key: cord-278186-t3izmz6n
authors: Le Naour, Julie; Galluzzi, Lorenzo; Zitvogel, Laurence; Kroemer, Guido; Vacchelli, Erika
title: Trial watch: TLR3 agonists in cancer therapy
date: 2020-06-02
journal: Oncoimmunology
DOI: 10.1080/2162402x.2020.1771143
sha: 
doc_id: 278186
cord_uid: t3izmz6n

Toll-like receptor 3 (TLR3) is a pattern recognition receptor that senses exogenous (viral) as well as endogenous (mammalian) double-stranded RNA in endosomes. On activation, TLR3 initiates a signal transduction pathway that culminates with the secretion of pro-inflammatory cytokines including type I interferon (IFN). The latter is essential not only for innate immune responses to infection but also for the initiation of antigen-specific immunity against viruses and malignant cells. These aspects of TLR3 biology have supported the development of various agonists for use as stand-alone agents or combined with other therapeutic modalities in cancer patients. Here, we review recent preclinical and clinical advances in the development of TLR3 agonists for oncological disorders. ABBREVIATIONS: cDC, conventional dendritic cell; CMT, cytokine modulating treatment; CRC, colorectal carcinoma; CTL, cytotoxic T lymphocyte; DC, dendritic cell; dsRNA, double-stranded RNA; FLT3LG, fms-related receptor tyrosine kinase 3 ligand; HNSCC, head and neck squamous cell carcinoma; IFN, interferon; IL, interleukin; ISV, in situ vaccine; MUC1, mucin 1, cell surface associated; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; polyA:U, polyadenylic:polyuridylic acid; polyI:C, polyriboinosinic:polyribocytidylic acid; TLR, Toll-like receptor

Toll-like receptors (TLRs) are an evolutionarily conserved family of pattern recognition receptors (PRRs) 1-4 that detect conserved molecular motifs in microbial and endogenous products, which are generally referred to as microbe-or damageassociated molecular patterns (MAMPs or DAMPs), respectively. [5] [6] [7] [8] [9] [10] [11] Since the initial discovery of Toll as a Drosophila melanogaster receptor with antifungal activity, [12] [13] [14] no less than 13TLRs have been characterized in mammalian organisms, 10 of which are also encoded by the human genome. 5, 15 Mammalian TLRs localize either to the cell surface (TLR1, TLR2, TLR4, TLR6, TLR10) or within endosomal compartments (TLR3, TLR7, TLR8, TLR9). 5, 16 Such endosomal TLRs are specialized in the recognition of potentially pathogenic nucleic acids, based on three general principles (1) availability (a function of initial concentration and degradation by endogenous nucleases), (2) localization (of both nucleic acids and TLRs) and (3) structural features (secondary nucleic acid conformations as well as chemical modifications). [17] [18] [19] On activation, nucleic acid-sensing TLRs initiate a signal transduction cascade that culminates with the secretion of numerous pro-inflammatory cytokines including type I interferon (IFN), which promote both innate and adaptive immune responses. [20] [21] [22] [23] [24] [25] [26] Double-stranded RNA (dsRNA) molecules are the prototypic ligands of TLR3, 27 and activation occurs upon dsRNA binding to TLR3 leucine-rich repeats (LLR) domain. 28, 29 Several mechanisms have been suggested to account for the accumulation of TLR3-activitory dsRNA molecules within endosomes, including clathrin-dependent endocytosis, 30, 31 uptake of apoptotic bodies from infected cells, 32, 33 autophagic uptake of dsRNA from the cytosol and trafficking to endosomes in the context of inhibited lysosomal degradation, [34] [35] [36] and formation of dsRNA complexes with cathelicidin antimicrobial peptide (CAMP). 37, 38 Upon ligand binding, the cytoplasmic Toll/IL-1 receptor (TIR) domain of TLR3 39,40 engages toll-like receptor adaptor molecule 1 (TIRAM1, best known as TRIF) and toll-like receptor adaptor molecule 2 (TIRAM2, best known as TRAM) to initiate a signal transduction cascade that culminates with the activation of TANK binding kinase 1 (TBK1) 41 and consequent derepression of interferon regulatory factor 3 (IRF3), 42 IRF7 43 and nuclear factor-kappa B (NF-κB). [44] [45] [46] Moreover, active TLR3 can signal via the mitogen-activated protein kinase (MAPK) system 5, 47, 48 to initiate transcriptional programs downstream of Jun protooncogene, AP-1 transcription factor subunit (JUN, best known as AP-1) 49 and cAMP responsive element binding protein 1 (CREB1). 50,51 Thus, TLR3 signaling favors the synthesis and secretion of a panoply of pro-inflammatory cytokines including not only type I IFN but also tumor necrosis factor (TNF), interleukin 6 (IL-6) and various chemokines such as C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1). 52-57 Of note, unlike other TLRs, TLR3 signaling appears to require tyrosine phosphorylation upon dsRNA recognition, 58 and operates exclusively via an MYD88 innate immune signal transduction adaptor (MYD88)-independent mechanism. 59, 60 Defective TLR3 activity contributes to numerous pathologies, including chronic inflammation, sepsis, autoimmune disorders and cancer. [61] [62] [63] [64] [65] [66] Specifically, loss-of-function TLR3 polymorphisms have been associated with an increased risk for breast carcinoma, 67 cervical cancer, 68 oral squamous cell carcinoma, 69 hepatocellular carcinoma (HCC), 70 and colorectal carcinoma (CRC); 71 as well as with poor disease outcome in patients with CRC 72 and non-small cell lung carcinoma (NSCLC). 73 Moreover, high expression levels of TLR3 or TRIF have been shown to convey positive prognostic value in patients with HCC, 74, 75 and neuroblastoma, 76 while TLR3 expression has attributed predictive value in a cohort of women with breast carcinoma treated with adjuvant radiotherapy plus a TLR3 agonist. 77, 78 Finally, several studies have demonstrated that the emission of DAMPs by dying cancer cells, either spontaneously or following treatment, enables the initiation of an efficient and durable anticancer immune response through the activation of TLRs and other PRRs on immune cells of the host. [79] [80] [81] [82] Thus, TLR3 stimulation stands out as a promising strategy to (re)instance cancer immunosurveillance 83 and demonstrated potential especially as an adjuvant to therapeutic tumor-targeting vaccines. [84] [85] [86] [87] [88] [89] [90] [91] However, whereas TLRs located at the plasma membrane can be actioned with small molecules and antibodies, targeting nucleicacid sensing TLRs, such as TLR3, require modified oligonucleotides. 87 Indeed, besides natural dsRNA molecules, TLR3 also recognizes synthetic dsRNA analogs, 48 such as polyriboinosinic:polyribocytidylic acid (polyI:C), 92 polyadenylic:polyuridylic acid (polyA:U), 93 polyriboinosinic-polyribocytidylic acidpolylysine carboxymethylcellulose (polyI:CLC, best known as Hiltonol™) 94 and polyI:C 12 U (best known as Ampligen™ or rintatolimod), 95, 96 all of which have been consistently used to induce TLR3 signaling in vitro and in vivo. [97] [98] [99] [100] [101] Over the past few years, numerous studies have confirmed the ability of TLR3 agonists to support the activation of tumorspecific immune responses in mice and patients, especially when combined with other therapeutic modalities. 90, [102] [103] [104] However, the clinical efficacy of this approach remains limited, potentially reflecting the existence of numerous, nonoverlapping immunosuppressive pathways that must be simultaneously disabled to allow for therapeutically relevant tumortargeting immune responses in patients. 83, 105, [105] [106] [107] 109 Here, we discuss recent progress on the development of TLR3 agonists for cancer therapy.

In this section, we summarize the key preclinical studies on the ability of TLR3 agonists to (re)instate anticancer immunosurveillance, which have been released since the publication of the latest Trial Watch dealing with this topic. 90

PolyI:C was originally synthesized in the mid-1960s by Hilleman and colleagues. 108 This synthetic dsRNA consists of an RNA duplex composed of one inosinic acid polymer and one cytidylic acid polymer. The treatment of immature dendritic cells (DCs) with TLR3 induces their functional maturation, as demonstrated by a reduction in phagocytic/pinocytic capacity coupled to increased expression of co-stimulatory molecules (e.g., CD80 and CD86), maturation markers (e.g., CD83) and immunostimulatory cytokines (e.g., IL-12). 109 Interestingly, TLR3 is highly expressed both by a subset of mouse (CD8α + ) 110, 112 and human (CD141 + ) 113, 114 DCs commonly known as type I conventional DCs (cDC1s). 86, [115] [116] [117] This basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DC lineage has been extensively studied for its ability to efficiently cross-prime CD8 + cytotoxic T lymphocytes (CTLs). [118] [119] [120] In line with these observations, Kline et al. have recently demonstrated that intraperitoneal injection of polyI:C elicits robust anti-leukemia T cell immunity and considerably prolongs survival of leukemia-bearing mice upon the engagement of CD8α + cDC1s. 121 Several combinatorial regimens have been developed to increase the antineoplastic effects of polyI:C, some of which demonstrated pronounced therapeutic activity in preclinical models of melanoma 122,123 as well as CRC, 124,125 mammary, 124 and squamous carcinoma. 122 In particular, systemic administration of the DC growth factor fms-related receptor tyrosine kinase 3 ligand (FLT3 LG) followed by intratumoral polyI:C injections improved magnitude and duration of response to B-Raf protooncogene, serine/threonine kinase (BRAF) and CD274 (best known as PD-L1) blockade in mouse B16 melanomas, via a mechanism involving cDC1s. 123, 126 Di and colleagues have recently evaluated the efficacy of polyI:C administered in combination with epithelial growth factor receptor (EGFR)vIIItargeted CAR-T cells, 127 both in vitro and in immunocompetent mice bearing subcutaneous CRC or orthotopic mammary cancer xenografts. In this setting, polyI:C significantly increased the levels of effector cytokines such as IL-2 and IFNγ, as well as the lytic activity of CAR-T cells while reducing the number and function of myeloid-derived suppressor cells (MDSC) in the peripheral blood and spleen. 124 Interestingly, Guinn and colleagues have recently reported that IFNγ synergizes with polyI:C in limiting the growth of mouse B16 melanoma and human UM-SCC1 squamous carcinoma cells in vitro, suggesting yet another mechanism through which polyI:C may mediate antineoplastic effects in vivo. 122 Along similar lines, polyI:C and the microtubular poison paclitaxel 128 have been reported to synergistically inhibit the growth of paclitaxel-resistant human CRC cells in vitro through a pathway that involves enhanced interferon beta 1 (IFNB1) expression downstream of TLR3. 125 These latter findings suggest that the ability of polyI:C (and potentially other TLR3 agonists) to activate innate immune pathways in malignant cells may contribute to its therapeutic efficacy, 129 which is generally attributed to the engagement of the host immune system. Further supporting this possibility, TLR3 is known to promote apoptosis 98, 130, 131 as well as a non-apoptotic form of cancer cell death known as necroptosis, [132] [133] [134] which (at least in some settings) has therapeutic value. 135, 136 Several laboratories have recently focused their attention on the design of innovative delivery platforms for polyI:C. Thus, Aznar and collaborators have developed a nanoplexed formulation of polyI:C complexed with polyethylenimine (BO-112), which induces the apoptotic demise of cancer cells accompanied by features of immunogenic cell death (ICD). 137 Intratumoral injection of BO-112 to mouse MC38 CRCs, 4T1 mammary carcinomas and B16 melanomas promoted tumor infiltration by CD8 + CTLs and established at least some degree of disease control dependent on IFNγ, 137 fostering clinical testing in patients with solid tumors (NCT02828098). Alongside, polyI:C has been delivered together with cancer cell lysates 138 with an injectable and self-assembled poly (L-valine) hydrogel. This vaccine formulation allowed for the recruitment, activation and maturation of DCs in vivo as it improved antigen persistence at the injection site and antigen drainage to lymph nodes. 139 Thus, subcutaneous administration of the hydrogel-based vaccine to melanoma-bearing mice mediated robust antineoplastic effects through a proficient CTL response. 139 Similar to polyI:C, polyA:U was synthesized by the Hilleman's laboratory in the mid-1960s. 110 This doublestranded polyribonucleotide, composed of equimolar polyadenylic acid and polyuridylic acid, was extensively investigated in the 1980s 140-142 as the first clinical trials investigating the safety and preliminary activity of polyI:C documented side effects including fever, nausea and hypotension on systemic administration. 142, 143 Even though polyI:C is more potent than polyA:U, 93,99,144 the latter is still used in several studies, at least in part because of its reduced toxicity. Supporting the ability of polyA:U to enhance anticancer immune responses, adjuvant polyA:U administration has been associated with a significant reduction in the risk for metastatic relapse amongst breast cancer patients with TLR3-expressing tumors. 77 Recently, Roselli et al. have reported that the intratumoral administration of naked polyA:U delays the growth of B16 melanomas in vivo, and significantly prolongs the survival of tumorbearing mice. 112 This effect appears to be orchestrated by multiple changes within the lymphoid compartment of the tumor microenvironment, encompassing an increased abundance of CD8 + CTLs expressing the effector molecule granzyme B (GZMB), 145 a reduction in the relative amount of tumor-infiltrating CD4 + CD25 + FOXP3 + regulatory T (T REG ) cells 105 (with respect to CD8 + cells), an improved proliferation of tumor-antigen specific CD8 + CTLs, as well as an enhanced expression of programmed cell death 1 (PDCD1, best known as PD-1) 146 and its main ligand CD274. These findings suggest that, similar to numerous chemotherapeutic agents that promote PD-L1 expression, 147, 148 polyA:U might be advantageously combined with immunotherapies blocking the PD-1/ PD-L1 axis. 112, 149, 150 Ampligen™, an analogue of polyI:C Ampligen™ (also known polyI:C 12 U, AMP-516 or rintatolimod) was synthesized in the 1970 s by William A. Carter by adding unpaired uracil and guanine bases to the classical polyI:C structure. 151 Ampligen™, which drives type I IFN production and IFNB1-dependent type I helper (T H 1) responses with reduced toxicity as compared to polyI:C, 151 was originally intended for the treatment of chronic fatigue syndrome (CFS), a complex disorder characterized by extreme fatigue, 152, 153 and only later it was used as a TLR3 agonist for other indications including cancer. 151 Recently, Tomasicchio et al. haver recruited 12 women with stage 1 to 3 breast carcinoma for ex vivo studies on their DC compartment, ultimately demonstrating that optimal DC maturation required a combinatorial treatment including Ampligen™, an autologous tumor cell lysate, the TLR7/8 agonist resiquimod, 154, 155 and a cytokine cocktail encompassing IFNγ, IFNα, IL-1β and CD40 ligand (CD40 L). DCs matured ex vivo under these conditions produced high levels of IL-12 and could enhance antigen-specific CD8 + CTL responses against erb-B2 receptor tyrosine kinase (ERBB2, better known as HER-2) 156 and mucin 1, cell surface associated (MUC1) 157 leading to the destruction of autologous breast cancer cells in vitro. 158 

Riboxxol (also known as RGIC®50) is a synthetic dsRNA containing cytosines, inosines and guanosines that potently stimulates the secretion of several pro-inflammatory cytokines and improves the ability of mouse and human cDC1s to stimulate T cell proliferation. 159 Schau and colleagues have recently designed a targeted delivery system consisting of neutravidin (a deglycosylated version of avidin) 160, 161 conjugated to monobiotinylated Riboxxol and a humanized anti-prostate stem cell antigen (PSCA) single-chain antibody derivative. These nanoparticle-like immunoconjugates, which were called "rapid inducer of cellular inflammation and apoptosis" (RICIA) were able to specifically deliver Riboxxol to PSCA-expressing tumor cells and induce a type I IFN response coupled to apoptotic cell death. 162 

ARNAX is a TLR3 agonist originally developed by Matsumoto and collaborators that consists of a phosphorothioate oligodeoxynucleotide (ODN)-guided dsRNA. 163 Unlike polyI:C, this chimeric molecule does not activate intracellular RNA sensors other than TLR3 164,165 such as DExD/H-box helicase 58 (DDX58 best known as RIG-I) and interferon induced with helicase C domain 1 (IFIH1, best known as MDA5) and hence presents reduced toxicity in vitro and in vivo. [166] [167] [168] Takeda and collaborators have recently demonstrated that ARNAX synergizes with a model peptide vaccine and a CD274 (best known as PD-L1) blockers in the eradication of various mouse tumors established in immunocompetent hosts. 169

Results from a number of translational and clinical studies addressing the safety and therapeutic potential of TLR3 agonists have been reported in the peer-reviewed literature since the publication of the latest Trial Watch on this topic (October 2018). 90 Here, we discuss some of these studies with a focus on findings and concepts that recapitulate the current state-of-the-art.

Recent immunohistochemical studies demonstrate that high TLR3 expression by tumor cells correlates with favorable disease outcome in a cohort of 194 patients with early-stage NSCLC, whereas TLR3 expression on immune cells, infiltrating the tumor bed, is associated with poor overall survival. 170 At least in part, these observations appear to reflect the ability of TLR3 activation to drive apoptotic cell death in cancer cells, as demonstrated in vitro as well as by the immunohistochemical quantification of active caspase 3 (CASP3), a key mediator of apoptosis, [171] [172] [173] in tumor biopsies. Tan and colleagues have recently reported that nasopharyngeal carcinoma (NPC) biopsies exhibit increased TLR3 mRNA levels as compared to healthy nasopharyngeal tissues, 54 which appears to constitute an actionable therapeutic target. Indeed, Hiltonol™ synergized with the endothelial growth factor receptor (EGFR)-specific antibody cetuximab in (1) maturation of DCs, (2) activation of natural killer (NK) cell-dependent antibody-dependent cellular cytotoxicity (ADCC) and cytotoxicity, and tumor infiltration by EGFR-specific CTLs. 54 None of these effects was affected by TLR3 polymorphisms (e.g., L412F or C829T), pleading in favor of a broad use of Hiltonol™ against NPC. 54 Finally, Hammerich et al. developed an in situ vaccine (ISV) approach, combining recombinant human FLT3LG, local radiotherapy, and Hiltonol™ that robustly activated an anticancer immune response amenable to boosting with PD-1 blockers in lymphoma bearing mice. 174 These results prompted the initiation of a hitherto ongoing clinical trial (NCT01976585) enrolling patients with advanced stage indolent non-Hodgkin's lymphoma (iNHL).

Preliminary results for the aforementioned NCT01976585 clinical trial (testing a Hiltonol™-based ISV approach in patients with iNHL) suggested that both responders and nonresponders to the ISV develop (at least some degree of) anticancer immunity, based on analysis of peripheral blood mononuclear cells (PBMCs) for maturation and exhaustion markers in DCs and CTLs. 174 However, it seems that a population of PD-1 + CD8 + T lymphocytes emerges in non-responders, potentially explaining why of 11 patients included in this preliminary analysis, no less than 8 experienced partial or complete lymphoma regression in the presence of a PD-1 blocker. 174 Conversely, only six patients showed stable disease or minor regression (lasting 3 to 18 months) at distant untreated tumors whereas two patients progressed. Altogether, these findings suggest that iNHL patients might benefit from a Hiltonol™based ISV approach combined with PD-1 blockers.

Weed et al. reported preliminary results from a Phase I clinical assay testing the safety and immunological efficacy of a MUC1-targeting peptide vaccine admixed with Hiltonol™ and combined with a phosphodiesterase type 5 (PDE5) inhibitor (tadalafil) 175 in subjects with head and neck squamous cell carcinoma (HNSCC) (NCT02544880). 176 While no severe side effects and treatment-limiting toxicities were documented, this regimen increased the amount of activated tumorinfiltrating lymphocytes (TIL) and reduced the levels of PD-L1 + macrophages at the tumor edge, 176 suggesting that the addition of a PD-1-or PD-L1-taregting immune checkpoint blockers may be useful also in this setting. Alongside, a pilot study on patients with metastatic HNSCC and melanoma who received intratumoral or intramuscular Hiltonol™ reported clinical benefits for at least one of the 8 individuals enrolled in this trial, coupled to moderate side effects (such as inflammation at the injection site and fatigue) as well as increased levels of CD4, CD8, PD-1, and PD-L1 in tumors, confirming the activation of systemic immunity. 177 Keskin and colleagues reported the results of a Phase Ib clinical trial in which newly diagnosed glioblastoma patients with unmethylated methylguanine methyltransferase (MGMT) received a personalized neoantigen vaccine, previously administered to melanoma patients, 178-180 admixed with Hiltonol™. 181 This regimen generated strong intratumoral T-cell responses even though glioblastoma is generally viewed as an immunological 'desert', 182 suggesting that robustly adjuvanted neoepitope-targeting vaccines may constitute a valid approach for the treatment of glioblastoma, especially in combination with immune checkpoint blockers. 183 Apparently at odds with this notion, Boydell and colleagues reported that a multipeptide vaccine (IMA950) admixed with Hiltonol™, administered prior to the vascular endothelial growth factor A (VEGFA)-targeting antibody bevacizumab, 184, 185 failed to improve the therapeutic activity of the latter in high-grade glioma patients, as assessed by progression-free and overall survival (NCT01920191). 186 Melssen et al. investigated the safety, immunogenicity and preliminary efficacy of a multipeptide vaccine 187,188 admixed with (1) Hiltonol™ and/or incomplete Freund's adjuvant (IFA), or (2) the mixed TLR2/TLR4 agonist lipopolysaccharide (LPS) 189, 190 and/or IFA in melanoma patients (NCT01585350). 191 Preliminary findings from this study indicate that Hiltonol™ plus IFA can induce durable peptide-specific CD8 + T cell responses in the absence of considerable side effects (doselimiting toxicities were documented in only 11% of the subjects). Finally, one Phase I study evaluated the therapeutic effect of TLR3 agonists in pediatric cancers (NCT01188096). 192 Of note, all six patients affected by type I neurofibromatosis (among the 23 enrolled in the trial) tolerated Hiltonol™ as a stand-alone intervention (mild side effects included fever, pain at site of injection, erythema and myalgias), 192 supporting the planification of a Phase II study for this specific oncological indication.

Overall, this translational and clinical literature supports the notion that TLR3 agonists may favor the ability of therapeutic vaccines to (re)activate immunosurveillance in (at least some) patients affected by solid tumors, although efficacy in the absence of immune checkpoint blockers remains limited.

Since the submission of the latest Trial Watch dealing with this topic (October 2018), 90 only 8 clinical studies encompassing the administration of TLR3 agonists to cancer patients have been initiated (source http://clinicaltrials.gov/), all of which involved either Hiltonol™ (4 studies), [193] [194] [195] or Ampligen™ (4 studies) 151 (Table 1) .

In particular, NCT04119830 aims at evaluating the toxicity of Ampligen™ in combination with the PD-1 blocker pembrolizumab, 196, 197 as well as the impact of this regimen on progression-free and overall survival in patients affected by metastatic, refractory or unresectable CRC. 198 Patients with CRC-derived liver metastases 199 are also being enrolled in NCT03403634, Phase II study involving the administration of a recombinant IFNα-2b (rIFNα-2b)-and Ampligen™-based cytokine modulating treatment (CMT) 200 plus the nonsteroidal anti-inflammatory drug celecoxib. 201 In this study, the impact of treatment on the immune microenvironment is evaluated by the immunohistochemical assessment of CTL/T REG cell ratio. [202] [203] [204] In an analogous manner, HLA-A2 + individuals with primary PD-1-resistant or refractory melanoma are being enrolled in NCT04093323, a Phase II study combining the aforementioned CMT (rIFNα-2b, Ampligen™, and celecoxib) with a vaccine in which cDC1s are loaded with tumor blood vessel-derived antigenic peptides. Twelve weeks after treatment initiation, patients with progressive disease may receive the cytotoxic T-lymphocyte associated protein 4 (CTLA4) blocker ipilimumab [205] [206] [207] [208] [209] with or without a PD-1/PD-L1 inhibitor. Patients experiencing complete responses or stable disease may receive PD-1/PD-L1 inhibitors or an appropriate alternative care. A modified variant of the rIFNα-2b-based CMT that involves aspirin 210, 212 instead of celecoxib is also being tested in prostate cancer patients scheduled for radical prostatectomy (NCT03899987). The objectives of this window-of-opportunity study aim at assessing safety, antitumor activity, and immunomodulatory effects.

Hiltonol™ is being tested as a stand-alone intervention only in a Phase I clinical assay involving the intravenous administration of the drug to patients affected by malignant pleural mesothelioma prior to surgical resection (NCT04345705). All the other clinical trials recently initiated to test Hiltonol™ in patients with cancer co-involve indeed either a PD-1 blocker 197,213 (NCT03789097 and NCT03835533) or an agonist for the co-stimulatory T cell receptor CD27 (varlilumab, also known as CDX-1127) 214,215 (NCT03617328) .

In particular, Hiltonol™ is being administered in combination with radiotherapy and rhFLT3LG as in situ vaccine supported by systemic pembrolizumab 216 to patients affected by metastatic breast cancer, HNSCC and NHL in the context of a Phase I/II assay (NCT03789097). This combinatorial regimen includes three therapies directed against a "target site": (1) rhFLT3LG, known also by the name of CDX-301, 217,218 that specifically recruits and expands DCs, (2) radiation therapy to the tumor and the draining lymph node (administered at a 10-20 times lower dose compared to the standard for patients with this specific type of neoplasm), 78 and (3) Hiltonol™, which should activate the immune cells recruited into the tumor by rhFLT3LG and radiation. Of note, pembrolizumab is already approved by the U.S. Food and Drug Administration (FDA) for the treatment of several neoplasms including HNSCC, but is not effective against metastatic breast carcinomas and NHL. 219, 220 Along similar lines, Hiltonol™ is currently being tested in individuals with metastatic castration-resistant prostate cancer 221-224 simultaneously receiving the PD-1 blocker nivolumab, 225 rhFLT3LG and stereotactic body radiation therapy (SBRT) 226 (NCT03835533). The principal purpose of this study is to monitor the safety and efficacy of different immunotherapy-based combinatorial regimens: one arm (cohort B) receives the aforementioned Hiltonol™-based regimen; a second arm (cohort A) receives nivolumab together with NKTR-214, an IL-2 agonist targeting interleukin 2 receptor subunit beta (IL2RB, also known as CD122) [227] [228] [229] ; and (3) a third arm (cohort C) receives nivolumab together with rhFLT3LG and INO-5151, a combined formulation of INO-5150 -a DNA vector expressing kallikrein-related peptidase 3 (KLK3, best known as PSA) 230 and folate hydrolase 1 (FOLH1, best known as PSMA) 231 -and INO-9012 -a DNA vector expressing IL-12. 232 Finally, the Phase I/II clinical study NCT03617328 evaluates the safety, efficacy and immunogenicity of a peptide vaccine comprised six class II MHC-restricted peptides (6MHP) in patients with melanoma. In this trial, vaccination is adjuvanted with Hiltonol™ and montanide ISA-51, 233 as well as with varlilumab. [234] [235] [236] The status of the following clinical trials discussed in our previous Trial Watches dealing with TLR3 agonists 90 has changed during the past 19 months: NCT02334735, NCT02544880, NCT02721043, NCT02826434, NCT02873819, NCT02897765, NCT03162562, NCT03358719, NCT03380871 and NCT03597282 which are now listed as "Active, not recruiting"; NCT02886065, which is listed as "Recruiting"; NCT02134925 which is currently listed as "Active, not recruiting with results"; NCT02149225, which is listed as "Completed"; NCT03206047 and NCT03300817, which have been "Suspended"; NCT02061449 which has been "Terminated"; as well as NCT02754362, which has been "Withdrawn" (source http:// clinicaltrials.gov/). NCT02134925 is a randomized Phase II study evaluating a MUC1-targeting peptide vaccine admixed with Hiltonol™ versus placebo in patients with newly diagnosed advanced colon polyps. Preliminary results from 110 patients enrolled in the study suggest that vaccination induces superior levels of circulating MUC1-specific IgG, and some degree of reduction in adenoma recurrence rate (56.3% versus 66.0%) (source http://clinicaltrials.gov/). To the best of our knowledge, the results of NCT02149225 (a Phase I study investigating the safety and preliminary efficacy of a Hiltonol™-adjuvanted vaccine in glioblastoma patients) have not been disseminated yet. NCT03206047 (a Phase I/II trial testing Hiltonol™-adjuvanted DC-targeting vaccine in women with recurrent ovarian, fallopian tube, or primary peritoneal cancer) and NCT03300817 (a Phase I study testing a Hiltonol™-adjuvanted, MUC1-targeting vaccine to prevent lung cancer in former and current smokers) have been suspended for undisclosed reasons or to ensure patient safety during the Covid19 epidemics, 237-239 respectively (source http://clinicaltrials.gov/). NCT02061449 (a Phase I study investigating Hiltonol™ plus radiation in patients with advanced cutaneous T cell lymphoma) has been terminated because of poor accrual. Finally, NCT02754362 (a Phase II trial testing Hiltonol™ and montanide ISA-51 in support of a multipeptide vaccine administered prior to bevacizumab in glioblastoma patients) has been withdrawn due to personnel changes (source http://clinicaltrials.gov/).

The blockade of co-inhibitory T cell receptors or their ligands, as achieved with immune checkpoint inhibitors targeting CTLA4, PD-1 and PD-L1, has been a major success in the treatment of patients with various tumors. However, at this stage, immunotherapies only provide long-term clinical benefits to a minority of patients, calling for a drastic amelioration of standard of care. In this context, numerous studies have been launched to identify additional immunosuppressive or immunostimulatory circuitries that can be drugged. As discussed in the present Trial Watch, TLR3 stands out as a promising target for the (re)elicitation of anticancer immunosurveillance. However, the existence of numerous immunosuppressive circuitries that enable tumor progression and resistance to conventional therapies considerably limits the efficacy of TLR3 agonists employed as stand-alone agents, as well as of vaccines adjuvanted with TLR3 agonists, to mediate clinically relevant effects.

We surmise that the development of properly scheduled combinatorial regimens involving multiple immunotherapeutic agents (notably, immune checkpoint blockers and agents that recruit and expand DCs) will be required for harnessing the full antineoplastic potential of TLR3 agonists. 

Toll-like receptors-taking an evolutionary approach

The evolution of vertebrate Toll-like receptors

Evolutionary origins of toll-like receptor signaling

Nucleic acid sensing in invertebrate antiviral immunity

Toll-like receptor signaling pathways

Toll-like receptors and innate immunity

Editorial: danger Signals Triggering Immune Response and Inflammation

Molecular and translational classifications of DAMPs in immunogenic cell death

DAMP-sensing receptors in sterile inflammation and inflammatory diseases

Immunogenic cell death and DAMPs in cancer therapy

Many faces of DAMPs in cancer therapy

The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults

A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

The Drosophila toll signaling pathway

Evolutionary dynamics of human Toll-like receptors and their different contributions to host defense

Regulation of endosomal TLRs

Human ADAR1 prevents endogenous RNA from triggering translational shutdown

Discriminating self from non-self in nucleic acid sensing

Pharmacological modulation of nucleic acid sensors -therapeutic potential and persisting obstacles

The role of nucleic acid sensing in controlling microbial and autoimmune disorders

Type I interferons and dendritic cells in cancer immunotherapy

Intracellular toll-like receptors

TLRs and innate immunity

Sensing of viral infection and activation of innate immunity by toll-like receptor 3

Regulation of antiviral T cell responses by type I interferons

Type I interferons in anticancer immunity

Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses

Toll-like receptors as molecular switches

Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from Toll-like receptor 3

The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-beta production

Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation

Viral immunity: cross-priming with the help of TLR3

Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

Autophagy-independent functions of the autophagy machinery

Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes

Membrane trafficking in autophagy

The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway

Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors

The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling

Identification of TBK1 complexes required for the phosphorylation of IRF3 and the production of interferon beta

IRF3 mediates a TLR3/TLR4-specific antiviral gene program

IRF7: activation, regulation, modification and function

Linking cellular stress responses to systemic homeostasis

NF-kappaB, inflammation, immunity and cancer: coming of age

Autophagy, inflammation, and immunity: a troika governing cancer and its treatment

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway

Cell-type specific MyD88 signaling is required for intestinal tumor initiation and progression to malignancy

Insights into the dynamic nature of the dsRNA-bound TLR3 complex

Small-molecule modulators of toll-like receptors

Impact of pattern recognition receptors on the prognosis of breast cancer patients undergoing adjuvant chemotherapy

Chemotherapyinduced antitumor immunity requires formyl peptide receptor 1

Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly

Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy

IRAK4 and TLR3 sequence variants may alter breast cancer risk among African-Aamerican women

Expressions of Toll-like receptors 3, 4, 7, and 9 in cervical lesions and their correlation with HPV16 infection in Uighur women

Association of TLR2, TLR3, TLR4 and CD14 genes polymorphisms with oral cancer risk and survival

Toll-like receptor 3 genetic variants and susceptibility to hepatocellular carcinoma and HBV-related hepatocellular carcinoma

Toll-like receptor genes and their association with colon and rectal cancer development and prognosis

TLR-3 polymorphism is an independent prognostic marker for stage II colorectal cancer

Host immune gene polymorphisms were associated with the prognosis of non-small-cell lung cancer in Chinese

Toll-like receptor 3 expressing tumor parenchyma and infiltrating natural killer cells in hepatocellular carcinoma patients

TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis

Toll-like receptor 3 expression inhibits cell invasion and migration and predicts a favorable prognosis in neuroblastoma

TLR3 as a biomarker for the therapeutic efficacy of double-stranded RNA in breast cancer

Optimising efficacy and reducing toxicity of anticancer radioimmunotherapy

Immunogenic cell death in cancer and infectious disease

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death

Activating the nucleic acid-sensing machinery for anticancer immunity

An RNA toolbox for cancer immunotherapy

Dendritic cells in cancer immunology and immunotherapy

Targeting Toll-like receptors: emerging therapeutics?

Trial watch: FDA-approved Toll-like receptor agonists for cancer therapy

Trial watch: peptide-based vaccines in anticancer therapy

Trial Watch: toll-like receptor agonists in cancer immunotherapy

Unleashing the potential of NOD-and Toll-like agonists as vaccine adjuvants

Polyinosinic acid is a ligand for toll-like receptor 3

Immunoadjuvant effects of polyadenylic: polyuridylicacids through TLR3 and TLR7

Therapeutic in situ autovaccination against solid cancers with intratumoral poly-ICLC: case report, hypothesis, and clinical trial

Clinical studies with ampligen (mismatched double-stranded RNA)

A clinical grade poly I:C-analogue (Ampligen) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro

Lymphocyte-polarized DC1s: effective inducers of tumor-specific CTLs

Exploiting poly(I:C) to induce cancer cell apoptosis

Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands

TLR3 agonists as immunotherapeutic agents

TLR3 agonists and proinflammatory antitumor activities

Toll-like receptor agonists in cancer therapy

Trial Watch: toll-like receptor agonists in oncological indications

Immune modulation of the tumor microenvironment for enhancing cancer immunotherapy

The regulation of immune tolerance by FOXP3

Regulatory T cells in cancer immunosuppression -implications for anticancer therapy

Cancer immunoediting and resistance to T cell-based immunotherapy

Immunological impact of cell death signaling driven by radiation on the tumor microenvironment

Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes

Targeting neoantigens to augment antitumour immunity

Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells

TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection

Deciphering the transcriptional network of the dendritic cell lineage

TLR3 activation of intratumoral CD103+ dendritic cells modifies the tumor infiltrate conferring anti-tumor immunity

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells

BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

Dendritic cell subsets and locations

Dendritic cell subsets in T cell programming: location dictates function

The role of dendritic cells in cancer

Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking and adoptive T cell therapy

Innate immune signaling and regulation in cancer immunotherapy

Antigen processing and presentation

CD8α+dendritic cells dictate leukemia-specific CD8+T cell fates

IFN-gamma synergism with poly I:C reduces growth of murine and human cancer cells with simultaneous changes in cell cycle and immune checkpoint proteins

Expansion and activation of CD103 + dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition

Combined adjuvant of poly I:C improves antitumor effects of CAR-T Cells

Toll-like receptor 3 agonist poly I:C reinforces the potency of cytotoxic chemotherapy via the TLR3-UNC93B1-IFN-beta signaling axis in paclitaxel-resistant colon cancer

CD103+ dendritic cells producing interleukin-12 in anticancer immunosurveillance

ErbB-targeted CAR T-cell immunotherapy of cancer

Cytosolic DNA sensing in organismal tumor control

Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell

Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif

Regulated necrosis: disease relevance and therapeutic opportunities

Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL

Necroptosis in development, inflammation and disease

Necroptosis: mechanisms and relevance to disease

PolyI:C-Induced, TLR3/ RIP3-dependent necroptosis backs up immune effector-mediated tumor elimination in vivo

Immunotherapeutic effects of intratumoral nanoplexed poly I:C

Transfected poly(I:C) activates different dsRNA receptors, leading to apoptosis or immunoadjuvant response in androgen-independent prostate cancer cells

Injectable polypeptide hydrogel for dual-delivery of antigen and TLR3 agonist to modulate dendritic cells in vivo and enhance potent cytotoxic T-lymphocyte response against melanoma

Adjuvant treatment with polyadenylic-polyuridylic acid (Polya.Polyu) in operable breast cancer

Adjuvant treatment with polyadenylic-polyuridylic acid in operable breast cancer: updated results of a randomised trial

A phase I clinical tolerance study of polyadenylic-polyuridylic acid in cancer patients

Phase I-II trials of poly IC stabilized with poly-L-lysine

A phase I-II trial of multiple-dose polyriboinosic-polyribocytidylic acid in patieonts with leukemia or solid tumors

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA

Granzyme B production distinguishes recently activated CD8(+) memory cells from resting memory cells

The diverse functions of the PD1 inhibitory pathway

Cisplatin and oxaliplatin induce similar immunogenic changes in preclinical models of head and neck cancer

PT-112 induces immunogenic cell death and synergizes with immune checkpoint blockers in mouse tumor models

Cancer Immunosurveillance by T Cells

Dissecting the mechanisms of immune checkpoint therapy

Ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer

Long term improvements in patients with chronic fatigue syndrome treated with ampligen

A double-blind, placebo-controlled, randomized, clinical trial of the TLR-3 agonist rintatolimod in severe cases of chronic fatigue syndrome

Haematological cancer: resiquimod-a topical CTCL therapy

Systemic administration of a TLR7 agonist attenuates regulatory T cells by dendritic cell modification and overcomes resistance to PD-L1 blockade therapy

HER2: biology, detection, and clinical implications

MUC1: a multifaceted oncoprotein with a key role in cancer progression

An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells

Activation of dendritic cells by the novel Toll-like receptor 3 agonist RGC100

Comparison of avidin, neutravidin, and streptavidin as nanocarriers for efficient siRNA delivery

Immobilization and surface characterization of NeutrAvidin biotin-binding protein on different hydrogel interlayers

Targeted delivery of TLR3 agonist to tumor cells with single chain antibody fragment-conjugated nanoparticles induces type I-interferon response and apoptosis

Defined TLR3-specific adjuvant that induces NK and CTL activation without significant cytokine production in vivo

Intracellular RNA sensing in mammalian cells: role in stress response and cancer therapies

RIG-I-like receptors: their regulation and roles in RNA sensing

Targeting Toll-like receptor 3 in dendritic cells for cancer immunotherapy

Tumor vaccines with dsRNA adjuvant ARNAX induces antigen-specific tumor shrinkage without cytokinemia

Adjuvant immunotherapy for cancer: both dendritic cell-priming and check-point inhibitor blockade are required for immunotherapy

Vaccine immunotherapy with ARNAX induces tumor-specific memory T cells and durable anti-tumor immunity in mouse models

Tolllike receptor 3 as a new marker to detect high risk early stage non-small-cell lung cancer patients

Caspases connect cell-death signaling to organismal homeostasis

Converging roles of caspases in inflammasome activation, cell death and innate immunity

Apoptotic caspases inhibit abscopal responses to radiation and identify a new prognostic biomarker for breast cancer patients

Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination

Tadalafil has biologic activity in human melanoma. Results of a pilot trial with Ta dalafil in patients with metastatic Melanoma (TaMe)

The reversal of immune exclusion mediated by tadalafil and an anti-tumor vaccine also induces PDL1 upregulation in recurrent head and neck squamous cell carcinoma: interim analysis of a phase I clinical trial

Therapeutic immune modulation against solid cancers with intratumoral poly-ICLC: a pilot trial

Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells

An immunogenic personal neoantigen vaccine for patients with melanoma

Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial

Tumor-infiltrating lymphocytes in glioblastoma are associated with specific genomic alterations and related to transcriptional class

Detection of neoantigen-specific T cells following a personalized vaccine in a patient with glioblastoma

Ten years of anti-vascular endothelial growth factor therapy

Exploratory study of the effect of IMA950/poly-ICLC vaccination on response to bevacizumab in relapsing high-grade glioma patients

Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting

Randomized multicenter trial of the effects of melanoma-associated helper peptides and cyclophosphamide on the immunogenicity of a multipeptide melanoma vaccine

Further observations on the inhibitory effect of myxoviruses on a transplantable murine leukemia

Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components

A multipeptide vaccine plus toll-like receptor agonists LPS or polyICLC in combination with incomplete Freund's adjuvant in melanoma patients

PDCT-03. A phase III trial of poly-ICLC in the management of recurrent or progressive pediatric low grade gliomas. Results for the neurofibromatosis 1 group. (NCT01188096)

Poly(I:C) as cancer vaccine adjuvant: knocking on the door of medical breakthroughs

A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates

The IL-20 subfamily of cytokinesfrom host defence to tissue homeostasis

Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model

Combinatorial immunotherapy with checkpoint blockers solves the problem of metastatic melanoma-An exclamation sign with a question mark

Metastatic colorectal cancer: therapeutic options for treating refractory disease

Colorectal liver metastases: an update on multidisciplinary approach

Long-term adjuvant therapy of high-risk malignant melanoma with interferon alpha 2b

Hydrogel dual delivered celecoxib and anti-PD-1 synergistically improve antitumor immunity

The ratio of CD8+/FOXP3 T lymphocytes infiltrating breast tissues predicts the relapse of ductal carcinoma in situ

An immunosurveillance mechanism controls cancer cell ploidy

The immune contexture in cancer prognosis and treatment

Nivolumab plus ipilimumab versus sunitinib in advanced renal-cell carcinoma

Audigier-Valette C. Nivolumab plus ipilimumab in lung cancer with a high tumor mutational burden

Development of anti-drug antibodies is associated with shortened survival in patients with metastatic melanoma treated with ipilimumab. Oncoimmunology

PD-L1 expression with immune-infiltrate evaluation and outcome prediction in melanoma patients treated with ipilimumab

Anti-CTLA-4 based therapy elicits humoral immunity to galectin-3 in patients with metastatic melanoma

Aspirin induces autophagy via inhibition of the acetyltransferase EP300

Aspirin-another caloric-restriction mimetic

Aspirin recapitulates features of caloric restriction

Mutational and antigenic landscape in tumor progression and cancer immunotherapy

PD-1 blockade and CD27 stimulation activate distinct transcriptional programs that synergize for CD8+T-cell-driven antitumor immunity

Trial watch: immunostimulatory monoclonal antibodies in cancer therapy

Pembrolizumab: living up to expectations

Efficacy and safety of CDX-301, recombinant human Flt3L, at expanding dendritic cells and hematopoietic stem cells in healthy human volunteers

CDX-301: a novel medical countermeasure for hematopoietic acute radiation syndrome in mice

The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC)

Breast cancer immunotherapy: facts and hopes

Randomized, double-blind, phase III trial of ipilimumab versus placebo in asymptomatic or minimally symptomatic patients with metastatic chemotherapy-naive castration-resistant prostate cancer

Immunotherapy for the treatment of prostate cancer

Prostate cancer as a model for tumour immunotherapy

Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial

Predictive role of plasmatic biomarkers in advanced non-small cell lung cancer treated by nivolumab

Stereotactic body radiation therapy: a novel treatment modality

A first-in-human study and biomarker analysis of NKTR-214, a novel IL2Rbetagamma-biased cytokine, in patients with advanced or metastatic solid tumors

NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models

The role of interleukin-2 during homeostasis and activation of the immune system

The discovery of prostate specific antigen as a biomarker for the early detection of adenocarcinoma of the prostate

Overview of prostate-specific membrane antigen

CD8+ T cells impact rising PSA in biochemically relapsed cancer patients using immunotherapy targeting tumor-associated antigens

Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines

Antibody tumor targeting is enhanced by CD27 agonists through myeloid recruitment

Safety and activity of varlilumab, a novel and first-in-class agonist anti-CD27 antibody, in patients with advanced solid tumors

New emerging targets in cancer immunotherapy: CD27 (TNFRSF7)

COVID-19 outbreak: an overview. Chemotherapy

Coronavirus infections: epidemiological, clinical and immunological features and hypotheses

The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak

LG is supported by a Breakthrough Level 

No potential conflicts of interest were disclosed. Lorenzo Galluzzi http://orcid.org/0000-0003-2257-8500 Laurence Zitvogel http://orcid.org/0000-0003-1596-0998 Guido Kroemer http://orcid.org/0000-0002-9334-4405