Available online at www.sciencedirect.com
ScienceDirect Dendritic cells and cancer immunotherapy Kristen J Radford1,2, Kirsteen M Tullett1,3,4 and Mireille H Lahoud4,5 Dendritic cells (DC) play an essential role in the induction and regulation of immune responses, including the generation of cytotoxic T lymphocytes (CTL) for the eradication of cancers. DC-based cancer vaccines are well tolerated with few side effects and can generate anti-tumour immune responses, but overall they have been of limited benefit. Recent studies have demonstrated that CD141+ DC play an important role in antitumour responses. These are now attractive targets for the development of vaccines that directly target DC in vivo. An understanding of the functional specialisations of DC subsets, strategies for the delivery of tumour Ag to DC and for enhancing immune responses, point to promising new avenues for the design of more effective DC-based cancer vaccines. Addresses 1 Mater Research Institute, University of Queensland, Translational Research Institute, Brisbane, Australia 2 University of Queensland, School of Biomedical Sciences, Brisbane, Australia 3 University of Queensland, School of Medicine, Brisbane, Australia 4 Centre for Biomedical Research, Burnet Institute, Melbourne, Australia 5 Department of Immunology, Monash University, Melbourne, Australia Corresponding author: Lahoud, Mireille H ([email protected])
Current Opinion in Immunology 2014, 27:26–32 This review comes from a themed issue on Tumour immunology Edited by Philip K Darcy and David S Ritchie
0952-7915/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coi.2014.01.005
Introduction Dendritic cells (DC) play a key role in initiating and maintaining immune responses. Since the discovery of DC by Ralph Steinman over 40 years ago [1] and the identification of their key function as mediators of T cellmediated immune responses, there has been a major focus on the use of DC in cancer immunotherapy. DC have been used to vaccinate cancer patients for nearly 20 years [2]. Until recently, most DC vaccines comprised DC or monocyte precursors of DC, isolated from the patient, loaded ex vivo with tumour antigen (Ag), and readministered to the patient. DC that migrated from the injection site to the draining lymph nodes were expected to prime naı¨ve, and or boost memory, tumour-specific T cells capable of eradicating the tumour. To date, the majority of trials have been Phase I studies on small cohorts of advanced cancer patients who had failed to respond to conventional therapies. These trials revealed Current Opinion in Immunology 2014, 27:26–32
that this approach: (1) is feasible in many malignancies; (2) is well tolerated with minimal toxicity; and (3) can induce tumour-specific immune responses in many patients. Whilst early DC therapies resulted in limited clinical benefits, recent advances in our understanding of DC biology and new knowledge obtained from clinical trials have identified new strategies that are expected to improve clinical outcomes. Harnessing the unique capacity of different DC subtypes to drive specific immune responses in combinations with approaches designed to overcome tumour-mediated immune suppression and immune regulation, are emerging as key strategies for the development of new generation DC vaccines.
Can DC vaccines be of clinical benefit? Early clinical studies showed a significant advantage of using DC vaccines over indirect vaccine approaches (e.g. naked peptides, recombinant proteins, tumour cells, viral vectors), with an objective response rate of 7.1% [3]. Since then we have learnt that immature DC generally induce tolerance rather than stimulate immunity, thus most trials now incorporate TLR ligands and or cytokines to specifically activate DC. The poor migratory capacity of early DC vaccines is being overcome by intranodal injection and optimising DC numbers. Recent analyses of trials in advanced melanoma, prostate cancer and renal cell cancer show objective clinical response rates ranging from 7.7% to 12.7%, with an overall clinical benefit rate of 30–54% when stable disease is included [4,5]. How clinical benefit is best evaluated is also being addressed. Overall survival (OS) may be a more appropriate measure of immunotherapy efficacy than measuring short term tumour regression [2]. Where OS has been monitored there is clear evidence that DC immunotherapy can have clinical benefit. The first DC therapy received FDA approval in 2010 having demonstrated a significant survival benefit in Phase III trials for metastatic prostate cancer (Sipuleucel T, Dendreon [6]). Phase III trials with survival as the primary outcome are underway for advanced glioma (DCVax-L UCI-08-16, NCT00045968, Northwest Biotherapeutics), renal cell cancer (NCT01582672, Argos Therapeutics) and melanoma (NCT01875653).
Which DC subsets are important for antitumour immunity? The emerging complexity of the DC network is an important consideration for the design of new generation DC vaccines. In human and mouse, multiple DC subsets exist that vary in location, phenotype and specialised function [7]. They can be broadly classified as conventional DC (cDC), plasmacytoid DC (pDC) and www.sciencedirect.com
Dendritic cells and cancer immunotherapy Radford, Tullett and Lahoud 27
inflammatory monocyte-derived (Mo) DC. The cDC can be further divided based on location into ‘lymphoid-resident’ and ‘migratory’ DC. The lymphoidresident DC capture Ag directly from the blood, lymph or other DC, whereas the migratory DC reside in the peripheral organs (e.g. lung, skin and gut) where they capture Ag then migrate to lymphoid tissues, and present Ag directly to T cells, or share Ag with lymphoidresident DC [7]. In both locations, cDC can be further segregated into multiple subsets with significant functional specialisation. There is now compelling evidence in mice that the lymphoid resident CD8+ cDC subset and the related migratory CD103+ cDC are essential for priming naı¨ve CD8+ T cell responses leading to the clearance of tumours [8–10]. In these models, tumour regression is also dependent on Type I IFN and accumulation of CD8+ DC at the tumour site [9,10]. The human equivalent of the mouse lymphoid CD8+/migratory CD103+ DC is the CD141+ (BDCA-3) DC subset found in lymphoid and peripheral tissues [11–16]. These DC excel at crosspresentation of cellular Ag, the process by which CD8+ T cell responses to tumour Ag, are widely considered to be generated. Human CD141+ DC and mouse CD8+/ CD103+ DC subsets share expression of chemokine receptor XCR1, and nectin-like molecule 2, Necl2/ CADM1, which are important for CTL priming, and Toll-receptor (TLR)-3, a known enhancer of cross-priming [7,12,13]. In particular, human CD141+ DC and mouse CD8+/CD103+ DC share expression of C-type lectin (CLR), CLEC9A, which recognises actin filaments exposed by dead cells, and mediates cross-presentation of Ag from dead cells [17,18,19]. These findings provide a strong rationale for utilising human CD141+ DC for immunotherapy. Whilst mouse CD8+/human CD141+ DC subsets are clearly important for tumour immunity a role for other DC subsets cannot be excluded. Evidence suggests that human CD141+ DC and mouse CD8+ DC are not the only DC subsets that can cross-present [20,21,22,23]. Harnessing DC subsets specialised in the induction of CD4+ T cell responses should also be an important consideration for generating tumour immunity [24]. The mouse CD11b+ subset may be most effective in this regard [7], but a similar role for its human CD1c+ cDC counterpart has not yet been established. The presence of pDC is generally associated with poor prognosis and tolerance in tumours [25,26]. However, activated Type I IFN-producing pDC have recently been shown to generate antitumour immunity in vitro [27,28] and in vivo, following intranodal injection in a Phase I trial in advanced melanoma patients [27]. The majority of DC clinical trials to date have employed DC derived in vitro in the presence of GM-CSF and IL-4 www.sciencedirect.com
(MoDC). Despite their long history in the clinic, there is a paucity of information on their physiological equivalent in humans and even less on their role in anti-cancer immune responses. MoDC resemble the inflammatory DC subtype, which develop rapidly from monocytes in response to inflammation or infection [29]. A DC subtype resembling MoDC in morphology and phenotype were recently found to be crucial for the induction of chemotherapy-induced anti-cancer immune responses within the tumour site in mice, providing the first evidence for a role of MoDC in tumour immunity [30]. Conversely, an equivalent subset recently identified in the ascitic fluid in human breast and ovarian cancer patients was proposed to contribute to disease pathogenesis [29].
Exploiting DC pattern recognition receptors for cancer immunotherapy DC subsets express a range of unique and shared pattern recognition receptors (PRR), including CLRs and TLRs that can be harnessed to enhance the efficacy of cancer immunotherapy (Figure 1) [31]. Monoclonal antibodies (mAbs) specific for CLRs can be used to target Ag directly to particular DC subset(s) in vivo [31–33]. This attractive approach circumvents the issues of poor DC migration and logistics associated with in vitro-manufactured, patient-specific vaccines, in addition to allowing precision in specifically targeting single or multiple DC subsets in vivo. DEC-205, a CLR that is highly expressed by mouse CD8+ DC, has been a major focus for the development of cancer vaccines. Delivery of tumour Ag using DEC205-specific mAb stimulated CD4+ and CD8+ T cell responses, resulting in rejection or delayed growth of established tumours, and was superior to ex vivo loaded DC [34–36]. Anti-DEC-205 mAbs are now being evaluated in Phase I/II clinical trials for advanced cancer patients expressing the tumour Ag NY-ESO-1 (CDX1401, CellDex Therapeutics; NCT00948961). In humans, MoDC, CD141+ DC, CD1c+ DC and pDC express DEC-205 and all have been shown to stimulate CD4+ and CD8+ T cell responses following DEC-205 mAb-mediated delivery of Ag in vitro [22,23,37,38,39]. Although formal side-by-side comparisons of all human DC subsets are yet to be done, emerging evidence suggests that it is the CD141+ DC subset, like their mouse counterpart, that is particularly effective at cross-presenting Ag delivered by DEC-205 to CD8+ T cells [22,39]. This is likely because of their superior ability compared to other DC to cross-present Ag from late endosomal compartments, to where DEC-205 traffics [22,39]. An attractive candidate for specifically targeting the CD141+ DC subset is the dead cell recognition receptor, CLEC9A. Anti-Clec9A mAbs are exceptional at delivering Current Opinion in Immunology 2014, 27:26–32
28 Tumour immunology
Figure 1
CD141DC FcR
CD1cDC
NECL2
DEC205
FcR DEC205
XCR1
CD11c CLEC9A CD40
CD11c CD40 DCIR TLR-8
TLR-8
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TLR-4
TLR-3
low
pDC FcR
TLR-3 low
LC/dermal DC CD303
DEC205
FcR
CD123
DCIR
DEC205 DCIR
BDCA-2
CD40
Langerin
CD40
TLR-7 TLR-9
TLR-3
MoDC FcR DEC205
DC-SIGN
CD11c CD40
MR
DCIR TLR-8 TLR-7 low
TLR-4 TLR-3 low Current Opinion in Immunology
Expression of antigen uptake receptors and TLR being evaluated for immunotherapy by human DC subsets. Antigen uptake receptors (including CLRs) common to multiple DC subsets are shown in blue and receptors unique to particular subsets are shown in green. TLRs expressed by multiple subsets are shown in red and those unique to particular subsets are shown in yellow.
Ag to mouse CD8+ DC in vivo for the induction of potent humoral and CTL responses and importantly, protective anti-tumour responses [40,41]. Whilst comparable to DEC-205 in terms of CTL induction, the specificity of Clec9A expression by mouse CD8+ DC, results in persistence of mAb in the bloodstream, prolonged Ag presentation by DC and superior humoral immune responses [42,43]. Targeting CLEC9A on human CD141+ DC induces both CD4+ and CD8+ T cell responses in vitro Current Opinion in Immunology 2014, 27:26–32
[44] and warrants further studies to determine the efficacy of this approach in vivo. One of the crucial lessons from early DC trials and mouse DC targeting studies is the requirement of DC activation for optimal CTL responses. DC express an array of TLRs that can be stimulated to enhance vaccine immunogenicity (Figure 1). Different TLR expression by DC subsets provides specificity for activation, but this differs www.sciencedirect.com
Dendritic cells and cancer immunotherapy Radford, Tullett and Lahoud 29
between mouse and man, complicating translation. In mice, TLR9 is expressed by all DC subsets and its ligand CpG is an effective vaccine adjuvant. In humans, clinical trials have demonstrated that CpG can boost humoral and cell-mediated vaccine responses, and is mostly tolerated, although some studies have reported increased adverse events [45]. However, TLR9 expression in humans is restricted to pDC and B cells. Thus CpG will likely be a more effective adjuvant in combination with human vaccines targeting pDC rather than cDC. The TLR3 ligand, poly I:C, is gaining popularity as a clinical vaccine adjuvant. It activates CD141+ DC, enhances cross-presentation and elicits Type I IFN responses [11,46]. Poly I:C derivatives Hiltonol and Ampligen, are well tolerated in humans, elicit potent Type I IFN responses [47] and are now being trialled in combination with DEC-205 targeting Ab (CellDex Therapeutics; NCT00948961). The TLR7/8 agonist, Resiquimod, activates multiple DC subtypes in humans and is also being trialled with DEC-205 Abs, alone or in combination with Hiltonol. Whilst current approaches consist of a DC targeting mAbtumour Ag fusion construct co-administered with TLR agonists, the development of nanoparticles, liposomes and nanoemulsions expressing the DC receptor mAb are emerging as attractive alternatives to incorporate multiple Ag, adjuvants and targeting specificity into a single delivery vehicle [31,48].
Enhancing immunogenicity of the tumour environment One of the major impediments to the induction of effective anti-tumour responses is overcoming the suppressive nature of the tumour environment. Tumours employ cellular and soluble factors that directly suppress DC and T cell activation [49,50], thus DC therapies need to be combined with mechanisms to boost the immunogenicity of the tumour environment, and enhance DC function. DC express PRR that recognise damage-associated molecular patterns (DAMPs), intracellular components revealed by dying or damaged tumour cells, including calreticulin, filamentous actin, high mobility group box 1 protein (HMGB1) and ATP. DAMPs can act to enhance DC migration, activation or facilitate processing of tumour-derived Ag [51,52]. Some chemotherapies and radiotherapy stimulate ‘immunogenic cell death’ of tumour cells by revealing DAMPS that enhance DC function [53]. Chemotherapy can also directly enhance DC functions including activation, migration and Ag presentation resulting in more potent anti-tumour responses [53]. This provides a strong rationale for combining DC vaccines with chemotherapy and/or radiotherapy. Early trials demonstrate that this approach is feasible, well tolerated, and induces clinical responses in advanced ovarian cancer [54], pancreatic cancer [55], colorectal, renal hepatocellular cancers, melanoma [56], and high grade glioma patients [57,58]. Notably in metastatic prostate cancer and high-grade glioma, combination www.sciencedirect.com
therapy is clinically beneficial when DC vaccines are administered before, rather than after, chemotherapy [6,59]. This highlights the need for careful scheduling and optimisation to maximise the benefits of combined therapies.
Overcoming immune regulation One of the most exciting recent advances in cancer immunotherapy has been the development of Ab against negative regulators of T cell function, CTLA-4 and PD-1 [60,61]. mAbs against both CTLA-4 and PD-1 induce strikingly high and durable clinical responses in melanoma and other tumours, but CTLA-4 treatment is accompanied by significant toxicity and adverse effects [60]. Combining DC immunotherapy with anti-CTLA-4 is feasible and well tolerated in advanced melanoma patients and induces a higher rate of clinical responses than could be expected with either treatment alone [61]. Abs against PD-1 are predicted to be similarly synergistic in combination with other vaccines [60]. In animal models, overcoming immune suppression is also being addressed by decreasing numbers and/or function of myeloid-derived suppressor cells and regulatory T cells, augmentation of T cell function with agonists to costimulatory molecules such as CD137, and blockade of inhibitory cytokines such as IL-10 and TGF-b [50,62]. Combining these strategies with DC immunotherapy will be a promising area for future investigation.
Concluding remarks In recent years, DC immunotherapy has moved from a simple concept of patient specific, in vitro generated vaccines towards a sophisticated approach of targeting Ag and activators to specialised DC subsets directly in vivo. The results of ongoing clinical trials using DEC-205 targeting in combination with TLR3 and TLR7/8 agonists are eagerly anticipated. Combining this approach with chemotherapy, radiotherapy or Ab against key immune checkpoint inhibitors will likely enhance efficacy by providing a more immunogenic environment for DC to function. Although our understanding of the complexity and subset specialisation of the DC network has increased exponentially, the role of specific DC subsets in tumour progression and immunity, particularly in humans, is still in its infancy. There is a strong rationale for targeting human CD141+ DC but more work is needed to establish the contribution of other DC subsets and how they can be best manipulated to improve therapeutic outcomes.
Acknowledgements MHL and KJR are supported by project grants from the National Health and Medical Research Council of Australia (NHMRC 604306 and 1025201) and the Prostate Cancer Foundation of Australia (PG2110). KJR holds a NHMRC CDF level 2 fellowship. KMT is the recipient of a University of Queensland International PhD Scholarship. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support Scheme. Current Opinion in Immunology 2014, 27:26–32
30 Tumour immunology
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to langerin, DEC205, and clec9a. Proc Natl Acad Sci U S A 2011, 108:2384-2389. 43. Lahoud MH, Ahmet F, Kitsoulis S, Wan SS, Vremec D, Lee CN, Phipson B, Shi W, Smyth GK, Lew AM et al.: Targeting antigen to mouse dendritic cells via Clec9A induces potent CD4 T cell responses biased toward a follicular helper phenotype. J Immunol 2011, 187:842-850. 44. Schreibelt G, Klinkenberg LJ, Cruz LJ, Tacken PJ, Tel J, Kreutz M, Adema GJ, Brown GD, Figdor CG, de Vries IJ: The C-type lectin receptor CLEC9A mediates antigen uptake and (cross)presentation by human blood BDCA3+ myeloid dendritic cells. Blood 2012, 119:2284-2292. 45. Bode C, Zhao G, Steinhagen F, Kinjo T, Klinman DM: CpG DNA as a vaccine adjuvant. Expert Rev Vaccines 2011, 10:499-511. 46. Meixlsperger S, Leung CS, Ramer PC, Pack M, Vanoaica LD, Breton G, Pascolo S, Salazar AM, Dzionek A, Schmitz J et al.: CD141+ dendritic cells produce prominent amounts of IFNalpha after dsRNA recognition and can be targeted via DEC205 in humanized mice. Blood 2013, 121:5034-5044. 47. Caskey M, Lefebvre F, Filali-Mouhim A, Cameron MJ, Goulet JP, Haddad EK, Breton G, Trumpfheller C, Pollak S, Shimeliovich I et al.: Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J Exp Med 2011, 208:2357-2366. 48. Zeng BJ, Chuan YP, O’Sullivan B, Caminschi I, Lahoud MH, Thomas R, Middelberg AP: Receptor-specific delivery of protein antigen to dendritic cells by a nanoemulsion formed using topdown non-covalent click self-assembly. Small 2013, 9:3736-3742. 49. Scarlett UK, Rutkowski MR, Rauwerdink AM, Fields J, EscovarFadul X, Baird J, Cubillos-Ruiz JR, Jacobs AC, Gonzalez JL, Weaver J et al.: Ovarian cancer progression is controlled by phenotypic changes in dendritic cells. J Exp Med 2012, 209:495-506. 50. Motz GT, Coukos G: Deciphering and reversing tumor immune suppression. Immunity 2013, 39:61-73. 51. Krysko O, Love Aaes T, Bachert C, Vandenabeele P, Krysko DV: Many faces of DAMPs in cancer therapy. Cell Death Dis 2013, 4:e631. 52. Kroemer G, Galluzzi L, Kepp O, Zitvogel L: Immunogenic cell death in cancer therapy. Annu Rev Immunol 2013, 31:51-72. 53. Galluzzi L, Senovilla L, Zitvogel L, Kroemer G: The secret ally: immunostimulation by anticancer drugs. Nat Rev Drug Discov 2012, 11:215-233. This review discusses the potential of chemotheraputic drugs to directly enhance immune cell function. 54. Chu CS, Boyer J, Schullery DS, Gimotty PA, Gamerman V, Bender J, Levine BL, Coukos G, Rubin SC, Morgan MA et al.: Phase I/II randomized trial of dendritic cell vaccination with or without cyclophosphamide for consolidation therapy of advanced ovarian cancer in first or second remission. Cancer Immunol Immunother 2012, 61:629-641. 55. Kimura Y, Tsukada J, Tomoda T, Takahashi H, Imai K, Shimamura K, Sunumura M, Yonemitsu Y, Shimodaira S, Koido S et al.: Clinical and immunologic evaluation of dendritic cellbased immunotherapy in combination with gemcitabine and/ or S-1 in patients with advanced pancreatic carcinoma. Pancreas 2012, 41:195-205. 56. Ellebaek E, Engell-Noerregaard L, Iversen TZ, Froesig TM, Munir S, Hadrup SR, Andersen MH, Svane IM: Metastatic melanoma patients treated with dendritic cell vaccination, interleukin-2 and metronomic cyclophosphamide: results from a phase II trial. Cancer Immunol Immunother 2012, 61:1791-1804. 57. Ardon H, Van Gool SW, Verschuere T, Maes W, Fieuws S, Sciot R, Wilms G, Demaerel P, Goffin J, Van Calenbergh F et al.: Integration of autologous dendritic cell-based immunotherapy in the standard of care treatment for patients with newly diagnosed glioblastoma: results of the
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HGG-2006 phase I/II trial. Cancer Immunol Immunother 2012, 61:2033-2044. 58. Phuphanich S, Wheeler CJ, Rudnick JD, Mazer M, Wang H, Nuno MA, Richardson JE, Fan X, Ji J, Chu RM et al.: Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma. Cancer Immunol Immunother 2013, 62:125-135. 59. Slovin SR: Toward maximizing immunotherapy in metastatic castration-resistant prostate cancer — rationale for combinatorial approaches using chemotherapy. Front Oncol 2012, 2:43.
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60. Hersey P, Gallagher S: A focus on PD-L1 in human melanoma. Clin Cancer Res 2013, 19:514-516. 61. Ribas A, Comin-Anduix B, Chmielowski B, Jalil J, de la Rocha P, McCannel TA, Ochoa MT, Seja E, Villanueva A, Oseguera DK et al.: Dendritic cell vaccination combined with CTLA4 blockade in patients with metastatic melanoma. Clin Cancer Res 2009, 15:6267-6276. 62. Vanneman M, Dranoff G: Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev Cancer 2012, 12:237-251.
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ScienceDirect Dendritic cells and cancer immunotherapy Kristen J Radford1,2, Kirsteen M Tullett1,3,4 and Mireille H Lahoud4,5 Dendritic cells (DC) play an essential role in the induction and regulation of immune responses, including the generation of cytotoxic T lymphocytes (CTL) for the eradication of cancers. DC-based cancer vaccines are well tolerated with few side effects and can generate anti-tumour immune responses, but overall they have been of limited benefit. Recent studies have demonstrated that CD141+ DC play an important role in antitumour responses. These are now attractive targets for the development of vaccines that directly target DC in vivo. An understanding of the functional specialisations of DC subsets, strategies for the delivery of tumour Ag to DC and for enhancing immune responses, point to promising new avenues for the design of more effective DC-based cancer vaccines. Addresses 1 Mater Research Institute, University of Queensland, Translational Research Institute, Brisbane, Australia 2 University of Queensland, School of Biomedical Sciences, Brisbane, Australia 3 University of Queensland, School of Medicine, Brisbane, Australia 4 Centre for Biomedical Research, Burnet Institute, Melbourne, Australia 5 Department of Immunology, Monash University, Melbourne, Australia Corresponding author: Lahoud, Mireille H ([email protected])
Current Opinion in Immunology 2014, 27:26–32 This review comes from a themed issue on Tumour immunology Edited by Philip K Darcy and David S Ritchie
0952-7915/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coi.2014.01.005
Introduction Dendritic cells (DC) play a key role in initiating and maintaining immune responses. Since the discovery of DC by Ralph Steinman over 40 years ago [1] and the identification of their key function as mediators of T cellmediated immune responses, there has been a major focus on the use of DC in cancer immunotherapy. DC have been used to vaccinate cancer patients for nearly 20 years [2]. Until recently, most DC vaccines comprised DC or monocyte precursors of DC, isolated from the patient, loaded ex vivo with tumour antigen (Ag), and readministered to the patient. DC that migrated from the injection site to the draining lymph nodes were expected to prime naı¨ve, and or boost memory, tumour-specific T cells capable of eradicating the tumour. To date, the majority of trials have been Phase I studies on small cohorts of advanced cancer patients who had failed to respond to conventional therapies. These trials revealed Current Opinion in Immunology 2014, 27:26–32
that this approach: (1) is feasible in many malignancies; (2) is well tolerated with minimal toxicity; and (3) can induce tumour-specific immune responses in many patients. Whilst early DC therapies resulted in limited clinical benefits, recent advances in our understanding of DC biology and new knowledge obtained from clinical trials have identified new strategies that are expected to improve clinical outcomes. Harnessing the unique capacity of different DC subtypes to drive specific immune responses in combinations with approaches designed to overcome tumour-mediated immune suppression and immune regulation, are emerging as key strategies for the development of new generation DC vaccines.
Can DC vaccines be of clinical benefit? Early clinical studies showed a significant advantage of using DC vaccines over indirect vaccine approaches (e.g. naked peptides, recombinant proteins, tumour cells, viral vectors), with an objective response rate of 7.1% [3]. Since then we have learnt that immature DC generally induce tolerance rather than stimulate immunity, thus most trials now incorporate TLR ligands and or cytokines to specifically activate DC. The poor migratory capacity of early DC vaccines is being overcome by intranodal injection and optimising DC numbers. Recent analyses of trials in advanced melanoma, prostate cancer and renal cell cancer show objective clinical response rates ranging from 7.7% to 12.7%, with an overall clinical benefit rate of 30–54% when stable disease is included [4,5]. How clinical benefit is best evaluated is also being addressed. Overall survival (OS) may be a more appropriate measure of immunotherapy efficacy than measuring short term tumour regression [2]. Where OS has been monitored there is clear evidence that DC immunotherapy can have clinical benefit. The first DC therapy received FDA approval in 2010 having demonstrated a significant survival benefit in Phase III trials for metastatic prostate cancer (Sipuleucel T, Dendreon [6]). Phase III trials with survival as the primary outcome are underway for advanced glioma (DCVax-L UCI-08-16, NCT00045968, Northwest Biotherapeutics), renal cell cancer (NCT01582672, Argos Therapeutics) and melanoma (NCT01875653).
Which DC subsets are important for antitumour immunity? The emerging complexity of the DC network is an important consideration for the design of new generation DC vaccines. In human and mouse, multiple DC subsets exist that vary in location, phenotype and specialised function [7]. They can be broadly classified as conventional DC (cDC), plasmacytoid DC (pDC) and www.sciencedirect.com
Dendritic cells and cancer immunotherapy Radford, Tullett and Lahoud 27
inflammatory monocyte-derived (Mo) DC. The cDC can be further divided based on location into ‘lymphoid-resident’ and ‘migratory’ DC. The lymphoidresident DC capture Ag directly from the blood, lymph or other DC, whereas the migratory DC reside in the peripheral organs (e.g. lung, skin and gut) where they capture Ag then migrate to lymphoid tissues, and present Ag directly to T cells, or share Ag with lymphoidresident DC [7]. In both locations, cDC can be further segregated into multiple subsets with significant functional specialisation. There is now compelling evidence in mice that the lymphoid resident CD8+ cDC subset and the related migratory CD103+ cDC are essential for priming naı¨ve CD8+ T cell responses leading to the clearance of tumours [8–10]. In these models, tumour regression is also dependent on Type I IFN and accumulation of CD8+ DC at the tumour site [9,10]. The human equivalent of the mouse lymphoid CD8+/migratory CD103+ DC is the CD141+ (BDCA-3) DC subset found in lymphoid and peripheral tissues [11–16]. These DC excel at crosspresentation of cellular Ag, the process by which CD8+ T cell responses to tumour Ag, are widely considered to be generated. Human CD141+ DC and mouse CD8+/ CD103+ DC subsets share expression of chemokine receptor XCR1, and nectin-like molecule 2, Necl2/ CADM1, which are important for CTL priming, and Toll-receptor (TLR)-3, a known enhancer of cross-priming [7,12,13]. In particular, human CD141+ DC and mouse CD8+/CD103+ DC share expression of C-type lectin (CLR), CLEC9A, which recognises actin filaments exposed by dead cells, and mediates cross-presentation of Ag from dead cells [17,18,19]. These findings provide a strong rationale for utilising human CD141+ DC for immunotherapy. Whilst mouse CD8+/human CD141+ DC subsets are clearly important for tumour immunity a role for other DC subsets cannot be excluded. Evidence suggests that human CD141+ DC and mouse CD8+ DC are not the only DC subsets that can cross-present [20,21,22,23]. Harnessing DC subsets specialised in the induction of CD4+ T cell responses should also be an important consideration for generating tumour immunity [24]. The mouse CD11b+ subset may be most effective in this regard [7], but a similar role for its human CD1c+ cDC counterpart has not yet been established. The presence of pDC is generally associated with poor prognosis and tolerance in tumours [25,26]. However, activated Type I IFN-producing pDC have recently been shown to generate antitumour immunity in vitro [27,28] and in vivo, following intranodal injection in a Phase I trial in advanced melanoma patients [27]. The majority of DC clinical trials to date have employed DC derived in vitro in the presence of GM-CSF and IL-4 www.sciencedirect.com
(MoDC). Despite their long history in the clinic, there is a paucity of information on their physiological equivalent in humans and even less on their role in anti-cancer immune responses. MoDC resemble the inflammatory DC subtype, which develop rapidly from monocytes in response to inflammation or infection [29]. A DC subtype resembling MoDC in morphology and phenotype were recently found to be crucial for the induction of chemotherapy-induced anti-cancer immune responses within the tumour site in mice, providing the first evidence for a role of MoDC in tumour immunity [30]. Conversely, an equivalent subset recently identified in the ascitic fluid in human breast and ovarian cancer patients was proposed to contribute to disease pathogenesis [29].
Exploiting DC pattern recognition receptors for cancer immunotherapy DC subsets express a range of unique and shared pattern recognition receptors (PRR), including CLRs and TLRs that can be harnessed to enhance the efficacy of cancer immunotherapy (Figure 1) [31]. Monoclonal antibodies (mAbs) specific for CLRs can be used to target Ag directly to particular DC subset(s) in vivo [31–33]. This attractive approach circumvents the issues of poor DC migration and logistics associated with in vitro-manufactured, patient-specific vaccines, in addition to allowing precision in specifically targeting single or multiple DC subsets in vivo. DEC-205, a CLR that is highly expressed by mouse CD8+ DC, has been a major focus for the development of cancer vaccines. Delivery of tumour Ag using DEC205-specific mAb stimulated CD4+ and CD8+ T cell responses, resulting in rejection or delayed growth of established tumours, and was superior to ex vivo loaded DC [34–36]. Anti-DEC-205 mAbs are now being evaluated in Phase I/II clinical trials for advanced cancer patients expressing the tumour Ag NY-ESO-1 (CDX1401, CellDex Therapeutics; NCT00948961). In humans, MoDC, CD141+ DC, CD1c+ DC and pDC express DEC-205 and all have been shown to stimulate CD4+ and CD8+ T cell responses following DEC-205 mAb-mediated delivery of Ag in vitro [22,23,37,38,39]. Although formal side-by-side comparisons of all human DC subsets are yet to be done, emerging evidence suggests that it is the CD141+ DC subset, like their mouse counterpart, that is particularly effective at cross-presenting Ag delivered by DEC-205 to CD8+ T cells [22,39]. This is likely because of their superior ability compared to other DC to cross-present Ag from late endosomal compartments, to where DEC-205 traffics [22,39]. An attractive candidate for specifically targeting the CD141+ DC subset is the dead cell recognition receptor, CLEC9A. Anti-Clec9A mAbs are exceptional at delivering Current Opinion in Immunology 2014, 27:26–32
28 Tumour immunology
Figure 1
CD141DC FcR
CD1cDC
NECL2
DEC205
FcR DEC205
XCR1
CD11c CLEC9A CD40
CD11c CD40 DCIR TLR-8
TLR-8
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TLR-4
TLR-3
low
pDC FcR
TLR-3 low
LC/dermal DC CD303
DEC205
FcR
CD123
DCIR
DEC205 DCIR
BDCA-2
CD40
Langerin
CD40
TLR-7 TLR-9
TLR-3
MoDC FcR DEC205
DC-SIGN
CD11c CD40
MR
DCIR TLR-8 TLR-7 low
TLR-4 TLR-3 low Current Opinion in Immunology
Expression of antigen uptake receptors and TLR being evaluated for immunotherapy by human DC subsets. Antigen uptake receptors (including CLRs) common to multiple DC subsets are shown in blue and receptors unique to particular subsets are shown in green. TLRs expressed by multiple subsets are shown in red and those unique to particular subsets are shown in yellow.
Ag to mouse CD8+ DC in vivo for the induction of potent humoral and CTL responses and importantly, protective anti-tumour responses [40,41]. Whilst comparable to DEC-205 in terms of CTL induction, the specificity of Clec9A expression by mouse CD8+ DC, results in persistence of mAb in the bloodstream, prolonged Ag presentation by DC and superior humoral immune responses [42,43]. Targeting CLEC9A on human CD141+ DC induces both CD4+ and CD8+ T cell responses in vitro Current Opinion in Immunology 2014, 27:26–32
[44] and warrants further studies to determine the efficacy of this approach in vivo. One of the crucial lessons from early DC trials and mouse DC targeting studies is the requirement of DC activation for optimal CTL responses. DC express an array of TLRs that can be stimulated to enhance vaccine immunogenicity (Figure 1). Different TLR expression by DC subsets provides specificity for activation, but this differs www.sciencedirect.com
Dendritic cells and cancer immunotherapy Radford, Tullett and Lahoud 29
between mouse and man, complicating translation. In mice, TLR9 is expressed by all DC subsets and its ligand CpG is an effective vaccine adjuvant. In humans, clinical trials have demonstrated that CpG can boost humoral and cell-mediated vaccine responses, and is mostly tolerated, although some studies have reported increased adverse events [45]. However, TLR9 expression in humans is restricted to pDC and B cells. Thus CpG will likely be a more effective adjuvant in combination with human vaccines targeting pDC rather than cDC. The TLR3 ligand, poly I:C, is gaining popularity as a clinical vaccine adjuvant. It activates CD141+ DC, enhances cross-presentation and elicits Type I IFN responses [11,46]. Poly I:C derivatives Hiltonol and Ampligen, are well tolerated in humans, elicit potent Type I IFN responses [47] and are now being trialled in combination with DEC-205 targeting Ab (CellDex Therapeutics; NCT00948961). The TLR7/8 agonist, Resiquimod, activates multiple DC subtypes in humans and is also being trialled with DEC-205 Abs, alone or in combination with Hiltonol. Whilst current approaches consist of a DC targeting mAbtumour Ag fusion construct co-administered with TLR agonists, the development of nanoparticles, liposomes and nanoemulsions expressing the DC receptor mAb are emerging as attractive alternatives to incorporate multiple Ag, adjuvants and targeting specificity into a single delivery vehicle [31,48].
Enhancing immunogenicity of the tumour environment One of the major impediments to the induction of effective anti-tumour responses is overcoming the suppressive nature of the tumour environment. Tumours employ cellular and soluble factors that directly suppress DC and T cell activation [49,50], thus DC therapies need to be combined with mechanisms to boost the immunogenicity of the tumour environment, and enhance DC function. DC express PRR that recognise damage-associated molecular patterns (DAMPs), intracellular components revealed by dying or damaged tumour cells, including calreticulin, filamentous actin, high mobility group box 1 protein (HMGB1) and ATP. DAMPs can act to enhance DC migration, activation or facilitate processing of tumour-derived Ag [51,52]. Some chemotherapies and radiotherapy stimulate ‘immunogenic cell death’ of tumour cells by revealing DAMPS that enhance DC function [53]. Chemotherapy can also directly enhance DC functions including activation, migration and Ag presentation resulting in more potent anti-tumour responses [53]. This provides a strong rationale for combining DC vaccines with chemotherapy and/or radiotherapy. Early trials demonstrate that this approach is feasible, well tolerated, and induces clinical responses in advanced ovarian cancer [54], pancreatic cancer [55], colorectal, renal hepatocellular cancers, melanoma [56], and high grade glioma patients [57,58]. Notably in metastatic prostate cancer and high-grade glioma, combination www.sciencedirect.com
therapy is clinically beneficial when DC vaccines are administered before, rather than after, chemotherapy [6,59]. This highlights the need for careful scheduling and optimisation to maximise the benefits of combined therapies.
Overcoming immune regulation One of the most exciting recent advances in cancer immunotherapy has been the development of Ab against negative regulators of T cell function, CTLA-4 and PD-1 [60,61]. mAbs against both CTLA-4 and PD-1 induce strikingly high and durable clinical responses in melanoma and other tumours, but CTLA-4 treatment is accompanied by significant toxicity and adverse effects [60]. Combining DC immunotherapy with anti-CTLA-4 is feasible and well tolerated in advanced melanoma patients and induces a higher rate of clinical responses than could be expected with either treatment alone [61]. Abs against PD-1 are predicted to be similarly synergistic in combination with other vaccines [60]. In animal models, overcoming immune suppression is also being addressed by decreasing numbers and/or function of myeloid-derived suppressor cells and regulatory T cells, augmentation of T cell function with agonists to costimulatory molecules such as CD137, and blockade of inhibitory cytokines such as IL-10 and TGF-b [50,62]. Combining these strategies with DC immunotherapy will be a promising area for future investigation.
Concluding remarks In recent years, DC immunotherapy has moved from a simple concept of patient specific, in vitro generated vaccines towards a sophisticated approach of targeting Ag and activators to specialised DC subsets directly in vivo. The results of ongoing clinical trials using DEC-205 targeting in combination with TLR3 and TLR7/8 agonists are eagerly anticipated. Combining this approach with chemotherapy, radiotherapy or Ab against key immune checkpoint inhibitors will likely enhance efficacy by providing a more immunogenic environment for DC to function. Although our understanding of the complexity and subset specialisation of the DC network has increased exponentially, the role of specific DC subsets in tumour progression and immunity, particularly in humans, is still in its infancy. There is a strong rationale for targeting human CD141+ DC but more work is needed to establish the contribution of other DC subsets and how they can be best manipulated to improve therapeutic outcomes.
Acknowledgements MHL and KJR are supported by project grants from the National Health and Medical Research Council of Australia (NHMRC 604306 and 1025201) and the Prostate Cancer Foundation of Australia (PG2110). KJR holds a NHMRC CDF level 2 fellowship. KMT is the recipient of a University of Queensland International PhD Scholarship. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support Scheme. Current Opinion in Immunology 2014, 27:26–32
30 Tumour immunology
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