Dendritic cell engineering for tumor immunotherapy - Future Medicine

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Dendritic cell engineering for tumor immunotherapy: from biology to clinical translation Dendritic cells (DCs) are the most potent APCs, with the ability to orchestrate a repertoire of immune responses. DCs play a pivotal role in the initiation, programming and regulation of tumor-specific immune responses, as they are poised to take up, process and present tumor antigens to naive or effector T lymphocytes. Although, to an extent, DC-based immunotherapeutic strategies have successfully induced specific anti-tumor responses in animal models, their clinical efficacy has rarely been translated into the clinic. This article attempts to present a complete picture of recent developments of DC-based therapeutic strategies addressing multiple components of tumor immunoenvironment. It also showcases certain practical intricacies in order to explore novel strategies for providing new impetus to DC-based cancer vaccination. KEYWORDS: cancer therapy n cellular vaccines n molecular medicine n translational oncology

Dendritic cells (DCs), the most potent APCs controlling the immune response, have emerged as an attractive and powerful tool for cancer immunot herapy. Ralph M Steinman and Zanvil A Cohn first discovered DCs in 1973, for which Steinman recently received the Noble Prize in Physiology or Medicine. DCs can be distinguished from other APCs by their abundant expression of costimulatory molecules and their ability to initiate a strong primary immune response. DCs recognize foreign antigens (Ags) through pattern-recognition receptors and secrete various proinflammatory cytokines in order to activate the host immune response [1] . The specialized ability of DCs to process and present Ags on MHC molecules play a central role in initiating and regulating antitumor immune response. During the past decade, several methods using different tumor Ags have been adapted to make an efficient clinical grade DC-based cancer vaccine. Clinical trials on different cancers have displayed the efficacy of this approach against established tumors. In this review, we describe the current progress in DC-based cancer therapy, including some recent clinical trials.

Ag processing & presentation DC-based cancer immunotherapy mainly focuses on removing tumor cells (TCs) by producing a strong antitumor immune response. However, the generation of such a response needs proper processing of tumor Ags, with increased expression of costimulatory signals (e.g., CD40, CD80

and CD86) and cytokines (e.g., IL‑12p70). The widely accepted model reveals that the tumor Ag is first acquired and then processed through the Ag-processing machinery of DCs. The exposure of Ag-processing machinery to danger signals results in conversion of proteasomes to immune proteasomes through upregulation of the proteasomes activator PA28 and a set of different catalytic subunits LMP2, LMP7 and multicatalytic endopeptidase complex subunit 1) [2] . This conversion accelerates generation of hydrophobic and basic C-terminal residue peptides, which preferentially binds to TAP1–TAP2 heterodimers and promotes peptide translocation into the endoplasmic reticulum. These processed peptides finally bind to the MHC class I peptide-binding cleft and are presented on the membrane of DCs for their recognition by lymphocytes (Figure 1) [3] . Following Ag processing, immature DCs undergo maturation into mature DCs (mDCs), which results in the upregulation of HLA, CD80, CD83, CD86, DC lysosome-associated membrane glycoprotein and CCR7. This increased expression helps mDCs to migrate through lymphatics into draining lymphoid tissues, where they have an optimal stimulatory capacity. Migration is one of the most important requirements for lymphocyte priming and is strongly influenced by the expression of CCR7. CCR7 through its interaction with transporter molecules such as TREM-2, LTC4 and LTD4 affect mDC’s migratory ability. CCR7+ DCs arrive at peripheral lymphatic vessels in response to a chemotactic gradient of CCR7

10.2217/IMT.12.40 © 2012 Future Medicine Ltd

Immunotherapy (2012) 4(7), 703–718

Arpit Bhargava1,2, Dinesh Mishra3, Smita Banerjee2 & Pradyumna Kumar Mishra*1 Division of Translational Research, Tata Memorial Centre, ACTREC, India 2 Department of Biotechnology, H.S.G. Central University, Sagar, India 3 Department of Pharmaceutical Sciences, G.G. Central University, Bilaspur, India *Author for correspondence: Tel.: +91 22 27405121 Fax: +91 22 27405061 [email protected] 1

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Bhargava, Mishra, Banerjee & Mishra

O Endocytic compartments

Endocytosis or phagocytosis

NH2

Ubiquinating enzyme complex and ubiquitin

NH2 COOH

ATP

Ubiquitin

C

Proteosomal complex

NH

AMP + PPi NH2

COOH

Peptides Class II MHC TAP

Peptides

Endoplasmic reticulum α β i

CLIP

Class I MHC

α β

Peptides

Peptide

Golgi complex

HLA DM

Exocytic vesicle Exocytic vesicle

Expression of peptide MHC II complex

CD4-mediated immune response

Expression of peptide MHC I complex

CD8-mediated immune response

Figure 1. Classical MHC class I and II antigen processing and presentation pathways of dendritic cells. CLIP: Class II-associated invariant chain peptide; PPi: Proton pump inhibitors; TAP: Transporter associated with Ag presentation.

ligands, CCL19 and/or CCL21, expressed by T‑zone reticular cells within secondary lymphoid organs and lymphatic vessels [4,5] . This role of DCs, recognizing and presenting Ags, has been utilized broadly for developing vaccines against cancer.

DC subtypes The increasing knowledge of DC biology has revealed the complexity of this cell system and has shown the importance of different DC subsets in controlling the type and quality of the elicited immune response [6] . DC systems mainly consists of two subsets – that is, myeloid DCs (myDCs) and plasmacytoid DCs (pDCs). Both DC subsets display different surface molecules and express different sets of cytokines or chemokines, which lead to distinct immunological results (Figure 2) . Human pDCs (DC2) are HLA-DR+ cells expressing high levels of the IL‑3Rα chain (CD123) along with some specific markers such as BDCA-2 and ILT7. They express Toll-like receptors (TLRs; 704

Immunotherapy (2012) 4(7)

TLR-7 and ‑9), and secrete large amounts of type I interferon while myDCs express a different set of TLRs (TLR-2, -3, -4 and -7). Human myDCs (DC1) are further divided into CD1c+, CD16 + and CD141+ subsets [7] . Studies on mouse CD8a- and CD8a+ myDC subsets further explained the ability of distinct DC subtypes to influence immuno logical results [8] . The CD8a- DC subset induces an effective Th2 response [9,10] , while the CD8a+ DC subset has the unique ability to cross-present exogenous Ags [11] and has an important role in inducing cytotoxic responses against cancer cells [12] . Although CD8- DCs can also cross-present exogenous Ags, the higher ability of CD8a+ DCs to internalize apoptotic cells makes them superior in comparison with CD8- DCs [13,14] . In spite of this groundbreaking subdivision, it is now elucidated that a large gene expression program is commonly shared by human and mouse DCs. Functionally, human CD141+ DCs strongly correspond to mouse CD8 + DCs and future science group

Dendritic cell-based cancer vaccines

have the strong ability to excel the cross-presentation in comparison with CD1c+, CD16 + and pDCs [15] . However, owing to the low frequency of primary DCs in circulation, Ag cross-presentation studies in humans is mainly done by using in vitro cultured monocytes [16] . A malignant counterpart (leukemic cells) of pDCs expressing the CD4 + CD56 + profile has been also reported to mimic their normal equivalent. Of course, future detailed analyses of these cells would be of particular interest for understanding DC biology and analyzing the importance of these malignant counterparts in vaccine development [17,18] . Different DC subsets are activated by different activation pathways that later play a pivotal role in generation of unique adaptive immune responses and in designing better vaccines.

DC generation & maturation DCs can be directly isolated from blood circulation by immune-magnetic separation; however,

high yields are not obtained by using such direct isolation, as the frequency of mDCs in peripheral blood is low [19] . Thus, for use in therapeutics, mDCs are mainly generated by in vitro culture of peripheral blood monocytes with suitable growth factors such as GM‑CSF and IL‑4, followed by maturation [20,21] . DCs can also be generated by expanding CD34 + cells in a medium containing autologous plasma with early-acting growth factors or in media containing FLT‑3 ligand, stem cell factor and thrombopoietin supplemented with IL‑7 and IL‑13 [22,23] . These methods are of prime importance as mDCs can be generated from frozen cord blood samples, providing an added source for allogeneic DCs in clinical settings. Standardization of protocols for the large-scale in vitro generation of homogeneous, mature and functional DCs is a precondition for DC vaccine preparations and CD34 + cells will surely play an important role in achieving this goal.

CCL21 CCR7 CD103 CD11b CD14 CD11c CD83 CRG-2 CD205 CD207 CLEC7A MIP-1α TROP1 MIP-3β EMR1 RANTES CXCL8 CD172a CD11b NALP3 CD11c TLR4 NALP1 TNF-α NALP2

CD45R CD205 Ly-6G IFN-α IFN-β IL-3Rα IL-6 TLR9 CD85g

Langerhans DCs

Inflammatory DCs

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CD11c IRF8 Neuropilin-1 CD172a Spi-B TLR7

Plasmacytoid DCs

CD80 CD86 CD40L CX3CR1 DC-LAMP DC-SIGN

CD16 CD56 B7-DC CD22 TSLPR

Other DC markers

DC lineages

Myeloid DCs

CD1c/BDCA-1+ CD1c CD172a CD11b TLR1 CD11c TLR6

CD141/BDCA-3 CLEC9a ID2 SynCAM1 IL-12 CD11c

IRF8 BDCA-3 TLR3 XCR1

CD4+CD8αCD4 IFN-γ IL-2 IL-6 CD11b

CD11c CD172a TLR1 TLR6

CD8α+

CD4+CD8αIFN-γ CD11c IL-2 CD172a IL-6 TLR1 CD11b TLR6

CD8 CLEC9a CD205 ID2 XCR1

CD103 CD11c IRF8 TLR3 IL-12

Figure 2. Detailed description of different dendritic cell lineages along with their cell surface markers. DC: Dendritic cell.

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With progress in the research of DC immunobiology, a mature immunophenotype is considered as the most important criterion for an ideal DC-based cancer vaccine (Table 1) . Defective DC maturation increases generation of CD4 + CD25 + Foxp3 + Tregs, induces Th2 polarization and indoleamine 2,3-dioxygenase (IDO), leading to a strong immunosuppressive state [24,25] . mDCs migrate more effectively to draining lymph nodes, prime naive T cells and induce an effective antitumor response in cancer patients. The common procedure for the generation of human monocyte-derived mDCs is to use a cytokine cocktail of TNF, IL‑1b, IL‑6 and PGE2. One of the major limits of this procedure is the use of PGE2, an important component for the migratory function of DCs. PGE2 results in the promotion of IL‑10-secreting cells, Th2 polarization and

recruitment of Tregs [26,27] . Active recruitment of Tregs upregulates IDO expression in human DCs through cytotoxic T lymphocyteassociated Ag–CD80 interaction, thus setting up a suppressive microenvironment. IDO is a tryptophan-degrading enzyme that inhibits the growth of low tryptophan-sensitive T cells [28] . It also begins in vitro differentiation of monocytes into regulatory DCs, with increased inhibitory receptors such as inhibitory receptor ILT3 and ILT4 [29] . An interesting study reported that IFN-a and polyinosinic:polycytidylic acid synergize with the ‘classical’ type 1-polarizing cytokine cocktail (TNF-a–IL‑1a–IFN-g) and results in the generation of fully mature type 1-polarized DCs also known as aDC1 [30] . Upon subsequent comparison with conventional the cytokine cocktail, it was observed that these aDC1 not only produce higher IL‑12p70

Table 1. Examples of agents in different types and characteristics used for dendritic cell maturation. Study (year)

Maturation agent

Characteristics

Ref.

Gautier et al. (2005) and Pedersen et al. (2005)

TNF, IL‑1b, IL‑6 and PGE2

Sustained Th1-polarization; IL‑12p70 secretion; IFN- b mRNA accumulation

Vandrlocht et al. (2010)

TLR2/4 agonist and IFN-g

Increased Th1 and Th17 polarization; superior inducers of functional antigen-specific T cells; increased chemokine secretion and effective migration

ten Brinke et al. (2010)

TLR agonist, MPLA, with IFN-g

IL‑12-producing mDCs; superior CD8 (+) CTL response

Dauer et al. (2008), Zobywalski et al. (2007) and Boullart et al. (2008)

TLR3 or TLR7/8 ligands with IL‑12p70-secreting DCs; stable maturation and substantial PGE2 and IFN recoveries of mDCs after cryopreservation

Anguille et al. (2009)

TLR7/8, GM‑CSF and IL‑15

Higher phenotypic expression of CD83 and costimulatory molecules (CD70, CD80 and CD86); higher migratory potential and IFN‑g, IL‑12p70 secretion; superior antigen-specific T‑cell responses

[43]

Roy et al. (2011)

NLGP

Upregulated maturation; higher expression of CD40, CD83, CD80 and CD86; increased Th1 cytokine secretions; substantial T‑cell allostimulatory capacity; promoted the generation of CTLs

[47]

Kushibiki et al. (2010) and Shixiang et al. (2010)

Photodynamic therapy

Elevated serum IL‑12 and TNF-a levels; decreased serum IL‑10 levels; high CTL activity; enhanced TC immunogenicity

Berger et al. (2010)

Photochemotherapy

DC differentiation, within a single day without added cytokines; large maturation

[150]

Gao et al. (2010)

Nicotine

Upregulated the expression of CD80, CD86, CD40, CD11b, MHC class I and II molecules in imDCs; imDC-dependent T‑cell priming and IL‑12 secretion; enhanced CD80 expression through PI3K activation

[151]

Bae et al. (2010)

Uncarinic acid C in combination with IFN-g

Higher expression levels of CD1a, CD83 and HLA-DR augmented the T‑cell stimulatory capacity; enhanced production of IL‑12p70; increased secretion of IFN-g; intermediate migratory capacity

[152]

Shen et al. (2004)

Negative regulatory pathways

Increased maturation; enhanced CTL response

[153]

[36,146] [42]

[147] [39,40,44]

[148,149]

CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; IFN: Interferon; imDC: Immature dendritic cell; mDC: Myeloid dendritic cell; MPLA: Monophosphoryl lipid A; NLGP: Neem leaf glycoprotein; TC: Tumor cell; TLR: Toll-like receptor.

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levels but also have efficient migration and T‑cell induction abilities. In addition, these cells have the ability to stably retain their type 1 phenotype in Treg-promoting immunosuppressive environments [31] . TLRs expressed on immature DCs are a family of conserved germline-encoded patternrecognition receptors and are widely known to mediate DC maturation [32] . TLRs recognize unique pathogen-associated molecular patterns and initiate a signaling cascade involving activation of different downstream proteins, such as MyD88, TRIF and IRAK. This subsequently activates transcription factors, such as NF-kB, resulting in the secretion of inflammatory cytokines to activate innate and adaptive immune cells [33,34] . This ability of TLRs has aroused interest in the use of TLR agonists as a pharmacological analog, as they not only increase phenotypic DC maturation, but also induce cross-presentation and promotes activation of T and NK cells [35,36] . The covalent conjugation of TLR agonists to Ags often promotes the generation of a CD8 + T‑cell response without affecting cellular uptake and Ag persistence in vivo. This ability of TLR agonist–Ag conjugates to elicit a CD8 + T‑cell response is mainly based on its engagement in DC crosspresentation pathways [37–39] . TLR3 or the TLR7/8 ligand in association with PGE2 results in DCs with ideal migratory properties and the ability to produce Th1-polarizing cytokines such as IL‑12p70 [40] . DCs stimulated in vitro with a TLR adjuvant CpG oligonucleotide showed enhanced maturation and IL‑12p70 secretion with a significant ability to stimulate Ag-specific T‑cell responses [41] . Besides, using combination strategies of TLR agonists with cytokines is an alternate method for the ideal generation of mDCs. One such approach is combining a TLR2/4 agonist and IFN-g [42] . Short-term culture of DCs with GM‑CSF and IL‑15 followed by activation using TLR7/8 fulfills all preclinical conditions of immune-stimulatory DCs [43] and can be used for designing anticancer strategies. In vivo Ag delivery to resident APCs via mannose receptors could improve T‑cell responses when delivered in the presence of GM‑CSF and TLR3 and TLR7/8 agonists. Combining monophosphoryl lipid, a TLR agonist, with IFN-g generated more IL‑12-producing mDCs, which polarize CD4 + T cells towards a Th1 phenotype and induce a superior CD8 + cytotoxic T‑cell response [44] . Another approach is through electroporation with mRNA encoding CD40, CD70 and TLR4. future science group

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It was reported that these DCs have an efficient T‑cell stimulatory capacity and, when coelectroporated with whole-tumor Ag mRNA, stimulate Ag-specific T‑cell responses [45] . Interestingly, epigenetic agents such as DNA methyltransferase or histone deacetylase inhibitors were also reported to have the potential for DC activation but warrant intense scrutiny before being established as antitumor agents [46] . Recently, neem leaf glycoprotein has emerged as a nontoxic agent inducing DC maturation. Neem leaf glycoprotein rescues DCs from a suppressed immature state to an active mature condition and actively induces Th1 polarization effects in the clinic [47] . These preclinical and early clinical data have confirmed the role of TLR agonists as an effective maturation agent for DC-based cancer immunotherapy; however, the use of neem leaf glycoprotein appears to be an emerging option.

Loading DCs with tumor Ags One of the most important aspects of a successful DC vaccine is loading DCs with a suitable Ag (Figure 3) . The major obstacle here is the lack of known tumor peptides in many HLA contexts that helps potential immune evasion of TCs. Significant efforts have been made in different aspects of generation and manipulation of DCs, including MHC-restricted synthetic peptides derived from the tumor Ags [48,49] , tumor RNA [50] , tumor lysates [51] , tumor-derived exosomes [52,53] and apoptotic TCs [54–56] . In addition, tumor Ag-loaded DCs are suggested to serve as adjuvant supporting chemotherapy for a more effective antitumor response [57] . However, controversy exists about the most efficient Ag to pulse DCs for an effective antitumor response [58–61] . DC–TC fusion is an alternate approach that not only breaks T‑cell tolerance to tumor associated Ags in animal models [62] , but also proved to be effective against a wide variety of established tumors [63–67] . However, there are several technical problems in producing such fusion products; inadequate recovery and lack of proper quality control has restricted their broadened clinical application. Using apoptotic TCs as a source of multivalent Ags to pulse DCs is another choice [68] , although the inability of these cells to produce a similar response in vivo is a major limit necessitating further improvements [69] . A growing body of evidence shows the use of tumor lysates as a better alternative, as the presence of multiple Ags reduces the risk of a TC escape [70,71] . Tumor lysates obtained mainly from freeze and thaw cycles induce DC maturation and www.futuremedicine.com

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Peripheral blood

Patient Vaccination

Monocytes GM-CSF IL-4 DC generation TNF CD40-L

Ag-pulsed DC

Tumor Ag Ag loading

DC maturation

Figure 3. Strategy for generating a tumor antigen-loaded dendritic cell-based cancer vaccine. Ag: Antigen; DC: Dendritic cell.

are widely used in the clinical setting for vaccination [72–74] . One major drawback of producing lysate-based DC vaccines is the weak maturationinducing effect, thus requiring cytokine cocktails to be used in combination [75] . The DC immunization approach for cancer therapy has gained limited success mainly because many patients have showed strong immune tolerance toward their cancer cells. Tumor lysate-loaded DC vaccination in melanoma patients results in increased frequencies of Tregs [76] . Thus, parallel application of adjuvants with tumor lysates is a precondition for immune stimulation, as TCs are poorly immunogenic owing to reduced expression of costimulating molecules and changes in the HLA class I and II Ag-processing pathways [77] . The successful encapsulation of the tumor lysates with poly(lactic-co-glycolic acid) nanoparticles for in vitro Ag delivery to DCs is an attractive strategy [78] . Sustained Ag release due to encapsulation improves vaccine efficiency and significantly increases the generation of TC-specific cytotoxic response. The use of a solid lipid nanoparticle drug delivery platform appears to be another promising approach, as solid lipid nanoparticles possess a combination of the advantages of high monodispersity, long temporal stability, low toxicity and relatively good stability for temperature, 708


possible dehydration and reconstitution. This makes solid lipid nanoparticles better to utilize in future anticancer immune therapeutics [79,80] . The evolution of gene transfer technology has moved towards developing DC vaccination protocols that aim to improve tumor Ag-specific immunity by genetically modifying autologous DCs. These genetically modified autologous DCs may be injected to stimulate an immune response against a protein expressed specifically on target cancer cells. DCs transduced with IL‑2 actively inhibit tumor growth by inducing a systemic antitumor response, demonstrating potential as a potent adjuvant in antitumor immunot herapy [81] . Viral vectors such as the HIV-1-based lentiviral vector and baculovirus are two promising delivery systems to induce antitumor immunity [82,83] . Gene silencing of the tolerance-inducing gene STAT3 by poly(lactic-coglycolic acid) nanoparticles or PD-L1 and PD-L2 augments DC maturation and function of tumor Ag-specific CD8 + T cells [84,85] . These findings of genetically engineered DCs have paved the way for using this technique as an effective tool for DC immunotherapy; however, to date, the application of tumor lysates with addition adjuvants seems to be perfect for using in clinical situation.

Clinical concerns Preclinical studies conducted during the past two decades have provided a compelling rationale for using DC-based vaccines in different cancers. The Cancer Vaccine Clinical Trial Working Group has recognized clinical considerations in the study design and protocol development as major problem in DC vaccine development [86] . Success of these vaccines is directly or indirectly influenced by several tumor- and host-related characteristics. Host-related factors such as age and immune unresponsiveness are reported to be associated with reduced DC numbers, making vaccines less effective [87] . Knowledge of HLA is essential for assessing epitope-specific T‑cell responses [88] . Moreover, the ability to generate an adequate immune response to DC-based vaccine is highly influenced by immune-suppressing conditions such as coinfection and other related medications [89] . Another vital factor that should be considered is the stage of disease, as DC vaccination like other forms of immunotherapy works efficiently when there is minimal residual disease in the body in comparison with the advanced disease with large tumor burden. Efficient vaccine delivery and postvaccination follow-up of the patients are other important issue for the success of any future science group


vaccine. It is well known that, in order to elicit an efficient immune responses, DCs must home to secondary lymphoid organs to prime T‑cell response. Results of recent investigations suggest that intradermal administration is superior in eliciting an effective antitumor Th1 response [90,91] . However, labeling studies have displayed significant differences in the distribution of DC-containing cell products administered via different routes [92–94] . Therapeutic DC vaccines are expected to induce a TC-specific immune response, and effective monitoring of this response is an important issue for estimating vaccine efficacy. Targeting specific molecules such as Th1/Th2 cytokines, direct measurement of CD4/CD8 cells, the enzyme-linked immunosorbent spot assay and HLA–peptide multimer staining are widely used strategies for monitoring therapeutic response [95] . Monitoring assays used should be established, reproducible and technically validated in the respective laboratories. In order to define the proportion of patients with a positive therapeutic outcome, cutoff values for an immune response should be identified prospectively. Using immunoassays for a minimum three different time points throughout clinical trials and use of two parallel tests for similar findings will also be helpful to adequately address the issue of interassay variability.

Clinical trials for DC-based therapy DC-based anticancer strategies have now developed from in vitro and murine studies to pre clinical and clinical trials. DC vaccination has occasionally led to successful tumor regression; however, the majority of clinical trials are only in the initial phase. Prostate cancer DC-based immunot herapy is a promising approach to augment tumor Ag-specific T‑cell responses; however, tumor escape with down regulation or complete loss of Ags may limit the susceptibility of TCs to the immune attack. Using strategies to target multiple Ags could be helpful in improving the treatment efficacy of DC vaccination. DC-based multiepitope immunotherapy (DCs loaded with PSCA, PAP, PSMA and pPSA) in hormone-refractory prostate cancer patients generated efficient antitumor responses [96] . Despite its therapeutic utility, the strategy was reported to be a safe and effective treatment alternative in hormone-refractory prostate cancer patients [97]. These results were in favor of the previous observations in which it was observed that PSCA and PSA peptideloaded autologous DCs induced clinical benefit future science group

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associated cellular responses mainly in immunecompetent patients [98,99] . The effectiveness of DC vaccination in multiple metastasis patients receiving a combination therapy of TC-loaded DCs and activated lymphocytes further suggested that DC therapeutics works betters in combination with other therapies [100] . Multiple myeloma Multiple myeloma patients who failed maintenance therapy after tandem autologous stem cell transplantation effectively tolerated DC therapy without any adverse affects, suggesting that it as a safe choice for treatment. An effective T‑cell response generated following vaccination with an autologous idiotype as a whole protein or an idiotype-derived class I-restricted peptide antiidiotype and keyhole limpet hemocyanin- loaded DCs proved the effectiveness and superiority of DC-based therapeutics [101] . These observations were further strengthened by the observations in advanced-stage cancer patients where vaccination with autologous DCs pulsed with MUC1-derived peptides induced TC lysis in an Ag-specific and HLA-restricted fashion [102] . Combinatorial approaches using DC vaccination with other anticancer therapies were also performed in metastatic melanoma patients. Clinical and immuno logic parameters demonstrated that treatment with tumor peptide and keyhole limpet hemocyanin-pulsed DCs in patients pretreated with daclizumab results in effective depletion of Tregs from the peripheral circulation and generation of TC-specific immune responses. The positive correlation of treatment-associated disease stability with prolonged survival demonstrated the clinical benefit and affirmed that the DC vaccination is effective, feasible and well tolerated in myeloma patients [103] . However, no such correlation was observed between the generated immune response and the disease stabilization [104] . Renal cancer Assessment of the safety, feasibility, induction of a T‑cell response and clinical response of metastatic renal cell carcinoma patients after DC-based vaccination was reported in different clinical trials. Significant TC-specific immunological responses with positive disease stabilization and no serious adverse events were reported in renal cell carcinoma patients vaccinated with a cocktail of survivin and telomerase peptides or tumor lysate and MUC1 peptide-loaded DCs [105,106] . Similarly, administration of allogeneic DC–TC fusion hybrids significantly increased percentage of T lymphocyte subsets in peripheral circulation www.futuremedicine.com

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with no serious adverse effects, toxicity or signs of autoimmune disease after vaccination in renal cell carcinoma patients [107] . Colorectal cancer A pilot study conducted in advanced colorectal cancer patients demonstrated that a DC-based vaccine targeting multiple tumor-associated Ags can induce immune responses to multiple tumor Ags in advanced colorectal cancer patients [108] . Initiation and regulation of T‑cell immunity by DCs is a key to optimize all DC-based cancer vaccines. In this viewpoint, Burgdorf, in a recently performed Phase I/II trial, evaluated clinical and immunological effects of autologous DCs pulsed with an allogeneic TC lysate and reported disease stability in four out of 17 patients with disseminated colorectal cancer. Analyses of the immuno logical consequences of the treatment showed a vaccine-associated rise in Th1 cytokine levels in patients achieving disease stability [109] . Ovarian cancer A DC-based vaccination approach is also suggested to overcome immune-tolerance in ovarian cancer of different stages. Pulsed DC administration to 21 patients with stage III, IV or recurrent ovarian cancer overexpressing the p53 protein reported an immunological response in 83% of patients with an increased overall survival and no serious systemic side affects [110] . In another Phase I/II trial with peptide-loaded DCs with or without low-dose intravenous cyclophosphamide, no signs of recurrence were reported after 36 months in six patients out of total 11, with an overall survival of 90% [111] . Breast cancer Investigations have shown that DC vaccination is an effective, safe and well-tolerated approach for breast cancer patients [112] . DC vaccination results in the marked expansion of activated tumor-specific T cells and induces effective antitumor immunity in metastatic breast carcinoma patients, and were observed to be associated with clinical response [113,114] . Data from a Phase I/II trial using a DC vaccine combined with IL‑2 suggested that combinatorial immunological approaches result in a decrease of inhibitory immunity, correlates with clinical outcome of the treated patients and is well tolerated by breast cancer patients without major side effects [115] . Other cancers DC vaccination with allogeneic tumor lystate-pulsed DCs along with keyhole limpet 710


hemocyanin as the adjuvant induces both humoral and cellular immunity in advanced urological cancer patients [116] . DCs pulsed with autologous tumor mRNA elicited effective T‑cell responses against Ags encoded by the transfected tumor mRNA [117] . Administration of WT1 RNAloaded DCs in a 46-year-old woman with endstage serous endometrial cancer resulted in generation of a vaccine-specific T‑cell response [118] . A pilot trial conducted on metastatic medullary thyroid carcinoma patients using autologous DCs pulsed with TC lysate showed that allogeneic TC lysate-based DC immunotherapy is safe and well tolerated [119] . In another Phase I/II clinical study, vaccination with a-galactosylceramide (a specific glycolipid Ag)-pulsed DCs was accompanied by the successful induction of T‑celldependent immune responses and prolonged survival of non-small-cell lung cancer patients [120,121] . These results are encouraging and warrant further evaluation for survival benefit of DC therapy in combination with adjuvants and other cancer therapies.

Combinational approaches A major challenge is to improve the clinical effectiveness by identifying novel and efficient approaches breaking tolerance of TCs and increasing their sensitivity to cytotoxic T lymphocytes. In light of this, different combination strategies using one or more therapies are proposed to be beneficial. One such anticancer strategy is combined chemoimmunotherapy consisting of alternate courses of chemotherapy and tumor-associated Ag-pulsed mDC vaccination. Synergization of these two strategies is useful as prior immunotherapy sensitizes TCs to subsequent chemot herapy. This has been validated through vaccination in glioblastoma multiforme patients receiving subsequent chemotherapy. The patients showed significantly longer postchemo therapy recurrence times and survival relative to patients receiving therapy in isolation [122] . Antonia et al. provided clinical support for an emerging paradigm in cancer immunotherapy, in which optimal use of vaccination was observed to be more effective, not as a separate modality, but in direct combination with chemotherapy [123] . Compelling evidence suggests that DCs also play an important role in chemotherapy-induced immunogenic cell death. Chemot herapeutic drugs such as cyclophosphamide directly affect DC homeostasis, leading to the preferential expansion of the CD8a(+) subset involved in the cross-presentation or they stimulate tumor infiltration, engulfment of tumor apoptotic material, future science group


and CD8 T‑cell cross-priming by CD8a(+) DCs. This stimulus can be boosted with interferon resulting in a strong antitumor response and tumor rejection [124] . Combined chemoimmunotherapy consisting of alternate courses of chemotherapy and vaccination with mDCs pulsed with LNCap prostate cancer cell line led to the marked improvement in the clinical and laboratory presentation, and to the decrease of PSA levels by more than 90%. Appropriate combination of tumor mass decline (by surgery and/or chemotherapy/radiotherapy) and neutralization of tumor-induced immune suppression might set the right conditions for inducting antitumor immune response by the active immunotherapy of choice. The immune response could then keep the residual TCs in check and restore the balance between the host and TC population [125] . Locher et al. reviewed the concept of recently emerged immunogenic chemotherapy and suggested that it relies on the capacity of a cytotoxic compound to trigger cell death. This modality elicits cross-priming by DCs of tumor Ag-specific T cells, which contributes to the tumoricidal activity of the compound and protects the host against disease relapse [126] . With rapid advances in understanding cancer biology, immunotherapy has emerged as another therapeutic approach with the potential to contribute to further improvements in the survival. Recently, the field of cancer immunotherapy received an important boost when the US FDA approved the first therapeutic cancer vaccine, sipuleucel‑T. An in-depth analysis showed the cost of sipuleucel‑T was similar to that of Taxotere® (Sanofi-Aventis, NJ, USA) the standard of care for hormonerefractory prostate cancer before sipuleucel‑T was approved for marketing [127] . Besides, the importance of chemotherapy as an antitumor strategy utilized as combined chemoi mmunot herapy consisting of alternate courses of chemotherapy and vaccination with mDCs pulsed with suitable tumor Ags might also prove to be beneficial.

Challenges in generating DC products for therapy Lacuna of DC-based cancer immunotherapy is the primary cause of failure to develop successful DC-based vaccinations. Even 38 years after the discovery of DCs, only a few FDA-approved DC-based vaccines (such as sipuleucel-T) have entered into the market [201] . The major challenge towards developing DC-based vaccine is the immune tolerance induced by TCs. These TCs form an immunosuppressive environment by releasing factors such as TGF-b, IDO future science group

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and IL‑10 [128] , and promote growth of type 2 macrophages, myeloid-derived suppressor cells, immature or tolerogenic DCs and Tregs, which downregulate the TC-specific cytotoxic immune responses [129] . The use of a nanoparticle-based particulate system such as poly(lactic-co-glycolic acid) nanoparticles and solid lipid nanoparticles seems to be a better option for TC Ag delivery, as they have low diffusivity, restricted movement, sustained delivery and the ability to activate phagocytic cells. These encapsulated Ags induce cross-presentation and are effectively presented by MHC-I molecules on DCs [130–132] . In comparison with other polymeric compounds, solid lipid nanoparticles seem to be a better option for DC immunotherapy owing to ease in drug release modulation, high drug payload and less toxicity. Importantly, lower exposure of water to the inner core provides a greater drug stability against chemical degradation [133–136] . Thus, using a nanoparticle-based approach for Ag delivery to DCs with adjuvants will target TCs and rescue tumor-induced immune suppression (Figure 4) . Another important aspect for the limited clinical response of DC-based vaccines approaches is the short lifespan of ex vivo engineered DCs and the small number of the injected DCs that migrate to the lymph nodes [137,138] . An alternate method to conquer this dilemma is to directly target DCs inside the patient’s body. Nanoparticle encapsulation can be helpful in achieving this task as they can easily be surface modified with DC-specific cell surface markers. Encapsulation protects tumor Ags from chemical degradation and helps them to effectively encounter peripheral or lymph node DCs, which results in sustained release of tumor antigens to overcome immune tolerance and activate a TC-specific cytotoxic immune response [139,140] . This surface-modified nanoparticle-based approach can be further improved by adding an adjuvant to activate immune cells [141,142] . The development of such novel nanoparticle-based approaches to target DCs in vivo in order to overcome tumor-induced immune suppression holds the key to design of future DC-based anticancer therapeutic modalities. Despite extensive data to prove its safety, DC immunot herapy remains an investigative approach, highlighting the necessity to take on more significant verification of principle studies and well-designed large efficiency clinical trials. It is mandatory that therapeutic-grade DCs should meet a standard criterion before administration, apart from source and culture methods. Standard operating procedures based on information gained from preclinical studies and the www.futuremedicine.com

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Size and shape Entrapment efficiency Percentage yield Nanoparticle encapsulation

Encapsulated Ags

Tumor Ags

Autologous DCs

Pulsing DCs with encapsulated Ags Increased maturation Effective Th1 polarization Tumor cell-specific cytotoxicity

Tumor Ag expression

Nanoparticle uptake

Figure 4. Pulse dendritic cells by nanoparticle encapsulation. Tumor Ags can be encapsulated using a biological polymer and autologous DCs are pulsed with this encapsulated Ag. Ag: Antigen; DC: Dendritic cell.

growing understanding of DC biology must be implemented to ensure a safe and effective DC vaccine. An important issue is stability – that is, the ability of DCs to retain viability and activity upon freezing and thawing, when the DC-based product is to be cryopreserved and thawed for multiple-dose administration to patients. Analyzing the costimulatory capacity of DCs is another critical issue, as it plays an important role in the generation of tumor-specific immunity [143] . A variety of functional assays is available, but they need to be standardized and validated in correlation with in vivo performance of the cells [144,145] . In addition, the measurement of diverse DC functions provides an opportunity for an improved control of the variability of DC products and for setting up a clinically functional profile for DCs. Standardization and selection of accurate and reliable processing approaches used in an established quality-control program setting is the key for developing a clinically successful DC-based anticancer immunotherapy.

Conclusion & future perspective DC-based cellular engineered vaccines offers the most prospective approach, particularly 712


where conventional therapy fails to deliver. It induces low toxicity and recognizes multiple target molecules, even newly arising Ags, to selectively eliminate the transformed cells. Vaccine approaches using tumor lysates as a whole-cell Ags seems to be more feasible because of its ability to present a broad spectrum of known and unknown Ags. The recent successful attempt of tumor lysate encapsulation increased the efficacy of a DC vaccine. Also, studies are now focusing on genetically modifying DCs with cDNAs encoding immunestimulatory cytokines or tumor-associated Ags to enhance the generation of more effective antitumor responses. In addition, unravelling the molecules and pathways that can influence the interactions between tumor microenvironment and DC maturation and function would be of key interest. Such strategies would help to predict the amenability of DC-based immunot herapy by virtue of an improvement in disease-free survival for patients when surgical/chemotherapy/radiotherapy interventions become impractical. Although DC therapy has been successful, to an extent, in inducing specific antitumor immune response in the future science group


animal tumor models, these benchside results have rarely translated to bedside practice owing to certain intricacies regarding quality assurance of clinical trials. Success could be achieved through well-organized, goal-oriented human trials, optimizing Ag delivery, organ-specific vaccine development and administration, followed by monitoring response to therapy in multiple cancers. This review attempts to present a complete picture of recent developments of DC-based therapeutic strategies, addressing multiple components of the tumor-immune system. It also showcases some pragmatic intricacies, as well as emphasizes the need to harness novel systemic avenues, in turn providing new impetus to DC-based cancer vaccination.

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Acknowledgements The authors are thankful to the Council of Scientific and Industrial Research (CSIR) and Department of Biotechnology (DBT), Government of India, New Delhi, for providing necessary assistance. A Bhargava is thankful to CSIR for providing a Senior Research Fellowship.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employ‑ ment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Background Dendritic cells (DCs) were first discovered by Ralph M Steinman and Zanvil A Cohn in 1973. Steinman received the Noble Prize in Physiology or Medicine in 2011. Antigen processing & presentation DCs acquire tumor antigens (Ags) and process them through Ag-processing machinery. Processed peptides finally bind to the MHC class I peptide-binding cleft and are presented on the membrane of DCs for their recognition by lymphocytes. Following Ag processing, immature DCs undergo maturation, which results in the upregulation of costimulatory molecules. This role of DCs has been utilized broadly for the development of vaccines against cancer. DC subtypes DC systems mainly consist of two subsets, myeloid DCs and plasmacytoid DCs, which display different surface molecules. Human myeloid DCs (DC1) are further divided into CD1c+, CD16 + and CD141+ subsets. Human CD141+ DCs strongly correspond to mouse CD8 + DCs and have strong ability to excel the cross-presentation in comparison with CD1c+, CD16 + and plasmacytoid DCs. Different DC subsets are activated by different activation pathways that will later play a pivotal role in designing better vaccines. Loading DCs with tumor Ags Tumor Ag-loaded DCs are suggested to serve as an adjuvant supporting chemotherapy for a more effective antitumor response. Sustained Ag release due to encapsulation improves vaccine efficiency and significantly increases the generation of a tumor cell-specific cytotoxic response. The solid lipid nanoparticle drug delivery platform appears to be a promising approach owing to high monodispersity, long temporal stability, low toxicity and a relatively good stability for temperature, possible dehydration and reconstitution. Clinical concerns Vaccine effectiveness is directly or indirectly influenced by tumor and host-related characteristics. Efficient vaccine delivery and postvaccination follow-up of the patients are other important issues for the success of any vaccine. Clinical trials for DC-based therapy Prostate cancer: DC-based multiepitope immunotherapy (DCs loaded with PSCA, PAP, PSMA and PSA) in HRPC patients generated efficient antitumor responses. Multiple myeloma: multiple myeloma patients who failed maintenance therapy after tandem autologous stem cell transplantation effectively tolerated DC therapy without any adverse affects, suggesting that it is a safe choice for treatment. Renal cancer: tumor cell specific immunological responses with positive disease stabilization and no serious adverse events were reported in renal cell carcinoma patients. Colorectal cancer: analyses of the immunological consequences of the treatment showed a vaccine-associated rise in Th1 cytokine levels in patients achieving disease stability. Ovarian cancer: the DC-based vaccination approach is suggested to overcome immune tolerance in ovarian cancer of different stages. Breast cancer: combinatorial immunological approaches result in a decrease of inhibitory immunity, which correlates with the clinical outcome of the treated patients without major side effects.



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Executive summary (cont.) Combinational approaches Combined chemoimmunotherapy consisting of alternate courses of chemotherapy and tumor-associated Ag-pulsed myeloid DC vaccination. The US FDA has approved the first therapeutic cancer vaccine, sipuleucel‑T. Challenges in generating DC products for therapy Lacuna of DC-based cancer immunotherapy is the primary cause of failure to develop successful DC-based vaccinations. The major challenge towards developing DC-based vaccines is the immune tolerance induced by tumor cells. Direct targeting through encapsulated tumor Ags is an alternate option. Conclusion & future perspective DC-based cellular engineered vaccines offer the most prospective approach, particularly where conventional therapy fails to deliver. Vaccine approaches using tumor lysates as a whole-cell Ag seems to be more feasible because of its ability to present a broad spectrum of known and unknown Ags. The recent successful attempt of tumor lysate encapsulation using a nanotechnology approach holds good promise with the potential of clinical translation.

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Website 201 Miller D. Dendreon’s Provenge Costs the

Same as Chemotherapy. www.minyanville.com/ businessmarkets/ articles/dendreon-provenge-cost-taxoteresanofi-aventis/7/29/2010/id/29371
