- Email: [email protected]
Antitumor dendritic cell–based vaccines: lessons from 20 years of clinical trials and future perspectives
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Antitumor dendritic cell–based vaccines: lessons from 20 years of clinical trials and future perspectives ~ CONSTANTINO, CELIA ~ JOAO GOMES, AMILCAR FALCAO, MARIA T. CRUZ, and BRUNO M. NEVES COIMBRA AND AVEIRO, PORTUGAL
Dendritic cells (DCs) are versatile elements of the immune system and are best known for their unparalleled ability to initiate and modulate adaptive immune responses. During the past few decades, DCs have been the subject of numerous studies seeking new immunotherapeutic strategies against cancer. Despite the initial enthusiasm, disappointing results from early studies raised some doubts regarding the true clinical value of these approaches. However, our expanding knowledge of DC immunobiology and the definition of the optimal characteristics for antitumor immune responses have allowed a more rational development of DC-based immunotherapies in recent years. Here, after a brief overview of DC immunobiology, we sought to systematize the knowledge provided by 20 years of clinical trials, with a special emphasis on the diversity of approaches used to manipulate DCs and their consequent impact on vaccine effectiveness. We also address how new therapeutic concepts, namely the combination of DC vaccines with other anticancer therapies, are being implemented and are leveraging clinical outcomes. Finally, optimization strategies, new insights, and future perspectives on the field are also highlighted. (Translational Research 2015;-:1–22) Abbreviations: APCs ¼ antigen-presenting cells; BMP4 ¼ bone morphogenetic protein 4; cDCs ¼ classical DCs; CDPs ¼ common DC precursors; CLPs ¼ common lymphoid precursors; CLRs ¼ C-type lectin receptors; CMPs ¼ common myeloid precursors; CTLs ¼ cytotoxic T cells; CTLA-4 ¼ cytotoxic T-lymphocyte–associated protein 4; DC ¼ dendritic cell; Dex ¼ dendritic cell–derived exosomes; GM-CSF ¼ granulocyte-macrophage colony–stimulating factor; HLA ¼ human leukocyte antigen; HSC ¼ hematopoietic stem cells; i.d. ¼ intradermal; IDO ¼ indoleamine2,3-dioxygenase; IL ¼ interleukin; IFN ¼ interferon; i.n. ¼ intranodal; iPSCs ¼ induced pluripotent stem cells; i.t. ¼ intratumoral; i.v. ¼ intravenous; LC ¼ Langerhans cells; mAb ¼ monoclonal antibody; M-CSF ¼ macrophage colony–stimulating factor; MDPs ¼ macrophage and DC precursors; MDSCs ¼ myeloid-derived suppressor cells; MHC ¼ major histocompatibility complex; MoDCs ¼ monocyte-derived DCs; NK ¼ natural killer; PAMPs ¼ pathogen-associated molecular
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From the Faculty of Pharmacy and Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Faculty of Medicine, Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI) and Center of Investigation in Environment, Genetics and Oncobiology (CIMAGO), University of Coimbra, Coimbra, Portugal; CNC, Institute for Biomedical Imaging and Life Sciences, University of Coimbra, Coimbra, Portugal; Department of Chemistry and QOPNA, Mass Spectrometry Centre, University of Aveiro, Aveiro, Portugal.
Submitted for publication May 19, 2015; revision submitted July 25, 2015; accepted for publication July 28, 2015. Reprint requests: Bruno M. Neves, Faculty of Pharmacy and Centre for Neuroscience and Cell Biology, University of Coimbra, 3000548 Coimbra, Portugal; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.07.008
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patterns; PAP ¼ prostatic acid phosphatase; PBMCs ¼ peripheral blood mononuclear cells; PD-1 ¼ programmed cell death 1; pDCs ¼ plasmacytoid DCs; PGE2 ¼ prostaglandin E2; PolyI:C ¼ polyinosinic:polycytidylic acid; PKR ¼ protein kinase RNA-activated receptors; RECIST ¼ response evaluation criteria in solid tumors; s.c. ¼ subcutaneous; SCF ¼ stem cell factor; TAAs ¼ tumor-associated antigens; TCR ¼ T-lymphocyte receptor; TGF ¼ transforming growth factor; TLR ¼ toll-like receptor; TRAIL ¼ TNF-related apoptosis-inducing ligand; Tregs ¼ regulatory T cells; WHO ¼ World Health Organization
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INTRODUCTION
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Dendritic cells (DCs), initially described by Steinman and Cohn in a series of pioneer studies,1 play a critical role at the interface between the innate and adaptive arms of the immune system. These cells induce primary immune responses, potentiate the effector functions of previously primed T lymphocytes, and orchestrate/ modulate the communication between other innate and adaptive immune cells.2 Although they represent a very small population of leukocytes, DCs are professional antigen-presenting cells (APCs) that are highly efficient in generating robust immune responses and maintaining tolerance to self or harmless foreign antigens. This functional plasticity renders DCs a very attractive tool for the design of immunotherapeutic approaches, namely to boost the immune system during cancer treatment. Since the first pilot study using ex vivo MAGE-1–pulsed DCs for the treatment of melanoma in 1995,3 more than 300 clinical trials on antitumor DC-based treatments have been completed or are currently ongoing (Fig 1, A). Ongoing or previously completed randomized phase 3 clinical trials have been performed across a wide range of malignancies, including melanoma, prostate cancer, malignant glioma, renal cancer, and lymphoma (Fig 1, B). The great expectations created by the early experimental results were not always followed by objective clinical responses, and some skepticism ensued. In fact, according to the World Health Organization criteria or the response evaluation criteria in solid tumors, antitumor DC-based vaccines rarely exceed 15% of the objective responses.4 However, the paradigm of antitumor activity assessment is changing, and overall survival (OS) is now viewed as one of the most relevant outcomes to measure therapeutic benefits.5 In 2010, this notion was reinforced by the Food and Drug Administration (FDA) approval of the first DC-based vaccine, sipuleucel-T (Dendreon, Washington), as a treatment for metastatic hormone-resistant prostate cancer. Despite the fact that only 5% of patients exhibited tumor regression in the phase 3 clinical trial, the vaccine was shown to significantly increase the OS by 22.5%.6 Meanwhile, the FDA and the European Medicines
Agency (EMA) granted a special regulatory framework, such as orphan drug designation, to several other antitumor DC-based vaccines. The ICT-107 vaccine (ImmunoCellular Therapeutics, California) for the Q6 treatment of glioblastoma multiforme and the AV0113 vaccine for malignant glioma from the Austrian biotech company Activartis received the orphan drug designation by the FDA and EMA. In 2012, the EMA also granted orphan drug designation to DCP-001, a vaccine for acute myeloid leukemia produced by the Dutch company DCPrime BV. Finally, antitumor DC-based Q7 vaccines have been tested in several countries as Hospital Exemption therapies, a special regime defined by the regulation on advanced therapy medicinal products. An example of a vaccine within this framework is the DCVax-L from Northwest Biotherapeutics (Bethesda, Maryland) that was recently approved by the German Paul Ehrlich Institute for treatment of glioma. In the present work, the current status of DC immunobiology is briefly reviewed and the characteristics of antitumor DC-based vaccines that have been tested in more than 300 clinical trials for more than the last 2 decades are then summarized. We specially address the practical aspects of DC vaccine production and the influence of the different procedures on clinical effectiveness. Finally, new therapeutic concepts, optimization strategies, and future perspectives on the area are also highlighted. DC IMMUNOBIOLOGY Origin and classification. DCs are a heterogeneous family of specialized APCs that ultimately share the same hematopoietic stem cell precursor. In bone marrow, hematopoietic stem cells give rise to common myeloid precursors and common lymphoid precursors, which then originate intermediate progenitors, such as macrophage and DC precursors. Macrophage and DC precursors further differentiate into common DC precursors that are restricted to the generation of the 2 known DC populations: classical DCs (cDCs), and plasmacytoid DCs (pDCs) (reviewed in Refs 7 and 8). It is now accepted that, independent of the precursor lineage, the differentiation and expansion of specific DC subsets is strongly regulated by different
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Fig 1. Twenty years of clinical trials using antitumor DC-based immunotherapies. (A) Clinical trials reporting antitumor DC-based vaccines registered at ClinicalTrial.gov until March 2015. (B) Frequencies distribution of clinical trials among the different malignancies and their actual status. In ‘‘Other’’ category are included bladder cancer, mesothelioma, urothelial carcinoma, and cervical cancer. DC, dendritic cell.
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hematopoietic cytokines such as Fms-like tyrosine kinase cytokine-3 ligand (Flt3L), macrophage colony– stimulating factor, and granulocyte-macrophage colony–stimulating factor (GM-CSF).8 On the basis of this knowledge, the ‘‘graded commitment’’ model was proposed.9 This model states that the different precursors are not restricted to give rise to one particular type of DC. Rather, there is a gradation of probability ranging from precursors that can give rise to all DC subtypes to those that can only produce the classical or plasmacytoid subsets. The classification of the different human DC subsets has been the subject of intense debate and constant evolution. DCs were for long classified on the basis of phenotypical and functional characteristics. However, given that these characteristics are often overlapping an unequivocal classification has been challenging. Recently, a new classification system based primarily on DC ontogeny and secondarily on their location, function, and phenotype-gained relevance.10 DCs found in lymphoid and nonlymphoid tissues are therefore classified into 2 main groups, cDCs and pDCs. Human skin hosts 3 major subtypes of cDCs differentiated by their expression of CD1a, CD14, and CD141: CD41/CD1ahi/CD142/DC-SIGN2/CD20611/CD162, CD41/CD1alow/CD141/DC-SIGN1/CD2061/CD162, and CD1411/XCR11 DCs.8,11 CD1411/XCR11 DCs have been shown to be extremely efficient in antigen cross-presentation and to produce significant amounts of tumor necrosis factor a (TNF-a), CXCL10, and interleukin 12 (IL-12)p70 on stimulation.12-14 These cells effectively interact with natural killer (NK) cells and CD81 T cells, an event facilitated by the
expression of the XCR1 chemokine receptor. The human epidermis is also populated by other specialized APCs termed Langerhans cells (LCs). LCs are characterized by low CD11c and high CD1a and CD2017 expressions, which distinguish them from cDCs subsets. In addition, unlike any other DC population, LCs derive from embryonic precursors and have the capacity to self-renew locally.15 LCs are capable to cross-present antigens and to evoke tolerogenic or immunogenic responses depending on the microenvironment and type of maturation stimulus.11 In blood and lymphoid and nonlymphoid tissues, 2 cDC populations have been described according to their expression of nonoverlapping markers CD141 and CD1c. CD1411 DCs are primarily present in the blood and lymph nodes, although they can also be found in the lungs and liver. CD1c1 DCs are the predominant cDCs in the blood, and are also found in lymph nodes, spleen, and mucosa. These cells express toll-like receptors (TLRs) 1–10 and some C-type lectin receptors, such as DC-SIGN, DEC-205, DCIR, mannose receptor, Cleac9A, and Dectin 1 and 2.16,17 CD1c1 DC Q10 subpopulation produces IL-8, IL-10, TNF-a, and IL23 on activation, and their major role appears to be related to the modulation of mucosal T-cell responses and immunity toward extracellular pathogens.18 Finally, besides the 2 cDCs subsets, blood and lymphoid tissues also host pDCs, cells phenotypically characterized as major histocompatibility complex (MHC)-II1/ CD11c2/CD1231/CD3031/CD3041. pDCs have an Q11 enormous functional plasticity and are able to polarize T cells into Th1, Th2, regulatory T cells (Tregs), and cytotoxic T cells (CTLs). These cells are primarily
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implicated in antiviral responses by producing large amounts of type 1 interferon (IFN-a/b) and by efficiently cross-presenting viral antigens to the CD81 T lymphocytes.20 Antigen recognition, processing, and presentation. DCs sample their microenvironment and capture antigens by receptor-mediated endocytosis, phagocytosis, and macropinocytosis. The antigen-processing mechanisms depend on the origin (endogenous or exogenous) and molecular nature of the antigen (protein or lipid).21 Therefore, 3 processing and presentation mechanisms have been characterized: (1) the exogenous (or endosomal) pathway, where antigenic peptides are generated and coupled to MHC-II molecules in phagolysosomes and then transported to the cell surface and presented to CD41 T cells; (2) the endogenous (or proteasome) pathway, where the intracellular proteins are degraded by the proteasome, and the resulting peptides are coupled to MHC-I molecules in the Golgi and then transported to plasma membrane and presented to CD81 T cells; and (3) mechanisms where lipid antigens are coupled to CD1 family molecules and presented to CD81 T cells, g/d T cells, or NK T cells.22 Exogenous antigens can also be presented to CD81 T cells via MHC-I complexes, a process termed crosspresentation. In cross-presentation, internalized proteins are retrotranslocated into the cytosol and degraded by the proteasome. The resulting antigenic peptides migrate to the ER, where they are coupled to MHC-I molecules and subsequently presented to the CD81 T lymphocytes, inducing either immunogenic responses (cross-priming) or tolerance (cross-tolerance).23 Cross-presentation is, therefore, critical for the priming and activation of CTLs against viruses, tumors, and intracellular bacteria. DC maturation. In the classical view, on contact with a ‘‘danger signal,’’ conventional immature DCs undergo a complex process of morphologic, phenotypic, and functional modifications referred to as maturation. These modifications allow the egress of DCs from the peripheral tissues to the marginal zones in the draining lymph nodes, where they present antigens to naive T lymphocytes. Several stimuli have been shown to trigger DCs maturation, including small reactive chemicals, proinflammatory cytokines, such as IL-1b, IL-6, IFN-g, and TNF-a, and pathogen-associated molecular patterns, such as lipopolysaccharides, bacterial DNA, and double-stranded RNA.24 One hallmark of DC maturation is the upregulation of costimulatory molecules, such as CD40, CD54, CD80, CD83, and CD86, and the shift in the chemokine receptor profile. The expression of costimulatory molecules is of great importance for the adequate stimulation of
T cells during antigen presentation, and the modification of chemokine receptors results in the acquisition of migration.24 Although immature DCs express the CCR1, CCR2, CCR5, CCR6, CXCR1, and CXCR2 receptors, mature DCs upregulate CXCR4 and CCR7 and gain responsiveness to the lymphoid chemokines CCL19 and CCL20.25 The profile of cytokines and che- Q12 mokines produced by the DCs also undergoes profound alterations during maturation, being fundamentally dependent on the DC subset and on the stimulus that triggers the maturation process. The expression of the cytokines TNF-a, IL-10, IL-1a/b, IL-12p70, IFN-g, IL-8, IL-6, and IL-23 is normally increased in mature DCs.26 After contact with the maturation stimulus, the DCs transiently produce chemokines, such as CCL2, CCL3, CCL4, CCL5, CCL8, and CXCL8, which are important for the recruitment of monocytes and neutrophils to the site of infection/inflammation. Later, the Q13 production of the lymphoid chemokines, such as CCL17, CCL18, CCL19, CCL22, and CXCL10 increases, promoting the recruitment of T and B lymphocytes and facilitating the interaction of DCs with these cells.27 DC–T cell interactions. During antigen presentation, DCs provide 3 signals that drive the activation and polarization of T cells into their effector and regulatory populations.28 Signal 1 is provided by the interaction of the T-lymphocyte receptor with the MHC-I or MHC-II– antigen complexes presented by the DCs. In the absence of costimulation, signal 1 is commonly associated with the inactivation of naive T lymphocyte by anergy or deletion, promoting tolerogenic responses. The second signal, costimulation, results from the interaction of the costimulatory molecules expressed by the DCs with the respective ligands on the surface of T cells. Together with signal 1, costimulation promotes T-cell survival and proliferation and stabilizes cytokine production. The increased expression of costimulatory molecules during DC maturation has long been associated with the transition from a tolerogenic to an immunogenic state.29 However, numerous data show that costimulatory molecules associated with immunogenic responses may also be involved in the induction of tolerance. In fact, the binding of the costimulatory molecules CD80 and CD86 to cytotoxic T-lymphocyte antigen 4 (CTLA-4) Q14 acts as a negative regulator of T-cell activation. Additionally, the interaction of CD80 and CD86 with CTLA-4 and CD28 has also been shown to be essential for Treg development, homeostasis, and suppressor activity.30 Similarly, the interaction between 4-1BBL and OX40L expressed on DCs with their receptors, 41BB and OX40, respectively, in T cells can promote immunogenic31 or tolerogenic responses.32 Therefore, Q15 signal 2 is the result of a complex balance between
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positive and negative costimulation during DC-T cell cross talk. DCs secrete soluble factors such as cytokines and chemokines (signal 3), which are crucial for the differentiation of CD81 T cells into CTLs33 and for the polarization of CD41 T cells into their different effectors (Th1, Th2, and Th17)34 or regulatory subpopulations (Treg, Tr1, and Th3).35 The presence of IL-12p70, IL-15, and IFN-a/b during the presentation of MHCI–antigen complexes to CD81 T cells enhances their expansion, promotes CTL differentiation, and regulates memory T-cell formation.36 Although IL-12p70 appears to be the critical ‘‘signal 3’’ for CD81 T-cell expansion, it was shown that it may negatively impact the development of both primary and secondary antigen-specific memory CD81 T cells.37 In the DC-mediated polarization of CD41 T cells, the Th1 phenotype is induced by IL-12p70, IL-18, and IL-27, whereas Th17 differentiation primarily depends on transforming growth factor b (TGF-b) and IL-6, although other cytokines, such as IL-1b, IL-21, and IL-23, may also be involved. Regarding Tregs polarization, it is now well established that it depends on the DC ontogeny and capacity to produce IL-2, IL-10, and TGF-b as well as on T-lymphocyte intrinsic factors and on the balance between positive and negative costimulatory signals.38 DESIRED PROPERTIES FOR ANTITUMOR IMMUNOTHERAPIES
Nearly 3 decades of experimental and clinical observations have allowed the scientific community to shed light on the optimal characteristics for antitumor immunotherapies. An important milestone was the identification of the key role played by CD81 T cells in immune responses to cancer.39,40 The induction of antigenspecific CD81 T cells and the subsequent generation of CTLs leads to the recognition of MHC-I–antigenic peptide complexes presented on the surface of tumor cells, triggering their destruction. Therefore, desirable antitumor vaccines should expand the numbers of circulating tumor-specific CTLs; improve their cytolytic abilities (increasing the production of granzymes and perforin); increase the CTLs’ avidity for MHC-I molecules on tumor cells and increase the expression of molecules, such as CXCR3 and CD103/CD49a, that favor the migration and persistence of CTLs into the tumor areas.41,42 It is also desirable that the elicited CD81 T cells express high levels of positive costimulatory molecules such as CD137 and low levels of the immune checkpoint proteins CTLA-4 and programmed cell death 1 (PD-1).43 Finally, antitumor vaccines are also expected to modulate the circulating effector and memory CD81 T cells.
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In addition to the CTL-driven responses, effective immunity against tumors is also strongly dependent on CD41 T cells and NK cells. CD41 T cells, particularly the Th1 subset, directly kill tumor cells,44 activate tumor infiltrating-macrophages and contribute to the differentiation and expansion of antigen-specific CTLs by producing cytokines such as IL-2 and IL-21.45,46 CD41 T cells are also required for the adequate formation of long-term memory CD81 T cells.47 The role of the NK cells in tumor immunosurveillance has gained substantial interest in recent years.48 In response to a balance between inhibitory/stimulatory signals from invariant receptors, NK cells directly kill tumor cells without prior immunization or MHC restriction. Q16 This is accomplished through at least 4 mechanisms: cytoplasmic granule release, effector molecule production, death receptor-induced apoptosis via the TNFrelated apoptosis-inducing ligand and Fas ligand, or antibody-dependent cellular cytotoxicity.49 In addition, NK cells indirectly contribute to tumor elimination by modulating the functions of other immune cells. Activated NK cells potentiate DC maturation and IL-12p70 production and favor immunogenic DC populations by killing immature DCs, whereas sparing fully activated DCs.50 Finally, the cell debris that result from the NK-induced destruction of tumor cells fuel the DCs with antigens, which enhance their cross-presentation to the CD81 T cells.51 However, some care must be taken with the elicitation of Th1 responses and the activation of NK cells in the context of cancer treatment. By producing IFN-g, these cells potently upregulate the expression of PD-1 ligands (PD-L1 and PD-L2) in tumors, which may contribute to tumor immune evasion.52 Other CD41 T-cell subsets, such as Th2 cells and Tregs, also counteract the CTLs antitumor activity. By secreting IL-4, Th2 cells limit the cytolytic activity of CTLs by decreasing the expression of granzymes and perforin,53 whereas Tregs negatively affect CTLs because of the production of IL-10 and TGF-b.54,55 In addition, Tregs can also impair CD81 T-cell expansion by competing with them for the cytokine IL-2.56 RATIONALE FOR ANTITUMOR DC-BASED VACCINES
DCs represent the perfect tool for immunotherapeutic interventions because of their unparalleled capacity for antigen presentation, their ability to modulate other immune players, and their functional plasticity.2 The effectiveness of DC vaccines is the subject of debate, mainly because the clinical outcomes addressed may not be the most adequate. However, there is no doubt that these approaches elicit strong tumor antigenspecific immune responses (CTLs and Th1 cells). A
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meta-analysis of antitumor DC-based vaccines for prostate cancer and renal cell carcinoma revealed that the interventions induced these immune responses in 77% and 61% of the patients, respectively.57 Moreover, in addition to boosting adaptive immunity, the DCs were shown to enhance the antitumor activity of NK cells by increasing their cytolytic abilities and IFN-g production.58 Following this seminal publication, a plethora of other studies revealed the crucial relevance of DC-NK cell cross talk for efficient tumor elimination (reviewed in Lion et al59). In fact, effective DC vaccine-mediated antitumor immunity is at least in part dependent on NK cell activity.51 This was elegantly demonstrated in murine models of melanoma and metastatic lung tumors, where the tumor eradication observed after DC vaccination was completely abrogated in animals that had been depleted of NK cells.60,61 Although only a limited number of DC-based cancer vaccination trials implemented NK cell motorization, the existing data indicate that nearly 50% of patients showed an increased frequency and/or an induction of NK cell activation.59 Therefore, it is now clear that the antitumor responses promoted by DC vaccination rely on their capacity to prime and activate CD81 T cells, to polarize CD41 T cells into Th1 populations, and to cross talk with NK cells.62-65 Another important argument favoring DC-based immunotherapies is their safety profile. Twenty years of observations in numerous phase I and II trials demonstrated that DC vaccines are generally well tolerated and induce minimal toxicity. The most common manifestations are local reactions at the injection sites, such as rash and pruritus, and occasionally systemic effects occur, including fever and malaise.4 Despite the initial concerns regarding the possibility of inducing autoimmunity, the DC-based therapies are rarely associated with severe immunotoxicity reactions. METHODOLOGIES FOR THE PRODUCTION OF DCBASED VACCINES
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The present review is focused on the diversity of approaches used to directly manipulate or target DCs in the context of cancer treatment, with a special emphasis on the impact of the different methodologies on vaccine effectiveness. The information systematized subsequently was collected from www.clinicaltrials.gov (until March 2015) and from key articles on the subject. Table I summarizes the different methodologies/approaches and the respective benefits and drawbacks. The complete list of clinical trials analyzed and their major characteristics can be found in the Supplementary material. Ex vivo DC manipulation. Ex vivo manipulation of DCs is by far the most explored strategy and is used in
approximately 97% of the clinical trials initiated to date (Fig 2, A). This approach requires obtaining DCs or DC precursors from patients, manipulating them (inducing maturation and loading antigens), and reinjecting them into the donor. From the time of the production of this type of DC-based vaccine according to good manufacturing procedures is a time- and costintensive procedure, individual aliquots are normally produced and cryopreserved at the beginning of the treatment and are released along the vaccination schedule. Cryopreservation, when performed under optimal conditions, does not significantly affect the viability, phenotype, or function of the DCs, and the thawed cells are suitable for clinical use.66 Currently, there is no standardized procedure for ex vivo manipulation, which results in a plethora of protocols that differ in the source of DCs, the maturation stimulus, the nature and procedure for antigen loading and, finally, the route of administration. DC source. The DCs used in tumor immunotherapies can be either autologous or allogenic. As shown in Fig 2, B, the use of autologous DCs has been by far the preferred approach. Although allogenic DCs are less frequently used, their usage is supported by experimental evidence demonstrating that allogeneic human leukocyte antigen (HLA) molecules represent potent immunogenic signals and boost antitumor immunity.67,68 Besides, it may be advantageous to use cells from normal donors because DCs and DC precursors from cancer-bearing patients are targeted by tumor immunosuppressive factors that render them functionally aberrant.69,70 The immunosuppressive factors that are usually overproduced by tumor cells include IL10, TGF-b, vascular endothelial growth factor (VEGF), IL-6, and prostaglandin E2 (PGE2), and dysfunctional DCs have been observed in patients with chronic lymphocytic leukemia, melanoma, ovarian, breast, renal, prostate, lung, and head and neck cancers.71-74 Allogenic DCs are commonly obtained by differentiation of peripheral blood mononuclear cells (PBMCs) of unrelated healthy donors. However, at least 2 clinical trials use DC-like cell lines (Fig 2, B). The use of the proprietary dendritic progenitor cell line DCone was tested in acute myeloid Q19 leukemia patients (NCT01373515), and the safety and tolerability of subcutaneous (s.c.) administration of an allogeneic pDC line was tested in patients with melanoma (NCT01863108). Given that circulating DCs represent less than 1% of PBMCs, the cells used in therapeutic cancer vaccines are typically differentiated from autologous leukapheresis-isolated CD141 monocytes or CD341 hematopoietic progenitors. Differentiation from monocytes involves the cell culture for 5–7 days in the
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Table I. Advantages and drawbacks of the different methodologies/approaches used for production of DCbased antitumor vaccines Advantages
Ex vivo DC manipulation Genetic source Autologous
- No risk of graft-vs-host disease
Allogenic
- Allogenic HLA molecules represent potent immunogenic signals67,68
DC source CD14+ monocytes
- Abundant source: CD14+ monocytes represent 10% of PBMCs
CD34+ hematopoietic progenitors Natural occurring DCs iPSC-derived DCs
Antigens and loading procedures Peptide pulsing
Protein pulsing
- CD34+-derived DCs stimulate more effective CTL responses than MoDCs75,76 - Low doses of plasmacytoid DCs effectively induce CD4+ and CD8+ T-cell responses77 - iPSCs may be continuously expanded in vitro153 - DCs can be tailored to a desired phenotype (eg, CD11c+CD141+XCR1+ DCs)154 - Peptides are synthesized and purified at low cost - Enables direct monitoring of T-cell responses - Short peptides are directly loaded into MHC molecules - Synthetic peptides allow for modifications - Full-length proteins may present more epitopes for immune recognition - Prolonged antigen presentation
Tumor lysates
- Encompasses multiple TAAs (even those not yet characterized)
Tumor apoptotic bodies
- Encompasses multiple TAAs (even those not yet characterized)
RNA transfection
- Tumor mRNA may be amplified as needed - Possibility to introduce mRNAs coding for immunostimulatory proteins
Viral transduction
- The vector may intrinsically activate DCs95 - Possibility to introduce genes coding for cytokines and costimulatory molecules94
DC-tumor cell hybrids
- Encompasses multiple TAAs - Antigen presentation is maintained for days
DC maturation protocols TNF-a + IL-1b + IL-6 + PGE2 TNF-a + IL-1b + IFN-a + IFN-g + polyinosinic: polycytidylic acid Routes of administration Intravenous Intradermal/subcutaneous
Drawbacks
- DCs from cancer-bearing patients are frequently dysfunctional69-74 - Survival of injected DCs may be shortened by T-cell–mediated rejection - CD14+-derived DCs (MoDCs) are not as efficient as other DC subsets in eliciting CTL responses - Limited number: represent 0.1% of PBMCs - Limited number: circulating DCs represent less than 1% of PBMCs - Laborious and expensive - Production according to GMP requirements is technically challenging - Limited number of known immunogenic TAAs - HLA restriction - Peptides with low affinity for MHC may be poorly immunogenic - Limited number of known immunogenic TAAs - Propensity of proteins to be targeted for MHC-II - Dependence on the availability of tumor cells - Immunologic responses more complex to monitor - Dependence on the availability of tumor cells - Immunologic responses more complex to monitor - Reagents and procedures to transfect DCs may affect their viability - Limited stability and short lifespan of mRNA - Immune responses to vector antigens may overwhelm those for tumor antigens95 - Viral vectors may perturb DC functions95 - Low expression of IL-12p70 and costimulatory molecules97
- PGE2 enhances CCR7 expression and DC migratory capacities106 - Efficiently induces IL-12p70 - aDC1s elicit potent CTL responses102,109-111
- PGE2 may reduce the capacity of DCs to produce IL-12p70107 - aDC1s may have reduced migratory capacities compared with standard matured DCs
- Induces antigen-specific humoral responses more efficiently than i.d. and i.n. routes117 - Elicit strong specific antitumor Th1 and CTL responses
- Injected DCs preferentially accumulate in the spleen and liver - Just 1%–5% of injected DCs reach the peripheral lymph nodes117,118 (Continued )
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Table I. (Continued ) Advantages
Intranodal Intratumoral In vivo DC targeting Cancer antigens fused to monoclonal antibodies targeting DC surface receptors
Drawbacks
- Substantially more DCs reach the T-cell areas in lymph nodes118,120 - May delay and reverse the tolerization of tumor-infiltrating effector T cells124
- Technically exigent - Great variability between patients - Technically exigent - Associated with high morbidity
- Bypasses the expensive and laborious ex vivo DC generation process - Allows targeting specific DC subsets128-130
- Limited to known TAAs - It requires the coadministration of adjuvants130 - Targeted receptor must be unambiguously expressed
Other strategies directly/indirectly involving DCs Nontargeted antigen-based - Easier to produce vaccines - Stable in many storage conditions - Induce CD4+ and CD8+ T-cell responses GM-CSF–secreting tumor cells - Potentiates TAAs presentation by endogenous DCs136 - Consistent induction of cellular and humoral antitumor immune responses137-139
Implantable DC-recruiting scaffolds
Dex
- Bypasses the expensive and laborious ex vivo DC generation process - The approach takes advantage of endogenous DCs - Preclinical studies show induction of specific and effective antitumor immunity141-143 - Dex are able to elicit CD4+ and CD8+ T-cell responses144-147 - Exosomes may carry additional mRNAs and miRNAs to enhance immune responses - Production under GMP conditions is well defined
- Limited to known TAAs - Immune responses may be transient and/ or of low magnitude - Prolonged GM-CSF production by tumor cells may cause immune tolerance140 - Dependence on the availability of tumor cells - Immunologic responses more complex to monitor - The design of biocompatible polymeric scaffolds incorporating functional proteins (chemokines) is technically challenging - Until present the results are only from preclinical studies - Existing clinical data indicate that T-cell responses elicited by Dex are limited149,150 - Requires large quantities of ex vivo generated DCs for exosome production
Abbreviations: CTL, cytotoxic T cell; DC, dendritic cell; Dex, dendritic cell–derived exosomes; GMP, good manufacturing procedures; GM-CSF, granulocyte-macrophage colony–stimulating factor; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; iPSC, induced pluripotent stem cell; MHC, major histocompatibility complex; MoDC, monocyte-derived dendritic cells; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell; PGE2, prostaglandin E2; TAA, tumor-associated antigen; TNF-a, tumor necrosis factor a.
presence of GM-CSF and IL-4, producing DCs with an immature phenotype that are commonly referred as monocyte-derived DCs (MoDCs). When CD341 hematopoietic precursors are used, the patients are frequently treated with GM-CSF before leukapheresis to mobilize precursors from the bone marrow. The harvested CD341 cells are then cultured with GM-CSF, TNF-a, Flt3L, TGF-b, and stem cell factor for 11– 12 days. This procedure leads to a heterogeneous mixture of APCs, namely MoDCs, LC-like cells, and other myeloid cells.75 Most clinical trials with a stated DC source use MoDCs (Fig 2, B). The preference for MoDCs instead of CD341-derived DCs is not related to their superior clinical efficacy, but rather to the limited number of CD341 precursors that can be isolated from apheresis products. In fact, CD341-derived DCs, and particularly their LC subset, were shown to stimulate in vitro more effective CTL responses than their counterpart MoDCs independent of the presence
of IL-12p70.75 Moreover, the efficacy of peptideloaded CD341-derived LCs and equivalent MoDCs was directly compared in stage III/IV melanoma patients (NCT00700167), and the results indicate that without exogenous IL-15 administration, the LCbased vaccines stimulate significantly more IFN-g–producing CTLs.76 Sipuleucel-T (Provange), the first FDA-approved cell-based antitumor vaccine, uses a different protocol to obtain DCs. Autologous PBMCs were incubated for 36–48 hours with a recombinant fusion protein consisting of human prostatic acid phosphatase and GM-CSF. GM-CSF targets the prostatic acid phosphatase antigen to APCs, namely DCs, promoting its uptake/processing and simultaneously inducing cellular maturation. The Q20 biologically active components of the vaccine are the CD541-matured APCs.6 Finally, among the clinical trials analyzed, only one uses ex vivo manipulated naturally occurring DCs (NCT01690377) (Fig 2, B). The
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Fig 2. Manipulation strategies and types of DCs used in antitumor vaccines. (A) Clinical trials with approaches directly exploring DCs: manipulation ex vivo followed by reinjection and in vivo DC targeting. (B) Frequencies distribution of clinical trials by DC genetic source (autologous or allogenic) and DC type. ‘‘Natural DCs’’ refer to primary cells directly isolated from the donor. ‘‘Not defined’’ indicates that the information regarding the parameter was not found in the clinical trial description or in the literature. DCs, dendritic cells; LCs, Langerhans cells; MoDCs, monocyte-derived dendritic cells.
authors77 of a study evaluated the efficacy of intranodal (i.n.) injection of tumor peptide–loaded pDCs in patients with metastatic melanoma. The results indicate that despite the limited number of pDCs administered, several patients mounted antivaccine CD41 and CD81 T-cell responses, demonstrating the feasibility of the approach. Selection of antigens and loading procedures. The choice of tumor antigens and the loading procedures are important parameters for DC-based vaccine production.78 A limited fraction (approximately 10%) of the tumor-associated antigens (TAAs) appear to be immunogenic, and among these, only a few are effectively associated with tumor rejection.79 TAAs are normally
unique mutated proteins, antigens derived from oncogenic viruses, or shared nonmutated self-antigens, such as tyrosinase, tyrosinase-related proteins, gp100, or MART-1.80 Q21 DCs have been pulsed with isolated/recombinant TAAs (full-length proteins or peptides), transfected with tumor messenger RNA (mRNAs), transduced Q22 with TAA-coding genes, and loaded with tumor cell lysates or apoptotic tumor cells (Fig 3, A). All these procedures were shown to elicit protective and therapeutic anticancer immune responses without inducing specific toxicity.81 Although pulsing DCs with short peptides lead to its direct loading onto MHC molecules at the cell surface, proteins and tumor lysates require
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Fig 3. Antigen loading procedures and routes of administration of ex vivo manipulated DCs. (A) Frequencies distribution of clinical trials by DC antigen loading strategies. (B) Administration routes for ex vivo manipulated DCs. In some cases, the same trial compares the efficiency of more than one administration route leading to count numbers (322) superior to the total number of respective trials (303). Not defined indicates that the information regarding the parameter was not found in the clinical trial description or in the literature. DCs, dendritic cells; i.d., intradermal; i.l., intralymphatical; i.n., intranodal; i.t., intratumoral; i.v., intravenous; s.c., subcutaneous.
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internalization, processing, and presentation. However, the use of defined peptides is limited to the low number of characterized TAAs and by the fact that some of them are HLA-A1 or HLA-A24 restricted, requiring therefore that the patient’s haplotype must be known and adequate. Regarding the loading of DCs with allogenic/autologous tumor cell lysates or apoptotic bodies, it is a strategy for long known to elicit antitumor immunity.82 Its major advantage relies on the use of whole tumor proteome, encompassing that way multiple TAAs. In terms of effectiveness, several studies indicate that DCs loaded with apoptotic tumor cells elicit stronger immune responses than lysate-loaded or RNApulsed DCs.83,84 The major drawback of using tumor lysates or apoptotic bodies as a source of antigens instead of defined peptides is the dependence on the availability of tumor cells. Moreover, immunologic responses are more complex to monitor, which make
the correlation between the interventions and the clinical outcomes difficult. Another common strategy is to transfect DCs with mRNA extracted from tumor cells or with in vitro synthesized mRNAs that encode particular TAAs. The ability of these loaded DCs to elicit strong antitumor CD41 and CD81 T-cell responses is well documented85-87 and is one of the most commonly used approaches in clinical trials (Fig 3, A). When compared with peptide loading, transfection with whole tumor mRNA avoids the limitation imposed by the use of known TAAs and matched HLA phenotypes. In addition, siRNAs targeting the immunoproteasome components (NCT00672542) or mRNA coding for maturation agents and costimulatory molecules (NCT01066390) could be introduced, enhancing the Q24 generation of TAA-derived peptides and eliminating the need for an additional DC maturation step. The latter
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approach was evaluated in melanoma patients using a vaccine composed of DCs transfected with mRNA encoding the immunostimulatory proteins CD40L, CD70, and constitutively active TLR4 in combination with mRNAs’ encoding tyrosinase, MAGE-A3, MAGE-C2, and gp100 (NCT01066390). The results demonstrated that most patients developed functional TAA-specific CD81 and CD41 T-cell responses, which were detected both at the skin and systemically.88,89 Among the strategies to transfect DCs, electroporation has shown to be the most efficient method, temporarily increasing cell membrane permeability, which facilitates the entry of mRNA without the need for additional reagents.90 DCs have also frequently been genetically engineered to stably express TAAs, such as mucin 1, p53, tyrosinase, melan-A, and gp100.91-93 The use of bacteria or viral vectors to deliver the DNA to DCs is safe, and the possibility of introducing genes encoding cytokines or costimulatory molecules is very appealing.94 Furthermore, the vector may intrinsically activate DCs by triggering endosomal or cytoplasmic sensors, such as TLRs, RIG, and protein kinase RNA-activated receptors, bypassing the need of a separate maturation step.95 Finally, the ex vivo fusion of DCs with tumor cells represents another approach for antigen loading. These cell hybrids, known as ‘‘dendritomes,’’ were shown to induce antitumor immune responses in vivo96-98 and have been tested in multiple clinical trials for more than the past few decades (Fig 3, A). Notably, dendritomes were found to be less effective in eliciting CTL responses than DCs loaded with apoptotic tumor cells,99 an observation that was attributed to the reduced expression of costimulatory molecules and IL-12p70 by the cell hybrids.97 DC maturation protocols. The DC maturation status and cytokine profile are parameters of great importance for the effectiveness of DC-based immunotherapies. Numerous clinical trials have shown a clear superiority of mature DCs over their immature counterparts.57,100 Immature DCs have a reduced ability to migrate from the injection sites to the lymph nodes,100 and can cause the inhibition of CTL functions by inducing IL-10– producing antigen-specific Tregs.101 In addition to the maturation status, the DC cytokine and chemokine profiles, particularly the expression of IL-12p70 and CCR7, are of great relevance for vaccine effectiveness. The cytokine IL-12p70 plays a central role in CD81 T-cell expansion and CTL differentiation.36 Clinically, the higher levels of IL-12p70 produced by DCs used in vaccines for glioma and melanoma patients were found to positively correlate with a more favorable clinical outcome.102,103 The chemokine receptor CCR7 governs the migration of DCs from the
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peripheral tissues to the draining lymph nodes under either homeostatic or inflammatory conditions. In the context of DC-based immunotherapies, the migration of DCs from the injection sites to the lymph nodes and the subsequent interaction with the T cells is a key feature for effective immunization. A growing body of evidence demonstrates that DCs expressing high levels of CCR7 elicit a more effective antigenspecific immune response in vivo, which lowers the required DC dosage.104,105 The appropriate DC maturation stimulus remains a matter of debate. On the basis of the literature, several maturation protocols have been tested, which consist of individual stimuli or cocktails that combine proinflammatory cytokines, CD40L, and TLR agonists. The maturation cocktail that was used most commonly across the clinical trials analyzed includes TNF-a, IL-1b, IL-6, and PGE2. The use of PGE2 is somehow paradoxical because although it enhances the DC migratory capacities by inducing CCR7 expression,106 it reduces IL-12p70 production.107 Protocols that combine exposure to the TLR3 ligand polyinosinic:polycytidylic acid and PGE2 were proposed to circumvent this problem and explore the exquisite capacity of TLR agonists to induce IL-12p70 expression.108 Another common DC maturation cocktail includes TNF-a, IL-1b, IFN-a, IFN-g and polyinosinic:polycytidylic acid. The resulting DCs, termed aDC1s, efficiently secrete IL-12p70 and were shown to elicit more potent CTL responses than the standard maturated DCs.102,109-111 From our analysis, aDC1-based vaccines were tested in at least 8 clinical trials (Fig 2, B). To increase their immunogenicity, DC vaccines are frequently administered along with adjuvants. The most frequently used adjuvants are GM-CSF, IL-2, IFN-a, and TLR agonists. GM-CSF strongly induces the proliferation of DC precursors and has a stronger chemotactic effect over DCs, promoting their recruitment and maturation. Interferon alpha enhances DC antigen cross-presentation and, therefore, promotes more effective CTL responses.112 Strong preclinical evidence has demonstrated that the cytokine IL-2 can improve DC vaccine efficacy.113 However, subsequent clinical trials have not shown superior antitumor immune responses in regimens combining DCs with IL-2.114 In fact, because of its stimulatory abilities toward Tregs and myeloid-derived suppressor cells (MDSCs), IL-2 can even negatively affect DC-based immunotherapies. In addition to their inclusion in maturation cocktails, TLR agonists are increasingly being used as adjuvants in DC vaccines. In the clinical trials analyzed, the most common are poly-ICLC, rintatolimod (TLR3 ligands), imiquimod (TLR7 ligand), resiquimod (TLR7/8 ligand), and DUK-CPG-001 (TLR9
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ligand). Another important question in the maturation protocols is the duration of DC exposure to the stimulus. The contact of DCs with maturation signals (danger signals) in vivo is transient because the cells rapidly leave the site of contact (normally the peripheral tissues) and reach the lymph nodes in 2–4 hours.115 However, most protocols in the literature indicate 24–48 hours of exposure. These time periods may be too long, exhausting the ability of the DCs to produce IL-12p70 and, therefore, decreasing their ability to elicit protective Th1 and CTL responses.116 Overall, we can conclude that the diversity of the maturation procedures used in the production of DCbased vaccines hampers the assessment of the real effects of the maturation stimuli because it is not the only parameter that differs among studies. It would be of great interest to design clinical trials that compare the effectiveness of DCs that have been matured with different cocktails to pursue a certain level of standardization. Routes of administration and cell doses. The route of administration, frequency of injections, and the number of cells injected are relevant aspects to consider in DC-based vaccinations, given that the efficacy of the intervention will strongly depend on the migration of sufficient numbers of DCs to T-cell areas in secondary lymphoid tissues. For more than the last 2 decades of clinical trials, DCs have been administered via different routes: intravenous (i.v.), intradermal (i.d.), s.c., i.n., and directly into the tumor (Fig 3, B). All these routes of administration elicit specific antitumor immune responses, but with different characteristics and efficiencies.116 The i.v. injected DCs are transiently detected in the lungs and then preferentially accumulate in the liver and spleen, with little distribution to the lymph nodes. In a clinical trial with prostate cancer patients, i.v. administration was shown to induce antigen-specific humoral responses more efficiently than the i.d. or i.n. routes.117 Regarding i.d. and s.c. routes, most DCs remain at the injection sites and are cleared by infiltrating macrophages.118 However, the 1%–5% of cells that actually reach the peripheral lymph nodes were shown to be sufficient to induce specific antitumor T cells.117,118 Furthermore, several lines of evidence suggest that the DCs retained at the injection site may act as adjuvants. They recruit, activate, and transfer antigens to resident DCs, namely dermal DCs and LCs, that then more effectively activate CD81 T cells.119 For these reasons and the fact that they are technically less exigent and less expensive, the s.c. and i.d. routes are used most commonly in trials for solid tumor treatments (Fig 3, B). Administration via i.n. route is performed under ultrasound guidance of the needle by an experienced radiologist. The
efficacy of the procedure heavily depends on the correct delivery and can, therefore, present great variability between patients. Surprisingly, although substantially more DCs migrate to the T-cell areas in the lymph nodes on correct i.n. vaccination, the antitumor T-cell responses are comparable or inferior to i.d. administration.118,120 Another route used in a few phase I/II clinical trials is intratumoral administration (Fig 3, B). The approach is technically exigent and associated with a high rate of intervention-associated morbidity. It has been tested in the treatment of several solid tumors, such as melanoma, breast, liver, and pancreatic cancers.121-123 An overall evaluation of the published studies demonstrates that the procedure is safe and could elicit antitumor immunity in at least some of the patients. Recently, using autochthonous prostate cancer animal models, Higham et al124 showed that one advantage of intratumor DC administration relies on the capacity of these cells to delay and reverse the tolerization of tumor-infiltrating effector T cells. As shown in Fig 3, B, currently, there is an increased tendency to administer DC vaccines via multiple routes, such as i.d. 1 s.c. or i.d. 1 i.v., with the aim of inducing more robust and broader immune responses. The minimal number of DCs required to induce an efficient immune response in humans has not yet been established. This is reflected in clinical trials by a plethora of DC doses and administration regimens. Doses ranging from as low as 0.3 3 106 naturally occurring pDCs injected via i.n. route every 2 weeks (NCT01690377) to 3 doses of 200 3 106 MoDCs administered via s.c. route (NCT00704938) can be found among analyzed trials. Verdijk et al determined that 0.5 3 106 DCs reaching the T-cell areas in the lymph nodes could be sufficient to induce de novo immune responses. Considering that a maximum of 4% of DCs administered via i.d/s.c. route migrate and only 80% remain viable after injection, the authors estimated that a minimal dose would consist of 15 3 106 cells.118 Experiments performed in lung cancer and neuroblastoma animal models showed that efficacy of DC vaccines is positively correlated with the dose used. Moreover, in one of the early clinical trials testing sipuleucel-T, the median time to disease progression was found to be 31.7 weeks for patients who received more than 100 3 106 cells per infusion compared with 12.1 weeks for patients who received fewer cells.125 However, the correlation between the number of administered DCs and vaccine effectiveness may vary with the selected administration route. Surprisingly, reducing the number of DCs injected via i.d. route was recently shown to improve their homing to the lymph nodes and, consequently, to potentially enhance vaccine efficacy.126,127 The authors suggested that
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multiple i.d. injections with small amounts of DCs would be a preferable strategy. In vivo DC targeting. Targeting antigens to DCs in vivo represents an enormous breakthrough for antitumor immunotherapies because it allows researchers to bypass the expensive and laborious ex vivo DC generation process.128 Antigens can be unambiguously targeted to different DC subsets, eliciting antigen-specific CD41 and CD81 T cell-mediated responses (reviewed in Caminschi et al129). The procedure involves the coupling of antigens to monoclonal antibodies (mAbs) that are specific for DC surface molecules, such as Fc receptors, CD40 and C-type lectin receptors, or the use lentiviral vectors with preferential tropism to DCs. Notably, the delivery of antigens via mAb induces tolerance in the absence of adjuvants, and requires the coadministration of DC maturation agents such as TLR3, TLR7/8, or CD40 agonists.130 On the basis of animal experiments, the delivery of antigens to DEC205, CD40, DC-SIGN, DCIR, mannose receptor, Cleac9A, and XCR1 showed promising results in inducing antitumor immunity (CD41 and CD81 T-cell responses, as well as humoral responses).129,131,132 The clinical application of the approach has been slow and has faced significant challenges. Several important factors must be considered in the development of human DC-targeting vaccines, including the biological functions of the targeted DCs, the pattern of expression and functions of the chosen receptors, and the adjuvants coadministered. Among the few in vivo DC-targeting therapies that are being tested in humans, DEC-205 is the most common targeted receptor (Fig 2, A). These vaccines consist of s.c. and i.d. administration of the cancer antigen NYESO-1 fused to a human anti–DEC-205 mAb in combination with the adjuvant poly-ICLC (NCT009 48961), decitabine (NCT01834248), the indoleamine2,3-dioxygenase (IDO) inhibitor INCB024360 (NCT0 2166905), or recombinant Flt3L (NCT02129075). The targeting of human chorionic gonadotropin beta protein to DCs via the mannose receptor is also being clinically evaluated for the treatment of breast, colorectal, pancreatic, bladder, and ovarian cancers. The fusion protein is injected via i.d. and s.c. routes in combination with adjuvants, such as GM-CSF and poly-ICLC (NCT0070 9462), or administered via i.v. route (NCT00648102). Using a different approach, a lentiviral vector encoding NY-ESO-1 and pseudotyped with Sindbis virus envelope proteins that are modified to target DC-SIGN on DCs (IDLV305) is under evaluation for the treatment of NY-ESO-1–positive solid tumors (NCT02122861). Although the clinical application of these nextgeneration DC-based vaccines is still in the early stages and, therefore, has several challenges that still have to
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be overcome, it represents an exciting and promising new field in antitumor immunotherapy and overall immune modulation. Other DC-based approaches. Apart from ex vivo manipulation and direct in vivo targeting, several other approaches exploit the immunogenic potential of DCs in cancer therapy. These include nontargeted antigenbased vaccines, GM-CSF–secreting tumor cell vaccines, implantable DC-recruiting/activating scaffolds, and DCderived exosomes (Dex). Q27 The nontargeted antigen-based vaccines are composed of peptides, proteins, or tumor nucleic acids. When injected, these antigens are captured and processed by the APCs, namely DCs, eliciting an antigen-specific immune response.131 Initial studies showed that this approach can induce CD41 and CD81 T-cell responses, although with low or no impact on the clinical course of the disease.133 The lack of clinical benefits in these cases was associated to the dominant polarization of CD41 T cells into IL-4– and IL-5–producing Th2 subsets and to the differentiation of tumor-specific Tregs. To overcome these immunosuppressive events and to potentiate antigen capture by the DCs, vaccines have begun to be administered in combination with GM-CSF, adjuvants, and low doses of lymphodepleting agents, such as cyclophosphamide.134,135 In phase II trials, vaccines composed of the MAGE-A3–recombinant protein and the AS15 adjuvant system were shown to cause clinical improvements in both metastatic melanoma (NCT00086866) and resected non-small cell lung cancer (NCT00290355). For the GM-CSF–secreting tumor cell vaccines, irradiated autologous tumor cells or allogenic tumor cell lines have been engineered to secrete GM-CSF and were then injected into patients. These engineered tumor cells strongly attract macrophages, granulocytes, T cells, and DCs, potentiating tumor antigen presentation.136 The clinical evaluation of this vaccination strategy revealed a consistent induction of cellular and humoral antitumor responses in melanoma,137 prostate cancer,138 and pancreatic cancer.139 As a major drawback, several animal models have shown that prolonged GM-CSF production by tumor cells may lead to disease progression as a result of immune tolerance caused by the recruitment of myeloid suppressor cells and the differentiation of tolerogenic DCs.140 A new approach for recruiting endogenous DCs to a source of tumor antigens recently garnered particular interest. Mooney et al141 showed that biocompatible polymers may be designed to incorporate and release a DC chemotactic agent, an adjuvant, and tumor antigens in a controlled manner. The authors tested the implantation of polylactide-co-glycolide scaffolds incorporating GM-CSF, various TLR agonists, and tumor lysates in mice in
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the context of preventive and therapeutic melanoma vaccination. The obtained results demonstrated that the mice receiving the scaffold mounted specific and protective antitumor immunity with an OS of 90% for the preventive vaccine and 33% long-term survival for the therapeutic vaccine.141,142 Using the same rationale, Wang et al143 developed a 2-step strategy to modulate the endogenous DCs in situ. In the first step, the DCs were recruited to an injectable GM-CSF– releasing thermosensitive mPEG2poly(lactic-co-glycolic acid) hydrogel. Then, the recruited DCs were loaded with cancer antigens through the use of viral or nonviral vectors. The strategy was shown to generate robust tumor-specific immune responses in both prophylactic and therapeutic models of murine melanoma.143 These encouraging results in animal models led to the clinical application of the strategy. Currently, an ongoing clinical trial is evaluating the feasibility of this approach for treatment of melanoma (NCT01753089). The matrix of the DC-activating scaffold is composed of poly(lactic-co-glycolic acid) and incorporates an autologous tumor cell lysate, GM-CSF, and CpG oligodeoxynucleotides. This strategy may represent the future of DC-based immunotherapies, given that it overcomes the survival and homing problems of the injected ex vivo–generated DCs and even surpasses the limitation of single antigen immunization observed in the in vivo DC–targeting approach. Dex have received great attention as potential immunotherapeutic agents because of the pioneer studies of Zitvogel et al,144 who showed that they inhibit tumor growth in an MHC- and CD81 T cell-dependent manner. The capacity of Dex to elicit CD81 and CD41 T-cell responses was attributed to the transfer of antigen–MHC-I and MHC-II complexes to the endogenous DCs.145 Similar to DCs, Dex were found to modulate NK cells, promoting their proliferation and activation in IL-15Ra–dependent and NKG2Dmediated processes, respectively.146 Recently, the activity of Dex against tumors was additionally shown to rely on B-cell–mediated mechanisms147 and on their capacity to enhance the immunogenicity of tumor cells.148 Despite the abundant experimental data on the use of Dex for antitumor immunotherapy, their clinical evaluation remains scarce (Fig 2, B). Results from 2 clinical trials on melanoma and non-small cell lung cancer patients demonstrated that Dex could be safely administered, nevertheless inducing only limited CTLs responses.149,150 Finally, although all clinical trials performed to date test antitumor DC-based vaccines in a therapeutic context, numerous preclinical experiments showed that preventive DC vaccination can significantly delay or even prevent the development of several tu-
mors.151,152 This supports the establishment of clinical trials that evaluate the safety and efficacy of DC-based vaccines for cancer prevention in high-risk groups in the near future. LESSONS FROM THE PAST AND FUTURE PERSPECTIVES
It is now clear that the effectiveness of antitumor DCbased vaccines depends on the ability of the DCs to induce antigen-specific CTLs and Th1 cells as well as NK cell cross talk and activation.51,131 These features must be achieved by selecting or targeting the adequate DC subset and by tailoring its maturation status and cytokine/chemokine profile. In accordance Q30 with this notion and based on their remarkable capacity to cross-present antigens, LC-like DCs derived from CD341 progenitors (NCT00700167 and NCT0 1456104) or CD141 monocytes (NCT01189383) are being evaluated in clinical trials for the treatment of melanoma. Similarly, the clinical effectiveness of other DC subsets, such as pDCs and Th1 polarizing-DCs, is under assessment in melanoma (NCT00390338), prostate cancer (NCT00970203), and glioma patients (NCT00766753). The recent characterization of a novel human DC subset (CD11c1CD1411XCR11) with superior antigen cross-presentation and NK cell activation capacities has excited great enthusiasm on its possible use.12,14 However, CD11c1CD1411XCR11 DCs represent less than 0.1% of PMBCs, a major drawback for their clinical application. In fact, a relevant obstacle for the progress of DC-based vaccines has been the lack of sufficient DC sources other than CD141 MoDCs. To overcome this issue, the emerging field of induced pluripotent stem cells (iPSCs) may represent a significant opportunity.153 Recently, Silk et al154 established a protocol for the differentiation of CD11c1CD1411XCR11 DCs by culturing human iPSCs with GM-CSF, stem cell factor, VEGF, and bone morphogenetic protein 4. These iPSC-derived DCs were shown to efficiently cross-present exogenously supplied peptides to naive CD81 T cells, inducing their expansion and activation. Because the iPSCs may be continuously expanded in vitro, they represent an unlimited source of autologous DCs that can be tailored to the desired phenotype, bypassing problems such as limited cell numbers and patient-topatient variability.153 Another fact that became evident is that the DC vaccine-induced antitumor immune effector cells must overcome multiple immunoescape/immunosuppressive mechanisms (reviewed in Topalian et al155). Solid tu- Q31 mors often generate an immunosuppressive microenvironment by producing soluble mediators such as adenosine, IDO, PGE2, TGF-b, and VEGF. These
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Fig 4. Frequency of therapeutic approaches combining DC-based vaccines and other anticancer treatments. (A) DC-based vaccines are increasingly being tested in combination with other treatments such as conventional chemotherapy, radiotherapy, and other immunotherapies. (B) Frequencies distribution of other therapies being tested with DC-based vaccines. Other cell therapies include stem cell transplantation, transgenic CTLs, ex vivo expanded T lymphocytes, and cytokine-induced killer cells. *In some cases, in the same trial, DC-based vaccines are tested in combination with multiple therapies leading to count numbers (149) superior to the total number of respective trials (119). DCs, dendritic cells; CTL, cytotoxic T cells; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; IDO, indoleamine-2,3-dioxygenase; mAb, monoclonal antibody; PD-1, programmed cell death 1; VEGF, vascular endothelial growth factor.
mediators inhibit the proliferation and differentiation of Th1 cells, suppress the activity of CTLs and DCs, and induce the differentiation of Tregs.156-158 Moreover, the tumor microenvironment is also prone to attract immunosuppressive cellular regulators such as circulating Tregs, MDSCs, and tumor-associated macrophages. Finally, immune checkpoints, an inhibitory network that normally maintains self-tolerance and prevents excessive and uncontrolled immune responses, can be co-opted by tumors to evade immune destruction.159,160 The major immune checkpoints that are dysregulated by tumors rely on ligand-receptor interactions, such as CD28–CTLA-4 and PD-1–PD-L1. There was a significant effort to establish strategies to bypass these barriers, and combination therapies emerged as a logical approach to potentiate DC vaccine effectiveness. The analysis of clinical trials for more than the last 2 decades reflects a clear tendency to adopt combination therapies instead of standalone DC vaccination (Fig 4, A). Some of the most common approaches involve the use of general lymphodepleting chemotherapeutic agents such as temozolomide and cyclophosphamide and fludarabine (Fig 4, B). By depleting the immune cells, these drugs eliminate negative regulators such Tregs and MDSCs, promoting a favorable environment for DC-induced expansion of antitumor effector cells in the recovery phase. In a pilot clinical trial, Ridolfi et al161 showed that the administration of low doses of temozolomide to melanoma patients before DC vaccination specifically reduced the CD41CD251Foxp31 Tregs. In turn, cyclophosphamide was shown to enhance antitumor immunity through
mechanisms that rely on Treg elimination and resetting DC homeostasis.162 The combination of DC vaccines with agents that preferentially depletes Tregs by directly targeting their abundantly expressed IL-2 receptor a chain (CD25) has also been tested. The agents most commonly used for this purpose are the anti-CD25 mAb basiliximab (NCT00626483) and daclizumab (NCT00847106) or denileukin diftitox (Ontak), a re- Q32 combinant IL-2 and diphtheria toxin fusion protein (NCT00703105, NCT00056134, and NCT00128622). Although some studies indicate that these combination therapies significantly increase DC vaccine efficacy as a result of increased antitumor-specific CTLs,163,164 others demonstrate that they induce tolerogenic DCs and promote the survival of resting Tregs.165 These paradoxical effects are partially because of CD25 expression not restricted to Tregs. As CD25 is also ex- Q33 pressed on effector T cells and activated NK cells, its blockade may compromise their antitumor activity. Another strategy for enhancing antitumor immunity consists of the combination of DC vaccines with immune checkpoint inhibitors. This approach is being tested in clinical experiments targeting the immune checkpoint receptors PD-1 (NCT01441765, NCT010 67287, NCT01096602, and NCT01753089) and CT LA-4 (NCT00090896 and NCT01753089) or the tryptophan degradation enzyme IDO (NCT01042535). Preliminary data obtained in patients with metastatic melanoma indicate that DC vaccines in combination with the anti–CTLA-4 mAb tremelimumab could be more effective than the same interventions in monotherapy (NCT00090896).166 Recently, combinations with
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Fig 5. Evolution of antitumor DC-based immunotherapies. First approaches were based on the administration of ex vivo manipulated DCs in monotherapy regimen, relying only on the capacity of DCs to generate antitumor effector cells (Th1, CTLs, and NK cells). Currently, DC vaccines are frequently combined to therapies aiming to reduce tumor burden such as chemotherapy, radiotherapy, anti-VEGF mAbs, and to therapies seeking to overcome tumor-associated immunosuppression. The later include broad lymphodepleting agents (temozolomide and cyclophosphamide); preferential regulatory T cells depleting agents (anti-CD25 mAbs and denileukin diftitox); blockers of immunosuppressive molecules (IDO inhibitors, anti–CTLA-4 and anti–PD-1 mAbs). Promising approaches to be explored in following years are based on in vivo delivery of antigens to DCs and on the development of implantable devices that will recruit, load, and mature DCs. CTLs, cytotoxic T cells; DCs, dendritic cells; IDO, indoleamine-2,3-dioxygenase; iPSCs, induced pluripotent stem cells; mAb, monoclonal antibody; MoDCs, monocyte-derived dendritic cells; NK, natural killer; PBMCs, peripheral blood mononuclear cells; PD-1, programmed cell death 1; PLGA, poly(lactic-co-glycolic acid); TAAs, tumor-associated antigens; VEGF, vascular endothelial growth factor.
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new anticancer drugs, such as the anti-VEGF mAb bevacizumab (Avastin) (NCT00683241, NCT00913913, and NCT02010606) and the tyrosine kinase inhibitors dasatinib and sunitinib (NCT01876212 and NCT00 678119), were also addressed. Moreover, by exploiting possible synergistic effects, DC-based vaccines have increasingly been used along with conventional radiotherapy and chemotherapy (NCT00639639, NCT01 973322, and NCT00617409). The positive effects of these therapeutic regimens are at least in part because of increased tumor cell apoptosis that fuels the DCs with relevant tumor antigens. From these 2 decades of clinical trials, it became evident that the classical objective responses, such as tumor shrinkage or tumor marker levels, are not adequate endpoints to evaluate the effectiveness of
DC-based antitumor immunotherapies. A systematic review conducted by Anguille et al4 on clinical trials evaluating DC vaccination in melanoma, prostate cancer, renal cell carcinoma, and glioma demonstrated that the clinical benefits in terms of the objective responses are real, but small. Objective responses were only observed in 8.5% of melanoma patients, 7.1% of prostate cancer patients, 11.5% of renal cell carcinoma patients, and 15.6% of glioma patients. In the same study, the authors found that although 2 trials on melanoma did not show survival benefits, all the other 38 studies demonstrated an increase in the median OS, ranging from 20% to 344%.4 These results reinforce the notion that survival is probably the most accurate endpoint to evaluate the therapeutic benefits of antitumor DC-based vaccines. However, clinical trials that
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monitor OS may be excessively long and costly.131 To surpass this limitation, alternative surrogate endpoints have been proposed. A growing number of studies are adopting immune response criteria, in which the effectiveness of the vaccination is evaluated by the expansion of antigen-specific effector T cells and NK cells.167 Finally, the selection of patients is also a relevant aspect to consider when designing an antitumor DC vaccination clinical trial. Optimally, patients should present a low tumor burden and be at an early stage of the disease, thus increasing the likelihood of developing effective antitumor immunity. CONCLUSIONS
Despite the variable rate of success in inducing clear beneficial outcomes, the first generation of antitumor DC-based vaccines has proven to be safe. Meanwhile, DC immunotherapies have substantially evolved because of our expanding knowledge of DC and tumor biology (Fig 5). The use of specific DC subsets that are tailored to a particular phenotype, the definition of adequate clinical endpoints and, particularly, the combination with other antitumor therapies have paved the way to the development of next-generation DC immunotherapies. Therefore, it is expected that these strategies may reveal the full therapeutic potential of DC-based vaccines for the treatment of malignant diseases in the future. UNCITED REFERENCE
19. ACKNOWLEDGMENTS
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Conflict of Interests: All the authors have read the journal’s policy on the disclosure of potential conflicts of interest and have none to declare. The authors thank FCT/MEC for the financial support to the QOPNA Research Unit (FCT UID/QUI/00062/ 2013), the national funds, and where applicable the FEDER within the PT2020 Partnership Agreement, and also the Portuguese RNEM Network. CNC is funded by FEDER funds through the Operational Program Competitiveness Factors-COMPETE, and the national funds are funded by the FCT-Foundation for Science and Technology under strategic project UID/ NEU/04539/2013. All authors have read the journal’s authorship agreement. Supplementary Data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.trsl.2015.07.008.
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Antitumor dendritic cell–based vaccines: lessons from 20 years of clinical trials and future perspectives ~ CONSTANTINO, CELIA ~ JOAO GOMES, AMILCAR FALCAO, MARIA T. CRUZ, and BRUNO M. NEVES COIMBRA AND AVEIRO, PORTUGAL
Dendritic cells (DCs) are versatile elements of the immune system and are best known for their unparalleled ability to initiate and modulate adaptive immune responses. During the past few decades, DCs have been the subject of numerous studies seeking new immunotherapeutic strategies against cancer. Despite the initial enthusiasm, disappointing results from early studies raised some doubts regarding the true clinical value of these approaches. However, our expanding knowledge of DC immunobiology and the definition of the optimal characteristics for antitumor immune responses have allowed a more rational development of DC-based immunotherapies in recent years. Here, after a brief overview of DC immunobiology, we sought to systematize the knowledge provided by 20 years of clinical trials, with a special emphasis on the diversity of approaches used to manipulate DCs and their consequent impact on vaccine effectiveness. We also address how new therapeutic concepts, namely the combination of DC vaccines with other anticancer therapies, are being implemented and are leveraging clinical outcomes. Finally, optimization strategies, new insights, and future perspectives on the field are also highlighted. (Translational Research 2015;-:1–22) Abbreviations: APCs ¼ antigen-presenting cells; BMP4 ¼ bone morphogenetic protein 4; cDCs ¼ classical DCs; CDPs ¼ common DC precursors; CLPs ¼ common lymphoid precursors; CLRs ¼ C-type lectin receptors; CMPs ¼ common myeloid precursors; CTLs ¼ cytotoxic T cells; CTLA-4 ¼ cytotoxic T-lymphocyte–associated protein 4; DC ¼ dendritic cell; Dex ¼ dendritic cell–derived exosomes; GM-CSF ¼ granulocyte-macrophage colony–stimulating factor; HLA ¼ human leukocyte antigen; HSC ¼ hematopoietic stem cells; i.d. ¼ intradermal; IDO ¼ indoleamine2,3-dioxygenase; IL ¼ interleukin; IFN ¼ interferon; i.n. ¼ intranodal; iPSCs ¼ induced pluripotent stem cells; i.t. ¼ intratumoral; i.v. ¼ intravenous; LC ¼ Langerhans cells; mAb ¼ monoclonal antibody; M-CSF ¼ macrophage colony–stimulating factor; MDPs ¼ macrophage and DC precursors; MDSCs ¼ myeloid-derived suppressor cells; MHC ¼ major histocompatibility complex; MoDCs ¼ monocyte-derived DCs; NK ¼ natural killer; PAMPs ¼ pathogen-associated molecular
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From the Faculty of Pharmacy and Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Faculty of Medicine, Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI) and Center of Investigation in Environment, Genetics and Oncobiology (CIMAGO), University of Coimbra, Coimbra, Portugal; CNC, Institute for Biomedical Imaging and Life Sciences, University of Coimbra, Coimbra, Portugal; Department of Chemistry and QOPNA, Mass Spectrometry Centre, University of Aveiro, Aveiro, Portugal.
Submitted for publication May 19, 2015; revision submitted July 25, 2015; accepted for publication July 28, 2015. Reprint requests: Bruno M. Neves, Faculty of Pharmacy and Centre for Neuroscience and Cell Biology, University of Coimbra, 3000548 Coimbra, Portugal; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.07.008
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patterns; PAP ¼ prostatic acid phosphatase; PBMCs ¼ peripheral blood mononuclear cells; PD-1 ¼ programmed cell death 1; pDCs ¼ plasmacytoid DCs; PGE2 ¼ prostaglandin E2; PolyI:C ¼ polyinosinic:polycytidylic acid; PKR ¼ protein kinase RNA-activated receptors; RECIST ¼ response evaluation criteria in solid tumors; s.c. ¼ subcutaneous; SCF ¼ stem cell factor; TAAs ¼ tumor-associated antigens; TCR ¼ T-lymphocyte receptor; TGF ¼ transforming growth factor; TLR ¼ toll-like receptor; TRAIL ¼ TNF-related apoptosis-inducing ligand; Tregs ¼ regulatory T cells; WHO ¼ World Health Organization
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INTRODUCTION
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Dendritic cells (DCs), initially described by Steinman and Cohn in a series of pioneer studies,1 play a critical role at the interface between the innate and adaptive arms of the immune system. These cells induce primary immune responses, potentiate the effector functions of previously primed T lymphocytes, and orchestrate/ modulate the communication between other innate and adaptive immune cells.2 Although they represent a very small population of leukocytes, DCs are professional antigen-presenting cells (APCs) that are highly efficient in generating robust immune responses and maintaining tolerance to self or harmless foreign antigens. This functional plasticity renders DCs a very attractive tool for the design of immunotherapeutic approaches, namely to boost the immune system during cancer treatment. Since the first pilot study using ex vivo MAGE-1–pulsed DCs for the treatment of melanoma in 1995,3 more than 300 clinical trials on antitumor DC-based treatments have been completed or are currently ongoing (Fig 1, A). Ongoing or previously completed randomized phase 3 clinical trials have been performed across a wide range of malignancies, including melanoma, prostate cancer, malignant glioma, renal cancer, and lymphoma (Fig 1, B). The great expectations created by the early experimental results were not always followed by objective clinical responses, and some skepticism ensued. In fact, according to the World Health Organization criteria or the response evaluation criteria in solid tumors, antitumor DC-based vaccines rarely exceed 15% of the objective responses.4 However, the paradigm of antitumor activity assessment is changing, and overall survival (OS) is now viewed as one of the most relevant outcomes to measure therapeutic benefits.5 In 2010, this notion was reinforced by the Food and Drug Administration (FDA) approval of the first DC-based vaccine, sipuleucel-T (Dendreon, Washington), as a treatment for metastatic hormone-resistant prostate cancer. Despite the fact that only 5% of patients exhibited tumor regression in the phase 3 clinical trial, the vaccine was shown to significantly increase the OS by 22.5%.6 Meanwhile, the FDA and the European Medicines
Agency (EMA) granted a special regulatory framework, such as orphan drug designation, to several other antitumor DC-based vaccines. The ICT-107 vaccine (ImmunoCellular Therapeutics, California) for the Q6 treatment of glioblastoma multiforme and the AV0113 vaccine for malignant glioma from the Austrian biotech company Activartis received the orphan drug designation by the FDA and EMA. In 2012, the EMA also granted orphan drug designation to DCP-001, a vaccine for acute myeloid leukemia produced by the Dutch company DCPrime BV. Finally, antitumor DC-based Q7 vaccines have been tested in several countries as Hospital Exemption therapies, a special regime defined by the regulation on advanced therapy medicinal products. An example of a vaccine within this framework is the DCVax-L from Northwest Biotherapeutics (Bethesda, Maryland) that was recently approved by the German Paul Ehrlich Institute for treatment of glioma. In the present work, the current status of DC immunobiology is briefly reviewed and the characteristics of antitumor DC-based vaccines that have been tested in more than 300 clinical trials for more than the last 2 decades are then summarized. We specially address the practical aspects of DC vaccine production and the influence of the different procedures on clinical effectiveness. Finally, new therapeutic concepts, optimization strategies, and future perspectives on the area are also highlighted. DC IMMUNOBIOLOGY Origin and classification. DCs are a heterogeneous family of specialized APCs that ultimately share the same hematopoietic stem cell precursor. In bone marrow, hematopoietic stem cells give rise to common myeloid precursors and common lymphoid precursors, which then originate intermediate progenitors, such as macrophage and DC precursors. Macrophage and DC precursors further differentiate into common DC precursors that are restricted to the generation of the 2 known DC populations: classical DCs (cDCs), and plasmacytoid DCs (pDCs) (reviewed in Refs 7 and 8). It is now accepted that, independent of the precursor lineage, the differentiation and expansion of specific DC subsets is strongly regulated by different
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Fig 1. Twenty years of clinical trials using antitumor DC-based immunotherapies. (A) Clinical trials reporting antitumor DC-based vaccines registered at ClinicalTrial.gov until March 2015. (B) Frequencies distribution of clinical trials among the different malignancies and their actual status. In ‘‘Other’’ category are included bladder cancer, mesothelioma, urothelial carcinoma, and cervical cancer. DC, dendritic cell.
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hematopoietic cytokines such as Fms-like tyrosine kinase cytokine-3 ligand (Flt3L), macrophage colony– stimulating factor, and granulocyte-macrophage colony–stimulating factor (GM-CSF).8 On the basis of this knowledge, the ‘‘graded commitment’’ model was proposed.9 This model states that the different precursors are not restricted to give rise to one particular type of DC. Rather, there is a gradation of probability ranging from precursors that can give rise to all DC subtypes to those that can only produce the classical or plasmacytoid subsets. The classification of the different human DC subsets has been the subject of intense debate and constant evolution. DCs were for long classified on the basis of phenotypical and functional characteristics. However, given that these characteristics are often overlapping an unequivocal classification has been challenging. Recently, a new classification system based primarily on DC ontogeny and secondarily on their location, function, and phenotype-gained relevance.10 DCs found in lymphoid and nonlymphoid tissues are therefore classified into 2 main groups, cDCs and pDCs. Human skin hosts 3 major subtypes of cDCs differentiated by their expression of CD1a, CD14, and CD141: CD41/CD1ahi/CD142/DC-SIGN2/CD20611/CD162, CD41/CD1alow/CD141/DC-SIGN1/CD2061/CD162, and CD1411/XCR11 DCs.8,11 CD1411/XCR11 DCs have been shown to be extremely efficient in antigen cross-presentation and to produce significant amounts of tumor necrosis factor a (TNF-a), CXCL10, and interleukin 12 (IL-12)p70 on stimulation.12-14 These cells effectively interact with natural killer (NK) cells and CD81 T cells, an event facilitated by the
expression of the XCR1 chemokine receptor. The human epidermis is also populated by other specialized APCs termed Langerhans cells (LCs). LCs are characterized by low CD11c and high CD1a and CD2017 expressions, which distinguish them from cDCs subsets. In addition, unlike any other DC population, LCs derive from embryonic precursors and have the capacity to self-renew locally.15 LCs are capable to cross-present antigens and to evoke tolerogenic or immunogenic responses depending on the microenvironment and type of maturation stimulus.11 In blood and lymphoid and nonlymphoid tissues, 2 cDC populations have been described according to their expression of nonoverlapping markers CD141 and CD1c. CD1411 DCs are primarily present in the blood and lymph nodes, although they can also be found in the lungs and liver. CD1c1 DCs are the predominant cDCs in the blood, and are also found in lymph nodes, spleen, and mucosa. These cells express toll-like receptors (TLRs) 1–10 and some C-type lectin receptors, such as DC-SIGN, DEC-205, DCIR, mannose receptor, Cleac9A, and Dectin 1 and 2.16,17 CD1c1 DC Q10 subpopulation produces IL-8, IL-10, TNF-a, and IL23 on activation, and their major role appears to be related to the modulation of mucosal T-cell responses and immunity toward extracellular pathogens.18 Finally, besides the 2 cDCs subsets, blood and lymphoid tissues also host pDCs, cells phenotypically characterized as major histocompatibility complex (MHC)-II1/ CD11c2/CD1231/CD3031/CD3041. pDCs have an Q11 enormous functional plasticity and are able to polarize T cells into Th1, Th2, regulatory T cells (Tregs), and cytotoxic T cells (CTLs). These cells are primarily
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implicated in antiviral responses by producing large amounts of type 1 interferon (IFN-a/b) and by efficiently cross-presenting viral antigens to the CD81 T lymphocytes.20 Antigen recognition, processing, and presentation. DCs sample their microenvironment and capture antigens by receptor-mediated endocytosis, phagocytosis, and macropinocytosis. The antigen-processing mechanisms depend on the origin (endogenous or exogenous) and molecular nature of the antigen (protein or lipid).21 Therefore, 3 processing and presentation mechanisms have been characterized: (1) the exogenous (or endosomal) pathway, where antigenic peptides are generated and coupled to MHC-II molecules in phagolysosomes and then transported to the cell surface and presented to CD41 T cells; (2) the endogenous (or proteasome) pathway, where the intracellular proteins are degraded by the proteasome, and the resulting peptides are coupled to MHC-I molecules in the Golgi and then transported to plasma membrane and presented to CD81 T cells; and (3) mechanisms where lipid antigens are coupled to CD1 family molecules and presented to CD81 T cells, g/d T cells, or NK T cells.22 Exogenous antigens can also be presented to CD81 T cells via MHC-I complexes, a process termed crosspresentation. In cross-presentation, internalized proteins are retrotranslocated into the cytosol and degraded by the proteasome. The resulting antigenic peptides migrate to the ER, where they are coupled to MHC-I molecules and subsequently presented to the CD81 T lymphocytes, inducing either immunogenic responses (cross-priming) or tolerance (cross-tolerance).23 Cross-presentation is, therefore, critical for the priming and activation of CTLs against viruses, tumors, and intracellular bacteria. DC maturation. In the classical view, on contact with a ‘‘danger signal,’’ conventional immature DCs undergo a complex process of morphologic, phenotypic, and functional modifications referred to as maturation. These modifications allow the egress of DCs from the peripheral tissues to the marginal zones in the draining lymph nodes, where they present antigens to naive T lymphocytes. Several stimuli have been shown to trigger DCs maturation, including small reactive chemicals, proinflammatory cytokines, such as IL-1b, IL-6, IFN-g, and TNF-a, and pathogen-associated molecular patterns, such as lipopolysaccharides, bacterial DNA, and double-stranded RNA.24 One hallmark of DC maturation is the upregulation of costimulatory molecules, such as CD40, CD54, CD80, CD83, and CD86, and the shift in the chemokine receptor profile. The expression of costimulatory molecules is of great importance for the adequate stimulation of
T cells during antigen presentation, and the modification of chemokine receptors results in the acquisition of migration.24 Although immature DCs express the CCR1, CCR2, CCR5, CCR6, CXCR1, and CXCR2 receptors, mature DCs upregulate CXCR4 and CCR7 and gain responsiveness to the lymphoid chemokines CCL19 and CCL20.25 The profile of cytokines and che- Q12 mokines produced by the DCs also undergoes profound alterations during maturation, being fundamentally dependent on the DC subset and on the stimulus that triggers the maturation process. The expression of the cytokines TNF-a, IL-10, IL-1a/b, IL-12p70, IFN-g, IL-8, IL-6, and IL-23 is normally increased in mature DCs.26 After contact with the maturation stimulus, the DCs transiently produce chemokines, such as CCL2, CCL3, CCL4, CCL5, CCL8, and CXCL8, which are important for the recruitment of monocytes and neutrophils to the site of infection/inflammation. Later, the Q13 production of the lymphoid chemokines, such as CCL17, CCL18, CCL19, CCL22, and CXCL10 increases, promoting the recruitment of T and B lymphocytes and facilitating the interaction of DCs with these cells.27 DC–T cell interactions. During antigen presentation, DCs provide 3 signals that drive the activation and polarization of T cells into their effector and regulatory populations.28 Signal 1 is provided by the interaction of the T-lymphocyte receptor with the MHC-I or MHC-II– antigen complexes presented by the DCs. In the absence of costimulation, signal 1 is commonly associated with the inactivation of naive T lymphocyte by anergy or deletion, promoting tolerogenic responses. The second signal, costimulation, results from the interaction of the costimulatory molecules expressed by the DCs with the respective ligands on the surface of T cells. Together with signal 1, costimulation promotes T-cell survival and proliferation and stabilizes cytokine production. The increased expression of costimulatory molecules during DC maturation has long been associated with the transition from a tolerogenic to an immunogenic state.29 However, numerous data show that costimulatory molecules associated with immunogenic responses may also be involved in the induction of tolerance. In fact, the binding of the costimulatory molecules CD80 and CD86 to cytotoxic T-lymphocyte antigen 4 (CTLA-4) Q14 acts as a negative regulator of T-cell activation. Additionally, the interaction of CD80 and CD86 with CTLA-4 and CD28 has also been shown to be essential for Treg development, homeostasis, and suppressor activity.30 Similarly, the interaction between 4-1BBL and OX40L expressed on DCs with their receptors, 41BB and OX40, respectively, in T cells can promote immunogenic31 or tolerogenic responses.32 Therefore, Q15 signal 2 is the result of a complex balance between
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positive and negative costimulation during DC-T cell cross talk. DCs secrete soluble factors such as cytokines and chemokines (signal 3), which are crucial for the differentiation of CD81 T cells into CTLs33 and for the polarization of CD41 T cells into their different effectors (Th1, Th2, and Th17)34 or regulatory subpopulations (Treg, Tr1, and Th3).35 The presence of IL-12p70, IL-15, and IFN-a/b during the presentation of MHCI–antigen complexes to CD81 T cells enhances their expansion, promotes CTL differentiation, and regulates memory T-cell formation.36 Although IL-12p70 appears to be the critical ‘‘signal 3’’ for CD81 T-cell expansion, it was shown that it may negatively impact the development of both primary and secondary antigen-specific memory CD81 T cells.37 In the DC-mediated polarization of CD41 T cells, the Th1 phenotype is induced by IL-12p70, IL-18, and IL-27, whereas Th17 differentiation primarily depends on transforming growth factor b (TGF-b) and IL-6, although other cytokines, such as IL-1b, IL-21, and IL-23, may also be involved. Regarding Tregs polarization, it is now well established that it depends on the DC ontogeny and capacity to produce IL-2, IL-10, and TGF-b as well as on T-lymphocyte intrinsic factors and on the balance between positive and negative costimulatory signals.38 DESIRED PROPERTIES FOR ANTITUMOR IMMUNOTHERAPIES
Nearly 3 decades of experimental and clinical observations have allowed the scientific community to shed light on the optimal characteristics for antitumor immunotherapies. An important milestone was the identification of the key role played by CD81 T cells in immune responses to cancer.39,40 The induction of antigenspecific CD81 T cells and the subsequent generation of CTLs leads to the recognition of MHC-I–antigenic peptide complexes presented on the surface of tumor cells, triggering their destruction. Therefore, desirable antitumor vaccines should expand the numbers of circulating tumor-specific CTLs; improve their cytolytic abilities (increasing the production of granzymes and perforin); increase the CTLs’ avidity for MHC-I molecules on tumor cells and increase the expression of molecules, such as CXCR3 and CD103/CD49a, that favor the migration and persistence of CTLs into the tumor areas.41,42 It is also desirable that the elicited CD81 T cells express high levels of positive costimulatory molecules such as CD137 and low levels of the immune checkpoint proteins CTLA-4 and programmed cell death 1 (PD-1).43 Finally, antitumor vaccines are also expected to modulate the circulating effector and memory CD81 T cells.
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In addition to the CTL-driven responses, effective immunity against tumors is also strongly dependent on CD41 T cells and NK cells. CD41 T cells, particularly the Th1 subset, directly kill tumor cells,44 activate tumor infiltrating-macrophages and contribute to the differentiation and expansion of antigen-specific CTLs by producing cytokines such as IL-2 and IL-21.45,46 CD41 T cells are also required for the adequate formation of long-term memory CD81 T cells.47 The role of the NK cells in tumor immunosurveillance has gained substantial interest in recent years.48 In response to a balance between inhibitory/stimulatory signals from invariant receptors, NK cells directly kill tumor cells without prior immunization or MHC restriction. Q16 This is accomplished through at least 4 mechanisms: cytoplasmic granule release, effector molecule production, death receptor-induced apoptosis via the TNFrelated apoptosis-inducing ligand and Fas ligand, or antibody-dependent cellular cytotoxicity.49 In addition, NK cells indirectly contribute to tumor elimination by modulating the functions of other immune cells. Activated NK cells potentiate DC maturation and IL-12p70 production and favor immunogenic DC populations by killing immature DCs, whereas sparing fully activated DCs.50 Finally, the cell debris that result from the NK-induced destruction of tumor cells fuel the DCs with antigens, which enhance their cross-presentation to the CD81 T cells.51 However, some care must be taken with the elicitation of Th1 responses and the activation of NK cells in the context of cancer treatment. By producing IFN-g, these cells potently upregulate the expression of PD-1 ligands (PD-L1 and PD-L2) in tumors, which may contribute to tumor immune evasion.52 Other CD41 T-cell subsets, such as Th2 cells and Tregs, also counteract the CTLs antitumor activity. By secreting IL-4, Th2 cells limit the cytolytic activity of CTLs by decreasing the expression of granzymes and perforin,53 whereas Tregs negatively affect CTLs because of the production of IL-10 and TGF-b.54,55 In addition, Tregs can also impair CD81 T-cell expansion by competing with them for the cytokine IL-2.56 RATIONALE FOR ANTITUMOR DC-BASED VACCINES
DCs represent the perfect tool for immunotherapeutic interventions because of their unparalleled capacity for antigen presentation, their ability to modulate other immune players, and their functional plasticity.2 The effectiveness of DC vaccines is the subject of debate, mainly because the clinical outcomes addressed may not be the most adequate. However, there is no doubt that these approaches elicit strong tumor antigenspecific immune responses (CTLs and Th1 cells). A
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meta-analysis of antitumor DC-based vaccines for prostate cancer and renal cell carcinoma revealed that the interventions induced these immune responses in 77% and 61% of the patients, respectively.57 Moreover, in addition to boosting adaptive immunity, the DCs were shown to enhance the antitumor activity of NK cells by increasing their cytolytic abilities and IFN-g production.58 Following this seminal publication, a plethora of other studies revealed the crucial relevance of DC-NK cell cross talk for efficient tumor elimination (reviewed in Lion et al59). In fact, effective DC vaccine-mediated antitumor immunity is at least in part dependent on NK cell activity.51 This was elegantly demonstrated in murine models of melanoma and metastatic lung tumors, where the tumor eradication observed after DC vaccination was completely abrogated in animals that had been depleted of NK cells.60,61 Although only a limited number of DC-based cancer vaccination trials implemented NK cell motorization, the existing data indicate that nearly 50% of patients showed an increased frequency and/or an induction of NK cell activation.59 Therefore, it is now clear that the antitumor responses promoted by DC vaccination rely on their capacity to prime and activate CD81 T cells, to polarize CD41 T cells into Th1 populations, and to cross talk with NK cells.62-65 Another important argument favoring DC-based immunotherapies is their safety profile. Twenty years of observations in numerous phase I and II trials demonstrated that DC vaccines are generally well tolerated and induce minimal toxicity. The most common manifestations are local reactions at the injection sites, such as rash and pruritus, and occasionally systemic effects occur, including fever and malaise.4 Despite the initial concerns regarding the possibility of inducing autoimmunity, the DC-based therapies are rarely associated with severe immunotoxicity reactions. METHODOLOGIES FOR THE PRODUCTION OF DCBASED VACCINES
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The present review is focused on the diversity of approaches used to directly manipulate or target DCs in the context of cancer treatment, with a special emphasis on the impact of the different methodologies on vaccine effectiveness. The information systematized subsequently was collected from www.clinicaltrials.gov (until March 2015) and from key articles on the subject. Table I summarizes the different methodologies/approaches and the respective benefits and drawbacks. The complete list of clinical trials analyzed and their major characteristics can be found in the Supplementary material. Ex vivo DC manipulation. Ex vivo manipulation of DCs is by far the most explored strategy and is used in
approximately 97% of the clinical trials initiated to date (Fig 2, A). This approach requires obtaining DCs or DC precursors from patients, manipulating them (inducing maturation and loading antigens), and reinjecting them into the donor. From the time of the production of this type of DC-based vaccine according to good manufacturing procedures is a time- and costintensive procedure, individual aliquots are normally produced and cryopreserved at the beginning of the treatment and are released along the vaccination schedule. Cryopreservation, when performed under optimal conditions, does not significantly affect the viability, phenotype, or function of the DCs, and the thawed cells are suitable for clinical use.66 Currently, there is no standardized procedure for ex vivo manipulation, which results in a plethora of protocols that differ in the source of DCs, the maturation stimulus, the nature and procedure for antigen loading and, finally, the route of administration. DC source. The DCs used in tumor immunotherapies can be either autologous or allogenic. As shown in Fig 2, B, the use of autologous DCs has been by far the preferred approach. Although allogenic DCs are less frequently used, their usage is supported by experimental evidence demonstrating that allogeneic human leukocyte antigen (HLA) molecules represent potent immunogenic signals and boost antitumor immunity.67,68 Besides, it may be advantageous to use cells from normal donors because DCs and DC precursors from cancer-bearing patients are targeted by tumor immunosuppressive factors that render them functionally aberrant.69,70 The immunosuppressive factors that are usually overproduced by tumor cells include IL10, TGF-b, vascular endothelial growth factor (VEGF), IL-6, and prostaglandin E2 (PGE2), and dysfunctional DCs have been observed in patients with chronic lymphocytic leukemia, melanoma, ovarian, breast, renal, prostate, lung, and head and neck cancers.71-74 Allogenic DCs are commonly obtained by differentiation of peripheral blood mononuclear cells (PBMCs) of unrelated healthy donors. However, at least 2 clinical trials use DC-like cell lines (Fig 2, B). The use of the proprietary dendritic progenitor cell line DCone was tested in acute myeloid Q19 leukemia patients (NCT01373515), and the safety and tolerability of subcutaneous (s.c.) administration of an allogeneic pDC line was tested in patients with melanoma (NCT01863108). Given that circulating DCs represent less than 1% of PBMCs, the cells used in therapeutic cancer vaccines are typically differentiated from autologous leukapheresis-isolated CD141 monocytes or CD341 hematopoietic progenitors. Differentiation from monocytes involves the cell culture for 5–7 days in the
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Table I. Advantages and drawbacks of the different methodologies/approaches used for production of DCbased antitumor vaccines Advantages
Ex vivo DC manipulation Genetic source Autologous
- No risk of graft-vs-host disease
Allogenic
- Allogenic HLA molecules represent potent immunogenic signals67,68
DC source CD14+ monocytes
- Abundant source: CD14+ monocytes represent 10% of PBMCs
CD34+ hematopoietic progenitors Natural occurring DCs iPSC-derived DCs
Antigens and loading procedures Peptide pulsing
Protein pulsing
- CD34+-derived DCs stimulate more effective CTL responses than MoDCs75,76 - Low doses of plasmacytoid DCs effectively induce CD4+ and CD8+ T-cell responses77 - iPSCs may be continuously expanded in vitro153 - DCs can be tailored to a desired phenotype (eg, CD11c+CD141+XCR1+ DCs)154 - Peptides are synthesized and purified at low cost - Enables direct monitoring of T-cell responses - Short peptides are directly loaded into MHC molecules - Synthetic peptides allow for modifications - Full-length proteins may present more epitopes for immune recognition - Prolonged antigen presentation
Tumor lysates
- Encompasses multiple TAAs (even those not yet characterized)
Tumor apoptotic bodies
- Encompasses multiple TAAs (even those not yet characterized)
RNA transfection
- Tumor mRNA may be amplified as needed - Possibility to introduce mRNAs coding for immunostimulatory proteins
Viral transduction
- The vector may intrinsically activate DCs95 - Possibility to introduce genes coding for cytokines and costimulatory molecules94
DC-tumor cell hybrids
- Encompasses multiple TAAs - Antigen presentation is maintained for days
DC maturation protocols TNF-a + IL-1b + IL-6 + PGE2 TNF-a + IL-1b + IFN-a + IFN-g + polyinosinic: polycytidylic acid Routes of administration Intravenous Intradermal/subcutaneous
Drawbacks
- DCs from cancer-bearing patients are frequently dysfunctional69-74 - Survival of injected DCs may be shortened by T-cell–mediated rejection - CD14+-derived DCs (MoDCs) are not as efficient as other DC subsets in eliciting CTL responses - Limited number: represent 0.1% of PBMCs - Limited number: circulating DCs represent less than 1% of PBMCs - Laborious and expensive - Production according to GMP requirements is technically challenging - Limited number of known immunogenic TAAs - HLA restriction - Peptides with low affinity for MHC may be poorly immunogenic - Limited number of known immunogenic TAAs - Propensity of proteins to be targeted for MHC-II - Dependence on the availability of tumor cells - Immunologic responses more complex to monitor - Dependence on the availability of tumor cells - Immunologic responses more complex to monitor - Reagents and procedures to transfect DCs may affect their viability - Limited stability and short lifespan of mRNA - Immune responses to vector antigens may overwhelm those for tumor antigens95 - Viral vectors may perturb DC functions95 - Low expression of IL-12p70 and costimulatory molecules97
- PGE2 enhances CCR7 expression and DC migratory capacities106 - Efficiently induces IL-12p70 - aDC1s elicit potent CTL responses102,109-111
- PGE2 may reduce the capacity of DCs to produce IL-12p70107 - aDC1s may have reduced migratory capacities compared with standard matured DCs
- Induces antigen-specific humoral responses more efficiently than i.d. and i.n. routes117 - Elicit strong specific antitumor Th1 and CTL responses
- Injected DCs preferentially accumulate in the spleen and liver - Just 1%–5% of injected DCs reach the peripheral lymph nodes117,118 (Continued )
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Table I. (Continued ) Advantages
Intranodal Intratumoral In vivo DC targeting Cancer antigens fused to monoclonal antibodies targeting DC surface receptors
Drawbacks
- Substantially more DCs reach the T-cell areas in lymph nodes118,120 - May delay and reverse the tolerization of tumor-infiltrating effector T cells124
- Technically exigent - Great variability between patients - Technically exigent - Associated with high morbidity
- Bypasses the expensive and laborious ex vivo DC generation process - Allows targeting specific DC subsets128-130
- Limited to known TAAs - It requires the coadministration of adjuvants130 - Targeted receptor must be unambiguously expressed
Other strategies directly/indirectly involving DCs Nontargeted antigen-based - Easier to produce vaccines - Stable in many storage conditions - Induce CD4+ and CD8+ T-cell responses GM-CSF–secreting tumor cells - Potentiates TAAs presentation by endogenous DCs136 - Consistent induction of cellular and humoral antitumor immune responses137-139
Implantable DC-recruiting scaffolds
Dex
- Bypasses the expensive and laborious ex vivo DC generation process - The approach takes advantage of endogenous DCs - Preclinical studies show induction of specific and effective antitumor immunity141-143 - Dex are able to elicit CD4+ and CD8+ T-cell responses144-147 - Exosomes may carry additional mRNAs and miRNAs to enhance immune responses - Production under GMP conditions is well defined
- Limited to known TAAs - Immune responses may be transient and/ or of low magnitude - Prolonged GM-CSF production by tumor cells may cause immune tolerance140 - Dependence on the availability of tumor cells - Immunologic responses more complex to monitor - The design of biocompatible polymeric scaffolds incorporating functional proteins (chemokines) is technically challenging - Until present the results are only from preclinical studies - Existing clinical data indicate that T-cell responses elicited by Dex are limited149,150 - Requires large quantities of ex vivo generated DCs for exosome production
Abbreviations: CTL, cytotoxic T cell; DC, dendritic cell; Dex, dendritic cell–derived exosomes; GMP, good manufacturing procedures; GM-CSF, granulocyte-macrophage colony–stimulating factor; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; iPSC, induced pluripotent stem cell; MHC, major histocompatibility complex; MoDC, monocyte-derived dendritic cells; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell; PGE2, prostaglandin E2; TAA, tumor-associated antigen; TNF-a, tumor necrosis factor a.
presence of GM-CSF and IL-4, producing DCs with an immature phenotype that are commonly referred as monocyte-derived DCs (MoDCs). When CD341 hematopoietic precursors are used, the patients are frequently treated with GM-CSF before leukapheresis to mobilize precursors from the bone marrow. The harvested CD341 cells are then cultured with GM-CSF, TNF-a, Flt3L, TGF-b, and stem cell factor for 11– 12 days. This procedure leads to a heterogeneous mixture of APCs, namely MoDCs, LC-like cells, and other myeloid cells.75 Most clinical trials with a stated DC source use MoDCs (Fig 2, B). The preference for MoDCs instead of CD341-derived DCs is not related to their superior clinical efficacy, but rather to the limited number of CD341 precursors that can be isolated from apheresis products. In fact, CD341-derived DCs, and particularly their LC subset, were shown to stimulate in vitro more effective CTL responses than their counterpart MoDCs independent of the presence
of IL-12p70.75 Moreover, the efficacy of peptideloaded CD341-derived LCs and equivalent MoDCs was directly compared in stage III/IV melanoma patients (NCT00700167), and the results indicate that without exogenous IL-15 administration, the LCbased vaccines stimulate significantly more IFN-g–producing CTLs.76 Sipuleucel-T (Provange), the first FDA-approved cell-based antitumor vaccine, uses a different protocol to obtain DCs. Autologous PBMCs were incubated for 36–48 hours with a recombinant fusion protein consisting of human prostatic acid phosphatase and GM-CSF. GM-CSF targets the prostatic acid phosphatase antigen to APCs, namely DCs, promoting its uptake/processing and simultaneously inducing cellular maturation. The Q20 biologically active components of the vaccine are the CD541-matured APCs.6 Finally, among the clinical trials analyzed, only one uses ex vivo manipulated naturally occurring DCs (NCT01690377) (Fig 2, B). The
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Fig 2. Manipulation strategies and types of DCs used in antitumor vaccines. (A) Clinical trials with approaches directly exploring DCs: manipulation ex vivo followed by reinjection and in vivo DC targeting. (B) Frequencies distribution of clinical trials by DC genetic source (autologous or allogenic) and DC type. ‘‘Natural DCs’’ refer to primary cells directly isolated from the donor. ‘‘Not defined’’ indicates that the information regarding the parameter was not found in the clinical trial description or in the literature. DCs, dendritic cells; LCs, Langerhans cells; MoDCs, monocyte-derived dendritic cells.
authors77 of a study evaluated the efficacy of intranodal (i.n.) injection of tumor peptide–loaded pDCs in patients with metastatic melanoma. The results indicate that despite the limited number of pDCs administered, several patients mounted antivaccine CD41 and CD81 T-cell responses, demonstrating the feasibility of the approach. Selection of antigens and loading procedures. The choice of tumor antigens and the loading procedures are important parameters for DC-based vaccine production.78 A limited fraction (approximately 10%) of the tumor-associated antigens (TAAs) appear to be immunogenic, and among these, only a few are effectively associated with tumor rejection.79 TAAs are normally
unique mutated proteins, antigens derived from oncogenic viruses, or shared nonmutated self-antigens, such as tyrosinase, tyrosinase-related proteins, gp100, or MART-1.80 Q21 DCs have been pulsed with isolated/recombinant TAAs (full-length proteins or peptides), transfected with tumor messenger RNA (mRNAs), transduced Q22 with TAA-coding genes, and loaded with tumor cell lysates or apoptotic tumor cells (Fig 3, A). All these procedures were shown to elicit protective and therapeutic anticancer immune responses without inducing specific toxicity.81 Although pulsing DCs with short peptides lead to its direct loading onto MHC molecules at the cell surface, proteins and tumor lysates require
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Fig 3. Antigen loading procedures and routes of administration of ex vivo manipulated DCs. (A) Frequencies distribution of clinical trials by DC antigen loading strategies. (B) Administration routes for ex vivo manipulated DCs. In some cases, the same trial compares the efficiency of more than one administration route leading to count numbers (322) superior to the total number of respective trials (303). Not defined indicates that the information regarding the parameter was not found in the clinical trial description or in the literature. DCs, dendritic cells; i.d., intradermal; i.l., intralymphatical; i.n., intranodal; i.t., intratumoral; i.v., intravenous; s.c., subcutaneous.
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internalization, processing, and presentation. However, the use of defined peptides is limited to the low number of characterized TAAs and by the fact that some of them are HLA-A1 or HLA-A24 restricted, requiring therefore that the patient’s haplotype must be known and adequate. Regarding the loading of DCs with allogenic/autologous tumor cell lysates or apoptotic bodies, it is a strategy for long known to elicit antitumor immunity.82 Its major advantage relies on the use of whole tumor proteome, encompassing that way multiple TAAs. In terms of effectiveness, several studies indicate that DCs loaded with apoptotic tumor cells elicit stronger immune responses than lysate-loaded or RNApulsed DCs.83,84 The major drawback of using tumor lysates or apoptotic bodies as a source of antigens instead of defined peptides is the dependence on the availability of tumor cells. Moreover, immunologic responses are more complex to monitor, which make
the correlation between the interventions and the clinical outcomes difficult. Another common strategy is to transfect DCs with mRNA extracted from tumor cells or with in vitro synthesized mRNAs that encode particular TAAs. The ability of these loaded DCs to elicit strong antitumor CD41 and CD81 T-cell responses is well documented85-87 and is one of the most commonly used approaches in clinical trials (Fig 3, A). When compared with peptide loading, transfection with whole tumor mRNA avoids the limitation imposed by the use of known TAAs and matched HLA phenotypes. In addition, siRNAs targeting the immunoproteasome components (NCT00672542) or mRNA coding for maturation agents and costimulatory molecules (NCT01066390) could be introduced, enhancing the Q24 generation of TAA-derived peptides and eliminating the need for an additional DC maturation step. The latter
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approach was evaluated in melanoma patients using a vaccine composed of DCs transfected with mRNA encoding the immunostimulatory proteins CD40L, CD70, and constitutively active TLR4 in combination with mRNAs’ encoding tyrosinase, MAGE-A3, MAGE-C2, and gp100 (NCT01066390). The results demonstrated that most patients developed functional TAA-specific CD81 and CD41 T-cell responses, which were detected both at the skin and systemically.88,89 Among the strategies to transfect DCs, electroporation has shown to be the most efficient method, temporarily increasing cell membrane permeability, which facilitates the entry of mRNA without the need for additional reagents.90 DCs have also frequently been genetically engineered to stably express TAAs, such as mucin 1, p53, tyrosinase, melan-A, and gp100.91-93 The use of bacteria or viral vectors to deliver the DNA to DCs is safe, and the possibility of introducing genes encoding cytokines or costimulatory molecules is very appealing.94 Furthermore, the vector may intrinsically activate DCs by triggering endosomal or cytoplasmic sensors, such as TLRs, RIG, and protein kinase RNA-activated receptors, bypassing the need of a separate maturation step.95 Finally, the ex vivo fusion of DCs with tumor cells represents another approach for antigen loading. These cell hybrids, known as ‘‘dendritomes,’’ were shown to induce antitumor immune responses in vivo96-98 and have been tested in multiple clinical trials for more than the past few decades (Fig 3, A). Notably, dendritomes were found to be less effective in eliciting CTL responses than DCs loaded with apoptotic tumor cells,99 an observation that was attributed to the reduced expression of costimulatory molecules and IL-12p70 by the cell hybrids.97 DC maturation protocols. The DC maturation status and cytokine profile are parameters of great importance for the effectiveness of DC-based immunotherapies. Numerous clinical trials have shown a clear superiority of mature DCs over their immature counterparts.57,100 Immature DCs have a reduced ability to migrate from the injection sites to the lymph nodes,100 and can cause the inhibition of CTL functions by inducing IL-10– producing antigen-specific Tregs.101 In addition to the maturation status, the DC cytokine and chemokine profiles, particularly the expression of IL-12p70 and CCR7, are of great relevance for vaccine effectiveness. The cytokine IL-12p70 plays a central role in CD81 T-cell expansion and CTL differentiation.36 Clinically, the higher levels of IL-12p70 produced by DCs used in vaccines for glioma and melanoma patients were found to positively correlate with a more favorable clinical outcome.102,103 The chemokine receptor CCR7 governs the migration of DCs from the
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peripheral tissues to the draining lymph nodes under either homeostatic or inflammatory conditions. In the context of DC-based immunotherapies, the migration of DCs from the injection sites to the lymph nodes and the subsequent interaction with the T cells is a key feature for effective immunization. A growing body of evidence demonstrates that DCs expressing high levels of CCR7 elicit a more effective antigenspecific immune response in vivo, which lowers the required DC dosage.104,105 The appropriate DC maturation stimulus remains a matter of debate. On the basis of the literature, several maturation protocols have been tested, which consist of individual stimuli or cocktails that combine proinflammatory cytokines, CD40L, and TLR agonists. The maturation cocktail that was used most commonly across the clinical trials analyzed includes TNF-a, IL-1b, IL-6, and PGE2. The use of PGE2 is somehow paradoxical because although it enhances the DC migratory capacities by inducing CCR7 expression,106 it reduces IL-12p70 production.107 Protocols that combine exposure to the TLR3 ligand polyinosinic:polycytidylic acid and PGE2 were proposed to circumvent this problem and explore the exquisite capacity of TLR agonists to induce IL-12p70 expression.108 Another common DC maturation cocktail includes TNF-a, IL-1b, IFN-a, IFN-g and polyinosinic:polycytidylic acid. The resulting DCs, termed aDC1s, efficiently secrete IL-12p70 and were shown to elicit more potent CTL responses than the standard maturated DCs.102,109-111 From our analysis, aDC1-based vaccines were tested in at least 8 clinical trials (Fig 2, B). To increase their immunogenicity, DC vaccines are frequently administered along with adjuvants. The most frequently used adjuvants are GM-CSF, IL-2, IFN-a, and TLR agonists. GM-CSF strongly induces the proliferation of DC precursors and has a stronger chemotactic effect over DCs, promoting their recruitment and maturation. Interferon alpha enhances DC antigen cross-presentation and, therefore, promotes more effective CTL responses.112 Strong preclinical evidence has demonstrated that the cytokine IL-2 can improve DC vaccine efficacy.113 However, subsequent clinical trials have not shown superior antitumor immune responses in regimens combining DCs with IL-2.114 In fact, because of its stimulatory abilities toward Tregs and myeloid-derived suppressor cells (MDSCs), IL-2 can even negatively affect DC-based immunotherapies. In addition to their inclusion in maturation cocktails, TLR agonists are increasingly being used as adjuvants in DC vaccines. In the clinical trials analyzed, the most common are poly-ICLC, rintatolimod (TLR3 ligands), imiquimod (TLR7 ligand), resiquimod (TLR7/8 ligand), and DUK-CPG-001 (TLR9
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ligand). Another important question in the maturation protocols is the duration of DC exposure to the stimulus. The contact of DCs with maturation signals (danger signals) in vivo is transient because the cells rapidly leave the site of contact (normally the peripheral tissues) and reach the lymph nodes in 2–4 hours.115 However, most protocols in the literature indicate 24–48 hours of exposure. These time periods may be too long, exhausting the ability of the DCs to produce IL-12p70 and, therefore, decreasing their ability to elicit protective Th1 and CTL responses.116 Overall, we can conclude that the diversity of the maturation procedures used in the production of DCbased vaccines hampers the assessment of the real effects of the maturation stimuli because it is not the only parameter that differs among studies. It would be of great interest to design clinical trials that compare the effectiveness of DCs that have been matured with different cocktails to pursue a certain level of standardization. Routes of administration and cell doses. The route of administration, frequency of injections, and the number of cells injected are relevant aspects to consider in DC-based vaccinations, given that the efficacy of the intervention will strongly depend on the migration of sufficient numbers of DCs to T-cell areas in secondary lymphoid tissues. For more than the last 2 decades of clinical trials, DCs have been administered via different routes: intravenous (i.v.), intradermal (i.d.), s.c., i.n., and directly into the tumor (Fig 3, B). All these routes of administration elicit specific antitumor immune responses, but with different characteristics and efficiencies.116 The i.v. injected DCs are transiently detected in the lungs and then preferentially accumulate in the liver and spleen, with little distribution to the lymph nodes. In a clinical trial with prostate cancer patients, i.v. administration was shown to induce antigen-specific humoral responses more efficiently than the i.d. or i.n. routes.117 Regarding i.d. and s.c. routes, most DCs remain at the injection sites and are cleared by infiltrating macrophages.118 However, the 1%–5% of cells that actually reach the peripheral lymph nodes were shown to be sufficient to induce specific antitumor T cells.117,118 Furthermore, several lines of evidence suggest that the DCs retained at the injection site may act as adjuvants. They recruit, activate, and transfer antigens to resident DCs, namely dermal DCs and LCs, that then more effectively activate CD81 T cells.119 For these reasons and the fact that they are technically less exigent and less expensive, the s.c. and i.d. routes are used most commonly in trials for solid tumor treatments (Fig 3, B). Administration via i.n. route is performed under ultrasound guidance of the needle by an experienced radiologist. The
efficacy of the procedure heavily depends on the correct delivery and can, therefore, present great variability between patients. Surprisingly, although substantially more DCs migrate to the T-cell areas in the lymph nodes on correct i.n. vaccination, the antitumor T-cell responses are comparable or inferior to i.d. administration.118,120 Another route used in a few phase I/II clinical trials is intratumoral administration (Fig 3, B). The approach is technically exigent and associated with a high rate of intervention-associated morbidity. It has been tested in the treatment of several solid tumors, such as melanoma, breast, liver, and pancreatic cancers.121-123 An overall evaluation of the published studies demonstrates that the procedure is safe and could elicit antitumor immunity in at least some of the patients. Recently, using autochthonous prostate cancer animal models, Higham et al124 showed that one advantage of intratumor DC administration relies on the capacity of these cells to delay and reverse the tolerization of tumor-infiltrating effector T cells. As shown in Fig 3, B, currently, there is an increased tendency to administer DC vaccines via multiple routes, such as i.d. 1 s.c. or i.d. 1 i.v., with the aim of inducing more robust and broader immune responses. The minimal number of DCs required to induce an efficient immune response in humans has not yet been established. This is reflected in clinical trials by a plethora of DC doses and administration regimens. Doses ranging from as low as 0.3 3 106 naturally occurring pDCs injected via i.n. route every 2 weeks (NCT01690377) to 3 doses of 200 3 106 MoDCs administered via s.c. route (NCT00704938) can be found among analyzed trials. Verdijk et al determined that 0.5 3 106 DCs reaching the T-cell areas in the lymph nodes could be sufficient to induce de novo immune responses. Considering that a maximum of 4% of DCs administered via i.d/s.c. route migrate and only 80% remain viable after injection, the authors estimated that a minimal dose would consist of 15 3 106 cells.118 Experiments performed in lung cancer and neuroblastoma animal models showed that efficacy of DC vaccines is positively correlated with the dose used. Moreover, in one of the early clinical trials testing sipuleucel-T, the median time to disease progression was found to be 31.7 weeks for patients who received more than 100 3 106 cells per infusion compared with 12.1 weeks for patients who received fewer cells.125 However, the correlation between the number of administered DCs and vaccine effectiveness may vary with the selected administration route. Surprisingly, reducing the number of DCs injected via i.d. route was recently shown to improve their homing to the lymph nodes and, consequently, to potentially enhance vaccine efficacy.126,127 The authors suggested that
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multiple i.d. injections with small amounts of DCs would be a preferable strategy. In vivo DC targeting. Targeting antigens to DCs in vivo represents an enormous breakthrough for antitumor immunotherapies because it allows researchers to bypass the expensive and laborious ex vivo DC generation process.128 Antigens can be unambiguously targeted to different DC subsets, eliciting antigen-specific CD41 and CD81 T cell-mediated responses (reviewed in Caminschi et al129). The procedure involves the coupling of antigens to monoclonal antibodies (mAbs) that are specific for DC surface molecules, such as Fc receptors, CD40 and C-type lectin receptors, or the use lentiviral vectors with preferential tropism to DCs. Notably, the delivery of antigens via mAb induces tolerance in the absence of adjuvants, and requires the coadministration of DC maturation agents such as TLR3, TLR7/8, or CD40 agonists.130 On the basis of animal experiments, the delivery of antigens to DEC205, CD40, DC-SIGN, DCIR, mannose receptor, Cleac9A, and XCR1 showed promising results in inducing antitumor immunity (CD41 and CD81 T-cell responses, as well as humoral responses).129,131,132 The clinical application of the approach has been slow and has faced significant challenges. Several important factors must be considered in the development of human DC-targeting vaccines, including the biological functions of the targeted DCs, the pattern of expression and functions of the chosen receptors, and the adjuvants coadministered. Among the few in vivo DC-targeting therapies that are being tested in humans, DEC-205 is the most common targeted receptor (Fig 2, A). These vaccines consist of s.c. and i.d. administration of the cancer antigen NYESO-1 fused to a human anti–DEC-205 mAb in combination with the adjuvant poly-ICLC (NCT009 48961), decitabine (NCT01834248), the indoleamine2,3-dioxygenase (IDO) inhibitor INCB024360 (NCT0 2166905), or recombinant Flt3L (NCT02129075). The targeting of human chorionic gonadotropin beta protein to DCs via the mannose receptor is also being clinically evaluated for the treatment of breast, colorectal, pancreatic, bladder, and ovarian cancers. The fusion protein is injected via i.d. and s.c. routes in combination with adjuvants, such as GM-CSF and poly-ICLC (NCT0070 9462), or administered via i.v. route (NCT00648102). Using a different approach, a lentiviral vector encoding NY-ESO-1 and pseudotyped with Sindbis virus envelope proteins that are modified to target DC-SIGN on DCs (IDLV305) is under evaluation for the treatment of NY-ESO-1–positive solid tumors (NCT02122861). Although the clinical application of these nextgeneration DC-based vaccines is still in the early stages and, therefore, has several challenges that still have to
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be overcome, it represents an exciting and promising new field in antitumor immunotherapy and overall immune modulation. Other DC-based approaches. Apart from ex vivo manipulation and direct in vivo targeting, several other approaches exploit the immunogenic potential of DCs in cancer therapy. These include nontargeted antigenbased vaccines, GM-CSF–secreting tumor cell vaccines, implantable DC-recruiting/activating scaffolds, and DCderived exosomes (Dex). Q27 The nontargeted antigen-based vaccines are composed of peptides, proteins, or tumor nucleic acids. When injected, these antigens are captured and processed by the APCs, namely DCs, eliciting an antigen-specific immune response.131 Initial studies showed that this approach can induce CD41 and CD81 T-cell responses, although with low or no impact on the clinical course of the disease.133 The lack of clinical benefits in these cases was associated to the dominant polarization of CD41 T cells into IL-4– and IL-5–producing Th2 subsets and to the differentiation of tumor-specific Tregs. To overcome these immunosuppressive events and to potentiate antigen capture by the DCs, vaccines have begun to be administered in combination with GM-CSF, adjuvants, and low doses of lymphodepleting agents, such as cyclophosphamide.134,135 In phase II trials, vaccines composed of the MAGE-A3–recombinant protein and the AS15 adjuvant system were shown to cause clinical improvements in both metastatic melanoma (NCT00086866) and resected non-small cell lung cancer (NCT00290355). For the GM-CSF–secreting tumor cell vaccines, irradiated autologous tumor cells or allogenic tumor cell lines have been engineered to secrete GM-CSF and were then injected into patients. These engineered tumor cells strongly attract macrophages, granulocytes, T cells, and DCs, potentiating tumor antigen presentation.136 The clinical evaluation of this vaccination strategy revealed a consistent induction of cellular and humoral antitumor responses in melanoma,137 prostate cancer,138 and pancreatic cancer.139 As a major drawback, several animal models have shown that prolonged GM-CSF production by tumor cells may lead to disease progression as a result of immune tolerance caused by the recruitment of myeloid suppressor cells and the differentiation of tolerogenic DCs.140 A new approach for recruiting endogenous DCs to a source of tumor antigens recently garnered particular interest. Mooney et al141 showed that biocompatible polymers may be designed to incorporate and release a DC chemotactic agent, an adjuvant, and tumor antigens in a controlled manner. The authors tested the implantation of polylactide-co-glycolide scaffolds incorporating GM-CSF, various TLR agonists, and tumor lysates in mice in
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the context of preventive and therapeutic melanoma vaccination. The obtained results demonstrated that the mice receiving the scaffold mounted specific and protective antitumor immunity with an OS of 90% for the preventive vaccine and 33% long-term survival for the therapeutic vaccine.141,142 Using the same rationale, Wang et al143 developed a 2-step strategy to modulate the endogenous DCs in situ. In the first step, the DCs were recruited to an injectable GM-CSF– releasing thermosensitive mPEG2poly(lactic-co-glycolic acid) hydrogel. Then, the recruited DCs were loaded with cancer antigens through the use of viral or nonviral vectors. The strategy was shown to generate robust tumor-specific immune responses in both prophylactic and therapeutic models of murine melanoma.143 These encouraging results in animal models led to the clinical application of the strategy. Currently, an ongoing clinical trial is evaluating the feasibility of this approach for treatment of melanoma (NCT01753089). The matrix of the DC-activating scaffold is composed of poly(lactic-co-glycolic acid) and incorporates an autologous tumor cell lysate, GM-CSF, and CpG oligodeoxynucleotides. This strategy may represent the future of DC-based immunotherapies, given that it overcomes the survival and homing problems of the injected ex vivo–generated DCs and even surpasses the limitation of single antigen immunization observed in the in vivo DC–targeting approach. Dex have received great attention as potential immunotherapeutic agents because of the pioneer studies of Zitvogel et al,144 who showed that they inhibit tumor growth in an MHC- and CD81 T cell-dependent manner. The capacity of Dex to elicit CD81 and CD41 T-cell responses was attributed to the transfer of antigen–MHC-I and MHC-II complexes to the endogenous DCs.145 Similar to DCs, Dex were found to modulate NK cells, promoting their proliferation and activation in IL-15Ra–dependent and NKG2Dmediated processes, respectively.146 Recently, the activity of Dex against tumors was additionally shown to rely on B-cell–mediated mechanisms147 and on their capacity to enhance the immunogenicity of tumor cells.148 Despite the abundant experimental data on the use of Dex for antitumor immunotherapy, their clinical evaluation remains scarce (Fig 2, B). Results from 2 clinical trials on melanoma and non-small cell lung cancer patients demonstrated that Dex could be safely administered, nevertheless inducing only limited CTLs responses.149,150 Finally, although all clinical trials performed to date test antitumor DC-based vaccines in a therapeutic context, numerous preclinical experiments showed that preventive DC vaccination can significantly delay or even prevent the development of several tu-
mors.151,152 This supports the establishment of clinical trials that evaluate the safety and efficacy of DC-based vaccines for cancer prevention in high-risk groups in the near future. LESSONS FROM THE PAST AND FUTURE PERSPECTIVES
It is now clear that the effectiveness of antitumor DCbased vaccines depends on the ability of the DCs to induce antigen-specific CTLs and Th1 cells as well as NK cell cross talk and activation.51,131 These features must be achieved by selecting or targeting the adequate DC subset and by tailoring its maturation status and cytokine/chemokine profile. In accordance Q30 with this notion and based on their remarkable capacity to cross-present antigens, LC-like DCs derived from CD341 progenitors (NCT00700167 and NCT0 1456104) or CD141 monocytes (NCT01189383) are being evaluated in clinical trials for the treatment of melanoma. Similarly, the clinical effectiveness of other DC subsets, such as pDCs and Th1 polarizing-DCs, is under assessment in melanoma (NCT00390338), prostate cancer (NCT00970203), and glioma patients (NCT00766753). The recent characterization of a novel human DC subset (CD11c1CD1411XCR11) with superior antigen cross-presentation and NK cell activation capacities has excited great enthusiasm on its possible use.12,14 However, CD11c1CD1411XCR11 DCs represent less than 0.1% of PMBCs, a major drawback for their clinical application. In fact, a relevant obstacle for the progress of DC-based vaccines has been the lack of sufficient DC sources other than CD141 MoDCs. To overcome this issue, the emerging field of induced pluripotent stem cells (iPSCs) may represent a significant opportunity.153 Recently, Silk et al154 established a protocol for the differentiation of CD11c1CD1411XCR11 DCs by culturing human iPSCs with GM-CSF, stem cell factor, VEGF, and bone morphogenetic protein 4. These iPSC-derived DCs were shown to efficiently cross-present exogenously supplied peptides to naive CD81 T cells, inducing their expansion and activation. Because the iPSCs may be continuously expanded in vitro, they represent an unlimited source of autologous DCs that can be tailored to the desired phenotype, bypassing problems such as limited cell numbers and patient-topatient variability.153 Another fact that became evident is that the DC vaccine-induced antitumor immune effector cells must overcome multiple immunoescape/immunosuppressive mechanisms (reviewed in Topalian et al155). Solid tu- Q31 mors often generate an immunosuppressive microenvironment by producing soluble mediators such as adenosine, IDO, PGE2, TGF-b, and VEGF. These
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Fig 4. Frequency of therapeutic approaches combining DC-based vaccines and other anticancer treatments. (A) DC-based vaccines are increasingly being tested in combination with other treatments such as conventional chemotherapy, radiotherapy, and other immunotherapies. (B) Frequencies distribution of other therapies being tested with DC-based vaccines. Other cell therapies include stem cell transplantation, transgenic CTLs, ex vivo expanded T lymphocytes, and cytokine-induced killer cells. *In some cases, in the same trial, DC-based vaccines are tested in combination with multiple therapies leading to count numbers (149) superior to the total number of respective trials (119). DCs, dendritic cells; CTL, cytotoxic T cells; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; IDO, indoleamine-2,3-dioxygenase; mAb, monoclonal antibody; PD-1, programmed cell death 1; VEGF, vascular endothelial growth factor.
mediators inhibit the proliferation and differentiation of Th1 cells, suppress the activity of CTLs and DCs, and induce the differentiation of Tregs.156-158 Moreover, the tumor microenvironment is also prone to attract immunosuppressive cellular regulators such as circulating Tregs, MDSCs, and tumor-associated macrophages. Finally, immune checkpoints, an inhibitory network that normally maintains self-tolerance and prevents excessive and uncontrolled immune responses, can be co-opted by tumors to evade immune destruction.159,160 The major immune checkpoints that are dysregulated by tumors rely on ligand-receptor interactions, such as CD28–CTLA-4 and PD-1–PD-L1. There was a significant effort to establish strategies to bypass these barriers, and combination therapies emerged as a logical approach to potentiate DC vaccine effectiveness. The analysis of clinical trials for more than the last 2 decades reflects a clear tendency to adopt combination therapies instead of standalone DC vaccination (Fig 4, A). Some of the most common approaches involve the use of general lymphodepleting chemotherapeutic agents such as temozolomide and cyclophosphamide and fludarabine (Fig 4, B). By depleting the immune cells, these drugs eliminate negative regulators such Tregs and MDSCs, promoting a favorable environment for DC-induced expansion of antitumor effector cells in the recovery phase. In a pilot clinical trial, Ridolfi et al161 showed that the administration of low doses of temozolomide to melanoma patients before DC vaccination specifically reduced the CD41CD251Foxp31 Tregs. In turn, cyclophosphamide was shown to enhance antitumor immunity through
mechanisms that rely on Treg elimination and resetting DC homeostasis.162 The combination of DC vaccines with agents that preferentially depletes Tregs by directly targeting their abundantly expressed IL-2 receptor a chain (CD25) has also been tested. The agents most commonly used for this purpose are the anti-CD25 mAb basiliximab (NCT00626483) and daclizumab (NCT00847106) or denileukin diftitox (Ontak), a re- Q32 combinant IL-2 and diphtheria toxin fusion protein (NCT00703105, NCT00056134, and NCT00128622). Although some studies indicate that these combination therapies significantly increase DC vaccine efficacy as a result of increased antitumor-specific CTLs,163,164 others demonstrate that they induce tolerogenic DCs and promote the survival of resting Tregs.165 These paradoxical effects are partially because of CD25 expression not restricted to Tregs. As CD25 is also ex- Q33 pressed on effector T cells and activated NK cells, its blockade may compromise their antitumor activity. Another strategy for enhancing antitumor immunity consists of the combination of DC vaccines with immune checkpoint inhibitors. This approach is being tested in clinical experiments targeting the immune checkpoint receptors PD-1 (NCT01441765, NCT010 67287, NCT01096602, and NCT01753089) and CT LA-4 (NCT00090896 and NCT01753089) or the tryptophan degradation enzyme IDO (NCT01042535). Preliminary data obtained in patients with metastatic melanoma indicate that DC vaccines in combination with the anti–CTLA-4 mAb tremelimumab could be more effective than the same interventions in monotherapy (NCT00090896).166 Recently, combinations with
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Fig 5. Evolution of antitumor DC-based immunotherapies. First approaches were based on the administration of ex vivo manipulated DCs in monotherapy regimen, relying only on the capacity of DCs to generate antitumor effector cells (Th1, CTLs, and NK cells). Currently, DC vaccines are frequently combined to therapies aiming to reduce tumor burden such as chemotherapy, radiotherapy, anti-VEGF mAbs, and to therapies seeking to overcome tumor-associated immunosuppression. The later include broad lymphodepleting agents (temozolomide and cyclophosphamide); preferential regulatory T cells depleting agents (anti-CD25 mAbs and denileukin diftitox); blockers of immunosuppressive molecules (IDO inhibitors, anti–CTLA-4 and anti–PD-1 mAbs). Promising approaches to be explored in following years are based on in vivo delivery of antigens to DCs and on the development of implantable devices that will recruit, load, and mature DCs. CTLs, cytotoxic T cells; DCs, dendritic cells; IDO, indoleamine-2,3-dioxygenase; iPSCs, induced pluripotent stem cells; mAb, monoclonal antibody; MoDCs, monocyte-derived dendritic cells; NK, natural killer; PBMCs, peripheral blood mononuclear cells; PD-1, programmed cell death 1; PLGA, poly(lactic-co-glycolic acid); TAAs, tumor-associated antigens; VEGF, vascular endothelial growth factor.
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new anticancer drugs, such as the anti-VEGF mAb bevacizumab (Avastin) (NCT00683241, NCT00913913, and NCT02010606) and the tyrosine kinase inhibitors dasatinib and sunitinib (NCT01876212 and NCT00 678119), were also addressed. Moreover, by exploiting possible synergistic effects, DC-based vaccines have increasingly been used along with conventional radiotherapy and chemotherapy (NCT00639639, NCT01 973322, and NCT00617409). The positive effects of these therapeutic regimens are at least in part because of increased tumor cell apoptosis that fuels the DCs with relevant tumor antigens. From these 2 decades of clinical trials, it became evident that the classical objective responses, such as tumor shrinkage or tumor marker levels, are not adequate endpoints to evaluate the effectiveness of
DC-based antitumor immunotherapies. A systematic review conducted by Anguille et al4 on clinical trials evaluating DC vaccination in melanoma, prostate cancer, renal cell carcinoma, and glioma demonstrated that the clinical benefits in terms of the objective responses are real, but small. Objective responses were only observed in 8.5% of melanoma patients, 7.1% of prostate cancer patients, 11.5% of renal cell carcinoma patients, and 15.6% of glioma patients. In the same study, the authors found that although 2 trials on melanoma did not show survival benefits, all the other 38 studies demonstrated an increase in the median OS, ranging from 20% to 344%.4 These results reinforce the notion that survival is probably the most accurate endpoint to evaluate the therapeutic benefits of antitumor DC-based vaccines. However, clinical trials that
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monitor OS may be excessively long and costly.131 To surpass this limitation, alternative surrogate endpoints have been proposed. A growing number of studies are adopting immune response criteria, in which the effectiveness of the vaccination is evaluated by the expansion of antigen-specific effector T cells and NK cells.167 Finally, the selection of patients is also a relevant aspect to consider when designing an antitumor DC vaccination clinical trial. Optimally, patients should present a low tumor burden and be at an early stage of the disease, thus increasing the likelihood of developing effective antitumor immunity. CONCLUSIONS
Despite the variable rate of success in inducing clear beneficial outcomes, the first generation of antitumor DC-based vaccines has proven to be safe. Meanwhile, DC immunotherapies have substantially evolved because of our expanding knowledge of DC and tumor biology (Fig 5). The use of specific DC subsets that are tailored to a particular phenotype, the definition of adequate clinical endpoints and, particularly, the combination with other antitumor therapies have paved the way to the development of next-generation DC immunotherapies. Therefore, it is expected that these strategies may reveal the full therapeutic potential of DC-based vaccines for the treatment of malignant diseases in the future. UNCITED REFERENCE
19. ACKNOWLEDGMENTS
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Conflict of Interests: All the authors have read the journal’s policy on the disclosure of potential conflicts of interest and have none to declare. The authors thank FCT/MEC for the financial support to the QOPNA Research Unit (FCT UID/QUI/00062/ 2013), the national funds, and where applicable the FEDER within the PT2020 Partnership Agreement, and also the Portuguese RNEM Network. CNC is funded by FEDER funds through the Operational Program Competitiveness Factors-COMPETE, and the national funds are funded by the FCT-Foundation for Science and Technology under strategic project UID/ NEU/04539/2013. All authors have read the journal’s authorship agreement. Supplementary Data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.trsl.2015.07.008.
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