Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70521—CD28, CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• the present invention relates to the field of autoimmune diseases.
• the present invention relates to methods of treating morphea with selective co- stimulation modulators binding to the CD80/CD86 ligands on an antigen- presenting cell.
• Morphea is an uncommon disorder. It is more frequent among women, and in young adults aged 20-40 years. The aetiology of morphea is unknown. It is believed that inflammatory processes in the skin induce increased synthesis of collagen from fibroblasts. At present, active superficial morphea can be treated with ultraviolet Al (UVA1) with good results. Meanwhile, there is no efficient therapy for the profound, progressive and destructive morphea variants.
• UVA1 ultraviolet Al
• WO 93/00431 (Bristol-Myers Squibb) identifies the CTLA4 receptor as ligand for the B7 antigen. WO 93/00431 further provides a CTLA4 immunoglobulin fusion protein comprising the extracellular domain of the CTLA4 receptor.
• recombinant chimeric fusion protein may be used to react with B7 positive cells to regulate immune responses mediated by T cell interactions with the B7 antigen positive cells.
• WO 01/92337 Bristol-Myers Squibb provides mutant molecules of the CTLA4 immunoglobulin fusion protein comprising the
• an object of the present invention relates to the provision of a medicament for the treatment of morphea and methods of treating morphea using said medicament.
• one aspect of the invention relates to method of treating or alleviating the symptoms of morphea comprising administration to a subject in the need thereof a therapeutically effective amount of a fusion protein, where said fusion protein is selected from the group consisting of:
• a fusion protein comprising a first amino acid sequence comprising an amino acid sequences corresponding to the extracellular domain of a cytotoxic T- lymphocyte-associated protein 4 (CTLA4) and a second amino acid sequence comprising amino acid residues corresponding to the constant region of an immunoglobulin molecule
• CTLA4 cytotoxic T- lymphocyte-associated protein 4
• the fusion protein of (a) having one or more mutations (preferable point mutations) in said extracellular domain of CTLA4 and/or one or more mutations (preferable point mutations) in said constant region of an immunoglobulin molecule Another aspect of the present invention relates to the use of fusion protein as defined herein for the manufacture of a medicament for the treatment or alleviation of the symptoms of morphea.
• Yet another aspect of the present invention is to provide the fusion protein as defined herein for use in the treatment or alleviation of the symptoms morphea.
• Figure 1 shows Patient No. 1 with disseminated morphea profunda (top picture) before treatment with abatacept and (bottom picture) after 16 treatments. For further details see Case 1 of Example 1.
• Figure 2 shows Patient No. 2 with disseminated morphea profunda (top picture) before treatment with abatacept and (bottom picture) after 5 treatments. For further details see Case 2 of Example 1.
• the present invention will now be described in more detail in the following. Detailed description of the invention
• identity is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
• sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
• the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned .
• the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
• the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
• Gapped BLAST may be utilised.
• PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules.
• sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
• the default settings with respect to e.g. "scoring matrix” and "gap penalty” may be used for alignment.
• the BLASTN and PSI BLAST default settings may be advantageous.
• the percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
• mutation refers to an alteration in the amino acid sequence of a reference (wildtype) polypeptide leading to a mutant polypeptide.
• the alteration is typically reflecting an underlying alteration in the nucleic acids sequence encoding wildtype (or reference) polypeptide.
• the reference polypeptide when referring to a mutated extracellular domain of human CTLA4 the reference polypeptide is the extracellular domain of human CTLA4 as set forth in SEQ ID NO : SEQ #3. Fusion protein
• fusion protein or "chimeric fusion protein” refers to a polypeptide comprising a least two heterologous protein sequences (subunits) joined to form a single polypeptide sequence. Fusion proteins are designed and prepared using standard techniques known the skilled person in the art. Typically, the least two nucleic acids sequences encoding the protein subunit are ligated in frame to generate a nucleic acid sequence comprising an open reading frame encoding said fusion protein. The nucleic acid sequence may be employed to produce the fusion protein by recombinant expression in a suitable host, for example CHO cells.
• Cytotoxic T lymphocyte associated antigen 4 (CTLA-4/CD152)
• Cytotoxic T lymphocyte associated antigen 4 (CTLA-4/CLTA4/CD152) is
• CTLA-4 was identified by differential screening of a murine cytolytic T cell cDNA library. Human CTLA-4 was cloned in 1988. Expression was first reported using a soluble genetic fusion of the extracellular domain to an Fc domain and this fusion protein was shown to be a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes (Linsley et al., J. Exp. Med. (1991) 174:561-569). The structure of CTLA-4 extracellular domain is characteristic of the immunoglobulin variable domain. The human CTLA-4 precursor sequence is identified by the Swiss-Prot primary accession number: P16410.
• CTLA-4 appears to be critical for the negative regulation of T cell responses.
• CTLA-4 aggregation of CTLA-4 has been found to provide inhibitory signals that down- regulate T cell responses (Allison and Krummel, (1995) Science 270:932).
• the B7 molecules have a higher affinity for CTLA-4 than for CD28 (Linsley et al., (1991) J. Exp. Med . 174: 561-569) and B7-1 and B7-2 have been found to bind to distinct regions of the CTLA-4 molecule and have different kinetics of binding to CTLA-4 (Linsley et al., (1994) Immunity 1 :793).
• the importance of the B7 :CD28/CTLA-4 co-stimulatory pathway has been demonstrated in vitro and in several in vivo model systems.
• Blockade of this co- stimulatory pathway results in the development of antigen specific tolerance in murine and human systems.
• expression of B7 by B7 negative murine tumor cells induces T-cell mediated specific immunity accompanied by tumor rejection and long lasting protection to tumor challenge.
• Activation of CTLA-4 for example, transmits a negative signal to a T cell.
• Engagement of CTLA-4 inhibits IL-2 production and can induce cell cycle arrest.
• mice that lack CTLA- 4 develop lymphoproliferative disease.
• the blockade of CTLA-4 with antibodies may remove an inhibitory signal, whereas aggregation of CTLA-4 with antibody transmits an inhibitory signal.
• the CTLA-4 sequence can, inter alia, have Ala or with Met as the N- terminal amino acid residue of the protein. Accordingly, the amino acid sequence corresponding to the extracellular domain of human CTLA-4 as set forth in SEQ ID NO: 3 is having the Met at position 1.
• the alternative version of the extracellular domain of human CTLA-4 having the Ala as the N-terminal amino acid residue of the protein is provided with SEQ ID NO: 4.
• Reference to amino acid residues in the extracellular domain of human CTLA-4 is, unless otherwise indicates, made using the extracellular domain of human CTLA-4 as set forth in SEQ ID NO : 3 as reference molecule.
• SEQ ID NO : 5 provides CTLA4Ig having a Met as the N- terminal amino acid residue of the protein.
• the alternative version having the Ala as the N-terminal amino acid residue is provided with SEQ ID NO : 6.
• SEQ ID NO: 7 provides the L104EA29Y variant having a Met as the N-terminal amino acid residue of the protein.
• the alternative version having the Ala as the N- terminal amino acid residue is provided with SEQ ID NO : 8.
• SEQ ID NO: 9 provides the L104E variant having a Met as the N-terminal amino acid residue of the protein.
• the alternative version having the Ala as the N- terminal amino acid residue is provided with SEQ ID NO: 10.
• the variants of the extracellular domain of human CTLA-4 having the sequences set forth in SEQ ID NO : 11, SEQ ID NO: 11.
• SEQ ID NO : 14 are in the form having a Met as the N-terminal amino acid residue of the protein. It follows the alternative version having Ala as the N-terminal amino acid residue may also be used.
• CTLA-4-Ig (CTLA4Ig, abatacept, Orencia,) is the extracellular domain of CTLA-4 fused to the Fc of IgGl, the resulting soluble protein is a dimer with a molecular weight of approximately 92 kDa. It is being developed by Bristol-Myers Squibb Co (BMS) for rheumatoid arthritis (RA). Abatacept contains in its CDR3-like domain the amino acid hexapeptide motif MYPPPY, which is shared between CD28 and CTLA-4 and is necessary for binding to the B7 ligands.
• SEQ ID NO : 18 comprises the amino acid hexapeptide motif MYPPPY.
• LEA-29Y A second generation form of abatacept has been, referred to as LEA-29Y (or belatacept), has being developed by Bristol-Myers Squibb as an
• This mutant molecule (set forth in SEQ ID NO : 7) having glutamic acid (E) instead of leucine (L) at position 104 and tyrosine (Y) instead of alanine (A) at position 29 of the extracellular domain of CTLA4 (SEQ ID NO : 3) exhibits approximately 2-fold greater binding avidity for CD80 (B7-1) and approximately 4-fold greater binding avidity for CD86 (B7-2) than abatacept.
• E glutamic acid
• L leucine
• Y tyrosine
• A alanine
• Scleroderma encompasses a spectrum of disorders characterized by thickening of the skin and subcutaneous tissue. Two d ifferent clinical categories generally are identified : systemic sclerosis, in which visceral changes are present, and localized scleroderma (morphea), in which lesions are limited to the skin .
• systemic sclerosis in which visceral changes are present
• morphea localized scleroderma
• morphea The aetiology of morphea is unknown . It is believed that inflammatory processes in the skin induce increased synthesis of collagen from fibroblasts. Several types of localized sclerosis or morphea may be d isting uished on the basis of clinical manifestations and levels of tissue involvement. However, morphea classification is hampered by common overlapping of the various types.
• a morphea classification have been proposed that includes the plaque (morphea en plaque, guttate morphea, atrophoderma of Pasini and Pierini, keloid morphea, and lichen sclerosus et atrophicus), generalized, bullous, linear (en coup de sabre, linear scleroderma, and prog ressive hemifacial atrophy), and deep (morphea profunda, eosinophilic fasciitis, and d isabling pansclerotic morphea of children) types and their correspond ing subtypes (in parenthesis) .
• Generalized morphea is a form of localized scleroderma .
• morphea skin plaques When morphea skin plaques are very widespread, it is referred to as generalized morphea .
• the skin patches become very hard and dark and spread over large areas of the body. Underlying muscles are often affected, causing them to tig hten and atrophy.
• Symptoms of generalized morphea are widespread morphea lesions over large areas of the body, which may sometimes cause limb contractures and atrophy.
• Antiphospholipid Syndrome Symptoms of Antiphospholipid Synd rome include deep vein thrombosis (DVT), chronic leg ulcers, recurrent miscarriages, headache, heart attacks, renal vein and artery thrombosis, pulmonary embolism, and pulmonary hypertension . It is identified by the presence of anticardiolipin antibodies, in a blood test.
• MBP major basic protein
• Linear scleroderma “en coup de sabre” (LSCS) and plaque morphea are both variants of localized scleroderma.
• LSCS presents as band-like sclerotic skin lesions, usually involving a single unilateral change in the frontoparietal area of the head. This is characterized by atrophy and a furrow of the skin. Progressive involution of the craniofacial bones may result in hemifacial atrophy, which appears to be identical to the Parry Romberg syndrome (idiopathic progressive facial hemiatrophy). A clear differentiation between Parry Romberg syndrome and LSCS is often not possible and the aetiology of the two conditions may be similar.
• Plaque morphea is characterized by circumscribed sclerotic plaques with ivory coloured centres and, in the active state of the disease, with violaceous borders. With further progression the centre becomes white or yellowed. Most commonly the lesions are single or few in number, but they may be multiple and are typically localized on the trunk and the extremities. Compared with plaque morphea, LSCS may be accompanied by neurological complications such as epileptic seizures and other focal neurological symptoms. Ipsilateral abnormalities of brain imaging have been reported in patients with LSCS.
• Morphea Profunda is a generalized or, more rarely, localized sclerotic process that mainly involves the deep dermis and subcutaneous tissue but also the fascia and superficial muscle.
• plaques are mildly inflamed, hyperpigmented, symmetrical, and somewhat ill-defined. The skin feels thickened and bound down to the underlying fascia and muscle. Plaques are smooth and shiny, but areas of both dermal and subcutaneous atrophy may be present, particularly in chronic lesions.
• Clinical findings may be difficult to distinguish from generalized morphea. The limits between morphea profunda and generalized morphea are not clear and may well be part of a spectrum including generalized morphea and eosinophilic fasciitis.
• Morphea profunda is more frequent in females than males.
• the onset of sclerosis is gradual and relatively rapid, usually occurring during a period of several months. Signs of acute inflammation, such as edema and erythema, are rarely observed . Increased physical exertion has been invoked as a possible precipitating factor.
• Eosinophilic fasciitis is an uncommon disease characterized by a symmetrical, scleroderma-like thickening of the skin of the limbs. Most patients start with an edematous phase that may be accompanied by pitting edema of the extremities.
• the upper extremities medial aspects typically show skin dimpling resulting from focal dermis-to-fascia tethering caused by sclerotic bands striding the panniculus, which is followed by a final stage consisting of induration and tightening of the skin.
• Lesions of morphea on different parts of the body are present in a significant proportion of cases (30%). Usually, these lesions are not synchronous with fasciitis and appear either before or after fascial inflammation.
• Hypergammaglobulinemia, peripheral eosinophilia, and increased ESR are features of eosinophilic fasciitis but, because they may be transient, normal laboratory findings do not rule out the diagnosis.
• eosinophilia may be seen on histological examination of the fascia (often in association with peripheral eosinophilia), its presence is not required for the diagnosis. Extracutaneous involvement most commonly presents as synovitis or tenosynovitis, arthritis, contractures, or carpal tunnel syndrome. Although uncommon and mild, other manifestations of visceral disease in eosinophilic fasciitis are pulmonary, muscle, esophageal, and even cardiac abnormalities.
• Disabling pansclerotic morphea of children is an aggressive mutilating variant of morphea with onset before the age of 14 years, although an adult onset has been described.
• This condition involves all layers of the skin, extending through the dermis and subcutaneous tissues to involve muscle, tendon, and bone.
• sclerotic plaques develop on the extensor aspects of the extremities and trunk to progressively involve the entire skin, including the neck, face, and scalp, with sparing of the fingertips and toes and lack of Raynaud's phenomenon.
• pansclerotic morphea Extensive, severe, unilateral lesions of pansclerotic morphea also have been described. Painful ulcerations and contracture deformities, often severe and disabling, occur as a result of pansclerotic changes. Soft-tissue calcification and subsequent ulceration are recognized complications posing significant
• localized scleroderma is still unsatisfactory. Unlike other less aggressive forms of localized scleroderma (plaque morphea), deep types of morphea showing evidence of progressive inflammatory and fibrotic lesions require treatment with anti-inflammatory drugs or other disease-modifying agents. Although their action mechanism is incompletely understood, daily antimalarial agents (chloroquine or hydroxychloroquine sulfate) are being used, presumably for their immunomodulating properties. Sometimes, in addition to antimalarials, more aggressive treatment may be necessary, including systemic corticosteroids (prednisone at a dose of approximately 1 mg/kg).
• systemic corticosteroids prednisone at a dose of approximately 1 mg/kg.
• UVA irradiation in the form of bath and oral psoralen, as well as high- and low-dose UVA1 therapy, has been applied to the treatment of localized scleroderma, even in extensive and deep forms such as pansclerotic morphea, with beneficial responses. UVA irradiation have been postulated to increase the activity of metalloproteinases and modulate the expression of cytokines participating in connective tissue
• a fusion protein comprising a first domain corresponding to the extracellular domain of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and a second domain corresponding to the constant region of an immunoglobulin molecule (exemplified by abatacept) may be successfully applies in the treatment or alleviating the symptoms of localized scleroderma (also referred to as morphea).
• CTLA4 cytotoxic T-lymphocyte-associated protein 4
• abatacept an immunoglobulin molecule
• the present invention employs recombinant chimeric fusion protein comprising a first protein domain comprising an amino acid sequences corresponding to the extracellular domain of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) joined to a second protein domain comprising amino acid residues corresponding to the constant region of an immunoglobulin molecule.
• the first protein domain is positioned N-terminal to the second protein domain in the fusion protein.
• the domain may be joined directly or separate by a spacer (or junction sequence).
• the fusion protein employed by the present invention is preferably obtained by recombinant expression using a mammalian producer cell.
• the fusion protein is expressed as an immature fusion protein having a signal peptide sequence located at the N- terminal end of the fusion protein.
• the fusion protein may be expressed with an upstream signal that is cleaved as the protein is secreted.
• Various signal peptides sequences may be employed for example the oncostatin M signal peptide as disclosed in WO 93/00431, which is hereby incorporated by reference. The choice of signal peptides sequences may depend on the expression system applied for recombinant production of the fusion protein .
• the sig nal peptide is cleaved of the immature fusion protein (post- translational processing) to obtain the mature fusion protein .
• the exact position of the cleavage may vary slightly (one or two amino acids) .
• the mature fusion protein may undergo further post-translational processing step which removes one or more amino acid resid ues from the N-term of the cleavage product.
• the mature form of the recombinant chimeric fusion protein is preferably used by the method of the present invention .
• the person skilled in the art in familiar with the art recombinant expression of proteins such as the above mentioned chimeric fusion proteins.
• WO 93/00431 and WO 01/92337 provide methods that may be used for producing the fusion proteins used by the present invention .
• WO 93/00431 and WO 01/92337 are hereby incorporated by reference.
• one aspect of the invention relates to method of treating or alleviating the symptoms of morphea comprising ad ministration to a subject in the need thereof a therapeutically effective amount of a fusion protein, where said fusion protein is selected from the group consisting of:
• a fusion protein comprising a first amino acid seq uence comprising an amino acid sequences correspond ing to the extracellular domain of a cytotoxic T- lymphocyte-associated protein 4 (CTLA4) and a second amino acid sequence comprising amino acid resid ues corresponding to the constant reg ion of an immunoglobulin molecule
• Fusion protein used by the method of the present invention may be a fusion protein sharing a hig h sequence identity with the fusion protein set forth in SEQ ID NO : 5 (abatacept) .
• said fusion protein is having at least 90% seq uence identity to the fusion protein set forth SEQ ID NO : 5, such as at least 95% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example at least 99% sequence identity sequence identity to the fusion protein set forth SEQ ID NO : 5.
• the size of fusion protein is comparable to or essentially the same as that of the fusion protein set forth SEQ ID NO: 5.
• the size of said fusion protein is in the range of 330 to 390 amino acids, such 340 to 380 amino acids.
• the fusion proteins are functionally equivalent to the fusion protein set forth in SEQ ID NO: 5 (abatacept) in the sense that they recognise and bind CD80 and/or CD86.
• the present invention provides the method described herein for the treatment of the various subtypes of morphea.
• said morphea is selected from the group consisting of plaque morphea, generalized morphea, bullous morphea, linear morphea and deep morphea.
• said morphea is selected from the group consisting of en plaque, guttate morphea, atrophoderma of Pasini and Pierini, keloid morphea, lichen sclerosus et atrophicus, en coup de sabre, linear scleroderma, progressive hemifacial atrophy, morphea profunda, eosinophilic fasciitis, and disabling pansclerotic morphea of children.
• en plaque guttate morphea
• atrophoderma of Pasini and Pierini keloid morphea
• lichen sclerosus et atrophicus en coup de sabre
• linear scleroderma progressive hemifacial atrophy
• morphea profunda morphea profunda
• eosinophilic fasciitis eosinophilic fasciitis
• said morphea is morphea profunda.
• CTL4 cytotoxic T-lymphocyte-associated protein 4
• extracellular domain of CTLA4 refers to a portion of CTLA4 that recognizes and binds CD80 and/or CD86.
• the extracellular domain includes fragments or derivatives of CTLA4 that bind CD80 and/or CD86.
• the CTLA-4 sequence can, inter alia, have Ala or with Met as the N-terminal amino acid residue of the protein.
• the extracellular domain of CTLA4 may be provided in alternative version of the extracellular domain of human CTLA-4 having, inter alia, Ala or with Met as the N- terminal amino acid residue of the protein.
• the extracellular domain preferably originates from a primate or more preferably a human cytotoxic T-lymphocyte-associated protein 4 (CTLA4).
• CTLA4 cytotoxic T-lymphocyte-associated protein 4
• said cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is of human orig in .
• the extracellular domain is the extracellular domain of the human cytotoxic T-lymphocyte-associated protein 4 (CTLA4) set forth in SEQ ID NO : 1.
• CTL4 human cytotoxic T-lymphocyte-associated protein 4
• the fusion protein may be provided in the formulation available as abatacept (CTLA-4-Ig, Orencia,) or Belatacept (trade name Nulojix) .
• said fusion protein is provided as abatacept (CTLA-4-Ig, Orencia,) .
• said fusion protein is provided as Belatacept (trade name Nulojix) .
• said extracellular domain comprises the amino acid sequence set forth in SEQ ID NO : 3.
• said extracellular domain is having or essentially having the amino acid seq uence set forth in SEQ ID NO : 3 or SEQ ID NO : 4.
• said fusion protein is having one or more mutations (preferably point mutations) in at least one region of said extracellular domain of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4), wherein said region is having an amino acid seq uence selected from the group consisting of SEQ ID NO : 17 and SEQ ID NO : 18.
• said fusion protein is having two, three, four or five mutations in the extracellular domain of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) .
• said extracellular domain comprises the amino acid seq uence set forth in SEQ ID NO : 3, wherein the sequence of SEQ ID NO : 3 have been mutated by one or more amino acid substitutions, wherein said amino acid substitution is independently selected from the group consisting of Ser25, Ala29, Thr30, Glu31, Arg33, Tyrl03, Leu l04 and Glyl05.
• the above fusion proteins are disclosed in further details in WO 93/00431 and WO 01/92337, which are incorporated by reference .
• the fusion protein comprising a said first amino acid seq uence and a second amino acid seq uence described herein may comprise a junction (spacer) sequence separating the two subunits.
• the said fusion protein further comprises a junction seq uence located between said first amino acid seq uence and said second amino acid sequence.
• the length of the junction seq uence may vary, e.g . the junction sequence may have a length of 50 amino acids or less, such as 40 amino acids or less, for example 30 amino acids or less, such as 20 amino acids or less, for example 10 amino acids or less, such as 5 amino acids or less.
• the junction amino acids sequence consists of a single amino acid .
• said junction amino acids sequence is a glutamine residue.
• the fusion protein use by the method of the present invention may be provided in the form of a molecule comprising a mutated CTLA4 molecule, such a molecule comprising a mutated extracellular domain of CTLA4, that binds CD80 and/or CD86 preferably with higher avid ity to CD80 and/or CD86 than abatacept (SEQ ID NO : 5) .
• a mutated CTLA4 molecule such as a mutated extracellular domain of CTLA4, that binds CD80 and/or CD86 preferably with higher avid ity to CD80 and/or CD86 than abatacept (SEQ ID NO : 5) .
• the fusion proteins are soluble proteins.
• said fusion protein comprises the amino acid seq uence set forth in SEQ ID NO : 5.
• said fusion protein is having or essentially having the amino acid sequence set forth in SEQ ID NO : 5 or SEQ ID NO : 6.
• the application of the abatacept (SEQ ID NO : 5) in the method of treating morphea is disclosed in Example 1.
• said fusion protein comprises the amino acid seq uence set forth in SEQ ID NO : 7.
• said fusion protein is having or essentially having the amino acid seq uence set forth in SEQ ID NO : 7 (referred to as the L104EA29Y variant) or SEQ ID NO : 8.
• said fusion protein comprises the amino acid sequence set forth in SEQ ID NO : 9.
• said fusion protein is having or essentially having the amino acid sequence set forth in SEQ ID NO : 9 (referred to as the L104E variant) or SEQ ID NO: 10.
• said cytotoxic T-lymphocyte-associated protein 4 comprises an amino acid selected from the group consisting of SEQ ID NO : 11 (referred to as the L104D variant), SEQ ID NO : 12 (referred to as the L104EA29L variant), SEQ ID NO : 13 (referred to as the L104EA29W variant) and SEQ ID NO: 14 (referred to as the L104EA29T variant).
• said extracellular domain of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is having or essentially having an amino acid selected from the group consisting of SEQ ID NO : 11, SEQ ID NO: 12, SEQ ID NO : 13 and SEQ ID NO : 14.
• the second subunit the fusion protein (the second amino acid sequence and Ig tail of the fusion protein) comprises amino acid residues corresponding to the constant region of an immunoglobulin molecule.
• the Ig constant region is a human or monkey Ig constant region, e.g ., human C(gamma)l, including the hinge, CH2 and CH3 regions.
• Modifications to the immunoglobulin molecule may be introduced to avoid unintended disulphide bridge formation and to reduce constant region-mediated biological effector functions and complement activation. Ways of providing such modified immunoglobulin molecule are disclosed in the prior art (e.g. in U .S. Patent Nos: 5,637,481; and 6,132,992).
• the fusion protein used by the method of the present invention as described herein comprises a second amino acid sequence comprising amino acid residues corresponding to the constant region of an immunoglobulin molecule.
• said constant region of an immunoglobulin molecule comprises a hinge, CH2 and CH3 regions of an immunoglobulin molecule.
• said constant region of an immunoglobulin is a human or monkey immunoglobulin constant region or a fragment thereof.
• said constant region of an immunoglobulin molecule comprises one or more mutations to reduce effector function.
• said constant region of an immunoglobulin molecule comprises a hinge and any or all of the cysteine residues within the hinge are substituted with serine.
• said fusion protein is having two, three, four or five mutations in the second amino acid sequence comprising amino acid residues corresponding to the constant region of an immunoglobulin molecule.
• said second amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, said second amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO : 15 or SEQ ID NO: 16.
• said fusion protein is soluble.
• said fusion protein has a great avidity to CD86 and/or CD80 than the fusion protein having the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.
• said fusion protein has a slower dissociation rate from binding CD86 than the fusion protein having the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO : 6.
• said fusion protein further comprises an amino acid sequence which alters the solubility, affinity or valency of said extracellular domain of CTLA4.
• said fusion protein is a selective co-stimulation modulator binding to the CD80/CD86 ligands on an antigen-presenting cell.
• the fusion protein used by the method of the present invention may be provided as a homodimer consisting of two identical fusion proteins as described herein, which are covalently linked by one disulphide bond.
• said fusion protein is provided in the form of a dimer of said fusion protein.
• the fusion protein used by the present invention may be provided in the form of a pharmaceutical composition.
• the pharmaceutical compositions may be provided in a variety of dosage forms, which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or micro-vesicles, liposomes, and injectable or infusible solutions.
• the preferred form depends upon the mode of administration and the therapeutic application.
• the most effective mode of administration and dosage regimen for the compositions of this invention depends upon the severity and course of the disease, the patients' health and response to treatment and the judgment of the treating physician. Accordingly, the dosages of the compositions should be titrated to the individual patient.
• the pharmaceutical compositions also preferably include suitable carriers and adjuvants which include any material which when combined with the molecule of the invention (e.g., a soluble CTLA4 mutant molecule, such as, L104EA29Y or L104E) retains the molecule's activity and is non-reactive with the subject's immune system.
• suitable carriers and adjuvants include, but are not limited to, human serum albumin; ion exchangers; alumina; lecithin; buffer substances, such as phosphates; glycine; sorbic acid; potassium sorbate; and salts or electrolytes, such as protamine sulfate.
• compositions comprising such carriers are formulated by well known conventional methods. Such compositions may also be formulated within various lipid compositions, such as, for example, liposomes as well as in various polymeric compositions, such as polymer microspheres.
• compositions used by the method of the present invention can be administered using conventional modes of administration including, but not limited to, intravenous (i.v.) administration, intraperitoneal (i.p.) administration, intramuscular (i.m.) administration, subcutaneous administration, oral
• administration administration as a suppository, or as a topical contact, or the implantation of a slow-release device such as a miniosmotic pump, to the subject.
• the fusion protein may be administered to a subject in an amount and for a time (e.g. length of time and/or multiple times) sufficient to block endogenous B7 (e.g., CD80 and/or CD86) molecules from binding their respective ligands, in the subject.
• Blockage of endogenous B7/ligand binding thereby inhibits interactions between B7-positive cells (e.g ., CD80- and/or CD86-positive cells) with CD28- and/or CTLA4-positive cells.
• Dosage of a therapeutic agent is dependent upon many factors including, but not limited to, the type of tissue affected, the type of morphea being treated, the severity of the disease, a subject's health, and a subjects response to the treatment with the agents.
• dosages of the agents can vary depending on the subject and the mode of administration.
• the soluble CTLA4 mutant molecules may be administered in an amount between 0.1 to 20.0 mg/kg weight of the patient/day, preferably between 0.5 to 10.0 mg/kg/day.
• Administration of the pharmaceutical compositions of the invention can be performed over various times, preferably every two weeks for the first month and monthly thereafter.
• the fusion protein provided in the form of a pharmaceutical composition can be administered for one or more hours.
• the administration can be repeated depending on the severity of the disease as well as other factors as understood in the art.
• the fusion protein (e.g. provided in the form of a pharmaceutical composition described herein) is administered by repeated intravenous injection at a frequency of every two weeks for the first month and monthly thereafter.
• the fusion protein (e.g . provided in the form of a pharmaceutical composition described herein) is being
• the present invention further provides the fusion protein described herein for use in the manufacture of a medicament for the treatment of localized scleroderma.
• a fusion protein selected from the group consisting of:
• a fusion protein comprising a first amino acid sequence comprising an amino acid sequences corresponding to the extracellular domain of a cytotoxic T- lymphocyte-associated protein 4 (CTLA4) and a second amino acid sequence comprising amino acid residues corresponding to the constant region of an immunoglobulin molecule
• said morphea is selected from the group consisting of plaque morphea, generalized morphea, bullous morphea, linear morphea and deep morphea.
• said morphea is selected from the group consisting of en plaque, guttate morphea, atrophoderma of Pasini and Pierini, keloid morphea, lichen sclerosus et atrophicus, en coup de sabre, linear scleroderma, progressive hemifacial atrophy, morphea profunda, eosinophilic fasciitis, and disabling pansclerotic morphea of children.
• fusion protein described herein and used by the method of the present invention may are also used for the also used for the manufacture of a medicament for the treatment or alleviation of the symptoms of localized scleroderma.
• a fusion protein selected from the group consisting of:
• a fusion protein comprising a first amino acid sequence comprising an amino acid sequences corresponding to the extracellular domain of a cytotoxic T- lymphocyte-associated protein 4 (CTLA4) and a second amino acid sequence comprising amino acid residues corresponding to the constant region of an immunoglobulin molecule
• said morphea is selected from the group consisting of plaque morphea, generalized morphea, bullous morphea, linear morphea and deep morphea.
• said morphea is selected from the group consisting of en plaque, guttate morphea, atrophoderma of Pasini and Pierini, keloid morphea, lichen sclerosus et atrophicus, en coup de sabre, linear scleroderma, progressive hemifacial atrophy, morphea profunda, eosinophilic fasciitis, and disabling pansclerotic morphea of children.
• the inventors provide the clinical data from the treatment of two patients suffering from chronic and progressive disseminated morphea profunda.
• the patients were received treatment with Abatacept, which is a recombinant fusion protein that selectively inhibits T-cell activation via
• the first patient an otherwise healthy 47-year-old woman, had first presented with clinical morphea lesions when she was 22 years old .
• a punch biopsy showed typical histological findings of morphea.
• the patient had no other organ-specific manifestations. Routine blood evaluation was normal.
• Antinuclear antibody (ANA) was positive.
• SSA, SSB, Scl-70, ENA, anti-cardiolipin and RNA-polymerase were all negative.
• a punch biopsy from an active lesion on the left thigh confirmed the diagnosis and showed a primarily lymphocytic inflammatory infiltrate around the superficial and deep blood vessels. Furthermore, inflammation at the junction between the dermis and the subcutaneous fat was observed, and the dermal collagen fibres were thickened.
• the treatment with abatacept was well tolerated .
• the disease activity was reduced, both when evaluating the whole body and the single lesions.
• the erythema around the lesions decreased ( Figure 1), and the older lesions became softer.
• the effect of the treatment was scored by modified Rodnan skin score (Clements et. al 1995). This total skin thickness score is commonly used as an outcome measure in trials of systemic sclerosis. Skin thickness is assessed by clinical palpation of 17 body areas on the front side on a 0-3 scale (normal, mild, moderate and severe).
• the modified Rodnan skin score is derived by summation of the scores from all 17 body areas.
• Table 1 Modified Rodnan skin score before and during treatment with abatacept in patient no. 1
• the second patient a 38-year-old woman developed diffuse progressive morphea profunda, when she was 8 years old.
• the skin on her shoulders, back and right lower leg has been mainly affected.
• the right leg is rotated outwards and the flexion of the right knee and foot is reduced. No other organ-specific involvements have been observed. All blood samples have been normal.
• the patient was treated with 7.5 mg prednisolone daily together with abatacept.
• the patient had symmetrical severe deep fibrotic lesions on her shoulders, upper arms, buttock and right thigh, with a modified Rodnan skin score of 13.
• the deep fibrotic lesions were atrophic and hyperpigmented, with a typical lilac ring in the periphery of the lesions ( Figure 2).
• abatacept 500 mg was given intravenously on days 1, 15, and 30 and thereafter every 4 weeks according to her weight (58 kg).
• the patient received a total of five treatments. She experienced an impressive improvement, and reported better movement of the shoulders, hips and knees. She could walk longer distances and had no adverse effects.
• abatacept treatment had to be stopped after 2.5 months, as the patient was diagnosed with breast cancer.
• the cancer is not believed to be related to the treatment with abatacept, but as a precaution it has been reported to the Danish Medicines Agency.
• Morphea profunda is a rare disease and often runs a progressive course with physical and psychological sequelae. Until now, no treatment has been proven effective on active disease or been able to soften old sclerotic lesions.
• the first patient is very satisfied with the abatacept treatment. She feels less itchy, has better joint movements and the treatment is well tolerated.
• the modified Rodnan skin score has reduced significantly during treatment. Thus, the disease activity has been diminished both when evaluating the whole body and the erythema around the single lesions. Even the older lesions feel softer on examination. Furthermore, it has been possible to taper the prednisolone treatment.
• the second patient was also satisfied, as abatacept improved her joint movements and walking distance. This patient was also able to stop prednisolone treatment. However, this patient had to stop after 5 treatments with abatacept. After the treatment was stopped, a further softening effect on the old lesions was observed.
• the inventors report here the first cases of diffuse morphea profunda, treated successfully with abatacept. The mechanism is unknown, but the expansion of T cells in the affected skin and circulation from patients with systemic sclerosis suggests antigen-specific activation. Th-17 and regulatory T- cells, which are subsets of effector T-cel Is, are key regulators of inflammation in several autoimmune diseases.
• WO 01/92337 (Bristol-Myers Squibb).

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