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# Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases

Vesna Risso, Elodie Lafont, Matthieu Le Gallo

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Vesna Risso, Elodie Lafont, Matthieu Le Gallo. Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases. Cell Death and Disease, 2022, 13 (3), pp.248. 10.1038/s41419-022-04688-x. hal-03631097

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# Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases

Vesna Risso ID <fnref n="1" />,², Elodie Lafont ID <fnref n="1" />,² and Matthieu Le Gallo ID <fnref n="1" />,²✉

© The Author(s) 2022

Cell death plays a pivotal role in the maintenance of tissue homeostasis. Key players in the controlled induction of cell death are the Death Receptors (DR). CD95 is a prototypic DR activated by its cognate ligand CD95L triggering programmed cell death. As a consequence, alterations in the CD95/CD95L pathway have been involved in several disease conditions ranging from autoimmune diseases to inflammation and cancer. CD95L-induced cell death has multiple roles in the immune response since it constitutes one of the mechanisms by which cytotoxic lymphocytes kill their targets, but it is also involved in the process of turning off the immune response. Furthermore, beyond the canonical pro-death signals, CD95L, which can be membrane-bound or soluble, also induces non-apoptotic signaling that contributes to its tumor-promoting and pro-inflammatory roles. The intent of this review is to describe the role of CD95/CD95L in the pathophysiology of cancers, autoimmune diseases and chronic inflammation and to discuss recently patented and emerging therapeutic strategies that exploit/block the CD95/CD95L system in these diseases.

*Cell Death and Disease* (2022)13:248; https://doi.org/10.1038/s41419-022-04688-x

## INTRODUCTION

The division, differentiation, and death of a cell are highly regulated events in every developing organism and, in the adult individual, the loss of single cells plays a primary role in the maintenance of tissue homeostasis. Cell death, considered as a physiological event, can be defined as a highly evolved and conserved cell elimination mechanism, which responds to homeostatic and morphogenetic stimuli. The cells have a genetically-encoded death program that is finely controlled at the transcriptional and post-transcriptional levels. The definition of “programmed or regulated cell death” (RCD) is appropriate for the description of this phenomenon. Amongst the different types of RCD [1], apoptosis remains the most studied. Two major apoptotic pathways have been described: the extrinsic pathway or Death Receptor (DR) pathway and the intrinsic or mitochondrial pathway, which are linked [2]. In both pathways, specific aspartyl cysteine proteases (caspases) are activated and cleave cellular substrates, ultimately leading to the disruption of multiple cellular processes and morphological changes, such as cell shrinkage or the formation of apoptotic bodies, typical of apoptosis. The crosstalk between the two apoptotic pathways is carried out by the fact that caspase-8, involved in the extrinsic pathway, is able to cleave BID, a Bcl-2 family protein involved in the intrinsic pathway, thus activating the latter after apoptotic stimulus via DR and eventually strengthening the apoptotic signal [3–5].

## Molecular bases of apoptotic signaling

The intrinsic mitochondrial-mediated apoptotic pathway. The intrinsic or mitochondrial pathway can be triggered by a variety of cellular stressors (e.g DNA-damaging agents, nutrient deprivation, hypoxia) and is tightly controlled by pro- and anti-apoptotic

members of the Bcl-2 family of proteins. These cellular stress primarily lead to the increased transcription and/or post-translational activation of pro-apoptotic members of the Bcl-2 family of proteins [6, 7]. The key event of this intrinsic RCD is the mitochondrial outer membrane permeabilization (MOMP) induced by the oligomerization of the pro-apoptotic effector members of this family (BAX, BAK, and in some cases BOK) at the MOM [8]. MOMP allows the release of several caspase activators, such as the cytochrome c, from the mitochondrial intermembrane space to the cytosol. Hence, understanding the molecular bases of the pore-forming capacity of the effectors and of the regulation of their activation is crucial [8–11]. In the cytosol, cytochrome c promotes the assembly of a caspase activation platform called the apoptosome that also includes caspase-9, the activation factor of apoptotic proteases-1 (Apaf-1) and dATP [12]. Indeed, in the absence of apoptotic stimuli, Apaf-1 exists in an inactive monomeric conformation while it undergoes heptameric oligomerisation upon binding to cytochrome c and dATP in apoptotic conditions [13]. The formation of the apoptosome triggers the activation of caspase-9 which in turn activates the effector caspases-3, -7 that drive cell demise [14, 15]. MOMP also promotes the release of anti-apoptotic factors, such as the second mitochondrial activators of caspase (Smac/Diablo) and Omi/HtrA2 (high temperature requirement A2) and endonuclease G (EndoG) [16]. The protein Smac [17, 18] interacts with the BIR2 and BIR3 domains of the X-linked inhibitor of apoptosis protein (XIAP), neutralizing the inhibitory effect of XIAP on caspases-3, 7, and 9 [19]. Omi/HtrA2 [20–23] is a serine protease which, once released into the cytosol, is also able to significantly increase the activity of caspases by inhibiting XIAP. Noteworthy, MOMP can also induce non-apoptotic cell death such as ferroptosis, necroptosis and pyroptosis as recently reviewed elsewhere [7, 24].

<footnote n="1">¹INSERM U1242, Oncogenesis Stress Signaling, University of Rennes, Rennes, France. ²Centre de lutte contre le cancer Eugène Marquis, Rennes, France.</footnote>

<footnote>✉email: matthieu.legallo@univ-rennes1.fr</footnote>
The extent of MOMP largely defines the propensity of a cell to
die or survive upon cell stress. The availability and activity of
the different Bcl-2 family members influences the cellular
readiness or "priming status" for MOMP. This priming status can
be determined through BH3-profiling [25, 26] that evaluates
MOMP upon incubation of permeabilized cells with BH3
peptides mimicking the action of some pro-apoptotic members
of the Bcl-2 family. This assay has mainly been used to predict
the sensitivity of cancer cells to various chemotherapeutic
agents (resistant cells usually display lower priming) and to
interrogate the sensitivity of cancer cells to the increasing
arsenal of BH3-mimetics (molecules mimicking the activity of
some pro-apoptotic Bcl-2 family members). MOMP is not
necessarily a complete process. Indeed, partial MOMP has been
observed when apoptotic induction is weak (minority MOMP) or
accompanied by caspase inhibition (incomplete MOMP). The
ability of cells to retain some non-permeabilized mitochondria,
ATP synthesis and to eliminate damaged mitochondria influ-
ences their propensity to survive upon incomplete MOMP.
Indeed, the remaining intact mitochondria can repopulate the
whole mitochondrial pool [27, 28]. In the case of minority
MOMP, caspase activation is insufficient to drive death but can
promote DNA damage and genomic instability [29]. In addition,
several reports indicate that MOMP can initiate multiple
inflammatory signaling, for example the cGAS/STING [30, 31]
or the NF-κB pathways [32]. Thereby MOMP can impact on the
cell and its microenvironment beyond its ability to promote
cell death.

Taken together, it appears that further understanding the
mechanisms dictating the extent of MOMP, its ability to induce
various types of cell death as well as non-death pathways in
different pathophysiological contexts (e.g., upon pathogen
infection, during tumor progression, etc.) and in different cell
types will be required to fully expand the therapeutic targeting
of the mitochondrial pathway. For further considerations on
this topic, we advise readers to explore the many recent reviews
available [7, 24, 33].

CD95 and CD95L: main structural features. The extrinsic apoptosis pathway takes its name from the extracellular signal molecules that bind to receptors exposed on the surface of target cells, leading to a different way of activating the apoptotic signal compared to the mitochondrial-mediated one. There is a family of receptors specialized in the transmission of the signal upon binding by their cognate ligand that leads to the extrinsic programmed cell death: the DR. The DR belong to the Tumor Necrosis Factor Receptor (TNFR) superfamily, which counts a total of 29 receptors associated with a smaller selection of 19 ligands of the corresponding TNF ligands superfamily. CD95, TNFR1, DR3, DR4, DR5, and DR6 are the most studied DR that, upon ligand binding, convey death signal by using a conserved intracellular region of ~80 amino acids called the "Death Domain" (DD) [34]. This review particularly focuses on the DR CD95, its physiological ligand CD95L and the current approaches developed to therapeutically target this pair. CD95, encoded by the FAS gene, is a 319aa type I glycoprotein devoid of enzymatic activity that signals through protein-protein interaction. Mature CD95 is composed of three cysteine-rich extracellular domains, CRD3, CRD2, and CRD1 starting from the transmembrane domain and moving towards the N-Terminal. CRD2 and partly CRD3 are used for the recognition and binding of the ligand, while CRD1, comprising a subdomain called PLAD (Pre-Ligand Assembly Domain) [35, 36], is needed for the preassembly of CD95 in homodimeric or homotrimeric forms at the plasma membrane. The cytosolic region is composed of the previously mentioned Death Domain (DD) [34], which is essential for the transduction of the apoptotic signal, and a Membrane Proximal Domain (MPD) which conveys non-apoptotic signaling (Fig. 1) [37]. CD95L, encoded by the FASLG

gene, consists of a total of 281aa, an extracellular region with a
C-terminus and an intracellular region with an N-terminus. This
protein is expressed at the plasma membrane in the form of a
homotrimer thanks to the preassembly between monomers that
takes place through an extracellular domain called TNF Homology
Domain (THD) [38]. The THD also mediates receptor binding. The
membrane-proximal extracellular stalk region is proteolytically
processed by several metalloproteases to release soluble forms of
CD95L (sCD95L), which generally display non-apoptotic activities
(see part 2). The cytosolic region is then composed of an 80 amino
acid tail containing a domain rich in proline, which is involved in
the reverse signaling induced by CD95L-CD95 interaction in CD4
and CD8 T cells (Fig. 1). This reverse signaling involves the co-
engagement of the TCR and co-stimulatory receptors along that of
CD95/CD95L [39–42]. The reported outcomes of this reverse
signaling depends on the cell type, with both proliferation and cell
cycle arrest being reported, but the knowledge on this subject is
still very partial.

Molecular bases of CD95-induced apoptotic signaling. CD95-
mediated extrinsic apoptotic signaling begins with the binding
of CD95L, via its THD on CRD2 and part of the CRD3 of CD95. In
addition to the pre-association of CD95 mediated by the PLAD
[35, 36], Fu et al. recently showed that proline motifs in the
transmembrane (TM) domain also contribute to the trimerization
of the receptor. Mutations of these motifs did not abrogate PLAD-
mediated preassembly of unliganded CD95 but reduced CD95L-
induced apoptosis, implying that these residues are important for
stabilizing signaling-active CD95 oligomers [43]. Binding of CD95L
has been proposed to trigger a reorganization of CD95 multimers
and a conformational change in CD95 intracellular domain,
allowing for the recruitment of the adaptor FADD (Fas-associated
protein with Death Domain) to CD95 via DD-mediated homotypic
interactions [44–47]. FADD is necessary for CD95L-induced
apoptosis [48, 49]. In addition to its DD, FADD contains a Death
Effector Domain and acts as a pivot for the assembly of DED
filaments which are chains of proteins formed through DED-
mediated interactions [50–52]. The DED chains nucleate from
FADD [51–54] and also comprise procaspase-8 and cellular FLICE-
like inhibitory proteins (c-FLIP) which are both key players in the
cell death network [51, 52]. Extensive work has been undertaken,
mainly in the past 15 years, to understand the mode of assembly
of these structures. Beyond CD95- and TRAIL-R1/2-associated
complexes, similar structures likely also nucleate from other death-
inducing complexes such as the ripoptosome, inflammasomes,
TNF-induced complex II, as well as the panoptosome [53, 55–57]
and could thus influence cell fate upon a plethora of signals. In the
case of CD95 signaling, the complex formed by CD95, FADD,
caspase-8 and cFLIP constitutes a platform for caspase-8 activation
which was first called the DISC (for Death-Inducing Signaling
Complex) [44]. Procaspase-8 contains two DEDs, DED1, and DED2,
located at its N-terminus and C-terminal large (p18) and small
(p10) catalytic subunits. As described below, the formation of the
DED filaments allows for the activation of caspase-8 which occurs
via dimerization and a serie of internal cleavages, leading to the
separation of the tandem DED from the catalytic subunits p18 and
p10 [53, 54, 58–60]. The active p10 and p18 subunits are released
into the cytoplasm to form mature active caspase-8 (Fig. 2). Fully
matured caspase-8, an heterotetramer of two p18 and two p10,
cleaves effector caspases-3, 6 and 7, which then cleave sub-
cellular substrates, ultimately inducing cell death [61]. Three
isoforms of cFLIP have been described: cFLIP long, short and
related (cFLIP<sub>L</sub>, cFLIP<sub>S</sub> and cFLIP<sub>R</sub>). cFLIP<sub>S</sub> and cFLIP<sub>R</sub> comprise
solely two tandem DED. In addition to the tandem DEDs, cFLIP<sub>L</sub>
comprises a small and a large caspase-like catalytically inactive
subunit. The initial DED-chain model, described by Inna Lavrik and
Marion MacFarlane's laboratories, proposed a nucleation of the
chain from FADD involving an interaction between the DED of
A

CD95

210

303

319

B

N-Term

<figure><img src="image_4.png" /></figure>

MPD

DD

C-Term

Intracellular

C-Term

<figure><img src="image_5.png" /></figure>

Stalk region
Cleavage

PRD

N-Term

CD95L

1

1

45

1

1

THD

137

103

<figure><img src="image_6.png" /></figure>

80

71

71

1

1

1

1

1

1

PRD

45

1

1

1

1

1

1

1

1

1

1

1

1

1

1

<caption>**Fig. 1** The CD95 receptor and its cognate ligand CD95L. Schematic representation of the functional domains of the CD95 Death Receptor (A) and its ligand CD95L in its membrane-bound form (B). (DD Death Domain, MPD Membrane Proximal Domain, CRD Cysteine-Rich Domain, PRD Proline-Rich Domain, THD TNF Homology Domain).</caption>

FADD with the DED1 of caspase-8, whilst further chain elongation implicated an interaction between the DED2 of FADD-associated caspase-8 with the DED1 of the incoming caspase-8, ultimately bringing the two catalytic domains of caspase-8 in close proximity [51, 52, 62, 63]. The molecular configuration of the DED filaments was further unveiled in 2016, by cryogenic electron microscopy (cryo-EM) analysis [53]. This study established that the orientation of the DED filaments actually relies on three different types of interactions (type I, II and III) between DEDs. Rather than a single linear chain nucleating from FADD through type I interactions, three strands of DED chains assemble via type II and III interactions to ultimately form a triple-helical structure [53, 54]. These different types of interactions define a hierarchy in the formation of the DED filaments, with FADD being rather poorly able to nucleate the DED of cFLIP, arguing against the theory of competition between procaspase-8 and c-FLIP for FADD. Thus, by affecting the conformation of caspase-8 and bringing in proximity the catalytic subunits of two procaspase-8, the DED-chain architecture works as a platform for the activation of this initiator caspase [64, 65].

With regard to cFLIP proteins, it was first thought that these act by competing with caspase-8 for FADD binding or by preventing FADD self-association, akin to the viral FLIP MC159 [65], but this view has been challenged. Multiple evidence now demonstrate

that cFLIP<sub>S/R</sub> actually precludes caspase-8 activation within the DISC. Indeed, reports highlighted that cFLIP<sub>S/R</sub> could limit DED-chain elongation and that cFLIP<sub>S/R</sub> incorporation into DED filaments actively prevented the formation of inter-strand assembly of caspase-8 catalytic domains [54, 63, 65]. Contrary to the small cFLIP isoforms, cFLIP<sub>L</sub> has been reported to possess a dual function, promoting or limiting caspase-8 activation and apoptosis. This is likely due to the fact that the cFLIP<sub>L</sub>/caspase-8 heterodimer does possess a catalytic activity, albeit DISC restricted, and that cFLIP<sub>L</sub> does not limit but promotes DED elongation. Hence, depending on the relative cellular amount of cFLIP<sub>L</sub> to caspase-8, cFLIP<sub>L</sub> might either facilitate the formation of filaments, and thereby of apoptosis-inducing caspase-8 homodimers (low cFLIP<sub>L</sub> to caspase-8 ratio) or, on the contrary (high cFLIP<sub>L</sub> to caspase-8 ratio), mainly result in formation of cFLIP<sub>L</sub>/caspase-8 heterodimers which, whilst able to cleave local substrates (e.g RIPK1), do not mediate apoptosis [63, 66–70].

Another initiator caspase, caspase-10, can be recruited to the TRAIL-R1/2 and CD95 DISC [66, 71, 72]. The role of this caspase in apoptosis induction by CD95L and TRAIL, and in particular its ability to substitute to caspase-8 loss, has been controversial. Caspase-10 is conserved in multiple other vertebrates [73] but lost in certain rodents (mice and rats) which has limited the study of its
<figure><img src="image_7.png" /><figcaption>**Fig. 2** CD95-dependent apoptotic signaling. Representation of the CD95-mediated conventional or apoptotic pathway. The interaction between CD95 and its membrane-bound ligand mCD95L, triggers the recruitment of the adaptor protein FADD, which then recruits procaspase-8 generating the oligomerized DISC. The oligomerisation and auto-cleavage of procaspase-8 into its active form induces then the activation of the effector caspases-3, -6, -7 leading to apoptosis. Active caspase-8 is also able to cleave Bid, generating t-Bid that promotes Mitochondrial outer membrane permeabilization (MOMP) and thus the apoptosome-mediated effector caspase activation.</figcaption></figure>

in vivo function. Some studies, mainly but not exclusively using Jurkat cells or primary T cells, reported that caspase-10 can contribute to DR-induced apoptosis, sometimes independently of caspase-8 [71, 74–79]. Interestingly, a recent study argued that this protease displays anti-apoptotic properties in certain cell lines [80]. Of note, this initiator caspase has been found as different splice variants in human cells, which have also been suggested to display opposing functions towards DR-mediated apoptosis [81]. How each of these isoforms and potentially their post-translational modifications (PTMs) impact on the DED-triple helix formation remains to be deciphered. Indeed, PTMs, most prominently glycosylation, phosphorylation and ubiquitination, of core components of the DISC proteins represent additional crucial checkpoints of DR signaling [82–84].

As mentioned above, caspase-8 also cleaves Bid, generating t-Bid that promotes MOMP and thus apoptosome-mediated effector caspase activation. Whether the engagement of the mitochondrial pathway downstream of CD95 is required for completion of apoptosis depends on the multiple variables described to influence DISC formation (e.g expression level of the DISC components, local lipid composition of the plasma membrane, etc.) as well as downstream regulators of the apoptosis pathway such as XIAP [85–87]. The discovery that caspase-8 is essential during embryonic development lead to the identification of its role as a regulator of necroptosis. Indeed, caspase-8, in concert with cFLIP<sub>L</sub>, is able to cleave RIPK1, along with other key components of the necroptotic cascade, which limits necroptosis induction, as reviewed in [61]. In addition, as further developed later, several of the players of the apoptotic pathway, and in particular DISC components, are also involved in non-cytotoxic signaling outputs.

# INVOLVEMENT OF CD95/CD95L IN CANCER AND AUTOIMMUNE DISEASES

## Cancer

Multiple defects in the DR-mediated pathway have been observed in human tumors [88–91]. In healthy individuals, extrinsic apoptosis plays a central role in the immune-mediated elimination

of infected or transformed cells. Therefore, defects in the extrinsic apoptotic pathway contribute to tumorigenesis primarily by limiting the efficiency of immune surveillance [92]. Cancer cells have different ways of escaping from apoptosis [93]. These include modification of the expression of pro- and anti-apoptotic proteins, such as inhibitors of apoptosis (IAPs) and the anti-apoptotic members of the Bcl-2 family among others, as well as the expression of CD95 itself at the membrane [94, 95]. Mutations in the FAS gene have been detected in both hematologic and solid tumor malignancies [96–99]. These mutations are mainly located in exon 8 and 9, which code for the DD, thus leading to resistance to CD95-mediated apoptosis [91, 93]. Accumulating evidence has shown that CD95 signaling cascades are often disrupted in several autoimmune diseases and malignant tumors [100–102], leading to the triggering of pro-tumorigenic cellular outcomes, rather than apoptosis [89, 103]. Considering the potential pro-tumorigenic effect of an incomplete induction of mitochondria-dependent death-signaling mentioned above, one could hypothesize that weak apoptotic signaling downstream of CD95 could also have tumor-promoting effects. Furthermore, the quality of cell death induced downstream of CD95 might also differentially impact on inflammation and tumor progression, even though this remains to be tested. In addition, several non-apoptotic pathways are also induced by CD95L, as detailed below, and contribute to its tumor-promoting and pro-inflammatory roles [88].

## Non-apoptotic CD95-mediated pathways (NF-κB, MAPK, PI3K/Akt)

**NF-κB pathway.** Several studies reported that CD95-mediated stimulation can induce the apoptotic pathway in some cells, while in others, the non-apoptotic NF-κB (nuclear factor kappa B) pathway is favored [104, 105]. NF-κB is a transcription factor playing an important role in the inflammatory responses as well as in the regulation of cell survival, differentiation and proliferation. A non-optimal regulation of this signaling pathway has been associated with a high incidence of pathological conditions, such as cancer and chronic inflammation [106]. At the cell population level, the stimulation of CD95 by CD95L has long been reported to concomitantly induce apoptotic signaling and NF-κB activation
<figure><img src="image_8.png" /><figcaption>**Fig. 3** CD95-dependent non-apoptotic signaling. Representation of the CD95-mediated unconventional or non-apoptotic pathways. The interaction between CD95 and its ligand CD95L recruits several adaptor proteins leading to the activation of the MAPK, NF-κB and PI3K pathways. The MAPK pathway requires a cascade of phosphorylations to eventually activate ERK, allowing its translocation to the nucleus where it induces the transcription of pro-survival/proliferation/pro-inflammatory genes. The NF-κB heterodimer is kept inactive by IκB, which after IKK-mediated phosphorylation releases NF-κB allowing its translocation to the nucleus where it promotes the transcription of pro-inflammatory/proliferation/migration genes. The PI3K/Akt and PLCy1 pathways are functionally linked in triggering the cell migration. Active PLCy1 participates in the elevation of cytoplasmic calcium levels, which then leads the activation of biochemical pathways that leads to cell proliferation, survival and migration through the phosphorylation and activation of Akt.</figcaption></figure>

[105, 107]. More recently, single cell studies have assessed if the apoptotic and NF-κB pathways were activated in the same cell [107, 108]. NF-κB was found to be activated in dying apoptotic cells, confirming the hypothesis that CD95-mediated NF-κB activation is correlated with the production of the so-called “find and eat me” pro-inflammatory cytokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF [104]. Some of these cytokines act as chemokines and are therefore able to affect the tumor immune microenvironment.

Mechanistically, it appears that CD95 mediates NF-κB activation through a FADD and caspase-8-involving pathway [104, 109–111]. The Death Domain of CD95, FADD, and caspase-8 were in fact reported as required for NF-κB activation by CD95L [110]. Experiments carried out inhibiting caspases prevented TRAIL/ anti-APO-1-induced apoptosis, but not NF-κB activation, indicating that both pathways bifurcate upstream of caspase-8 full activation [112]. Furthermore, the ability of DR to induce NF-κB activation was drastically reduced in a FADD-deficient CD95<sup>pos</sup> cell line (e.g., Jurkat cells) [112]. Caspase-8 participates in CD95L- and TRAIL- induced inflammatory signaling as a scaffold for assembly of a Caspase-8-FADD-RIPK1-containing complex, leading to NF-κB- dependent inflammation [109, 113]. Whilst this has not been studied for CD95 yet, it is tempting to speculate that NF-κB activation could also be ignited from the CD95 DISC, as recently shown for TRAIL [114]. Contrary to FADD and caspase-8 which seem to be essential for NF-κB activation upon CD95L, the role of RIPK1 in this process seems to be less pronounced and depends on the cell type [104, 109]. Recently, Horn et al. described a new role for caspase-10 that would negatively regulate the caspase-8- induced cell death, thus activating the cell survival induced by the NF-κB pathway [80]. TRADD, which is essential for the TNF-alpha- induced NF-κB activation, was not involved in the CD95L-induced NF-κB activation [110]. Experiments performed on cell lines resistant to CD95-mediated apoptosis, reported TRAF2 as a key player in pancreatic cancer pathophysiology [115]. This group also

observed that the stimulation of TRAF2-overexpressing cells with CD95L led to induction of NF-κB, enhanced IL-8-secretion, and a further increased invasiveness. In fact, several E3 ligases contribute to NF-κB activation upon CD95 stimulation, namely cIAP1/2 and the Linear Ubiquitin chain Assembly Complex (LUBAC), likely in a manner similar to their roles in TNF and TRAIL-induced gene-activation [104, 114, 116]. Downstream of these different actors, the activation of NF-κB relies on IκBα degradation, the protein responsible for constitutively inhibiting NF-κB. In a manner similar to TNF and TRAIL signaling, it is likely that several components of the CD95 DISC and/or secondary complex modified with ubiquitin allow the recruitment and activation of the IKK complex and potentially the TAB/TAK1 complex. The IKK complex is composed of three subunits (i.e., IKKα, IKKβ, IKKγ). The IKKβ subunit can then phosphorylate IκBα, marking it for lysine-48 ubiquitination and degradation by the proteasome. This leads to the translocation of NF-κB into the nucleus which promotes the expression of multiple genes including pro-inflammatory cytokines as well as anti-apoptotic proteins, such as cIAP1, cIAP2, and XIAP (Fig. 3) [117, 118]. Moreover cFLIP can be upregulated in some cell lines under critical involvement of the NF-κB pathway [119, 120] also resulting in increased resistance to CD95L or TNF.

**MAPK pathway.** The MAPK family includes six main groups in humans, among which JNK (Jun N-terminal Kinase), ERK1/2 and the p38 isoform must be mentioned for their involvement in CD95-mediated pro- and anti-apoptotic signaling pathways [121–123]. The induction of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, which regulates growth, proliferation, differentiation, survival, innate immunity and cellular development is involved in tumorigenesis in multiple tumor types [124]. In the latent state, the inactive MAPKs are cytosolic. The activation of the different MAPKs takes place according to a common general scheme, which provides for a series of sequential phosphorylations catalyzed by
different kinases activated in succession. MAPK is phosphorylated by a MAPK kinase (MKK), itself phosphorylated by a MAPK kinase kinase (MKKK), in turn activated by an activator protein.

CD95-mediated stimulation has been suggested early on to
induce the JNK pathway through the DAXX adapter protein (Death
domain associator protein 6), which after fixation with CD95-DD
induces the apoptotic pathway [125, 126]. c-FLIP can block this
pathway by inhibiting DAXX [127]. CD95-mediated JNK activation
also appears to occur rather slowly, compared to other cell stress
stimuli, such as inflammatory cytokines and oxidative stress [128].
Indeed, expression of cFLIP variants or use of different caspase
inhibitors in primary human keratinocytes, blocked late death
ligand-induced JNK or p38-MAPK activation, suggesting that these
responses are secondary to caspase activation [129]. This may be
due to the fact that caspase-3 can cleave and thereby activate the
MAP3K MEKK1 [128]. Of note, the signal induced by soluble CD95L
rather results in a rapid and transient phosphorylation of ERK1/2
[130]. This MAPK protein is widely involved in enhancing growth
and proliferation upon CD95 activation [121]. Stimulation of CD95
on primary sensory neurons triggers neurite growth through
sustained activation of the extracellular signal-regulated kinase
(ERK) pathway and subsequent upregulation of p35, a neurite
growth mediator [131]. Of note, in pancreatic apoptosis-resistant
tumor cells, CD95L- and TRAIL-induced upregulation of pro-
inflammatory genes was found to be partially depend on the ERK
signaling pathway via caspase-mediated activation [132]. The
same group suggested that the stimulation of the ERKs pathway
must probably depend on a caspase-dependent factor operating
downstream of the DISC complex. According to another group,
CD95L can also induce the autocrine production of EGFR
(Epidermal Growth Factor Receptor) ligands and the consequent
activation of EGFR followed by ERK1 and ERK2 mitogen-activated
protein kinases [133]. In primary fetal astrocytes, blocking ERK
phosphorylation with specific inhibitors resulted in a significant
reduction of CD95-induced proliferation [134]. In this context, ERK
phosphorylation is also caspase-dependent. Noteworthy, cFLIP_L
can also contribute to ERK activation. Indeed, caspase-8 can cleave
cFLIP_L into different cleavage products. One of these cleavage
products is identified with the name of p43-FLIP [135], which
associates with Raf-1 activating the phosphorylation cascade,
leading to ERK activation and ultimately to ERK translocation into
the nucleus, where it exerts proliferative or pro-inflammatory
effects through downstream transcription factor targets (Fig. 3).

PI3K pathway. As mentioned previously, mCD95L can be cleaved by various metalloproteases to produce several soluble forms of the ligand, together referred here as sCD95L [136–140]. Soluble CD95L has been shown to be accumulated in the serum of patients suffering from various diseases [141, 142], whilst the exact cleavage form(s) accumulating in most of these cases remains to be determined. sCD95L was initially believed to be a competitor of its membrane-bound counterpart (mCD95L) in the interaction with CD95 and the consequent induction of the apoptotic signal. It is only in the last decade that it has been reported that not only sCD95L failed in the induction of programmed cell death [143, 144], but that its interaction with CD95 led to the induction of a different type of signal, including engagement of ERK, NF-KB, and PI3K/Akt [145, 146]. Gene-targeted mice selectively lacking either metalloprotease-dependent soluble CD95L (sCD95L) or membrane-bound CD95L (mCD95L) were generated [147]. Mice lacking sCD95L appeared normal and their T cells were able to kill target cells, whereas T cells lacking mCD95L could not kill cells through CD95 activation. Furthermore, mice lacking mCD95L displayed SLE-like symptoms and histiocytic sarcoma. Of note, one group has described that the stimulation of CD95 with sCD95L can induce a calcium-dependent process that leads to the activation of a c-yes/PLCy1/PI3K/Akt pathway promoting Triple Negative Breast Cancer (TNBC) cell migration [141] (Fig. 3).

mCD95L has also been shown to activate the PI3K/Akt pathway. There appears to be a crosstalk between the two signaling paths PI3K and NF-κB under mCD95L stimulation. Indeed in mutant PIK3CA (PI3K alpha catalytic subunit), but not WT PIK3CA-expressing Hct116 cells, TRAIL, and CD95L stimulated NF-κB activation [148]. It is now clear that caspase-8 not only mediates the cell death signal initiated by CD95L, but also contributes to the induction of apoptosis-independent pathways, such as cell migration and adhesion. Caspase-8 was found to be a substrate of Src kinase c-yes [149]. It has been observed that the stimulation of motility through the EGF, first activates c-yes, and then triggers the phosphorylation of caspase-8 on Tyrosine-380 in the linker region between the two subunits (i.e., p18 and p10) of the procaspase-8 converting it from a pro-apoptotic factor to a cell motility factor. The Y380 phosphorylation prevented downstream activation of the caspase cascade proving a valuable path to explore for sensitization of CD95-resistant tumors to extrinsic apoptotic stimuli [150]. The catalytic domain of caspase-8 is in fact not required for the induction of the migration signal. Once phosphorylated, caspase-8 interacts with the p85 alpha subunit of PI3K (Fig. 3) [151].

In a mouse cell model of glioblastoma (GBM) the c-yes/PI3K-p85
interaction was reported to signal cell invasion via glycogen
synthase kinase 3-beta pathway and subsequent expression of
matrix metalloproteinases [152]. Blockade of CD95-mediated
activity in this cellular model drastically reduced the number of
invading cells. In the same context CD95 expression associates
with stemness and EMT features and poorer overall survival. CD95-
mediated activation of the PI3K p85 also maintained the
expression of EMT-related transcripts. The authors therefore
suggested that CD95 would be a potential prognostic biomarker
in GBM [153].

Systemic autoimmune diseases

To date, more than 80 diseases in which the etiology is certainly,
or most likely, autoimmune have been described [154]. Around
the 1960s/70s the distinction was made between systemic
autoimmune diseases, with general signs and symptoms and
the involvement of multiple organs and systems, and organ-
specific autoimmune diseases, where the immuno-pathological
damage is localized to an organ and the clinical picture closely
linked to the dysfunction of the organ itself. The self and non-self
recognition functions are carried out through an elaborate
identification system that involves T and B lymphocytes. The
central selection process eliminates the vast majority of auto-
reactive lymphocytes at an immature stage during their develop-
ment, through Bcl-2-interacting mediators of cell death, such as
Bim [155]. However, despite the numerous central tolerance
mechanisms, many mature B and T lymphocytes, generated in the
central lymphoid organs, then reach the peripheral lymphoid
organs and undergo activation, turning into their self-reactive
form [156, 157]. The Bim-dependent apoptotic pathway is
required both for the killing of self-reactive immature B and T
lymphocytes during their development and for the elimination of
auto-reactive mature B and T lymphocytes in peripheral lymphoid
organs [158]. However, except in the thymus, most of the TCR-
related mature T-cell apoptosis is induced by the extrinsic
pathway via membrane DR, and in particular one of the most
important elements of this regulation is apoptosis activated by the
CD95/CD95L system [159]. The importance of CD95 and CD95L in
eliminating activated T cells is underlined by the anomalies
observed when mutations in the genes coding for CD95 or CD95L
occur. The CD95/CD95L system has a dual role in immune
regulation [160–163]. It constitutes one of the mechanisms by
which cytotoxic lymphocytes kill the target, but is also involved in
the process of turning off the response. Activation of the
lymphocytes leads to an increase in CD95L expression and the
ability to trigger apoptosis. Recently Heikenwälder's group
reported that blocking CD95L could prevent auto-aggression of hepatocytes by CD8<sup>pos</sup> T cells in the precancerous context of Non-alcoholic steatohepatitis (NASH). The liver cells coming into contact with aberrantly activated CD8<sup>pos</sup> T cells die by apoptosis due to contact with the CD95L overexpressed in these reactive T cells [164]. The same process could occur and damage other organs as well. This observation, made on a mouse model, could be useful for the design of future immunotherapies without affecting the antigen-specific T-cell immunity.

Peripheral T-cell CD95-induced apoptosis eliminates over-
activated and self-reactive T cells via a mechanism called
"Activation-Induced Cell Death" (AICD) [165]. T-cell activation is
also associated with CD95L expression at the cell surface, thus
representing a specific aspect of the immune system. AICD is
induced through the interaction between CD95 and CD95L, both
expressed on activated T cells surface [166]. Similarly to T cells, it
has been reported that not only B cells are capable of expressing
CD95L but that the level of CD95L expression correlates with the
level of activity of B cells, thus making them capable of killing
CD95 expressing cells [167]. Consequently, the abnormal activa-
tion of these CD95L-expressing B cells is implicated in the immune
modulation of various diseases and thus constitutes a therapeutic
target [168, 169]. The constitutive expression of CD95L in some
"immunologically privileged" tissues (e.g., the Sertoli cells, the
testes, or the anterior chamber of the eye), has suggested that
CD95L plays also an important role in reducing the activity of
immune cells in these tissues. Several studies exploring mutations
in the genes encoding CD95 and CD95L have allowed to better
understand the pathogenesis of autoimmune diseases, such as
ALPS (Autoimmune LymphoProliferative Syndromes) or SLE
(Systemic Lupus Erythematous).

ALPS: Autoimmune lymphoproliferative syndromes. Some FAS gene mutations impair the function of the molecule, leading either to a reduced expression on the membrane, or to the impairment of the ability to transmit the apoptotic signals [170]. The defective shutdown of the immune response resulting from the defective function of CD95 can be the cause of both the progressive accumulation of lymphocytes in the peripheral lymphatic organs and the development of autoimmune reactions [171]. The most common trigger of ALPS is due to autosomal dominant mutations of the FAS gene [172, 173], and, less frequently, of FASLG, the gene encoding the CD95 ligand [174]. Much less common forms of autoimmune lymphoproliferation are due to mutations in another factor in the T-cell apoptosis pathway, caspase-10. Controversial studies have been carried out in this regard as several heterozygous CASP10 variants have been identified along with variants known to be polymorphic. It has recently been observed that said CASP10 mutations are capable of impaired apoptosis [175]. In ALPS patients lacking germline mutations in FAS, some dominant somatic mutations in the DR and notably in the Death Domain were found. These somatic mutations were identified as missense variants likely to change the normal structure and impact the oligomerization and functionality of CD95 [176]. Of note, a large study in a cohort of 100 ALPS patients with CD95 DD mutations reported that the risk of non-Hodgkin and Hodgkin lymphomas, respectively, was 14 and 51 times greater than expected [177]. Collectively, all diseases associated with abnormal lymphocyte apoptosis, lymphoproliferation, and autoimmunity, are named Autoimmune Proliferative Syndromes. The syndromes can be classified according to the mutated gene(s) responsible for the defect and they are usually characterized by lymphadenomegaly and hepatosplenomegaly associated with autoimmune manifestations, mainly of the hematological type, such as hemolytic anemia, thrombocytopenia, and neutropenia, as well as the presence of cell-type-specific autoantibodies [178–180]. Furthermore, the accumulation of a minority population of self-reactive CD3<sup>pos</sup> TCRα<sup>pos</sup> CD4<sup>neg</sup> CD8<sup>neg</sup>

T cells called double negative (DNT) has been reported in the early
1990s as a major feature of ALPS. Despite their similarity to normal
differentiated T cells, DNTs are remarkably proliferative, particu-
larly in the paracortical region [181]. Last year Kimberly Gilmour's
group carried out a study on 215 patients with clinical evidence of
ALPS, intending to define the most useful and predictable
biomarkers for a better ALPS diagnosis. Among the several
subgroups of patients, levels of different biomarkers, including
DNTs and sCD95L, were observed significantly higher in the ALPS-
FAS patients than in the "unknown ALPS" (ALPS-U), cases for
which the genetic determinant is not identified. They developed a
diagnostic protocol for the potential identification of patients with
presymptomatic or mild disease. The combination of such
biomarkers could be useful in the process of confirming or
excluding the ALPS diagnosis [182]. Today the diagnosis requires
performing a cell apoptosis test and molecular type analysis, and
the choice of therapy is guided by the severity and nature of the
symptoms, but generally it is based on the intake of immuno-
suppressants such as rituximab. The increased sCD95L serum
levels are now part of the new diagnostic criteria procedure for an
ALPS [183, 184]; these high levels have indeed been associated
with the pathology without their pathophysiological role being
elucidated [185]. Curiously, some groups observed a change in
sCD95L levels in correlation with aging, and age-related condi-
tions and/or diseases with an increase in molecular signals due to
aging oxidative stress [186]. Furthermore, oxidative stress seems
to promote CD95L cleavage through activation of MMPs, and
more interestingly this MMP activation seems to increase as a
function of aging [187].

SLE: Systemic lupus erythematous. In both humans and mice, mutations in the genes coding for CD95 or CD95L are also strongly associated with certain forms of lupus disease. The defects in apoptosis described in autoimmunity and lymphoproliferative syndromes correspond to the human equivalent of the *MRL/lpr* mouse model (*Murphy Roths Large/lymphoproliferation*), deeply investigated as a murine SLE-like model [188, 189]. This model was generated following the identification of an autosomal recessive modification on chromosome 19 [190]. The mentioned mutation was found on the gene encoding CD95 protein. Similar to this model, a second model was designed and generated after the discovery of a second autosomal recessive mutation, on chromosome 1, corresponding to the gene coding for CD95L [191]. The latter model took the name of *MRL/gld* for generalized lymphoproliferative disease. In addition to those two mouse models, a wider selection of mouse models is available to sift genetic and cellular aspects of SLE [192, 193]. Since the etiology of SLE is multifactorial and multigenetic, some of these models, such as those mentioned above, derive from spontaneous genetic factors, while others assume a SLE-like phenotype after exposure to certain chemicals such as intraperitoneal injections of pristane (2, 6, 10, 14 tetramethylpentadecane) [194], or overexpression of cytokines (ie IL-6, IL-12, INF-I) [195–197]. Others, similarly to induced graft-versus-host disease models, develop a lupus-like syndrome following donor cell injection [198]. Despite their numerous limitations, over the years these SLE-like mouse models have been widely used to screen numerous potential therapies, pointing out their importance in the study of this disease and in the therapeutic advancement in this field [199]. Systemic lupus erythematosus is a rare systemic autoimmune disease, more severe in women, especially of childbearing age. Very recently Lars Rönnblom's group has observed that there is a correlation between the cumulative genetic risk and survival, organ damage, renal dysfunctions, in patients affected by SLE, introducing Genetic Risks Score (GRS) as a potential tool for predicting outcomes in patients with SLE [200]. The term "systemic" means that the disease affects several organs. Genetically speaking, germline heterozygous mutations in the *FAS* gene have been
<table><caption>**Table 1.** Table listing the 127 patents published in the last 25 years concerning CD95 or its cognate ligand CD95L or the entire CD95/CD95L interaction system.</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>1</td><td>WO2003079750</td><td>2003.10.02</td><td>WO</td><td>Autoimmune diseases/ inflammatory diseases/cancer</td><td>Antibodies</td><td>Antagonistic anti-hfs ligand human antibodies and fragments thereof</td><td>LANCASTER Joanne Sloan</td><td>[350]</td></tr><tr><td>2</td><td>US20200102397/ WO2017051002</td><td>2020.04.02/ 2017.03.30</td><td>WO</td><td>Cancer</td><td>Antibodies</td><td>Anti-cd95I antibody</td><td>GIEFFERS Christian, HILL Oliver, THIEMANN Meinolf et al.</td><td>[221]</td></tr><tr><td>3</td><td>WO2015165973</td><td>2017.03.08</td><td>WO</td><td>Cancer</td><td>Antibodies</td><td>Diagnostic anti-cd95I antibody</td><td>FRICKE, Harald GIEFFERS, Christian SYKORA, Jaromir</td><td>[221]</td></tr><tr><td>4</td><td>WO2008080623</td><td>2016.04.28</td><td>WO</td><td>Cancer</td><td>Antibodies</td><td>Neutralization of cd95 activity blocks invasion of glioblastoma cells in vivo</td><td>MARTIN-VILLALBA Ana, KLEBER Susanne, WIESTLER Benedikt et al.</td><td>[325]</td></tr><tr><td>5</td><td>US20150274833/ EP2920210/ WO2014076292</td><td>2015.10.01/ 2015.09.23/ 2014.05.22</td><td>WO</td><td>Cancer</td><td>Antibodies</td><td>Recombinant bispecific antibody binding to cd20 and cd95</td><td>HERRMANN Andreas, GROSSE-HOVEST Ludger</td><td>[271, 272, 351]</td></tr><tr><td>6</td><td>EP2717911/ WO2012168259</td><td>2014.04.16/ 2012.12.13</td><td>WO</td><td>Cancer</td><td>Antibodies</td><td>Protein tyrosine phosphatase, non-receptor type 11 (ptpn11) and triple-negative breast cancer</td><td>BENTIRES-ALJ Mohamed, ACETO Nicola, STADLER Michael</td><td>[352]</td></tr><tr><td>7</td><td>US20060083738/ EP1506237/ WO2003097698</td><td>2006.04.20/ 2005.02.16/ 2003.1.27</td><td>WO</td><td>Cancer</td><td>Antibodies</td><td>Treatment of cancer by the use of anti-fas antibody</td><td>JOHNSTON Patrick Gerard, LONGLEY Daniel</td><td>[269, 353-355]</td></tr><tr><td>8</td><td>WO2010066914</td><td>2011.10.19</td><td>WO</td><td>Others</td><td>Antibodies</td><td>Remedies for pemphigus containing anti-fas-ligand antibodies</td><td>PINCELLI Carlo, MARCONI Alessandra</td><td>[356]</td></tr><tr><td>9</td><td>US20030082180/ WO2001041803</td><td>2003.05.01/ 2001.06.14</td><td>WO</td><td>Others</td><td>Antibodies</td><td>Combination of compounds that inhibit the biological effects of tnf-α and cd95I in a medicament</td><td>KRAMMER Peter, MARTIN-VILLALBA Ana</td><td>[357]</td></tr><tr><td>10</td><td>US20110300113/ EP2064235/ WO2008034608</td><td>2011.12.08/ 2009.06.03/ 2008.03.27</td><td>WO</td><td>Others</td><td>Antibodies/cells</td><td>The death receptor cd95 controls neurogenesis of adult neural stem cells in vivo and in vitro</td><td>MARTIN-VILLALBA Ana, CORSINI Nina, LETELLIER Elisabeth et al.</td><td>[358]</td></tr><tr><td>11</td><td>WO2010006772</td><td>2011.08.04</td><td>WO</td><td>Inflammatory diseases</td><td>Antibodies/fusion proteins</td><td>Use of cd95 inhibitors for the treatment of inflammatory disorders</td><td>MARTIN-VILLALBA Ana, LETELLIER Elisabeth, SANCHO-MARTINEZ Ignacio</td><td>[359]</td></tr><tr><td>12</td><td>US20170166648/ EP3150224/ WO2014013036</td><td>2017.06.15/ 2017.04.05/ 2014.01.23</td><td>WO</td><td>Others</td><td>Antibodies/fusion proteins</td><td>Inhibitors of the cd95 signaling pathway for treatment of mds</td><td>FRICKE Harald, FONTENAY Michaela, KUNZ Claudia</td><td>[348, 349]</td></tr><tr><td>13</td><td>US20060234968/ WO2004071528</td><td>2006.10.19/ 2004.08.26</td><td>WO</td><td>Others</td><td>Antibodies/fusion proteins</td><td>Inhibition of the cd95 ligand/ receptor system for the treatment of neurological disorders and injuries</td><td>MARTIN-VILLALBA Ana, KRAMMER Peter, DEMJEN Deana</td><td>[360]</td></tr><tr><td>14</td><td>US20030170244</td><td>2003.09.11</td><td>US</td><td>Cancer/others</td><td>Antibodies/fusion proteins</td><td>Inhibition of fas signaling</td><td>PLUENNEKE John D, CONNOR Timothy</td><td>[361]</td></tr><tr><td>15</td><td>US6846637/ WO1999065935</td><td>2005.01.25/ 1999.12.23</td><td>WO</td><td>Cancer/ autoimmune diseases/others</td><td>Antibodies/ polypeptides</td><td>Fas peptides and antibodies for modulating apoptosis</td><td>CHIODI Francesca</td><td>[362]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>Nº</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>16</td><td>WO2016170027</td><td>2016.10.27</td><td>WO</td><td>Inflammatory diseases</td><td>Antibodies/peptides</td><td>Methods and pharmaceutical compositions for the treatment of th17 mediated diseases</td><td>LEGEMBRE Patrick, BLANCO Patrick, FLYNN Robin</td><td>[101]</td></tr><tr><td>17</td><td>US20120294856/ EP2502069/ WO2011058175</td><td>2012.11.22/ 2012.09.26/ 2011.05.19</td><td>WO</td><td>Cancer</td><td>Antobodies/enzymes/ RNA interfering molecules</td><td>Compounds inhibiting cd95 signaling for the treatment of pancreatic cancer</td><td>MARTIN-VILALBA Ana, HERHAUS Peter, SANCHO-MARTINEZ Ignacio et al.</td><td>[363]</td></tr><tr><td>18</td><td>US20200407728/ WO2016069282</td><td>2020.12.31/ 2016.05.06</td><td>WO</td><td>Autoimmune diseases</td><td>Cells</td><td>Altering gene expression in modified t cells and uses thereof</td><td>ZHAO Yangbing, REN Jiangtao, LIU Xiaojun, JUNE Carl H.</td><td>[364]</td></tr><tr><td>19</td><td>US20150104428/ EP2833896/ WO2013149211</td><td>2015.04.16/ 2015.02.11/ 2013.10.03</td><td>WO</td><td>Autoimmune diseases</td><td>Cells</td><td>Compositions and treatment methods for mesenchymal stem cell-induced immunoregulation</td><td>SHI Songtao, AKIYAMA Kentaro, CHEN Chider</td><td>[330, 331]</td></tr><tr><td>20</td><td>WO2015038665</td><td>2015.03.19</td><td>WO</td><td>Autoimmune diseases/ inflammatory diseases</td><td>Cells</td><td>A composition of stem cells having highly expressed fas ligand</td><td>SHI Songtao, LIU Shiyu, CHEN Fa-ming</td><td>[365]</td></tr><tr><td>21</td><td>US20200121719/ EP3565888/ WO2018129332</td><td>2020.04.23/ 2019.11.13/ 2018.07.12</td><td>WO</td><td>Cancer</td><td>Cells</td><td>Expansion of tumor-infiltrating lymphocytes (tis) with tumor necrosis factor receptor superfamily (tnfrsf) agonists and therapeutic combinations of tis and tnfrsf agonists</td><td>LOTZE Michael, T, RITTHIPICHAL Krit</td><td>[277, 366, 367]</td></tr><tr><td>22</td><td>EP3569700/ WO2011052545</td><td>2019.11.20/ 2011.05.05</td><td>WO</td><td>Cancer</td><td>Cells</td><td>Method for producing antigen-specific b-cell population</td><td>KITAMURA Daisuke, NOJIMA Takuya</td><td>[368]</td></tr><tr><td>23</td><td>US20180008670</td><td>2018.01.11</td><td>US</td><td>Cancer</td><td>Cells</td><td>Chimeric antigen receptor targeting of tumor endothelium</td><td>WAGNER Samuel C., ICHIM Thomas E, MINEV Boris</td><td>[280]</td></tr><tr><td>24</td><td>WO2015161276</td><td>2015.10.22</td><td>WO</td><td>Cancer</td><td>Cells</td><td>Crispr-cas-related methods, compositions and components for cancer immunotherapy</td><td>WELSTEAD G. Grant, FRIEDLAND Ari E, MAEDER Morgan L, BUMCROT David A</td><td>[369]</td></tr><tr><td>25</td><td>WO2014039044</td><td>2014.13.03</td><td>US</td><td>Cancer</td><td>Cells</td><td>Methods of producing t memory stem-cell populations</td><td>GATTINONI Luca, LUGLI Enrico, ROEDERER Mario, RESTIFO Nicholas P</td><td>[281, 370, 371]</td></tr><tr><td>26</td><td>US20040131599/ WO20200272798</td><td>2004.07.08/ 2002.09.19</td><td>WO</td><td>Cancer</td><td>Cells</td><td>Fas ligand-expressing hematopoietic cells for transplantation</td><td>CIVIN Curt, I, DRACHMAN Daniel, WHARTENBY Katherine, PARDOLL Drew M</td><td>[372]</td></tr><tr><td>27</td><td>US20090191167/ EP2046351/ WO2008014470</td><td>2009.07.30/ 2009.04.15/ 2008.01.31</td><td>WO</td><td>Cancer/ autoimmune diseases/others</td><td>Cells</td><td>Adult sertoli cells and uses thereof</td><td>WHITE David J</td><td>[373]</td></tr><tr><td>28</td><td>WO2018227286</td><td>2018.12.20</td><td>WO</td><td>Inflammatory diseases/others</td><td>Cells</td><td>Allograft tolerance without the need for systemic immune suppression</td><td>NAGY Andras, HARDING Jeffrey, NAGY Kristina</td><td>[374]</td></tr><tr><td>29</td><td>WO2018130679</td><td>2019.11.20</td><td>WO</td><td>Cancer</td><td>Chemicals</td><td>Methods and pharmaceutical compositions for reducing cd95- mediated cell motility</td><td>LEGEMBRE Patrick, VACHER Pierre, POISSONNIER Amanda, BLANCO Patrick</td><td>[101]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>30</td><td>US20190084987</td><td>2019.03.21</td><td>US</td><td>Cancer</td><td>Chemicals</td><td>Small molecule histone methyltransferase suv39h1 inhibitor and uses thereof</td><td>LU Chunwan, LEBEYEA Ifyna, LIU Kébin</td><td>[301]</td></tr><tr><td>31</td><td>US20150343024/ EP2931375/ WO2014090224</td><td>2015.12.03/ 2015.10.21/ 2014.06.19</td><td>WO</td><td>Cancer</td><td>Chemicals</td><td>Use of active substance combinations for inducing tumor senescence</td><td>RÖCKEN Martin, WIEDER Thomas, HAHN Matthias et al.</td><td>[375]</td></tr><tr><td>32</td><td>WO2008036244</td><td>2008.03.27</td><td>WO</td><td>Cancer</td><td>Chemicals</td><td>Use of cyclosporin a to sensitize resistant cancer cells to death receptor ligands</td><td>REED John C, THOMAS Michael P</td><td>[376]</td></tr><tr><td>33</td><td>WO2019206834</td><td>2019.10.31</td><td>WO</td><td>Cancer/ autoimmune diseases</td><td>Chemicals</td><td>Compounds and pharmaceutical compositions for reducing cd95-mediated cell motility</td><td>VACHER Pierre, LEGEMBRE Patrick, JEAN Mickael et al.</td><td>[142]</td></tr><tr><td>34</td><td>WO2002047728</td><td>2002.06.20</td><td>WO</td><td>Others</td><td>Chemicals</td><td>Treatment of posterior capsule opacification</td><td>ALLAN Bruce Duncan Samuel</td><td>[377]</td></tr><tr><td>35</td><td>WO2019141862</td><td>2019.07.25</td><td>WO</td><td>Inflammatory diseases</td><td>Chemicals/antobodies/ fusion proteins/ nucleotide complexes</td><td>Combination therapeutics</td><td>WALCZAK Henning, TARABORRELLI Lucia, PELTZER Nieves</td><td>[321]</td></tr><tr><td>36</td><td>US20090142369</td><td>2009.06.04</td><td>US</td><td>Others</td><td>Cosmetic composition</td><td>Method for preventing skin- cellular injury by using green algae extract and cosmetic composition containing green algae extract</td><td>SHIH Meng-Han, SHIH Mei-Fen</td><td>[378]</td></tr><tr><td>37</td><td>US20080118466</td><td>2008.05.22</td><td>US</td><td>Autoimmune diseases</td><td>Drug delivery system</td><td>Treatment of rheumatoid arthritis with soluble fas-ligand cross-linkers</td><td>HUREZ Vincent Jacques, MICHELSON Seth G, SHODA Lisl Katharine et al.</td><td>[379, 380]</td></tr><tr><td>38</td><td>EP0930890/ WO1998017305</td><td>1999.07.28/ 1998.04.30</td><td>WO</td><td>Autoimmune diseases</td><td>Drug delivery system</td><td>Use of fas1 or fas1 transfected cd47+ / fas1- (th1-cell lines for the treatment of th1/th2 diseases</td><td>HAHNE Michael, TSCHOPP Juerg, DA CONCEIÇÃO-Silva Fatima, SCHROETER Michael</td><td>[381]</td></tr><tr><td>39</td><td>US20200046780/ EP3592392</td><td>2020.02.13/ 2020.01.15</td><td>US/EP</td><td>Autoimmune diseases/others</td><td>Drug delivery system</td><td>Fas-engineered biomaterials with immunomodulatory function</td><td>SHIRWAN Haval, GARCIA Andres J, YOLCU Esma S et al.</td><td>[136, 382]</td></tr><tr><td>40</td><td>US20050214311/ EP1478390/ WO2003070271</td><td>2005.09.29/ 2004.11.24/ 2003.08.28</td><td>WO</td><td>Cancer</td><td>Drug delivery system</td><td>Novel complexes for inducing an immune response</td><td>SCREATON Gavin R, SIMON Katharina A, GALLIMORE Awen M</td><td>[383]</td></tr><tr><td>41</td><td>US20200108117/ WO2019246130</td><td>2020.04.09/ 2019.12.26</td><td>US</td><td>Cancer/others</td><td>Drug delivery system</td><td>Drug delivery systems comprising an intraocular pressure lowering agent, a neurotrophic agent, a c-type natriuretic peptide, a natriuretic peptide receptor-b, an apoptosis signaling fragment inhibitor or a fas- ligand inhibitor for treating glaucoma or ocular hypertension</td><td>SCHIFFMAN Rhett M, SCHEIBLER Lukas</td><td>[384]</td></tr><tr><td>42</td><td>US20200246432/ WO2019040372</td><td>2020.08.06/ 2019.02.28</td><td>WO</td><td>Others</td><td>Drug delivery system</td><td>Nitric oxide- and fas ligand- eluting compositions and devices and methods of treatment using same</td><td>KURAL M Hamdi, GUI Liqiong, NIKLASON L Elizabeth, SALTZMAN William Mark</td><td>[385]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>43</td><td>WO2019246141</td><td>2019.12.26</td><td>WO</td><td>Others</td><td>Drug delivery system</td><td>Drug delivery systems comprising a neurotrophic agent, an apoptosis signaling fragment inhibitor (fas) or fas-ligand (fas) inhibitor, a tumor necrosis factor-α (tnf-α) or tnf receptor inhibitor, a mitochondrial peptide, an oligonucleotide, a chemokine inhibitor, or a cysteine-aspartic protease</td><td>SCHIFFMAN Rhett M, SCHEIBLER Lukas</td><td>[384]</td></tr><tr><td>44</td><td>WO2019246130</td><td>2019.12.26</td><td>WO</td><td>Others</td><td>Drug delivery system</td><td>Sustained-release drug delivery systems comprising an intraocular pressure lowering agent, a cnp compound, an npr-b compound, a tie-2 agonist, or neurotrophic agent for use for treating glaucoma or ocular hypertension</td><td>SCHIFFMAN Rhett M, SCHEIBLER Lukas</td><td>[384]</td></tr><tr><td>45</td><td>US20040096450/ WO2000040263</td><td>2004.05.20/ 2000.07.13</td><td>WO</td><td>Others</td><td>Drug delivery system</td><td>Methods and compositions for treating diseases associated with increased fas-ligand titers</td><td>FRENCH Lars E, VIARD Isabelle, TSCHOPP Jurg</td><td>[386]</td></tr><tr><td>46</td><td>US20190330305/ WO2014121085</td><td>2019.10.31/ 2014.08.14</td><td>WO</td><td>Autoimmune diseases/ inflammatory diseases/cancer</td><td>Fusion proteins</td><td>Pd-11 and pd-12-based fusion proteins and uses thereof</td><td>TYKOCINSKI Mark L</td><td>[387]</td></tr><tr><td>47</td><td>EP1481687</td><td>2004.12.01</td><td>EP</td><td>Autoimmune diseases/ inflammatory diseases/cancer</td><td>Fusion proteins</td><td>Use of multimeric ligands of the tnf family with reduced toxicity for treating cell proliferative diseases</td><td>ROSAT Jean-Pierre</td><td>[294]</td></tr><tr><td>48</td><td>US20070269449/ WO2004085478</td><td>2007.11.22/ 2004.10.07</td><td>WO</td><td>Autoimmune diseases/ inflammatory diseases/others</td><td>Fusion proteins</td><td>Cd95-fc fusion proteins</td><td>WALCZAK Henning</td><td>[388]</td></tr><tr><td>49</td><td>EP1214411/ WO2001018202</td><td>2002.06.19/ 2001.03.15</td><td>WO</td><td>Autoimmune diseases/ inflammatory diseases/others</td><td>Fusion proteins</td><td>Flint analog compounds and formulations thereof</td><td>ATKINSON Paul Robert, TIAN Yu, WITCHER Derrick Ryan</td><td>[389-391]</td></tr><tr><td>50</td><td>WO2001090382</td><td>2001.11.29</td><td>WO</td><td>Autoimmune diseases/ inflammatory diseases/others</td><td>Fusion proteins</td><td>Fas ligand-fused proteins</td><td>TOUMA Jyunko</td><td>[392]</td></tr><tr><td>51</td><td>US20180318394/ EP3313429/ WO2016205714</td><td>2018.11.08/ 2018.05.02/ 2016.12.22</td><td>WO</td><td>Autoimmune diseases/others</td><td>Fusion proteins</td><td>Immunomodulation for the long-term prevention and treatment of autoimmune diseases and foreign tissue rejection</td><td>SHIRWAN Haval</td><td>[382, 393, 394]</td></tr><tr><td>52</td><td>US20110081369/ EP1250055</td><td>2011.04.07/ 2002.10.23</td><td>US/EP</td><td>Autoimmune diseases/others</td><td>Fusion proteins</td><td>Methods of immune modulation with death receptor-induced apoptosis</td><td>SHIRWAN Haval</td><td>[382, 393, 394]</td></tr><tr><td>53</td><td>US2011003385/ EP0804561</td><td>2011.01.06/ 1997.11.05</td><td>US/EP</td><td>Autoimmune diseases/others</td><td>Fusion proteins</td><td>Regulated transcription of targeted genes and other biological events</td><td>CRABTREE Gerald R, SCHREIBER Stuart L, SPENCER David M</td><td>[395]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>54</td><td>US20090239240/<br>EP2039768</td><td>2009.09.24/<br>2009.03.25</td><td>US</td><td>Autoimmune<br>diseases/others</td><td>Fusion proteins</td><td>Mutant forms of fas ligand and<br>uses thereof</td><td>KETING Chu</td><td>[266]</td></tr><tr><td>55</td><td>US20180148512/<br>WO2014121093</td><td>2018.05.31/<br>2014.08.07</td><td>WO</td><td>Cancer</td><td>Fusion proteins</td><td>Fusion proteins that facilitate<br>cancer cell destruction</td><td>TYKOCINSKI Mark L</td><td>[289]</td></tr><tr><td>56</td><td>WO2015197874</td><td>2017.05.03</td><td>WO</td><td>Cancer</td><td>Fusion proteins</td><td>Combination of cd95/cd95I<br>inhibition and cancer<br>immunotherapy</td><td>KUNZ Claudia,<br>FRICKE Harald,<br>HÖGER Thomas,<br>GÄMIER Juergen</td><td>[266, 396-398]</td></tr><tr><td>57</td><td>US20150297745/<br>EP2897642/<br>WO2014045022</td><td>2015.10.22/<br>2015.07.23/<br>2014.03.27</td><td>WO</td><td>Cancer</td><td>Fusion proteins</td><td>Agents and methods</td><td>COBBOLD Mark,<br>MILLAR David</td><td>[399]</td></tr><tr><td>58</td><td>WO2012170072</td><td>2014.04.17</td><td>WO</td><td>Cancer</td><td>Fusion proteins</td><td>Engineered antibody-tnfsf<br>member ligand fusion<br>molecules</td><td>GREWAL Iqbal,<br>KHARE Sanjay D,<br>GRESSER Michael,<br>SYED Rashid</td><td></td></tr><tr><td>59</td><td>US20120177575/<br>EP2456468/<br>WO2011010156</td><td>2012.07.12/<br>2012.05.30/<br>2011.01.27</td><td>WO</td><td>Cancer</td><td>Fusion proteins</td><td>Fas (apo-1,cd95) targeted<br>platforms for intracellular drug<br>delivery</td><td>ATEH Davidson D,<br>MARTIN Joanne E</td><td>[400]</td></tr><tr><td>60</td><td>US20110008842</td><td>2011.01.13</td><td>US</td><td>Cancer</td><td>Fusion proteins</td><td>Chimeric nucleic acids<br>encoding polypeptides<br>comprising cd70 and fas-<br>ligand domains</td><td>PRUSSAK Charles E,<br>KIPPS Thomas J,<br>CANTWELL Mark J</td><td>[401]</td></tr><tr><td>61</td><td>EP3406630/<br>US20180186856/<br>WO2014013037</td><td>2018.11.28/<br>2018.02.22/<br>2014.01.23</td><td>WO</td><td>Cancer/<br>autoimmune<br>diseases/<br>inflammatory<br>diseases/others</td><td>Fusion proteins</td><td>Nucleic acids encoding<br>artificial signal peptides and<br>methods of production thereof</td><td>HILL Oliver,<br>GIEFFERS Christian,<br>THIEMANN Meinolf</td><td>[324, 325, 346, 348, 402]</td></tr><tr><td>62</td><td>WO2014013039</td><td>2014.01.23</td><td>WO</td><td>Cancer/<br>autoimmune<br>diseases/<br>inflammatory<br>diseases/others</td><td>Fusion proteins</td><td>Composition comprising a<br>mixture of cd95-fc isoforms</td><td>HILL Oliver,<br>GIEFFERS Christian,<br>THIEMANN Meinolf</td><td>[324, 325, 402]</td></tr><tr><td>63</td><td>WO2013060864</td><td>2014.09.03</td><td>WO</td><td>Cancer/<br>autoimmune<br>diseases/others</td><td>Fusion proteins</td><td>Chimeric molecule involving<br>oligomerized fasI<br>extracellular domain</td><td>TAUPIN Jean-Luc,<br>DABURON Sophie,<br>MOREAU Jean-François,<br>CAPONE Myriam</td><td>[292, 293]</td></tr><tr><td>64</td><td>WO2008025516</td><td>2011.02.03</td><td>WO</td><td>Cancer/<br>inflammatory<br>diseases/others</td><td>Fusion proteins</td><td>Cd95I or trail fusion proteins</td><td>HILL Oliver,<br>GIEFFERS Christian,<br>THIEMANN Meinolf</td><td>[324, 325, 402]</td></tr><tr><td>65</td><td>US20190016782/<br>WO2014106839</td><td>2019.01.17/<br>2014.07.10</td><td>WO</td><td>Cancer/others</td><td>Fusion proteins</td><td>Stable form of signal<br>converting protein fusion<br>proteins, and methods of use<br>and preparation thereof</td><td>DRANITZKI<br>ELHALEL Michal,<br>SHANI Noam</td><td>[266]</td></tr><tr><td>66</td><td>EP2042509/<br>US20070154905/<br>WO1997003998</td><td>2009.04.01/<br>2007.07.05/<br>1997.02.06</td><td>WO</td><td>Cancer/others</td><td>Fusion proteins</td><td>Modulators of the function of<br>fas receptors and other<br>proteins</td><td>WALLACH David,<br>BOLDIN Mark,<br>GONCHAROV Tanya,<br>GOLSTEV Yury V</td><td>[403]</td></tr><tr><td>67</td><td>WO2007022273</td><td>2007.02.22</td><td>WO</td><td>Cancer/others</td><td>Fusion proteins</td><td>Vegf-activated fas ligands</td><td>QUINN Timothy P</td><td>[404, 405]</td></tr><tr><td>68</td><td>US20040147447/<br>WO1999066039</td><td>2004.07.29/<br>1999.12.23</td><td>WO</td><td>Cancer/others</td><td>Fusion proteins</td><td>Tnfr-like protein with<br>death domain</td><td>LU Jian J,<br>GOMES Bruce C,<br>FIELES William E</td><td></td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>69</td><td>US20110171212</td><td>2011.07.14</td><td>US</td><td>Inflammatory diseases</td><td>Fusion proteins</td><td>Methods and compositions for preventing radiation-induced pneumonitis</td><td>BELKA Claus HERBST Jörg</td><td>[406]</td></tr><tr><td>70</td><td>US20040176279/ WO2002060949</td><td>2002.08.08</td><td>WO</td><td>Inflammatory diseases/others</td><td>Fusion proteins</td><td>Glycoforms a fas-ligand inhibitory protein analog</td><td>JENKINS Nigel, WITCHER Derrick R WROBLEWSKI Victor J</td><td>[407]</td></tr><tr><td>71</td><td>US20160340409/ EP2621514/ WO2012042480</td><td>2016.11.24/ 2013.08.07/ 2012.04.05</td><td>WO</td><td>Others</td><td>Fusion proteins</td><td>Compositions and methods for treatment of hematological malignancies</td><td>DRANITZKI ELHALEL Michal</td><td>[266]</td></tr><tr><td>72</td><td>US20040018170</td><td>2004.01.29</td><td>US</td><td>Others</td><td>Fusion proteins</td><td>Fas ligand-avidin/streptavidin fusion proteins</td><td>SHIRWAN Haval</td><td>[382, 393, 394]</td></tr><tr><td>73</td><td>EP1097226/ WO2000003023</td><td>2001.05.09/ 2000.01.20</td><td>WO</td><td>Cancer/ autoimmune diseases/others</td><td>Fusion proteins</td><td>Usurpin, a mammalian ded-caspase homologue that precludes caspase-8 recruitment and activation by the cd95 (fas, apo-1) receptor complex</td><td>NICHOLSON Donald, W, RASPER Dita M, XANTHOUDAKIS Steve, ROY Sophie</td><td>[408]</td></tr><tr><td>74</td><td>EP3337509/ US20170051352</td><td>2018.06.27/ 2017.02.23</td><td>US/EP</td><td>Autoimmune diseases</td><td>Method</td><td>Methods of treating autoimmune conditions in patients with genetic variations in dcr3 or in a dcr3 network gene</td><td>HAKONARSON Hakon, KAO Charly, CARDINALE Christopher et al.</td><td>[409]</td></tr><tr><td>75</td><td>US20160194714</td><td>2016.07.07</td><td>US</td><td>Autoimmune diseases</td><td>Method</td><td>Biomarkers for predicting relapse in multiple sclerosis</td><td>RUS Horea TEGLA Cosmin</td><td>[306–309]</td></tr><tr><td>76</td><td>US20160208332</td><td>2016.04.07</td><td>US</td><td>Autoimmune diseases</td><td>Method</td><td>Diagnosis and prognosis of multiple sclerosis</td><td>RUS Horea CUDRICI Cornelia TEGLA Cosmin</td><td>[306–309]</td></tr><tr><td>77</td><td>US20100285600/ EP1891233/ WO2006116602</td><td>2010.11.11/ 2008.02.27/ 2006.11.02</td><td>WO</td><td>Autoimmune diseases</td><td>Method</td><td>Markers associated with the therapeutic efficacy of glatiramer acetate</td><td>LANCET Doron BECKMANN Jacques AVIDAN Nili et al.</td><td>[410]</td></tr><tr><td>78</td><td>US20090074870/ WO2002002751</td><td>2009.03.19/ 2002.01.10</td><td>WO</td><td>Autoimmune diseases/others</td><td>Method</td><td>Alteration of cell membrane with fasl</td><td>SHIRWAN Haval</td><td>[411]</td></tr><tr><td>79</td><td>WO2015107105</td><td>2016.11.23</td><td>WO</td><td>Cancer</td><td>Method</td><td>Method of predicting the responsiveness of a cancer disease to treatment on the basis of dna methylation</td><td>FRICKE Harald</td><td>[297–299]</td></tr><tr><td>80</td><td>WO2015104284</td><td>2016.11.16</td><td>WO</td><td>Cancer</td><td>Method</td><td>Methods and pharmaceutical compositions for preventing or reducing metastatic dissemination</td><td>LEGEMBRE Patrick, SEGUI Bruno, LEVADE Thierry, MICHEAU Olivier</td><td>[296, 412]</td></tr><tr><td>81</td><td>WO2014118317</td><td>2015.12.31</td><td>WO</td><td>Cancer</td><td>Method</td><td>Methods for predicting and preventing metastasis in triple-negative breast cancers</td><td>LEGEMBRE Patrick, MALLETER Marine, TAUZIN Sébastien et al.</td><td>[141]</td></tr><tr><td>82</td><td>US20150098924/ WO2006037762</td><td>2015.04.09/ 2006.04.13</td><td>WO</td><td>Cancer</td><td>Method</td><td>Method for ex-vivo purging in autologous transplantation</td><td>DUPUIS Marc, GREANEY Peter, DUCHOSAL Michel</td><td>[413]</td></tr><tr><td>83</td><td>EP1668360/ US20050069963</td><td>2006.06.14/ 2005.03.31</td><td>US/EP</td><td>Cancer</td><td>Method</td><td>Multifactorial assay for cancer detection</td><td>LOKSHIN Anna E. GORELIK Elieser</td><td>[414]</td></tr><tr><td>84</td><td>US20050158807/ WO2003056340</td><td>2005.07.21/ 2003.07.10</td><td>WO</td><td>Cancer</td><td>Method</td><td>Fadd proteins, phosphorylated p38-mapk and fasl as tumor markers</td><td>CHIOCCHIA Gilles, TOURNEUR Lea, FEUNTEUN Jean et al.</td><td>[415]</td></tr><tr><td>85</td><td>WO2005053739</td><td>2005.06.16</td><td>WO</td><td>Cancer</td><td>Method</td><td>Combination therapy</td><td>JOHNSTON Patrick G, LONGLEY Daniel</td><td>[416]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>86</td><td>EP1127075/<br/>WO2000027883</td><td>2001.08.29/<br/>2000.05.18</td><td>WO</td><td>Cancer</td><td>Method</td><td>A method of treating tumors using fas-induced apoptosis</td><td>DONG Jian-Yun<br/>NORRIS James S</td><td>[417]</td></tr><tr><td>87</td><td>WO2001048238</td><td>2001.07.05</td><td>WO</td><td>Cancer</td><td>Method</td><td>Chemotherapeutant screening method</td><td>KRAMMER Peter,<br/>EICHHORST Sören,<br/>LI-WEBER Min,<br/>MÜLLER-SCHILLING<br/>Martina</td><td>[418]</td></tr><tr><td>88</td><td>WO1999003998</td><td>1999.01.28</td><td>WO</td><td>Cancer</td><td>Method</td><td>Methods and compositions for tumor reduction</td><td>NABEL Gary J</td><td>[419]</td></tr><tr><td>89</td><td>US6153385/<br/>WO1997020067</td><td>2000.11.28/<br/>1997.06.05</td><td>WO</td><td>Cancer/<br/>autoimmune diseases/others</td><td>Method</td><td>Process for detecting the expression of cd95 ligand in cells</td><td>DEBATIN Klaus-Michael<br/>HERR Ingrid</td><td>[420]</td></tr><tr><td>90</td><td>EP0876503/<br/>WO1997020064</td><td>1998.11.11/<br/>1997.06.05</td><td>WO</td><td>Cancer/<br/>autoimmune diseases/others</td><td>Method</td><td>Process for assessing the activity of drugs</td><td>DEBATIN Klaus-Michael,<br/>FRIESEN Claudia,<br/>KRAMMER Peter,<br/>HERR Ingrid</td><td>[420]</td></tr><tr><td>91</td><td>EP0689600/<br/>WO1994020625</td><td>1996.01.03/<br/>1994.09.15</td><td>WO</td><td>Cancer/others</td><td>Method</td><td>Process to induce the death of tumor cells</td><td>WONG Grace H W</td><td>[421]</td></tr><tr><td>92</td><td>WO1999003999</td><td>1999.01.28</td><td>WO</td><td>Inflammatory diseases</td><td>Method</td><td>Methods and compositions for inhibiting the pro-inflammatory response</td><td>NABEL Gary J,<br/>CHEN Jian-Jun</td><td>[422]</td></tr><tr><td>93</td><td>US20020127233</td><td>2002.09.12</td><td>US</td><td>Inflammatory diseases/cancer/ others</td><td>Method</td><td>Method for inhibiting inflammation in immune privileged sites using fas-ligand fragments</td><td>ZHU Bing,<br/>CYNADER Max S,<br/>PATY Donald W,<br/>LUO Liqing</td><td>[423]</td></tr><tr><td>94</td><td>US20180369380</td><td>2018.12.27</td><td>US</td><td>Others</td><td>Method</td><td>Methods and compositions for treating conditions of the eye</td><td>GRAGOUDAS Evangelos S,<br/>POULAKI Vasiliki,<br/>MILLER Joan W</td><td>[424]</td></tr><tr><td>95</td><td>US20140045198/<br/>EP2678688<br/>WO2012113760</td><td>2014.02.13/<br/>2014.01.01/<br/>2012.08.30</td><td>WO</td><td>Others</td><td>Method</td><td>Method of predicting the evolution of a patient suffering of a neurovascular disease</td><td>MONTANER<br/>VILLALONGA Joan,<br/>ROSELL NOVEI Anna<br/>NAVARRO SOBRINO Miriam</td><td>[357, 425]</td></tr><tr><td>96</td><td>US20110294690/<br/>EP2338058/<br/>WO2010031821</td><td>2011.12.01/<br/>2011.06.29/<br/>2010.03.25</td><td>WO</td><td>Others</td><td>Method</td><td>Differential diagnostic biomarkers of stroke mimicking conditions and methods of use thereof</td><td>MONTANER<br/>VILLALONGA Joan</td><td>[357, 425]</td></tr><tr><td>97</td><td>US20060241150/<br/>WO2005000405</td><td>2006.10.26/<br/>2005.01.06</td><td>WO</td><td>Others</td><td>Method</td><td>P38 kinase inhibitor compositions and methods of use</td><td>WEINER David B,<br/>MUTHUMANI Karuppiah</td><td>[426]</td></tr><tr><td>98</td><td>WO2006077232</td><td>2006.07.27</td><td>WO</td><td>Others</td><td>Method</td><td>Multimeric soluble fas-ligand for eliminating alloreactive t lymphocyte in allogenic harmatopoietic stem-cell transplantation</td><td>DUPUIS Marc,<br/>DEMOTZ Stéphane,<br/>GREANEY Peter et al.</td><td>[413, 427]</td></tr><tr><td>99</td><td>US20050129684</td><td>2005.06.16</td><td>US</td><td>Others</td><td>Method</td><td>Methods for preserving the viability of photoreceptor cells by anti-fas-ligand/anti-fas-receptor antibodies</td><td>ZACKS David,<br/>MILLER Joan W</td><td>[428]</td></tr><tr><td>100</td><td>US20030224403</td><td>2003.12.04</td><td>US</td><td>Others</td><td>Method</td><td>Lethal toxin cytopathogenicity and novel approaches to anthrax treatment</td><td>POPOV Serguei G,<br/>CARRON Edith G,<br/>CARDWELL Jennifer</td><td>[429]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>101</td><td>US6524821/<br/>WO2000007618</td><td>2003.02.25/<br/>2000.02.17</td><td>WO</td><td>Others</td><td>Method</td><td>Anti-apoptotic compositions comprising the r1 subunit of herpes simplex virus ribonucleotide reductase or its n-terminal portion; and uses thereof</td><td>LANGELIER Yves,<br/>MASSIE Bernard</td><td>[430]</td></tr><tr><td>102</td><td>US6485929/<br/>WO1999036091</td><td>2002.11.26/<br/>2000.07.22</td><td>WO</td><td>Others</td><td>Method</td><td>Method for inhibiting cd95-independent apoptosis in aids</td><td>KRAMME, Peter H,<br/>BERNDT Christina</td><td>[431]</td></tr><tr><td>103</td><td>WO2015189236</td><td>2015.12.17</td><td>WO</td><td>Cancer</td><td>Method</td><td>Methods and pharmaceutical compositions for reducing cd95-mediated cell motility</td><td>LEGEMBRE Patrick,<br/>COUNILLON Laurent,<br/>LAGADIC-GOSSMANN<br/>Dominique</td><td>[295]</td></tr><tr><td>104</td><td>US2003011865/<br/>WO2000059538</td><td>2002.06.06/<br/>2000.10.12</td><td>WO</td><td>Autoimmune<br/>diseases/others</td><td>Nucleotide complexe</td><td>Antigen-specific induction of peripheral immune tolerance</td><td>AUGUST Thomas J.<br/>LEONG Kam W.<br/>GEORGANTAS Robert</td><td>[432]</td></tr><tr><td>105</td><td>WO2001051503</td><td>2001.07.19</td><td>WO</td><td>Cancer</td><td>Nucleotide complexe</td><td>Polynucleotides for inhibiting metastasis and tumor cell growth</td><td>BARBERA-GUILLEM Emilio</td><td>[433]</td></tr><tr><td>106</td><td>US20020042064/<br/>EP1121438/<br/>WO2000023583</td><td>2002.04.11/<br/>2001.08.08/<br/>2000.04.27</td><td>WO</td><td>Cancer/<br/>autoimmune<br/>diseases/others</td><td>Nucleotide complexe</td><td>PS3 binding areas</td><td>KRAMMER, Peter,<br/>MÜLLER-SCHILLING<br/>Martina,<br/>OREN Moshe</td><td>[251]</td></tr><tr><td>107</td><td>US20040033979/<br/>EP1176965</td><td>2004.02.19/<br/>2002.02.06</td><td>US/EP</td><td>Cancer/<br/>autoimmune<br/>diseases/<br/>inflammatory<br/>diseases</td><td>Nucleotide complexe</td><td>Antisense modulation of fas mediated signaling</td><td>DEAN Nicholas M,<br/>MARCUSSON Eric G,<br/>WYATT Jacqueline,<br/>ZHANG Hong</td><td>[434]</td></tr><tr><td>108</td><td>US20030119776/<br/>EP1313853</td><td>2003.06.26/<br/>2003.05.28</td><td>US/EP</td><td>Cancer/<br/>autoimmune<br/>diseases/others</td><td>Nucleotide complexe</td><td>Modulation of fas and fasl expression</td><td>PHILLIPS Nigel C,<br/>FILION Mario C</td><td>[435]</td></tr><tr><td>109</td><td>US20070190607/<br/>WO2001058953</td><td>2007.08.16/<br/>2001.08.16</td><td>WO</td><td>Autoimmune<br/>diseases</td><td>Polypeptides</td><td>Inhibitors of pre-ligand assembly domain and function of the tumor necrosis factor receptor family</td><td>LENARDO Michael J,<br/>CHAN Francis Ka-Ming,<br/>SIEGEL Richard M</td><td>[36]</td></tr><tr><td>110</td><td>US7097972/<br/>WO1996025501</td><td>2006.08.29/<br/>1996.08.22</td><td>WO</td><td>Autoimmune<br/>diseases/<br/>inflammatory<br/>diseases/cancer</td><td>Polypeptides</td><td>Method and composition for regulating apoptosis</td><td>DIXIT, Vishva M</td><td>[436]</td></tr><tr><td>111</td><td>US20120245081</td><td>2012.09.27</td><td>US</td><td>Autoimmune<br/>diseases/<br/>inflammatory<br/>diseases/others</td><td>Polypeptides</td><td>Fas peptide mimetics and uses thereof</td><td>GREENE Mark I,<br/>MURALI Ramachandran,<br/>HASEGAWA Akihiro</td><td>[437]</td></tr><tr><td>112</td><td>US20070184522/<br/>EP1737483/<br/>WO2005117940</td><td>2007.08.09/<br/>2007.01.03/<br/>2005.12.15</td><td>WO</td><td>Autoimmune<br/>diseases/others</td><td>Polypeptides</td><td>Cell death modulation via antagonists of fasl and fas activation</td><td>ZARNEGAR Abdolreza,<br/>DEFRANCES Marie C,<br/>ZOU Chun-Bin</td><td>[438]</td></tr><tr><td>113</td><td>US6451759/<br/>WO1999036079</td><td>2002.09.17/<br/>1999.07.22</td><td>WO</td><td>Autoimmune<br/>diseases/others</td><td>Polypeptides</td><td>Noncleavable fas ligand</td><td>KANG, Sang-Mo<br/>BRAAT, Dries<br/>BAEKKESKOV, Steinunnn<br/>STOCK, Peter, G.</td><td>[439]</td></tr><tr><td>114</td><td>WO2020132465</td><td>2020.06.25</td><td>WO</td><td>Cancer</td><td>Polypeptides</td><td>Methods and compositions related to therapeutic peptides for cancer therapy</td><td>BECKER Lev,<br/>CUI Chang</td><td>[286]</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>N°</th><th>Publication Id</th><th>Publication date</th><th>Country</th><th>Disease</th><th>Approach</th><th>Title</th><th>Inventors</th><th>Ref.</th></tr></thead><tbody><tr><td>115</td><td>EP2102234/ WO2008067305</td><td>2009.09.23/ 2008.05.06</td><td>EP</td><td>Cancer</td><td>Polypeptides</td><td>Polypeptides comprising intracytoplasmic death domain and nkg2d ligand domain</td><td>WAGNER Thomas E, WEI,Yanzhang</td><td>[440]</td></tr><tr><td>116</td><td>US20190085050/ WO2015158810</td><td>2019.03.21/ 2015.10.22</td><td>WO</td><td>Cancer/ autoimmune diseases/ inflammatory diseases</td><td>Polypeptides</td><td>Polypeptides and uses thereof for reducing cd95-mediated cell motility</td><td>LEGEMBRE Patrick, VACHER Pierre, SANSEAU Doriane et al.</td><td>[146]</td></tr><tr><td>117</td><td>EP1225908/ WO2001028582</td><td>2002.07.31/ 2001.04.26</td><td>WO</td><td>Cancer/ inflammatory diseases/others</td><td>Polypeptides</td><td>Therapeutic applications of flint polypeptides</td><td>BUMOL Thomas F COHEN Fredric J</td><td>[441]</td></tr><tr><td>118</td><td>US20100041596/ WO2007002633</td><td>2010.02.18/ 2007.01.04</td><td>WO</td><td>Inflammatory diseases</td><td>Polypeptides</td><td>Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain (plad) of tumor necrosis factor receptors</td><td>LENARDO Michael, DENG Guo-Min, CHAN Francis Ka-Ming, ZHENG Lixen</td><td>[442]</td></tr><tr><td>119</td><td>US20100041596/ WO2009027350</td><td>2017.08.31/ 2009.03.05</td><td>WO</td><td>Inflammatory diseases/others</td><td>Polypeptides</td><td>Use of sco-spondin peptides for inhibiting or preventing neuronal apoptosis mediated by cell death receptor ligands</td><td>MEINIE, Annie, LALLOUE Fabrice, JAUBERTEAU Marie-Odile</td><td>[443]</td></tr><tr><td>120</td><td>US20130288979/ EP2982685/ WO2012066103</td><td>2017.11.14/ 2016.02.10/ 2012.05.24</td><td>WO</td><td>Others</td><td>Polypeptides</td><td>Inhibitors of apoptosis and uses thereof</td><td>BARRERE Stéphanie, NARGEOТ Joël, LEBLEU Bernard et al.</td><td>[443]</td></tr><tr><td>121</td><td>US20010018416</td><td>2001.08.30</td><td>US</td><td>Others</td><td>Polypeptides</td><td>Compositions and methods for treating hepatitis-c</td><td>SLESAREV Vladimir I, DIMITROV Todor</td><td>[444]</td></tr><tr><td>122</td><td>US20060089491</td><td>2004.06.07</td><td>US</td><td>Cancer</td><td>Polypeptides/nucleotide complexes</td><td>Fas-ligand derived polypeptides</td><td>NAGATA Shigekazu, SUDA Takashi, TAKAHASHI Tomohiro, NAKAMURA Norio</td><td>[445]</td></tr><tr><td>123</td><td>US20090169599</td><td>2009.07.02</td><td>US</td><td>Others</td><td>Reprogrammed virus</td><td>Scientifically modulated and reprogrammed treatment (smart) fas/fasI virus technology intended to neutralize t-helper cells infected with the human immunodeficiency virus</td><td>SCHEIBER Lane Bernard SCHEIBER II Lane Bernard</td><td>[283]</td></tr><tr><td>124</td><td>EP2355833/ US20100324116</td><td>2011.08.17/ 2010.12.23</td><td>US/EP</td><td>Cancer</td><td>RNA interfering molecules</td><td>Fas/fasI or other death receptor targeted methods and compositions for killing tumor cells</td><td>KRUSE Carol, TRITZ Richard</td><td>[446]</td></tr><tr><td>125</td><td>US20050119212</td><td>2004.06.18</td><td>US</td><td>Autoimmune diseases/others</td><td>RNA interfering molecules</td><td>Rna interference mediated inhibition of fas and fasI gene expression using short interfering nucleic acid (sina)</td><td>HAEBERLI Peter, MCSWIGGEN James</td><td>[447, 448]</td></tr><tr><td>126</td><td>US20070004666</td><td>2007.01.04</td><td>US</td><td>Cancer/ autoimmune diseases/others</td><td>Transcription factors</td><td>Methods for modulating apoptotic cell death</td><td>LASHAM Annette, WATSON James D</td><td>[447, 448]</td></tr><tr><td>127</td><td>WO1998008965</td><td>1998.03.05</td><td>WO</td><td>Autoimmune diseases/others</td><td>Transcription factors/ nucleotide complexes</td><td>Cd95 regulatory gene sequences and transcription factors</td><td>WATSON James, D, RUDERT Fritz</td><td>[447, 448]</td></tr></tbody></table>

The source of information used is the Patentscope of the WIPO IP Portal and the search was carried out by selecting only the patents published in the United States Patent Office and European Patent Office. Furthermore, the selection was made using keywords and selecting only the patents with these keywords in the title of the publication or the corresponding abstract on the front page. The keywords used are CD95, CD95L, CD95 ligand, Fas, FasL, Fas ligand.
absent together with its expression level (WO2017140826). In 2019, the soluble metalloprotease-cleaved CD95L, associated with a large number of immune infiltrate cells, has been identified as a possible biomarker for tumor immune infiltration (CD3<sup>pos</sup> and CD8<sup>pos</sup>, and also CD4 and FoxP3 T cells) in advanced HGSOC (High-Grade Serous Ovarian Cancer) [235]. These biomarkers can facilitate the identification of cancer patients prone to respond or resist to proposed immunotherapy and therefore to select an appropriate and personalized chemotherapy treatment. In the last 20 years, the strategies adopted in cancer immunotherapy can be classified into the two large families of active and passive immunotherapy.

## Active approaches

Active immunotherapy is based on the principle that the drug stimulates the patient's immune response against the tumor, thus acting indirectly. On the contrary, in the case of passive immunotherapy, the drug is directly capable of destroying the tumor cell. Among the active forms of immunotherapy recognized by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), should be mentioned the immunomodulatory mAbs, which mostly inhibit the immunosuppressive receptors expressed by activated T cells (e.g., Ipilimumab inhibiting CTLA-4 and Pembrolizumab inhibiting PD-1), the immunostimulatory cytokines, generally used as adjuvants of other anticancer immunotherapies (e.g., IL-2/ Proleukin + Ipilimumab), the immunogenic cell death (ICD) inducers, which exert their antitumoral effect through cytostatic and cytotoxic mechanisms.

**Immunomodulatory mAbs.** In some pathological conditions, the so-called immune system checkpoints act directly as a "brake" in the immune response against cancer. The role of immunomodulatory monoclonal antibodies mAbs is precisely to lift these inhibitions by "removing the brake" of the immune system. To date, the most common and most widely used are the inhibitors of checkpoint Cytotoxic T Lymphocyte-Associated Antigen-4 (CTLA-4), Programmed Death 1 (PD-1) and PD-L1 [236]. There are six drugs targeting PD-1 or PDL-1 and only one targeting CTLA-4 currently approved for use in therapy of different types of cancer. Recently, the combination of two inhibitory checkpoints (i.e., ipilimumab (anti-CTLA-4 and nivolumab (anti-PD-1) has also joined the list of approved drugs, showing good therapeutic efficacy in several studies and thus paving the way for new clinical trials in different types of cancers [237]. However, other immune checkpoints are targeted in preliminary stages of clinical development [238]. In recent years, bispecific antibodies have also been developed with the aim of targeting multiple checkpoints simultaneously (e.g., CTLA-4 and PD-1), thus amplifying the signal [239]. However, this double-targeting has so far showed a higher toxicity as compared to its corresponding single therapies. Some of these double-targeting systems will be described in later sections of this review.

**Immunostimulatory cytokines.** With a counterbalancing action, immunostimulatory cytokines act instead as "stimulators" of the immune response. Cytokines can promote the activation, proliferation and survival of lymphocytes (T, B, NK) so as to obtain an antitumor response. Interleukins, interferons and chemokines belong to this large family. The protagonists in the field of immuno-oncology are certainly interleukin-2 (IL-2), the first cytokine FDA approved for therapeutic purposes, IL-12, -15, -21, and interferon alpha (IFN-α), for a long time used for the treatment of hematological neoplasms, for renal carcinoma and melanoma [240]. Having a short half-life, the efficacy of these drugs is limited following their systemic administration. They also induce severe adverse effects before reaching therapeutic doses [241–243]. Today the new engineered generation of these cytokines is

making its way into the world of oncological immunotherapy, with improved half-life, antitumor efficacy and toxicity [244].

**Immunogenic cell death inducers.** One of the most widely used ICD agents is Doxorubicin (DOX), a drug discovered in the late 1960s that acts as a DNA intercalating agent and induces apoptosis. As above-mentioned, it has been observed by several groups that some tumor cell lines express CD95L on their surface [245–249] and more importantly, that DOX-induced apoptosis is mediated by the expression of CD95L with the consequent induction of cell death by binding to CD95 [250]. This observation introduced a new perspective on the use of the targeted CD95/CD95L system. In the following years, several other cytotoxic drugs showed the ability to up-regulate the expression of CD95L in cancer cells. In addition to doxorubicin, among the many, we mention Cisplatin, Etoposide, 5-Fluorouracile, Methotrexate, and Bleomycin [251–253]. A parallel mechanism by which these DNA-damaging chemotherapy agents lead to autocrine or paracrine apoptosis of the cell involves the activation of the p53 system, which once activated acts as a transcription factor that regulates the expression of pro-apoptotic genes such as PUMA and BAX [254, 255]. Several studies have been conducted on the implication of p53 in the regulation of dose-dependent heavy side effects, the first of which is cardiotoxicity [256, 257]. Other studies explored the combination of DOX with different drugs with the aim to reduce both acute and chronic DOX-induced cardiotoxicity without affecting its p53-mediated anticancer activity. These studies mention beta-blockers (e.g., Carvedilol), iron-chelating agents (e.g., Dexrazoxane, DEX), angiotensin-converting enzyme inhibitors -ACEI- (e.g., Zofenopril) or even Flavonoids (e.g Frederine), used in combination with DOX for the attenuation of cardiotoxicity [258–261]. Very recently, Todorova et al. carried out a study in which a new combination of DOX and Dantrolene is proposed. Dantrolene appears to mitigate the cardiotoxicity of DOX without affecting its antitumor action in a breast cancer model [262]. Also in 2020, a team from China has developed a new method of co-administration of the DOX, according to which by pre-treating the triple-negative breast cancer cells MDA-MB-231 with Quercetin, followed by the DOX, it is possible to hinder the multidrug resistance of this aggressive cell line [263]. Taken together, these observations are promising for future development from a clinical point of view.

**Cancer vaccines.** The concept of cancer vaccines was first introduced in the 1990s when Bacillus Calmette-Guerin was approved by the FDA for the treatment of early-stage bladder cancer. To date, only three cancer vaccines have been approved by the FDA, due to the poor results often obtained in phases III and IV of the trials [264]. The cancer vaccine approach does not aim to prevent the cancer onset but to activate the immune system against cancer cells. The patent WO2015197874, published by a German team in 2017, proposes a combination of inhibition of the CD95/CD95L complex and cancer immunotherapy, such as a cancer vaccine [265–268]. As previously mentioned, CD95L expression on the tumor endothelium promotes an immunosuppressive environment through preferential killing of tumor-reactive CD8<sup>pos</sup> cells. Thus, the cancer vaccine would try to get the immune system to mount an attack against cancer cells by using a simultaneous inhibition of the CD95L/CD95 signaling system. More specifically, this cancer vaccine would contain cancer antigens in the form of a protein, a fragment thereof, or as RNA or DNA encoding that protein, to stimulate the immune system against this antigen.

## Passive approaches

**Monoclonal antibodies.** Passive forms of immunotherapy include mAbs that specifically target the receptors on the surface of neoplastic cells expressing "tumor-associated antigens" (TAA), by
altering their functions. Some of these antibodies can be
administered in combination with chemotherapeutic agents so
that the antibodies deliver these agents specifically to cancer cells.
An example of such a system is represented by the combination of
anti-CD95 antibodies with a chemotherapeutic agent such as
5-Fluorouracil or Tomudex (WO2003097698) [269, 355]. The goal is
to synergize the pro-apoptotic effect of anti-CD95 mAbs with
cancer chemotherapeutic agents to kill cancer cells. A different
combination approach consists in genetically fusing a full-length
monoclonal antibody targeting the cancer cell, such as rituximab,
with a more biological component represented by a TNF
superfamily ligand, such as CD95L, in its full-length, or truncated
form, or a fragment thereof, thus offering two approaches to kill
cancer cells. The antibody-TNFSF ligand fusion molecules would
combine the specificity of the antibodies to the target antigen
with the potent death-inducing properties of the TNFSF member
ligand, thus providing improved efficacy and safety. The two
combined killing approaches are thus performed through ADCC-
independent apoptosis (Ab-dependent cellular cytotoxicity), and
the second through the recruitment of effector cells to kill tumor
targets (WO2012170072) [270]. Another technique involves the
use of bispecific antibodies, the method of which consists of
linking an antibody that reacts with the tumor cell to a second
antibody that reacts with a cytotoxic effector cell. This is the case
of patent WO2014076292 published by Baliopharm AG concern-
ing a bispecific antibody with a first binding site for the CD95
receptor and a second binding site for the CD20 antigen
[271, 272, 351]. This strategy aims to improve the treatment
carried out with rituximab, an antibody able to target and kill
CD20 expressing malignant and normal B cells unspecifically, thus
showing significant side effects. This technique brings the effector
cell into close opposition to the tumor cell, producing increased
tumoricidal activity. Overall, the results of preclinical tests
performed on antibody systems referring to CD95 have been
encouraging, but to date, none of these CD95-related molecules
are currently in clinical trials.

ACT adoptive cell transfer. Adoptive cell transfer is another very
promising type of passive immunotherapy, which involves the re-
introduction of specific effector cells into the patient bloodstream
[273, 274]. Among them, Lymphokine Activated Killer Cells (LAK),
Tumor-infiltrating lymphocytes (TILs) and Chimeric Antigen
Receptors (CAR)-T cells are to be mentioned. Lymphokine
Activated Killer Cells are obtained from the patient's endogenous
T cells, which are extracted, cultured in the presence of the
lymphokine interleukin-2 (IL-2) and reinfused into the patient's
blood [275]. Tumor-infiltrating lymphocytes may have greater
tumoricidal activity than LAKs, as they are isolated from resected
tumor tissue, thus originating cells with greater tumor specificity
than those obtained from blood [276]. An interesting method has
recently been published by Iovance Biotherapeutics, Inc., con-
cerning the expansion of TILs from tumor cells using, among
others, CD95 agonists, for the treatment of diseases such as cancer
(WO2018129332) [277, 278, 366]. A more recent strategy has been
developed on the idea of genetically modifying T cells to express
TAA-specific T-Cell Receptors, or Chimeric Antigen Receptors that
recognize specific proteins on the cancer cells surface. Lately, it
has been shown that CAR-T cells up-regulate the expression of
CD95 and CD95L resulting in activation of the cell death program
independently of TCR or CAR antigen-mediated activation [279].
The work of the Donda's team highlights the importance of the
role of the CD95/CD95L system in CAR-T cells-induced apoptosis
by demonstrating the rescue of CAR-T cells upon in vivo blockade
of this death-signaling pathway by CD95-Fc recombinant proteins.
Patent US20180008670, published in 2018 in the U.S., concerns a
method using CAR-T cells to stimulate immunity towards tumor
endothelial cells. It is known that one of the limitations of CAR-T
cells includes the lack of ability for the T cells to infiltrate deep into

tumor tissue. In this formulation CAR-T cells would be able to
destroy the CD95L-positive tumor endothelial cells, but also
survive in their presence [280]. A year later an American group
made the observation that CD95 is highly expressed on patient-
derived T cells used for clinical ACT (adoptive cell transfer). They
elaborated a T-cell co-engineered system including CD95 DNR
(Dominant Negative Receptor) and either a T-cell receptor or
Chimeric Antigen Receptor. These cells were genetically modified
to express a defective CD95 variant, impairing the induction of
apoptotic signal, together with a Chimeric Antigen Receptor,
resulting in superior antitumor efficacy, greater longevity and no
observed autoimmunity [281]. An interesting observation was
recently made by Joshua D Brody's team, who found that the
CD95/CD95L system, in addition to its antigen-specific T-cell killing
capability, mediates off-target "bystander" killing of antigen-
negative tumor cells. They propose that CD95-mediated bystander
elimination of Ag-loss variants may already be occurring in CAR-T
treated patients. This process appears to be induced by CD95
upregulation on tumor cells after exposure to T-cell-secreted IFN-
γ. They developed a CAR-T mouse model showing an improve-
ment in tumor clearance when CD95 signaling is intact [282].
Overall, these observations open the door to promising new
therapeutic opportunities exploiting the CD95/CD95L system in
the cancer immunotherapy context

THERAPEUTIC PERSPECTIVES IN CANCER

It is well described that CD95 can promote pro-apoptotic and anti-apoptotic activities according to the physiological context [90]. Some previous studies have shown that down-regulating CD95 via shRNA in cancerous cells activates a death program by the induction of DNA damage and the activation of apoptotic effectors. One of these studies has been carried out and exposed in the US20100324116 patent, in which the inventors set out a siRNA-agent with the aim to reduce the amount of RNA encoding a CD95/CD95L gating polypeptides (e.g., FAPP2 or PATZ1 polypeptides) in significant quantities to sensitize brain tumor cells to CD95-mediated apoptosis [283, 284]. Over the past 25 years, several different potential therapeutic strategies related to the CD95/CD95L interaction have been studied. Among them, polypeptide systems, fusion proteins, methods, chemicals, antibodies and drug delivery systems are probably the most extensively studied.

Antitumoral polypeptides

Few patents describing CD95-related polypeptides have been
published to provide a different approach in cancer treatment. In
2015 it was observed that blood polymorphonuclear neutrophils
(PMNs) could kill cancer cells with a mechanism that remains to be
elucidated [285]. Last year, a new method for reducing the toxicity of
anticancer treatment on normal or non-cancerous cells has been
registered as a patent at the University of Chicago (WO2020132465).
This invention showed that ELANE, identified as the major
anticancer protein released by PMNs, could cleave the CD95
receptor, releasing an intracellular proteolytic fragment containing
the Death Domain and selectively killing a wide range of cancer cells
[286]. The invention is a combination of specific CD95 peptides and
the DNA encoding these peptides to treat different types of cancer.
As previously mentioned, metalloproteases-cleaved CD95L (sCD95L)
can exert a pro-oncogenic activity, through its interaction with CD95,
promoting the survival and proliferation of cancer cells, but also
their dissemination [141]. Therefore, a group proposed
(WO2015158810) the use of polypeptides composed of the amino
acid sequence encompassing the intracellular domain of CD95
which they previously identified as inducing a calcium-dependent
cell motility process in T lymphocytes [145]. These inventors
reported that the use of such peptides prevented the activation of
PLCy1 and the consequent calcium response that leads to cell
migration. The same group reported a few years later that five
molecules selected from the FDA/EMA-approved chemical library,
namely Ritonavir, Diflunisal, Anethole, Rosiglitazone and Daunorubi-
cin, could all block the recruitment of PLCγ1 to CD95 and reduce T
lymphocyte motility (WO2018130679). Less recently, Wagner and
Wei published a method related to the use of a combination of
polypeptides and their encoding polynucleotides (WO2008067305)
[287, 288]. They proposed a polypeptide composed of a ligand
domain for a stimulatory Natural Killer receptor (e.g., the extracellular
domain of MULT-I, which binds the NK cells receptor NKG2D) and the
CD95 intracytoplasmic death domain. This method is supposed to
activate the NK cells through the NKG2D receptor after contact with
the tumor cells expressing the polypeptidic fusion compound so that
not only the engaged tumor cells will be killed via CD95 induced-
mechanisms but also are lysed directly by the activated NK cells.

CD95-related chimeric proteins

Another similar therapeutic approach related to the CD95/CD95L system for the treatment of cancer is the use of fusion proteins or chimeric proteins. Since the years 2000s, the fusion protein system has been perhaps the most widely studied. Patent WO2014121093 should be mentioned among the most recent of them [289]. Here, the inventors elaborated a chimeric system composed of a component capable of inducing the CD95-mediated apoptotic signal, and a component capable of blocking the CD47 receptor expressed at the tumor cell membrane and involved in the suppression of macrophage phagocytosis of the tumor cells.

The approach concerning fusion proteins that provide a
physiologically similar oligomerized form of CD95L was studied by
two different groups. One exposed a bi-component protein
comprising the CTLA-4 extracellular domain and the CD95L
extracellular domain, present in the form of a covalently bound
and stable homo-hexamer, suitable for the treatment of a patient
with cancer (WO2014106839) [290, 291]. If said patient has a tumor
expressing the B7 receptor (e.g., B-cell lymphoma), this compound
should be administered to exploit the double affinity of the bi-
protein for the B7 and CD95 receptors, and finally inducing apoptosis
of the malignant cells. The second group instead describes a
chimeric protein composed of the extracellular domain of CD95L and
a domain capable of inducing the oligomerization in this chimeric
system (WO2013060864) [292–294]. Said domain is represented by
the Ig-like domain of the Leukemia Inhibitory Factor (LIF) receptor
gp190, which self-associates in the context of the chimeric protein
giving rise to a dodecameric form with cytotoxic activity towards the
cells expressing CD95. This system could therefore have various
applications in the clinical field for the treatment of various diseases,
such as cancer, autoimmune diseases and others.

Innovative antitumoral methods

The innovative methods approached in the context of the
treatment of cancer patients are numerous and varied, among
the most recent of which are the patents WO2015189236,
WO2015104284, and WO2014118317, all filed and published by
the same group. The first concerns a method aimed to reduce
CD95-induced cell migration (WO2015189236). NHE1 is a NA⁺/H⁺
exchanger channel which this group reported to be indispensable
for the CD95-induced cell motility process in fibroblasts [295]. This
invention provides pharmaceutical compositions of compounds
with NHE1 inhibiting properties to be administered if the subject
shows elevated blood levels of sCD95L. This group also reported
that in triple-negative breast cancer (TNBC), the serum level of
CD95L could constitute an important parameter for the prognosis
of the survival time and/or the relapse-free survival time. The same
group therefore patented the invention WO2015104284, which
aims to first determine the expression of sCD95L in the serum of
subjects with triple-negative breast cancer (TNBC) and then to
compare this level of expression to a predetermined standard
value. The concluding step involves the administration to said

subject of an effective therapeutic dose of plasma membranes
structural components. The goal is to reduce the fluidity of the
plasma membrane, a factor that this group reported as involved in
the induction of cell migration by CD95 [296]. Subsequently, this
same research group developed another patent, this time
concerning the prediction and prevention of metastases in TNBC
(WO2014118317). The authors describe a method for identifying
serum levels of sCD95L in TNBC patients, stating that these
patients develop a high risk of relapse if the level of sCD95L is
significantly higher than a standard expression level [141]. In the
same couple of years, another inventor published a method for
predicting the sensitivity of tumor cells for a given treatment
targeting inhibition of the CD95/CD95L system (WO2015107105)
[297-299]. The invention more specifically concerns the analysis of
the methylation levels of a DNA sequence of a gene belonging to
this apoptotic signaling cascade obtained directly from a subject
suffering from cancer, and consequent observation on the
possible responsiveness of said cancer cells to a specific
treatment. DNA and histone modifications remain the two major
mechanisms of epigenetic regulation of gene expression [300].
Some inhibitors of these mechanisms, such as Decitabine and
Vorinostat, are currently in clinical use to inhibit DNA methylation
and histone acetylation respectively. An equally important role in
the regulation of gene expression is played by the methylation of
histone lysine residues through the action of Histone Methyl-
transferase (HMTase), for which to date only two chemical
inhibitors (Verticillin A and Chaetocin) have been generated and
found to be toxic in vivo. The Augusta University Research
Institute, Inc. has developed a new inhibitor for HMTase SUV39H1
that appears to be useful in activating certain cytotoxic T-cell
effectors, such as CD95L, thereby reversing cancer-induced
immune suppression and promoting the killing of cancer cells
by cytotoxic T cells (US20190084987) [301].

CURRENTLY USED THERAPIES AND THERAPEUTIC PERSPECTIVES IN AUTOIMMUNE DISEASES

Despite our growing knowledge of the immunological abnorm-
alities that can lead to autoimmunity, the etiologies of most
human autoimmune diseases remain unclear. This is probably
because human autoimmune diseases are generally heteroge-
neous and multifactorial, not only between different diseases but
also within the same disease [302]. They can, within a single
disease, present a wide variety of clinical manifestations and
severity, for instance the propagation speed, the number of
affected joints, as well as a vast phenotypic heterogeneity.
Besides, autoimmune diseases can clinically manifest long after
the autoimmune reactions have been induced. Autoimmune
diseases are often characterized by a severe imbalance between
pro and anti-inflammatory mechanisms and by a vast diversity of
signaling pathways and of cells and cytokines such as interleukins,
interferons, and Treg cells that play a crucial role in immune
tolerance. In recent decades, enormous progress has been made
to identify the mechanisms associated with the activation and
inactivation of T cells and to improve techniques based on the
study of selective immune suppression in human autoimmune
diseases. To date, the techniques used to counteract the
mechanisms of autoimmunity are varied and include different
peptide analogs, immunosuppressants, anti-inflammatories,
monoclonal antibodies, inducers of immune tolerance, therapies
targeting certain autoantigens, often used in conjunction with
immunosuppressants to reduce their doses. Several groups
around the world have carried out studies for which patents have
been filed.

Multiple sclerosis

MS is a chronic autoimmune neurodegenerative disease that
affects the central nervous system (CNS). It is characterized by an
abnormal reaction of the immune defenses towards certain
components of the CNS, damaging myelin and oligodendrocytes
[303]. The symptoms are varied but CNS defective functions are
frequent, with recurrent remissions and exacerbations. MS is
suspected in patients with optic neuritis, especially if the deficits
are multifocal or intermittent. In such cases, magnetic resonance
imaging (MRI) scans of the brain and spinal cord and cerebrospinal
fluid (CSF) analyses are performed, as this techniques allow to
exclude other treatable pathologies that can mimic MS [304]. At
the moment there is no definitive cure, but numerous therapies
are available to modify its course, slowing its progression. The
most severe form of MS is undoubtedly represented by relapsing-
remitting multiple sclerosis (RRMS). Subjects with RRMS tend to
have more brain lesions with widely varying localization and very
different symptoms [305]. To date, the diagnosis to confirm the
presence of the disease is given by tests resulting positive at least
on two areas of myelin lesions in the CNS. These tests are not only
painful but also risky and highly expensive. It is, therefore,
necessary to develop additional methods for the diagnosis of this
disease. The inventors of US20160194714 offer a new method for
detecting relapse in RRMS patients using biomarkers, such as
CD95L, sirtuin 1 (SIRT1), RGC-32 and IL-21, in a population of cells
(e.g., PBMCs, CD4<sup>pos</sup>, CD8<sup>pos</sup>, glial cells, neurons, etc.) [306–309].
They noted a decrease in CD95L, SIRT1, and RGC-32 in relapsing
RRMS patients, while an increase in IL-21 occurs. Overall, these
four proteins can be used as markers to highlight the activity of
this disease.

Systemic lupus erythematosus

The involvement of CD95L has been extensively studied in
different chronic inflammatory autoimmune diseases, such as MS,
SLE and RA. Several groups have observed differences in the
frequency of the T-helper cells (Th) subgroups in SLE patients
versus HCs (Healthy Controls), which also differ in their sensitivity
to TCR-mediated cell death [310–313]. This could explain the
discordant results on CD95L expression levels in total lymphocytes
from healthy donors and patients with chronic inflammatory
disease. A few years ago, it was noted that transcription of CD95L
is a crucial step for the regulation of T-helper cell death sensitivity.
This group found that human Th1 cells express higher mRNA
levels of CD95L than Th17 cells. Resistance of Th17 cells to AICD
was associated with lower expression of CD95L and overexpres-
sion of the anti-apoptotic caspase-8 inhibitory protein (FLIP) [314].
In the mid-2000s, an important role was attributed to these IL-17A
and IL-17F producing lymphocytes in the context of autoimmune
diseases. Th17 cells orchestrate autoimmune inflammation, in
addition to their function as eliminators of extracellular pathogens
[315–317]. Yang et al. observed that SLE patients exhibit
significant infiltration of Th17 lymphocytes secreting cytokines
in their skin [318]. It is therefore possible to hypothesize that by
modulating their trafficking to the organs, the pathogenesis of the
SLE disease could consequently be modulated. In the context of
this chronic inflammatory disease, soluble CD95L (sCD95L) has
been shown to be involved in promoting the trafficking of Th17
lymphocytes into damaged organs, at the expense of Treg
lymphocytes in a CD95-driven murine model of SLE [142]. Blocking
the CD95/CD95L system could thus represent an attractive
approach for the treatment of Th17 cell-mediated diseases. This
was the intent of the authors of the WO2016170027 patent, who
proposed to use CD95 antagonist antibodies, having specificity for
CD95 or sCD95L, with the potential to prevent the endothelial
transmigration of Th17 cells in the organs and the consequent
damage given by the accumulation of the activated T cells in said
organs [142]. DR-mediated cell death is essential for the
differentiation, growth and function of lymphocytes. In 2017,
Croft and Siegel discussed the implication of some of these
receptors in inducing inflammation and their potential in future
therapies for rheumatoid diseases [319]. Interestingly, the

combined blockade of TNFR1, TRAIL-R and CD95 seems to give
excellent results in the prevention of inflammation caused by the
respective ligands, whereas targeting these receptors individually
did not have that effect (WO2019141862) as demonstrated in a
murine model of dermatitis [320]. Such observations lead to the
conclusion that different cell DR systems may act in combination
to contribute to the pathogenesis of autoimmune inflammatory
diseases. Importantly, uncontrolled induction of cell death down-
stream of DR, rather than increased DR-induced gene-activatory
signaling pathways, could actually be key in driving inflammation
in such contexts [320–322]. Interestingly, the Decoy Receptor 3
(DcR3), encoded by the *TNFRSF6B* gene, was found to act as a
regulator of the amplification of the immune response by binding
with stimulatory cytokines, such as CD95L, TL1A and LIGHT,
limiting the interaction of the latter with their own receptor [323].
It, therefore, seems deductible that genetic modifications of the
*TNFRSF6B* gene, involving a reduced expression of DcR3, or a
lower binding activity for the aforementioned cytokine, or even
the suppression of its expression, could contribute to cause
inflammatory signals. With this idea in view, the inventors of
US20170051352 have developed a method for treating auto-
immune conditions in patients carrying alterations of the gene
encoding the DcR3 protein, or of a DcR3 network gene, by
administering to said patient an effective amount of DcR3 ligands
inhibitors [323].

Fusion proteins in the context of autoimmune diseases

As in the context of cancer, one of the widely adopted strategies
in studying new potential treatments for autoimmune diseases is
represented by the use of fusion proteins and nucleotides that
encode them. In 2018, APOGENIX AG published a patent relating
to a nucleotide sequence encoding an isolated chimeric
compound formed by the extracellular domain of CD95 and an
immunoglobulin domain or a functional fragment thereof. The
inventors intend to generate a stable system to inhibit the
extrinsic apoptotic signal initiated by CD95L for the prophylaxis or
treatment of various diseases, including autoimmune diseases and
solid cancers (US20180186856) [324, 325, 402]. A few years earlier
the same inventors developed a mixture of fusion protein
isoforms having the same composition as the aforementioned
system with the difference that this patent does not mention any
nucleotide sequence encoding the chimeric protein, as well as the
cell hosting the nucleotide sequence (WO2014013039). A different
chimeric system is represented by the invention WO2016205714,
which exposes an immune tolerance inducer "medicament"
comprising a CD95L moiety together with a streptavidin or avidin
moiety [326–328]. The claimed compound is to be administered
alone or mixed with the IL-2 protein to achieve sequential or
simultaneous action in inducing long-term and specific immuno-
suppression. CD95L is then part of another fusion compound, the
one described by the patent WO2014121085, in which the
extracellular domain of CD95L corresponds to half of the fusion
protein. The other half is the extracellular domain of a PD-1
receptor-activating factor, such as its ligand PD-L1 and PD-L2
[329]. This system aims to inhibit the differentiation and
proliferation of a selection of cells, including activated T cells on
which the PD-1 receptor is widely expressed, thus the induction of
PD-1 ligation by its ligands mediates an inhibitory signal that
results in reduced cytokine production and reduced T-cell survival.
Thus, in the setting of autoimmune and inflammatory diseases,
the fusion protein of this invention could reduce autoimmune and
inflammatory manifestations.

Cells engineering modifications

Some other groups have then explored the field of cells
engineering modification by proposing methods of isolating
these cells from a patient sample, treating/modifying these cells
and reintroducing the said modified cells by systemic infusion or
transplantation. This is the case of the patent WO2013149211, which describes a method using modified mesenchymal stem cells (MSCs) to overexpress CD95, CD95L as well as the CD95-regulated monocyte chemotactic protein 1 (MCP-1) which seems to play an important role in the recruitment of T cells to MSCs [330, 331]. It has previously been hypothesized that such MSCs play an important role in reducing T-cell proliferation through a mechanism involving T-cell apoptosis [332]. Therefore, this invention offers a potential therapeutic method for the treatment of autoimmune diseases, and more specifically of Systemic Sclerosis. Similarly, patent WO2015038665 relates to a system composed of modified MSCs to overexpress CD95L after exposure of these cells to a salicylate, such as common aspirin [333]. The authors offer a method aimed at increasing survival rates in patients suffering from autoimmune and inflammatory diseases. In 2016, another modified cell-related strategy was developed by the Trustees of the University of Pennsylvania, which involves the use of genetically modified effector cells to downregulate endogenous CD95 using the CRISPR system to treat autoimmune diseases (WO2016069282) [364].

## DISCUSSION AND CONCLUSION

For nearly three decades, members of the TNF superfamily, and the signal cascades they trigger, have been targeted by researchers and pharmaceutical companies to develop new therapies for the treatment of cancer and autoimmune diseases [334–338]. These molecules are widely involved in multiple cellular mechanisms such as apoptosis, proliferation, survival, tumor growth and differentiation. Since their role in mediating immune surveillance as well as protection from infections is essential, prolonged inhibition of these molecules could be dangerous. The progenitor of the TNF superfamily (i.e., TNF) remains the most studied and the most promising in terms of therapeutic potential [337, 338]. Among the members of the TNF superfamily, the research carried out on the TNF system is the most funded, with sales revenues exceeding 25 billion USD [338] followed by DR4/DR5 (Trail) systems and finally by the CD95 complex. Currently, five anti-TNF biologics have been clinically approved for the treatment of autoimmune diseases, namely Infliximab, Adalimumab, Etanercept, Golimumab, and Certolizumab Pegol, all with a specific structure for TNF-alpha recognition and blockade [339]. Despite the evident efficacy of these drugs, not all treated patients respond as expected and some seem to develop adverse reactions

associated with these drugs, such as effects on the neurological and dermatological levels [340–342]. There is therefore a growing need for new pharmacological systems with better specificity and greater safety.

## CD95-related therapeutic perspectives

Despite the evident role of CD95/CD95L in cancer and chronic inflammatory autoimmune diseases, since 1990 only a little over a hundred patents targeting the CD95/CD95L system have been conceived and published (Fig. 4). In the past, the complexity of the multiple CD95/CD95L-mediated signaling systems found in cancer and autoimmune diseases, the lack of specificity of the previously proposed strategies tested in vivo and the consequent severe side effects found [219, 220], have diminished the pharmacological interest for this target. Recent study report strategies focusing on more challenging compounds and delivery methods, with a particular attention to circumventing the severe adverse effects associated with the systemic activation of CD95. The extensively studied CD95-Fc fusion proteins, for instance, represent an interesting way to inhibit CD95L. However, these chimeric proteins, compared to those used in the TNF-TNFR2 system [343], exhibit a relatively low affinity for the corresponding CD95L and far less efficacy in inhibiting death induced by ligand interaction with CD95. A possible explanation is given by the fact that the interactions between these proteins occur through a complex mechanism of oligomerization given by the association of multiple trimers of both counterparts [35, 294, 344]. A better neutralization or stimulation of these proteins might therefore be achieved by a neutralization/stimulation system in which the binding protein is in a stable physiological-like form consisting of at least one trimer, if not an oligomer thereof. It seems that the oligomerization of the binding protein improves the stability of the therapeutic compound, consequently increasing its affinity for the target and the final system specificity [345]. Such oligomerized-related strategies have exhibited more efficient results, compared to previous systems generations. Fortunately, some of the newly proposed strategies appear to give encouraging preclinical results and so far, only one of these is currently in clinical trials. APG101 is the best prototype of future therapeutic approaches involving the CD95 system. It is an 84 kDa CD95L-neutralizing CD95 trimer fusion protein, able to pass the blood-brain barrier. Asunercept, the trade name for APG101, is now the subject of a controlled phase II clinical trial in patients with relapsed glioblastoma multiforme (GBM) (NCT01071837). The

<figure><img src="image_9.png" /><figcaption>**Fig. 4** Contribution of the CD95L/CD95 system in therapeutic-end studies. Graphic representation of the distribution of the number of patents targeting the most studied cell Death Receptors CD95, TNFR1 and -R2, TRAILR1 and -R2 and their respective ligands CD95L, TNF alpha, and TRAIL.</figcaption></figure>
glioblastoma model was chosen in accordance with several in vivo and in vitro non-clinical studies, which extensively described the involvement of CD95L in the growth, invasiveness and migration of glioblastoma cells [152, 324]. Merz et al. observed a decreased invasiveness on two cellular models of GBM after knockdown of the FASLG gene, without however affecting the viability of the cells sensitive to apoptosis [325]. They also reported a restored invasiveness following the administration of soluble recombinant CD95L, which was blocked by the addition of the APG101 fusion protein. This formulation, consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1, was in fact designed to specifically bind CD95L, thus disrupting the CD95L/CD95 signal cascade and the resulting cellular invasiveness. The collected results show a remarkable survival prolongation in patients with GBM, which makes it interesting for a possible transfer to other types of cancer [324, 346]. Furthermore, some experiments carried out on a cohort of 84 patients, showed greater efficacy of this compound when it is administered in combination with radiotherapy, observing a significant reduction in tumor growth compared to radiotherapy treatment alone [347]. Other preclinical studies, conducted on patients suffering from a lower risk myelodysplastic syndrome (MDS), have then highlighted a possible role of Asunercept in the treatment of anemia, a characteristic feature of this pathological condition [348]. In low risk MDS the administration of erythropoiesis-stimulating agents (ESAs) is widely used to correct cytopenia. However, some patients show resistance to ESA, thus requiring alternative treatments to contain the anemia associated with low risk MDS. CD95 is overexpressed in two-thirds of MDS patients, and is thought to be negatively implicated in the regulation of erythrocyte production [348]. The blocking of CD95 signal cascade can therefore increase erythropoiesis in MDS patients. The use of APG101 in this context seems to be particularly promising, as the neutralization of CD95L allows the blocking of the CD95L/CD95L signal and finally the restoration of erythropoiesis. A phase I clinical study (NCT01736436) conducted on 20 patients with low and intermediate MDS treated with intravenous APG101 is currently underway [349]. In said patients APG101 showed good tolerance and safety, promising prerequisites for use on a larger scale of this drug in the future.

DATA AVAILABILITY

All data generated or analyzed during this study are included in this published article and Supplementary Materials.

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