associate editor: michael g. rosenblum dendritic...
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1521-0081/67/4/731–753$25.00 http://dx.doi.org/10.1124/pr.114.009456PHARMACOLOGICAL REVIEWS Pharmacol Rev 67:731–753, October 2015Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
ASSOCIATE EDITOR: MICHAEL G. ROSENBLUM
Dendritic Cells as Pharmacological Tools for CancerImmunotherapys
Sébastien Anguille, Evelien L. Smits, Christian Bryant, Heleen H. Van Acker, Herman Goossens, Eva Lion, Phillip D. Fromm,Derek N. Hart, Viggo F. Van Tendeloo, and Zwi N. Berneman
Faculty of Medicine and Health Sciences, Vaccine & Infectious Disease Institute, Laboratory of Experimental Hematology, TumorImmunology Group (S.A., H.H.V.A., H.G., E.L., V.F.V.T., Z.N.B.), and Faculty of Medicine and Health Sciences, Center for Oncological
Research (E.L.S.), University of Antwerp, Antwerp, Belgium; Center for Cell Therapy & Regenerative Medicine, Antwerp University Hospital,Antwerp, Belgium (S.A., E.L.S., Z.N.B.); and ANZAC Research Institute, Dendritic Cell Biology and Therapeutics Group, University of
Sydney, Sydney, New South Wales, Australia (C.B., P.D.F., D.N.H.)
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732I. Cancer Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732II. Dendritic Cell–Based Cancer Immunotherapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
A. Immunobiological Aspects of Dendritic Cell–Based Cancer Immunotherapy . . . . . . . . . . . . . . . 734B. Pharmacological Aspects of Dendritic Cell–Based Cancer Immunotherapy. . . . . . . . . . . . . . . . . 736
III. Toward the Delivery of Effective Dendritic Cell–Based Cancer Immunotherapy: The AcuteMyeloid Leukemia Showcase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739A. Using Dendritic Cells with Optimal Immunocompetence (Condition Number 1) . . . . . . . . . . . 740
1. Source of DC Precursor Cell.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7402. Culture Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7403. Culture Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7404. Differentiation Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7405. Maturation Protocol.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7406. Choice of Target Antigen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7427. Source of Antigen(s).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7428. Antigen-Loading Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7439. Designer Dendritic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74410. Route of Administration.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
B. Enhancing the Strength of the Antitumor Immune Effector Cells (Condition Number 2) . . . 7441. Enhancing T-Cell–Mediated Antitumor Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
a. Cytotoxic T-lymphocyte antigen‐4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745b. Programmed death-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745c. B and T lymphocyte attenuator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745d. Immunosuppressive cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745
2. Enhancing Natural Killer Cell–Mediated Antitumor Immunity.. . . . . . . . . . . . . . . . . . . . . . . . 745C. Making Tumor Cells Susceptible to Immune Attack (Condition Number 3) . . . . . . . . . . . . . . . . 746
1. Targeting Programmed Death-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7462. Targeting Indoleamine 2,3-Dioxygenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7463. Targeting CD200. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
This work was supported by grants of the Research Foundation Flanders (FWO Vlaanderen, Grant G039914N); the Methusalem programof the Flemish Government attributed to Goossens; the Belgian Foundation against Cancer (Stichting tegen Kanker); the Belgian PublicUtility Foundation VOCATIO; and the Belgian Hercules Foundation. S.A. is a former PhD fellow of the Research Foundation Flanders anda former holder of an Emmanuel van der Schueren Fellowship granted by the Flemish League against Cancer (Vlaamse Liga tegen Kanker).S.A. was awarded a 2012 Endeavour Research Fellowship by the Department of Industry, Innovation, Science, Research and TertiaryEducation of the Australian Government.
Address correspondence to: Dr. Sébastien Anguille, Antwerp University Hospital, Center for Cell Therapy & Regenerative Medicine(U111), Wilrijkstraat 10, 2650 Edegem, Belgium. E-mail: [email protected]
dx.doi.org/10.1124/pr.114.009456.s This article has supplemental material available at pharmrev.aspetjournals.org.
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4. Mobilizing Acute Myeloid Leukemia Cells from Immune-Privileged Bone MarrowNiches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
5. Undoing Tumor Cells’ Cloak of Invisibility.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7476. Reducing Tumor Burden. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Abstract——Although the earliest—rudimentary—attempts at exploiting the immune system for cancertherapy can be traced back to the late 18th Century,it was not until the past decade that cancerimmunotherapeutics have truly entered mainstreamclinical practice. Given their potential to stimulate bothadaptive and innate antitumor immune responses,dendritic cells (DCs) have come under intensescrutiny in recent years as pharmacological tools forcancer immunotherapy. Conceptually, the clinicaleffectiveness of this form of active immunotherapyrelies on the completion of three critical steps: 1) theDCs used as immunotherapeutic vehicles must properlyactivate the antitumor immune effector cells of thehost, 2) these immune effector cells must be receptive to
stimulation by the DCs and be competent to mediatetheir antitumor effects, which 3) requires overcomingthe various immune-inhibitorymechanisms used by thetumor cells. In this review, following a brief overviewof the pivotal milestones in the history of cancerimmunotherapy, we will introduce the reader tothe basic immunobiological and pharmacologicalprinciples of active cancer immunotherapy usingDCs. We will then discuss how current research istrying to define the optimal parameters for each ofthe above steps to realize the full clinical potential ofDC therapeutics. Given its high suitability forimmune interventions, acute myeloid leukemia waschosen here to showcase the latest research trendsdriving the field of DC-based cancer immunotherapy.
I. Cancer Immunotherapy
Although the idea of exploiting the immune systemfor cancer therapy has been around for more than twocenturies, immunotherapy has only recently becomea mainstream cancer treatment (Rosenberg, 1999).This journey culminated in immunotherapy beingnominated by the journal Science as Breakthrough ofthe Year for 2013 (Couzin-Frankel, 2013). The timelinepresented in Fig. 1 provides a nonexhaustive overviewof the pivotal milestones in the history of cancerimmunotherapy. The first known attempt to immunizeagainst cancer was made in 1777 by the surgeon to theDuke of Kent, James Nooth, who injected himself withmalignant tissue to prevent the development of cancer(Rosenberg, 1999; Ichim, 2005). Cancer immunother-apy formally started in 1891 (Ichim, 2005) (Fig. 1),when William Coley —regarded by many as the Fatherof Immunotherapy— described the regression of cancerafter injection of a bacterial preparation and postu-lated that this was due to the induction of antitumorimmunity (Coley, 1891). In 1909, Paul Ehrlich hypoth-esized that the immune system could protect the hostagainst developing cancers, thereby laying the founda-tion for the “cancer immune surveillance” theory(Ehrlich, 1909). It took another 50 years, however,before the concept of cancer immune surveillance was
developed by Lewis Thomas (Thomas, 1959) and FrankMacFarlane Burnet (Burnet, 1967). Thomas’ andBurnet’s views on the critical role of the immunesystem in preventing the development of cancer weresupported by a series of mouse tumor transplantationexperiments conducted during the mid-1940s to early-1960s (Fig. 1), which led to the hypothesis that tumorcells express specific “antigens” that permit theirrecognition and elimination by the immune system(Gross, 1943; Foley, 1953; Baldwin, 1955; Prehn andMain, 1957; Klein et al., 1960). In the followingdecades, the interactions between the immune systemand cancer cells were defined, resulting in the centraltenet of cancer immunotherapy: the finding that CD8+
effector T cells (also called cytotoxic T lymphocytes[CTLs]) are capable of selectively killing tumor cellsafter recognition of specific antigens expressed on thetumor cell surface in association with major histocom-patibility complex (MHC) molecules (Zinkernagel andDoherty, 1974a,b; Coulie et al., 2014). More recently,other immune components, such as natural killer (NK)cells, have been shown to contribute to the host defenseagainst cancer (Marcus et al., 2014).
Most currently available cancer immunotherapeuticsaim to harness the T-cell (adaptive) immune responseagainst cancer (Coulie et al., 2014). These can broadlybe divided into passive and active therapies (Mellman
ABBREVIATIONS: ACT, adoptive cell transfer; AML, acute myeloid leukemia; APC, antigen-presenting cell; BTLA, B and T lymphocyteattenuator; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T-lymphocyte antigen‐4; CXCR-4, C-X-C chemokine receptor type-4; DC, dendriticcell; GM-CSF, granulocyte-macrophage colony-stimulating factor; HMGB1, high mobility group box 1; HOCl, hypochlorous acid; HSP, heat-shock protein; IDO, indoleamine 2,3-dioxygenase; IFN, interferon; IL, interleukin; LC, Langerhans cell; mAb, monoclonal antibody; mDC,myeloid dendritic cell; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; moDC, monocyte-derived dendriticcell; NK, natural killer; PBMC, peripheral blood mononuclear cell; PD-1, programmed death-1; pDC, plasmacytoid dendritic cell; PD-L1/2,programmed death-ligand 1 or 2; PGE2, prostaglandin E2; poly(I:C), polyinosinic:polycytidylic acid; TCR, T-cell receptor; TH, T helper type;TLR, Toll-like receptor; TNF, tumor necrosis factor; Treg, regulatory T cell; WT1, Wilms’ tumor 1.
732 Anguille et al.
et al., 2011; Topalian et al., 2011). Passive immuno-therapies have been in clinical practice for some timein the form of T-cell immunomodulatory monoclonalantibodies (mAbs) and adoptive cell transfer (ACT)(Mellman et al., 2011). Ipilimumab (Yervoy; Bristol-Myers Squibb, New Brunswick, NJ) is the prototype ofthe rapidly growing family of T-cell–potentiatingmAbs. It targets cytotoxic T-lymphocyte antigen‐4(CTLA-4), which serves as an immune checkpointmolecule on T cells (i.e., a molecule involved in thenegative regulation of T-cell function) (Hodi et al.,2010; Pardoll, 2012). ACT involves the transfusion ofallogeneic or autologous tumor-reactive T cells (and/orother antitumor immune effector cells) (June, 2007).The past decade has witnessed several major advance-ments in the field of ACT, including the use of T cellsgenetically engineered to express T-cell receptors(TCRs) or chimeric antigen receptors to enable target-ing of predefined, clinically relevant tumor antigens(Park et al., 2011; Restifo et al., 2012). For detailedinformation on mAb-mediated immune checkpointblockade and ACT, we refer the reader to otheroverview articles on these topics (Pardoll, 2012;Kershaw et al., 2013).This review will focus on active immunotherapy
using tumor antigen–loaded dendritic cells (DCs) foranticancer “vaccination.” Although it is increasinglyevident that at least part of its pharmacologicalmechanism-of-action relies on activation of innateantitumor effectors (in particular NK cells) (Lionet al., 2012a; Van Elssen et al., 2014; Van Ackeret al., 2015), the basic concept behind DC therapy is toexploit the intrinsic antigen-presenting properties of
DCs to elicit a potent tumor antigen-specific T-cell–driven immune response (Anguille et al., 2014; Dattaet al., 2014). In contrast to the two aforementionedpassive cancer immunotherapy strategies, DC therapyaspires to “train” the host’s immune system to recognizeand attack cancer cells and, in so doing, enable thegeneration of immunologic memory (Rescigno et al.,2007; Melero et al., 2014). As shown in the timeline, theDC story began more than four decades ago in 1973(Fig. 1). In that year, Zanvil Cohn and Ralph Steinman(who was awarded the 2011 Nobel Prize in Medicineand Physiology for his seminal work on DCs) describeda rare population of mouse splenocytes with a distinc-tive tree-like morphology (Steinman and Cohn, 1973).The cells were therefore named dendritic cells, after theGreek word for tree (dendreon) (Steinman, 2007).Interest in the therapeutic exploitation of DCs grewafter it became apparent that DCs, in their role ashighly specialized antigen-presenting cells (APCs), areinstrumental in the development of adaptive antitumorimmunity (Palucka and Banchereau, 2012; Mellman,2013). The first clinical trial of DC-based vaccinetherapy was published in 1996 (Hsu et al., 1996) (Fig. 1).Since then, numerous clinical studies have beenconducted across a wide range of cancer types, in-cluding phase III clinical trials in melanoma, prostatecancer, glioblastoma, and renal cell carcinoma (Anguilleet al., 2014). These studies provided ample evidencethat DC therapy is safe and capable of mountingprotective antitumor immunity without major toxicity(Anguille et al., 2014). Until recently, the clinicalbenefit of DC-based cancer immunotherapy wasdeemed modest. However, this field was reinvigorated
Fig. 1. Milestones in the history of cancer immunotherapy. The timeline depicts some of the pivotal milestones in the history of cancerimmunotherapy, with a particular focus on dendritic cell (DC)-based cancer immunotherapy. References: 1(Rosenberg, 1999); 2(Coley, 1891); 3(Ehrlich,1909); 4–8(Gross, 1943; Foley, 1953; Baldwin, 1955; Prehn and Main, 1957; Klein et al., 1960); 9(Thomas et al., 1957); 10, 11(Thomas, 1959; Burnet, 1967);12(Coulie et al., 2014); 13(Steinman and Cohn, 1973); 14, 15(Zinkernagel and Doherty, 1974a,b); 16(Kiessling et al., 1975); 17(van der Bruggen et al., 1991);18(Hsu et al., 1996); 19(Kantoff et al., 2010); 20(Pardoll, 2012). *Allogeneic hematopoietic stem-cell transplantation, which is nowadays widely used inthe treatment of hematologic malignancies including acute myeloid leukemia, is considered as a form of immunotherapy because its primarymechanism-of-action relies on the transfusion of lymphocytes endowed with antitumor activity (the so-called graft-versus-leukemia or graft-versus-tumor activity) (Topalian et al., 2011). #Ipilimumab (Yervoy; Bristol-Myers Squibb), an mAb directed against CTLA-4, was the first-in-class immunecheckpoint mAb to be approved by the U.S. Food and Drug Administration (FDA) and by the European Medicines Agency for patients with metastaticmelanoma (in 2011). Nivolumab (Opdivo; Bristol-Myers Squibb/Ono Pharmaceutical) and pembrolizumab (Keytruda; Merck, Kenilworth, NJ), twomAbs directed against the immune checkpoint molecule PD-1, have gained recent approval by the Japanese health authorities and the FDA,respectively, for use in patients with advanced melanoma (Galluzzi et al., 2014).
Dendritic Cell–Based Cancer Immunotherapy 733
after the demonstration of survival gains in DC-treatedcancer patients in a number of trials (Anguille et al.,2014; Cao et al., 2014; Wang et al., 2014). The foremostexample is sipuleucel-T (Provenge; Dendreon Corpora-tion, Seattle, WA), the first and currently the only DC-based immunotherapeutic that received approval fromthe U.S. Food and Drug Administration. Despitelimited therapeutic benefit based on the assessment ofthe prostate-specific antigen tumor marker response,sipuleucel-T was found to improve overall survival by4.1 months compared with placebo (25.8 versus 21.7months) in a phase III clinical trial for metastatichormone-refractory prostate cancer (Kantoff et al.,2010). The field of DC-based cancer immunotherapycontinues to evolve rapidly (Anguille et al., 2014).Current research efforts are driven by a constant desireto optimize this form of immunotherapy and to increaseits antitumor activity (Datta et al., 2014). There arenow a diverse range of DC vaccine products withimproved therapeutic activity (Anguille et al., 2014).The ever-expanding portfolio of immune checkpointmAbs and other cancer (immunotherapy) drugs has setthe stage for combination therapies, which will likelyfurther improve the efficacy of DC-based cancerimmunization (Anguille et al., 2014). Following a briefintroduction into the basics of the biology of DCs(section II.A) and the various DC vaccine formulationscurrently used for immunotherapy (section II.B), wewill review the different avenues that are currentlybeing pursued to maximize the immunologic potencyand therapeutic activity of this exciting and promisingimmunotherapeutic modality (section III).
II. Dendritic Cell–Based Cancer Immunotherapy
A. Immunobiological Aspects of Dendritic Cell–BasedCancer Immunotherapy
Dendritic cells are a rare population of APCs thatoriginate from hematopoietic progenitor cells in thebone marrow (Collin et al., 2013). They are specializedin antigen uptake, processing, and presentation toT cells (MacDonald et al., 2002). DCs can be foundthroughout the body, in particular at sites of potentialantigenic encounter (e.g., the skin) (Benteyn et al.,2015). The taxonomy of the DC system is complex andwill be only briefly discussed here. The reader isdirected to MacDonald et al. (2002), Ziegler-Heitbrocket al. (2010), Collin et al. (2013), Merad et al. (2013),Cohn and Delamarre (2014), and Mody et al. (2015), fora comprehensive review of the DC nomenclature. Thehuman DC family consists of several distinct subsets,which differ from each other with respect to theirlocalization, cell-surface phenotype, and functionalspecialization (Collin et al., 2013). For example, thehuman skin hosts two main types of DCs: epidermalLangerhans cells (LCs) and dermal DCs (Mody et al.,2015). Langerhans cells are characterized by expression
of the cell-surface markers CD1a and CD207/Langerinand are functionally endowed with a potent CTL-stimulatory activity, especially when compared withCD14+ dermal DCs (Ueno et al., 2010; Banchereauet al., 2012). Blood DCs, which constitute less than 1%of the total peripheral blood mononuclear cell (PBMC)fraction (Jongbloed et al., 2010), are usually dividedinto two main subsets based on their hematopoieticcell-lineage origin (MacDonald et al., 2002): myeloidDCs (mDCs; also termed conventional DCs; myeloidlineage) and plasmacytoid DCs (pDCs; lymphoidlineage). The myeloid DC population is characterizedby the phenotypic expression of CD11c and containstwo distinct subsets (MacDonald et al., 2002; Collin et al.,2013): CD1c/BDCA-1+ mDCs and CD141/BDCA-3+
mDCs. Plasmacytoid DCs can be phenotypicallydiscriminated from mDCs based on the lack of CD11cexpression and based on their positivity for CD123,CD303/BDCA-2, and CD304/BDCA-4 (MacDonaldet al., 2002; Collin et al., 2013). The functionaldifferences between these individual DC subsets, inparticular the subroles they play in antitumor immu-nity, remain to be fully elucidated. CD141+ mDCs havebeen postulated to be superior over other DCs subsetsat (tumor) antigen cross-presentation (Jongbloed et al.,2010) [i.e., the ability of APCs to present exogenousantigens—which are normally directed into the MHCclass II pathway—via MHC class I molecules to CD8+
T cells (Nierkens et al., 2013; Cohn and Delamarre,2014)], although more recent studies have shown thatall blood DC subsets have fairly similar cross-presentation capacities (Cohn et al., 2013; Seguraet al., 2013; Tel et al., 2013b).
Dendritic cells can also be subdivided into two maintypes based on their developmental stage: immatureDCs and mature DCs (Datta et al., 2014; Benteynet al., 2015). Immature DCs continuously sample theperipheral tissues for foreign antigens. They are highlyspecialized in uptake of these antigens and areequipped with a wide range of phagocytic andendocytic receptors (e.g., C-type lectins, such as themannose receptor) to fulfill this function (Sallustoet al., 1995). Immature DCs have rather low expressionof MHC and T-cell costimulatory molecules on theircell surface and a limited capacity for secretingproinflammatory cytokines (Benteyn et al., 2015). Asa result, they are largely ineffective at stimulatingT-cell responses (Benteyn et al., 2015). Moreover,immature DCs can lead to T-cell tolerance by inducingT-cell deletion/anergy and by promoting the generationof T cells with intrinsic immunosuppressive properties[also called regulatory T (Treg) cells] (Mahnke et al.,2002). When antigen uptake occurs in the presence ofinflammatory cytokines or danger signals (such asthose provided by infectious organisms or dying cancercells), immature DCs will undergo a maturation pro-cess that is characterized by decreased capacity for
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antigen uptake, increased expression of MHC and T-cell costimulatory molecules (such as CD40, CD70,CD80, and CD86), and enhanced production of proin-flammatory cytokines and chemokines (Benteyn et al.,2015). DCs express different families of patternrecognition receptors that enable them to sense suchdanger signals; among these, the family of the so-calledToll-like receptors (TLRs) is perhaps the best charac-terized (Medzhitov, 2001). As will be discussed later inthis review, TLR ligands are being increasinglyadopted in DC vaccine manufacturing protocols tostimulate DC maturation. Examples are polyinosinic:polycytidylic acid [poly(I:C), a TLR3 agonist] (Ammiet al., 2015), picibanil (OK-432, a TLR4 agonist)(Kitawaki et al., 2011a), and resiquimod (R-848, a dualTLR7/8 agonist) (Smits et al., 2008). The conversion ofimmature DCs into mature DCs is also accompaniedby an upregulation of chemokine receptors, such as theC:C chemokine receptor type-7 (Sallusto et al., 1998).
This enables the DCs to migrate via the afferentlymphatics to the T-cell–rich areas of the lymph nodes(De Vries et al., 2003; Wimmers et al., 2014) (seeSupplemental Video for an animation illustrating theprocess of DC migration to the lymph nodes).
In the lymph nodes, DCs will present the antigensthey captured via MHC molecules to T cells, whichenter the T-cell–rich areas via so-called high-endothelial venules (Topalian et al., 2011; Vasaturoet al., 2013) (Supplemental Video). Effective stimula-tion of antigen-specific T cells is a hallmark feature ofmature DCs and requires the delivery of three signals(Kalinski and Okada, 2010; Kirkwood et al., 2012) (Fig.2, inset, and Supplemental Video):
1. Signal 1: Presentation of the antigen in thecontext of MHC molecules to a compatible TCR.Antigenic peptides bound to MHC class IImolecules are presented to CD4+ T cells [which
Fig. 2. The clinical success of DC-based therapies against AML and, by extension, other cancer types relies on three key conditions. Improvements inthe therapeutic efficacy of DC vaccine strategies against AML will require 1) a further enhancement of the immunogenicity of the DC products toensure optimal stimulation of the immune effector cells (such as T and NK cells); 2) augmenting the strength of the antileukemic immune response,e.g., by overcoming Treg-mediated immunosuppression; and 3) increasing the responsiveness of AML cells to immune attack by making these cellsvisible to the immune system and by overcoming their immune escape mechanisms. The figure inset shows a close-up of the immunological synapsebetween DCs and T cells. The outcome of DC–T-cell interaction, i.e., T-cell activation or inhibition, is determined by the balance between variouspositive (+) and negative (2) signals delivered via distinct cell surface receptors and their ligands, cytokines (e.g., IL-10 and IL-12) and enzymes (e.g.,IDO). Targeting immune checkpoints, such as CTLA-4, PD-1 or its ligands, and BTLA, can help to tip the balance in favor of protective anti-tumorimmunity. HVEM, herpes virus entry mediator; TNF(R)SF, TNF (receptor) super family; SOCS-1, suppressor of cytokine signaling-1.
Dendritic Cell–Based Cancer Immunotherapy 735
can differentiate into T helper type 1 (TH1), type2 (TH2), or type 17 (TH17) subsets] (Datta et al.,2014), whereas those bound to MHC class Imolecules are recognized by CD8+ CTLs (Cohnand Delamarre, 2014) (Supplemental Video).
2. Signal 2: Delivery of appropriate costimulatorysignaling. The outcome of this process is de-termined by the balance between the differentactivating (positive) and inhibiting (negative)signals that the T cells receive from the DCs(Vasaturo et al., 2013). These signals are de-livered through interaction of costimulatory andcoinhibitory molecules expressed on the DCsurface with their respective counter-receptorson the T cells (Vasaturo et al., 2013) (Supple-mental Video). As shown in Fig. 2, multiplecostimulatory and coinhibitory receptor/ligandpairs have been identified. Most of these mole-cules are either immunoglobulin superfamilymembers or tumor necrosis factor (TNF) andTNF receptor superfamily members (Murphyet al., 2006). CD80, CD86, and programmeddeath-ligand 1 or 2 (PD-L1/2) are examples ofimmunoglobulin superfamily members expressedon the DC surface, whereas CD28, CTLA-4,programmed death (PD)-1, and B andT lymphocyte attenuator (BTLA) belong to thesame family but are expressed on the T cells(Greaves and Gribben, 2013). CD40, CD70,OX40L, 4-1BBL, and herpes virus entry media-tor, which are expressed on the DC surface, andCD40L, CD27, OX40, and 4-1BB, which areexpressed on the T cells, are all examples ofTNF (receptor) superfamily members (Vinay andKwon, 2009) (Fig. 2, inset).
3. Signal 3: Production of T-cell–stimulatory cyto-kines. The third signal or “polarization” signal,involves the secretion of proinflammatory cyto-kines by DCs, including interferons (IFNs) andinterleukin-12 (IL-12) (Kalinski and Okada,2010; Vasaturo et al., 2013). These cytokinesstimulate the expansion, effector function, andmemory development of CTLs (Curtsinger andMescher, 2010). IL-12, which can be producedafter interaction of CD40 with CD40L, also playsa decisive role in determining the type of CD4+
T-cell immunity (Kalinski and Okada, 2010); itpolarizes the differentiation of naive CD4+ T cellsinto TH1 cells, which in turn provide “help” to theCTLs (Kalinski and Okada, 2010; Wimmerset al., 2014).
Recently, the addition of a fourth signal to the above“three-signal theory” has been proposed. This signalcovers all DC-related factors that enable the activatedCTLs to home to the tumor site (Kalinski and Okada,2010). Once arrived at the tumor site, these CTLs will
look for tumor cells that express their cognate antigenin the form of peptide fragments bound to MHC class Imolecules (Anguille et al., 2013). After interaction ofthe MHC/antigen complex with the TCR, CTLs will betriggered to release cytotoxic effector molecules (suchas perforins and granzymes), resulting in lysis of therecognized tumor cells (Trapani and Smyth, 2002;Anguille et al., 2013).
B. Pharmacological Aspects of Dendritic Cell–BasedCancer Immunotherapy
Over the past few decades, a great deal of researchhas been committed to translating the above immuno-biological principles into clinically applicable pharma-ceutical approaches. From the pharmacological point ofview, DC-based immunotherapy is regarded as a vacci-nation strategy whereby tumor antigen-loaded DCs areadministered to the patient with the intent of elicitingprotective and specific CTL immunity toward thetumor cells (Cohn and Delamarre, 2014). From theregulatory point of view, DC vaccines are classified as“human cell, tissue, or cellular or tissue-based prod-ucts” (U.S. Food and Drug Administration terminology)or as “advanced therapy medicinal products” (EuropeanMedicines Agency terminology), which are a specialgroup of therapeutics including gene therapies,tissue-engineered products, and somatic cell therapies(Maciulaitis et al., 2012). Sipuleucel-T is the first, andso far only, DC-based advanced therapy medicinalproduct that received regulatory approval for thetreatment of metastatic, hormone-refractory prostatecancer (Melero et al., 2014). The manufacturing of thisautologous blood cell product starts with a so-calledleukapheresis procedure to obtain large numbers ofautologous PBMCs. These PBMCs, which containAPCs including, but not restricted to, DCs, are thencultured ex vivo for 36–44 hours in the presence ofPA2024, a recombinant fusion protein consisting of thetumor antigen prostatic acid phosphatase and theimmunostimulatory cytokine granulocyte-macrophagecolony-stimulating factor (GM-CSF). After this simul-taneous antigen loading and activation step, the cellsare washed and suspended for intravenous reinfusioninto the patient (Kantoff et al., 2010). In addition tothis transfusion-like protocol, DCs can also be isolateddirectly from the blood and used for immunotherapeu-tic purposes (Bol et al., 2013; Wimmers et al., 2014).The use of native blood DCs has, however, beenhampered by the relatively low frequency of DCs inperipheral blood (as discussed earlier in this section,DCs represent only 1% of the total PBMC fraction) (Bolet al., 2013; Wimmers et al., 2014). Hence, only fewstudies exist that have attempted the clinical trans-lation of this strategy. One example involves thecollection of large numbers of PBMCs (through leuka-pheresis) and subsequent isolation of blood DCs usinga mAb against CMRF-56, an antigen that is expressed
736 Anguille et al.
on the DC surface after a short overnight in vitroincubation (Lopez et al., 2003; Radford et al., 2005).The CMRF-56 isolation platform can be used to obtaina blood DC-enriched PBMC fraction that containsroughly one-third of DCs including both CD1c+ andCD141+ mDC subsets as well as pDCs (Vari et al.,2008). Directly isolated, highly purified native bloodCD1c+ DCs (Schreibelt et al., 2014) as well as pDCs(Tel et al., 2013a) have been used to vaccinatemelanoma patients. Just recently, a similar strategywas adopted by Prue et al. (2015) using CD1c+ mDCsdirectly isolated by immunomagnetic selection fromleukapheresis products of prostate cancer patients.Despite the overall limited experience, these studiesconfirm the technical feasibility of using naturallycirculating DCs as vaccine vehicles for cancer immu-notherapy (Wimmers et al., 2014).Nevertheless, rather than using primary DCs, most
clinical experience so far has been obtained with DCscultured ex vivo starting from precursor cells that areavailable in much greater quantities than naturallycirculating blood DCs (Anguille et al., 2012c). Celltypes frequently used as starting material for DC
vaccine manufacturing are monocytes (Anguille et al.,2012c) and CD34+ hematopoietic progenitor cellsderived from bone marrow (Lardon et al., 1997),peripheral blood (Mu et al., 2004), or cord blood (Chenet al., 2013). Monocyte-derived DCs (moDCs) are by farthe most commonly used DC type in DC vaccineformulations (Anguille et al., 2014; Chiang et al.,2015). This type of DC vaccine is usually produced byisolating CD14+ monocytes from leukapheresis prod-ucts and by culturing them in vitro for 5–6 days intoimmature DCs with GM-CSF and IL-4 (Tacken et al.,2007) (Fig. 3; Supplemental Video). Because immatureDCs are considered to be poor stimulators of immunity,the next step in DC vaccine manufacturing is to triggertheir activation/maturation by exposing the immatureDCs for another 2 days to a proinflammatory cytokinecocktail composed of TNF-a, IL-1b, IL-6, and prosta-glandin E2 (PGE2), commonly referred to as the“Jonuleit” cytokine cocktail (Jonuleit et al., 1997)(Fig. 3). To complete the DC vaccine production process,DCs must be loaded with disease-relevant tumorantigen(s), a step that can be performed either at the im-mature or at themature stage (Tacken et al., 2007) (Fig. 3).
Fig. 3. The gold-standard protocol for DC vaccine manufacturing. DC-based immunotherapeutics are classically prepared using an in vitro cultureprocedure, in which 1) peripheral blood monocytes are allowed to differentiate into immature DCs in the presence of GM-CSF and IL-4 for 5–6 days andthen 2) exposed for another 2 days to a proinflammatory cytokine cocktail to induce DC maturation. Before administration to the patient, DCs are firstloaded with tumor antigen “cargo”; this antigen-loading step can be performed either at the immature or mature DC stage. Each of these phases in theDC manufacturing process can have a major impact on the immunostimulatory potency of the final DC product. Therefore, much ongoing research isdevoted to modifying and optimizing the culture conditions for DC generation to ensure that DCs with the “best possible” immunostimulatorycapacities are being used for immunotherapy and, ultimately, to maximize the chances of therapeutic success. Examples of such modifications arelisted in the text box insets.
Dendritic Cell–Based Cancer Immunotherapy 737
Although being considered as the “gold-standard”DCs, these so-called IL-4 moDCs might be suboptimalfor inducing TH1-mediated anticancer T-cell immu-nity because they resemble in vivo occurring CD14+
dermal DCs known to stimulate humoral immunity(Palucka et al., 2010; Palucka and Banchereau, 2012).As discussed above, LCs, presumably via a mechanismthat involves IL-15, are far more superior than dermalDCs (Banchereau et al., 2012) and moDCs (Romanoet al., 2012; Hutten et al., 2014) in terms of theircapacity to stimulate antigen-specific CTL immunity.This explains the impetus behind the recent efforts todesign new protocols for the generation of DCs withLC-like features (Hutten et al., 2014). Such LC-type,IL-15–expressing DCs can be generated from CD34+
hematopoietic progenitor cells using specific differen-tiation factors (Romano et al., 2011) or from CD14+
monocytes by replacing IL-4 with IL-15 (Mohamadzadehet al., 2001; Anguille et al., 2009, 2013).As may be clear from the previous section on the
immunobiology of DCs, the immunogenicity of DCvaccines will not only depend on the type of DC used inthe vaccine formulation, but also on the functionalstate of the DCs induced by maturation signals(Kirkwood et al., 2012; Datta et al., 2014). The conventionalJonuleit cytokine cocktail implemented in most DCvaccine production protocols has several importantlimitations, which will be discussed in detail in thenext section. Hence, there is currently a great deal ofresearch into developing alternative DC maturationprocedures (Butterfield, 2013; Anguille et al., 2014).Within this context, TLR agonists have come underintense scrutiny given their ability to endow DCs withpotent TH1-polarizing capacity, which is the type ofimmune response that is most desired in cancerimmunotherapy (Kirkwood et al., 2012). Such TLRstimuli can either be administered concomitantly withthe DC vaccine or can be incorporated into the vaccineitself, for example as ex vivo maturation agents(Anguille et al., 2014). Various clinical-grade TLR-based protocols for maturing DCs have been pub-lished in recent years, but, for the sake of brevity, onlytwo will be discussed here: a-type-1–polarized DCsand TriMix DCs. a-Type-1–polarized DCs, whichderive their name from the fact that they are highlyspecialized at inducing TH1 immune responses(Kirkwood et al., 2012), are generated by exposingimmature DCs to a combination of TNF-a, IL-1b, IFN-g,IFN-a, and the TLR3 agonist poly(I:C) (Okada et al.,2011; Hansen et al., 2013). Okada et al. (2011) recentlyreported on the results of a clinical trial of a-type-1–polarized DC vaccinations with concurrent systemicadministration of a TLR3 agonist in patients withrecurrent malignant glioma. Although single arm andearly phase, the study by Okada et al. (2011) hinted ata possible clinical benefit and provided the impetus forother, currently ongoing clinical trials investigating
the clinical efficacy of this particular DC vaccineformulation (Kirkwood et al., 2012; Anguille et al.,2014). Instead of exogenous pulsing with maturation-inducing cytokines, DCs with an activated/maturephenotype can also be obtained using genetic engi-neering techniques such as mRNA electroporation(Benteyn et al., 2015). Thielemans’ group at the FreeUniversity of Brussels (Brussels, Belgium) has pio-neered a method to generate clinical-grade, matureDCs by electroporating immature DCs ex vivo witha mixture of three mRNA molecules (TriMix) coding fora constitutively active TLR4, CD40L, and CD70,respectively (Benteyn et al., 2015). These so-calledTriMix DCs have been used with promising results tovaccinate melanoma patients (Van Lint et al., 2014).
Another pharmacological aspect that deserves to bementioned here relates to the route of administrationof DC vaccines. To encounter T cells and induce tumorantigen-specific T-cell immunity, DCs must berecruited to the lymph nodes (Verdijk et al., 2008).DCs that are injected intravenously are believed to beineffective because they localize in lungs, liver, spleen,and bone marrow, rather than in the lymph nodes(Fong et al., 2001; Mullins et al., 2003). Nevertheless,in a recently published clinical trial (Wilgenhof et al.,2013), signs of clinical benefit have been obtained afterintravenous TriMix DC vaccination, corroboratingearlier studies that already indicated that DCs caninduce immunity even when administered via theintravenous route (Fong et al., 2001; Bedrosian et al.,2003). Most often, DC vaccines are delivered byintradermal injection nearby superficial lymph nodes(Wimmers et al., 2014). After injection into the skin,DCs will use their chemokine receptors (e.g., C:Cchemokine receptor type-7) to migrate from the skinvia the afferent lymphatics to the draining lymphnodes (Wimmers et al., 2014) (Supplemental Video).Importantly, however, trafficking studies revealed thatmost of the intradermally injected DCs die at theinjection site and that only a minority (less than 5%)effectively reach the lymph nodes (Verdijk et al., 2009).To obtain higher numbers of DCs in the lymph nodes,intranodal vaccination has been tested, but its superi-ority over the intradermal administration route hasnot been unequivocally established (Bedrosian et al.,2003; Lesterhuis et al., 2011). Variable results ofintranodal injection might be related to the complexityof the procedure, so that even when performed withultrasound guidance by a highly experienced radiolo-gist, a high portion of the injections will not or onlypartly be delivered intranodally (de Vries et al., 2005;Verdijk et al., 2009; Lesterhuis et al., 2011).
In addition to the administration route, the optimaldose of DC vaccination is another pharmacologicalvariable that still remains to be fully established despitethe numerous phase I clinical trials that have alreadybeen performed (Figdor et al., 2004; Butterfield, 2013).
738 Anguille et al.
Many of these trials did not show any evidence of dose-limiting toxicity (Draube et al., 2011; Butterfield,2013). Maximum tolerated doses are generally notreached in DC vaccine studies, and the highestachievable dose levels are usually defined by thetotal number of DCs that can be produced from oneleukapheresis (i.e., the maximum feasible dose)(Butterfield, 2013; Tel et al., 2013a). A meta-analysis of29 DC vaccine trials in prostate cancer and renal cellcancer, including a total of 906 patients, revealeda positive association between total DC dose andclinical outcome, indicating that appropriate dosageis critical for therapeutic success (Draube et al., 2011).Unfortunately, this meta-analysis does not permit todraw firm conclusions regarding the minimum effec-tive dose required per vaccination. Despite the generalbelief that high numbers of DCs are required tostimulate a potent antitumor immune response invivo, the above-mentioned study by Tel et al. (2013a)using naturally circulating pDCs revealed that clinicalresponses can be obtained even with relatively smallnumbers of DCs (0.3–3 � 106 DCs per vaccine). Extraevidence that higher DC numbers do not necessarilyresult in higher benefit was provided by Aarntzen et al.(2013), who observed a paradoxical increase in DCsreaching the lymph nodes if the number of intrader-mally injected DCs was reduced. Imaging data from thesame group also revealed that antitumor T-cellresponses can be induced with as few as 5 � 105 DCsin the T-cell areas of the draining lymph nodes (Verdijket al., 2009). In line with this, using a computationalmodel, Celli et al. (2012) estimated that less than 100antigen-bearing DCs are sufficient to initiate a T-cellresponse in the lymph node. Overall, these dataindicate that DC vaccines may be effective even atvery low doses.New routes of DC-based cancer therapy are cur-
rently being explored, including in vivo targeting ofDCs (Caminschi et al., 2012; Datta et al., 2014) and theuse of artificial APCs (Eggermont et al., 2014). Thesetopics have been excellently reviewed by others
(Caminschi et al., 2012; Eggermont et al., 2014; Modyet al., 2015) and will not be covered in this review.
III. Toward the Delivery of Effective DendriticCell–Based Cancer Immunotherapy: The Acute
Myeloid Leukemia Showcase
A growing body of evidence indicates that DCvaccination can be of clinical benefit to cancer patients(Draube et al., 2011; Anguille et al., 2014; Cao et al.,2014; Wang et al., 2014), encouraging the furtherdevelopment of this form of immunotherapy. Neverthe-less, there is a general agreement among cancerresearchers that the true clinical potential of DC-based cancer immunotherapy has not been attained yet(Bot et al., 2013; Datta et al., 2014). Conceptually, thegeneration of antitumor immunity by DCs requiresthree sequential steps (Mellman et al., 2011), asschematically represented in Fig. 2:
1. The DCs used for therapy must be immunosti-mulatory and induce the right type of immuneresponse (Anguille et al., 2014).
2. The antitumor immune effector cells of the host,most notably CTLs and NK cells, must becapable of mounting protective immunity (Lizeeet al., 2013).
3. The tumor cells must be susceptible to immuneattack, which requires overcoming the tumorimmune escape mechanisms (Kirkwood et al.,2012).
Hence, the key to unlocking the full clinical potential ofDC-based cancer immunotherapeutics is to establishhow these three conditions can be optimally fulfilled.In this section, we will provide an overview of theresearch efforts that are currently being undertaken toenable successful completion of each of these threesteps. The focus here lies on the optimization of DC-based immunotherapy for AML, which, for the reasonsspecified in Table 1, is a particularly useful tumor type
TABLE 1Why AML is a useful tumor type to optimize and evaluate the efficacy of DC-based immunotherapeutics
Reasons
• AML is an immunogenic malignancy known to be susceptible to lysis by leukemia antigen-specific CD8+ CTLs as well as NK cells (Schurch et al.,2013). The so-called graft-versus-leukemia effect associated with allogeneic hematopoietic stem cell transplantation (HSCT) elegantly illustratesthe therapeutic role of the immune system in AML (Schurch et al., 2013). AML is therefore believed to be particularly amenable toimmunotherapeutic approaches (Anguille et al., 2012c; Martner et al., 2013; Schurch et al., 2013).
• Conventional chemotherapy followed by allogeneic HSCT is associated with significant toxicity and is therefore not feasible for older patients withAML, who represent the majority of AML patients (Anguille et al., 2012c). Their favorable toxicity profile makes immunotherapy approaches, suchas DC-based immunotherapy, worthy of investigation to fill this treatment void (Anguille et al., 2011a).
• DC therapy has already shown to be potentially useful in AML, especially when applied in a minimal residual disease (MRD) setting(i.e., a situation in which a small amount of tumor cells persists after therapy and that is responsible for tumor recurrence) (Van Tendeloo et al.,2010; Anguille et al., 2012c). This corroborates the notion that immunotherapy is most optimally applied as an adjunct therapy at early stages ofcancer when only few immunosuppressive tumor cells are present (Gulley et al., 2011; Anguille et al., 2014).
• Standard response evaluation criteria used in solid malignancies (e.g., RECIST) do not appear to capture the true clinical benefit ofimmunotherapy approaches (Schlom, 2012; Anguille et al., 2014). In AML, numerous surrogate end points for evaluation of treatment efficacy(e.g., molecular MRD markers) have been developed and validated for use in clinical trials, facilitating translational research (Cilloni et al., 2009;Hourigan and Karp, 2013).
Dendritic Cell–Based Cancer Immunotherapy 739
to evaluate the clinical efficacy of DC-based immuno-therapy. Although AML was chosen to showcase howthe DC immunotherapy field is evolving, many of thestrategies that will be discussed here are broadlyapplicable and are currently under active investigationin other tumor types as well.
A. Using Dendritic Cells with OptimalImmunocompetence (Condition Number 1)
The efficacy of any DC-based therapeutic strategydepends, in the first place, on the immunostimulatoryquality of the DCs used (Anguille et al., 2014) (Fig. 2).As discussed earlier, the DC family is very heteroge-neous, consisting of different DC subsets capable ofeliciting different types of immunity. Hence, selectingthe “right” DC subset for immunotherapy is ofparamount importance (Gilboa, 2007; Anguille et al.,2009; Frankenberger and Schendel, 2012; Kirkwoodet al., 2012; Chiang et al., 2015). Much ongoingresearch is concentrated on optimizing the DC productdesign to ensure that DCs with the best possibleimmunogenic characteristics are being used (Anguilleet al., 2014; Datta et al., 2014; Chiang et al., 2015).There are several aspects of the DC manufacturingprocess that determine the immunogenicity of the finalDC therapeutic product, each of which is beingevaluated for possible improvements (Fig. 3) (Figdoret al., 2004; Gilboa, 2007; Tacken et al., 2007; Anguilleet al., 2009; Tesfatsion, 2014).1. Source of DC Precursor Cell. The first aspect
relates to the choice of precursor cell for ex vivo DCpreparation. As discussed above, most clinical trialsperformed hitherto have relied on monocytes as pre-cursor cells for DC vaccine manufacturing (Supple-mental Video) (Gilboa, 2007; Tacken et al., 2007;Chiang et al., 2015). Monocytes can be isolated fromleukapheresis harvests by various techniques, includ-ing centrifugal elutriation, adherence, or immunomag-netic selection using anti-CD14 mAb-coated magneticbeads (Table 2) (Nicolette et al., 2007). Given the factthat moDCs are by far the most frequently used DCtype in clinical trials, we will only address here thecurrent strategies for optimizing moDC culture proto-cols (Fig. 3). Table 2 provides a compilation of theclinical trials of moDC-based vaccination in AML. Foreach clinical trial listed, details on the logisticalaspects related to moDC vaccine manufacturing anda summary of the key clinical findings are provided(Table 2).2. Culture Time. The gold-standard protocol for
moDC generation encompasses 1 week of culture,including ;5 days for differentiation of monocytes intoimmature DCs and ;2 days for converting theseimmature DCs into mature DCs (Fig. 3; SupplementalVideo) (Caux et al., 1992; Anguille et al., 2009). Anumber of reports have demonstrated the feasibility ofreducing the total time of in vitro moDC culture from 7
to ;3 days without affecting the phenotype or functionof the resultant DCs (Czerniecki et al., 1997, 2001;Dauer et al., 2003; Anguille et al., 2009; Beck et al.,2011; Chiang et al., 2011). Moreover, these so-calledFast DCs have been shown to possess a bettercostimulatory phenotype than their 7-day counterparts(Lichtenegger et al., 2012) and have a lower expressionof the T-cell coinhibitory molecule PD-L1 (Frankenbergerand Schendel, 2012). In addition, the use of Fast DCsoffers the advantage of reducing the time-consumingprocess of ex vivo DC manufacturing (Anguille et al.,2009). As a consequence, Fast DCs are increasinglyfinding their way into clinical trials, a trend that isalso observed in the field of DC therapy for AML(Subklewe et al., 2014) (Table 2).
3. Culture Media. The type of medium used for exvivo DC culture is an often overlooked parameter thatmay influence the functional capacity of the culturedDCs (da Silva Simoneti et al., 2014). All aspects of theDC vaccine manufacturing process, including theculture media and reagents used, must comply to goodmanufacturing practice requirements that are enforcedby health regulatory agencies to ensure the quality andsafety of the DC product (Figdor et al., 2004). Differentgood manufacturing practice-grade DC culture mediahave been marketed, each yielding DCs with differentimmune-stimulatory properties (da Silva Simonetiet al., 2014), indicating that the choice of cell culturemedium determines the potency of the final DC vaccineproduct. Serum, which is often added to the culturemedium, is postulated to have a negative influence onDC vaccine functionality in vitro (da Silva Simonetiet al., 2014; Kolanowski et al., 2014), but the in vivoimpact of using serum remains to be studied.
4. Differentiation Protocol. As discussed earlier, themost commonly used cytokines for inducing moDCdifferentiation are GM-CSF and IL-4 (Gilboa, 2007;Anguille et al., 2009) (Fig. 3). The choice of DCdifferentiation regimen is an important determinantof the immunogenic quality of moDCs (Ueno et al.,2010). This is supported by the observation that theability of moDCs to stimulate antitumor immunity canbe dramatically improved by replacing IL-4 with IL-15(Dubsky et al., 2007; Anguille et al., 2009, 2012a) orIFN-a (Papewalis et al., 2008). Such alternativelydifferentiated DCs can also exert direct cytotoxicitytoward AML cells (Papewalis et al., 2008; Anguilleet al., 2012a), further increasing their attractivenessfor clinical exploitation (Roothans et al., 2013; Telet al., 2014).
5. Maturation Protocol. The second step in moDCmanufacturing involves the induction of DC matura-tion, a step that is usually performed by exogenousadministration of a cytokine cocktail composed of TNF-a, IL-1b, IL-6, and PGE2 (Jonuleit et al., 1997; Gilboa,2007) (Fig. 3; Supplemental Video). The major limita-tion to using this particular mixture is the inability of
740 Anguille et al.
TABLE
2Clinicaltrials
ofmoD
C-bas
edva
ccinationin
AML
Cas
erepo
rtsareex
clude
dfrom
this
overview
.
Ref.
Log
istics
NClinical
Resultsa
Sou
rce/Isolation
Method
Culture
Tim
eCulture
Med
ium
Differentiation
Matur
ation
Reg
imen
Targe
tAntige
nAntige
nSou
rce
Loa
dingMethod
Other
Asp
ects
Adm
in.
Rou
te
Lee
etal.,20
04PBSC
harvest/
CD14
+
isolation
8–9da
ysAIM
VGM-C
SF+IL
-4TNF-a
NA
AML
cell
lysa
teb
Pulsing
/SC/IV
2Disea
sepr
ogression
(2)
Kitaw
akiet
al.,
2011
aLeu
kaph
eresis/
Elutriation
3or
6da
ysCellG
roGM-C
SF+IL
-4OK-432
WT1,
hTERT,
etc.
Apo
ptotic
AMLcellsc
Pulsing
adjuva
nt
OK-432
ID4
Disea
sestab
ilization
(2)
↓AML
blas
tcount(1)
Oga
sawaraan
dOta,20
14;
Ota
and
Oga
sawara,
2014
Leu
kaph
eresis/
Adh
eren
ceND
ND
GM-C
SF+IL
-4OK-432
+PGE2
WT1
Pep
tide
Pulsing
adjuva
nt
OK-432
ID7d
↓WT1mRNA
leve
l(2)
DiPersioet
al.,
2009
;Khou
ryet
al.,20
10
Leu
kaph
eresis/
Adh
eren
ceND
ND
Uns
pecified
cytokine
mix
Uns
pecified
cytokine
mix
hTERT
mRNA
Electropo
ration
+LAMP
ID21
DFSof
79%
[12m
]
Van
Ten
deloo
etal.,20
10;
Bernem
anet
al.,20
14
Leu
kaph
eresis/
CD14
+
isolation
8da
ysCellG
ro+1%
hAB
GM-C
SF+IL
-4TNF-a+IL
-1+IL
-6+PGE2
WT1
mRNA
Electropo
ration
+/2LAMP
ID30
Indu
ctionof
CR
(2/3)
Indu
ctionof
CMR
(8/23)
Con
tinued
CR
[63m
](8)
Disea
sestab
ilization
(3)
Kitaw
akiet
al.,
2011
bLeu
kaph
eresis/
Elutriation
ND
ND
GM-C
SF+IL
-4TNF-a+PGE2
WT1
Pep
tide
Pulsing
+ZOL
ID/IV
3Disea
sestab
ilization
(1)
↓AML
blas
tcount(1)
Rosen
blattet
al.,
2014
Leu
kaph
eresis/
Adh
eren
ce7–
10da
ysND
GM-C
SF+IL
-4TNF-a
MUC1,
WT1,
PRAME,etc.
AML
cells
Fus
ionhy
brids
(PEG)
adjuva
nt
GM-C
SF
SC
13Con
tinued
CR
[23m
](9)
Sch
norfeilet
al.,
2014
;Subk
lewe
etal.,20
14
Leu
kaph
eresis/
Adh
eren
ce3da
ysND
GM-C
SF+I
L-4
R-848
+poly(I:
C)+othe
reWT1,
PRAME
mRNA
Electropo
ration
/ID
2fND
(ongo
ing
stud
y)
CD14
+isolation,mag
netic
isolation
ofCD14
mon
ocytes;C(M
)R,complete(m
olecular)
remission
;DFS,diseas
e-free
survival;hAB,hu
man
AB
seru
m;hT
ERT,hu
man
telomeras
ereve
rsetran
scriptas
e;LAMP,inclusion
oflysosome-as
sociated
mem
bran
epr
oteinin
mRNAconstru
ct;M
UC1,
mucin1;
ND,n
oda
ta;P
BSC,p
eripheral
bloo
dstem
cell;P
EG,p
olye
thylen
eglycol;P
RAME,p
referentially
expr
essedan
tige
nin
melan
oma;
ZOL,incombination
withzoledr
onate.
aThenumbe
rin
parentheses
indicatesthenumbe
rof
patien
tsin
whom
thede
sign
ated
clinical
effect
was
observed
.Thenumbe
rin
bracke
tsindicatesthefollow
up-tim
e(inmon
ths).
bPrepa
redby
repe
ated
free
ze-thaw
cycles.
c Prepa
redby
g-irrad
iation
.dIn
cluding2pa
tien
tswithacutelymph
oblastic
leuke
mia.
e IncludingTNF-a,IL
-1b,IF
N-g,PGE2.
f Sch
edulesto
include
20pa
tien
ts.
Dendritic Cell–Based Cancer Immunotherapy 741
the resultant DCs to produce IL-12p70 (Kalinski et al.,2001). The lack of IL-12 secretion can mainly beattributed to the presence of PGE2 (Kalinski et al.,2001). In addition, PGE2 can induce indoleamine 2,3-dioxygenase (IDO) expression in moDCs (Braun et al.,2005), which promotes the activation of Treg cells(Chung et al., 2009) (Fig. 2, inset) and can preventDCs from attracting naive T cells (Muthuswamy et al.,2010). However, because PGE2 is considered manda-tory for licensing DCs to migrate to the lymph nodes, itcannot simply be omitted from the maturation regimen(Scandella et al., 2002). The incorporation of TLRagonists into the DC maturation cocktail may help tocircumvent this problem. TLR-matured DCs have beenshown to combine migratory capacity with high IL-12production, even with PGE2 in the maturation cocktail(Boullart et al., 2008; Anguille et al., 2009; Beck et al.,2011). In addition, TLR maturation stimuli also enableDCs to overcome Treg-mediated immunosuppression(Pasare and Medzhitov, 2003; Lichtenegger et al.,2012) and to activate innate antitumor effectors, inparticular NK cells (Beck et al., 2011; Lichteneggeret al., 2012). It is therefore not surprising to witness anincreased use of TLR-based maturation regimens inclinical trials of DC-based cancer immunotherapy(Galluzzi et al., 2012). In AML, several DC vaccinestudies have already incorporated TLR agonists[i.e., poly(I:C), OK-432, and R-848] to promote DCmaturation (Table 2).6. Choice of Target Antigen. The primary immuno-
logic goal of DC-based cancer therapy is to immunize orvaccinate the patients by inducing a (memory) tumorantigen-specific T-cell immune response via the in-trinsic antigen-presenting ability of DCs (Gilboa,2007). As a consequence, the choice of antigenic targetprobably represents one of the most critical aspects inDC vaccine design (Gilboa, 2007). Since the discoveryof the first cancer antigen by Boon and colleagues in1991 (van der Bruggen et al., 1991) (Fig. 1), more than100 tumor antigens have been identified and theirnumber is still increasing (Cheever et al., 2009).Selecting the right target antigen has thus becomea challenging task (Cheever et al., 2009; Anguille et al.,2012b). To facilitate and rationalize this antigenselection process, the Translational Research WorkingGroup of the National Cancer Institute has proposeda number of criteria for assessing the suitability ofa given tumor antigen for immunotherapeutic target-ing (Cheever et al., 2009). Based on this report,preference should be given to antigens that: i) areoverexpressed in the tumor cell compartment but not(or only minimally) expressed in normal cells andtissues; ii) are consistently overexpressed amongpatients with a given tumor type and, within eachpatient, expressed in most to all tumor cells; iii) play arole in the malignant phenotype; iv) contain both CD8+
and CD4+ T-cell epitopes and are immunologically
relevant (i.e., demonstration of immunity toward theantigen in cancer patients); and v) hold clinicalrelevance (i.e., demonstration of clinical responses inimmunotherapy studies targeting the given antigen)(Cheever et al., 2009; Anguille et al., 2012b). Oneexample of a tumor-associated antigen that fulfils allthese requirements and that is known to be overex-pressed in AML is the Wilms’ tumor 1 (WT1) protein(Anguille et al., 2012b), which has already been shownto be an excellent choice for DC-based immunotherapyin AML (Van Tendeloo et al., 2010; Van Driesscheet al., 2012). An upcoming trend is to target antigensthat are preferentially expressed in the cancer stemcell compartment, to enable eradication of the malig-nant cell population that is responsible for tumorpropagation and/or recurrence (Ruben et al., 2013;Snauwaert et al., 2013).
7. Source of Antigen(s). The best source of antigenicmaterial remains another unresolved issue in DCproduct design (Gilboa, 2007) (Fig. 3). Tumor antigenscan be derived from either “undefined” or “defined”antigen sources (Nicolette et al., 2007). Undefinedantigen preparations contain both known and un-known epitopes (Nicolette et al., 2007); examples usedin the setting of DC therapy for AML are peptideseluted from the tumor cell surface, whole AML cellpreparations for fusion with DCs, necrotic AML celllysates (e.g., prepared by repeated freeze-thaw cycles),apoptotic AML cells (e.g., prepared by g-irradiation),total tumor RNA extracted from the AML cells, andAML cell-derived extracellular vesicles (van der Polet al., 2012) including exosomes (small vesicles ofendosomal origin released from live cells) and apoptoticblebs (large vesicles released from cells undergoingapoptosis) (van den Ancker et al., 2010; Anguille et al.,2012c; Yao et al., 2014). Sources of defined antigens aresynthetic peptides or nucleic acids encoding a specifictumor antigen (van den Ancker et al., 2010; Anguilleet al., 2012c). Several of these undefined and definedantigen sources have already been used in moDC-based vaccine trials in AML (Table 2).
The main advantage of using undefined antigenpreparations is that they cover the full repertoire ofantigens expressed by the tumor cells (Schui et al.,2002; Nicolette et al., 2007; Tesfatsion, 2014). At thesame time, this is also considered to be their majordisadvantage. Indeed, with an undefined antigenicpayload, there is a high likelihood of deliveringimmunologically irrelevant antigens or even self-antigens, which may lead to T-cell tolerance orautoimmunity (Schottker and Schmidt-Wolf, 2011).For example, some concern has been recently raisedover two tumor antigens, survivin and receptor forhyaluronic acid–mediated motility, because of theirexpression in activated T lymphocytes imposing a riskfor T-cell fratricide (Leisegang et al., 2010; Snauwaertet al., 2012). Furthermore, using tumor cells or tumor
742 Anguille et al.
cell elements as antigen source entails the risk ofdelivering other tumor-derived factors that haveimmunosuppressive effects (e.g., IL-10 and transform-ing growth factor-b) (Schui et al., 2002; Schottker andSchmidt-Wolf, 2011; Szczepanski et al., 2011; Rubenet al., 2013; Hong et al., 2014). Nevertheless, severalstudies have shown that an effective CTL immuneresponse can be triggered by DCs charged with eithernecrotic leukemic cell lysates or apoptotic leukemiccells or blebs (Galea-Lauri et al., 2002; Spisek et al.,2002; Weigel et al., 2006; Ruben et al., 2014). Oneplausible explanation for this observation is that thepotential immunosuppressive effects of tumor cells canbe overcome by the various immunostimulatory factorsreleased from tumor cells upon necrotic or apoptoticcell death induction (Chiang et al., 2010). For example,tumor cells undergoing necrosis have been shown torelease heat-shock proteins (HSPs) and high mobilitygroup box 1 (HMGB1), factors that are known topromote the maturation of DCs via a TLR4-dependentmechanism (Chiang et al., 2010). Apoptotic tumor cellscan also release HSPs and HMGB1 and exposecalreticulin on their cell surface. Calreticulin acts asan “eat me signal,” facilitating phagocytic uptake byDCs (Chiang et al., 2010; Guo et al., 2014; de Bruynet al., 2015). Oxidation by hypochlorous acid (HOCl)treatment is a relatively novel strategy to producehighly immunogenic tumor cell preparations for DCantigen loading. In addition to inducing necrotic celldeath (with the related advantage of eliciting therelease of HSPs, HMGB1, and other DC-activatingsignals), exposure of tumor cells to HOCl gives rise tooxidized biomolecules, which can lead to direct DCactivation and promote DC antigen processing and(cross-) presentation. Moreover, it has been proposedthat HOCl reduces the production of immunosuppres-sive cytokines (e.g., IL-10) by tumor cells (Chiang et al.,2010, 2013, 2015). To the best of our knowledge, HOCl-treated leukemic cells have not yet been used asantigen source in moDC-based immunotherapy proto-cols in AML.8. Antigen-Loading Method. The method used for
delivering the antigenic payload to the DCs is anotheraspect that requires careful consideration, because thishas a major impact on the antigen-presenting qualityof the final DC therapeutic product (Gilboa, 2007)(Fig. 3). So far, most DC-based immune interventionsagainst cancer, including AML, have relied on theuse of peptide pulsing or pulsing with lysed orapoptotic tumor cells for antigen loading (Figdoret al., 2004; van den Ancker et al., 2010) (Table 2).Other antigen-loading methods, such as chemical orelectrical fusion of DCs with tumor cells and the use oftumor-derived extracellular vesicles (exosomes andapoptotic blebs) as antigen-delivery carriers, are in-creasingly finding their way into clinical trials(Galluzzi et al., 2012; Ruben et al., 2014; Gu et al., 2015).
Although full-scale comparisons of these variousapproaches are lacking, there are some data favoringthe use of certain antigen-delivery methods overothers. For example, pulsing of moDCs with apoptoticAML cells has a higher antigen-loading efficiencycompared with pulsing with lysates (Galea-Lauriet al., 2002; van den Ancker et al., 2011). Fusion ofDCs with AML cells also seems to be superior toloading with lysed or apoptotic leukemic cells (Galea-Lauri et al., 2002; Klammer et al., 2005), although onereport found no clear difference in functionalitybetween lysate-pulsed DCs and AML-DC fusionhybrids (Weigel et al., 2006). The use of leukemia-derived exosomes and apoptotic blebs also seems to besuperior to pulsing with lysates (Gu et al., 2015) andapoptotic tumor cells (Ruben et al., 2014), respectively.A relatively new method for antigen loading of DCsinvolves the use of antigen-containing synthetic nano-or microparticles (Zhang et al., 2013). This strategy notonly allows for delivering antigen but also for providingDCs with immunostimulatory adjuvants (Zhang et al.,2013). Zhang et al. (2013) recently showed that poly-meric microparticles containing both WT1 and theTLR9 agonist CpG can be used to generate DCs withpotent capacity to stimulate WT1-specific antileukemicimmunity. DCs can also be genetically modified tosynthesize and express predefined tumor antigen(s),e.g., by using lentiviral vectors encoding a given tumorantigen (Sundarasetty et al., 2013) or by electroporat-ing tumor antigen-encoding mRNA into the cells(Smits et al., 2009). The latter technique has gainedincreasing popularity over the past decade (Gilboa andVieweg, 2004) and has also been adopted in moDC-based vaccine trials in patients with AML (Anguilleet al., 2012c) (Table 2). The attractiveness of the mRNAelectroporation strategy lies in its demonstrated in vivoability to induce a broad (i.e., multiepitope) leukemia-specific immune response without the need for takinginto account the human leukocyte antigen constitutionof the patient; this is in contrast with tumor antigenpeptide pulsing, which is restricted to a particularhuman leukocyte antigen (Van Tendeloo et al., 2010).
Although each loading strategy has its own specificadvantages and disadvantages, perhaps the mostdecisive argument for selecting a particular method iswhether it allows for delivering the antigen in thecontext of both MHC class I and II molecules (Gilboaand Vieweg, 2004). Indeed, the simultaneous pre-sentation of MHC class I and class II epitopes torecruit both CD8+ and CD4+ T cells is considered to bea critical determinant for effective and long-lastingantitumor immunity (Wang, 2002) (SupplementalVideo). When DCs are pulsed with lysates or apoptotictumor cells, the antigens will predominantly enter theexogenous, MHC class II–processing pathway; theseantigen-loading methods rely on cross-presentation toenable class I presentation (Decker et al., 2006).
Dendritic Cell–Based Cancer Immunotherapy 743
Although cross-presentation has been shown to occurin vivo (Kitawaki et al., 2011a), this process may notalways be effective (Decker et al., 2006). One way toovercome this is to combine different antigen-loadingmethods (e.g., pulsing with lysates in conjunction withmRNA loading) to allow the antigen(s) to access boththe MHC class I– and II–processing pathways (Deckeret al., 2009; Decker et al., 2006). Another appealingstrategy is to use tumor antigen-encoding mRNA thatis appended with an MHC class II–targeting sequenceof an endosomal/lysosomal protein, such as DC-lysosome-associated membrane protein (Benteynet al., 2013).9. Designer Dendritic Cells. The advent of electro-
poration technology and other genetic engineeringmethods has now also opened up the possibility tofine-tune the phenotype and function of DCs towardthe desired immunostimulatory direction (Smits et al.,2009). DCs with an enhanced immunostimulatoryprofile can be obtained by genetically enforcing theexpression of costimulatory molecules, e.g., OX40L(Yanagita et al., 2004) or the TH1-polarizing cytokineIL-12 (Curti et al., 2005) (Fig. 2, inset). Short-interfering RNA technology has been used effectivelyto silence genes that have a negative impact on theimmunostimulatory potency of DCs, e.g., genes codingfor coinhibitory molecules, such as PD-L1/2 (Hoboet al., 2010, 2013), genes coding for suppressivecytokines, such as IL-10 (Iversen et al., 2009; Iversenand Sioud, 2015), or genes coding for signalingmolecules regulating cytokine expression in DCs, suchas signal transducer and activator of transcription-3(Brady et al., 2013; Sanseverino et al., 2014) andsuppressor of cytokine signaling-1 (Zhou et al., 2006)(Fig. 2, inset).10. Route of Administration. The classic parenteral
routes of drug administration, e.g., intravenous, areconceptually less appropriate for DC vaccine adminis-tration, because DCs administered through theseroutes are not primarily distributed to their main siteof action, the lymph node (Malik et al., 2014). To enabledelivery to the lymph node, DCs are usually adminis-tered by subcutaneous or intradermal injection nearbyperipheral lymph nodes or by intralymphatic or directintranodal injection (Pyzer et al., 2014). A meta-analysis of clinical trials of DC-based immunotherapyin prostate cancer and renal cell cancer revealed thatthe choice of administration route may indeed in-fluence the therapeutic efficacy, with lymph node-targeting administration routes being superior overthe intravenous route (Draube et al., 2011). Neverthe-less, intravenous DC vaccination has been reported toelicit durable clinical responses in melanoma (e.g.,intravenously injected TriMix DCs) (Wilgenhof et al.,2013) and to prolong overall survival in prostate cancer(e.g., sipuleucel-T) (Kantoff et al., 2010), allowing us toconclude that intravenous administration can be an
effective route to deliver DC vaccines. Focusing onAML, most clinical trials of moDC-based vaccinationhave employed the intradermal administration route;only limited experience has been obtained with sub-cutaneous or intravenous DC vaccine administration inthe context of AML (Table 2). One study combinedintradermal and intravenous administration in anattempt to target DCs to both the lymph nodes andthe bone marrow (Kitawaki et al., 2011b), which is theprimary site in AML where high-avidity antileukemiaT cells reside (Melenhorst et al., 2009). However,because of the small sample size of this study, it isimpossible to conclude whether combined intradermal/intravenous administration is superior over intrader-mal administration alone from a clinical standpoint.
B. Enhancing the Strength of the Antitumor ImmuneEffector Cells (Condition Number 2)
For any DC strategy to be effective, it is imperativethat the immune cells of the host are responsive tostimulation and functionally capable of mediatingtumor rejection (Mellman et al., 2011). However, veryoften the functioning of the immune system isdisturbed in cancer patients (Finn, 2012). This obser-vation also applies to patients with AML, whofrequently have a compromised immune status thatprevents them from mounting protective antileukemicimmune responses (Anguille et al., 2011a; Schurchet al., 2013). For instance, patients with AML oftenspontaneously develop leukemia-specific CTLs(Anguille et al., 2012b), but these are every so oftenanergic (Narita et al., 2001; Beatty et al., 2009) ordysfunctional (Le Dieu et al., 2009; Florcken et al.,2013) and thereby fail to recognize and kill theirtargets. Similarly, at the innate immunity level, NKcells of AML patients are often functionally disabled(Lion et al., 2012b). This impaired immune competencecan dramatically hamper the efficacy of DC therapy inpatients with AML. Therefore, to maximize the chanceof treatment success, future DC studies for AML mustnot only focus on enforcing the intrinsic immunosti-mulatory properties of the DCs used in the therapeuticformulation, but also on harnessing the capacity of thepatients’ immune cells to respond to DC stimulationand to properly perform their cytotoxic effector func-tions against AML cells.
1. Enhancing T-Cell–Mediated Antitumor Immunity.The adaptive immune response against cancer can beharnessed by modulating the signals that govern T-cellstimulation or inhibition at the DC/T-cell immunologicsynapse (Vasaturo et al., 2013) (Fig. 2, inset). Althoughin some studies the focus has been on engagingcostimulatory pathways to enhance T-cell function(Houtenbos et al., 2007), most research is aimed attargeting immune checkpoint pathways, such as theB7/CTLA-4 axis and the PD-1/PD-L axis (Vasaturoet al., 2013; Anguille et al., 2014; Datta et al., 2014).
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a. Cytotoxic T-lymphocyte antigen‐4. Combining DCtherapy with CTLA-4 blockade represents an appeal-ing strategy to potentiate DC vaccine-induced T-cellimmunity (Anguille et al., 2014; Datta et al., 2014).Zhong et al. (2006) showed that the concomitant use ofan mAb against CTLA-4 enhances the in vitro capacityof DCs to induce functional CTL responses againstAML cells. The feasibility of using the anti–CTLA-4mAb ipilimumab in patients with hematologic malig-nancies, including AML patients, was recently con-firmed (Bashey et al., 2009). It is important, however,to mention that ipilimumab treatment is associatedwith a high percentage of immune-related adverseevents (Norde et al., 2012). Furthermore, conflictingdata exist regarding the effect of ipilimumab on Treg
cells with some studies showing inhibition of Treg cells(Norde et al., 2012) and other studies showing Treg cellproliferation (Kavanagh et al., 2008).b. Programmed death-1. A similar approach
revolves around the use of DCs in combination withtherapeutic blockade of the PD-1/PD-L coinhibitorypathway (Norde et al., 2012). Blockade of PD-1 onT cells has been shown to promote the capacity of DCsto stimulate leukemia antigen-specific T cells (Hoboet al., 2012). The observed promotion of antileukemicimmunity after interference with the PD-1/PD-Lsignaling pathway is not only due to enhancedactivation of effector T cells but also in part due toreducing Treg cell numbers and attenuating theirimmunosuppressive activity (Ge et al., 2009; Zhouet al., 2010). A phase II clinical trial investigating thecombined use of DC therapy with the anti–PD-1 mAbpidilizumab (CT-011; CureTech, Yavne, Israel) iscurrently underway in remission patients with AML(Rosenblatt et al., 2011). A clinical trial of PD-L1/2–silenced DC vaccination in combination withdonor lymphocyte infusions for the treatment ofpost-transplant leukemia relapse has also beenregistered (CCMO identifier, NL37318) (Norde et al.,2012).c. B and T lymphocyte attenuator. Although CTLA-4
and PD-1 are by far the best characterized T-cellcoinhibitory molecules, other negative regulators of T-cell function, such as BTLA, have been identified(Norde et al., 2012) (Fig. 2, inset). BTLA, which belongsto the same family of coinhibitory receptors as CTLA-4and PD-1, has been implicated in the functionalinhibition of tumor antigen-specific T cells (Nordeet al., 2012). As demonstrated for PD-1, blockade ofBTLA has been shown to enable in vitro expansion ofleukemia antigen-specific T cells upon DC stimulation(Hobo et al., 2012). In contrast to CTLA-4 and PD-1,mAbs targeting BTLA have not yet entered the clinicaltrial arena (Hobo et al., 2012).d. Immunosuppressive cells. Another attractive
way to empower the antitumor effector response isto inhibit immunosuppressive cell populations, such
as Treg cells and myeloid-derived suppressor cells(MDSCs) (Anguille et al., 2014). Treg cells, which occurwith increased frequency in patients with AML, arewell known to have suppressive effects on DCs as wellas on CTLs and NK cells and thus constitute animportant barrier to effective DC-based immunother-apy (Ustun et al., 2011; Anguille et al., 2014; Chianget al., 2015; Vasaturo et al., 2015). In a murine AMLmodel, Delluc et al. (2009) observed that Treg depletionfacilitates the induction of antileukemic immunity bysubsequent DC immunization, providing a strongrationale for implementing Treg-targeted therapy inDC-based immunotherapy programs (Ustun et al.,2011). Depletion or blockade of Treg cells can beachieved with a variety of pharmacological agents,including but not limited to the anti-CD25 mAbdaclizumab (Zinbryta; Biogen Idec, Cambridge, MA/AbbVie, North Chicago, IL), the IL-2 immunotoxindenileukin diftitox (Ontak; Eisai Medical Research),low-dose cyclophosphamide or IDO inhibitors such as1-methyl-tryptophan (Ustun et al., 2011; Anguilleet al., 2014). Several of these agents are available forclinical use and are currently being tested in AMLpatients (Ustun et al., 2011).
MDSCs, a heterogeneous population of immaturemyeloid cells with immunosuppressive properties,have recently drawn increasing attention as potentialtargets for improving the efficacy of DC-based cancerimmunotherapies (Anguille et al., 2014; Vasaturoet al., 2015). Although the precise role and significanceof these cells in AML remains largely elusive (Teagueand Kline, 2013; De Veirman et al., 2014), the numberof MDSCs appears to be inversely correlated withclinical outcome in DC vaccine-treated AML patients(Ogasawara and Ota, 2014), underscoring their poten-tial importance.
2. Enhancing Natural Killer Cell–Mediated Antitu-mor Immunity. Although much focus is being placedon augmenting T-cell immunity, it is important not tolose sight of the important role of NK cells inantitumor immunity (Lion et al., 2012a,b). As dis-cussed above, the efficacy of DC therapy relies in parton the presence of a competent innate immune system(Lion et al., 2012a; Van Elssen et al., 2014). In thiscontext, NK cell activation after DC administration inAML patients has been shown to correlate withfavorable clinical outcome (Van Tendeloo et al.,2010; Lion et al., 2012a). In view of this, it is likelythat the efficacy of DC-based immunotherapy can befurther improved by harnessing the NK-cell stimula-tory potential of DCs or by combining DCs withtherapies that potentiate NK-cell immunity (Anguilleet al., 2011a, 2013, 2014). DCs can be endowed withthe ability to stimulate NK cell cytotoxicity towardAML cells by using IL-15 instead of IL-4 during moDCdifferentiation and/or by using TLR ligands for in-ducing their maturation (Anguille et al., 2013, 2015;
Dendritic Cell–Based Cancer Immunotherapy 745
Okada et al., 2015). DCs genetically engineered toexpress NK cell–activating cytokines, such as IFN-a(Anguille et al., 2011b), can also be used to harnessthe antileukemic activity of NK cells (Willemen et al.,2015). Another conceptually attractive approach is touse these cytokines as adjuvants during DC therapy(Anguille et al., 2014). IL-15, which recently becameavailable for clinical use (Conlon et al., 2015), may beof particular interest in this regard given its potentNK-cell stimulatory properties (Van den Bergh et al.,2015). In addition, IL-15 can sensitize AML cells forNK cell–mediated killing by upregulating activatingNK cell receptors on the AML cell surface (Szczepanskiet al., 2010). Endogenous IL-15 is induced in AMLpatients after lymphodepleting chemotherapy andleads to in vivo expansion of NK cells (Miller et al.,2005; Bachanova et al., 2014). This postchemotherapysurge in IL-15 provides a strong rationale forscheduling the administration of DCs in AMLpatients after the initial chemotherapy (Barrett andLe Blanc, 2010).
C. Making Tumor Cells Susceptible to Immune Attack(Condition Number 3)
Whether DC-induced antitumor immunity will lead tocancer eradication ultimately depends on the strengthof the immune system to overcome the various immuneescape mechanisms used by the tumor cells (Anguilleet al., 2014; Vasaturo et al., 2015) (Fig. 2). AML cellsalso exploit a myriad of mechanisms to defend them-selves against immune attack (Barrett and Le Blanc,2010; Teague and Kline, 2013). A comprehensive reviewon immune escape in AML has been provided elsewhere(Lion et al., 2012b; Teague and Kline, 2013) and willtherefore not be discussed in detail here. In this section,we will only mention some examples of immune escapemechanisms for which treatment solutions are readilyapplicable and that can be targeted to synergisticallyenhance the efficacy of DC-based cancer immunother-apy in AML.1. Targeting Programmed Death-1. One well rec-
ognized mechanism by which AML cells inhibitantileukemic immune responses is through expres-sion of ligands for PD-1 (Zhang et al., 2009; Zhouet al., 2010; Teague and Kline, 2013). As discussed inthe previous section, PD-L1/2 provides an inhibitorysignal to T cells during DC–T-cell interaction (Fig. 2,inset). Because PD-L1 can also be expressed on AMLblasts and can be used by these cells to inhibit CTLfunction directly (Saudemont and Quesnel, 2004) aswell as indirectly via Treg cells (Zhou et al., 2010),conjoint targeting of the PD-1/PD-L pathway can helpto improve the efficacy of DC therapy on three fronts:1) by favoring effective antigen presentation at theDC–T-cell immunologic synapse, 2) by restoring AML-induced suppression of CTL function, and 3) bycounteracting Treg cells.
2. Targeting Indoleamine 2,3-Dioxygenase.Another example of an immunosuppressive moleculethat can be expressed by both DCs and AML cells isIDO (Curti et al., 2007, 2010; Chamuleau et al., 2008;Sorensen et al., 2009; Teague and Kline, 2013) (Fig. 2).In view of its central role in Treg biology, targeting IDOhas been proposed as an attractive strategy to over-come Treg-mediated immune escape in AML (Ustunet al., 2011; Teague and Kline, 2013; Arpinati andCurti, 2014). A clinical trial using the IDO inhibitor 1-methyl tryptophan in combination with DC therapywas recently initiated for patients with metastaticbreast cancer (ClinicalTrials.gov identifier: NCT01042535).It should, however, be taken into account that the adaptiveimmune system can react against IDO-expressing tumorcells by targeting IDO (Sorensen et al., 2009). Indeed,spontaneous T-cell responses against IDO have beendetected in cancer patients, and these IDO-reactiveCTLs have been shown capable of killing IDO-expressing AML blasts (Sorensen et al., 2009). Froma theoretical point of view, IDO inhibitors could thushave a negative impact on antileukemic immunity.Other Treg-targeting strategies may therefore bepreferred.
3. Targeting CD200. The CD200 surface moleculewas recently recognized as a key mediator of immuneescape in AML (Martner et al., 2013). CD200 contrib-utes to AML-induced immunosuppression througha multifaceted mode of action, which includes activeinhibition of TH1 responses (Coles et al., 2012a),induction of Treg cells (Coles et al., 2012b), andsuppression of NK cell function (Coles et al., 2011). Inan in vitro study of AML, Memarian et al. (2013)showed that abrogation of CD200/CD200R interactionenhances the T-cell-stimulatory capacity of DCs. Theseobservations provide a compelling rationale for com-bining DC vaccination with anti-CD200 agents. Sama-lizumab (Alexion Pharmaceuticals, Cheshire, CT), ananti-CD200 mAb, is currently under investigation ina phase I clinical trial in patients with chronic lympho-cytic leukemia and multiple myeloma (Mahadevanet al., 2010), but, to the best of our knowledge, hasnot yet been studied in the context of AML or incombination with DC therapy.
4. Mobilizing Acute Myeloid Leukemia Cells fromImmune-Privileged Bone Marrow Niches. AML cellsare also capable of hiding from immune detection, atleast in part by residing in immune-privileged bonemarrow niches (Barrett and Le Blanc, 2010; Peled andTavor, 2013). One conceptually attractive strategy toincrease their visibility to the immune system is tomobilize AML cells out of these protective bone marrowniches (Zeng et al., 2009; Uy et al., 2012). Antagonistsfor C-X-C chemokine receptor type-4 (CXCR-4) can beused for this purpose as they disrupt the interactionbetween CXCR-4 expressed on AML cells with stromalcell–derived factor-1a expressed on bone marrow
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stromal cells (Zeng et al., 2009; Uy et al., 2012; Zhanget al., 2012; Peled and Tavor, 2013). A recent phase Iand II clinical study in AML patients has confirmedthat plerixafor (Mozobil; Genzyme/Sanofi, Cambridge,MA), a CXCR-4 antagonist used for hematopoietic stemcell mobilization, can disengage AML cells from thebone marrow and increase their sensitivity to chemo-therapy (Uy et al., 2012). Histone deacetylase inhib-itors, which are increasingly being used in thetreatment of AML, may have a similar effect byrepressing CXCR-4 expression in AML cells (Gulet al., 2010; Mandawat et al., 2010).5. Undoing Tumor Cells’ Cloak of Invisibility.
In addition to residing in protective bone marrowniches, AML cells are notorious for creating an“invisibility cloak” that prevents them from beingrecognized by T cells and NK cells (Teague and Kline,2013). Elements of this “invisibility cloak” includedownregulation of leukemia antigen presentation, re-duced expression of MHC and T-cell costimulatorymolecules, or shedding of surface molecules involved inimmune recognition and activation (e.g., NKG2Dligands) (Barrett and Le Blanc, 2010; van Luijn et al.,2010; Lion et al., 2012b; Teague and Kline, 2013).These immune evasion mechanisms can clearly affectthe efficacy of active specific immunotherapyapproaches such as DC therapy. Therefore, strategiesfor potentiating immune recognition and destructionof AML cells may be exploited in combination with
DC-based immunotherapy to increase its therapeuticactivity (Anguille et al., 2014). A number of pharma-cological agents can be used for this purpose, includingcytokines, TLR ligands, hypomethylating agents (e.g.,azacitidine and decitabine), histone deacetylase in-hibitors, and kinase inhibitors (for references, seeTable 3).
6. Reducing Tumor Burden. Finally, AML cellsmay not only evade the immune system by hidingfrom immune recognition but also by being present insuch large numbers that the immune system is simplyoutweighed (Emens and Jaffee, 2005; Drake, 2010;Schlom, 2012). This provides a compelling rationale tofirst reduce the tumor burden with conventionalchemotherapy before applying immunotherapy strate-gies, such as DC therapy. Support for this concept hasbeen obtained in different tumor models (Choudhuryet al., 2006; Drake, 2010; Schlom, 2012), includingAML, where the administration of DCs after standardremission induction chemotherapy (i.e., in the settingof minimal residual disease) has shown encouragingresults (Van Tendeloo et al., 2010). Although chemo-therapy is traditionally being considered to be immu-nosuppressive (e.g., by tilting the balance betweenantitumor immune effector cells and Treg cells in favorof the latter) (Lichtenegger et al., 2014), manychemotherapeutic agents used for AML appear to haveparadoxical immune-potentiating effects (Obeid et al.,2007; Liseth et al., 2010; Wemeau et al., 2010; van de
TABLE 3Examples of nonchemotherapeutic antileukemic agents capable of reinstating the immunogenicity of AML cells
Drug Effects on AML Cell Immunogenicity Effects on DCs (and Other Antitumor Immune Effectors)
Cytokines (e.g., interferon-a) • Immunogenic cell death (Rizza et al., 2015) • ↑ DC activation and function (Anguille et al.,2011b)
• ↑ MHC and/or costimulatory molecule expression(Anguille et al., 2011b)
• ↑ direct antileukemic cytotoxicity of DCs (Telet al., 2014)
• ↑ AML antigen expression expression (Anguilleet al., 2011b)
• ↑ activity of T cells and NK cells (Anguille et al.,2011b)
TLR ligands [e.g., poly(I:C), R-848] • Immunogenic cell death (Smits et al., 2007; Lionet al., 2011)
• ↑ DC activation and function (Smits et al., 2007;Lion et al., 2011)
• ↑ MHC and/or costimulatory molecule expression(Smits et al., 2007)
• ↑ activity of T cells and NK cells (Smits et al.,2010)
Hypomethylating drugs (e.g.,azacitidine, decitabine)
• Immunogenic cell death (Tang et al., 2013) • ↑ DC function (Frikeche et al., 2011)• Derepression of AML antigen expression (e.g.,CTAs, WT1) (Almstedt et al., 2010; Goodyearet al., 2010; Atanackovic et al., 2011; Kwon et al.,2012; Yao et al., 2013)
• ↑ Treg cells (conflicting data) (Goodyear et al.,2012; Gang et al., 2014)
• ↑ MHC and/or costimulatory molecule expression(Wang et al., 2013)
• ↓ activity of NK cells (Schonefeldt et al., 2013;Gang et al., 2014)
• ↑ NKG2D ligand expression (Rohner et al., 2007)Histone deacetylase inhibitors • Immunogenic cell death (Fredly et al., 2011) • ↓ DC function (Falkenberg and Johnstone, 2014)
• ↑ differentiation of AML cells toward a DCphenotype (Moldenhauer et al., 2004)
• ↑ IDO expression in DCs (Reddy et al., 2008)
• Derepression of AML antigen expression (e.g.,CTAs) (Goodyear et al., 2010; Yao et al., 2013)
• ↑ MHC and/or costimulatory molecule expression(Maeda et al., 2000; Yao et al., 2013)
• ↑ NKG2D ligand expression (Rohner et al., 2007;Diermayr et al., 2008; Poggi et al., 2009)
Protein kinase C inhibitor (bryostatin) • ↑ differentiation of AML cells toward a DCphenotype (Roddie et al., 2002)
• ↑ DC activation and function (Do et al., 2004;Ariza et al., 2011)
• ↑ NKG2D ligand expression (Rohner et al., 2007)
CTAs, cancer-testis antigens.
Dendritic Cell–Based Cancer Immunotherapy 747
Ven et al., 2012). For example, cytarabine andanthracyclines (the two standard chemotherapeuticagents in AML) cause AML cells to undergo immuno-genic apoptosis (Wemeau et al., 2010; Fredly et al.,2011), thereby favoring the development of antileuke-mia immunity (Barrett and Le Blanc, 2010; Rezvaniet al., 2012). These observations provide strong supportfor the concept of chemoimmunotherapy in AML. DC-based vaccination strategies are also being increas-ingly applied in the context of allogeneic hematopoieticstem-cell transplantation for AML to boost the graft-versus-leukemia reaction. The reader is referred toPlantinga et al. (2014) for an update on DC-basedimmunotherapy in the postallogeneic hematopoieticstem-cell transplantation setting.
IV. Conclusions
Because they lie at the heart of the immune system’sability to recognize and eliminate cancer cells, DCshave attracted a great deal of interest for antitumorimmunotherapy in recent decades (Palucka andBanchereau, 2012; Anguille et al., 2014). Current effortsin the field of DC-based cancer immunotherapy aregeared toward identifying the key factors responsiblefor therapeutic success/failure to unleash the fullclinical potential of DCs for cancer immunotherapy(Kirkwood et al., 2012; Anguille et al., 2014; Chianget al., 2015). AML can be a good study model for thispurpose, in particular because it allows us to examinethe efficiency of this form of immunotherapy in a lowtumor burden context (i.e., in the setting of minimalresidual disease), which is probably the most appro-priate clinical situation to apply immunotherapy(Gulley et al., 2011). In recent years, many effortshave been made to refine and optimize the protocols forDC manufacturing (condition no. 1), but, at the sametime, we should be aware that therapeutic success islikely not to result from a single intervention. Thefuture of DC therapy lies in combining this form ofactive specific immunotherapy with other therapies toincrease the antitumor activity of the immune effectorcells (condition no. 2) and to overcome the myriad ofimmune escape mechanisms used by cancer cells(condition no. 3) (Anguille et al., 2014; Chiang et al.,2015). The past decade has witnessed the advent ofa new generation of DC vaccines with improved potency.This, together with the ever-expanding portfolio ofimmunomodulatory and immune-potentiating therapiesavailable for combinatorial use, may allow us toconclude that we now stand on the brink of realizingthe true potential of DCs for cancer immunotherapy.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Anguille,Smits, Bryant, Van Acker, Goossens, Lion, Fromm, Van Tendeloo,Berneman.
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