Onto better TRAILs for cancer treatment

OPEN REVIEW Onto better TRAILs for cancer treatment D de Miguel* ,1,2 , J Lemke 3,5 , A Anel 1,2 , H Walczak 3 and L Martinez-Lostao* ,1,2,4,6 Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-Rtargeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists. Cell Death and Differentiation (2016) 23, 733–747; doi:10.1038/cdd.2015.174; published online 4 March 2016 Facts On its discovery, TRAIL was described to be capable of inducing apoptosis selectively in cancer cells. However, soon afterwards it was found that many cancer cell lines as well as primary cancer cells are either intrinsically TRAILresistant, or become resistant upon TRAIL treatment. The results from TRAIL using clinical trials have been disappointing, showing little antitumor efficacy. All these clinical trials have used a soluble form of the protein, which is known to be rather unstable and to have poor physicochemical properties. TRAIL has four receptors that are expressed at the plasma membrane, of which two can trigger apoptosis. Little is known about the relative contribution or differential roles of these two pro-apoptotic TRAIL receptors (TRAIL-Rs). Physiologically, TRAIL is expressed as a transmembrane protein. This fact may be exploitable therapeutically since membrane-bound as well as artificially cross-linked TRAIL is by several orders of magnitude more active than conventional soluble trimeric TRAIL. New TRAIL formulations with increased bioactivity due to improved stability and/or cross-linking efficiency have been developed. Besides, new approaches trying to combine inherent TRAIL pro-apoptotic ability with delivery systems based on nanoparticles are also being explored. Open Questions Could new forms of TRAIL or other TRAIL-R agonist formulations with increased bioactivity, improved pharmacokinetic and targeting properties contribute to overcoming TRAIL resistance without causing systemic toxicity? Could such novel TRAIL-R-targeting biotherapeutics exert improved synergy with known TRAIL-sensitizing agents, over TRAIL-R agonists used clinically to date? Despite remarkable advances in understanding the biology of cancer and the development of novel diagnostic and therapeutic strategies, cancer still remains one of the major causes of death. To date, in addition to surgical resection of the tumor, conventional radioand chemotherapy constitute the central pillars of cancer treatment. These therapies aim to limit proliferation and/or induce the death of cancer cells. However, they mostly lack cancer specificity and, therefore, also damage normal, healthy tissues resulting in often severe side effects that constitute the dose-limiting toxicities. In addition, many cancers acquire resistance to these therapies, rendering them ineffective in consecutive treatment rounds. Hence, during the past decades great efforts have been made to develop new therapeutic approaches, aiming to improve the specific targeting of cancer cells and to overcome resistance to current therapies. 1,2 1 Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; 2 Instituto de Investigación Sanitaria de Aragón, Zaragoza, Spain; 3 UCL Cancer Institute, Faculty of Medical Sciences, University College London, London, UK and 4 Instituto de Nanociencia de Aragón, Zaragoza, Spain *Corresponding author: D de Miguel or L Martinez-Lostao, Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza, Spain. Tel: +34 976 76 12 87; Fax: +34 976 76 21 23; E-mail: diego_demig[email protected] or [email protected] 5 Current address: Clinic of General and Visceral Surgery, Universität Ulm, Ulm, Germany 6 Current address: Servicio de Inmunología, Hospital Clínico Universitario Lozano Blesa, Avda/San Juan Bosco 15, Zaragoza 50009, Spain Received 21.8.15; revised 11.12.15; accepted 17.12.15; Edited by G Melino; published online 04.3.16 Cell Death and Differentiation (2016) 23, 733–747 & 2016 Macmillan Publishers Limited All rights reserved 1350-9047/16 www.nature.com/cdd The better understanding of tumor biology, tumor immunology and how cancer cells interact with the tumor microenvironment, sparked the development of cancer immune-therapeutics as well as so-called targeted cancer therapeutics. 2–5 The identification of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also referred to as Apo-2 ligand (Apo2L), 6,7 and most importantly, the discovery of TRAIL’s capacity to kill cancer cells while sparing all the vital normal cells, 8 appeared to represent a promising step forward in the development of targeted anticancer therapies. TRAIL belongs to the TNF superfamily (SF) of cytokines and is capable of inducing apoptosis in cells by binding to either of two cognate death receptors (DRs), TRAIL-R1/DR4 (ref. 9) and TRAIL-R2/DR5. 10–14 Physiologically, TRAIL has been implicated in the function of cytotoxic effector cells 15,16 and the homeostasis of the lymphoid compartment by being a mediator of activation-induced cell death (AICD) in effector immune cells. 17 Given the cancer-selective apoptosis-inducing potential of TRAIL and the fact that TRAIL-R1 and, even more so, TRAIL-R2 are often highly expressed in different malignancies, 9,13,14,18–23 the use of TRAIL or other agonists for TRAIL-R1/R2 for cancer therapy appeared an attractive concept. Consequently, TRAIL-R agonists were developed for clinical application. The results of the clinical studies performed with these first-generation TRAIL-R agonists so far have been rather disappointing, however, with limited patient benefit despite promising pre-clinical results. 24–26 The fact that many human tumors are partially or completely resistant to monotherapy with TRAIL and other TRAIL-R agonists likely contributed to the limited therapeutic activity observed in these studies. However, another—perhaps decisive—factor for the lack of clinical efficacy of the specific TRAIL-R agonists that have been tested clinically most likely is that their agonistic capacity was simply not sufficiently potent. This is exemplified by a recent study in which it was shown that, only when used in combination, two of the abovementioned clinically developed TRAIL-R agonists exerted virtually the same agonistic activity as isoleucine zipperTRAIL (iz-TRAIL), 27 a highly active form of TRAIL that has been in use for some time 28–30 and is based on the original leucine-zipper form of TRAIL (LZ-TRAIL) used in the study in which TRAIL’s tumor-selective apoptosis-inducing potential was discovered, importantly, in the absence of systemic toxicity. 8 Unfortunately, this fact went largely unnoticed and because of safety concerns with certain more potent forms of TRAIL, 31 several TRAIL-R agonists with, as it turned out, insufficient agonistic activity and consequently pro-apoptotic potency were developed for clinical use. Yet, the fact that to date no sufficiently potent TRAIL-R agonist that lacks systemic toxicity has been clinically validated, has led to the development of novel formulations of TRAIL and other TRAIL-R agonists with improved bioactivity, with the aim to overcome TRAIL resistance in combination with improved sensitization strategies and patient-selection criteria. 32 This review summarizes the main novel formulations of such TRAIL-R agonists that are currently being tested or developed to improve biological attributes such as stability, delivery, targeting and cytotoxic activity against tumor cells as well as their potential for applications in cancer therapy. TRAIL signaling Physiologically, TRAIL is expressed as a type 2 transmembrane protein that can be cleaved, resulting in the release of a 24 kDa extracellular portion comprising amino acids 114–281 of the protein. The C-terminal extracellular domain of TRAIL shares high homology with other members of the TNF SF and is composed of two anti-parallel β-sheets. 33–35 As shown by the crystal structure of TRAIL interacting with TRAIL-R2, TRAIL forms a trimer and each receptor molecule interacts with the crevice formed by two monomers of the trimer. Thereby, the TRAIL trimer can engage three receptors simultaneously. Interestingly, unlike other TNF SF members, the ligand trimer appears to be stabilized by an internal zinc atom, which interacts non-covalently with three cysteine residues, one from each TRAIL monomer. This interaction is thought to be crucial for the stability, solubility and bioactivity of trimeric TRAIL. 33–35 TRAIL can bind to four transmembrane receptors: TRAILR1, TRAIL-R2, TRAIL-R3, also known as decoy receptor 1 (DcR1) and TRAIL-R4 (DcR2), as well as to the soluble receptor osteoprotegerin (OPG). 9,13,36–39 Among them, only TRAIL-R1 and TRAIL-R2 are able to trigger apoptosis as TRAIL-R3, TRAIL-R4 and OPG lack the functional cytoplasmic death domain (DD) that is required for apoptosis induction. 40,41 On the basis of overexpression experiments, TRAIL-R3 and TRAIL-R4 have been suggested to act as decoy receptors that inhibit apoptosis induction by TRAIL as a consequence of ligand scavenging. 23,42 In addition, TRAIL-R4 has been proposed to be capable of inhibiting TRAIL-induced apoptosis by forming ligand-independent inactive complexes with TRAIL-R2 or the induction of pro-survival pathways such as NF-κB. 43–45 However, there is still controversy concerning the physiological role of TRAIL-R3 and TRAIL-R4, and their function might depend on the cell type. For example, and in contrast to the mentioned studies, these receptors have also been described not to function as DcRs in the human hepatocellular carcinoma cell lines Hep3b and a TRAILresistant variant of HepG2 (HepG2-TR). 46 TRAIL triggers the extrinsic apoptosis pathway upon binding of the TRAIL trimer to TRAIL-R1 and/or TRAIL-R2, resulting in receptor trimerization, which in turn leads to recruitment of the adaptor protein Fas-associated DD (FADD) via homotypic DD-DD interaction between the DDs of the ligand cross-linked receptors and FADD, respectively. FADD, in turn, recruits pro-caspase-8 and pro-caspase-10 via homotypic interactions of death-effector domains (DED) present both in FADD and caspase-8 and -10, respectively. This multi-protein complex formed by TRAIL-DRs, FADD and caspase-8/10 is called death-inducing signaling complex (DISC). 47–51 On recruitment to the DISC, the pro-caspases-8 and -10 form homodimers. This induces a conformational change that exposes their proteolytically active sites, resulting in auto-activation and subsequent cleavage of additional procaspase-8 and -10 molecules leading to full caspase activation at the DISC. 52–55 TRAIL can activate both branches of the apoptosis pathway by caspase-8-mediated cleavage and activation of the effector caspase-3 and the BH3-only protein Bid. In so-called type I cells, cleavage and activation of caspase-3 by activated New TRAIL formulations for cancer treatment D de Miguel et al 734 Cell Death and Differentiation caspase-8 is sufficient to induce apoptosis, whereas in type II cells, activation of the mitochondrial pathway is required for apoptosis induction as a consequence of TRAIL DISC activation. 56,57 The latter is triggered by caspase-8-mediated cleavage of Bid, which results in the formation of truncated Bid (tBid) as the active fragment of this protein. 58–61 Subsequently, tBid activates the mitochondrial pathway by enabling the pro-apoptotic Bcl2-family members Bax and Bak to insert in the mitochondrial outer membrane (MOM), resulting in MOM permeabilization (MOMP) and release of cytochrome Cand Smac/DIABLO (second mitochondrial activator of caspases/ direct inhibitor of apoptosis-binding protein with low pI) 62,63 from the mitochondrial intermembrane space into the cytosol. 64,65 Although TRAIL-R1 and TRAIL-R2 bear high structural similarity and both are able to trigger apoptosis upon TRAIL-induced cross-linking, functional differences between them have been reported. First, TRAIL-R2 has higher affinity for TRAIL than TRAIL-R1. 66 Yet, higher affinity does not necessarily result in enhanced DISC activation as although TRAIL-R2 can be engaged by the soluble ligand, this interaction only triggers a comparably weak DISC formation. 67 This result supports the notion that TRAIL-R2 may require further cross-linking of soluble TRAIL (sTRAIL), whereas stimulation of TRAIL-R1 by sTRAIL appears to be able to trigger apoptosis independently of further crosslinking. 68,69 However, recently it was shown that oligomerized TRAIL versions can also activate TRAIL-R1 more efficiently than sTRAIL. 70 Altogether, it seems clear that TRAIL presents a much stronger activity when it is presented in its transmembrane form than their soluble counterparts, and this enhanced activity is directly linked to its ability to cluster and arrange their specific receptors in supramolecular structures. In line with this, several studies showed that clustering of two trimers was sufficient to improve their activity to optimal levels for the other members of the TNF family ligands. 71,72 The requirement for oligomerization for optimal agonistic activity has also been proposed for the other members of the TNF SF, including CD95L (also known as FasL or APO-1L) whose ability to induce apoptosis is dramatically increased (up to 1.000-fold) on clustering of soluble trimers. 73,74 Once clustered, the receptors adopt a supramolecular hexagonal organization, similar to a ‘honeycomb’structure. 75 In line with this, several studies showed that dimerization of two trimers was sufficient to improve their activity to optimal or nearoptimal levels. 71,72 The clustering of DRs achieved thereby most likely facilitates and stabilizes DISC assembly. 75–77 Along these lines, a new way to improve sTRAIL bioactivity by enhancing TRAIL-R2 clustering was described very recently. 27,78 In these studies, sTRAIL was used in combination with the TRAIL-R2-specific agonistic antibody AMG-655/ Conatumumab. Of note, both sTRAIL and AMG-655 had been developed to be used individually as novel anticancer biotherapeutics and had already been tested in clinical trials as discussed in more detail below. Co-administration of AMG-655 and sTRAIL was able to greatly enhance the inherent ability of sTRAIL to activate TRAIL-R2, even sensitizing certain cancer cell lines that are resistant to sTRAIL. This synergistic effect was due to secondary TRAIL-R2 crosslinking exerted by the antibody, which acted in cooperation with the normal engagement of TRAIL-R2 exerted by sTRAIL. In a similar way, a recent work has used another specific TRAIL-R2 antibody in combination with sTRAIL, obtaining the same synergistic effect. 67 It is, however, still largely unresolved what the relative contribution of the two individual TRAIL-DRs to apoptosis induction in a given cancer is. Although TRAIL-R1 has been described to mediate cell death in chronic lymphocytic leukemia cells, acute myelogenous leukemia cells and pancreatic tumors, 79–82 TRAIL-R2 appears to be the main contributor to apoptosis induction in several other epithelialderived cancers. 83,84 This differential pro-apoptotic performance of TRAIL-R1 and TRAIL-R2 depending on the cell/ cancer type may be exploitable therapeutically by specifically targeting the receptor that is preponderant at inducing apoptosis in the particular cancer type in question. Such targeting may increase the specific cytotoxic effect by sparing non-apoptotic interactions with other TRAIL-Rs. Apart from antibody-based biotherapeutics, such receptor-specific TRAIL constructs can be generated by inducing point mutations in residues within the TRAIL sequence that are required for interaction with particular TRAIL-Rs and not others. A number of such TRAIL variants have been devised and have become valuable tools for assessing specific roles of the different TRAIL-Rs, and, moreover, have recently been shown to bear the potential of improving the efficacy of specifically activating TRAIL-R1 and TRAIL-R2, respectively. 45,80,83,85–87 TRAIL-induced apoptosis is tightly regulated at different stages to prevent excessive cell death in normal cells. These mechanisms are exploited by tumor cells to evade TRAILinduced apoptosis. At the level of expression of the TRAIL-Rs it has been suggested that, as mentioned above, the nonapoptotic receptors TRAIL-R3, -R4 and/or OPG may modulate sensitivity to TRAIL. At the DISC level, the main regulator protein is cellular FLICE-Like Inhibitory Protein (cFLIP), that closely resembles caspase-8 but lacks the protease activity required for apoptosis induction. 88,89 Two main variants of cFLIP are expressed on the protein level: a short isoform (cFLIP S ) and a long isoform (cFLIP L ). 90 Both cFLIP isoforms contain two DEDs that are structurally similar to the DEDs present in the N-terminal portion of pro-caspase-8 and -10 and allow recruitment to the DISC. The cFLIP S isoform can inhibit caspase-8 activation in a dominant-negative manner by competing with it for binding to FADD. The role of cFLIP L is, however, more complex and seemingly depends on the ratio between caspase-8 and cFLIP L . 91–93 Although cFLIP L was first reported to act as an anti-apoptotic protein in a manner similar to cFLIP S , 88 later studies demonstrated that the cFLIP L /caspase-8 heterodimer, apart from retaining enzymatic activity, also displays an enhanced and more localized activity toward certain substrates when compared with the caspase-8 homodimer, somehow modulating caspase-8 substrate specificity. 94–96 In fact, the activity of the FLIP L / caspase-8 heterodimer is required to prevent necroptosis. 91–93,97,98 Nevertheless, it should be noted that, when expressed at high levels, cFLIP L can also completely prevent DR-induced apoptosis. Several studies have demonstrated that cancer cells exploit overexpression of cFLIP to evade TRAIL-induced apoptosis 99–101 and, consequently, New TRAIL formulations for cancer treatment D de Miguel et al 735 Cell Death and Differentiation downregulation of cFLIP may sensitize certain cancers to TRAIL-induced apoptosis. 46,102–105 Another important checkpoint in the apoptotic cascade is exerted by XIAP (X-linked inhibitor of apoptosis protein), a molecule that can bind caspases 3, 7 and 9, thereby inhibiting their pro-apoptotic activity. 106 Several additional mechanisms of different nature can modulate TRAIL signaling. Posttranslational modifications such as O-glycosylation, which promotes ligand-stimulated clustering of TRAIL-DRs and recruitment/activation of procaspase-8, 107 ubiquitination regulating the full activation of caspase-8 upon TRAIL stimulation 108 and endocytosis of the DISC upon TRAIL binding 109 are just a few examples of several mechanisms proposed to be implicated in the modulation of TRAIL signaling. Apart from inducing apoptosis, TRAIL can also trigger nonapoptotic signaling such as necroptosis and the activation of pro-inflammatory pathways (via NF-κB, Akt, MAPK and JNK activation). Induction of these non-apoptotic pathways depends on the cell type and is often triggered in scenarios when apoptosis induction is inhibited. 40,110–112 The induction of pathways resulting in gene activation has been suggested to be mediated by the formation of a secondary complex following DISC activation. This secondary complex also contains the DISC components FADD, caspase-8 and cFLIP 113 and, additionally, recruits receptor interacting protein 1 (RIP1), TNF receptor-associated factor 2 (TRAF2) and the NF-κB essential modulator (NEMO). 114 Initially, TRAILinduced activation of pro-inflammatory pathways was proposed to be mainly a mechanism to negatively regulate apoptosis induction by TRAIL. However, activation of these pathways, such as NF-κB, AKT and MAP kinases can also enhance the malignancy of cancer cells by increasing their proliferation, migration, invasion and/or metastasis. 115–117 In addition, both exogenous TRAIL and FasL were shown to induce proliferation and to promote migration in KRASmutated cancer cells upon external administration. 118 These findings led to the recent discovery of a pro-invasive role for endogenous TRAIL in KRAS-mutated cells. In these cells, autocrine endogenous TRAIL stimulates cancer cellexpressed TRAIL-R2 to activate Rac1 which, in turn, activates PI3K to induce cell migration. 119 Interestingly, activation of this signaling pathway was independent of TRAIL-R2’sDDbut instead required its membrane proximal domain (MPD). 119 TRAIL-R agonists as anticancer therapeutics So far, two main TRAIL-DR-targeting therapeutic strategies have being pursued in clinical trials: (i) a recombinant form of human sTRAIL (Apo2L.0 or AMG-951/Dulanermin) and (ii) agonistic antibodies that specifically target TRAIL-R1 or TRAIL-R2. 32 Although these TRAIL-R agonists have been shown to be safe and well tolerated in patients, their respective anticancer activities have been largely disappointing 24–26 (extensively reviewed by Lemke et al. 32 and Holland 120 ). The fact that most primary tumor cells are intrinsically resistant to TRAIL or may acquire resistance during the course of treatment 121–125 has most likely contributed to this failure. In addition to these considerations, non-apoptotic signaling induced by TRAIL has been shown to be exploited by tumor cells, at least in certain cases, to their own advantage. For example, it has been shown that TRAIL promotes the development of liver metastasis in a pancreatic adenocarcinoma xenograft model, pointing toward potentially harmful effects of monotherapy with TRAIL-R agonists. 116 In this context, it is noteworthy to mention that TRAIL-R1 expression positively correlates with tumor grade in patients with breast cancer. 126,127 To avoid the undesired pro-tumorigenic effects of monotherapy with TRAIL-R agonists in TRAIL-resistant cancers, it has been proposed to combine them with sensitizing agents (reviewed in Lemke et al, 32 ). However, regardless of the promising results obtained with such combinatorial approaches, careful evaluation, both pre-clinically and in early clinical testing, is needed as it may bear the risk of sensitizing a vital normal cell type to TRAIL-induced cell death. 28–30,46 It is now clear that, besides adding more potent sensitizing agents to a TRAIL-R-agonist-comprising therapy, improvement of the agonistic capacities of TRAIL-R agonists is imperative to render TRAIL-based therapies effective. To enhance the therapeutic potential of TRAIL, different shortcomings of currently used TRAIL-R agonists need to be addressed. In the specific case of Apo2L.0/AMG-951/Dulanermin, the disappointing results obtained in clinical trials are most likely due to the combination of its short plasma half-life and rapid clearance from circulation 128,129 with its limited ability to cluster TRAIL-DRs. It should be noted that antibodies directed to TRAIL-DRs have a comparably long half-life in serum, whereas their in vivo activity is hampered by the fact that they require external cross-linking to induce effective TRAIL-DR clustering and, hence, TRAIL-DRmediated apoptosis. 130,131 To overcome these shortcomings and pharmacological downsides, novel TRAIL formulations have been developed with the aim to increase the efficiency of TRAIL-DR-targeting therapies (Figure 1). These novel formulations improve the activity of TRAIL-R agonists by tackling the following two main aspects: (i) increasing stability and valency and (ii) enhancing cancerspecific delivery. A wide variety of experimental approaches are currently engineered to address these aspects, resulting in novel versions of TRAIL-R agonists with promising attributes, which will hopefully prove useful to cancer treatment in the future. Increasing the stability of TRAIL The correct conformation and stability of TRAIL has a crucial role for its biological activity since trimerization of TRAIL monomers is pivotal to induce TRAIL-R clustering on the cell surface. The physical and chemical changes can, however, result in the collapse of TRAIL’s trimeric structure. 31,33,34,132 Furthermore, TRAIL monomers can easily form disulfidelinked dimers that impairs its apoptotic potential by up to 90fold. 34 The first recombinant versions of TRAIL comprised the extracellular portion of the protein or its TNF homology domain (THD) with an N-terminally added poly-Histidine tag (His-TRAIL 6 ) or FLAG epitope tag (FLAG-TRAIL 7 ). These tags were added merely to facilitate the purification process. Noteworthy, FLAG-TRAIL alone was poorly active, and New TRAIL formulations for cancer treatment D de Miguel et al 736 Cell Death and Differentiation required further cross-linking by the FLAG-specific antibody M2. 7 These constructs rendered promising results in vitro and also provided promising in vivo safety profiles in the different animal models, mainly rodents and nonhuman primates. 8,31,133 However, both His-TRAIL and cross-linked FLAG-TRAIL were capable of killing freshly isolated primary human hepatocytes (PHH) in vitro. 30,134,135 Most likely, the main reason for this hepatotoxicity was the formation of aberrant supramolecular aggregates owing to the interactions between the added tags. In particular, in the case of HisTRAIL, metal analysis showed an abnormally low molar ratio between zinc and the TRAIL trimer, implying that anomalous supramolecular structures may have formed. 31 These findings suggested that TRAIL trimer stability may impact hepatotoxicity in vivo and turned the focus on potential liver toxicity of systemic TRAIL administration. Figure 1 TRAIL formulations with increased bioactivity for cancer treatment. Different formulations of TRAIL using distinct experimental approaches have been developed to increase its therapeutic potential. These formulations are mainly based in fusion proteins with single-chain variable antibody fragments (scFv), conjugation with nanoparticles and, cell-based methods to express and/or secrete Apo2L/TRAIL. The main properties improved with these highly bioactive formulations are the increase of the molecule stability, tumor targeting and the possibility of combination with other antitumor agents in a unique formulation. References: 1: leucine zipper-TRAIL; 8,142 2: Isoleucine zipper-TRAIL homotrimer; 30 3: PEG-HZ-TRAIL; 150,152 4: APG350; 209 5: Fn14:TRAIL; 192,193 6: TRAIL HSA-NPs; 153 7: PEG-TRAIL microspheres; 152,169 8: TRAIL-PEG-NPs; 154 9: TRAILLPs; 173,174,176 10: PEG-TRAIL/Dox microspheres; 151 11: TRAIL/Dox HSA-NPs; 167 12: magnetic NPs-TRAIL; 170 13: LUV-TRAIL; 67,171,172,212 14: LUV-Apo2L.0; 213 15: sTRAILtargeted stealth liposome; 179 16: TRAIL/Tf/Dox HSA-NPs; 168 17: immuno-LipoTRAIL; 177 18: Anti-CD3:TRAIL K12:TRAIL; 196 19: leukocytes coated with LUV-TRAIL-ES; 178 20: granulocytes coated with CLL1:TRAIL; 202 21: MBOS4:TRAIL; 69 22: scFv425:sTRAIL; 189,190 23: scFvCD19:sTRAIL; 185 24: Db αEGFR -scTRAIL; 145 25: scFvCD33:sTRAIL; 186 26: Anti-MCSP:TRAIL; 188 27: scFv-EHD2-scTRAIL; 211 28: scFvG28:TRAILmutRs; 195 29: scFvCD70:TRAILmutRs; 70 30: RGD-L-TRAIL; 203 31: CD40ed:TRAILed; 214 32: MSC. scFvCD20-sTRAIL; 187 33: ANG-CLP/PTX/pEGFP-hTRAIL; 176 34: sTRAIL-expressing E. coli DH5α 215 New TRAIL formulations for cancer treatment D de Miguel et al 737 Cell Death and Differentiation The only recombinant form of TRAIL approved for use in clinical trials to date has been an untagged version of the molecule comprising residues 114–281 of TRAIL. This version, known as the aforementioned Apo2L.0 or AMG-951/ Dulanermin, appeared to be both active and safe as it worked well in several xenotransplant cancer models 8,84,128,133 but did not kill freshly isolated PHH and was well tolerated by cynomolgus monkeys and chimpanzees. 30,31,128,136 Consequently, Apo2L.0/Dulanermin was tested in the cancer patients where it indeed proved to be safe, though also disappointingly inactive. 25,26,137–141 Apo2L.0/Dulanermin is rather unstable, presenting low pharmacokinetic profiles, especially concerning its serum half-life with an extended distribution half-life (t 1/2 α) of only 3–5 min and an elimination half-life (t 1/2 β) of 20 min. 8,128 In addition, as previously mentioned, Apo2L.0/Dulanermin mainly induces activation of TRAIL-R1 and appears to be unable to potently activate TRAIL-R2. 68,69 To address these issues, improved versions of TRAIL have been engineered to enhance its stability while retaining the proper trimer structure. The first approach that, interestingly, even predated the engineering of Apo2L.0/Dulanermin, was the inclusion of a specific trimerization domain, a modified leucine zipper motif (LZ-TRAIL) 8 followed by the use of an isoleucine zipper (iz-TRAIL) 30 at the N terminus of the extracellular domain. The addition of these trimerization motifs achieves robust stabilization of the TRAIL trimer by specific interactions between the modified leucine or isoleucine zipper domains that form stable triple helices. These first high-activity recombinant forms of TRAIL were significantly more active than Apo2L.0/Dulanermin, both in vitro and in vivo, and also exhibited better pharmacokinetic profiles in rodents with an extended distribution half-life (t 1/2 α) of 1.3 h and an elimination half-life (t 1/2 β) of 4.8 h. Most importantly, however, these proteins showed neither specific toxicity on PHH ex vivo nor systemic toxicity in vivo in mice. 8,142 More recently, Berg et al. 143 developed a new highly stable version of TRAIL by the incorporation of the tenascin-C (TNC) oligomerization domain (TNC-TRAIL), which stabilized the trimeric conformation in a similar fashion to LZ-TRAIL and izTRAIL. Besides, several groups recently developed novel versions of highly stable TRAIL trimers that build upon a single-chain TRAIL (scTRAIL) trimer. 144,145 Contrarily to ‘classic’approaches in which TRAIL is expressed from a monomer-encoding cDNA, scTRAIL is expressed as a single amino-acid sequence encoding a TRAIL trimer as three consecutive extracellular TRAIL domains that are fused in a head-to-tail configuration, inserting a short linker between each domain. Hence, once correctly folded, scTRAIL already forms an active TRAIL trimer, reducing the risk of unspecific aggregation of the monomers. The common feature of these constructs is their more stable trimerization, which enhances their pro-apoptotic potential so that they are even able to kill some of the cancer cell lines that are resistant to the lessactive Apo2L.0/Dulanermin. 8,30,142–145 In addition, these forms of recombinant TRAIL also exhibit increased in vivo half-lives, whereas the formation of higher-order, aberrant protein oligomers that can result in hepatotoxicity and systemic toxicity 31 appears not to occur. 8,30,142,144,145 Another strategy to improve the in vivo performance of TRAIL is based on covalently linking TRAIL to molecules known to have favorable pharmacokinetic properties such as human serum albumin (HSA) 146 or polyethylene glycol (PEG). PEGylation is a process by which polymer chains of PEG are added covalently to biomolecules such as peptides, proteins or antibodies. The resulting PEGylated biomolecules usually present improved pharmacokinetic properties and, consequently, enhanced therapeutic efficacy. 147–149 Hence, PEGylated versions using site-specific N-terminal PEGylation of izTRAIL showed widely improved pharmacokinetic profiles in vivo and, furthermore, greatly augmented stability and solubility under physiological conditions. 150–154 In addition, PEGylation improved TRAIL’s efficacy at targeting cancer cells owing to the enhanced permeability and retention (EPR) effect, which will be discussed in more detail below. Targeting TRAIL to cancer cells An important obstacle when treating primary tumors effectively with TRAIL is that they are often intrinsically TRAIL-resistant, or acquire resistance when treated with TRAIL. Several studies have shown that co-administration of certain chemotherapeutic drugs can sensitize the cancer cells to TRAILinduced apoptosis. 155–159 However, chemotherapeutics lack cancer cell selectivity and cause severe adverse effects by also targeting normal cells. Thus, this obstacle could be overcome by improving the specificity of TRAIL for cancer cells when used in combination with chemotherapeutics or other sensitizing compounds. Furthermore, targeted delivery of TRAIL specifically to the tumor would increase the local concentration and minimize dilution of the drug in circulation. Mainly two approaches of targeting methods have been pursued: (i) passive targeting based on the EPR effect and (ii) active targeting by using antibody fragments or peptides that target TRAIL to specific tumor-enriched antigens. Passive targeting: combining TRAIL with nanoparticles. The nanoparticle (NP)-based systems have emerged as a promising means to improve drug delivery in vivo. 160–162 Structurally, NPs have a diameter in the range of 50–150 nm and can be composed of a wide variety of compounds, including lipids and polymers. These compounds can be combined with different therapeutic molecules trapped inside the NPs and/or presented on the NP surface. Independent of the NP composition, they possess interesting and desirable general features such as improved pharmacokinetics, pharmacodynamics and in vivo stability of the therapeutic molecules encapsulated by them (Figure 2). Another important characteristic of NPs is the aforementioned EPR effect. Depending on the size and surface property of the NP in question, and given that blood and lymph vessel systems in tumors are thought to be leaky to macromolecules, NPs readily spill from capillaries and lymph vessels that vascularize tumor tissue. Consequently, the EPR effect allows the NPs to better target tumors than the therapeutic molecules alone. 162–166 The optimal diameter of the NPs to take advantage of the EPR effect is in the range of 10–150 nm. Regarding the EPR effect, many anticancer drug-containing nano-systems such as micelles, microspheres and liposomes New TRAIL formulations for cancer treatment D de Miguel et al 738 Cell Death and Differentiation have been developed, and several NP products such as Doxil (Centocor Ortho Biotech Products, Horsham, PA, USA), DaunoXome (Diatos, Paris, France) and Genexol-PM (Samyang, Seoul, Korea) have already been approved for clinical use or are currently tested in clinical trials. As summarized in Table 1, a number of TRAIL-containing NPs are currently being developed. To engineer the NP-core, different chemical compositions have been used such as human serum albumin, 153,167,168 poly (lactic-co-glycolic) acid (PLGA) microspheres, 151,152,169 a combination of PEGylated heparin and poly-L-lysine, 154 magnetic ferric oxide 170 or liposomes. 67,171–179 Among them, thanks to their versatility, liposomes have emerged as the most versatile of these platforms. Moreover, liposomes can be easily modified sizeand composition-wise depending on the desired physicochemical properties. In addition, they represent a safe choice as liposomes have been widely studied and used in the clinic as drug carriers. 180,181 Concerning the manner in which TRAIL is integrated with the NPs, there are two different strategies: (i) to encapsulate TRAIL inside the particles so that they are released from the particle in a constant and stable manner; 151–154,169,173–176 or (ii) to attach TRAIL to the surface of the nanoparticles so that TRAIL gets immobilized, resembling the physiological membrane-bound protein, increasing its bioactivity. 168,170–172,177–179 An additional benefit of both strategies is the possibility to load NPs with additional drugs that could act in concert with TRAIL thereby enhancing its pro-apoptotic effect. In fact, the combination of TRAIL with doxorubicin 151,168,173,174 or paclitaxel 76 in NPs has already been reported. In all the cases, the therapeutic effect was greatly enhanced by co-delivery of the chemotherapeutic agents with TRAIL, whereas no systemic toxicity was detected in vivo. Besides the EPR effect, some authors have boosted the intrinsic tumor-targeting ability of NPs by functionalizing them with targeting molecules such as single-chain variable fragments (scFv), 177 transferrin, allowing transferrin-mediated endocytosis of the NPs, 168 or angiopep-2, 176 a molecule that specifically targets the low-density lipoprotein receptor-related protein, which is highly expressed on the blood–brain barrier and glioma cells. 182 Furthermore, angiopep-2 has recently not only been used for enhanced delivery across the blood–brain barrier, but also for targeting brain tumors by the so-called ‘dual targeting effect’. 176 Active targeting: antigen-restricted activation of TRAIL receptors. An additional strategy to enhance TRAIL targeting is the use of domains or motifs that specifically target cancer cells or cells of the tumor stroma. Several groups have developed novel TRAIL constructs that have been fused to such domains. The resulting fusion proteins are intrinsically bivalent, maintaining the ability to engage TRAIL-DRs and simultaneously combining this with the specific targeting of an antigen expressed on the surface of particular tumor cells or cells in the tumor microenvironment. Although antibodies would be an obvious choice to provide such targeting ability, whole immunoglobulins have a molecular weight of approximately 150 kDa, rendering them sterically less than ideal to be used as fusion domains. Single-chain variable-fragment (scFv) domains, by contrast, bear the advantage of maintaining antigen-specificity of full immunoglobulins, while presenting a much smaller size (around 25 kDa) allowing them to be readily fused recombinantly to other biotherapeutic such as TRAIL. 183,184 Various such scFv:TRAIL constructs have been developed (Table 2), targeting surface antigens known to be highly expressed by the cells of certain tumor types. These include FAP, 69 CD19, 185 CD33, 186 CD20, 187 MCSP (melanoma-associated chondroitin sulfate proteoglycan), 188 ErbB2 (ref. 144) or epidermal growth factor receptor (EGFR). 145,189–191 A variant of this experimental approach is the use of the Fn14:TRAIL fusion protein. 192,193 In this case, the protein fused with TRAIL is not an scFv, but a peptide corresponding to the extracellular domain of Fn14, the receptor for TWEAK/Apo3L (TNF-related weak inducer of apoptosis/Apo3L). TWEAK is a multifunctional cytokine involved in many cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis and inflammation, which is not only expressed by normal cells but also in tumor tissue (reviewed in ref. 194). An Fn14: Figure 2 Main effects of nanoparticle-based formulations of TRAIL. Different formulations of TRAIL using nanoparticle-based methods have been recently developed, including liposomes. These experimental approaches show a variety of advantages that help to improve the therapeutic potential of TRAIL in cancer. Conjugation with nanoparticles increases the stability of TRAIL therefore increasing its half-life and allowing a sustained release in the tumor. The so-called enhanced permeability and retention (EPR) effect allows the nanoparticles to be more specific targeting tumors than the antitumor molecules alone. This passive targeting may be improved including different molecules in the nanoparticle composition that specifically target them to the tumor. Finally, nanoparticles loaded with other drugs than TRAIL, which specifically sensitize tumor cells to TRAIL and enhance its proapoptotic effect, may have a synergistic effect killing tumor cells New TRAIL formulations for cancer treatment D de Miguel et al 739 Cell Death and Differentiation Table 1 Main formulations of TRAIL based on nanoparticles Formulation Type of platform TRAIL location Combined formulation Main effects Experimental testing Ref. TRAIL HSA-NPs Human serum albumin NPs Inside —Increased biological half-life Increased drug bioavailability Passive targeting Pharmacokinetic studies in vivo 153 PEG-TRAIL microspheres PLGA microspheres Inside —Increased biological half-life Sustained delivery Pharmacokinetic studies in vivo 152 Increased antitumor activity Absence of side effects Tumor xenograft model (CRC) in vivo 169 PEG-TRAIL/Dox microspheres PLGA microspheres Inside Doxorubicin Increased antitumor activity CRC and prostate cell lines in vitro Tumor xenograft model (CRC and prostate) in vivo 151 TRAIL-PEG-NPs PEGylated heparin and poly-Llysine NPs Inside —Increased biological half-life Increased antitumor activity Absence of side effects Pharmacokinetic studies in vivo Tumor xenograft model (CRC) in vivo 154 ANG-CLP/PTX/pEGFPhTRAIL Angiopep-2 modifed cationic liposome Inside (cDNA) Placlitaxel Sustained delivery Passive targeting Increased antitumor activity GBM cell line in vitro Tumor xenograft model (GBM) in vivo 176 Liposomes TRAIL-LPs Inside —Increased antitumor activity Tumor xenograft model (NSCLC) in vivo 175 GBM cell line in vitro Tumor xenograft model (GBM) in vivo 173 Doxorubicin Passive targeting NSCLC cell line in vitro 174 LUV-TRAIL Surface —Increased antitumor activity Absence of side effects Leukemic, lymphoma and multiple myeloma cell lines in vitro 172 Increased DISC recruitment Leukemic and lymphoma cell lines in vitro 67 Increased antitumor activity Leukemic cells in vitro and primary leukemic cells ex vivo 212 Increased anti-inflammatory effect Rheumatoid arthritis experimental model in vivo 171 E-selectin Increased antitumor activity Tumor xenograft model (CTC) in vivo 178 Immuno-LipoTRAIL Surface scFv αEGFR Increased antitumor activity Active targeting Tumor xenograft model (CRC) in vivo 177 sTRAIL-targeted stealth liposomes Surface —Improved pharmacokinetics Increased antitumor activity Neuroblastoma orthotopic model in vivo 179 Apo2L.0-LPs Surface —Improved pharmacokinetics Increased antitumor activity Multiple cell lines in vitro Tumor xenograft model (CRC) in vivo 213 Magnetic NPs-TRAIL Magnetic ferric oxide NPs Surface —Passive targeting Increased antitumor activity GBM cell line in vitro Tumor xenograft model (GBM) in vivo 170 TRAIL/Tf/Dox HSA-NPs Human serum albumin NPs with transferrin Surface Doxorubicin Sustained delivery Active targeting Increased antitumor activity CRC, pancreas and BC cell lines in vitro Tumor xenograft model (CRC) in vivo 168 TRAIL/Dox HSA-NPs Human serum albumin NPs with transferrin Surface Doxorubicin Sustained delivery Active targeting Increased antitumor activity Lung carcinoma cell line in vitro Tumor xenograft model (lung carcinoma) in vivo 167 Abbreviations: ANG-CLP, angioprep-2 modified cationic liposome; BC, breast cancer; CRC, colorectal cancer; CTC, circulating tumor cell; Dox, doxorubicin; EGFR, epidermal growth factor receptor; GBM, glioblastoma multiforme; HSA, human serum albumin; LPs, liposomes; NPs, nanoparticles; NSCLC, non small cell lung cancer; PEG, polyethylene glycol; pEGFP, plasmid enhanced green fluorescence protein; PLGA, poly (lactic-coglycolic) acid; PTX, paclitaxel; scFv, single-chain variable fraction; Tf, transferrin. New TRAIL formulations for cancer treatment D de Miguel et al 740 Cell Death and Differentiation TRAIL fusion protein that showed increased bioactivity in an experimental model of multiple sclerosis 193 also showed enhanced antitumor activity in vitro and in vivo against hepatocellular carcinoma. 192 This activity relies on the ability of Fn14:TRAIL to interfere with TWEAK-Fn14 signaling in cancer cells and simultaneously trigger TRAIL-induced apoptosis. It is worth pointing out that some authors have constructed such fusion proteins using novel versions of TRAIL such as TNC-TRAIL and scTRAIL to improve trimer stability. 70,144,145,195 An interesting additional variation to this approach, which has again been developed by several groups independently, is the targeting of TRAIL not to the surface of tumor cells but to that of immune cells via specific antigens expressed on their Table 2 Main formulations of TRAIL fusion proteins Fusion protein Target Main effects Experimental testing Ref. MBOS4:TRAIL FAP Increased bioactivity Active targeting Fibrosarcoma cell lines in vitro 69 CD40ed:TRAILed CD40 Increased bioactivity Active targeting Fibrosarcoma cell lines in vitro 214 scFv425:sTRAIL EGFR Increased drug bioavailability Active targeting Absence of side effects Pharmacokinetic studies in vivo Tumor xenograft model (RCC) in vivo 189 Increased antitumor activity Hematologic and solid tumor cell lines in vitro 190 scFvCD19:sTRAIL CD19 Active targeting Absence of side effects Increased antitumor activity Hematologic tumor cell lines and B-CLL primary cells in vitro Tumor xenograft model (B-ALL) in vivo 185 scFvCD33:sTRAIL CD33 Active targeting Increased antitumor activity Hematologic tumor cell lines and AML primary cells in vitro 186 Anti-MCSP:TRAIL MCSP Active targeting Absence of side effects Increased antitumor activity Melanoma cell lines and normal primary cells in vitro Tumor xenograft model (melanoma) in vivo 188 Db aEGFR -scTRAIL EGFR Active targeting Absence of side effects Increased antitumor activity HCC and CRC cell lines in vitro Tumor xenograft model (CRC) in vivo 145 Anti-CD3:TRAIL K12:TRAIL CD3 CD7 Enhanced T-cell activity Increased antitumor activity Hematologic, solid tumor cell lines and tumor primary cells in vitro Tumor xenograft model (CRC) in vivo 196 scFvCD70:TRAILmutRs CD70 Increased bioactivity Active targeting Hematologic and solid tumor cell lines in vitro 70 scFv:G28-TRAIL CD40 Increased bioactivity Active targeting Induction of DC maturation Fibrosarcoma cell lines in vitro 195 MSC.scFvCD20-sTRAIL CD20 Active targeting Absence of side effects Increased antitumor activity Hematologic tumor cell lines and normal primary cells in vitro Tumor xenograft model (NHL) in vivo 187 CLL1:TRAIL CLL1 Enhanced T-cell activity Increased antitumor activity Absence of side effects Hematologic and solid tumor cell lines in vitro 202 RGD:TRAIL Integrins Active targeting Increased antitumor activity BC and CRC cell lines in vitro Tumor xenograft model (NHL) in vivo 203 scTRAIL: Fc (APG350) —Increased antitumor activity Several cell lines in vitro Tumor xenograft model (CRC) in vivo 209 scFv-EHD2-scTRAIL —Increased antitumor activity Active targeting Several cell lines in vitro Tumor xenograft model (CRC) in vivo 211 FN14:TRAIL TWEAK Increased antitumor activity Absence of side effects HCC cell lines in vitro Tumor xenograft model (HCC) in vivo 192 Increased anti-inflammatory effect Multiple sclerosis experimental model in vivo 193 Abbreviations: AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; BC, breast cancer; B-CLL, B-cell chronic lymphocytic leukemia; CRC, colorectal carcinoma; DC, dendritic cell; EGFR, epidermal growth factor receptor; HCC, hepatocellular carcinoma; MCSP, melanoma-associated chondroitin sulfate proteoglycan; MSC, mesenchymal stem cells; NHL, non-Hodgkin’s lymphoma; RCC, renal cell carcinoma; RGD, peptide with the sequence ACDCRGDCFC; scFv, single-chain variable region. New TRAIL formulations for cancer treatment D de Miguel et al 741 Cell Death and Differentiation