Title:  Interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders

United States Patent:  6,830,752

Issued:  December 14, 2004

Inventors:  Buyse; Marie-Ange (Merelbeke, BE); Sablon; Erwin (Merchtem, BE)

Assignee:  Innogenetics N. V. (BE)

Appl. No.:  071485

Filed:  February 7, 2002

Abstract

The present invention concerns molecules which bind and neutralize the cytokine interferon-gamma. More specifically, the present invention relates to sheep-derived antibodies and engineered antibody constructs, such as humanized single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies which can be used to treat diseases wherein interferon-gamma activity is pathogenic. Examples of such diseases are: septic shock, cachexia, multiple sclerosis and psoriasis.

Description of the Invention

FIELD OF THE INVENTION

The present invention concerns molecules which bind and neutralize the cytokine interferon-gamma. More specifically, the present invention relates to sheep-derived antibodies and engineered antibody constructs, such as humanized single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies and peptabodies which can be used to treat diseases wherein interferon-gamma activity is pathogenic. Examples of such diseases are: septic shock, cachexia, multiple sclerosis and psoriasis.

BACKGROUND OF THE INVENTION

Interferon-gamma (IFN.gamma.) is a member of the interferon family of immunomodulatory proteins and is produced by activated T helper type-1 cells (Th1 cells) and natural killer cells (NK cells). Apart from its potent antiviral activity, IFN.gamma. is known to be involved in a variety of immune functions (for a review, see Billiau, 1996) and inflammatory responses. Indeed, IFN.gamma. is the primary inducer of the expression of the major histocompatibility complex (MHC) class-II molecules (Steinman et al., 1980) by macrophages and other cell types and stimulates the production of inflammatory mediators such as tumor necrosis factor-alpha (TNF.alpha.), interleukin-1 (IL-1) and nitric oxide (NO) (Lorsbach et al., 1993). In this respect, IFN.gamma. is shown to be important in the macrophage-mediated defence to various bacterial pathogens. Furthermore, IFN.gamma. is also shown to be a potent inducer of the expression of adhesion molecules, such as the intercellular adhesion molecule-1 (ICAM-1, Dustin et al., 1988), and of important costimulators such as the B7 molecules on professional antigen presenting cells (Freedman et al., 1991). Moreover, IFN.gamma. induces macrophages to become tumoricidal (Pace et al., 1983) and provokes Ig isotype switching (Snapper and Paul, 1987).

The anti-viral, tumoricidal, inflammatory- and immunomodulatory activity of IFN.gamma. clearly has beneficial effects in a number of clinical conditions. However, there are a number of clinical situations in which IFN.gamma.-activity has deleterious effects. These include cancer cachexia (Denz et al., 1993; Iwagaki et al., 1995), septic shock (Doherty et al., 1992), skin disorders such as psoriasis and bullous dermatoses (Van den Oord et al., 1995), allograft rejection (Landolfo et al., 1985; Gorczynski, 1995), chronic inflammations such as ulcerative colitis and Crohn's disease (WO 94/14467 to Ashkenazi & Ward), and autoimmune diseases such as multiple sclerosis (M S, Panitch et al., 1986), experimental lupus (Ozmen et al., 1995), arthritis (Jacob et al., 1989; Boissier et al., 1995) and autoimmune encephalomyelitis (Waisman et al., 1996).

Cachexia is a phenomenon often seen in cancer patients and is associated with losses of lean body mass, and altered carbohydrate and lipid metabolism. This so called `chronic wasting syndrom` is often the immediate cause of death. In recent years, interest has focused on the role of proinflammatory cytokines in cancer related cachexia. Current data support the concept that cachexia is linked to the presence of certain cytokines among which IFN.gamma. seems to play a central role. Denz et al. (1993) reported that increased neopterin and decreased tryptophan concentrations--which are closely related to IFN.gamma.-activity--are detected in cachectic patients suffering from hematological disorders. Neopterin is synthesized and secreted by monocytes/macrophages upon stimulation by IFN.gamma. from activated T cells. Tryptophan is an indispensable amino acid which can be catabolized by indoleamine 2,3-dioxygenase, an enzyme induced by IFN's, and which absence initiates mechanisms responsible for cachexia (Brown et al., 1991). The correlation between high neopterin levels, decreased tryptophan levels and weight loss was confirmed by Iwagaki et al. (1995). In experimental models, cancer-induced cachexia can be altered by the administration of IFN.gamma. neutralizing antibodies (Matthys et al., 1991; Langstein et al., 1991) Septic shock is the result of a severe bacterial infection, and remains a common cause of death among critically ill, hospitalized patients despite improvements in supportive care (Bone et al., 1992). Although septic shock may be associated with gram-positive infections, attention has focused on the more common pathogenesis of gram-negative sepsis and the toxic role of endotoxin (=lipopolysaccharide or LPS), a component of the outer membrane of gram-negative and some gram-positive bacteria. Many of the effects of LPS are mediated through the release of cytokines such as TNF.alpha. (Tracey, 1991), IL-1 (Wakabayashi et al., 1991) and IFN.gamma. (Bucklin et al., 1994). Much of the evidence supporting the role of these cytokines as mediators of septic shock comes from lethality studies involving the blockade of individual cytokines, resulting in protection of experimental animals from otherwise lethal doses of endotoxin or gram-negative bacteria. One of the first events in septic shock is the activation of T cells by antigen presenting cells onto which bacterial superantigen is bound (Miethke et al., 1993). Upon activation, for which co-stimulation of CD28 is essential (Saha et al., 1996), these T cells proliferate and produce a surge of proinflammatory cytokines such as IL-2, TNF.alpha. and IFN.gamma. eventuating in the clinical syndrome. Also, it is hypothesized that LPS induces the expression of the .alpha.1/.beta.1 integrin (VLA-1) heterodimer on activated monocytes which then display an increased capacity to adhere to the endothelial basement membrane. Similar effects can be induced by incubation of monocytes with IFN.gamma. (Rubio et al., 1995). VLA-1 might also contribute to further monocyte activation and potentiation of the production of monocyte-derived pro-inflammatory cytokines during sepsis (Rubio et al., 1995). Although very promising results were obtained with antibodies neutralising TNF.alpha. in experimental animal models, clinical trials with anti-TNF.alpha. antibodies revealed only a slight reduction or even no reduction in mortality rate of patients with septic shock (Wherry et al., 1993; Reinhart et al., 1996). A fusion protein containing the extracellular portion of the TNF receptor and the Fc portion of IgG1 also did not affect mortality (Fisher et al., 1996). Pentoxifylline (PTX), a methyl xanthine derivative, is currently being tested for its effect on the outcome of septic shock. PTX is known to lower the serum concentrations of at least TNF.alpha., IL-1 and IFN.gamma. (Bienvenu et al., 1995; Zeni et al., 1996). Initial data reveal that PTX leads to an improvement of the clinical status of septic patients (Mandi et al., 1995). There is evidence that IFN.gamma. is a mediator of lethality during sepsis. Antibodies that either neutralize IFN.gamma. or block the IFN.gamma.-receptor are protecting against lethality (Bucklin et al., 1994; Doherty et al., 1992). A synergistic effect between IFN.gamma. and TNF.alpha. has also been suggested (Doherty et al., 1992; Ozmen et al., 1994). Although not in itself lethal, IFN.gamma. has been shown to be essential for the manifestation of TNF-induced lethality in the generalized Shwartzman reaction (Ozmen et al., 1994).

Bullous, inflammatory and neoplastic dermatoses are a heterogenous group of skin disorders during which IFN.gamma. may play a pathogenic role. Bullous dermatoses encompass epidermolysis bullosa acquisita, bullous penihigoid, dermatitis herpetiformes Duhring, linear IgA disease, herpes gestationis, cicatricial pemhigoid, bullous systemic lupus erythematosis, epidermolysis bullosa junctionalis, epidermolysis bullosa dystrophicans, porphyria cutanea tarda and Lyell-Syndrome (Megahed, 1996). Also erythema exsudativum multiform major (Kreutzer et al., 1996), IgG-mediated subepidermal bullous dermatosis (Chan & Cooper, 1994), bullous lichen planus (Willsteed et al., 1991) and paraneoplastic bullous dermatosis (Pantaleeva, 1990) can be classified among the bullous dermatoses. A pathogenic role of IFN.gamma. during bullous dermatoses has been suggested by Van den Oord et al. (1995). The role of IFN.gamma. during inflammatory and neoplastic dermatoses, compared to bullous dermatoses, has been more extensively investigated. Indeed, it has been demonstrated that IFN.gamma. is involved during the pathogenesis of verrucosis (Asadullah et al., 1997), eosinophilic pustular folliculitis (Teraki et al., 1996), cutaneous T cell lymphoma (Wood et al., 1994), granuloma faciale (Smoller & Bortz, 1993), Sweet's syndrome (Reuss-Borst et al., 1993), atopic eczema (Arenberger et al., 1991), follicular mucinosis (Meisnerr et al., 1991), lichen-planus and psoriasis (Vowels et al., 1994). One of the most extensively studied inflammatory dermatoses is psoriasis. Psoriasis is a hyperproliferative skin disorder affecting approximately 2% of the population. Evidence is accumulating that the disease has a T-cell mediated autoimmune etiology. The role of T-cells in psoriasis has been demonstrated by Gottlieb et al. (1995). The latter authors suggested that, in most of the patients, clinical and histopathological features of psoriasis are primarily linked to skin infiltration by IL-2 receptor-positive leukocytes. Disease improvement can be induced by the administration of a fusion protein composed of human interleukin-2 and fragments of diphteria toxin, which selectively blocks the growth of activated lymphocytes. Other effective anti-psoriatic, T-cell suppressing agents include the immunosuppressive drugs cyclosporin and FK506 (Griffiths, 1986) and anti-CD4 monoclonal antibodies (Morel et al., 1992). More direct evidence for the role of T cells in the induction of the complex tissue alterations seen in psoriasis has been generated by Schon et al. (1997) using a model with scid/scid mice in which they transferred naive, minor histocompatibility mismatched CD4⁺ T-cells, resulting in the development of a skin disorder that resembles psoriasis. The autoimmune character of the disease has been proposed by Valdimarsson et al. (1995) who stated that products of activated T-cells can induce keratinocytes of individuals with psoriatic predisposition to express determinants that are recognized by T cells specific for epitopes on .beta.-haemolytic streptococci. Several data suggest that IFN.gamma. may play a crucial role in the pathogenesis of psoriasis. IFN.gamma., produced by activated T cells would be involved in the recruitment of lymphocytes (Nickoloff, 1988), in the induction of activation and adhesion molecules on epidermal keratinocytes (Dustin et al., 1988), as well as in the abnormal keratinocyte proliferation (Barker et al., 1993). Not only enhanced levels of IFN.gamma. has been detected in psoriatic epidermis (Kaneko et al., 1990), also de novo suprabasal expression of IFN.gamma. receptor in psoriasis has been demonstrated (Van den Oord et al., 1995).

Inflammatory bowel disease (IBD), which encompasses ulcerative colitis and Crohn's disease, is characterized by the appearance of lesions of unknown aetiology in most parts of the gut. IBD is rather common, with a prevalence in the range of 70-170 in a population of 100,000. The current therapy of IBD involves the administration of anti-inflammatory or immunosuppressive agents, which usually bring only partial results, and surgery. In view of the apparent shortcomings of the present treatment, Ashkenazi and Ward (WO 94/14467) suggested the usage of a bispecific antibody construct targeting IFN.gamma. and another molecule, such as IL-1 and TNF.alpha., to treat IBD. However, the exact role of IFN.gamma. during IBD is not well understood.

MS is a severely disabling progressive neurological disease of unknown aetiology, but probably involving autoimmune responses and resulting in the appearance of focal areas of demyelinisation (Williams et al., 1994). MS affects 1 in 1000 persons in the USA and Europe, but due to improved diagnosis that number is increasing. Onset of disease is usually around 30 years of age and, on average, patients are in need of treatment for another 28 years. MS is among the most expensive chronic diseases of western society, based on duration and intensity of care. However, diagnosis of exacerbations and early identification of onset of exacerbations has improved greatly, allowing design of novel treatment strategies. Active multiple sclerosis lesions feature T-lymphocyte and monocyte-macrophage accumulations at plaque margins where myelin is being destroyed. The inflammatory cells that invade the white matter and the soluble mediators that they release are held primarily responsible for myelin breakdown. Population-based studies indicate that certain HLA-antigens occur with higher frequency in patients with MS (with predominant MHC being the Dw2(DR2)DQ1.2 haplotype (Olerup et al., 1991). Similar associations of class I and class II haplotypes have also been detected in other autoimmune disorders such as rheumatoid arthritis and insulin dependent diabetes (Nepom, 1993). The lesions of MS are comparable to those found in chronic relapsing experimental allergic encephalitis (EAE), an autoimmune disease that can be induced in animals by immunization with e.g. whole myelin (Allen et al., 1993) or with the myelin/oligodendrocyte glycoprotein (Genain et al., 1995b). The lesions associated with EAE are similar in appearance as the ones occurring in MS and also contain inflammatory infiltrates of T-cells and macrophages (Genain et al., 1995b). Furthermore, in adoptive transfer experiments, T cells sensitized to specific myelin antigens can transfer the disease state of EAE (Genain et al., 1995b; Waldburger et al., 1996). A few years ago, the American FDA approved the use of the immunosuppressive drug interferon (trade name Betaseron) for treatment of chronic relapsing MS. The effect of this drug--although modest--clearly demonstrates the involvement of the cytokine network in the pathophysiology of MS. In the last few years, a large number of studies have addressed the molecular mechanism by which Betaseron exerts its beneficial effects. Lately, it was shown that IFN.beta. dose-dependently inhibited T-cell proliferation, expression of IL-2 receptors and secretion of IFN.gamma., TNF.alpha. and IL-13 (Rep et al., 1996). Furthermore, it was demonstrated that IFN.beta. could specifically prevent the IFN.gamma.-induced up regulation of MHC class II antigens and adhesion molecules on antigen-presenting cells (Jiang et al., 1995) and human brain microvessel endothelial cells (Huynh et al., 1995).

One of the earliest events in MS is damage of the blood brain barrier (BBB) by activated, encephalitogenic T-cells (Tsukada et al., 1993). The mechanism by which these cells destruct locally the BBB, which is mainly constituted of endothelial cells, is not elucidated, but it is known that at the systemic level, local production of certain cytokines such as IFN.gamma. enhance the capability of lymphocytes to adhere to endothelial cells (Yu et al., 1985; Tsukada et al., 1993). Also, on choroid plexus epithelial cells of EAE animals, an increased expression of ICAM-1 and VCAM-1 (Steffen et al., 1994), for which LFA-1 and VLA-4 are the natural ligands on lymphocytes, has been observed. Mc Carron et al. (1993) reported that adhesion of MBP-specific T lymphocytes was significantly up regulated when cerebral endothelial cells were treated with IL-1, TNF.alpha. or IFN.gamma.. That the adhesion of encephalitogenic T-cells to the endothelium is an early and very important event in the onset of MS is shown by the finding that anti LFA-1 therapy can completely block the induction of EAE (Gordon et al., 1995). Additional circumstantial evidence for a stimulatory role of IFN.gamma. in the pathophysiology of MS comes from observations that disease exacerbations are induced by viral upper respiratory infections, known to stimulate the secretion of IFN.gamma. by type-2 helper T cells (Panitch, 1994). The proinflammatory role of IFN.gamma. in autoimmune disease is strengthened by an earlier finding that treatment of MS patients with hIFN.gamma. resulted in an aggravation of the symptoms (Panitch et al., 1986). The role of IFN.gamma. as proinflammatory cytokine in autoimmune disorders has been studied in several experimentally induced forms of autoimmunity. In experimental neuritis, induced by myelin or antigen-specific T cells in rat, IFN.gamma. clearly acted as pro-inflammatory cytokine and administration of a monoclonal antibody to IFN.gamma. suppressed the disease (Hartung et al., 1990). In the case of experimental autoimmune thyroiditis (EAT) in mice, induced by the injection of thyroglobulin, treatment of the animals with anti-IFN.gamma. at 4 weeks after induction of EAT proved to be beneficial, since characteristic features of EAT such as the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to thyroglobulin, were significantly reduced (Tang et al., 1993).

In the mouse EAE model for MS, where the disease can be induced by injection of either spinal cord homogenate or myelin basic protein, elevated concentrations of several cytokines, including IFN.gamma. were observed both in serum and in the lesions in the CNS (Willenborg et al., 1995). However, administration of anti-IFN.gamma. at the initiation of the disease, resulted in an exacerbation of the disease (Billiau et al., 1988; Duong et al., 1994; Willenborg et al., 1995). It must be noted, however, that in these experiments the effect of anti-IFN.gamma. was determined at the onset of acute EAE rather than at the time of chronic relapse of the disease, which in fact is the only relevant situation for MS. Pathologically, typical acute EAE differs substantially from MS in that prominent inflammation occurs in gray, white and meningeal structures, but demyelisation is scant or absent (Genain et al., 1995b). In order to explain the findings with anti-IFN.gamma.antibodies, the authors suggest a different action of IFN.gamma. at the systemic level (anti-inflammatory action) compared to the local level (inflammatory action) (Billiau et al., 1988), or suggest an early role (within 24 h after immunization) of IFN.gamma. in disease resistance (Duong et al., 1994). Willenborg et al. (1995) conclude that the time of treatment plays a critical role on the outcome and suggest this to be the explanation for conflicting results in different autoimmune processes. Recently, Heremans et al. (1996) described facilitation of spontaneous relapses in chronic relapsing EAE in Biozzi ABH mice by administration of anti-IFN.gamma. during the remission phase. The onset of relapses was delayed when animals were treated with IFN.gamma. during the remission phase, results which are in contradiction to the excacerbation seen in humans who were treated with hIFN.gamma..

An experimental EAE model that more closely resembles the disease course and symptomatology of MS in humans can be found in marmosets. Indeed, in these animals a chronic relapsing-remitting form of EAE can be induced which is characterized by an initial, acute phase with clinically mild neurological signs, followed by recovery. A late spontaneous relapse occurs in these animals and chronic lesions resemble active plaques of chronic MS (Massacesi et al., 1995). This unique model can efficiently be employed to evaluate a prospective therapy for MS. In this model, a critical role for TNF.alpha. in demyelisation is suggested by the observation that rolipram, a selective inhibitor of the type IV phosphodiesterase, suppressed TNF.alpha. secretion and demyelisation (Genain et al., 1995a; Sommer et al., 1995) when administered shortly after immunization, thus interfering with acute EAE. The effect of anti-IFN.gamma. on acute EAE or on disease relapse has to our knowledge never been investigated in marmoset.

Taken together, it is well established that there are a number of clinical situations in which IFN.gamma.-activity has deleterious effects. Consequently, several potential therapies to neutralize IFN.gamma.-activity have been proposed. Among the latter proposals are the use of: anti-IFN.gamma. antibodies (Ozmen et al., 1995; Bucklin et al., 1994), recombinant anti-IFN.gamma. Fv fragments (EP 0528469 to Billiau & Froyen), bispecific molecules (WO 94/14467 to Ashkenazi and Ward), drugs such as pentoxifylline (Bienvenu et al., 1995), synthetic polypeptides which inhibit binding of IFN.gamma. to its receptor (U.S. Pat No. 5,451,658 to Seelig; U.S. Pat. No. 5,632,988 to Ingram et al.), Epstein-Barr virus derived proteins (U.S. Pat. No. 5,627,155 to Moore & Kastelein), soluble IFN.gamma. receptors (EP 0393502 to Fountoulakis et al.; U.S. Pat. No. 5,578,707 to Novick & Rubinstein) and oligonucleotides which bind to IFN.gamma.(WO95/00529 to Coppola et al.). However, these compounds are faced with problems such as suboptimal stability, affinity and clearance rates, lack of specificity, efficacy and tissue penetrance, toxic side effects and unwanted carrier effects. Indeed, the carrier effect of antibodies can limit their efficiency to block the target cytokine. For example, Montero-Julian et al. (1995) showed that during treatment of myeloma patients with anti-IL-6, accumulation of IL-6 in the serum in the form of monomeric immune complexes occurred, hereby stabilizing the cytokine. Furthermore, it has also been shown that the therapeutic efficacy of a cytokine can be prolonged by the formation of cytokine/antibody complexes, since the efficacy of recombinant human IL-2 treatment could be increased by prolonging its in vivo half-life by complexing with an anti-IL-2 antibody (Courtney et al., 1994). The carrier-effect of anti-cytokine antibodies can be overcome by the construction of monovalent scFv fragments, although their low MW (V30.000) and the associated fast clearance rate, make them less suitable candidates for long-term treatment. However, the undesirable carrier effect can be avoided by the formation of higher immune complexes, as such increasing the clearance of the cytokine-antibody complexes (Montero-Julian et al., 1995). The use of monoclonal antibodies for diagnostic or therapeutic purposes in vivo is, besides the carrier effect, also limited because of their nature (i.e. the majority are murine mAb's and administration of antibodies of mouse origin inevitably results in a human anti-mouse antibody [HAMA] response), their suboptimal efficacy, stability and affinity and their large molecular size. Proposed solutions to some of these problems involve the use of F(ab')2, F(ab) and scFv derivatives or of humanized versions of the parent antibody, either by CDR grafting (Kettleborough et al., 1991) or by resurfacing of the antibodies (Roguska et al., 1994). Another proposed solution is the development of several modified antibodies or antibody constructs by bioengineering or chemical methods. Indeed, some mAb's were made more effective by conjugating chemotherapeutic drugs and other toxins to the antibodies (Ghetie and Vitetta, 1994) or by developing bispecific and/or multivalent antibody constructs capable of simultaneously binding several--or two different epitopes on the same--or different antigens. These antibody constructs have been produced using a variety of methods: a) antibodies of different specificities or univalent fragments of pepsin-treated antibodies of different specificities have been chemically linked (Fanger et al., 1992); b) two hybridomas secreting antibodies of different specificity have been fused and the resulting bispecific antibodies from the mixture of antibodies were subsequently isolated; c) genitically engineered single chain antibodies have been used to produce non-covalently linked bispecific antibodies (e.g. diabodies (Holliger et al., 1993), minibodies (Kostelny et al., 1992) and tetravalent antibodies (Pack et al; 1995; WO 96/13583 to Pack) or covalently-linked bispecific antibodies (e.g. chelating recombinant antibodies (Kranz et al., 1995), single chain antibodies fused to protein A or Streptavidin (Ito and Kurosawa, 1993; Kipriyanov et al., 1996) and bispecific tetravalent antibodies (EP 0517024 to Bosslet and Deeman). Recently, also trivalent antibody constructs, named triabodies (Kortt et al., 1997), and pentavalent constructs, named peptabodies (Terskikh et al., 1997), have been described. These constructs may have a higher avidity in comparison to bivalent constructs and may be useful for diagnostic or therapeutic purposes in vivo.

However, and despite the fact that several potential therapies to neutralize IFN.gamma.-activity have been proposed, no prior art exists regarding the production and existence of engineered antibody constructs, such as humanized single-chain Fv fragments, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies, and ruminant-derived antibodies such as sheep antibodies which overcome the above-indicated problems and which can efficiently be used to treat diseases wherein interferon-gamma activity is pathogenic.

SUMMARY OF THE INVENTION

It is clear from the prior art as cited above that problems such as suboptimal stability, affinity, clearance rate, specificity, efficacy, and an unwanted carrier effect and HAMA response hamper the successful usage of several therapeutics which, potentially, could neutralize the activity of IFN.gamma.. Also suggested solutions to overcome some of these problems did not result in the development of effective products. Thus, unpredictable and unknown factors still appear to determine the success of these biologicals. Despite these unknown factors, the present inventors have been able to design and develop useful constructs which effectively neutralize IFN.gamma.-activity. Indeed, the constructs have all a surprisingly high affinity for IFN.gamma., they do not provoke a HAMA or related response, and they do not result in a carrier effect. In addition, some of the constructs pass the blood brain barrier, whereas others have a very good clearance rate. Therefore, the present invention aims at providing a molecule which binds and neutralizes interferon-gamma and which is chosen from the group consisting of:

a scFv comprising the humanized variable domain of the monoclonal antibody D9D10

a chimeric antibody comprising the humanized variable domain of the monoclonal antibody D9D10

a diabody comprising the humanized variable domain of the monoclonal antibody D9D10

a multivalent antibody

a ruminant antibody.

The present invention further aims at providing a multivalent antibody chosen from the group consisting of triabodies, tetravalent antibodies, peptabodies and hexabodies.

The present invention also aims at providing a triabody, tetravalent antibody, peptabody and hexabody which comprise 3, 4, 5 and 6 variable domains, respectively, of different anti-interferon-gamma antibodies.

The present invention further aims at providing a triabody as described above which comprises 3 identical variable domains of an anti-interferon-gamma antibody. A preferred variable domain used in the latter constructs is derived from the mouse anti-interferon-gamma antibody D9D10 which is described by Sandvig et al. (1987) and Froyen et al. (1993) or from the sheep anti-interferon-gamma antibody described in the present application. Therefore, the present invention aims at providing a triabody as described above which comprises 3 identical D9D10 scFv's, 3 identical humanized D9D10 scFv's, 3 identical sheep-derived anti-interferon-gamma scFv's or 3 identical humanized sheep-derived anti-interferon-gamma scFv's.

The present invention further aims at providing a tetravalent antibody (called MoTAb I) as described above which comprises 4 identical domains of an anti-interferon-gamma antibody. More specifically, the present invention aims at providing a tetravalent antibody as described above which comprises either 4 identical D9D10 scFv's or 4 identical sheep-derived anti-interferon-gamma scFv's in the format of a homodimer of 2 identical molecules, each containing 2 D9D10 scFv's or 2 humanized D9D10 scFv's or 2 sheep-derived anti-interferon-gamma scFv's or 2 humanized sheep-derived anti-interferon-gamma scFv's, and a dimerization domain, or, a full-size humanized D9D10 antibody or sheep-derived anti-interferon-gamma antibody to which 2 humanized D9D10 scFv's or 2 humanized sheep-derived anti-interferon-gamma scFv's, respectively, are attached at the carboxyterminus (called MoTAb II) (see FIG. 1).

The present invention further aims at providing a peptabody and hexabody as described above which comprise 5 and 6 identical variable domains of an anti-interferon-gamma antibody, respectively. A preferred variable domain used in the latter constructs is derived from the mouse anti-interferon-gamma antibody D9D10 which is described above or from the sheep anti-interferon-gamma antibody described in the present application. Therefore, the present invention aims at providing a peptabody and hexabody as described above which comprises 5 or 6 identical D9D10 scFv's, 5 or 6 identical humanized D9D10 scFv's, 5 or 6 identical sheep-derived anti-interferon-gamma scFv's, or, 5 or 6 identical humanized sheep-derived anti-interferon-gamma scFv's, respectively.

The present invention further aims at providing a molecule as described above, wherein said ruminant antibody is a sheep antibody.

The present invention also aims at providing a molecule as described above, wherein said sheep antibody is a monoclonal antibody. Furthermore, the present invention aims at providing a humanized antibody, a single-chain fragment or any other fragment which is derived from said monoclonal antibody and which has largely retained the specificity of said monoclonal antibody.

Moreover, the present invention aims at providing methods for producing the above-described molecules.

The present invention further aims at providing a pharmaceutical composition comprising a molecule as described above, or a mixture of said molecules, in a pharmaceutically acceptable excipient.

The present invention also aims at providing a molecule or a composition as described above for use as a medicament.

Furthermore, the present invention aims at providing a molecule or a composition as described above for preventing or treating septic shock, cachexia, immune diseases such as multiple sclerosis and Crohn's disease and skin disorders such as bullous, inflammatory and neoplastic dermatosis.

Finally, the present invention aims at providing a molecule as described above for determining interferon gamma levels in a sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference.

The present invention is based on the finding that a molecule which binds and neutralizes human interferon-gamma and which is chosen from the group consisting of:

a scFv comprising the humanized variable domain of the monoclonal antibody D9D10

a chimeric antibody comprising the humanized variable domain of the monoclonal antibody D9D10

a diabody comprising the humanized variable domain of the monoclonal antibody D9D10

a multivalent antibody

a ruminant antibody is useful to treat diseases where IFN.gamma. activity is pathogenic.

As used herein the terms "molecule which binds and neutralizes IFN.gamma." refer to a molecule which recognizes and binds any particular epitope of IFN.gamma. resulting in the neutralization of any bioactivity of IFN.gamma.. Particular epitopes of IFN.gamma. relate to the so-called E2 epitope recognized and bound by the mAb D9D10, the so-called E1 epitope (Kwok et al., 1993) or any other epitope. IFN.gamma. specifically relates to human IFN.gamma. but may also relate to non-human primate, mouse, rat, sheep, goat, camel, cow, llama or any other IFN.gamma.. Furthermore, the term "bioactivity of IFN.gamma." relates to the antiviral activity (Billiau, 1996), the induction of the expression of MHC-class-II molecules by macrophages and other cell types (Steinman et al., 1980), the stimulation of the production of inflammatory mediators such as TNF.alpha., IL-1 and NO (Lorsbach et al., 1993), the induction of the expression of adhesion molecules such as ICAM-1 (Dustin et al., 1988) and of important costimulators such as the B7 molecules on professional antigen presenting cells (Freedman et al., 1991), the induction of macrophages to become tumoricidal (Pace et al., 1983), the induction of Ig isotype switching (Snapper and Paul, 1987), any pathological and/or clinical activity during diseases where IFN.gamma. is pathogenic (Billiau, 1996) or any other known bioactivity of IFN.gamma.. In this regard, it should be clear that any assay system demonstrating the IFN.gamma.-neutralizing capacity of a molecule, such as the ones described by Novelli et al. (1991), Lewis (1995) and Turano et al. (1992) can be used. Some of these assays are also described in the subsection Evaluation of anti-IFN.gamma. neutralizing molecules in the Examples section of the present application (see further). It should be noted that the molecules which bind and neutralize IFN-.gamma. as described above neutralize at least one bioactivity, but not necessarily all bioactivities, of IFN-.gamma..

The present invention further relates to a scFv comprising the humanized variable domain of the monoclonal antibody D9D10. As used herein, the term single-chain Fv, also termed single-chain antibody, refers to engineered antibody constructs prepared by isolating the binding domains (both heavy and light chain) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function. This forms, in essence, a radically abbreviated antibody, having only the variable domain necessary for binding the antigen. Determination and construction of single chain antibodies are described in U.S. Pat. No. 4,946,778 to Ladner et al. and in the Examples section of the present application (see further). The term "humanized" means that at least a portion of the framework regions of an immunoglobulin or engineered antibody construct is derived from human immunoglobulin sequences. It should be clear that any method to humanize antibodies or antibody constructs, as for example by variable domain resurfacing as described by Roguska et al. (1994) or CDR grafting or reshaping as reviewed by Hurle and Gross (1994), can be used. The humanization of the scFv comprising the variable domain of the monoclonal antibody D9D10 is described further in the Examples section of the present application. The monoclonal antibody D9D10 was prepared essentially as described by Sandvig et al. (1987) and Froyen et al. (1993). It should also be noted that the process of humanization of an antibody or antibody construct is regularly accompanied by a significant loss in binding affinity of this antibody or antibody construct (Kettleborough et al., 1991; Park et al., 1996 and Mateo et al., 1997). In contrast, and surprisingly, the constructs humanized by the present inventors were not characterized by a significant loss in binding affinity in comparison to their non-humanized counterparts.

The present invention also relates to a chimeric antibody comprising the humanized variable domain of the monoclonal antibody D9D10. The term "chimeric antibody" refers to an engineered antibody construct comprising variable domains of one species (such as mouse, rat, goat, sheep, cow, llama or camel variable domains), which may be humanized or not, and constant domains of another species (such as non-human primate or human constant domains) (for review see Hurle and Gross (1994)). It should be clear that any method known in the art to develop chimeric antibodies or antibody constructs can be used. The generation of a chimeric antibody comprising the humanized variable domain of the monoclonal antibody D9D10 is described further in the Examples section of the present application.

The present invention also concerns a diabody comprising the humanized variable domain of the monoclonal antibody D9D10. The term "diabody" relates to two non-covalently-linked scFv's, which then form a so-called diabody, as described in detail by Holliger et al. (1993) and reviewed by Poljak (1994). It should be clear that any method to generate diabodies, as for example described by Holliger et al. (1993), Poljak (1994) and Zhu et al. (1996), can be used. The generation of diabodies comprising the variable domain of the monoclonal antibody D9D10 is described further in the Examples section of the present application.

It should also be clear that the scFv's, chimeric antibodies and diabodies described above are not limited to comprise the variable domain of the monoclonal antibody D9D10 but may also comprise variable domains of other anti-IFN.gamma. antibodies, such as the sheep anti-IFN.gamma. antibody described further in the present application, which efficiently neutralize the bioactivity of IFN.gamma..

Furthermore, the diabodies described above may also comprise two scFv's of different specificities. For example, the latter diabodies may simultaneously neutralize IFN.gamma. on the one hand and may target another molecule, such as TNF-.alpha., IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-b, CD40, CD40L, IL-6, tumour growth factor-beta (TGF-.beta.), transferrin receptor, insulin receptor and prostaglandin E2 or any other molecule, on the other hand.

The present invention also concerns multivalent antibodies which bind and neutralize IFN.gamma.. As used herein, the term multivalent antibody refers to any IFN.gamma.-binding and IFN.gamma.-neutralizing molecule which has more than two IFN.gamma.-binding regions. Examples of such multivalent antibodies are triabodies, tetravalent antibodies, peptabodies and hexabodies which bind and neutralize IFN.gamma. and which have three, four, five and six IFN.gamma.-binding regions, respectively.

The present invention thus relates, as indicated above, to triabodies which bind and neutralize IFN.gamma.. As used herein, the term "triabody" relates to trivalent constructs comprising 3 scFv's, and thus comprising 3 variable domains, as described by Kortt et at (1997) and Iliades et al. (1997). A method to generate triabodies is described by Kortt et al. (1997) and the generation of triabodies comprising the variable domain of the monoclonal antibody D9D10 is described further in the Examples section of the present application. It should be noted that the triabodies of the present invention may comprise: 3 variable domains of 3 different anti-IFN.gamma. Ab's (i.e. 3 anti-IFN.gamma. Ab's which recognize and bind a different epitope on IFN.gamma. [see also above]), 3 variable domains of 3 identical anti-IFN.gamma. Ab's such as 3 variable domains of D9D10 or 3 variable domains of humanized D9D10 or 3 variable domains of sheep anti-IFN.gamma. Ab's or 3 humanized variable domains of sheep anti-IFN.gamma. Ab's, 1 or 2 variable domain(s) of anti-IFN.gamma. Ab's in combination with 2 or 1 variable domain(s) of an Ab which binds to any other molecule than IFN.gamma., respectively. Examples of such other molecules comprise TNF-.alpha., IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6, tumour growth factor-beta (TGF-.beta.), transferrin receptor, insulin receptor and prostaglandin E2.

The present invention further relates to tetravalent antibodies which bind and neutralize IFN.gamma.. As used herein, the term "tetravalent antibody" refers to engineered antibody constructs comprising 4 antigen-binding regions as described by Pack et al. (1995) and Coloma & Morrison (1997). Methods to generate these tetravalent antibody constructs are also described by the latter authors. The generation of the following 2 different tetravalent antibodies comprising the variable domain of the monoclonal antibody D9D10 are described further in the Examples section of the present application: MoTabI which consists of 4 identical humanized D9D10 scFv's in the format of a homodimer of two identical molecules each containing two D9D10 scFv's which are linked together using a dimerization domain; the latter domain also drives the homodimerization of the molecule, and, MoTab II which consists of a full-size humanized D9D10 molecule to which two humanized D9D10 scFv's are attached at the carboxyterminus (CH3-domain). It should be noted that the tetravalent antibodies of the present invention may comprise: 4 variable domains of 4 different anti-IFN.gamma. Ab's (i.e. anti-IFN.gamma. Ab's which recognize and bind to a different epitope on IFN.gamma.), 4 variable domains of 4 identical anti-IFN.gamma. Ab's such as 4 variable domains of D9D10 or 4 variable domains of humanized D9D10 or 4 variable domains of sheep anti-IFN.gamma. Ab's or 4 humanized variable domains of sheep anti-IFN.gamma. Ab's, 2 variable domain(s) of one anti-IFN.gamma. Ab in combination with 2 variable domain(s) of another anti-IFN.gamma. Ab, 2 variable domain(s) of anti-IFN.gamma. Ab's in combination with 2 variable domain(s) which binds to any other molecule than IFN.gamma.. Examples of such other molecules comprise TNF-.alpha., IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6, TGF-.beta. transferrin receptor, insulin receptor and prostaglandin E2.

Furthermore, the term "dimerization domain" of MoTab I refers to any molecule known in the art which is capable of coupling the two identical molecules. Examples of such domains are the leucine zipper domain (de Kruif & Logtenberg, 1996), the helix-turn-helix motif described by Pack et al. (1993), the max-interacting proteins and related molecules as described in U.S. Pat. No. 5,512,473 to Brent & Zervos and the polyglutamic acid-polylysine domains as described in U.S. Pat. No. 5,582,996 to Curtis.

The present invention thus relates, as indicated above, to peptabodies and hexabodies which bind and neutralize IFN.gamma.. As used herein, the term "peptabodies" relates to pentavalent constructs as described in detail by Terskikh et al. (1997). The term "hexabodies" relates to hexavalent constructs which are similar to the pentavalent constructs as described in detail by Terskikh et al. (1997) but wherein the pentamerization domain is replaced by any hexamerization domain known in the art. A method to generate peptabodies is also described by Terskikh et al. (1997) and a method to generate hexabodies can be derived from the description by the latter authors. It should be noted that the peptabodies and hexabodies of the present invention may comprise: 5 (relating to the peptabodies) or 6 (relating to the hexabodies) variable domains of 5 or 6 different anti-IFN.gamma. Ab's (i.e. 5 or 6 anti-IFN.gamma. Ab's which recognize and bind a different epitope on IFN.gamma. [see also above]), 5 or 6 variable domains of identical anti-IFN.gamma. Ab's such as 5 or 6 variable domains of D9D10, or, 5 or 6 variable domains of humanized D9D10, or, 5 or 6 variable domains of sheep anti-IFN.gamma. Ab's, or, 5 or 6 humanized variable domains of sheep anti-IFN.gamma. Ab's, less than 5 or 6 variable domain(s) of any anti-IFN.gamma. Ab's in combination with less than 5 or 6 variable domain(s) of an Ab which binds to any other molecule than IFN.gamma., respectively. Examples of such other molecules comprise TNF-.alpha., IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6, TGF-.beta., transferrin receptor, insulin receptor and prostaglandin E2.

The present in invention further relates to ruminant antibodies which bind and neutralize IFN.gamma.. The term "ruminant" relates to animals belonging to the suborder Ruminantia of even-toed hoofed mammals (as sheep, goats, cows, giraffes, deer, llama, vicunas and camels) that chew the cud and have a complex 3- or 4-chambered stomach.

More specifically, the present invention relates to sheep antibodies which bind and neutralize IFN.gamma.. The term "sheep" relates to any of numerous ruminant mammals belonging to the genus Ovis. The generation of sheep anti-IFN.gamma. antibodies is described in the Examples section of the present application. The present invention also relates to sheep monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. Indeed, the monoclonal sheep antibodies of the present invention can be generated by any method known in the art. It should be noted that also humanized antibodies, scFv's or any other fragment thereof which has largely retained the specificity of said sheep antibody or sheep monoclonal antibody are covered by the present invention. As used herein, the term "fragment" refers to F(ab), F(ab')2, Fv, and other fragments which retain the antigen binding function and specificity of the parent antibody. It should also be understood that the variable domains of the sheep anti-IFN.gamma.(monoclonal) antibodies or scFv of the sheep anti-IFN.gamma. (monoclonal) antibodies may be part of the chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies as described above.

The present invention further relates to scFv's, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies, hexabodies and sheep antibodies which bind and neutralize IFN.gamma. and which are produced by the methods as described above and in the Examples section of the present application.

The present invention further relates to a composition comprising scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. in a pharmaceutically acceptable excipient, possibly in combination with other drugs or other antibodies, antibody derivatives or constructs for use as a medicament to prevent or treat septic shock, cachexia, immune diseases such as multiple sclerosis and Crohn's disease and skin disorders such as bullous, inflammatory and neoplastic dermatoses. Examples of such other drugs or other antibodies, antibody derivatives or constructs are, with regard to septic shock: an isotonic crystalloid solution such as saline, dopamine, adrenaline and antibiotics; with regard to cachexia: anti-TNF-alpha antibodies; with regard to multiple sclerosis: ACTH and corticosteroids, interferon beta-1b (Betaseron), interferon beta-1a (Avonex), immunosuppressive drugs such as azathioprine, methotrexate, cyclophosphamide, cyclosporin A and cladribine (2-CdA), copolymer 1 (composed of 4 amino acids common to myelin basic proteins), myelin antigens, roquinimex A, the mAb CAMPATH-1H and potassium channel blockers; with regard to Crohn's disease: sulfasalazine, corticosteroids, 6 mercaptopurine/azathioprine and cyclosporin A; with regard to psoriasis: cyclosporin A, methotrexate, calcipotriene (Dovonex), zidovudine (Retrovir), histamine2 receptor antagonists such as ranitidine (Zantac) and cimetidine (Tagamet), propylthiouracil, acitretin (Soriatane), fumaric acid, vitamin D derivates, tazarotene (Tazorac), IL-2 fusion toxin, tacrolimus (Prograf), CTLA4Ig, anti-CD4 mAb's and T-cell receptor peptide vaccines. It should also be clear that any possible mixture of the above-indicated IFN-.gamma.-binding molecules may be part of the above-indicated pharmaceutical composition.

As used herein, the term "composition" refers to any composition comprising as an active ingredient scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. according to the present invention possibly in the presence of suitable excipients known to the skilled man. The scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. of the invention may thus be administered in the form of any suitable composition as detailed below by any suitable method of administration within the knowledge of a skilled man. The preferred route of administration is parenterally. In parenteral administration, the compositions of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with a pharmaceutically acceptable excipient. Such excipients are inherently nontoxic and nontherapeutic. Examples of such excipients are saline, Ringer's solution, dextrose solution and Hank's solution. Nonaqueous excipients such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.

The scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. of the invention are administered at a concentration that is therapeutically effective to treat or prevent septic shock, cachexia, immune diseases such as multiple sclerosis and Crohn's disease and skin disorders such as bullous, inflammatory and neoplastic dermatoses. The dosage and mode of administration will depend on the individual. Generally, the compositions are administered so that the scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. are given at a dose between 1 .mu.g/kg and 10 mg/kg, more preferably between 10 .mu.g/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg for each IFN-.gamma.-binding molecule. Preferably, they are given as a bolus dose. Continuous short time infusion (during 30 minutes) may also be used. If so, the scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. or compositions comprising the same may be infused at a dose between 5 and 20 .mu.g/kg/minute, more preferably between 7 and 15 .mu.g/kg/minute (for each IFN-.gamma.-binding molecule).

According to the specific case, the "therapeutically effective amount" of a scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. needed should be determined as being the amount sufficient to cure the patient in need of treatment or at least to partially arrest the disease and its complications. Amounts effective for such use will depend on the severity of the disease and the general state of the patient's health. Single or multiple administrations may be required depending on the dosage and frequency as required and tolerated by the patient.

The present invention further relates to scFv's and/or chimeric antibodies and/or diabodies and/or triabodies and/or tetravalent antibodies and/or peptabodies and/or hexabodies and/or sheep antibodies which bind and neutralize IFN.gamma. for determining IFN.gamma. levels in a biological sample, comprising:

1) contacting the biological sample to be analysed for the presence of IFN.gamma. with a scFv and/or chimeric antibody and/or diabody and/or triabody and/or tetravalent antibody and/or peptabodies and/or hexabodies and/or sheep antibody as defined above,

2) detecting the immunological complex formed between IFN.gamma. and said scFv and/or chimeric antibody and/or diabody and/or triabody and/or tetravalent antibody and/or peptabodies and/or hexabodies and/or sheep antibody.

As used herein, the term "a method to detect" refers to any immunoassay known in the art such as assays which utilize biotin and avidin or streptavidin, ELISA's and immunoprecipitation, immunohistochemical techniques and agglutination assays. A detailed description of these assays is given in WO 96/13590 to Maertens & Stuyver. The immunohistochemical detection of IFN.gamma. in cryosections of spinal cord and brain of non-human primates suffering from experimental autoimmune encephalomyelitis is described in detail in the Examples section of the present application. The term "biological sample" relates to any possible sample taken from a mammal including humans, such as blood (which also encompasses serum and plasma samples), sputum, cerebrospinal fluid, urine, lymph or any possible histological section, wherein IFN.gamma. might be present.

Claim 1 of 18 Claims

What is claimed is:

1. A method for neutralizing interferon-gamma activity in a mammal comprising administering to the mammal a pharmaceutically effective amount of a molecule that binds and neutralizes interferon-gamma, said molecule selected from the group consisting of:

a scFv comprising a humanized variable domain, wherein said variable domain comprises amino acids 1-117 and 133-239 of SEQ ID NO: 85;

a chimeric antibody comprising:

a) a humanized heavy chain variable domain, said heavy chain variable domain having an amino acid sequence as shown in positions 1-117 of SEQ ID NO: 85, and

b) the humanized light chain variable domain, said light chain variable domain having an amino acid sequence as shown in positions 133-239 of SEQ ID NO: 85;

a diabody comprising:

a) a humanized heavy chain variable domain, said heavy chain variable domain having an amino acid sequence as shown in positions 1-117 of SEQ ID NO: 85, and

b) a humanized light chain variable domain, said light chain variable domain having an amino acid sequence as shown in positions 133-239 of SEQ ID NO: 85; and,

a multivalent antibody, wherein said multivalent antibody is selected from the group consisting of a triabody, a tetravalent antibody, a peptabody, and a hexabody, and wherein said multivalent antibody comprises:

a) a humanized heavy chain variable domain, said variable domain comprising amino acids 1-117 of SEQ ID NO: 85; and

b) a humanized light chain variable domain, said variable domain comprising amino acids 133-239 of SEQ ID NO: 85.

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