Accepted Manuscript

Title: Death receptor agonist therapies for cancer, which is theright TRAIL?

Author: <ce:author id="aut0005" biographyid="vt0005">Pamela M. Holland

PII: S1359-6101(13)00107-XDOI: http://dx.doi.org/doi:10.1016/j.cytogfr.2013.12.009Reference: CGFR 762

To appear in: Cytokine & Growth Factor Reviews

Received date: 22-11-2013Accepted date: 15-12-2013

Please cite this article as: Holland PM, Death receptor agonist therapies forcancer, which is the right TRAIL?, Cytokine & Growth Factor Reviews (2013),http://dx.doi.org/10.1016/j.cytogfr.2013.12.009

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Death receptor agonist therapies for cancer, which is the right TRAIL?

Pamela M. Holland

1Therapeutic Innovation Unit, Amgen Inc., 360 Binney St., Cambridge, MA 02142

Correspondence: Pamela M. Holland, Amgen Inc., 360 Binney St., Cambridge, MA 02142,

Office: (617) 444-5530, E-mail: hollandp@amgen.com

Keywords: Apoptosis, Apo2L/TRAIL, death receptor, cancer

Conflict of interest statement: The author is an employee of Amgen Inc. and has received stock/stock options from Amgen Inc.

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Abstract

The activation of cell-surface death receptors represents an attractive therapeutic strategy to

promote apoptosis of tumor cells. Several investigational therapeutics that target this extrinsic

pathway, including recombinant human Apo2L/TRAIL and monoclonal agonist antibodies

directed against death receptors -4 (DR4) or -5 (DR5), have been evaluated in the clinic.

Although Phase 1/1b studies provided encouraging preliminary results, findings from

randomized Phase 2 studies failed to demonstrate significant clinical benefit. This has raised

multiple questions as to why pre-clinical data was not predictive of clinical response. Results

from clinical studies and insight into why current agents have failed to yield robust responses are

discussed. In addition, new strategies for the development of next generation death receptor

agonists, are reviewed.

Introduction

Apoptosis is integral to normal, physiological processes that regulate cell number, and results in

the removal of unnecessary or damaged cells. Evasion of apoptosis by tumor cells is key to the

pathogenesis and progression of cancer, and advancements in our understanding of the regulation

of programmed cell death pathways has led to the development of novel agents to reactivate

apoptosis in malignant cells. Activation of cell-surface death receptors by tumor necrosis factor-

related apoptosis inducing ligand (Apo2L/TRAIL, TNFSF10) and death-receptor agonists is one

approach aimed at promoting apoptosis of tumor cells via activation of the extrinsic pathway.

The early observation that Apo2L/TRAIL preferentially triggers apoptosis in tumor cells over

normal cells highlighted its potential as a candidate therapeutic in cancer. Several investigational

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therapeutics that target this pathway, including soluble recombinant human Apo2L/TRAIL

(dulanermin) and agonist monoclonal antibodies directed against death receptors 4 (DR4) or 5

(DR5), have been developed and evaluated in phase 1 and 2 trials, either as single agents or in

combination with cytotoxic chemotherapy or other targeted agents. These studies demonstrated

that this class of agents is well tolerated and provided preliminary evidence of activity.

However, findings from randomized Phase 2 studies have not demonstrated strong clinical

activity, and no death receptor agonist therapies have advanced into Phase 3 [1-6].

Why have death receptor agonist therapies underperformed in the clinic? One possibility

is that clinically, tumors are inherently resistant to death receptor agonism, despite showing

potent activity in pre-clinical models. In this case, resistance might be attributed to extrinsic

pathway-specific issues, and agonists with distinct features with respect to their receptor

selectivity, cross-linking requirements or pharmacokinetics, might all be expected to yield poor

activity. On the other hand, it is possible that modality-specific issues have contributed to the

weak clinical findings. In this case, liabilities associated with the unique characteristics of

agonist antibodies or soluble Apo2L/TRAIL may have independently resulted in similarly weak

outcomes. It is likely that multiple factors have influenced the clinical findings generated to

date, and the feasibility and supporting data for these alternatives is discussed. A greater

understanding of these factors should provide insight into why death receptor agonist therapies

have not lived up to their potential in the clinic to date, and may provide approaches to

reconsider extrinsic pathway activation as a cancer therapeutic.

1.1 Apo2L/TRAIL and its receptors

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Apo2L/TRAIL is a member of the TNF ligand superfamily, and its physiological role is to

modulate immune responses. Apo2L/TRAIL is expressed on many cells of the innate and

adaptive immune system in a stimulus dependent manner [7, 8]. Apo2L/TRAIL has potent anti-

viral activity in vitro and in mice can function as a key effector molecule in NK cell mediated

cytotoxicity [8]. Apo2L/TRAIL induction on NK cells also plays a critical role in the anti-

metastatic effects mediated by IFNγ in vivo [7].

Apo2L/TRAIL was identified based on its sequence homology to the extracellular

domains of TNF and CD95/FasL [9, 10]. Although the sequence identity across the family is

only 25-30%, all ligands are type II transmembrane proteins that exist in the membrane or can be

shed from the cell surface and are active as self-assembling non-covalent trimers [11]. Like

TNF, lymphotoxin, and CD40L, the structure of Apo2L/TRAIL is a homotrimeric jelly roll

protein formed by antiparallel β-pleated sheets, that binds the extracellular portion of three

cysteine-rich receptors, thereby inducing oligomerization of intracellular death domains [12, 13].

Crystallography studies have also highlighted the presence of a unique zinc binding site buried at

the trimer interface which is critical for maintaining the native structure, stability and biological

activity of Apo2L/TRAIL [14]. Preparations of recombinant Apo2L/TRAIL lacking zinc have

been shown to have reduced solubility, and tend to aggregate, potentially explaining the

associated toxicity reported by some in early studies [15, 16].

Apo2L/TRAIL binds multiple receptors with high affinity, presumably to enhance

regulatory flexibility and signaling complexity in the physiological setting. Two of these

receptors, DR4 (TR-1, TNFRSF10A) and DR5 (TR-2, TNFRSF10B), recruit adaptor proteins via

death domain interactions and initiate the formation of the death inducing signaling complex

(DISC), leading to the induction of apoptosis [17] (Fig. 1). Increasing evidence has suggested

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that optimal signal transduction mediated by DR4 and DR5 occurs when the receptors are

clustered and aggregated into lipid rafts, which can be enhanced by multiple agents [18-20].

Therefore, receptor architecture may be an important component of facilitating DISC formation

and optimal caspase activation.

Apo2L/TRAIL binds additional receptors that share close homology within the

extracellular domain but do not signal cell death. DcR1 (TR-3, TNFRSF10C) lacks a

cytoplasmic tail and is membrane anchored via a glycophosphatidylinositol (GPI) moiety, and

DcR2 (TR-4, TNFRSF10D) has a truncated, nonfunctional death domain [17]. In some settings

DcR1 and DcR2 may use distinct mechanisms to attenuate Apo2L/TRAIL-induced apoptosis.

For example, DcR2 can be co-recruited to DR5 in a ligand dependent manner to prevent initiator

caspase activation, and may also sequester DR5 through a pre-ligand assembly domain (PLAD)

shared by both receptors, independent of ligand engagement [21, 22]. A distinct functional role

for DcR1 other than competing for Apo2L/TRAIL binding has not been described.

Apo2L/TRAIL also binds to osteoprotegerin (OPG), a soluble decoy receptor for

RANKL that blocks the RANK-RANKL interaction and limits osteoclastogenesis [23, 24]. The

issue of whether Apo2L/TRAIL can influence osteoclastogenesis remains controversial, although

Apo2L/TRAIL deficient mice show no evidence of altered bone density and no alterations in

frequency or in vitro differentiation of bone marrow precursor osteoclasts [25-27].

1.2 Apo2L/TRAIL signaling

Binding of homotrimeric Apo2L/TRAIL to DR4 and DR5 induces oligomerization of the

receptors and initiation of a pathway mediated by proteases called caspases [17]. This pathway,

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sometimes referred to as the extrinsic pathway to denote its responsiveness to extracellular

signals, is initiated by the recruitment of the initiator caspases-8 and -10 via the adaptor protein

FADD to generate a death-inducing signaling complex (DISC) [28] (Fig. 1). The initiator

caspases in turn activate the downstream effector caspases-3, -6 and -7, which cleave a variety of

cellular substrates in order to execute the apoptotic program (Fig. 1).

In some cells, referred to as type 1, the death receptor-initiated extrinsic pathway

generates a signal strong enough to initiate apoptosis by itself. However, in the majority of cells,

referred to as type 2, the death receptor-initiated signal needs to be amplified in order to induce

apoptosis. This amplification can be achieved by cross talk between the extrinsic pathway and

the Bcl-2-regulated mitochondrial, or intrinsic, pathway [17]. One mechanism by which the

extrinsic pathway recruits the intrinsic pathway involves caspase-8-mediated cleavage of the pro-

apoptotic BH3-only Bcl-2 family member, Bid, to generate active truncated Bid (tBid). Active

tBid antagonizes the function of pro-survival Bcl-2 family members, such as Bcl-2, Bcl-XL and

Mcl-1, triggering a sequence of events that culminates in release of apoptotic factors from the

mitochondrion [28]. These apoptotic factors mediate activation of caspase-9 and antagonize the

activity of inhibitors of apoptosis (IAPs) that otherwise function to suppress caspase activity.

Thus, the intrinsic pathway can function together with the extrinsic pathway to promote caspase

activation and apoptosis.

Whereas the predominant feature of Apo2L/TRAIL signaling is the induction of apoptosis,

it is also capable of weakly activating NFkB and the MAP kinase pathways [29, 30].

Apo2L/TRAIL induced IKK and JNK activation is dependent on the protein kinase RIP, which

is dispensable for Apo2L/TRAIL induced apoptosis, but forms a secondary intracellular complex

with FADD, Traf2 and caspase 8, leading to kinase activation [29, 30]. The physiological

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significance of Apo2L/TRAIL-induced activation of these pathways is unclear. Some reports

have suggested that Apo2L/TRAIL-induced NFkB activation promotes survival and proliferation

of apoptosis-resistant tumor cells and vascular smooth muscle cells [31-33]. Others have shown

that the NFkB subunits RelA (p65) and c-Rel mediate opposing effects on Apo2L/TRAIL

survival or death [34]. The current data is complex and controversial, yet suggests that the

biological outcome of Apo2L/TRAIL-induced NFkB activation is likely to be context dependent

and may positively and negatively influence cellular outcomes. It is worth noting that compared

to other cytokines such as TNF, the magnitude of NFkB or JNK induction by Apo2L/TRAIL is

considerably smaller. An increased understanding of the molecular mechanisms that determine

Apo2L/TRAIL-mediated apoptotic and non-apoptotic signaling is warranted. To this end, the

identification of novel factors that influence Apo2L/TRAIL signaling may provide insight.

Ubiquitination, which is a known critical regulator of many TNF induced immune and apoptotic

responses, including NFkB regulation, has recently been shown to also play a role in

Apo2L/TRAIL signaling [35, 36]. Apo2L/TRAIL can promote ubiquitination of caspase 8 via

the E3 ligase CUL3 in the DISC, leading to caspase 8 aggregation and increased apoptosis [36].

This raises the possibility that the ubiquitination states of proteins such as caspase 8 and RIP1

within Apo2L/TRAIL signaling complexes could influence cellular outcomes in response to

Apo2L/TRAIL.

1.3 Pre-clinical validation of death receptor agonists

Extensive pre-clinical validation evaluating the anti-tumor effects of death receptor agonists has

been performed. Apo2L/TRAIL as well as agonist antibodies targeting either DR4 or DR5 have

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shown activity in vitro and in vivo against a wide range of tumor cell lines, including lung,

colon, pancreatic, non-Hodgkin’s lymphoma (NHL), multiple myeloma, glioma and breast [28,

37]. In addition, Apo2L/TRAIL has shown activity against primary tumor explants derived from

patient pancreatic and colorectal tumors [38, 39]. Not all cancer cells are sensitive to

Apo2L/TRAIL, and anti-tumor activity is enhanced when Apo2L/TRAIL is combined with

chemotherapy, radiotherapy, IFNγ or other targeted therapies [37]. Apo2L/TRAIL resistance

appears to be multi-factorial and can be influenced by the level of pro- and anti-apoptotic factors

within a cell. For example over-expression of anti-apoptotic Bcl-2 family proteins or XIAP

diminishes Apo2L/TRAIL induced apoptosis and cells may be re-sensitized by Bcl-2 or IAP

antagonists [28]. In many combination settings, including those with apoptosis pathway

regulators, growth factor inhibitors, or proteasome and histone deacetylase inhibitors,

cooperativity is directly or indirectly achieved by simultaneous activation of the extrinsic and

intrinsic pathways. However, positive interactions may also be achieved in combination with

agents that kill tumors cells via independent mechanisms, such as ADCC, or via targeting cells

within the tumor microenvironment, such as osteoclast activity in a bone metastasis setting [40,

41]. Collectively, all of these agents may lead to increased stress of tumor cells, which may

already be preferentially poised to die over normal cells due to multiple genetic alterations.

Death receptor agonist antibodies, either alone or with conventional chemotherapeutics and

targeted agents, also yielded promising preclinical results, warranting the evaluation of these

agents in a clinical setting [42-47].

1.4 Clinical experience of death receptor agonist therapies

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Several agents for agonizing Apo2L/TRAIL-death receptors have been evaluated in the clinic

(Table 1). Dulanermin is an optimized, zinc-coordinated, homotrimeric recombinant

Apo2L/TRAIL protein consisting of amino acids 114-281 of the endogenous polypeptide, and is

the only agonist that engages both Apo2L/TRAIL death receptors, DR4 and DR5.

Conatumumab, drozitumab, tigatuzumab, and lexatumumab are all monoclonal agonist

antibodies selectively targeting DR5, whereas mapatumumab is a fully human agonist antibody

against DR4 [42-47].

Several phase 1, monotherapy, dose-escalation, safety studies in patients with advanced

solid tumors have been completed. These trials demonstrated that death receptor agonists were

tolerable as monotherapies, and concerns regarding fulminant hepatotoxicity, as observed with

FasL [48], have not been substantiated. No consistent dose-limiting toxicities have been

reported. The pharmacokinetics of the antibody agonists maintain drug concentrations that

predict activity in preclinical models, with less frequent dosing compared to dulanermin. Single

agent anti-tumor activity, however, has been modest, with only a few durable partial responses

reported across different tumor histologies, including chondrosarcoma [1, 49], follicular

lymphoma [50] and non-small cell lung cancer [2, 51-54](Table 1) . Increases in circulating cell

death markers after treatment have suggested that there was pharmacodynamic activity but have

not been useful as predictive markers of response, in part due to the small number of clinical

responses observed [55].

Several phase 1b safety studies of death receptor agonists in combination with

chemotherapy and/or targeted agents, which would be predicted to enhance the anti-tumor

activity of the class through cross-talk between the intrinsic and extrinsic pathways, have also

been reported in studies of advanced solid tumors and specific tumor histologies [3, 56-65]

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(Table 1). Death receptor agonists were safely combined with standard doses of cancer

therapeutics in small cohorts of patients. These combinations include single-agent cytotoxics

(gemcitabine, doxorubicin, and pemetrexed), cytotoxic combinations (FOLFIRI,

carboplatin/paclitaxel, and cisplatin/gemcitabine), targeted agents (rituximab, panitumumab,

bortezomib, and vorinostat), and cytotoxic targeted agent combinations (FOLFOX/bevacizumab,

carboplatin/paclitaxel/bevacizumab, and irinotecan/cetuximab) (Table 1). Importantly, in these

studies, the combinations did not seem to significantly further sensitize normal cells to apoptosis,

and no significant drug-drug pharmacokinetic interactions were reported. However, efficacy in

small single-arm trials is difficult to formally assess in combination with another agent.

Single-agent phase 2 trials of the fully human DR4 agonist antibody mapatumumab have

been completed in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and NHL.

Two partial responses and one complete response among 40 patients with pretreated follicular

NHL were observed [50]; however, there were no objective responses in treatment-refractory

NSCLC and CRC patients [66, 67]. Randomized Phase 2 studies in NSCLC with either

dulanermin or drozitumab showed no improved response rates or progression-free survival

benefit [6, 68]. In randomized Phase 2 studies with conatumumab and chemotherapy in

pancreatic and non-small cell lung cancer, the addition of conatumumab failed to improve

outcomes [3, 69]. Results from Phase 2 studies with Tigatuzumab have not been reported.

1.5 Potential limitations of current death receptor agonist therapies

The limited activity observed in Phase 1/2 studies with death receptor agonist therapies has not

warranted initiation of Phase 3 trials. This has raised questions as to why compelling pre-clinical

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findings from multiple agents did not translate into more robust clinical efficacy. For agonist

antibodies, one possible reason may be attributed to the cross-linking requirement for effective

receptor engagement and clustering. In vitro, this may be achieved by the addition of exogenous

cross-linking agents. In vivo, cross-linking is mediated by engagement with activating and

inhibitory Fcγ receptors (FcγR) expressed on the surface of immune cells. The DR5 agonist

antibody conatumumab is dependent on cross-linking for activity in vitro and in vivo [42].

Similarly, drozitumab also requires FcγR binding to trigger tumor-cell apoptosis [70]. In a

patient setting, the presence of infiltrating immune cells expressing the relevant FcγR in the

tumor microenvironment would be required to facilitate cross-linking and subsequent agonist

antibody activity. Therefore, reduced numbers of FcγR-expressing immune cells within a tumor

could contribute to limited antibody cross-linking and diminished anti-tumor response mediated

by death receptor agonist antibodies.

The effects of antibody cross-linking may be multi-factorial. Importantly, cross-linking

enhances death receptor clustering on the tumor cell surface to generate robust caspase

activation. In addition, antibody effector function might also contribute to the mechanism of

action of death receptor agonist antibodies. For example, the DR4 antibody mapatumumab was

reported to mediate antibody-dependent cellular cytotoxicity (ADCC) against DR4 expressing

target cells in vitro [71]. The DR5 antibody conatumumab can also induce ADCC of target cells

in vitro, and this is influenced by the allelic polymorphism of FcγR [72], which leads to high or

low affinity forms of the receptor. Collectively, this raises the possibility that death receptor

agonist antibodies may bring additional immune-mediated mechanisms to bear on the tumor. In

this respect, an agonist antibody directed against the mouse TRAIL death receptor can also

induce tumor specific effector and memory T cells [73]. Interestingly, in randomized phase 2

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trials conducted with conatumumab in patients with both advanced non-small cell lung cancer

and metastatic colorectal cancer, trends toward a stronger treatment effect on overall survival

were observed in patients carrying a high affinity allele of the FcγRIIIa receptor, suggesting that

the high affinity form of the receptor may be associated with enhanced responsiveness to

conatumumab [72]. However, no similar trends were observed in a phase 2 pancreatic cancer

study, therefore the predictive value of FcγRIIIa polymorphisms for conatumumab is unclear and

requires additional studies [72].

The limited efficacy observed with dulanermin could be attributed to its limited exposure.

Dulanermin has a half-life of less than 1 hour in humans [1]. In addition, some soluble forms of

TNF family ligands have been shown to have different activity profiles compared to their

corresponding membrane form. For example, the soluble form of FasL has limited apoptosis

inducing activity [74-76]. The soluble form of Apo2L/TRAIL has been reported to retain

receptor activating potential for DR4, but is a weak inducer of apoptosis mediated through DR5

[77-80].

1.6 New strategies to target the extrinsic pathway

The results from clinical studies have prompted investigators to explore alternative

methodologies to improve the efficacy of death receptor agonists. Several approaches have been

employed to promote oligomerization or improve the stability of Apo2L/TRAIL, whose poor

pharmacokinetic properties might influence its anti-tumor activity [1]. For example, fusion of

Apo2L/TRAIL to a tenascin C (TNC) oligomerization domain, or fusion of two single-chain

Apo2L/TRAIL molecules to the Fc portion of a human IgG to generate a “hexameric”

Apo2L/TRAIL (www.apogenix.com), have been reported to increase apoptosis inducing-

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activity[80] (Table 2). Fusion to human serum albumin (HSA), or PEGylation was also shown

to improve the stability and activity of Apo2L/TRAIL [81-83]. The use of poly (lactic-co-

glycolic acid) (PLGA) microspheres or nanoparticles for Apo2L/TRAIL encapsulation and

delivery to tumor sites has also been described, and shown to have superior antitumor activity in

vivo compared to free recombinant Apo2L/TRAIL [84, 85]. Other mechanisms to promote

oligomerization or mimic a membrane-anchored form of Apo2L/TRAIL have been achieved by

the fusion of soluble ligand to a single chain fragment of a variable region (scFv) antibody

fragment. Fusion to a tumor-specific target antigen can also make these agents tumor selective.

The scFv-targeted ligand is inactive but becomes activated upon antibody mediated binding and

cross-linking to the tumor cell surface (reviewed in [86, 87]). In order to also enhance T-cell

responsiveness to tumors, a gene immunotherapy approach using Apo2L/TRAIL-overexpressing

lymphocytes combined with an EpCAMxCD3 bi-specific antibody to enhance anti-tumor

responsiveness has been described [88]. Other delivery methods reported to selectively express

Apo2L/TRAIL in the tumor environment include oncolytic viruses and stem cell delivery. A

recombinant oncolytic herpes simplex virus (oHSV) type-1 bearing a secretable Apo2L/TRAIL

was shown to inhibit tumor growth and invasiveness of mice bearing Apo2L/TRAIL- and oHSV-

resistant intracerebral glioblastoma tumors [89]. Stem cell delivery would provide a cell-based

delivery system to continually produce Apo2L/TRAIL within the tumor, thereby increasing its

efficacy. By taking advantage of the inherent tropism of stem cells to malignant lesions and

modifying them to express antitumor agents, a variety of adult stem cell types have been

engineered to express soluble Apo2L/TRAIL, and successfully applied in a variety of pre-

clinical cancer models (reviewed in [90]). Two recent reports also described the use of

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Apo2L/TRAIL-expressing stem cells encapsulated in biodegradable, synthetic extracellular

matrix which would promote more sustained therapeutic release [91, 92].

Methodologies to overcome the limitations of bivalent death receptor agonist antibodies

have also been an area of active investigation (Table 2). Although some studies have shown that

monovalent anti-DR5 scFv antibodies can directly activate DR5 and induce cell death [93],

multimerization of scFv antibody fragments can improve pharmacokinetic properties and

increase affinity. Wange et al engineered a molecule designed to mimic the affinity of naturally

occurring pentavalent molecules, termed a “combody”. This pentameric structure is composed

of a scFv directed against DR5 and fused to the coiled-coil domain of COMP (cartilage

oligomeric protein 48), which significantly increased its affinity [94]. The DR5 combody was

shown to have antitumor activity in vitro and in vivo. In another example, tetranectin, an

abundant C-type lectin domain (CTLD)-containing protein found in serum and tissue, was used

as a platform to specifically engineer DR4-specific “Atrimer” complexes [95]. Loop regions

within the CTLD were randomized to create a phage display library used to select DR4 binding

complexes with subnanomolar affinity to DR4, and showed DR4-specific tumor cell killing in

vitro. Similarly, a scaffold based on the third fibronecting type III domain of tenascin C (Tn3)

that resembles an antibody variable region was used to generate a phage display library, and

panned against DR5-Fc [96]. Linear fusion of DR5-specific Tn3 modules into multivalent

constructs and additional optimization resulted in increased agonist activity in vitro and in vivo.

In a separate study, a DR5 agonist antibody decorated on PLGA nanoparticles which

encapsulated camptothecin was shown to have activity on tumor cells in vitro [97]. Although the

majority of engineered death receptor agonist therapies have only been evaluated pre-clinically

to date, one agent, TAS266, was reported to have entered clinical trials in late 2012

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(www.clinicaltrials.gov), but the study was quickly terminated for unknown reasons. TAS266 is

a tetrameric nanobody, consisting of a single, heavy chain domain (VHH) antibody, which occurs

naturally in the camelid species. TAS266 is humanized and linked to form a tetrameric structure,

which should not be dependent on cross-linking by Fcγ receptors in the tumor microenvironment

for activity [98]. Collectively, these examples demonstrate that generation of more potent and

tumor selective death receptor agonist therapies is feasible and begin to pave the path toward

how these approaches might lead to clinical applications. It is worth noting that although these

agents have shown activity in pre-clinical models, the clinical therapeutic potential for more

potent next generation death receptor agonists will be influenced by their safety index, potential

for immunogenicity or aggregation, and ease of manufacturability.

 

1.7 Conclusion and Perspective

The last decade has brought forth significant advances in our understanding of the

mechanisms of cell death and evasion of apoptosis as a tumor survival mechanism. Many novel

therapeutics aimed at restoring apoptosis induction in cancer cells have emerged and undergone

clinical evaluation. Although a foundation to support apoptosis induction as a therapeutic

strategy is in place, much remains to be learned about the underlying molecular intricacies and

regulatory components. Importantly, it is difficult to distinguish the observed weak clinical

activity from either insufficient pathway activation by current death receptor agonist therapies, or

a lack of significance of the Apo2L/TRAIL extrinsic pathway in human tumors, and the

development of more potent death receptor agonist therapies may help to address this. Our

understanding of mechanisms of resistance in cell lines suggests that multiple factors contribute

to a resistant phenotype. These factors include receptor expression, the ability of the receptor to

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initiate a signal, and the threshold sensitivity of the cell to the initiating signal. The same factors

are likely to contribute to the resistance of primary human tumors but it is unclear whether the

contribution of these individual factors to the resistant phenotype is different in cell lines versus

primary human tumors. In addition, identifying patients with the greatest likelihood of response

by the application of informative, validated markers will be key to the success of these strategies.

Ongoing research continues to provide additional insight into the functions of proteins that

regulate cell death. Our increased knowledge into the regulation of TNF-family death receptor

signaling, in combination with advances in methodologies to promote receptor clustering in a

tumor selective and receptor selective manner, provides the potential for superior clinical activity

over first-generation approaches to target death receptors that have been tested to date. The

clinical development of next generation death receptor agonist therapies is eagerly awaited.

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108. Belada D, Mayer J, Czuczman MS, Flinn IW, Durbin-Johnson B, Bray GL. Phase II study of dulanermin plus rituximab in patients with relapsed follicular non-Hodgkin's lymphoma (NHL). J Clin Oncol. 2010;28:Abstract 8104. 109. Sharma S, de Vries EG, Infante JR, Oldenhuis CN, Gietema JA, Yang L, et al. Safety, pharmacokinetics, and pharmacodynamics of the DR5 antibody LBY135 alone and in combination with capecitabine in patients with advanced solid tumors. Invest New Drugs. 2013:April ePub. 110. Plummer R, Attard G, Pacey S, Li L, Razak A, Perrett R, et al. Phase 1 and pharmacokinetic study of lexatumumab in patients with advanced cancers. Clin Cancer Res. 2007;13:6187-94. 111. Merchant MS, Geller JI, Baird K, Chou AJ, Galli S, Charles A, et al. Phase I trial and pharmacokinetic study of Lexatumumab in pediatric patients with solid tumors. J Clin Oncol. 2012;30:4141-7. 112. Trarbach T, Moehler M, Heinemann V, Kohne CH, Przyborek M, Schulz C, et al. Phase II trial of mapatumumab, a fully human agonistic monoclonal antibody that targets and activates the tumour necrosis factor apoptosis-inducing ligand receptor-1 (TRAIL-R1), in patients with refractory colorectal cancer. Br J Cancer. 2010;102:506-12. 113. Sun W, Nelson D, Alberts SR, Poordad F, Leong S, Teitelbaum UR, et al. Phase Ib study of mapatumumab in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC) and chronic viral hepatitis. J Clin Oncol. 2011;29:Abstract 261. 114. Belch A, Sharma A, Spencer A, S T, Bahlis N, Doval D, et al. A multicenter randomized Phase II Trial of Mapatumumab, a TRAIL-R1 agonist monoclonal antibody, in combination with Bortezomib in patients with relapsed/refractory Multiple Myeloma (MM). ASH Annual Meeting 2010;116:Abstract 5031. 115. Von Pawel J, Harvey JH, Spigel DR, Dediu M, Reck M, Cebotaru CL, et al. A randomized phase II trial of mapatumumab, a TRAIL-R1 agonist monoclonal antibody, in combination with carboplatin and paclitaxel in patients with advanced NSCLC. J Clin Oncol. 2010;28:Abstract LBA7501. 116. Kim G, Borad M, Pitot H, Rubin J, Greenberg J, McCroskery P, et al. Pilot study of Tigatuzumab (CS-1008) in combination with FOLFIRI in patients with metastatic colorectal cancer (CRC). ESMO. 2012:Abstract 2284. 117. Reck M, Krzakowski M, Chmielowska E, Sebastian M, Hadler D, Fox T, et al. A randomized, double-blind, placebo-controlled phase 2 study of tigatuzumab (CS-1008) in combination with carboplatin/paclitaxel in patients with chemotherapy-naive metastatic/unresectable non-small cell lung cancer. Lung Cancer. 2013:Oct ePub. 118. Holland PM. Targeting Apo2L/TRAIL receptors by soluble Apo2L/TRAIL. Cancer Lett. 2013;332:156-62.

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Figure Legend

Figure 1. Apo2L/TRAIL apoptosis signaling.

Apo2L/TRAIL-mediated apoptosis is triggered upon binding to pro-apoptotic receptors

DR4 and DR5 on the surface of a target cell. Apoptosis may be attenuated by ligand binding to

DcR1, DcR2 or OPG, as these receptors do not induce cell death. Binding of death receptor

agonists to the receptors DR4 and/or DR5 results in DISC (death-inducing signaling complex)

formation. DISC formation involves recruitment of the adaptor protein FADD to the receptor

via the death domain (DD) and the inactive pro-caspases 8 and 10. This facilitates activation and

self-processing of caspase 8 and 10, leading to their release into the cytoplasm, where they

activate effector caspases 3 and 7. c-FLIP is a negative regulator and the ratio of caspase 8 to c-

FLIP in the DISC is an important determinant of response to death receptor engagement. Once

activated, caspases 3 and 7 cleave intracellular substrates, resulting in cell death. XIAP is

another negative regulator of the extrinsic pathway and functions to bind and sequester active

caspase 3. The intrinsic, or Bcl-2 regulated mitochondrial pathway, is responsive to a variety of

cellular stresses and DNA damaging agents, leading to activation of the tumor suppressor p53

and upregulation of the pro-apoptotic Bcl-2 members Puma and Noxa. These facilitate Bax and

Bak activation, and the release of cytochrome C and Smac/DIABLO from the mitochondrion.

Cytochrome C complexes with Apaf-1 and caspase 9 to form the apoptosome and further

activate caspase-3, -6 and -7, thereby providing signal amplification. Extrinsic and intrinsic

pathway cross-talk is mediated by Bid, a caspase-8 substrate that translocates to the mitochondria

and activates Bax and Bak upon cleavage. Figure adapted from Holland, Cancer Letters [118].

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Table 1. Clinical Trials of Death Receptor Agonist Therapies

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Agent/Target Phase/Indication Combination agents Results

Bevacizumab/FOLFOX No improvement over chemo, 63 pts [61]

FOLFIRI, mutant KRAS Trend to improved 6.5 month PFS [99] Ph 2, Colorectal cancer

Panitumumab No evidence of activity, 52 pts [60]

Ph2, Non-small cell lung cancer Carboplatin/paclitaxel

Trend to improved OS for pts on maintenance monotherapy among FcγRIIIa V-allele carriers[69]

Ph 2, Pancreatic cancer Gemcitabine Trend to improved 6 month PFS, 41 pts [3]

Ph 1/2, Sarcoma Doxorubicin No improvement over dox, 86 pts [56]

Monotherapy PR in 1/37 pts (NSCLC), metabolic PR in 1/37 pts (CRC) [2]

Bortezomib or vorinostat CR in 3/33 pts (1 CR bortezomib arm, 2 CR vorinostat arm) [65]

Conatumumab Fully human DR5 agonist antibody (Amgen)

Ph 1b, Solid tumors or lymphoma

Ganitumab SD in 3/9 pts [100]

Bevacizumab/FOLFOX PR in 5/9 pts, SD in 3/9 pts, 1 no assessment [101] Ph 1b, Colorectal cancer

Cetuximab/irinotecan or FOLFIRI in mCRC

PR in 3/20 pts, SD in 13/20 pts, PD in 4/20 pts [102]

Ph2, Non-Hodgkin’s lymphoma Rituximab No improvement over rituximab, 40 pts [103]

Ph 2, Non-small cell lung cancer

Bevacizumab/carboplatin/ paclitaxel No improvement over backbone, 120 pts [68]

Ph 2, Sarcoma Monotherapy No objective responses [104]

Drozitumab Fully human DR5 agonist antibody (Genentech/Roche)

Ph 1, Solid tumors Monotherapy No objective responses, 50 pts [53]

Bevacizumab/FOLFOX PR in 13/23 pts, SD in 7/23 pts, PD in 3/23 pts [105]

Ph 1b, Colorectal cancer Cetuximab/irinotecan or FOLFIRI in mCRC

PR in 6/27 pts, SD in 17/27 pts, PD in 3/27 pts, 1 no assessment [63, 106]. PR in 1 pt with BRAF mutant mCRC [107]

Ph 1b/2, Non-Hodgkins lymphoma Rituximab No improved overall response [64, 108]

Ph 2, Non-small cell lung cancer

Bevacizumab/carboplatin/ paclitaxel

No improved RR or progression free survival [6]

Dulanermin Recombinant human ligand DR4, DR5 dual agonist (Amgen/Genentech)

Ph 1, Solid tumors, NHL Monotherapy 2 PR in chondrosarcoma [1] LBY135 Recombinant chimeric DR5 agonist antibody (Novartis)

Ph 1, Solid tumors Monotherapy, capecitabine SD in 21/38 pts monotherapy, PR in 2/27 pts combination, SD in 12/27 pts combination. Immunogenicity in 16/73 pts [109]

Monotherapy, adult or pediatric pts

SD in 12/37 pts (3 sarcoma), DLT in 3/7 pts at 20 mg/kg [110] SD in 5/24 pts, no PR, CR [111]

Multiple chemotherapy regimens Unspecified PR with FOLFIRI, doxorubicin combinations [62]

Lexatumumab Fully human DR5 agonist antibody (Human Genome Sciences/GSK)

Ph 1/1b, Solid tumors or lymphoma

Interferon gamma No results reported

Ph 1/2, Cervical cancer Cisplatin/radiation Ongoing Ph 2, Colorectal cancer Monotherapy No activity, 38 pts [112]

Ph 1b, Hepatocellular carcinoma Sorafenib PR in 2/19 pts, SD in 4/19 pts [113]

Ph 2, Multiple myeloma Bortezomib No improvement over bortezomib, 104 pts [114]

Ph 1b/2, Non-Hodgkin lymphoma Monotherapy CR in 2/40 pts, PR in 1/40 pts, both follicular

lymphoma [50]

Ph2, Non-small cell lung cancer

Monotherapy Carboplatin/paclitaxel

SD in 9/32 pts [66] No improvement over chemo, 111 pts [115]

Mapatumumab Fully human DR4 (Human Genome Sciences/GSK) Dagonist antibody

Monotherapy SD in 19/49 pts [51]

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Abbreviations: CR, complete response, DLT, dose-limiting toxicity, OS, overall survival, PD, progressive disease, PFS, progression-free survival, PR, partial response, Pts, patients, SD, stable disease.

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Table 2. Apo2L/TRAIL and death receptor antibody modifications to improve agonist stability and activity.

 

Target Multimerization Agent Activity Reference

TNC oligomerization domain In vitro [80]

APG 350, hexameric TRAIL-Fc In vitro, in vivo www.apogenix.com

HSA-TRAIL In vitro, in vivo [81]

Peg-TRAIL In vivo [82, 83]

TRAIL-loaded PLGA microspheres In vivo [84]

Nanoparticle TRAIL In vitro, in vivo [85]

scFv-TRAIL bi-functional fusion proteins In vitro, in vivo Rev. in [86, 87] TRAIL-over expressing lymphocytes with EpCAMxCD3 bi-specific antibody In vitro, in vivo [88]

oHSV type-1 with secretable TRAIL In vitro, in vivo [89]

Apo2L/TRAIL

TRAIL stem cells In vivo [90-92]

DR4 Atrimer In vitro [95]

Combody-pentameric DR5-directed scFV fused to COMP In vitro, in vivo [94]

Tn3 tandem repeats In vitro, in vivo [96]

PLGA nanoparticles In vitro [97]

DR5

TAS 266, tetrameric camelid nanobody In vitro, in vivo, clinical trials [98], www.clinical.trials.gov

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Pamela M. Holland biosketch 

Pamela Holland received her BS in Animal Physiology from the University of California, San Diego, in June 1985.  Before attending graduate school, she worked at Cetus Corporation in Emeryville, CA on early PCR applications, and made significant contributions to the development of TaqMan.  She began her graduate studies in the Biochemistry Department at the University of WA in 1992, and was the recipient of a National Science Foundation Pre‐Doctoral Fellowship.  She received her PhD in 1999 from the Fred Hutchinson Cancer Research Center, where she cloned and characterized the JNK‐activating kinase MKK7.  She then joined Immunex Corporation in Seattle, WA as a post‐doctoral fellow and characterized the function of the RIP‐family kinase RIP4.  Upon appointment to a scientist position at Immunex (later Amgen) in 2002, she supported pre‐clinical and clinical development of the death receptor agonist therapies AMG 655 (conatumumab) and Apo2L/TRAIL (dulanermin).  Since that time she has worked on the development of large and small molecule therapeutics aimed at promoting tumor cell apoptosis.  Her current research interests also include tumor immunology and the development of novel agents to enhance immune‐mediated clearance of tumor cells. 

 

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*Author Photo

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Fig. 1 Apo2L/TRAIL signaling

c-FLIP

Caspase-8, 10

Caspase-3, 7

Cellular

substrates

DR4/DR5

Apoptosis

FADD

DISC

Apo2L/TRAIL

DcR1

DcR2

OPG

XIAP

Caspase-9 Apaf

Apoptosome Cyto C

Bcl-2 Bid

Bax/Bak

Smac

Stress

DNA damage

p53

Puma/Noxa

Intrinsic pathway

Extrinsic pathway

Figure