Abbreviations: AdV, adenovirus; TAA, tumor-associated antigen; gp100, glycoprotein 100; MART-1, melanoma antigen recognized by T cells; hTyr, human tyrosinase; KLH, keyhole limpet hemocyanin; LAMP, lysosome-associated membrane protein; TriMix, triad of CD40 ligand, CD70, and TLR4; PSA, prostate-specific antigen; hTERT, human telomerase reverse transcriptase; CEA, carcinoembryonic antigen; MUC-1, mucin-1; TriCom, triad of costimulatory molecules CD80 (B7.1), CD54 (ICAM-1), CD58 (LFA-3); Treg, regulatory T cells; ONTAK, denileukin diftitox; WT1, Wilms' tumor antigen-1; DTH, delayed-type hypersensitivity; I, phase I study design; II, phase II study design; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; TC, tumor cells; DFS, disease-free survival; N/A, not available or assessable.
Last, because the laborious and time-consuming process of ex vivo DC generation poses significant scientific, business, and regulatory hurdles for large-scale DC-based cancer vaccination, in vivo DC-targeting strategies are gaining increasing attention (Yang et al., 2008). Such an in vivo gene therapy approach would target DCs by binding to a typical DC surface marker, such as DC-SIGN (dendritic cell-specific iCMA3-grabbing nonintegrin), and deliver its cargo in situ in order to provide the DCs with one or more antigens and necessary activation signals (Tacken et al., 2006, 2007). For this purpose, lentiviral vectors have been carefully examined as gene transfer vehicles for in vivo modification of DCs and have been demonstrated to induce potent T cell-mediated immune responses that can control tumor growth (Lopes et al., 2006; Breckpot et al., 2008).
Molecules Used for Genetic Modification of Dendritic Cells
For optimal activation and generation of productive T cell immunity, it is believed that TAA-specific T cells must receive three coordinated signals. The first signal is provided to the T cell receptor (TCR) by MHC molecules harboring a tumor antigen-derived peptide. The second signal is called the costimulatory signal, delivered to the appropriate receptors on T cells by their ligands expressed on APCs. The third signal refers to a polarization signal, brought about by cytokines and/or chemokines from the APCs to naive T cells, that determines their differentiation into effector cells. Genetic engineering of DCs would harness DCs to provide one or more of these signals to increase their immune-stimulatory capacity. In addition, molecules can be introduced into DCs to prolong their life span, so that antigens would be presented for an extended period of time.
Tumor-associated antigens: Signal 1
DCs can be engineered with defined TAAs, using cDNA or RNA, either synthetically derived or directly isolated from the tumor cells. The advantage of transfecting DCs with a cDNA-encoding TAA (Ribas et al., 2000; Chan et al., 2006) or mRNA-encoding TAA (Van Tendeloo et al., 2001a; Heiser et al., 2002; Ponsaerts et al., 2002; Su et al., 2002; Van Driessche et al., 2005a; Bontkes et al., 2007) over peptide pulsing is that various epitopes of the full-length TAA will be presented at the same time, resulting in a so-called multiepitope vaccine, independent of the HLA haplotype of the patient. In addition, using whole tumor cell-derived mRNA (Su et al., 2003; Benencia et al., 2008) or DNA (Artusio et al., 2006), the entire array of patient-specific TAAs can be presented without the need for prior identification of antigens. To date, most known TAAs are self-antigens, for example, Wilms' tumor protein-1 (WT1) (Van Driessche et al., 2005a), which is overexpressed in a wide variety of tumors. To elicit an antitumor immune response against these antigens, T cell tolerance needs to be broken by potent immune-stimulatory APCs, with the potential risk of inducing autoimmunity. Therefore, signs of autoimmunity need to be carefully examined in clinical DC trials.
An interesting strategy is to exploit the capacity of nucleic acids, aside from their antigen-encoding properties, to exert innate immune-stimulatory effects on DCs by acting as ligands for highly conserved pattern recognition receptors (Kariko et al., 2004; Ceppi et al., 2005; Coban et al., 2005). In this way, by using coding RNA or DNA, DCs can both present TAAs and obtain increased immune-stimulatory capacities.
Costimulatory and adhesion molecules: Signal 2
Both costimulatory and adhesion molecules play a key role in the interaction between DCs and T cells. Although DCs intrinsically express both classes of molecules, additional expression of activating costimulatory molecules and adhesion molecules that favor the interaction with T cells further enhances their ability to generate antitumor immune responses. Activating costimulatory molecules that have been upregulated on DCs by genetic engineering include CD40 ligand (CD40L) (Kikuchi et al., 2000; Gonzalez-Carmona et al., 2008; Ziske et al., 2009), CD70 (Bonehill et al., 2009), 4-1BBL (Wiethe et al., 2003b), OX40 ligand (OX40L) (Dannull et al., 2005a), and receptor activator of NF-κB (RANK)/RANK ligand (RANKL) (Wiethe et al., 2003a). To further improve adhesion of DCs and T cells, promising in vitro and clinical studies have been reported in which triple expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1 (CD54), lymphocyte function-associated antigen (LFA)-3 (CD58), and costimulatory molecule B7.1 (CD80), a triad of molecules referred to as TRICOM, was obtained in DCs after transduction with recombinant poxviruses (Hodge et al., 1999; Morse et al., 2005).
Cytokines and chemokines: Signal 3
Cytokines also play a vital role in the polarization and skewing of the adaptive immune responses (signal 3) and hence in the activation of killer T cells by DCs. Two strategies have been reported to augment cytokine secretion by DC, that is, modification of DCs with (1) genes encoding one or multiple cytokines and (2) genes encoding a domain of the transcription factor NF-κB (Lee et al., 2002), resulting in enhanced secretion of multiple proinflammatory cytokines. The impact of cytokines on the induction of antitumor T cell responses has been tested in human and mouse models for interleukin (IL)-12 (helper T type 1 [Th1]-skewing, important for antitumor immune responses) (Nishioka et al., 1999; Ribas et al., 2001, 2002; Vujanovic et al., 2006; Bontkes et al., 2007, 2008; Tsai et al., 2009), IL-18 (Th1-skewing cytokine) (Tatsumi et al., 2002; Vujanovic et al., 2006; Tong et al., 2008), interferon (IFN)-γ (Th1-skewing cytokine) (Xue et al., 2007), IL-2 (T cell proliferation factor) (Ribas et al., 2001), IL-7 (for generation and maintenance of memory T cells) (Westermann et al., 1998; Miller et al., 2000; Ribas et al., 2001; Sharma et al., 2003), tumor necrosis factor (TNF)-α (proinflammatory cytokine and maturation factor for DCs) (Chen et al., 2002; Ye et al., 2006), IFN-α (stimulation of cross-presentation and antitumor immune response) (Tuting et al., 1998), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (DC growth and differentiation factor) (Cao et al., 1998; Ojima et al., 2006). Although most studies stated that the produced cytokines positively influenced the induction of an antitumor immune response, there was also a report on an IL-12-induced dose-dependent inhibition of the antitumor response by a yet undefined mechanism in a mouse melanoma model (Ribas et al., 2002). In other studies (Nishioka et al., 1999; Bontkes et al., 2007, 2008; Tsai et al., 2009), however, IL-12 has been shown to have strong immune-stimulatory effects, also on other cells than T cells. Bontkes and colleagues (2008) showed that cotransfection of DCs with TAA and IL-12 resulted in an antitumor immune response, mediated both by T cells and natural killer (NK) cells, demonstrating that IL-12 enhances the cytotoxic effect of NK cells. Moreover, secretion of IL-12 by DCs activates IFN-γ production by NK cells, being part of the cross-talk between DCs and NK cells that leads to increased reciprocal activation of DCs and NK cells. In this context, the importance of the presence of NK cells and the cross-talk between NK cells and (gene-modified) DCs for the induction of antitumor immune responses has been established using IL-12-secreting DCs (Miller et al., 2003; Wargo et al., 2005). It is likely that also other hematopoietic cells (e.g., macrophages and granulocytes) contribute to tumor regression, when they are attracted to tumor sites by cytokines and chemokines released after activation of innate and adaptive immune cells. To this end, DCs can be gene-modified to express chemokines, such as lymphotactin (Cao et al., 1998) and secondary lymphoid tissue chemokine (SLC or CCL-21) (Kirk et al., 2001a,b; Matsuyoshi et al., 2004; Yang et al., 2004), in order to attract T cells to facilitate their activation. Another strategy to bring DCs into contact with T cells is through gene transfer of homing molecules, such as E/L-selectin, in order for them to migrate to the T cell zones of peripheral lymph nodes where activation could take place (Robert et al., 2003; Dorrie et al., 2008).
Other approaches to enhance DC function
To enhance their life span, DCs have been modified to overexpress various antiapoptotic molecules (Pirtskhalaishvili et al., 2000; Balkir et al., 2004). Protection of DCs from apoptosis resulted in enhanced antitumor immune responses in a mouse model of prostate cancer (Pirtskhalaishvili et al., 2000).
Another strategy to further enhance the immune-stimulatory potential of DCs is modification of DCs with small interfering RNA (siRNA) targeting the suppressor of cytokine signaling-1 (SOCS1), a negative regulator of signaling. Silencing SOCS1 in DCs strongly enhanced antigen-specific antitumor immunity in a mouse model (Shen et al., 2004). Downregulation of SOCS1 might also be combined with upregulation of immune-stimulatory molecules through gene transfer.
Combination of strategies
To mimic in vivo DC activation, it is likely that a combination of different molecules will result in the ultimate immune-stimulatory DCs and the induction of a more potent antitumor immune response. A new strategy was developed, in which DCs were electroporated both with mRNA encoding the costimulatory molecules CD40L and CD70 and with mRNA encoding a constitutively active form of Toll-like receptor-4 (TLR4), called TriMix DCs (Bonehill et al., 2009). Toll-like receptors have been shown to bind pathogen-associated molecular patterns, and activation of these receptors on DCs (e.g., by modification of DCs with a constitutively active form [Cisco et al., 2004; Abdel-Wahab et al., 2005]) results in highly immune-stimulatory DCs. By using gene-modified DCs instead of systemic cytokine administration or microbial products, localized expression limits toxicity. Also by others, it was shown that transduction of more than one gene is superior over single-gene transfer for murine DCs modified to express ICAM-1, LFA-3, and B7.1 (Hodge et al., 1999) and murine DCs expressing IL-12 and IL-18 (Vujanovic et al., 2006). However, Bontkes and colleagues (2008) reported that IL-18 did not further increase the positive effects of IL-12 on stimulation of CTLs and NK cell effector functions when human DCs were modified to express both cytokines, thereby demonstrating that results obtained with murine DCs cannot always be extrapolated to human DCs.
Clinical Trials Using Gene-Modified Dendritic Cells
Since the results of the first DC vaccine trial were published in 1996 (Hsu et al., 1996), the exploitation of DCs for cancer immunotherapy has been the subject of increasing investigation. To date, most translational research has focused on the implementation of a nongenetic modification approach using DCs loaded with tumor antigen epitopes, whole tumor cell lysates, or necrotic/apoptotic neoplastic cells. The field of DC-based cancer gene therapy is relatively new, although this treatment modality is gaining considerable impetus as evidenced by the growing number of registered clinical trials (see
Salient characteristics of completed cancer vaccine trials
The clinical trials conducted so far, which were mainly of phase I/II study design, covered a broad range of targeted tumor types. The use of gene-modified DCs has been examined both in solid and hematological malignancies, with a predominant focus on melanoma and prostate cancer (Table 1) (Van Tendeloo et al., 2007; Morrison et al., 2009).
Viral as well as nonviral approaches for the genetic modification of DCs have been clinically studied. Viral gene transfer methods have traditionally found difficult application in DC trials in view of the biosafety issues arising from the use of viral constructs (Van Driessche et al., 2005b). Therefore, mostly nonintegrating viral vectors have been tested in a translational research setting, most of which were recombinant adenoviral vectors (Tsao et al., 2002; Mazzolini et al., 2005; Antonia et al., 2006; Butterfield et al., 2008). In two studies, replication-defective poxviruses (vaccinia and fowlpox, respectively) were used to transduce DCs with genes encoding tumor-associated antigens (TAAs) (Di Nicola et al., 2004; Morse et al., 2005). In contrast, nonviral gene delivery, either in the form of DNA or mRNA transfection, has been more widely implemented. However, DNA-based gene therapy has not gained much interest in translational research, because it is associated with low transfection efficiency rates (Ponsaerts et al., 2003). In this regard, a study by Pecher and colleagues (2002) using DCs transfected with cDNA of the multi-TAA mucin (MUC)-1, showed heterogeneous expression of MUC-1 in only 2–53% of transfected DCs (Table 1). Therefore, most clinical trials have been adopting a nonviral, non-DNA transfer approach, based on the transfection of DCs with translatable mRNA (Van Driessche et al., 2005b). Various sources of mRNA have been used for this purpose, notably whole tumor cell-extracted mRNA, either of allogeneic or autologous origin, and mRNA encoding a specific TAA (Table 1). In addition, several RNA transfection methods have been clinically employed, among which electroporation is gaining the fastest popularity (Van Tendeloo et al., 2007).
In addition to the heterogeneity in targeted tumor type and adopted genetic modification approach, most clinical trials also differ in the vaccine formulation (monocyte-derived vs. bone marrow-derived DCs, immature vs. mature DCs) and the mode of administration (intradermal, intravenous, intranodal, intratumoral) (Van Tendeloo et al., 2007; Morrison et al., 2009). The optimal administration route remains to be determined, although intradermal inoculation has been attributed favorable efficacy with regard to DC migration (Morse et al., 1999) and the induction of antigen-specific cellular immune responses (Mu et al., 2005; Kyte and Gaudernack, 2006). However, these conclusions should be treated with some reservation, because biases in study design might have influenced the accurate interpretation of the results (Kyte and Gaudernack, 2006).
Safety and immunological and clinical outcomes of DC-based cancer gene therapy
The safety and tolerability of DC-based cancer gene therapy have been amply documented in all phase I/II clinical trials performed hitherto. Vaccination with gene-modified DCs is well tolerated and related adverse events are usually limited to CTC (common toxicity criteria) grade 1 or 2 (Van Tendeloo et al., 2007). Local inflammatory signs at the injection site and the draining lymph nodes are among the most common side effects (Heiser et al., 2002; Di Nicola et al., 2004; Mu et al., 2005; Su et al., 2005; Kyte and Gaudernack, 2006). Systemic, flulike symptoms have been sporadically documented (Heiser et al., 2002; Mazzolini et al., 2005; Su et al., 2005). In addition, hematological toxicity (lymphocytopenia, thrombocytopenia) has been reported, although it is generally not dose-limiting (Mazzolini et al., 2005; Van Tendeloo et al., 2009). As with all immunotherapeutic treatment modalities, particular attention is being paid to the induction of autoimmune phenomena. Transient occurrence of autoantibodies (Morse et al., 2003; Mazzolini et al., 2005; Su et al., 2005; Antonia et al., 2006) and vitiligo-like skin changes (Tsao et al., 2002; Mazzolini et al., 2005; Kyte and Gaudernack, 2006) are the most frequently noted autoimmune side effects.
The immunological outcome of DC-based cancer gene therapy has been assessed for several end points such as directional migration to regional lymph nodes, induction of antigen-specific cellular immune responses, and DC-mediated NK cell activation (summarized in Table 1). A limited number of studies addressed the migratory capacity of gene-modified DCs, which is a prerequisite for the induction of tumor-specific immunity. Effective localization of DCs in the T cell areas of the lymph nodes largely depends on the route of administration; intradermal (Morse et al., 1999) or intranodal (Schuurhuis et al., 2009) injections have been shown optimal in that regard. Intratumoral administration was suggested to be associated with poor lymphatic transfer of DCs to the draining lymph nodes, possibly contributing to impaired antitumoral activity (Feijoo et al., 2005). Irrespective of the study design, most clinical trials provided evidence of tumor-specific CD8+ and/or CD4+ T cell responses, using a variety of immunological monitoring assays (delayed-type hypersensitivity [DTH] responses, IFN-γ enzyme-linked immunospot [ELISPOT] assay, tetramer staining, assessment of CTL killing activity) (Onaitis et al., 2002; Morrison et al., 2009). Innate NK cell-mediated immune responses have been documented in only three published clinical trials (Mazzolini et al., 2005; Osada et al., 2006; Butterfield et al., 2008), although the pivotal role of NK cells in DC-based cancer vaccination is being increasingly underscored. Phenotypic NK cell changes, such as increased expression of activation markers and granzyme B, were noted in 7 of 10 tested melanoma patients during the course of a vaccination trial with melanoma antigen recognized by T cells (MART)-1 adenovirally transduced DCs (Butterfield et al., 2008). In addition, post-hoc analysis of a study using DCs transduced to express carcinoembryonic antigen (CEA) and costimulatory molecules revealed an increment in NK cell activity and/or frequency in nearly half of the evaluable patients after vaccination. Strikingly, the presence of NK cell responses appeared to be correlated with beneficial clinical outcome (Morse et al., 2005; Osada et al., 2006).
Strategies to optimize the clinical efficacy of DC-based cancer gene therapy: results of the first clinical trials
In view of the limited clinical benefit, alternative approaches to lever the therapeutic efficacy of gene-modified DCs are currently at the center of attention. Because the induction of antigen-specific CD4+ T cells is of the utmost importance for the generation of potent antitumor immunity, one promising way is to ensure MHC class II presentation by coupling the TAA with a lysosomal-associated membrane protein (LAMP)-encoding sequence (Su et al., 2005; Bonehill et al., 2009). The efficacy of this approach to induce enhanced CD4+ T cell and high-avidity CTL responses with clinical benefit has already been proven in patients with metastasized prostate cancer (Su et al., 2005). We are currently preparing a phase I/II clinical trial for patients with acute myeloid leukemia to evaluate the therapeutic efficacy of DCs electroporated with mRNA encoding LAMP-coupled leukemia-associated antigens and IL-12p70. Genetic modification of DCs to stimulate expression of the pivotal Th1-polarizing cytokine IL-12p70 is another elegant strategy to promote antitumor immunity. This concept has been translated into a phase I clinical study of patients with metastasized gastrointestinal malignancies using non-antigen-loaded DCs, capable of high IL-12p70 production after ex vivo adenoviral transfer of the corresponding IL-12 gene. From an immunological point of view, intratumoral injection of these gene-modified DCs resulted in systemic production of IFN-γ and IL-6 in all patients, as well as in an increment of NK cell activity (5 of 15 patients) and tumor-infiltrating CD8+ T cells (3 of 11 patients). Preliminary data on the clinical efficacy showed a partial tumor mass reduction in 1 of 11 evaluable patients and transient disease stabilization in 2 patients (Mazzolini et al., 2005). Other clinically tested approaches to enhance the immune-stimulatory potential of DCs include genetic modification to express costimulatory molecules and/or DC maturation stimuli in addition to the tumor antigens (Morse et al., 2005; Osada et al., 2006; Bonehill et al., 2009) (Table 1).
Because cancer is generally associated with increased numbers of regulatory T (Treg) cells that might hamper the immune-stimulatory competence of DCs, adjuvant strategies aimed at reducing the Treg population might improve the clinical outcome of DC-based immunotherapy. Depletion of CD4+CD25+ Treg cells can be achieved with a recombinant fusion protein of IL-2 and diphtheria toxin (denileukin diftitox, ONTAK), which specifically targets cells expressing high levels of the IL-2 receptor-α subunit (CD25) (Finn, 2008). An early clinical trial combining anti-CD25 therapy and tumor mRNA-transfected DC vaccination showed encouraging immunological potency in patients with metastasized renal cell carcinoma, as reflected by the fact that vaccine-induced CTL frequencies increased by up to 16-fold when using a concomitant Treg cell depletion tactic (Dannull et al., 2005b). Similar results were obtained in one study, demonstrating the efficacy of multidose denileukin diftitox administration in reducing the number of circulating CD4+CD25+FoxP3+ Treg cells and prolonging the persistence of DC vaccine-induced tumor-specific immune responses (Table 1) (Morse et al., 2008).
Combination of DC-based cancer gene therapy with standard treatment modalities, such as chemotherapy, represents another promising therapeutic strategy that merits further investigation. One study addressed the clinical outcome of chemotherapy in association with p53-transduced DC therapy in a setting of advanced small-cell lung cancer. Patients with progressive disease after having completed DC vaccination, showed an unpredicted high clinical response rate to second-line chemotherapy (>60%) as compared with historic control subjects. Moreover, immunological responders to DC vaccination (evidenced by the induction of p53-specific T cells) were most prone to the effects of subsequent chemotherapy, suggestive of a correlation between both phenomena (Table 1) (Antonia et al., 2006).
Future Considerations
The use of ex vivo-generated DCs for cancer therapy entails several advantages with regard to tumor-specific stimulation of the patient's immune system. DCs express a variety of costimulatory and adhesion molecules that are critical for priming of naive T cells and for breaking immune tolerance to the tumor. In addition, DCs can migrate to the T cell areas of the lymphoid organs to present TAAs in an immune-stimulatory context. Various genetic modification approaches can be applied to load DCs with relevant TAAs and immune-modulatory molecules, providing the necessary characteristics for optimal DC functioning. As discussed previously, mRNA electroporation has emerged as a promising method for manipulation of DCs as it allows high transgene expression combined with high cell viability. Given the variety of available molecules to manipulate DC functionality and/or survival as well as the complexity of the DC system with different DC subsets, it is obvious that the use of distinct gene-modified DCs should be deliberated rationally and that future research is needed to analyze the immune response induced in patients by these DCs. Overall, in clinical trials mRNA-transfected DCs were shown to be safe and well tolerated, and resulted in detectable immune responses in more than 50% of vaccinated patients (Van Tendeloo et al., 2007; Morrison et al., 2009). However, despite the indications that DC-based cancer gene therapy may be able to induce tumor-specific T cells, the clinical responses observed were sporadic and/or limited. Notwithstanding the fact that even minor clinical responses must be considered valuable and encouraging, especially in the enrolled group of predominantly late-stage cancer patients in whom other treatment options have been exhausted (Finn, 2008; Morrison et al., 2009), the reasons for the apparent discrepancy between immunological changes and clinical efficacy are incompletely understood. Possible explanations could be the relatively short follow-up times (Schuurhuis et al., 2009), the lack of validated immunological surrogate markers indicative of clinical benefit (Gilboa and Vieweg, 2004; Van Tendeloo et al., 2007), quantitative and/or qualitative limitations of the induced T cells, or the immune-suppressive microenvironment of the tumor.
Therefore, the immune-compromised state associated with advanced cancer patients and the presence of negative immune regulators are additional factors that need to be taken into account in therapeutic cancer vaccines. Indeed, as discussed previously, several preclinical studies (Dannull et al., 2005b; Morse et al., 2008) have demonstrated that overcoming immune suppression is an auspicious tool to improve the efficacy of immunotherapy. On the other hand, gene modification of DCs, resulting in a phenotype that is less prone to immune suppression, might be a valuable alternative. To this end, it was reported by Kortylewski and colleagues (2005, 2009) that through the use of CpG-Stat3 siRNA, DCs were able to increase the number of tumor-specific CD8+ T cells and NK cells, and decrease the number of Treg cells in the tumor microenvironment. The challenge for cancer immunotherapy research will be to determine which combination of approaches leads to a favorable clinical response and outcome. For this, more translational and clinical research is needed to examine the full potential of gene-modified DCs as cellular vaccines and to target multiple immune effector cells of the complex cellular response.
Finally, the ultimate goal is to target antigens to DCs directly in vivo. The strategies discussed in the present review will help to identify all attributes necessary for therapeutic manipulation of the immune system in order to improve the otherwise insufficient antitumor response and for targeting DCs in vivo, which represents the next step in the development of DC-based cancer therapy.
Footnotes
Acknowledgments
This work was supported in part by research grants of the Fund for Scientific Research–Flanders (G.0370.08 and G.0082.08), the Foundation against Cancer (Stichting tegen Kanker), the Antwerp University Concerted Research Action (BOF-GOA, grant 802), the Methusalem Financement Program of the Flemish Government attributed to Prof. Dr. Herman Goossens (Antwerp University, Vaccine and Infectious Disease Institute, VAXINFECTIO), and the Interuniversity Attraction Pole Financement Program (IAP P6/41) of the Belgian Government. E.L.J.M.S. was supported by a Stichting Emmanuel van der Schueren research grant of the Flemish League against Cancer (VLK). S.A. is a Ph.D. fellow and N.C. is a postdoctoral research fellow of Fund for Scientific Research–Flanders (FWO–Vlaanderen).
Author Disclosure Statement
No competing financial interests exist.