Patent Application
20110038867
The present invention refers to the use of antagonists of human Fas ligand (also named CD95L or Apo1L and hereinafter abbreviated as FasL), more particularly to the use of humanized antibodies against FasL for the prevention and/or treatment of skin diseases associated with keratinocyte acantholysis, particularly for the prevention and/or treatment of pemphigus.
Pemphigus is an autoimmune bullous skin condition characterized by loss of keratinocyte adhesion due to autoantibodies directed against desmosomal proteins (desmogleins, dsg). Pemphigus has a world-wide distribution with an incidence of approximately 1-5 per 100.000 per year, and with a female predominance.
Pemphigus is characterized by loss of adhesion of suprabasal keratinocytes that round up in a process known as acantholysis. Pathogenesis of pemphigus is undergoing a major revision, mostly because, in addition to anti-desmoglein antibodies, a new group of anti-cholinergic receptor antibodies can induce acantholysis (Kalish, 2000). Further, it has been demonstrated that steric hindrance alone cannot account for blister formation upon antigen-antibody binding (Kitajima, 2002). Further, it was shown that desmosome formation is not inhibited by pemphigus autoantibodies (PVIgG) binding to pemphigus, while the desmosomal connections are dissociated 24-36 hrs after treatment with PvIgG. During this time, a series of signal transduction steps triggered by PVIgG take place.
These observations suggest a role for apoptosis in pemphigus. Indeed, pemphigus is a disease due to lack of cell adhesion (Payne A S et al, 1978) and apoptosis can occur in association with cell detachment (Marconi et al, 2004). These concepts are fully confirmed by the detection of apoptosis in lesional pemphigus skin. In particular, acantholytic cells express the major markers of apoptosis (Wang X et al, 2004). More interestingly, TUNEL-labeling nuclei are also found attached to the blister roof, suggesting the presence of apoptotic cells in perilesional skin before detachment. Moreover, apoptotic cells expressing Ig and activated caspase-8 are detected at the edge of the lesion, in areas where no disruption of cell-cell contacts is visible (Wang X et l, 2004). These data definitely demonstrate that in pemphigus, apoptosis takes place in keratinocytes before acantholysis.
Apoptosis plays a fundamental role in the regulation of cellular homeostasis and is involved in many pathophysiologic processes. Apoptosis can follow both cell to cell detachment (Rezgui et al, 2000; Bergin et al, 2000) and loss of cell-matrix interaction (Giancotti and Ruoslahti, 1999).
Fas (or FasR) is a member of the TNF-receptor superfamily which, upon binding with Fas ligand (FasL), triggers apoptosis in many cell systems (Sharma et al, 2000). Intracellular signaling of Fas-FasL-induced apoptosis operates via recruitment of a number of adaptor molecules such as FADD (Fas-associated death domain) and FLICE (FADD-like ICE, caspase 8), which in turn is inhibited by FLIP (FLICE inhibitory protein) (Juo et al, 1998). Fas-FasL interaction is involved in the pathomechanisms of several immune-inflammatory and infectious conditions, such as AIDS (Bahr et al, 1997) and systemic lupus erythematosus (Kovacs et al, 1997). Cutaneous diseases characterized by an implication of Fas-FasL pathway include acute graft versus host disease, toxic epidermal necrolysis and melanoma (Wehrli et al, 2000). Further, it was reported that acantholytic-like lesions are observed in cultured keratinocytes treated with both PVIgG and with anti-FasR, while PvIgG induce the clustering of FasR, FasL and caspase-8 on the cell membrane several hours before the formation of the lesions (Wang X et al, 2004). Moreover, it was reported that the caspase-1-like inhibitor significantly blocked the blister formation in a pemphigus mouse model (Li et al, 2006). Taken together, these data indicate that FasL and the extrinsic apoptotic pathway play a critical role in the mechanisms underlying acantholysis.
Whatever the nature of the pemphigus autoantibodies, exposure to PVIgG up-regulates the expression of several pro-apoptotic genes, including FasL, many hours before acantholysis. Moreover, intravenous IgG (IVIgG) prevents PvIgG-induced up-regulation of FasL and apoptosis in keratinocytes. IVIgG also prevents acantholysis and apoptosis in vivo (Arredondo J et al, 2005).
Without treatment, the mortality of pemphigus vulgaris approaches 100%. Early systemic therapy is required to control pemphigus, but side effects from systemic therapy are a major complication. Treatment includes administration of corticosteroids, medications containing gold, the anti-inflammatory drug dapsone, or medications that suppress the immune system (such as azathioprine, methotrexate, cyclosporin, cyclophosphamide, or mycophenolate mofetil). The most common treatment for pemphigus is nowadays steroids which need to be administered for life and can cause severe side effects. Most pemphigus patients die by these side effects. Some antibiotics are also effective, particularly minocycline and doxycycline. Intravenous immunoglobulin (IVIg) is occasionally used. Plasmapheresis is a process whereby antibody-containing plasma is removed from the blood and replaced with intravenous fluids or donated plasma. Plasmapheresis may be used in addition to the systemic medications to reduce the amount of antibodies in the bloodstream. A number of new molecules are also under development: mycophenolate mofetil, PI-0824, PRTX-100, anti-CD20 are immunesuppressive drugs which act on T and B cells.
The current mortality rate still ranges from 5 to 25%; infection is the most frequent cause of death and long-term immunosuppressive therapy (mainly corticosteroid) is one of the significant factors still provoking a high mortality rate. Immunoglobulin can produce some short-term improvement, but does not seem to induce lasting remissions, and its cost makes it impractical for long-term use.
There are several caveats also in the use of plasmapheresis. Patients suppressed with prednisone or others immunosuppressive agents and receiving plasmapheresis, are at higher risk of sudden death from sepsis. In addition, the treatment is very expensive, and a 2-week hospitalization period is necessary to administer the therapy. Therefore, because of the risk of sudden death from sepsis and the high cost, plasmaphaeresis is indicated only in the most refractory cases where the patient is clearly at risk of dying from the disease itself.
It is an object of the present invention to provide a therapeutic agent for pemphigus. The development of a new drug which blocks FasL would allow to completely prevent cell detachment and the formation of the skin lesion.
In general, the present invention refers to the treatment of a skin disease associated with keratinocyte acantholysis by administering a FasL antagonist. FasL antagonists may be selected from anti FasL antibodies, particularly humanized or human anti FasL antibodies, nucleic acid effector molecules of Fas expression such as antisense molecules or molecules capable of RNA interference such as siRNA molecules, soluble Fas receptor molecules, antagonistic FasL muteins, and low molecular weight chemical compounds inhibiting the Fas-FasL interaction. FasL antagonists prevent keratinocyte apoptosis and subsequent cell-cell detachment (acantholysis). Thus, FasL antagonists are particularly suitable for the prevention and/or treatment of pemphigus, e.g. for the prevention and/or treatment of mucocutaneous pemphigus.
The present invention relates to a medicament containing at least one compound inhibiting the biological effects of FasL. The expression “compound inhibiting the biological effects of FasL” used herein relates to all the compounds which can fully or at least substantially inhibit or neutralize the biological effects of FasL. For example, the inhibitory or neutralizing effect may be based on suppressing the binding of FasL to its natural receptor and therefore the thus caused signal transmissions. This can be achieved e.g. by using antibodies binding to FasL per se or soluble receptors mimicking Fas or antagonistic FasL muteins, thus blocking the binding of FasL to the cellular receptors. Interfering with Fas or FasL expression by siRNA will block Fas/FasL system.
FasL antagonist therapy is either a monotherapy or be given in combination with other medicaments suitable for the treatment of pemphigus or other skin diseases, particularly as described above. For example, a combination therapy of FasL antagonists and steroids might allow a drastic reduction of the steroid doses.
In a preferred embodiment of the present invention, there is provided a therapeutic agent for pemphigus, comprising an antibody against a human Fas ligand, or an active fragment thereof as an active ingredient. The antibody is preferably a chimeric, humanized or human anti-FasL antibody or an antigen-binding fragment or derivative, e.g. a recombinant single chain antibody. If desired, the antibody may be conjugated to effector molecules, e.g. cytostatic, cytotoxic and/or radioactive compounds.
Preferred humanized antibodies suitable for the treatment of skin disease associated with keratinocyte acantholysis, in particular pemphigus according to the present invention, are described in WO 1997/002290A1 (“Anti-Fas ligand antibodies and assay method using the same antibody”) or in WO 1998/010070 A1 (“Humanized immunoglobulin reacting specifically with Fas Ligand or active fragments thereof and region inducing apoptosis originating in Fas Ligand humanized antibodies”), the contents of which are herein incorporated by reference. Further preferred humanized antibodies suitable for the treatment of skin disease associated with keratinocyte acantholysis, in particular pemphigus, according to the present invention, are described in U.S. Pat. No. 7,262,277 (“Antagonistic Anti-hFas ligand human antibodies and fragments thereof”), the content of which is herein incorporated by reference, too.
Human or humanized antibodies have at least three potential advantages over mouse and in some cases chimeric antibodies for use in human therapy: 1) because the effector portion is human, it may interact better with the other parts of the human system; 2) the human immune system should not recognize the framework or C region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody; 3) injected humanized antibodies will presumably have a half-life more like that of naturally occurring human antibodies, allowing smaller and less frequent doses to be given.
Further preferred FasL antagonists are soluble FasR molecules comprising the extracellular soluble part of the Fas receptor or modified antagonistic FasL molecules which have a competitive or non-competitive antagonistic activity. These molecules inhibit FasL/FasR interactions in that FasL binds to the soluble receptor analogue or the antagonistic FasL molecule binds to the natural receptor thereby reducing or fully eliminating the binding of biologically active FasL to the natural receptor. During treatment with siRNA, an analysis of Fas and/or FasL protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of Fas and/or FasL protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain Fas and/or FasL proteins associated with a trait, condition, or disease.
In an especially preferred aspect, the present invention refers to the use of
In a second especially preferred aspect, the present invention refers to the use of
In a third especially preferred aspect, the present invention refers to the use of
In a fourth preferred aspect, the present invention refers to the use of
In an especially preferred embodiment, the present invention is directed to the use of 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 U.S. Pat. No. 7,262,277, the content of which is herein incoporated by reference. In particular, the preferred anti-FasL human antibodies suitable for the treatment of skin disease associated with keratinocyte acantholysis according to the present invention comprise a light chain variable region comprising a polypeptide with the sequence shown in SEQ ID NO 2 of U.S. Pat. No. 7,262,277 (incorporated herein by reference) and further comprising a heavy chain variable region comprising a polypeptide with the sequence shown in SEQ ID NO 10 or 18 of U.S. Pat. No. 7,262,277 (incorporated herein by reference). More particularly, the invention refers to the use of the anti-hFas human antibody 3E1 and/or 4G11 as described in U.S. Pat. No. 7,262,277 (incorporated herein by reference) for the manufacture of a medicament for the prevention and/or treatment of skin disease associated with keratinocyte acantholysis, particularly of pemphigus.
In a still further preferred aspect, the present invention refers to the use of
In a finally further preferred aspect, the present invention refers to the use of
The medicament of the present invention may be provided as a pharmaceutical composition together with a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is administered by injection or infusion, e.g. intravenously, intraarterially, subcutaneously, intraperitoneally or by other suitable means. The composition may be administered locally or systemically. Preferably, the composition is administered systemically.
Pharmaceutical compositions suitable for use in the invention comprise the active agent in an effective amount to achieve the intended purpose. An effective dose of a medicament of the present invention may be in the range of 0.1 μg to 100 mg, up to a total dose of about 1 g depending upon the route of administration. The pharmaceutical compositions may be administered daily, e.g. once or several times, or every two to four days, every week or once in two weeks. The medicament may be administered in a single treatment cycle consisting of one or several medicament administrations or in several treatment cycles each consisting of one or several medicament administrations. Each treatment cycle may have a duration of one day up to several weeks, months, or even years.
Hence, according to the present invention the medicament may also be used in a combination therapy with at least one further therapy effective against a skin disease associated with keratinocyte acantholysis and in particular against pemphigus. The medicament will be used either alone or in combination with other immunosuppressive drugs, particularly with steroids, in order to reduce their dose and/or to minimize their chronic side effects. When used in combination, the medicament will preferably be administered on a monthly basis. When used alone, the medicament will preferably be administered continuously within a time frame depending on the individual case.
Further, the present invention shall be explained in more detail by the following examples.
We first evaluated the presence of apoptosis in epidermis from perilesional skin in frozen sections from untreated pemphigus patients by TUNEL staining. Fluorescent specimens were analyzed by confocal scanning laser microscopy. In suprabasal layers from perilesional epidermis most keratinocytes are apoptotic, as compared to normal skin (
In order to confirm apoptosis in pemphigus lesions we used formalin-fixed and paraffin embedded biopsies and detected the active form of caspase-3. Staining protocol was performed by UltraVision LP Detection System AP Polymer and Fast Red Chromogen (Lab Vision Corporation, CA, USA) according to manufacturer's instruction. Visualization was obtained with Fast Red tablets in naphthol phosphate substrate. In pemphigus samples we found that caspase-3 fragment is located both in the roof and in the floor of the blister, with some cells being positive in perilesional epidermis (
As apoptotic keratinocytes are abundantly expressed in pemphigus, we wanted to explore whether pemphigus sera are capable of inducing apoptosis in normal human keratinocytes. To this purpose, keratinocytes were plated in chamber slides and cultured in serum-free medium (KGM) up to preconfluence. Cells were then cultured in keratinocyte basal medium and treated for 48 h with the addition of 25% serum from either untreated patients or patients treated with systemic corticosteroids. Sera from healthy subjects were used as controls. Apoptosis was evaluated by TUNEL staining in situ. Approximately 100 cells were evaluated, in randomly selected fields, and the percentage of TUNEL-positive cells was counted. Sera from pemphigus but not from healthy subjects or patients undergoing steroid treatment induced apoptosis in human keratinocytes (
As the Fas/FasL system is implicated in many apoptotic processes also at the skin level (Wehrli et al, 2000), we measured FasL levels in sera from pemphigus patients by a two-site enzyme immunoassay (ELISA). Serum concentration was determined by absorbance at 450 nm against recombinant human FasL standard protein. FasL levels were very high in sera from untreated patients and below the limit of detection in sera from patients treated with corticosteroids or in sera from healthy subjects. Sera from HBV patients were used as positive control (
As FasL is contained in high amounts in pemphigus sera, we looked at the expression of its cognate receptor FasR. To this purpose we used formalin-fixed and paraffin embedded biopsies. Staining protocol was performed by UltraVision LP Detection System AP Polymer and Fast Red Chromogen (Lab Vision Corporation, CA, USA) according to manufacturer's instruction. Visualization was obtained with Fast Red tablets in naphthol phosphate substrate. While FasR is expressed only in basal keratinocytes in normal skin, in active pemphigus lesions, FasR is detected both in the basal and in the suprabasal cells. Even more intriguing, in mucocutaneous pemphigus (PMC) FasR seems to be expressed throughout the epidermal layers and even before blister formation (
FasL is one of the major triggers of the caspase-8 activated extrinsic apoptotic pathway. Therefore, we wanted to evaluate whether this pathway plays a role in pemphigus apoptosis. To this purpose, patient sera were pretreated with anti-FasL neutralizing antibody or caspase-8 inhibitor, and added to keratinocyte cultures. Keratinocytes were cultured in KGM and treated with pemphigus sera or with sera from untreated patients. Sera were pretreated with anti-FasL neutralizing antibody (2.5 mg per ml for 30 min) or caspase-8 inhibitor Z-IETD-FMK (100 μM for 30 min). Apoptosis was evaluated by TUNEL staining. Addition of anti-FasL neutralizing antibody or caspase-8 inhibitor partially prevented pemphigus sera-induced keratinocyte apoptosis (
In addition, Keratinocytes were treated as in
Taken together, these data suggest that pemphigus sera induce keratinocyte apoptosis through the extrinsic apoptotic pathway triggered by the Fas/FasL system.
Recent studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis (Weiske et al, 2001). In order to evaluate whether Fas/FasL-induced apoptotic pathway is also responsible for desmosomal separation, we treated for 72 hrs confluent keratinocytes, cultivated in KGM in presence of 1.8 mM CaCl2, with pemphigus sera with or without therapy. Protein extracts from the culture were analyzed by Western blotting using anti-Dsg1 and anti-Dsg3 antibodies. β-actin was used as internal control. We found that pemphigus sera can cleave Dsg1 and Dsg3. In particular, sera from untreated patients, but not from patients under steroid therapy strikingly cleave Dsg1 and Dsg3. (
Most importantly, treatment of keratinocytes with increasing amounts of FasL (0.1, 10, 100 ng/ml) for 72 hrs, cleaved dsgs in a dose-dependent manner. Protein extracts from the culture were analyzed by Western blotting using anti-Dsg1 and anti-Dsg3 antibodies. β-actin was used as internal control. These doses are consistent with the ones detected in pemphigus sera (
Given that FasL exerts an important role in the pathogenesis of pemphigus, we have tested an anti-FasL antibody (NOK2, antibody produced by the hybridoma cell line NOK2, accession number No. FERM BP-5045). Confluent keratinocytes, cultivated in KGM with 1.8 mM CaCl2, were treated for 72 hrs with: 1. KGM alone; 2. anti-FasL (NOK2, 15 μg/ml) Ab; 3. FasL (50 ng/ml); 4. FasL+anti-FasL Ab. We present evidence that anti-FasL Ab (NOK2) prevents FasL-induced dsg cleavage. We also show that anti-FasL Ab (NOK2) inhibits caspase-8-induced apoptosis (
In order to further confirm the central role of FasL, we have used other anti-FasL antibodies (F918-7-3, antibody produced by the hybridoma cell line with accession number No. FERM BP-5533; F918-7-4, antibody produced by the hybridoma cell line with accession number No. FERM BP-5534; F919-9-18, antibody produced by the hybridoma cell line with accession number No. FERM BP-5535). Confluent keratinocytes, cultivated in KGM with 1.8 mM CaCl2, were treated for 72 hrs with: KGM alone; recombinant FasL (50 ng/ml); hybridoma medium diluted 1:1 in KGM; FasL+hybridoma medium at different dilution in KGM. We present evidence that anti-FasL antibodies prevents FasL-induced dsg3 cleavage in a dose-dependent manner (
To investigate whether the extrinsic apoptotic pathway is responsible for dsg cleavage, we pretreated confluent keratinocytes with caspase-8 inhibitor Z-IETD-FMK (100 μM for 30 min) or with anti-FasL (NOK2, 15 μg/ml) Ab. Then cells were incubated for 72 hrs with healthy or untreated pemphigus sera. Protein extracts from the culture were analyzed by Western blotting using anti-Dsg3 antibodies and anti-caspase-8 Ab. Vinculin was used as internal control. (
In conclusion we have shown that FasL exert dual activity, by both activating the caspase-8 mediated extrinsic apoptotic pathway and Dsg cleavage. In agreement with our work, Wang and coworkers (Wang et al, 2004) have suggested that apoptosis could be the cause of the acantholytic phenomenon. They showed that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) coaggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation.
A well established animal model has been long and widely used for studying pemphigus. Passive transfer of PVIgG into neonatal mice induce cell detachment and the formation of the bulla. This model has been used to assess the involvement of apoptosis and FasL in the pathogenesis of pemphigus. We injected subcutaneously PVIgG (5 mg/g/BW) purified from patients sera in newborn C57BL/6N Crl mice. Normal newborn mice treated with IgG purified from sera of healthy individuals (NIgG) will be used as controls. Animals were sacrificed 20 hours after injection.
Hematoxilin and eosin staining shows that blister develop only in mice treated with PVIgG, but not in mice treated with normal human IgG (
In order to evaluate the role of FasL in vivo, mice were treated with PVIgG or PVIgG plus anti-FasL antibody (MFL3 clone, specific for mouse). Anti-FasL (40 μg/mouse) was administered 3 hrs after PVIgG injection and prevented blister formation in mice, as shown by H & E staining. In addition, the length of clefts in anti-FasL treated mice was markedly reduced (
In conclusion we have shown that FasL exert dual activity, by both activating the caspase-8-mediated extrinsic apoptotic pathway and Dsg cleavage. Most importantly, blocking FasL protects from acantholysis in vitro and in vivo.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP08/10597 | 12/12/2008 | WO | 00 | 9/21/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61013147 | Dec 2007 | US | |
| 61051801 | May 2008 | US |
