New target affinity and specificity of anti-sfas-l antibody pc111

Abstract

The present disclosure relates to antibodies having high affinity and specificity for soluble form of human Fas ligand (sFasL; also known as CD95L or CD178 or Apo1L) and uses thereof.

Description

Technical Field

[0001] This disclosure relates to antibodies with high affinity and specificity for soluble human Fas ligands (sFasL; also known as CD95L, CD178, or Apo1L) and their uses. Background Technology

[0002] Fas ligand is a 40 kDa homotrimeric type II transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily. FasL is primarily expressed on activated immune cells such as T cells, B cells, dendritic cells, and NK cells. Immune cells expressing FasL recognize the Fas death receptor (Fas) on the surface of target cells, which triggers apoptosis in many cellular systems upon binding to Fas ligand (FasL) (Sharma et al., 2000).

[0003] FasL exists in both a membrane-bound form (mFasL) and a soluble form (sFasL). The soluble Fas ligand (sFasL) is produced and released through proteolytic cleavage of the extracellular domain of mFasL expressed at the plasma membrane by a zinc-regulated matrix metalloproteinase (MMP). The soluble form (sFasL) exhibits both apoptotic and non-apoptotic activities.

[0004] The Fas-FasL interaction and the resulting signaling pathways have been shown to play important roles in the physiological regulation of the immune system and have been involved in the pathogenesis and progression of various malignancies and immune-inflammatory and infectious conditions. For example, the involvement of the Fas-FasL pathway has been shown to be a key mediator of keratinocyte apoptosis and acantholysis in pemphigus (Lotti et al., 2018). Further studies have demonstrated that the Fas-FasL system plays a crucial role in the development of severe skin conditions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) (Viard et al., 1998; Abe et al., 2003; Chang et al., 2004; Murata et al., 2008).

[0005] However, the Fas / Fas ligand system also plays a crucial role in immune cell homeostasis, prevention of autoimmunity, and cancer progression. To maintain homeostasis in the immune system, membrane-bound mFasL, expressed on cytotoxic lymphocytes, binds to the Fas receptor (Fas) and induces the lysis of infected cells, overproliferating T lymphocytes, and cancer cells via apoptosis (O'Reilly et al., Nature, October 1, 2009, 461(7264):659-63). Defects in the Fas / FasL pathway may limit the efficiency of immune surveillance and contribute to lymphoproliferative disorders, autoimmunity, and cancer (Tuomela et al., FrontImmunol. March 23, 2022; 13:867098).

[0006] Cell-to-cell interactions between activated immune cells carrying membrane-bound FasL and target cells carrying membrane-bound Fas are necessary for maintaining homeostasis in the immune system. Inhibition of these Fas-mFasL interactions may therefore limit the efficiency of immune surveillance and lead to the initiation and / or progression of lymphoproliferative disorders, autoimmune diseases, and / or cancers.

[0007] Therefore, while inhibiting the Fas-FasL apoptosis pathway is a key target for treating various diseases, the cell-to-cell interaction between mFasL and the Fas receptor is also essential for maintaining immune system homeostasis. Thus, inhibitors that suppress mFasL-Fas interaction may lead to undesirable physiological effects.

[0008] Therefore, it is crucial to develop inhibitors that target FasL without affecting the monitoring and homeostatic pathways regulated by the Fas / mFasL system.

[0009] Accordingly, there is a need for a reagent that can selectively inhibit the interaction between Fas and the soluble form of FasL (sFasL) without inhibiting the interaction between Fas and the membrane-bound form of FasL (mFasL).

[0010] Therefore, the purpose of this disclosure is to provide agents that target the soluble form of FasL (sFasL) with high affinity and selective specificity for the prevention and treatment of conditions associated with and / or mediated by soluble FasL (sFasL). Summary of the Invention

[0011] This disclosure provides agents specifically targeting soluble forms of Fas ligand protein (sFasL), particularly human Fas ligand protein (hsFasL), as active agents for treating subjects, particularly human subjects, affected by diseases mediated and / or triggered by soluble FasL (sFasL-mediated conditions).

[0012] In the first aspect, subjects, particularly human subjects, are affected by sFasL-mediated conditions and additionally suffer from immune homeostasis dysregulation and / or have an increased risk of developing immune homeostasis dysregulation.

[0013] Secondly, the subjects, especially human subjects, were affected by sFasL-mediated conditions and also suffered from secondary comorbidities.

[0014] In the third aspect, the subjects, particularly human subjects, are characterized by a combination of the first and second aspects, namely, that the subjects, particularly human subjects, are affected by sFasL-mediated conditions, have immune homeostasis dysregulation and / or have an increased risk of developing immune homeostasis dysregulation, and further have secondary comorbidities.

[0015] In certain embodiments, the reagent is an antibody against FasL, particularly human FasL, or an antigen-binding fragment thereof, exhibiting selective high affinity binding to the soluble form of FasL while substantially not inhibiting the binding of mFasL to Fas. This selective binding to sFasL allows for targeted inhibition of the pathological form of sFasL without substantially inhibiting the homeostatic form of mFasL. Therefore, the interaction between mFasL and the Fas receptor is substantially unaffected, thus avoiding undesirable effects on immune homeostasis. In some embodiments, undesirable effects on immune homeostasis include inhibiting the function of activated immune cells carrying mFasL, which can lead to lymphoproliferative disorders, autoimmune diseases, cancer, and aberrant immune responses at sites of immune forgiveness.

[0016] Therefore, this disclosure provides a highly selective and targeted therapy for soluble FasL-mediated diseases with an improved safety profile that minimizes the risk of unintended effects triggered by the inhibition of the interaction between mFasL and Fas. This risk may include inducing and / or increasing immune homeostasis dysregulation, which can lead to the occurrence and / or progression of lymphoproliferative disorders, autoimmune diseases, and / or cancers.

[0017] The term “immune homeostasis” specifically refers to the adequate and / or balanced function of the immune system of the subject being treated. The term “dysregulation” specifically refers to a disorder of the immune system, such as a disorder of the function of immune cells carrying membrane-bound FasL, and more particularly to a disorder of the ability of those immune cells to interact with target cells carrying Fas receptors.

[0018] According to certain aspects, this disclosure relates to an effective and safe treatment for subjects suffering from soluble FasL-mediated conditions such as pemphigus and other blistering skin diseases, wherein the subjects also suffer from or have an increased risk of developing immune system-related disorders such as immune-inflammatory diseases, autoimmune diseases, and / or cancer.

[0019] In some embodiments, the subject suffers from an immunocompromising disorder such as HIV infection, HBV infection, and / or HCV infection, and / or receives immunosuppressive drugs such as cyclosporine. In further embodiments, the subject suffers from an immune-inflammatory disorder, such as autoimmune lymphoproliferative syndrome (ALPS). In further embodiments, the subject suffers from an autoimmune disorder, such as Hashimoto's thyroiditis. In further embodiments, the subject suffers from cancer, such as lymphoma. In further embodiments, the subject has an increased risk of developing the conditions described above.

[0020] According to a further preferred aspect of the invention, this disclosure relates to an effective and safe treatment for subjects suffering from sFasL-mediated conditions such as pemphigus or another blistering skin disease, wherein the subjects also suffer from secondary comorbidities.

[0021] The term "comorbidity" specifically refers to a secondary condition that simultaneously affects the subject along with sFasL-mediated comorbidity. In some embodiments, comorbidity is an additional sFasL-mediated condition. In some embodiments, comorbidity is a non-sFasL-mediated condition. In some embodiments, comorbidity is triggered by sFasL-mediated comorbidity. In some embodiments, comorbidity is unrelated to and / or independent of soluble FasL-mediated comorbidity. In some embodiments, comorbidity is iatrogenic comorbidity, i.e., comorbidity caused and / or induced by treatment (e.g., prior or concomitant treatment for sFasL-mediated comorbidity, particularly immunosuppressive treatment for sFasL-mediated comorbidity).

[0022] The therapeutic efficacy of antibodies or antigen-binding fragments is based on a combination of high binding affinity and selectivity to soluble forms of human FasL.

[0023] In specific embodiments of this disclosure, the reagent is a monoclonal antibody or an antigen-binding fragment thereof, comprising a VH region having complementarity-determining regions CDR H1, CDR H2, and CDR H3 as specified in SEQ ID NOs: 1, 3, and 5, and a VL region having complementarity-determining regions CDR L1, CDR L2, and CDR L3 as specified in SEQ ID NOs: 7 to 9. In a more particular embodiment, the antibody has an IgG heavy chain constant region, such as the human IgG heavy chain constant region, preferably an IgG1 or IgG4 heavy chain constant region.

[0024] This disclosure also relates to nucleic acid molecules encoding monoclonal antibodies or antigen fragments thereof as disclosed herein, for the treatment of subjects, particularly human subjects, affected by diseases mediated and / or triggered by sFasL. In a first aspect, the subject additionally suffers from immune homeostasis and / or has an increased risk of developing immune homeostasis. In a second aspect, the subject additionally suffers from secondary comorbidities. In a third aspect, the subject is characterized by a combination of the first and second aspects described above.

[0025] A further aspect of this disclosure is a pharmaceutical composition comprising an antibody or antigen-binding fragment or nucleic acid molecule disclosed herein and one or more pharmaceutically acceptable carriers for use in treating a subject, particularly a human subject, affected by a disease mediated and / or triggered by soluble FasL. In a first aspect, the composition is used in a subject who also suffers from immune homeostasis dysregulation and / or has an increased risk of developing immune homeostasis dysregulation. In a second aspect, the composition is used in a subject who also suffers from secondary comorbidities. In a third aspect, the subject is characterized by a combination of the first and second aspects described above. Detailed Implementation

[0026] The purpose of this disclosure is to provide a therapeutic agent for diseases mediated by soluble FasL (sFasL-mediated conditions). The development of agents that specifically block the activity of sFasL allows for targeted therapies that minimize the risk of triggering or enhancing immune homeostasis dysregulation associated with the Fas / mFasL system.

[0027] Therefore, this disclosure relates to sFasL antagonists that selectively inhibit the biological effects of the soluble form of FasL (sFasL), particularly selected from anti-sFasL antibodies, and even more particularly from human or humanized anti-sFasL antibodies. The expression "sFasL antagonist that selectively inhibits the biological effects of sFasL" as used herein refers to an antagonist capable of completely or at least substantially inhibiting or neutralizing the biological effects of sFasL without substantially affecting the interaction between mFasL and Fas. For example, the inhibition or neutralization effect may be based on blocking and / or inhibiting the binding of sFasL to its natural cellular receptor, thereby preventing and / or reducing the physiological signals generated by such binding.

[0028] sFasL antagonists can be used as monotherapy or in combination with further medications, such as those for the treatment of sFasL-mediated conditions.

[0029] In certain embodiments, this disclosure provides therapeutic agents for sFasL-mediated conditions comprising an antibody against a human sFas ligand or an antigen-binding fragment thereof as the active ingredient. The antibody may comprise a monoclonal antibody, such as a chimeric, humanized, or human anti-sFasL antibody or an antigen-binding fragment thereof, such as a recombinant single-chain antibody. The antibody or antibody fragment may be monovalent or multivalent and may contain modifications, such as specific glycosylation patterns, or modifications to the Fc region for modulating its physiological characteristics, including antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). The antigen-binding fragment of the anti-FasL antibody disclosed herein may be a recombinant single-chain antibody or a single-chain variable fragment. In certain embodiments, the antibody is a human or humanized full-length sFasL antibody. If desired, the antibody may be conjugated to an effector molecule, such as a cellular inhibitory, cytotoxic, and / or radioactive compound.

[0030] Preferred humanized antibodies suitable for the prevention and / or treatment of sFasL-mediated conditions are described in US 7,262,277 ("Antagonistic Anti-hFas ligand human antibodies and fragments thereof"), the contents of which are incorporated herein by reference. US 7,262,277 discloses isolated anti-hFasL human antibodies with high affinity for hFasL peptides and which can be used to neutralize hFasL activity. US 7,262,277 specifically describes anti-hFas human antibodies 3E1 (produced by hybridoma cells with accession number ATCC PTA-4017) and 4G11 (produced by hybridoma cells with accession number ATCC PTA-4018). Hybridoma cells with accession numbers ATCC PTA-4017 and ATCC PTA-4018 were deposited on January 29, 2002, at the American Center for Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209 (USA).

[0031] Surprisingly, antibody 3E1, disclosed in US 7,262,277, has been found to have high target affinity and specificity for soluble FasL, but it cannot bind to membrane-bound FasL (mFasL).

[0032] This discovery provides novel therapeutic uses for these antibodies, particularly for their administration to subjects suffering from immune homeostasis dysregulation and / or at an increased risk of developing immune homeostasis dysregulation. In a particular embodiment, this disclosure provides the use of the antibodies in subjects suffering from or at an increased risk of developing immune system-related dysregulations, such as immune-inflammatory diseases, autoimmune diseases, and / or cancer. Furthermore, this discovery provides advantageous novel therapeutic uses for these antibodies, particularly for their administration to subjects also suffering from secondary comorbidities.

[0033] In a particularly preferred embodiment, this disclosure relates to anti-FasL human antibodies or antigen-binding portions thereof for the prevention and / or treatment of sFasL-mediated conditions, said anti-FasL human antibodies or antigen-binding portions thereof comprising a light chain variable region and / or a heavy chain variable region as described in US 7,262,277, the contents of which are incorporated herein by reference. Specifically, preferred anti-FasL human antibodies according to this disclosure for the prevention and / or treatment of sFasL-mediated conditions comprise a light chain variable region having a polypeptide having the sequence shown in SEQ ID NO: 2 of US 7,262,277 (incorporated herein by reference), and further comprise a heavy chain variable region having a polypeptide having the sequence shown in SEQ ID NO: 10 or 18 of US 7,262,277 (incorporated herein by reference). More specifically, this disclosure relates to the use of anti-hFas human antibodies 3E1 (produced by hybridoma cells with accession number ATCC PTA-4017) and / or 4G11 (produced by hybridoma cells with accession number ATCC PTA-4018) as described in US 7,262,277 (incorporated herein by reference) in the prevention and / or treatment of sFasL-mediated conditions.

[0034] In the context of this disclosure, the term "sFasL-mediated disease prevention" means treating a disease at diagnosis (e.g., in its early stages) to prevent its progression to a more severe spectrum. The term "sFasL-mediated disease treatment" means managing a disease to alleviate and / or improve its symptoms, preferably to the point of complete cure.

[0035] Therefore, according to a first aspect, this disclosure relates to a monoclonal antibody or antigen-binding fragment thereof specifically targeting a soluble form of human Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from:

[0036] (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3:

[0037] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or

[0038] (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2);

[0039] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3) or

[0040] (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln LysLeu Gln Gly (SEQ ID NO: 4); and

[0041] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or

[0042] (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6),

[0043] It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3:

[0044] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0045] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0046] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0047] or

[0048] (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human soluble Fas ligand protein (sFasL);

[0049] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects also suffer from immune homeostasis dysregulation and / or have an increased risk of developing immune homeostasis dysregulation, such as immune system-related disorders, such as immune-inflammatory diseases, autoimmune diseases, and / or cancer.

[0050] In a further aspect, this disclosure relates to monoclonal antibodies or antigen-binding fragments thereof specifically targeting a soluble form of human Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from:

[0051] (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3:

[0052] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or

[0053] (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2);

[0054] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3) or

[0055] (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln LysLeu Gln Gly (SEQ ID NO: 4); and

[0056] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or

[0057] (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6),

[0058] It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3:

[0059] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0060] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0061] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0062] or

[0063] (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human sFas ligand protein (sFasL).

[0064] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects additionally suffer from lymphoproliferative disorders, autoimmune disorders and / or cancer, and / or are at risk of developing lymphoproliferative disorders, autoimmune disorders and / or cancer.

[0065] According to a second aspect, this disclosure relates to monoclonal antibodies or antigen-binding fragments thereof specifically targeting a soluble form of human Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from:

[0066] (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3:

[0067] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or

[0068] (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2);

[0069] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3) or

[0070] (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln LysLeu Gln Gly (SEQ ID NO: 4); and

[0071] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or

[0072] (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6),

[0073] It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3:

[0074] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0075] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0076] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0077] or

[0078] (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human soluble Fas ligand protein (sFasL);

[0079] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects who also suffer from secondary comorbid conditions.

[0080] Furthermore, according to a third aspect, this disclosure relates to a monoclonal antibody or antigen-binding fragment thereof specifically targeting a soluble form of human Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from:

[0081] (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3:

[0082] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or

[0083] (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2);

[0084] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3) or

[0085] (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln LysLeu Gln Gly (SEQ ID NO: 4); and

[0086] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or

[0087] (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6),

[0088] It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3:

[0089] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0090] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0091] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0092] or

[0093] (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human soluble Fas ligand protein (sFasL);

[0094] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in a subject, wherein the subject is characterized by a combination of the first and second aspects described above.

[0095] As described above, this disclosure also relates to monoclonal antibodies or antigen-binding fragments that compete with the monoclonal antibodies or antigen-binding fragments disclosed above for binding to the human sFasL protein (sFasL). Preferably, the antibody competes with the monoclonal antibodies described above that have heavy chain CDR sequences H1 to H3 comprising the amino acid sequences of SEQ ID NO: 1 to 5 and light chain CDR sequences L1 to L3 comprising the amino acid sequences of SEQ ID NO: 7 to 9. In a very preferred embodiment, the antibody competes with the monoclonal antibodies described above that have heavy chain CDR sequences H1 to H3 comprising SEQ ID NO: 1, 3, and 5 and light chain CDR sequences L1 to L3 comprising SEQ ID NO: 7 to 9.

[0096] In some embodiments, as disclosed herein, a competitive antibody binds to the same or overlapping epitopes of human sFasL with monoclonal antibodies having the CDRs defined in SEQ ID NO: 1 to 9. Competition can be determined by a standard assay in the art that can quantify the relative and absolute binding affinity of an antibody to a protein, particularly against human sFasL, relative to a given reference antibody.

[0097] The quantitative binding of sFasL antibody to sFasL protein can be determined using one of various surface plasmon resonance (SPR) measurement platforms. Examples include Biacore and Forte Octet. The recombinant target protein sFasL (e.g., His-tagged FasL protein Acro Bio FAL-H5241) is tethered to a capture chip, allowing the sFasL antibody to flow through the chip while changes in molecular interactions are recorded in real time. Measurements are performed in some embodiments, such as those described in Yakes BJ et al., Talanta, August 15, 2016; 156-157:55-63. The binding rate (on-rate) (Ka; M) is measured. -1 s -1 ), dissociation rate (off-rate) (Kd; s -1 The binding affinity (KD, µM) can be calculated by adding and removing sFasL from the flow. The measurement of binding affinity can be determined by dividing the dissociation rate by the binding rate. Therapeutic applications typically require a KD value less than 10 µM. A KD value of 10 nM to 1 pM is optimal.

[0098] According to the first aspect, this disclosure specifically relates to monoclonal antibodies or antigen-binding fragments thereof that specifically target human sFas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein the amino acid sequences of the heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3 are as follows:

[0099] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1),

[0100] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3), and

[0101] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5),

[0102] Furthermore, the amino acid sequences of the light chain complementarity-determining regions (CDRs) CDR L1, CDR L2, and CDR L3 are as follows:

[0103] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0104] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0105] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0106] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects additionally suffer from and / or have an increased risk of developing immune homeostasis, such as immune system-related disorders, such as immune-inflammatory diseases, autoimmune diseases, and / or cancer.

[0107] or

[0108] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects additionally suffer from lymphoproliferative disorders, autoimmune diseases, and / or cancer, and / or are at risk of developing lymphoproliferative disorders, autoimmune diseases, and / or cancer.

[0109] or

[0110] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects also suffer from immune homeostasis dysregulation and / or have an increased risk of developing immune homeostasis dysregulation, such as immune inflammatory diseases, autoimmune diseases, and / or cancer.

[0111] According to the second aspect, this disclosure specifically relates to monoclonal antibodies or antigen-binding fragments thereof that specifically target human sFas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein the amino acid sequences of the heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3 are as follows:

[0112] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1),

[0113] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3), and

[0114] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5),

[0115] Furthermore, the amino acid sequences of the light chain complementarity-determining regions (CDRs) CDR L1, CDR L2, and CDR L3 are as follows:

[0116] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0117] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0118] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0119] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects also suffer from secondary comorbidities.

[0120] According to the third aspect, this disclosure specifically relates to monoclonal antibodies or antigen-binding fragments thereof that specifically target human sFas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein the amino acid sequences of the heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3 are as follows:

[0121] (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1),

[0122] (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln LysVal Gln Gly (SEQ ID NO: 3), and

[0123] (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5),

[0124] Furthermore, the amino acid sequences of the light chain complementarity-determining regions (CDRs) CDR L1, CDR L2, and CDR L3 are as follows:

[0125] (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ IDNO: 7),

[0126] (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and

[0127] (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9),

[0128] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in a subject, wherein the subject is characterized by a combination of the first and second aspects described above.

[0129] In specific embodiments of this disclosure, at least one amino acid of the aforementioned CDR1, CDR2, or CDR3 amino acid sequence of the VH and / or VL chains as defined in SEQ ID Nos. 1 to 9 is replaced by another amino acid while maintaining the structural integrity of the antibody and epitope binding. These exchanges can be conserved (i.e., by similar amino acids) or non-conserved.

[0130] Specifically, at least one amino acid in the VH and VL CDR1, CDR2, or CDR3 sequences as defined in SEQ ID NO: 1 to 9 can be replaced by a conserved amino acid substitution, i.e., the amino acid is replaced by another amino acid with similar biochemical properties, for example, replacing another aliphatic amino acid with an aliphatic amino acid such as Gly, Ala, Val, Leu, or Ile; replacing a basic amino acid such as His, Lys, or Arg with another basic amino acid or Met; replacing an acidic amino acid or its amide such as Asp, Glu, Asn, or Gln with another acidic amino acid or its amide; replacing an aromatic amino acid such as Phe, Tyr, or Trp with another aromatic amino acid. In some preferred embodiments, amino acids 1, 2, 3, 4, or 5 of SEQ ID NO: 1 to 9 are replaced by another conserved or non-conserved amino acid.

[0131] In a preferred embodiment, this disclosure relates to the use of anti-FasL human antibodies or antigen-binding portions thereof, said antibodies or antigen-binding portions comprising light chain variable regions and / or heavy chain variable regions as described in US 7,262,277 (SEQ ID NO: 2 and SEQ ID NO: 10 or 18, respectively), the contents of which are incorporated herein by reference.

[0132] Therefore, in a further preferred embodiment, this disclosure relates to an anti-sFasL antibody or an antibody-binding fragment thereof, wherein the light chain variable region of said antibody or antibody-binding fragment comprises a polypeptide having the following amino acid sequence:

[0133] EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO: 10),

[0134] Furthermore, the heavy chain variable region of the antibody or its antibody-binding fragment contains a polypeptide having the following amino acid sequence:

[0135] QVQLVQSGAEVKKPGASVKVSCKASGYIFI RHGIT WVRQAPGQGLEWMG WINAYNGNTNYAQKVQG RVTMTTDKSTSTAYMELRRSLRSDDAAVYYCAR ETMVRGVPLDY WGQGTLVTVSS (SEQ ID NO: 11)

[0136] or

[0137] QVQLVQSGAEVKKPGASVKVSCKASGYIFI SHGIS WVRQAPGQGLEWMG WINAYSGNTNYAQKLQG RVTMTTDRSTSTAYMELRSLRSDDTAVYYCAR ETMVRGVPCDY WGQGTLVTVSS (SEQ ID NO: 12).

[0138] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects additionally suffer from and / or have an increased risk of developing immune homeostasis, such as immune system-related disorders, such as immune-inflammatory diseases, autoimmune diseases, and / or cancer.

[0139] or

[0140] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects additionally suffer from lymphoproliferative disorders, autoimmune diseases, and / or cancer, and / or are at risk of developing lymphoproliferative disorders, autoimmune diseases, and / or cancer.

[0141] or

[0142] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects also suffer from secondary comorbidities.

[0143] or

[0144] Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects also suffer from immune homeostasis and / or have an increased risk of developing immune homeostasis, such as immune system-related disorders, such as immune-inflammatory diseases, autoimmune diseases and / or cancer, and further have secondary comorbidities.

[0145] Monoclonal sFasL antibodies or their antigen-binding fragments may contain, along the entire length of the protein, a VL amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 10 and a VH amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 11 or 12.

[0146] The "percentage (%) amino acid sequence identity" of a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve maximum percentage sequence identity. Alignment for determining percentage amino acid sequence identity can be performed in various ways within the art, for example, using publicly available computer software such as BLAST.

[0147] Antibodies used for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects as disclosed above may be selected from partially or fully human antibodies, chimeric antibodies, and / or humanized antibodies. Preferably, the antibody is a human antibody. Furthermore, the antibodies according to this disclosure may be monospecific and / or bispecific. Optionally, the antibody may be multivalent and multispecific. Additionally, the antigen-binding fragment of the antibody according to this disclosure may be selected from Fab, Fab', and / or F(ab')2 and / or single-chain Fv fragments.

[0148] Antibodies can be of any suitable class. The term "class" refers to the type of constant domains or constant regions possessed by its heavy chain. As used herein, "constant domain" or "constant region" refers to the sum of the domains of an antibody excluding the variable region. Constant regions do not directly participate in antigen binding but exhibit a variety of effector functions. The antibodies of this disclosure can be any of the five major antibody classes (IgA, IgG, IgGE, IgG, and IgM), or any of their subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. According to this disclosure, antibodies or fragments of the IgG, IgA, or IgM classes are particularly suitable.

[0149] According to a highly preferred embodiment, the antibody is selected from IgG classes, such as its subclasses IgG1, IgG2, IgG3, or IgG4, or IgM classes or IgA classes, or antigen-binding fragments thereof. In a most preferred embodiment of this disclosure, the antibody has an IgG heavy chain constant region, preferably a heavy chain constant region of subclass IgG1 or IgG4. In a most preferred embodiment of the invention, the antibody has an IgG4 heavy chain constant region. In another most preferred embodiment, the antibody has a kappa (κ) light chain.

[0150] Therefore, the most preferred antibody of the present invention is a monoclonal IgG4 anti-sFasL antibody or its antigen-binding fragment, comprising a VH region having complementarity-determining regions CDR H1, CDR H2, and CDR H3 as specified in SEQ ID NOs: 1, 3, and 5 above, and a VL region having complementarity-determining regions CDR L1, CDR L2, and CDR L3 as specified in SEQ ID NOs: 7 to 9 herein. In a more preferred embodiment of the invention, the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) comprises a polypeptide having a sequence as specified in SEQ ID NO: 10 herein, and the heavy chain variable region (VH) comprises a polypeptide having a sequence as specified in SEQ ID NO: 11 or 12 above.

[0151] In a more preferred embodiment of the present invention, the above-mentioned anti-sFasL antibody comprises a light chain constant region (LC) of a polypeptide having the following amino acid sequence:

[0152] RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC (SEQ ID NO: 13)

[0153] And the heavy chain constant region (HC) of a polypeptide having the following amino acid sequence:

[0154] ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 14).

[0155] In yet another embodiment, this disclosure relates to the use of anti-hFas ligand human antibodies or antigen-binding fragments thereof produced by hybridoma cells of accession number ATCC PTA-4017, and / or to the use of anti-hFas ligand human antibodies or antigen-binding fragments thereof produced by hybridoma cells of accession number ATCC PTA-4018 (as described in US 7,262,277, as antibodies 3E1 and 4G11, respectively), for the prevention and / or treatment of subjects suffering from the aforementioned sFasL-mediated conditions. In the most preferred embodiment of this disclosure, the use of anti-hFas ligand human antibody 3E1 (produced by hybridoma cells of accession number ATCC PTA-4017) or its antigen-binding fragments thereof is provided for the prevention and / or treatment of subjects suffering from the aforementioned sFasL-mediated conditions.

[0156] In a final and further preferred aspect, this disclosure relates to the use of a monoclonal antibody or an antigen-binding fragment thereof that recognizes the same human sFasL epitope as the antibodies described above.

[0157] The antibody or antigen-binding fragment of this disclosure binds specifically to soluble human FasL (sFasL) with high affinity. Furthermore, the antibody or antigen-binding fragment does not bind efficiently to membrane-bound FasL. The binding properties of the antibody allow for selective targeting of sFasL without the undesirable side effects resulting from binding to mFasL. In particular, administration of the antibody of this disclosure can lead to a reduced risk of triggering physiological and / or pathological processes mediated and / or triggered by inhibition of the mFasL pathway.

[0158] As used herein, the terms “binding” and “specific binding” refer to the binding of an antibody or a fragment thereof to human sFasL. A measure of the strength of antibody binding is called affinity. Methods for determining such binding and / or affinity using in vitro assays are known to those skilled in the art. Detection using flow cytometry, immunohistochemistry, and / or fluorescence is described and particularly preferred herein, according to this disclosure.

[0159] The binding affinity between an antibody and an antigen is defined by the terms ka (the rate constant of association between the antibody and the antigen), kd (the dissociation rate constant), and KD (the equilibrium dissociation constant).

[0160] When measured by surface plasmon resonance (SPR), the antibodies and their antigen-binding fragments according to this disclosure preferably exhibit a dissociation constant KD with human soluble FasL of about 1000 pM or less, preferably about 750 pM or less, about 500 pM or less, or about 400 pM or less, most preferably about 350 pM or less, or about 200 pM or less. In a very preferred embodiment, when measured by SPR, the dissociation constant KD of the antibodies and their antigen-binding fragments with human soluble FasL is in the range of about 100 to 500 pM, preferably in the range of about 200 to 400 pM, more preferably in the range of about 200 to 300 pM. In a very preferred embodiment, when measured by SPR, the dissociation constant KD with human soluble FasL is about 250 pM. In an alternative preferred embodiment, when measured by flow-induced dispersion analysis (FIDA), the dissociation constant KD with human soluble FasL is about 340 pM.

[0161] Furthermore, the antibodies and antigen-binding fragments of the present disclosure do not exhibit effective binding affinity for membrane-bound human FasL (mFasL). In some embodiments, when measured under comparable conditions, the binding affinity of the antibody or its antigen-binding fragment for mFasL is at least about 100 times or at least about 1000 times lower than that for sFasL. In particular, the antibodies and their antigen-binding fragments of the present disclosure exhibit a dissociation constant KD of at least about 500 nM, preferably at least about 1000 nM, for human membrane-bound FasL (mFasL).

[0162] In the context of this disclosure, the term "soluble FasL (sFasL)-mediated condition" refers to a condition in which a reduction in the level of soluble FasL in the body, particularly measurable levels of soluble FasL in the patient's blood circulation, is required to improve and / or cure the condition. In some embodiments, the level of soluble FasL is upregulated compared to healthy subjects. In the serum of healthy subjects, the mean concentration of sFasL is below about 120 to 125 pg / mL (Puviani et al., The Journal of Investigative Dermatology, Vol. 120, No. 1, January 2003, 164-167). In one embodiment, the level of sFasL in the serum of patients affected by soluble FasL (sFasL)-mediated condition is significantly elevated compared to healthy subjects. According to a preferred embodiment, the serum soluble FasL level in patients affected by soluble FasL (sFasL)-mediated disease according to the present invention is at least 2 times higher, preferably 3 times higher, more preferably 4 or 5 times higher, or even still higher than that in healthy subjects. Even more preferably, the average concentration of sFasL in the blood of patients according to the present disclosure is greater than 124 pg / ml or significantly greater than 124 pg / ml. According to a further preferred embodiment of the present disclosure, the serum sFasL level in patients is correlated with the severity and / or progression of sFasL-mediated disease.

[0163] In a preferred embodiment, sFasL-mediated conditions are selected from immune-inflammatory and / or infectious conditions and / or skin conditions associated with keratinocyte acantholysis, apoptosis, and / or necrosis. In a more preferred embodiment, the skin condition is selected from bullous and / or blistering skin conditions, most preferably from pemphigus, toxic epidermal necrolysis (TEN), and / or Stevens-Johnson syndrome (SJS). According to a particularly preferred embodiment, the sFasL-mediated condition is pemphigus. Other examples of sFasL-mediated blistering skin conditions showing elevated sFasL in plasma according to this disclosure are selected from drug-induced hypersensitivity syndrome (DIHS), drug response with eosinophilia and systemic symptoms (DRESS), systemic lupus erythematosus, and erosive oral and genital lichen planus. Further preferred sFasL-mediated non-dermatological conditions are selected from acute respiratory distress syndrome and rheumatoid arthritis.

[0164] According to the first aspect, subjects to be treated according to this disclosure suffer from immune homeostasis dysregulation and / or have an increased risk of developing immune homeostasis dysregulation, particularly mFasL-mediated conditions. In the context of this disclosure, mFasL-mediated conditions are conditions triggered by defects and / or regulated homeostasis of the immune system due to blockage of membrane-bound FasL activity. The Fas / mFasL pathway is involved in the pathological mechanisms of several immune-inflammatory diseases, autoimmune diseases, and cancers.

[0165] Therefore, in this disclosure, membrane-bound FasL (mFasL)-mediated diseases are preferably selected from lymphoproliferative diseases, autoimmune diseases, and / or cancers. A preferred example of a lymphoproliferative disease caused by blockage of the mFasL pathway is autoimmune lymphoproliferative syndrome (ALPS). An example of an autoimmune disease caused by dysregulation of the mFasL pathway is Hashimoto's thyroiditis. Furthermore, examples of cancers caused by and / or associated with dysregulation of the mFasL pathway are lymphomas.

[0166] Therefore, this disclosure relates to the use of the above-described antibodies and their antigen-binding fragments in treating subjects who suffer from sFasL-mediated conditions and are at risk of developing and / or suffering from mFasL-mediated conditions, particularly selected from the immune inflammatory conditions, autoimmune diseases, and cancers described above.

[0167] According to a preferred embodiment of this disclosure, subjects with sFasL-mediated disease have received cancer treatment within the past 10 years, the past 5 years, the past 3 years, or the past 1 year, and are therefore at an increased risk of cancer recurrence.

[0168] Furthermore, this disclosure relates to the use of the aforementioned antibody and its antigen-binding fragment in treating subjects who suffer from primary sFasL-mediated disease and additional secondary comorbidities.

[0169] Preferably, the sFasL-mediated disease is selected from pemphigus, toxic epidermal necrolysis (TEN), and / or Stevens-Johnson syndrome (SJS), with pemphigus being the most preferred. In a preferred aspect, the secondary comorbid disease is triggered by a primary sFasL-mediated disease, particularly by pemphigus, toxic epidermal necrolysis (TEN), and / or Stevens-Johnson syndrome (SJS), with pemphigus being the most preferred.

[0170] Preferred examples of secondary comorbidities according to the invention that are associated with or triggered by pemphigus are selected from Cushing's syndrome, adrenal insufficiency, myasthenia gravis, mucositis, herpes infection, fungal infection, insomnia, hidradenitis suppurativa, type 2 diabetes, hypertension, cataracts, benign prostatic hyperplasia, and bone disorders such as osteoporosis, osteoporosis, and pathological or fragility fractures due to decreased bone mineral density. Most preferred secondary comorbidities are selected from type 2 diabetes, hypertension, benign prostatic hyperplasia, cataracts, myasthenia gravis, and bone disorders such as osteoporosis, osteoporosis, and pathological or fragility fractures due to decreased bone mineral density.

[0171] In a further preferred aspect, the secondary comorbidity is an iatrogenic comorbidity caused and / or induced by the treatment of the primary sFasL-mediated condition (particularly the treatment of pemphigus, toxic epidermal necrolysis (TEN), and / or Stevens-Johnson syndrome (SJS), most preferably the treatment of pemphigus). Treatment can be prior to or concomitant to the sFasL-mediated condition. Prior to or concomitant to sFasL therapy is preferably selected from immunosuppressive therapies, such as those using corticosteroids, rituximab, azathioprine, mycophenolate mofetil, cyclophosphamide, and intravenous immunoglobulin (IVIg).

[0172] According to this disclosure, iatrogenic comorbidities may include hypertension, dyslipidemia, heart disease, diabetes, lung infection, and bone conditions such as osteoporosis, osteoporosis, and pathological or fragility fractures due to decreased bone mineral density.

[0173] In a specific aspect, the present invention relates to the use of the aforementioned antibody and its antigen-binding fragment in treating subjects suffering from sFasL-mediated conditions, particularly pemphigus, and

[0174] (i) In addition, suffering from the comorbid conditions described above;

[0175] (ii) In addition, suffering from the lymphoproliferative disorders, immune inflammatory disorders, immunological and / or autoimmune disorders as described above;

[0176] (iii) In addition, he / she suffers from cancer;

[0177] (iv) Having a comorbid condition before the start of any sFasL-mediated treatment for a condition, especially before the start of pemphigus treatment (in untreated patients);

[0178] (v) Having additional comorbidities acquired during prior or concurrent sFasL-mediated disease treatment (especially during prior or concurrent immunosuppressive pemphigus treatment (e.g., treatment with rituximab, steroids, azathioprine, mycophenolate mofetil, cyclophosphamide, and intravenous immunoglobulin (IVIg)); and

[0179] (vi) In addition, suffer from the comorbid conditions mentioned above, with or without immunosuppressive co-treatment.

[0180] Further aspects involve nucleic acid molecules, such as DNA molecules, that encode antibody VH or antibody VL regions, or that encode complete monoclonal antibodies or antibody fragments thereof as described above, for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects as described above.

[0181] Nucleic acid molecules can be DNA carriers or RNA molecules, which are typically formulated into lipid nanoparticle encapsulations. When the nucleic acid molecule encoding the antibody of this disclosure is injected into a patient, the cells express the antibody and enter circulation. The use of DNA and RNA delivery technologies, particularly the use of mRNA encoding therapeutic antibodies, in antibody therapy is a well-known therapeutic approach in the art (e.g., Van Hoecke and Roose, J Trans Med (2019) 17:54 and Deal et al., Vaccines (2021), 9, 2018).

[0182] In therapeutic applications, anti-sFasL antibodies or fragments thereof are administered in an effective amount to subjects in need, particularly human subjects. The dosage will depend on the specific antibody type, the severity and stage of the disease, and the route of administration.

[0183] In a preferred embodiment of this disclosure, the subject who requires the anti-sFasL antibody of this disclosure due to the effects of soluble FasL (sFasL)-mediated disease is a subject whose serum sFasL concentration is elevated compared to that of a healthy subject.

[0184] Typically, anti-sFasL antibodies or their antigen-binding fragments are administered as pharmaceutical compositions comprising an active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well known in the art. The effective dose of the pharmaceuticals disclosed herein can range from 0.1 μg to 100 mg, up to a total dose of about 1 g, depending on the route of administration.

[0185] Examples of suitable carriers and excipients for formulating antibodies or antibody fragments include saline and aqueous buffers, and are well known in the art. The effective dose of the drug disclosed herein can range from 0.1 μg to 100 mg, up to a total dose of about 1 g, depending on the route of administration.

[0186] Therefore, a further aspect of this disclosure is a pharmaceutical composition comprising, as described above, an anti-sFasL antibody or an antigen-binding fragment thereof or a nucleic acid molecule as described above, and one or more pharmaceutically acceptable carriers or excipients, for use in human medicine, preferably for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects as disclosed above.

[0187] Depending on the stage and severity of the condition, the pharmaceutical composition may be administered once or multiple times during the course of the condition. For example, it may be administered once or several times daily, every other day, twice a week, or once a week, for an appropriate duration. The pharmaceutical composition may be administered in a single treatment cycle consisting of one or more administrations, or in several treatment cycles consisting of one or more administrations. Each treatment cycle may have a duration ranging from one day to several weeks, months, or even years.

[0188] In some embodiments, the pharmaceutical composition is administered parenterally, such as by subcutaneous, intramuscular, or intravenous injection or infusion. In further embodiments, the pharmaceutical composition is administered locally, such as topically, orally, intranasally, or intrapulmonaryly, such as by aerosol inhalation. Preferably, the composition is administered systemically.

[0189] Anti-sFasL antibodies or antibody fragments thereof can be used for monotherapy or combination therapy. Therefore, anti-sFasL antibodies or antibody fragments thereof can be administered alone or in combination with at least one further active agent that is effective for treating soluble FasL (sFasL)-mediated conditions, preferably for skin conditions selected from bullous and / or blistering skin diseases, and most preferably for pemphigus, toxic epidermal necrosis (TEN), and / or Stevens-Johnson syndrome (SJS). In particular, the anti-sFasL antibodies of this disclosure can be used in combination with other active ingredients, particularly selected from steroids, cyclosporine, IVIg, TNF inhibitors, IL17 or IL23 inhibitors, rituximab, anti-CD20 antibodies, and / or plasma exchange.

[0190] The present disclosure will be further described in more detail with reference to the following figures and embodiments. Attached Figure Description

[0191] Figure 1The binding curves of FasL (sFasL) for a constant concentration of PC111 at 15 nM (A) are shown, with data points involving higher-order oligomers marked in orange. The binding isotherms of the curves in subplot A have had three points marked in orange removed to allow for fitting (B).

[0192] Figure 2 Overview of membrane proteome arrays.

[0193] Figure 3 : Membrane proteome array (MPA): Optimization of ligand concentrations for screening. Serial dilutions of each test ligand were measured by incubation with known targets, and target binding was measured by flow cytometry. The optimal ligand concentration for screening was selected based on a combined assessment of binding strength and background signal (left) and high background event rate (right).

[0194] Figure 4 MPA screening results. Binding of each ligand to MPA was detected by flow cytometry at previously determined optimal concentrations. The binding interactions confirmed in downstream validation studies are shown, and any proteins that failed validation were removed.

[0195] Figure 5 The level of PC111 in mouse plasma at 2 hours in a concanavalin A (Con A) liver injury model.

[0196] Figure 6 : The level of sFasL in mouse plasma at 2 hours in a concanavalin A (Con A) liver injury model.

[0197] Example

[0198] An anti-FasL human IgG4, κ, monospecific bivalent antibody was used, comprising the heavy chain variable (VH) region of SEQ ID NO: 11 (with complementarity-determining regions CDR H1, CDR H2, and CDR H3 according to SEQ ID NO: 1, 3, and 5) and the light chain variable (VL) region of SEQ ID NO: 10 (with complementarity-determining regions CDR L1, CDR L2, and CDR L3 according to SEQ ID NO: 7-9). This antibody is internally and hereinafter referred to as "antibody PC111".

[0199] Example 1 – Surface Plasmon Resonance (SPR) Analysis of the Binding Affinity between PC111 and sFasL

[0200] The binding affinity of PC111 to sFasL was determined for three different batches of PC111 using sFasL from different sources and different SPR instruments. The mean binding affinity (equilibrium dissociation constant; KD) of the six measurements shown in Table 1 is 238 pM, ranging from 650 pM to 93 pM.

[0201] Table 1

[0202]

[0203] Example 2 – Flow-induced dispersion analysis

[0204] The following experiments tested the binding of the antibody PC111 to the FasL protein. In these studies, flow-induced dispersion analysis (FIDA) was used, which utilizes dispersion phenomena in pressure-driven flow to provide an accurate assessment of molecular diffusivity and hydrodynamic radius (Rh) after signal analysis. Changes in the hydrodynamic radius of the binder reflect the strength of the interaction.

[0205] 2.1 Methods and Materials

[0206] Capillary: Coated capillary

[0207] Detector: 480nm fluorescence detection of riboflavin

[0208] Indicator (labeled molecule) consumption per measurement: 39 nL

[0209] Analyte consumption per measurement: 12 μL

[0210] Analysis time: 5.5 minutes per measurement

[0211] PC111-Alexa Concentration: 100nM

[0212] FasL concentration: 0.01 to 320 nM

[0213] Assay buffer: PBS pH 7.45, BSA 0.1% (1 mg / ml)

[0214] Mixing principle: Premix (>10 minutes)

[0215] Temperature: 25℃

[0216] 2.2 Results

[0217] PC111 was used as an indicator at a constant concentration of 15 nM and titrated with FasL from 0.01 to 320 nM. The Rh of PC111 alone was measured to be 4.8 nm, and 6.8 nm when complexed with FasL. Since FasL is a homotrimer, PC111 could theoretically bind to FasL in a stoichiometric ratio of 1 FasL:3 PC111, depending on steric hindrance. Multiple stoichiometric combinations are possible.

[0218] PC111 was flagged, and an increase in its Rh was detected with the addition of sFasL. Figure 1 A). Higher levels of Rh were detected when the sFasL concentration reached the range of 10 to 80 nM. Figure 1 The orange squares in A strongly indicate the presence of higher-order oligomers. When sFasL > 100 nM, the Rh of PC11 drops to 6.8 nm, indicating a 1:1 stoichiometry. No fitting model has yet been developed that also includes higher-order oligomers, as they may differ for each system; therefore, by removing… Figure 1 The three points marked in orange in A were fitted using a standard 1:1 excess indicator model. Figure 1 B). The dissociation constant KD of PC111 and sFasL in solution is 340 pM.

[0219] Example 3 – Membrane Proteome Array

[0220] Membrane proteome array (MPA) technology was used to screen for binding targets of PC111 to the human membrane proteome in order to determine antibody target specificity and potential off-target binding. This technique uses flow cytometry to directly detect the binding of the antibody to membrane proteins expressed in unfixed cells. Figure 2 All target proteins have native conformations and appropriate post-translational modifications.

[0221] 3.1 Determination of screening conditions

[0222] To optimize the ligand concentration and cell line used for screening, HEK-293T cells (ATCC CRL-3216) and QT6 cells (ATCC CRL-1708) were transfected with plasmids at a density of 18,000 cells / well in 384-well cell culture plates. The plasmids encoded known ligand targets, protein A (binding antibody Fc; positive control), or vector-only (pUC; negative control). The transfected cells were then incubated for 36 hours at 37°C and 5% CO2 in a medium consisting of Corning DMEM, 10% FBS, 2 mM L-alanyl-L-glutamine, penicillin-streptomycin (PenStrep), MEM NEAA, and 10 mM HEPES. After incubation, four 4-fold dilutions of each ligand, starting at 20 μg / ml, were added quadruplicately to transfected cells, and the bound ligands were detected in a high-throughput immunofluorescence flow cytometry assay using a single-dose fluorescently labeled secondary antibody.

[0223] The mean fluorescence intensity (MFI) value for each test ligand dilution in each cell line was determined using ForeCyt software (Intellicyt), and plotted using Excel (Microsoft). The high background rate for each assay condition was calculated as the percentage of positive events exceeding the set fluorescence threshold in cells transfected with negative controls. The optimal screening concentration and cell lines for each test ligand (Table 2 below) were determined by background signal (MFI) and high background rate in the vector control. Figure 3 Molecules are preferably screened on HEK-293T cells at the highest concentrations that produce acceptable low background (<50,000 MFI) and the lowest high background rate (<1%). If acceptable screening conditions are not identified on HEK-293T cells, molecules are screened on QT6 cells at the highest acceptable concentrations.

[0224] Table 2

[0225]

[0226] 3.2 Screening using membrane proteome arrays

[0227] Plasmids containing cDNA clones of ~6,000 membrane proteins (representing over 94% of the human membrane proteome) were transfected into HEK-293T cells (18,000 cells / well) in 384-well cell culture plates (one plasmid containing a unique cDNA per well) and incubated at 37°C and 5% CO2 in a medium consisting of Corning DMEM, 10% FBS, 2 mM L-alanyl-L-glutamine, penicillin / strepmycin (Penn / strep), MEM NEAA, and 10 mM HEPES. Each 384-well plate contained wells independently transfected with plasmids encoding either GFP or membrane-binding protein-A constructs, serving as controls for transfection efficiency and antibody binding for fluorescence labeling, respectively. After 36 hours of incubation, cells were dissociated using a CellStripper and reformed into a two-dimensional matrix in new 384-well plates using a JANUS automated workstation. Each well on the matrix plate contained 48 different overexpressed protein components, each represented by a unique combination of two distinct wells on the matrix plate, as it was contained in both the "row" and "column" pools. Therefore, each protein in the MPA was tested twice to determine reactivity. Test ligands were added to the membrane proteomics array matrix plates at predetermined concentrations (Table 2 above), washed in 1×PBS, and detected by flow cytometry using fluorescently labeled antibodies. All flow cytometry data were captured using ForeCyt software (Intellicyt).

[0228] The test ligand targets were then identified by detecting binding to overlapping merged matrix wells from the same transfection plate, allowing for specific deconvolution. Individual targets showing binding above 3 standard deviations above the background in both wells were selected for downstream validation experiments.

[0229] To present individual values ​​of binding of the test ligand to each protein in the membrane proteome array, the two-dimensional binding data were transformed using standard matrix deconvolution. In short, each point (representing a single overexpressed protein) was converted to radians, then transformed using the formula r.sin(2θ), and plotted as target binding ( Figure 4 Any transformed value that is less than three standard deviations below the calculated background fluorescence was identified as nonspecific fluorescence. All data and analyses were performed in Excel (Microsoft).

[0230] 3.3 Target Validation

[0231] To validate any identified off-target interactions, cells were transfected in a 384-well format with plasmids encoding the identified target, protein A, or vector-only. After incubation at 37°C and 5% CO2 for 36 hours, four 4-fold dilutions of each test ligand, starting at 20 μg / ml, were added to the transfected cells, and ligand binding was then detected using high-throughput immunofluorescence flow cytometry (under the same conditions described in Table 2 above).

[0232] If the known target of the molecule is the only validated protein in the MPA screening, validation is not displayed because the assay setup results have already demonstrated specific reactivity in a screening equivalent to the validation screening.

[0233] 3.4 Results

[0234] During the MPA study, in addition to Fc receptors FCGR1A and FCGR2B, PC111 was also identified to bind to Fc-binding control protein A. No binding of PC111 to membrane-bound FASLG was detected. No off-target binding was identified for PC111.

[0235] Example 4 – Pharmacokinetic / Pharmacodynamic Relationship of PC111 and sFasL in Humanized FasL Mice

[0236] This study was conducted to obtain data on the PC111 concentration required to neutralize plasma sFasL. A concanavalin A (Con A) liver injury model was used as a model capable of inducing acute sFasL release into the bloodstream (Heymann et al., 2015). Based on preliminary experiments, it was demonstrated that in WT, a dose of 12 mg / kg Con A induced the highest level of sFasL release 2 hours post-injection, compared to a saline control. In subsequent treatment studies, humanized FasL mice were administered different doses of PC111 or a solvent (PBS) intravenously (iv), and 15 minutes later, they received Con A (12 mg / kg; iv). The study design is shown in Table 3 below (n = 2 animals per group).

[0237] Table 3

[0238]

[0239] Two hours after Con A administration, mice were anesthetized and terminally bled. Total human IgG (representing PC111) and human sFasL were quantified by ELISA. IgG and sFasL were measured three times for each sample. An approximately linear increase in human IgG was observed from PC111 levels of 0.33 mg / kg (0.6 μg / mL) to 10 mg / kg (17 μg / mL). Figure 5 ).

[0240] sFasL was analyzed in the same plasma samples. Administration of Con A caused a significant 7-fold induction of sFasL to >200 pg / mL. Administration of PC111 showed an approximately linear dose-dependent decrease in human sFasL from 0.33 mg / kg (200 pg / mL) to 10 mg / kg (50 μg / mL). Figure 6 ).

[0241] The PC111 dose that produced a 50% reduction in sFasL (ED50) was determined to be 1.25 mg / kg, at a plasma exposure of 5 μg / mL over 2 hours.

[0242] In summary, a positive correlation was observed between increased PC111 dosage and blood levels, leading to a decrease in circulating sFasL levels. Notably, this confirms that PC111 can neutralize human sFasL in the in vivo environment.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof specifically targeting human soluble Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from: (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3: (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2); (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys ValGln Gly (SEQ ID NO: 3) or (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys LeuGln Gly (SEQ ID NO: 4); and (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6), It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3: (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ ID NO: 7), (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9), or (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human Fas ligand protein (FasL); Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects also suffer from immune homeostasis dysregulation and / or have an increased risk of developing immune homeostasis dysregulation.

2. A monoclonal antibody or antigen-binding fragment thereof specifically targeting human soluble Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from: (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3: (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2); (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys ValGln Gly (SEQ ID NO: 3) or (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys LeuGln Gly (SEQ ID NO: 4); and (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6), It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3: (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ ID NO: 7), (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9), or (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human Fas ligand protein (FasL); Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects, wherein the subjects additionally suffer from lymphoproliferative disorders, autoimmune disorders and / or cancer, and / or are at risk of developing lymphoproliferative disorders, autoimmune disorders and / or cancer.

3. A monoclonal antibody or antigen-binding fragment thereof specifically targeting human soluble Fas ligand protein (sFasL), comprising at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein said antibody or antigen-binding fragment is selected from: (i) An antibody or antigen-binding fragment comprising a VH region having heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3: (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1) or (a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO: 2); (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys ValGln Gly (SEQ ID NO: 3) or (b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys LeuGln Gly (SEQ ID NO: 4); and (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5) or (c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO: 6), It also includes a VL region with the following light chain complementarity determination regions (CDRs): CDR L1, CDR L2, and CDR L3: (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ ID NO: 7), (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9), or (ii) Antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment of (i) when binding to human Fas ligand protein (FasL); Use for the prevention and / or treatment of soluble FasL (sFasL)-mediated conditions in subjects who also suffer from secondary comorbid conditions.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, for use according to any one of claims 1 to 3, comprises at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein the amino acid sequences of the heavy chain complementarity-determining regions (CDRs) CDR H1, CDR H2, and CDR H3 are as follows: (a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO: 1), (b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys ValGln Gly (SEQ ID NO: 3), and (c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO: 5), Furthermore, the amino acid sequences of the light chain complementarity-determining regions (CDRs) CDR L1, CDR L2, and CDR L3 are as follows: (a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ ID NO: 7), (b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO: 8), and (c3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 9).

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, for use according to any one of claims 1 to 3, wherein the VL region of said antibody or antigen-binding fragment comprises an amino acid sequence: EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO:10) Furthermore, the VH region of the antibody or its antigen-binding fragment contains an amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYIFI RHGIT WVRQAPGQGLEWMG WINAYNGNTNYAQKVQG RVTMTTDKSTSTAYMELRSLRSDDAAVYYCAR ETMVRGVPLDY WGQGTLVTVSS (SEQ ID NO:11) or QVQLVQSGAEVKKPGASVKVSCKASGYIFI SHGIS WVRQAPGQGLEWMG WINAYSGNTNYAQKLQG RVTMTTDRSTSTAYMELRSLRSDDTAVYYCAR ETMVRGVPCDY WGQGTLVTVSS (SEQ ID NO:12)。 6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, for use according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is the same as that of the antibody or antigen-binding fragment thereof according to claims 1(i), 2(i) or 3, and recognizes the same human soluble FasL epitope.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, used for the purpose according to any one of claims 1 to 3, wherein, When measured by SPR, the dissociation constant of the antibody or antigen-binding fragment with human soluble FasL is less than about 1000 pM, preferably less than about 750, less than about 500 pM or less than about 400 pM, more preferably less than about 350 pM or less than about 200 pM, and most preferably 250 pM.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, for use according to any one of claims 1 to 3, wherein the dissociation constant of the antibody or antigen-binding fragment with membrane-bound FasL (mFasL) is at least about 500 nM, preferably at least about 1000 nM.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, for use according to any one of claims 1 to 3, wherein the antibody is selected from partially or fully human antibodies, chimeric antibodies and / or humanized antibodies, and wherein the antigen-binding fragment of the antibody is selected from Fab, Fab' and / or F(ab')2 and / or single-chain Fv fragments.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, for use according to any one of claims 1 to 3, wherein the antibody has an IgG heavy chain constant region, preferably an IgG1 or IgG4 heavy chain constant region.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 1 to 3, wherein the soluble FasL (sFasL) mediated condition is selected from immune inflammatory and / or infectious conditions and / or skin conditions associated with keratinocyte acantholysis, apoptosis and / or necrosis.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 1 to 3 and 11, wherein the skin condition is selected from bullous and / or blistering skin conditions, preferably selected from pemphigus, toxic epidermal necrolysis (TEN) and / or Stevens-Johnson syndrome (SJS), and most preferably selected from pemphigus.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 1 to 3 and 11 to 12, wherein immune homeostasis dysregulation is a membrane-bound FasL (mFasL) mediated condition, preferably selected from lymphoproliferative disorders, autoimmune diseases and / or cancer.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 1 to 3 and 11 to 13, wherein the lymphoproliferative disorder is selected from autoimmune lymphoproliferative syndrome (ALPS), the autoimmune disorder is selected from Hashimoto's thyroiditis, and the cancer is selected from lymphoma.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 1 to 3 and 11 to 14, wherein the subject has received cancer treatment in the past 5 to 10 years, preferably in the past 3 years, more preferably in the past 1 year.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 3 and 11 to 13, wherein the comorbid condition is selected from Cushing's syndrome, adrenal insufficiency, myasthenia gravis, mucositis, herpes infection, fungal infection, insomnia, hidradenitis suppurativa, type 2 diabetes, hypertension, cataracts, benign prostatic hyperplasia and bone diseases such as osteoporosis, osteoporosis and pathological or fragility fractures.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 3 and 11 to 13, wherein the comorbidity is iatrogenic comorbidity and is preferably selected from hypertension, dyslipidemia, heart disease, diabetes, lung infection and bone diseases such as osteoporosis, osteoporosis and pathological or fragility fractures.

18. A nucleic acid molecule encoding a monoclonal antibody or an antigen fragment thereof according to any one of claims 1 to 10, for use according to any one of claims 1 to 3 and 11 to 17.

19. The nucleic acid molecule of claim 18, used for any one of claims 1 to 3 and 11 to 17, wherein the nucleic acid molecule is a DNA carrier or an RNA molecule.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the nucleic acid molecule according to any one of claims 18 to 19, for use in monotherapy according to any one of claims 1 to 3 and 11 to 17.

21. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a nucleic acid molecule according to any one of claims 18 to 19, for use according to any one of claims 1 to 3 and 11 to 17, in combination with at least one further active ingredient, said further active ingredient being effective against soluble FasL (sFasL) mediated conditions, preferably effective against skin conditions selected from bullous and / or blistering skin conditions, most preferably effective against pemphigus, toxic epidermal necrolysis (TEN), and / or Stevens-Johnson syndrome (SJS).

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the nucleic acid molecule according to any one of claims 18 to 19, for use according to claim 21, wherein the further active ingredient is selected from at least one of steroids, cyclosporine, IVIg, TNF inhibitors, IL17 or IL23 inhibitors and / or plasma exchange.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the nucleic acid molecule according to any one of claims 18 to 19, for use in human therapy according to any one of claims 1-3 and 11-17.

24. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10 or a nucleic acid molecule according to any one of claims 18 to 19, and one or more pharmaceutically acceptable carriers, for use according to any one of claims 1 to 3 and 11 to 17.

25. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a nucleic acid molecule according to any one of claims 18 to 19, or a pharmaceutical composition according to claim 24, administered systemically and / or locally.

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