Covarcin for the treatment of autoimmune bullous diseases

Abstract

Methods for treating and preventing autoimmune bullous diseases (AIBD) are provided. The present invention provides a pharmaceutical agent containing the tick protein covarcin and its functional equivalents as an active ingredient, which inhibits complement activation, and a method for treating and preventing AIBD in combination with a second AIBD therapeutic agent selected from systemic corticosteroid therapy, topical corticosteroid therapy, immunosuppressive therapy, and immunosuppressive biological therapy.

Description

[Technical field]

[0001] The present invention relates to methods for treating and preventing autoimmune bullous diseases.

[0002] All documents mentioned in the text and listed at the end of this specification are hereby incorporated by reference. It is built in. [Background technology]

[0003] complement The complement system is an essential part of the body's natural defense mechanisms against foreign invaders. It is involved in inflammatory processes. More than 30 proteins in serum and on the cell surface are involved. In recent years, they have been implicated in both beneficial and pathological processes. There are approximately 35 known components of the complement system that can be linked to the complement pathway, as well as the complement system itself, which plays a key role in regulating angiogenesis, platelet activation, and cell proliferation. At least 85 biological molecules with functions as diverse as fertilization, glucose metabolism, and spermatogenesis. It was found that it interacts with the target pathway.

[0004] The complement system is activated by the presence of foreign antigens. (1) IgM and IgG complexes (2) the classical pathway, which is activated by recognition of non-self surfaces (specific (lacking regulatory molecules) and an alternative pathway activated by bacterial endotoxin; and (3) Mannan-binding lectin (MBL) binds to mannose residues on the surface of pathogens. There are three activation pathways, the lectin pathway, which is activated by the , similar to C3 on the cell surface 1 Complement activation by formation of C5 convertase and C6 convertase This involves a parallel cascade of events that results in the production of acute phase inflammatory mediators This results in the release of C3a and C5a and the formation of the membrane attack complex (MAC). The parallel linkages involved in the mitochondrial and alternative pathways are shown in Figure 1. 1 The components of the complement pathway are identified by the letters " The complement components are represented by a number such as "C" followed by the number "3", so that "C3" represents the third component of the complement system. Some of these components are cleaved upon activation of the complement system, resulting in the production of cleaved products. Items are designated by a number followed by a lowercase letter. Thus, C5 is conventionally referred to as C5a and C5b. Complement proteins do not necessarily act in numerical order. The numbers do not necessarily indicate an order of action. This naming convention is used throughout this application. Used.)

[0005] The classical, alternative and lectin complement pathways are collectively referred to herein as C5b initiates the "late" events of complement activation. These are: The final complement components interact with each other to form the MAC, which is a series of polymerization reactions. It creates holes in the cell membrane of pathogens, which may lead to their death. These include C5b (which initiates assembly of the membrane attack system), C6, C7, C8, and C9. can be.

[0006] LTB4 Leukotriene B4 (LTB4) is the most potent chemotactic and chemokinetic agent described. It is an eicosanoid that mediates neutrophil intravasation via upregulation of integrins. [1] LTB4 is also a complete secretagogue for neutrophils. LTB4 induces the aggregation of IL-1 and increases microvascular permeability. LTB4 recruits and activates superoxide dismutase (SAD)-dependent inflammatory cytokines, including monocytes and eosinophils. [2] and may enhance and prolong tissue inflammation. Modulating gene expression, including the production of pro-inflammatory cytokines and mediators [3, 4]. LTB4 also plays a role in the induction and management of adaptive immune responses. For example, they regulate the trafficking of dendritic cells to draining lymph nodes [5, 6] and promote the Th2 proliferation of lung T cells. production of the inhibitor IL-13 [7] and recruitment of antigen-specific effector CD8+ T cells [8 ] and human B lymphocyte activation and proliferation [9].

[0007] Leukotriene B4 (LTB4) and hydroxyeicosanoids act as BLT1 and They mediate their effects via the BLT2 G protein-coupled receptor [10, 11]. Human BLT1 is a high-affinity receptor specific for LTB4 (Kd 0.39-1.5n M;

[12] ), and competitive binding studies have shown that 20-hydroxyLTB4 and 12-epiLTB4 Human BLT2 has a higher activity than BLT1 and can replace LTB4. 12-epi-LTB4, 2 0-Hydroxy LTB4, 12(S)-HETE, 15(S)-HETE, 12(S)- Binds a broader range of eicosanoids, including HPETE and 15(S)-HPETE Human BLT2 is activated by the IL-1 receptor

[13] . Human BLT2 is a cytoplasmic receptor that is involved in the activation of the IL-1 receptor and is involved in the activation of the IL-1 receptor

[14] . The amino acid identities between human BLT2 and mouse BLLT2 are 45.2 and 44.6%, respectively. It shares 92.7% identity with T2.

[11]

[0008] Although human BLT1 has recently been described to be expressed in endothelial cells and vascular smooth muscle cells, It is primarily expressed on the surface of leukocytes. Human BLT2 is expressed in a broader range of tissues and cell types. It inhibits the activation, extravasation and apoptosis of human neutrophils

[14] and reduces inflammation. In mouse models of chronic arthritis

[15] and renal ischemia-reperfusion

[16] , neutrophil infiltration Several specific functions of BLT1 and BLT2 reduce symptoms associated with Both BLT1 and BLT2 have been shown to inhibit LTB4 and Although it can mediate pathological effects via hydroxyeicosanoids

[17] , BL T1 undoubtedly plays a dominant role in several pathologies, such as collagen-induced arthritis in mice. A growing number of studies have demonstrated that BLT1- / - The deficient mice also demonstrated that BLT1 is important in directing neutrophil migration during inflammatory responses. In particular, they used a 5LO-deficient mouse strain to investigate the expression of BLT1 on neutrophils. Autocrine activation has been shown to be necessary for the recruitment of neutrophils to arthritic joints.

[19]

[0009] Several commercially available drugs target eicosanoids. These include phospholipase A2 (PLA2), thereby modulating the eicosanoid precursor arachidonic acid Glucocorticoids, which inhibit the release of AA

[20] , as well as prostaglandins and Nonsteroidal anti-inflammatory drugs (NSAIDs) and other drugs that prevent the synthesis of thromboxane These include COX2 inhibitors

[21] , which inhibit the 5-LO enzyme required for LTB4 synthesis (dihydrochloride). Lawton;

[22] ) or CysL, which mediates the effects of cysteinyl leukotrienes. either by antagonizing the T1 receptor (zafirlukast and montelukast)

[23] or by There are also several LK modulators available that are orally available, e.g. It is approved by the FDA for use in treating asthma. There are currently no drugs on the market that act specifically on this condition.

[0010] Autoimmune bullosa disease (AIBD) The skin, the largest organ of the body, is made up of five distinct layers. The epidermis is the outermost layer of the skin. This is the protective layer between the epidermis (which together with the dermis make up the outer skin) and the subcutaneous tissue. The dermis is made up mainly of dense irregular connective tissue and is attached to the surface via a basement membrane. The dermis and epidermis are tightly bound together by special proteins and structures that form the bond between them. It is necessary and separation of these layers causes blisters or bullas to develop.

[0011] Under normal circumstances, blisters develop in response to skin irritation or injury, but autoimmune blisters In AIBD, the blisters attack desmosome or hemidesmosome structural proteins. These proteins are necessary for the proper functioning of the basement membrane. In AIBD, attacks caused by autoantibodies against specific structures or proteins occur. As a result, the adhesion between the epidermis and the dermis is damaged, which ultimately results in the separation of the epidermis and the dermis. and blisters form.

[0012] Thus, AIBD is characterized by blistering lesions that primarily affect the skin, as a result of autoantibodies against skin antigens. In some AIBDs, the blisters appear on the mucous membranes ( for example, in the esophagus, anus, mouth, nasal cavity, genital tract and pharynx be.

[0013] Risk factors for the development of AIBD include advanced age, drug therapy, viral infections, and UV or X-ray exposure. This includes exposure to radiation.

[0014] The specific signs and severity of blister disease vary among individuals, and in some cases, the blister lesions appear on the skin. It can affect large areas of the skin. There is no cure for AIBD, but treatments do exist. Without such treatment, the disease can lead to life-threatening complications.

[0015] Pemphigus, pemphigoid, IgA-mediated skin diseases, and epidermolysis bullosa acquisita (EBA) There are several different classes of AIBD. Pemphigus, pemphigoid, and IgA-mediated skin diseases are It can be further divided into additional subtypes.

[0016] Pemphigus is a group of diseases caused by an antibody-mediated autoimmune reaction against desmoglein. Pemphigus vulgaris is a general term for related AIBD. The two main types of pemphigus are pemphigus vulgaris and It is pemphigus foliaceus.

[0017] Pemphigus vulgaris is characterized by autoantibodies against Dsg3 (approximately 50% of patients have autoantibodies against Dsg1). It is characterized by blisters that easily rupture and cause painful sores. Pemphigus vulgaris is the most common form of pemphigus that is seen in most cases. It begins in the cavity, followed by blistering of the skin, but any area can potentially be affected.

[0018] Pemphigus foliaceus produces itchy, scaly, crusted lesions that quickly break down and affect the top layer of the skin. It is characterized by the formation of numerous small blisters on the skin. The scalp and face are usually affected first. Eventually the chest and upper back may be affected. The lesions are usually painless. Mucous membranes are usually affected. These patients have autoantibodies against Dsg1.

[0019] Additional disorders include paraneoplastic pemphigus (arising from tumors, e.g. Dsg1 and Dsg3 AIBD with autoantibodies against desmocollin) and IgA pemphigus (autoantibodies against desmocollin) It is sometimes classified as a subtype of pemphigus, including those with ulcerative colitis (having a granular body).

[0020] "Pemphigoid" is used to describe a group of related diseases characterized by a blistering skin rash. The main forms of pemphigoid are bullous pemphigoid, mucous membrane pemphigoid, and pemphigoid gestationis. It is a sore.

[0021] Bullous pemphigoid is a chronic skin disease characterized by pruritus and tense subepidermal blisters. The antigen BP180 (also called dystonin) and its NC16A domain Autoantibodies against collagen XVII (localized in collagen XVII) have been observed. The blisters develop within a few weeks on the skin of the groin, axillae, abdomen, and flexors. It often spreads, causing the lesions to cover a large area of ​​skin and forming blisters in the mouth. In most cases, mucous membranes are not affected, and if they are, the blisters tend to heal quickly. The lesions of bullous pemphigoid are often accompanied by intense itching.

[0022] Mucous membrane pemphigoid (MMP), also called cicatricial pemphigoid (CP), also affects primarily the mucous membranes. Affects the skin (mainly the mouth and eyes, but can also affect the nose, throat, genitals, and anus), epidermis, The manifestations of MMP vary depending on the specific site involved and the progression of the disease. The autoantigens are also directed against BP180, but vary among individuals. Autoantigens have been identified.

[0023] Epidermolysis bullosa acquisita is relatively rare. In this condition, BP The separation of the epidermis and dermis is also observed. Autoantibodies against the BMZ (which form anchoring fibers that connect the BMZ to the papillary dermis) are typically This autoimmune skin disorder is characteristic of this condition, which affects the elderly and middle-aged.

[0024] Most forms occur in middle-aged individuals, usually those in their 50s and 60s. However, Autoimmune bullous diseases can affect individuals of any age, including children. These symptoms The overall incidence and prevalence of bullous pemphigoid varies depending on the particular study population. Acne is the most common immunological blistering disease in Western Europe, affecting 1 million people in the UK each year. Incidence rate of 43 per capita and 7-13 per million people annually in the rest of Europe has been reported

[24] .

[0025] The diagnosis of AIBD is based on clinical evaluation and a detailed medical history of the patient, as well as on blood or skin tests. Immunofluorescence assays, based on the identification of characteristic autoantibodies in skin biopsies, are the preferred diagnostic method. be.

[0026] Currently, there is no cure for these disorders, but they can be treated with antidepressants, such as prednisone. Treatment is not well tolerated and may be managed with corticosteroids such as For example, systemic corticosteroid therapy (e.g., prednisolone) may be associated with toxicity. and prednisolone) may be effective, but this is not effective in all cases. Long-term treatment with high doses of corticosteroids can cause serious side effects. Steroids are common, non-specific anti-inflammatory agents. Subjects were treated with long-term, high-dose steroids. In many of these cases, other immunosuppressants are used as a "bridge" because patients cannot continue taking them. will be done.

[0027] Topical corticosteroids are also used, but their use in a wide range of diseases is limited. These may be limited by factors such as the patient's ability to apply the treatment, systemic absorption, and may be associated with adverse events. Immunosuppressants (e.g., dapsone, methylprednisolone, mycophenolate, azathioprine or cyclophosphamide) and immunosuppressive biologics Therapies (e.g., rituximab and intravenous immunoglobulin G (IVIG)) have also been used. However, they can have adverse effects.

[0028] Therefore, new therapies to treat and prevent AIBD are needed.

[0029] Complement inhibitors WO2004 / 106369 (Evolutec Limited

[25] ) discloses a method for the preparation of a complement A particular subset of the disclosed complement inhibitors are directed against C5 and inhibit complement activation. This prevents C5 from being cleaved into C5a and C5b by either of the somatic activation pathways. A specific example of such a C5 cleavage inhibitor is the mammary gland of the species Ornithdoros moubata. This protein is produced by mites and is described in WO2004 / 1063 It is a protein consisting of amino acids 19 to 168 of the amino acid sequence shown in Figure 4 of 69. In WO2004 / 106369, this protein is designated as "EV576" and "Om These proteins are known as "CI proteins" and, more recently, "Coversins." (See, for example, Jor e et al., Nature Structural & Molecular Biology , Structural basis for therapeutic inhibition tion of complement C5 - doi:10.1038 / nsmb (See, e.g., 3196). This protein is referred to herein as "coversin." will be done.

[0030] In ticks, covercin is expressed by the nucleotide sequence ##STR00002## at the N-terminus of the mature covercin protein. A leader sequence containing amino acids 1 to 18 of the amino acid sequence shown in Figure 4 of / 106369 The leader sequence is cleaved off after expression. The protein may be any of the amino acids shown in FIG. 4 of WO2004 / 106369 and FIG. 2 of the present application. It has a sequence consisting of amino acids 19 to 168 of the amino acid sequence.

[0031] Covarcin also has the ability to inhibit leukotriene B4 (LTB4) activity. The ability to bind LTB4 can be determined using standard in vitro assays known in the art. For example, the binding of Coversin to an anti-LTB4 antibody competes for binding to labeled LTB4. The assay was carried out by competitive ELISA, by isothermal titration calorimetry, or by fluorescence titration. It can be proved.

[0032] WO2007 / 028968, WO2008 / 029167, WO2008 / 0291 69, WO2011 / 083317 and WO2016 / 198133. Several further patents have been issued relating to the use of Covarcin or its functional equivalents in WO2015 / 185760 discloses that Covarcin and its structural equivalents can be used in, e.g. This study was carried out to determine whether the C5 polymorphisms prevent cleavage, which reduces the therapeutic efficacy of the commercially available C5 complement inhibitor eculizumab. These applications disclose that the drug is effective in treating AIBD. The use of Covarcin or any functional equivalent thereof is not disclosed.

[0033] The potential role of the complement pathway in relation to AIBD has been debated, but complement-targeting AI There is no current treatment for BD, and the pathophysiology of these disorders is incompletely understood. Moreover, there is evidence that blister formation can be induced directly and without complement activation

[26] . Based on this, we have developed a new approach that targets blocking autoantibody binding rather than preventing complement activation. A novel therapeutic approach has been suggested in the art

[26] . IL-17A inhibits pemphigoid disease-like skin inflammation in Ltb4r1 mice Resistance cannot be overcome.

[27]

[0034] Similarly, the role of LTB4 in AIBD, specifically in neutrophil motility in the disease, is unclear. The currently approved treatments for these conditions are discussed in relation to , does not target this molecule.

[0035] In contrast, the studies leading to the present invention have demonstrated that IL-1 binds LTB4 and also binds C5, as described above. Conversen, a molecule that inhibits the complement pathway by inhibiting the mitochondrial endothelial cell wall, has been shown to be effective in mouse models of AIBD. It has been shown to reduce blister-affected body surface area (ABSA). 5) and also LTB4, and thus , alone or in combination with other AIBD treatments, are particularly useful in the prevention and treatment of AIBD. It is beneficial. Summary of the Invention [Problem to be solved by the invention]

[0036] Covarcin significantly reduced the percentage of affected body surface area (ABSA) in a mouse model of EBA. In Example 1, it was shown that the inflammatory bowel disease was reduced by the induction of the disease. Administration of Covarcin significantly reduced ABSA percentage compared to mice that were not treated with Covarcin. Determine the area of ​​skin showing erythema, blisters, erosions, crusts, or hair loss, and (assessed by calculating the percentage of total body surface area affected by lesions (ABSA) It has been shown that other drugs (zileuton and methylprednisolone) can reduce A greater effect was observed with Covaxin administration compared with a similar study using This is especially true at higher concentrations (e.g., 2.5 mg / kg and 0.25 mg / kg). Therefore, Covarcin is a novel systemic immunotherapy that is currently used to treat AIBD. It is more effective than immunosuppressants (methylprednisolone) and less effective than LTB4 inhibitors (zileuton). Administration of Covaxin after induction of disease also appears to be more effective than ABSA. It has also been shown that this can lead to a decrease in the percentage of C5 and The dual inhibitory activity of Covarcin, which targets both LTB4 and LTB5, is particularly advantageous for the treatment of AIBD. It appears to be.

[0037] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity Prevention experiments were performed using a modified Covasin polypeptide that retained the ability to bind to the IL-1 receptor. A decrease in the percentage of ABSA was also observed using C5 binding activity and L Agents with both TB4 binding activity and reduced C5 binding activity are preferred. or C5 binding activity, but retains or has reduced LTB-4 binding ability. 4 binding activity or drugs that have no LTB4 binding activity but retain C5 binding ability Agents can be used in the present invention. [Means for solving the problem]

[0038] The inventors therefore identified the tick protein covarcin (described in the art and herein). Administration of EV576 and OmCI (also known as EV576 and OmCI in clinical trials

[25] ) has been shown to treat or alleviate AIBD. demonstrated that it can be used to prevent

[0039] The present invention therefore relates to a compound comprising amino acids 19 to 16 of the amino acid sequence in FIG. Therapeutic or prophylactic use of a drug that is a protein containing 8 or a functional equivalent of this protein. The present invention provides a method for treating or preventing AIBD comprising administering an effective amount of

[0040] The present invention also relates to a method for treating or preventing AIBD, comprising administering to said patient a therapeutically effective amount of A protein comprising amino acids 19 to 168 of the amino acid sequence (SEQ ID NO: 2) in The present invention provides an agent that is a functional equivalent of a protein.

[0041] The present invention also relates to a method for the preparation of a peptide comprising the amino acid sequence 19 to 168 of the amino acid sequence in FIG. of a drug that is a protein containing the nucleotide sequence of ... The present invention provides a method for treating or preventing AIBD, comprising administering a therapeutically or prophylactically effective amount of do.

[0042] The present invention also relates to a method for treating or preventing AIBD, comprising administering to said patient a therapeutically effective amount of A protein comprising amino acids 19 to 168 of the amino acid sequence (SEQ ID NO: 2) in The present invention provides agents that are nucleic acid molecules that encode functional equivalents of the proteins.

[0043] The present invention also relates to a method for the preparation of a peptide comprising: (a) a peptide sequence comprising amino acids 19-1 of the amino acid sequence in FIG. Treatment or prevention of diseases that involve the use of a drug that is a protein containing 68 or a functional equivalent of this protein and (b) administering a second AIBD treatment. Provide a method of prevention.

[0044] The present invention also provides a method for treating or preventing AIBD comprising: A protein comprising amino acids 19 to 168 of the amino acid sequence (SEQ ID NO: 2) in 2, or and (b) providing a second AIBD treatment. .

[0045] The present invention also relates to a method for the preparation of a peptide comprising: (a) a peptide sequence comprising amino acids 19-1 of the amino acid sequence in FIG. 68 or a nucleic acid molecule encoding a functional equivalent of this protein. a therapeutically or prophylactically effective amount of an agent; and (b) administering a second AIBD treatment. Methods for treating or preventing AIBD are provided.

[0046] The present invention also provides a method for treating or preventing AIBD comprising: A protein comprising amino acids 19 to 168 of the amino acid sequence (SEQ ID NO: 2) in 2, or an agent that is a nucleic acid molecule that encodes a functional equivalent of the protein; and (b) a second A Providing IBD treatment.

[0047] The present invention also provides a method for treating or preventing an AIBD, comprising administering to said patient a second AIBD. Reduce the amount of treatment required to treat or prevent AIBD 2. A method for reducing the duration of treatment with AIBD therapy comprising administering to a subject an amino acid sequence as shown in FIG. A protein comprising amino acids 19 to 168 of SEQ ID NO:2 or the function of this protein and a therapeutically or prophylactically effective amount of a drug or a nucleic acid molecule encoding said drug, administering said second AIBD treatment.

[0048] The present invention is also a method for reducing autoantibody titers in a subject with AIBD. and a protein comprising amino acids 19 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2). or a drug which is a functional equivalent of this protein, or a nucleic acid molecule encoding said drug. The present invention provides a method for treating a chronic inflammatory bowel disease comprising administering to a patient a therapeutically or prophylactically effective amount of a compound comprising administering to said ... The method may include administering a second AIBD treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] disease The subject may have, be suspected of having, or be at risk of developing AIBD. .

[0050] The AIBD is preferably pemphigus, pemphigoid, IgA-mediated skin disease and acquired epidermidis. Pemphigus can be pemphigus vulgaris or pemphigus foliaceus. "Pemphigoid" refers to bullous pemphigoid, mucous membrane pemphigoid, and / or gestational pemphigoid. Preferably, the AIBD is EBA or bullous pemphigoid. The mouse model presented is a model for EBA, but may also be used for other AIBDs, particularly bullous idiopathies. It is also beneficial for pemphigus. The causative mechanisms are similar for all AIBDs: That is, immune complex formation and immune responses, particularly those involving neutrophils and / or eosinophils, are strongly involved. Assimilation of epidemic response.

[0051] The presence of these diseases can be diagnosed by routine diagnostic techniques well understood in the art. can be determined by the

[0052] Subjects at risk of developing AIBD may be administered the methods described herein to prevent AIBD. Risks for AIBD, especially bullous pemphigoid, include: Factors that may contribute to BP include genetic factors and other inducing factors.

[29] Major histocompatibility complex class II (MHC II) genes in patients with HLA -There are reports of a genetic predisposition to the presence of DQB1*0301, which is encoded in mitochondria. Polymorphisms in the ATP synthase 8 gene (MT-ATP8), which is known to be involved in the pathogenesis of BP, have been implicated. These include CYP2D6 gene polymorphisms and FC gamma RIIIa polymorphisms. .

[0053] Other inducers include 1. Drugs: Most BP-inducing drugs contain or release sulfhydryl groups (penicillin, Lamin, captopril, penicillin and its derivatives, furosemide, and some Cephalosporins. Phenol ring-containing drugs (some cephalosporins and acetylsalicylic acid Licylic acid), angiotensin-converting enzyme inhibitors other than captopril, most nonsteroidal anti-inflammatory drugs, Anti-inflammatory drugs, immunomodulators such as vaccines, dipeptidyl peptidase IV inhibitors (Gl lipin), especially vildagliptin, and TNF-α blockers have also been reported to induce BP. It has been done. 2. Viruses: Post-vaccination 3. UV or X-ray exposure Includes:

[0054] Approximately one-third of BP patients have a neuropsychiatric disorder; stroke, dementia, Parkinson's disease, etc. do.

[0055] Subjects with one or more of these risk factors are candidates for the treatment or prevention of AIBD. It is preferable in this respect.

[0056] Administration results The subject may experience a reduction in the incidence of symptoms, a reduction in symptoms, or an absence of symptoms or recurrence as a result of treatment. Preferably, the treatment includes inhibiting or delaying the onset of typical This results in a reduction in the symptoms of the disease. For example, this can include a reduction in the size of the blisters, a reduction in the number of blisters, , a reduction in the percentage of body surface area affected, a reduction in the degree of blister exudation, a reduction in pruritus, and This may manifest in a reduction in the incidence and / or severity of infections resulting from the blisters. , with complete resolution of symptoms and no further recurrences.

[0057] Clinical scoring was based on the Bullous Pemphigoid Disease Activity Index (PBMI). Using the BPDAI (BPDAI)

[30] , The total BPDAI is calculated using two scores: total BPDAI activity and BPDAI loss. The total BPDAI activity score is made up of skin blisters / erosions, skin wheals / redness, and skin lesions. It is the arithmetic sum of three subcomponents: plaques, and mucosal blisters / erosions.

[0058] The BPDAI damage score was significantly higher for more permanent lesions such as post-inflammatory hyperpigmentation, scarring, and other lesions. The BPDA is the arithmetic sum of the items that are assessed in a domain-specific manner to assess the damage caused by certain characteristics. I quantifies lesion count and size threshold. Lesions are graded based on affected area. The BPDAI gives additional weight to skin sites that are primarily affected in BP, such as the extremities. and less emphasis on the scalp and face to better distinguish clinical responses in BP. Not bad.

[0059] The total BPDAI score can range from 0 to 372. Large 360 ​​(Maximum 240 for total skin activity [120 for erosion / blister, 120 for wheal / erythema 120] and mucosal activity 120), as well as BPDAI damage It ranges from 0 to 12, with higher scores indicating greater disease activity or damage. The BPDAI score will be used to determine subject inclusion.

[0060] The BPDAI also has another subjective measure known as the BPDAI-Pruritis Index. The BPDAI pruritus component was scored on the following scales: past 24 hours (0-10), past week (0-10), and past The severity of itching over the past month was measured on a visual analog scale (0-10) with a total score of 30. Based on.

[0061] Preferably, the treatment is at least 1 or 2 days after onset of pemphigoid disease as measured by the Bullous Pemphigoid Disease Activity Index (BPDAI). Preferably, the treatment results in a reduction in the score of 2 or more findings. Any one or more of the following (e.g., two or three): skin wheals / erythema and mucosal blisters / erosions ) score.

[0062] In one embodiment, treatment results in a decrease in skin blister / erosion scores. In one embodiment, the treatment results in a reduction in the skin wheal / erythema score. In one embodiment, the treatment results in a reduction in the mucosal blister / erosion score. In one embodiment, the treatment results in a reduction in the scores for erosions and skin wheals / erythema. In one embodiment, the subject is treated with the sedative inhibitor, which causes a reduction in the scores of skin wheals / erythema and mucosal blisters / erosions. Treatment resulted in a decrease in the scores for skin blisters / erosions and mucosal blisters / erosions. In embodiments, treatment results in a decrease in a score on the BPDAI-Pruritis Index.

[0063] In some embodiments, the effect is a reduction or prevention of neutrophil and / or eosinophil involvement. Thus, it can be mediated.

[0064] Treatment may also be administered prior to the onset of one or more stages of the disease or after progression of the disease stages. This may result in an increased latency period between blister formation. In some embodiments, blister formation may be prevented.

[0065] Treatment may also result in a decrease in autoimmune antibody titers.

[0066] The treatment may also result in a reduction in the amount or duration of a second AIBD treatment required.

[0067] Thus, in a further embodiment of the present invention, the suppression of blisters in subjects with AIBD is or to reduce the size and / or number of affected body surfaces in subjects with AIBD. The method comprises the steps of: or a functional equivalent of this protein. Administering a therapeutically or prophylactically effective amount of a drug or a nucleic acid molecule encoding said drug. Methods are provided for treating AIBD, which may occur alone or in conjunction with a second AIBD treatment.

[0068] The present invention also provides a method for reducing the size and / or number of blisters in a subject with AIBD. or reduce the percentage of affected body surface in a subject with AIBD. 2 (SEQ ID NO:2) for use in the method of A protein comprising ~168 or a functional equivalent of this protein, or Nucleic acid molecules encoding the agents are provided, which may be used alone or as a second AIBD treatment. may be accompanied by.

[0069] The agents of the invention can be used in combination with other AIBD treatments, as described above. Combination of the Agents of the Invention with Other (Herein Referred to as "Secondary") AIBD Treatments The amount of the second AIBD is reduced by 100% compared to the amount used in the absence of treatment with the agent of the present invention. The amount is reduced or compared to the treatment period used in the absence of treatment with the agent of the present invention. This may result in a reduction in the duration of treatment with the second AIBD. Certain diseases, such as infections, diabetes, osteoporosis, thrombosis, and gastrointestinal ulcers, It is advantageous to take into account the side effects of known treatments. Reduce the amount of second AIBD treatment used for or reduce the severity of symptoms caused by the second AIBD. Methods for reducing the duration of treatment are also provided.

[0070] Preferably, the second AIBD treatment is systemic corticosteroid therapy, topical corticosteroid therapy, or The therapeutic options are selected from immunosuppressive therapy, immunosuppressive therapy and immunosuppressive biological therapy.

[0071] Preferably, the corticosteroid is selected from prednisone and prednisolone. Preferably, the immunosuppressive therapy is methylprednisolone, mycophenolate, azathioprine, or cyclosporine. Selected from purines, anti-inflammatory antibiotics (e.g., dapsone) and cyclophosphamide Preferably, the immunosuppressive biological therapy is rituximab and intravenous immunoglobulin G. (IVIG).

[0072] When an agent of the invention and a second AIBD therapy are used, they may be administered together or separately. The agent of the invention may be administered first, and a second AIBD treatment may be administered second. It may be administered to the eye, or vice versa.

[0073] Thus, for example, in the above methods, the agent of the invention may be administered in combination with one or more other AIBs. When used in combination with D treatment, this is a second AIBD treatment that can be used to treat AIBD. or the amino acid sequence in FIG. 2 (sequence A protein containing amino acids 19 to 168 of number 2) or a functional equivalent of this protein A second A for use in a method of treating or preventing AIBD with a drug Amino acids 19 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2) for the treatment of IBD or a drug that is a functional equivalent of this protein. can.

[0074] The treatment results in a reduction in the amount of, or duration of, a second AIBD treatment. If a reduction occurs, the reduction will be compared to the amount of the second AIBD treatment used in the absence of the agent of the present invention. Compared to, at most or at least 10, 20, 30, 40, 50, 60, 70, 80% It could be.

[0075] subject Preferred subjects, agents, dosages and the like are as disclosed herein.

[0076] Any reference to any decrease or increase refers to the disease state compared to said subject in the absence of treatment. The decrease or increase in a disease parameter is preferably a quantifiable In this case, the increase or decrease is preferably statistically significant. The decrease is compared to a parameter in the absence of treatment (e.g., before the treatment is initiated). At least 3, 5, 10, 15, 20, 30, 40, 50% or more good.

[0077] The subject to which the agent is administered in the practice of the present invention is preferably a mammal, and preferably The subject to whom the agent is administered is a human. The subject to whom the agent is administered is a human being at risk for or has AIBD. It is the subject of

[0078] The methods of the invention also include determining whether a subject is at risk for or has AIBD. (ii) to determine whether or not the patient has a history of and determining the severity of the AIBD. do.

[0079] Drugs to be used in the present invention According to one embodiment of the invention, the agent is Covarcin itself or a functional equivalent thereof. In the following, the term "coversin-type protein" refers to a protein having the amino acid sequence shown in FIG. "A protein comprising amino acids 19 to 168 of sequence number (SEQ ID NO: 2) or a functional equivalent thereof" It is used as an abbreviation for

[0080] Conversen was isolated from the salivary glands of the tick Ornithodoros moubata. Covasin is a distant member of the lipocalin family and inhibits complement activation. Covaxin is the first member of the lipocalin family shown to bind C5. by binding to and preventing the cleavage of C5 into C5a and C5b by the C5 convertase. It inhibits the classical, alternative, and lectin complement pathways, thereby preventing activation of Inhibits both the production of the (e.g., pro-inflammatory) peptide C5a and the formation of MAC Conversen binds to C5 in rat, mouse and human serum and is involved in the C5 convertase. It has been demonstrated that it prevents the cleavage of C5 by erythropoietin with an IC50 of approximately 0.02 mg / ml.

[0081] Thus, a covercin-type protein has the amino acid sequence (SEQ ID NO:2) in FIG. Amino acids 19 to 168 or amino acids 1 to 1 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2) 68. The first of the protein sequences shown in FIG. The 18 amino acids of this signal sequence are not required for C5 binding or LTB4 binding activity. This can be reduced if necessary, for example for the efficiency of recombinant protein production. It can be.

[0082] Conversen protein was determined to be 1 nM using surface plasmon resonance (SPR). It has been demonstrated that covercin-type peptides (e.g. (functional equivalent of the covasin protein) is preferably less than 360 nM, more preferably More preferably less than 300 nM, most preferably less than 250 nM, preferably less than 200 nM, More preferably less than 150 nM, most preferably less than 100 nM, even more preferably binds C5 with a Kd of less than 50, 40, 30, 20, or 10 nM, advantageously less than 5 nM wherein the Kd is preferably determined by the method described in

[31] : Determined using surface plasmon resonance.

[0083] Covarcin inhibits the classical, alternative and lectin complement pathways. Preferably, the covercin-type protein stabilizes the overall conformation of C5. However, they do not directly block the C5 cleavage sites targeted by the C5 convertases of the three activation pathways. Conversen binds to C5 so as to prevent its disruption. Binding of Conversen to C5 modulates the overall conformation of C5. This leads to stabilization of the convertase but does not block the convertase cleavage site. Equivalents preferably also share these properties.

[0084] C5 is cleaved by the C5 convertase enzyme (Figure 1). The product of this cleavage is analyte. It contains the phylatoxin C5a and the lytic complex C5b, which acts as a membrane attack complex (MAC C promotes the formation of the complex of C5b, C6, C7, C8 and C9, also known as C 5a induces chemotaxis of neutrophils and eosinophils, activation of neutrophils, increased capillary permeability and eosinophil activation. Highly pro-inflammatory with implications in many pathological inflammatory processes, including inhibition of neutrophil apoptosis It is a steroid peptide

[32] .

[0085] Monoclonal antibodies and small molecules that bind and inhibit C5 have been used to treat a variety of diseases, particularly to treat rheumatoid arthritis, PNH, psoriasis, systemic lupus erythematosus and transplant rejection

[33] However, some of these monoclonal antibodies have been developed to block the C5 polymorphism. does not bind to certain C5 proteins from subjects with is ineffective

[34] . Preferably, the covercin-type protein should be able to inhibit not only wild-type C5 but also , C5 polymorphisms (e.g., which may render treatment with eculizumab ineffective or prevent eculizumab It also binds to and inhibits the cleavage of C5 from subjects with C5 polymorphisms that reduce the efficacy of treatment with The term "C5 polymorphism" refers to any one or more C5 variants that are different from wild-type C5. Possible insertions, deletions, amino acid substitutions, frameshifts, truncations, or any one of these changes or any version of C5 altered by two or more combinations. The wild-type C5 was identified under the accession number NP_001726.2; version GI:3801694. 7. Examples of C5 polymorphisms include the Polymorphisms at amino acid position 885, e.g., Arg885Cys (c. 2653C>T), p.Arg885His (c.2654G>A (encoded by) and Arg885Ser

[34] .

[0086] C5 polymorphism, e.g., that renders treatment with eculizumab ineffective or prevents eculizumab and agents that bind to C5, including C5 from subjects with C5 polymorphisms that reduce the efficacy of treatment with The ability of the antibody to bind to the antibody can be determined by standard in vitro assays known in the art, e.g., surface Plasmon resonance, or after incubation of proteins with labeled C5 on a gel The covasin protein can be determined by Western blot analysis. C5 genotype and / or C5 polymorphisms, e.g., ineffectiveness of treatment with eculizumab, if or from subjects with C5 polymorphisms that reduce the efficacy of treatment with eculizumab. C5 is either 360 nM or less, more preferably 300 nM or less, and most preferably 25 Less than 0 nM, preferably less than 200 nM, more preferably less than 150 nM, most preferably is less than 100 nM, and even more preferably less than 50, 40, 30, 20, or 10 nM. and preferably binds with a Kd of less than 5 nM, wherein the Kd is determined using surface plasmon resonance. and is preferably determined by the method described in

[31] .

[0087] This may result in wild-type C5 and C5 polymorphisms, such as ineffective treatment with eculizumab, or to C5 from subjects with C5 polymorphisms that reduce the efficacy of treatment with eculizumab. It may show higher, lower or the same affinity for

[0088] The ability of the covercin-type proteins to inhibit complement activation also indicates that the drugs inhibit complement activation in serum. For example, the activity of the complement in serum can be determined by measuring its ability to inhibit activation. may be measured by any means known in the art or described herein. This can be done.

[0089] Conversen-type proteins are also defined as having the function of inhibiting eicosanoid activity. Conversen has also been demonstrated to bind to LTB4. A functional equivalent of the protein also binds LTB4 with an affinity similar to that of the covercin protein. If the covercin-type protein does not retain the C5-binding ability, Such covercin-type proteins should retain significant LTB-4 binding ability.

[0090] The ability of covercin-type proteins to bind LTB4 has been demonstrated using standard methods known in the art. Convenient in vitro assays, e.g., competing with covercin for binding to labeled LTB4 Determined by competitive ELISA, isothermal titration calorimetry or fluorescence titration between anti-LTB4 antibodies Data obtained using fluorescence titrations show that Covarcin inhibits LTB4 by 200 to 400 ng / L. It has been shown that the antibody binds to the IgG antibody with a Kd between 300 and 300 pM. ) in LTB4 (Caymen Chemicals, Ann Arbor, MI, US) A) was measured using a spectrofluorometer, e.g., an LS 50 B spectrofluorometer (PerkinElmer). The quantification can be performed using a fluoroscopy (Nelmer, Norwalk, CT, USA). This can be carried out as follows.

[0091] A 100 nM purified covercin solution in 2 mL of PBS was placed in a quartz tube equipped with a magnetic stirrer. In addition, the bed (10 mm path length; Hellma, Muhlheim, Germany) The temperature was adjusted to 20°C and equilibrated, after which the fluorescence of Tyr / Trp in the protein was measured for 28 h. The excitation was at 340 nm (slit width: 15 nm). The fluorescence emission corresponding to the maximum emission was taken at 340 nm. The measurement was performed at 1000 nm (slit width: 16 nm). A 30 μM LTB4 ligand solution in PBS was Add a maximum volume of 20 μL (1% of the total sample volume) stepwise and incubate for 30 seconds. The steady-state fluorescence was measured after incubation. To calculate the KD value, the initial fluorescence intensity was set to 100%. The data was normalized using a titration of a 3 μM solution of N-acetyl-tryptophanamide. The inner filter effect was corrected using the FTIR method and the data were plotted against the corresponding ligand concentration. Next, we used nonlinear least-squares regression based on the law of mass action for the formation of bimolecular complexes. Using the published formula (Breustedt et al., 2006)

[35] , we calculated the origin of the n software version 8.5 (OriginLab, Northampton, MA The data was fitted using a method called FT-IR ...

[0092] Covarcin is preferably less than 1 nM, more preferably less than 0.9 nM, and most preferably less than 0.8 nM. less than 0.7 nM, more preferably less than 0.6 nM, and most preferably less than 0.5 LT with a Kd of less than 0.5 nM, even more preferably less than 0.4 nM, advantageously less than 0.3 nM. B4, wherein the Kd can be determined using fluorescence titration, preferably by the method described above. Coversin-type proteins preferably share these properties.

[0093] Conversen-type proteins (e.g., having reduced C5 binding activity or C5 binding A modified covasin polypeptide that does not have LTB-4 binding activity but retains LTB-4 binding ability is, for example, For example, less than 5 nM, less than 2 nM or less than 1 nM, more preferably less than 0.9 nM, and most preferably For β-aminobutyric acid, the concentration is less than 0.8 nM, preferably less than 0.7 nM, more preferably less than 0.6 nM, and most preferably less than 0.8 nM. Preferably less than 0.5 nM, even more preferably less than 0.4 nM, advantageously less than 0.3 nM and wherein the Kd is preferably determined using fluorescence titration. is determined by the method described above.

[0094] According to one embodiment of the present invention, the covercin protein is a wild type C5 and a C5 polymorphism, For example, to render treatment with eculizumab ineffective or to reduce the efficacy of treatment with eculizumab Both C5 from subjects with a C5 polymorphism that reduces LTB4 can bind to LTB4.

[0095] Thus, the covercin-type proteins are involved in the conversion of complement C5 to complement C5 by C5 convertase. a and complement C5b, and also to inhibit LTB4 activity. It would be particularly advantageous to use an agent that binds to both C5 and LTB4. Both the C5 and eicosanoid pathways may be involved in the pathology observed in AIBD. Therefore, the use of a single agent that inhibits multiple pathways involved in AIBD may Therefore, it is desirable to develop agents that inhibit only a single pathway involved in the inflammatory effects of complement-mediated diseases and disorders. In addition, enhanced efficacy can be achieved compared to using a single molecule. There are practical advantages associated with administering

[0096] Preferably, the agent of the present invention is derived from a blood-sucking arthropod. The term refers to insects, ticks, lice, fleas and mites, etc., that feed by sucking blood from a suitable host. Preferably, the agent is for ticks, preferably the tick Ornithias niger. Derived from thodoros moubata.

[0097] The functional equivalent of Covarcin is that it is derived from subjects with wild-type C5 or C5 polymorphisms. The ability of C5 to bind to either C5 and the conversion of C5 to C5a and C5b by C5 convertase It may be a homologue or fragment of covercin that retains the ability to prevent cleavage into C5b. The homologue or fragment may also retain the ability to bind LTB4.

[0098] Functional equivalents of covercin include, in particular, wild-type C5 or C5 polymorphisms, e.g., e.g. Invalidating or reducing the efficacy of eculizumab treatment C5 from subjects with the 5 polymorphism, and / or LTB4. The active site environment of one or more of the covasins is structurally similar to that of covasin. or a molecule, such as a synthetic molecule, that contains a similar or identical tertiary structure. The exact amino acid residues in covercin required for binding to C5 and LTB4 are , as shown in the Jore et al. reference cited above.

[0099] Homologs include, for example, Rhipicephalus appendiculatus, R .sanguineus, R.bursa, A.americanum, A.cajen nense, A.hebraeum, Boophilus microplus, Ba nnulatus, B. decoloratus, Dermacentor retic ulatus, D.andersoni, D.marginatus, D.variab ilis, Haemaphysalis inermis, Ha.leachii, Ha .punctata, Hyalomma anatolicum anatolicum , Hy.dromedarii, Hy.marginatum marginatum, Ixodes ricinus, I. persulcatus, I. scapulari s, I.hexagonus, Argas persicus, A.reflexus, Ornithodoros erraticus, O.moubata moubata , Omporcinus, and other tick species including O. savignyi. Paralogs and coversin sequences explicitly identified in FIG. 2, including the versin protein sequence. and orthologs.

[0100] The term "homologue" also refers to mosquitoes of the genera Culex, Anopheles, eles and Aedes species, especially Culex quinquefas ciatus, Aedes aegypti and Anopheles gambiae Mosquito species, including; flea species, e.g. Ctenocephalides felis (cat flea) ; horseflies; sandflies; blackflies; tsetse flies; lice; mites; leeches; and flatworms. It is meant to include the covercin protein sequence. is thought to exist in three separate forms of approximately 18 kDa in O. moubata. and the term "homologue" is meant to include these alternative forms of covercin. .

[0101] Methods for identifying homologues of the covercin sequence shown in Figure 2 will be apparent to one of skill in the art. For example, homologs can be identified by homology matches in both public and non-public sequence databases. They may be identified by searching, preferably, publicly available databases. However, non-publicly or commercially available databases may be subject to restrictions, especially if they are publicly available. A primary database is equally useful if it contains data not represented in the primary database. A depository site for primary nucleotide or primary amino acid sequence data, either publicly or commercially available Examples of publicly available primary databases include GenBan. k database (http: / / www.ncbi.nlm.nih.gov / ), E.M. BL database (http: / / www.ebi.ac.uk / ), DDBJ database (http: / / www.ddbj.nig.ac.jp / ), SWISS-PRO T Protein Database (http: / / expasy.hcuge.ch / ), PI R(http: / / pir.georgetown.edu / ), TrEMBL(htt p: / / www.ebi.ac.uk / ), TIGR database (http: / / www w.tigr.org / tdb / index.html), NRL-3D database (http: / / www.nbrfa.georgetown.edu), Prote in Data Base (http: / / www.rcsb.org / pdb), NR DB database (ftp: / / ncbi.nlm.nih.gov / pub / nrdb / README), OWL database (http: / / www.biochem.uc l.ac.uk / bsm / dbbrowser / OWL / ) and secondary database PR OSITE(http: / / expasy.hcuge.ch / sprot / prosi te.html), PRINTS(http: / / iupab.leeds.ac.uk / bmb5dp / prints.html), Profiles(http: / / ulr ec3.unil.ch / software / PFSCAN_form.html), P fam(http: / / www.sanger.ac.uk / software / pfa m), Identify(http: / / dna.stanford.edu / iden tify / ) and Blocks (http: / / www.blocks.fhcrc. org databases. Commercially available databases or non-public data Examples of bases include PathoGenome (Genome Therapeutics Inc.) and PathoSeq (formerly Incyte Pharmaceutica ls Inc.

[0102] Typically, the amino acid sequence is between two polypeptides (preferably over a specific region such as the active site). An identity of greater than 30% is an indication of functional equivalence and thus of the two proteins. is considered to be an indication of homology. Preferably, the homologous protein is The identified covasin protein sequence (SEQ ID NO: 2) had a sequence identity of greater than 60%. A more preferred homologue has the coversin protein sequence shown in FIG. ) and a degree of identity greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively The percentage identity referred to herein is based on BLAST version 2.1.3 Using NCBI (the National Center for Biotechnology Information;http: / / www.ncb The default parameters [Blos um 62 matrix; gap opening penalty = 11 and gap extension penalty = 1 The percent identity is determined using a related reference sequence (e.g., the sequence of SEQ ID NO:2). The amino acid sequence may span the entire length of the nucleic acid sequence (amino acids 1 to 168 or amino acids 19 to 168 of SEQ ID NO:2).

[0103] Thus, a covercin-type protein may be a reference sequence, e.g., amino acid sequence of FIG. 2, SEQ ID NO:2. 2, amino acids 19 to 168 or certain specific amino acids 1 to 168 of SEQ ID NO:2. Reference to percent amino acid sequence identity includes, for example, amino acids 19-168 of FIG. 2, SEQ ID NO:2, 2, amino acids 1 to 168 of SEQ ID NO: 2 and at least 60%, 70%, 80%, 90% 95%, 98% or 99% identity to the sequence of the target gene. When a covercin-type protein contains the sequence: The covercin-type protein is a fusion protein (e.g., a fusion protein containing another protein, e.g., a heterologous protein). Suitable second proteins are described below.

[0104] Functional equivalents of the covercin protein sequence shown in FIG. 2 include sequences from the wild-type sequence, e.g. For example, 1, 2, 3, 4, 5, 7, 10 or more amino acids, or up to 1, 2 , 3, 4, 5, 7 or 10 amino acid substitutions, insertions or deletions (e.g., N or C-terminal deletions), provided that such mutants are Live C5 and / or C5 polymorphisms (e.g., ineffective treatment with eculizumab, if or C5 polymorphisms that reduce the efficacy of treatment with eculizumab. and / or LTB4. This is consistent with related reference sequences (e.g. , amino acids 1 to 168 of SEQ ID NO:2 or amino acids 19 to 168 of SEQ ID NO:2) Thus, the variants include those that do not adversely affect the function or activity of the protein. The term also includes proteins containing conservative amino acid substitutions. (e.g., allelic variants or geographical location within the species from which the covercin protein is derived) It is intended to include wild-type C5 and / or C5 polymorphisms (e.g., Invalidating or reducing the effectiveness of eculizumab treatment Improved ability to bind C5 and / or LTB4 from subjects with Mutants having the same structure can also be generated by systematic or directed mutation of specific residues in the protein sequence. It can be designed in a similar way.

[0105] Functional equivalents of covasin include fragments of the covasin protein, provided that Such fragments are expressed as wild-type C5 and / or C5 polymorphisms (e.g., treatment with eculizumab). C5 polymorphisms that render eculizumab treatment ineffective or reduce its efficacy The fragments may include, for example, For example, a covercin protein sequence having less than 150 amino acids, less than 145 amino acids (also or homologues), provided that these fragments are not intended to be construed as limiting the scope of the present invention. and / or C5 polymorphisms (e.g., those that render eculizumab treatment ineffective or C5 and / or C5 polymorphisms that reduce the efficacy of treatment with rituximab or retains the ability to bind LTB4. acid, derived from the covercin protein sequence (or homologue) which is at least 145 amino acids The fragments may include polypeptides that bind to complement wild-type C5 and / or C5 polymorphisms (e.g., those that render eculizumab treatment ineffective or prevent eculizumab treatment) C5 and / or LTB from subjects with a C5 polymorphism that reduces the efficacy of treatment with Retains the ability to combine with 4.

[0106] Any functional equivalent or fragment thereof preferably corresponds to the system found in covasin. For example, the functional equivalents may have the amino acid sequence shown in FIG. From the amino terminus to the carboxyl terminus of the sequence consisting of amino acids 1 to 168 of the sequence (SEQ ID NO:2) When aligned, the amino acids are spaced 32 amino acids apart, 62 amino acids apart, 28 amino acids apart, and 1 amino acid apart. It contains six cysteine ​​residues spaced from each other by 21 amino acids and 10 amino acids. Exemplary fragments of the covercin protein include SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, The corresponding fragments are disclosed in SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. The DNAs are SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: This is disclosed in No. 13.

[0107] Fragments of the O. moubata covercin protein explicitly identified in FIG. 2 herein Such fragments include not only the above-mentioned homologues of this protein, but also fragments of the above-mentioned homologues of this protein. Such fragments of homologues are typically of the form Although the fragment has greater than 60% identity to the above-mentioned sequence, the more preferred fragment of the homologue is the fragment shown in FIG. The fragments of the covercin protein sequence were 70%, 80%, 90%, 95%, and 98%, respectively. or greater than 99% identity. Preferably, such fragments are prepared using the above-mentioned system. Fragments with improved properties can of course be obtained by systematically varying the wild-type sequence. Fragments can be rationally designed by purification or fragmentation followed by appropriate activity assays. against either the wild type or polymorphic variants of C5 and / or LTB4 These fragments may show similar or greater affinity to covercin. The size of the protein fragment may be as described above for the protein fragment.

[0108] As described above, the covercin-type protein is preferably a wild-type C5 and / or C 5 Polymorphisms (e.g., those that render eculizumab treatment ineffective or ineffective The results showed that the IL-16 binding assay was performed in subjects with a C5 polymorphism that reduces efficacy of the treatment. They have reduced or no C5 binding capacity but LTB4 binding capacity. Coversin-type proteins which retain activity may also be used in the present invention.

[0109] Conversen-type proteins that do not retain C5 binding ability but retain LTB-4 binding activity See, for example, co-pending UK patent application GB170640 filed on April 21, 2017. 6.4 (Applicant Reference Number P070475GB) and issued on the same day as this application was filed. International application PCT / EP2018 / xxxxxx (applicant reference number P070475 WO), the entire contents of which are incorporated herein by reference. Reduced or no C5 binding activity, but LTB-4 binding ability Such covercin-type proteins carrying the above structure may be used in all aspects of the present invention.

[0110] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity Such a covercin-type protein that retains the function of the covercin-type protein may contain or be composed of the following sequence: It may be configured as follows. SEQ ID NO:22 (SEQ ID NO:5 in GB1706406.4) is a sequence of SEQ ID NO:4, Met114 to Gln, Met116 to Gln, Leu117 to Ser, Asp118 to Asn , Ala119 to Gly, Gly120 to Ser, Gly121 to Ala, Le Change u122 to Asp, Glu123 to Asp, and Val124 to Lys. This is the amino acid sequence of the modified covercin (covercin variant 1). SEQ ID NO:23 (SEQ ID NO:6 in GB1706406.4) is a sequence similar to SEQ ID NO:4, except that Ala44 is Asn, Met116 to Gln, Leu117 to Ser, Gly121 to Ala , changing Leu122 to Asp, Glu123 to Ala, and Asp149 to Gly 1 shows the amino acid sequence of a modified covercin that has been modified to act as a covercin variant 2. ) SEQ ID NO:24 (SEQ ID NO:7 in GB1706406.4) is a sequence similar to SEQ ID NO:4, except that Ala44 is Asn, Met116 to Gln, Leu122 to Asp, and Asp149 to Gl y is modified to modify the amino acid sequence of the modified covercin. Ant 3) SEQ ID NO:25 (SEQ ID NO:8 of GB1706406.4) is a sequence similar to SEQ ID NO:4 except that Ala44 is 1 shows the amino acid sequence of modified covercin, which has been modified to change the Asn to 1. Variant 4) SEQ ID NO:26 (SEQ ID NO:9 in GB1706406.4) corresponds to amino acid position 1 of SEQ ID NO:4 14-124 (amino acid positions 132-142 of SEQ ID NO:2) between beta H and alpha 2 The amino acid sequence of the loop between the SEQ ID NO:27 (SEQ ID NO:10 in GB1706406.4) corresponds to covercin variant 1 Beta H and alpha H at amino acid positions 114 to 124 of SEQ ID NO: 4 (SEQ ID NO: 22) 2 is the amino acid sequence of the loop between SEQ ID NO:28 (SEQ ID NO:11 in GB1706406.4) corresponds to covercin variant 2 Beta H and alpha H at amino acid positions 114 to 124 of SEQ ID NO: 4 (SEQ ID NO: 23) 2 is the amino acid sequence of the loop between SEQ ID NO:29 (SEQ ID NO:12 in GB1706406.4) corresponds to covercin variant 3 Beta H and alpha H at amino acid positions 114 to 124 of SEQ ID NO: 4 (SEQ ID NO: 24) 2 is the amino acid sequence of the loop between

[0111] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity A coversin polypeptide that retains the function of the coversin polypeptide is a modified coversin polypeptide (e.g., Exhibits cotriene or hydroxyeicosanoid binding activity and reduced C5 binding "Modified Coversin Polypeptides" Reference to any modified version of SEQ ID NO:2 or SEQ ID NO:4, i.e., the sequence Covastatin with or without the 18 amino acid signal sequence found at the N-terminus of no. 2 This should be understood as a reference to a synthase polypeptide.

[0112] Such polypeptides have leukotriene or hydroxyeicosanoid binding activity. and may exhibit reduced or no C5 binding, and may have a C5 binding activity of 1 to 30 SEQ ID NO:4, with the amino acid substitution: (i) at positions 114 to 124 of SEQ ID NO: 4, the following substitutions (a) to (j): Met114 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A substituted with La, Pro, His, or Thr; b. Met116 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A substituted with La, Pro, His, or Thr; c. Leu117 is Ser, Asp, Asn, Glu, Arg, Lys, Gly, A la, or substituted with Pro; d. Asp118 is Asn, Gln, Arg, Lys, Gly, Ala, Leu, S Substituted with Er, Ile, Phe, Tyr, Met, Pro, His, or Thr r; e. Ala119 is Gly, Asp, Asn, Glu, Arg, Lys, Leu, I substituted with Le, Phe, Tyr, Met, Pro, or His; f. Gly120 is Ser, Asp, Asn, Glu, Arg, Lys, Leu, I substituted with Le, Phe, Tyr, Met, Pro, or His; g. Gly121 is Ala, Asp, Asn, Glu, Arg, Lys, Leu, I substituted with Le, Phe, Tyr, Met, Pro, or His; h. Leu122 is Asp, Glu, Asn, Ala, Gln, Arg, Lys, P ro, or substituted with His; i. Glu123 is Asp, Ala, Gln, Asn, Arg, Lys, Gly, L Substituted with eu, Ser, Ile, Phe, Tyr, Pro, His, or Thr r; j. Val124 is Lys, Gln, Asn, Arg, Lys, Gly, Ala, P Substituted with ro, His, or Thr or / and (ii) Ala44 in SEQ ID NO: 4 is Asn, Asp, Gln, Glu, Arg, Ly Substituted with s, Leu, Ile, Phe, Tyr, Met, Pro, or His ] or a modified covercin polypeptide having up to five amino acids deleted from the N-terminus thereof. It may comprise a fragment of

[0113] As used herein, LK / E binding activity refers to the binding activity of LTB4, B4 isoleukotrienes and Any hydroxylated derivatives thereof, including HETE, HPETE, and EET, The ability to bind to leukotrienes and hydroxyeicosanoids, including but not limited to, LTB4 binding is of particular interest.

[0114] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity A modified covasin polypeptide that retains the function of the covasin polypeptide may also be modified as described below with reference to SEQ ID NO:2. or 4, or comprising SEQ ID NO: 2 or 4 modified as described below. It's okay to do that.

[0115] The unmodified covercin polypeptide in SEQ ID NO: 2 and SEQ ID NO: 4 is Beta H at amino acid positions 114 to 124 (amino acid positions 132 to 142 of SEQ ID NO: 2) The present invention is characterized by a loop between alpha 1 and alpha 2. This loop has the sequence shown below: -Met-Trp-Met-Leu-Asp-Ala-Gly-Gly-Leu-Glu -Val- (SEQ ID NO:26) The first Met is at position 114 of SEQ ID NO:4 and at position 132 of SEQ ID NO:2.

[0116] Reduced C5 binding activity or no C5 binding activity but LTB-4 binding ability In the modified covercin polypeptide having the formula The synth polypeptide has the following substitutions (a) to (j) at positions 114 to 124 of SEQ ID NO:4: Met114 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A substituted with La, Pro, His, or Thr, preferably Gln or Ala ; b. Met116 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A substituted with La, Pro, His, or Thr, preferably Gln or Ala ; c. Leu117 is Ser, Asp, Asn, Glu, Arg, Lys, Gly, A la, or Pro, preferably substituted with Ser or Ala; d. Asp118 is Asn, Gln, Arg, Lys, Gly, Ala, Leu, S Er, Ile, Phe, Tyr, Met, Pro, His, or Thr, preferably A Substituted by sn; e. Ala119 is Gly, Asp, Asn, Glu, Arg, Lys, Leu, I Le, Phe, Tyr, Met, Pro, or His, preferably Gly or Asn has been replaced with; f. Gly120 is Ser, Asp, Asn, Glu, Arg, Lys, Leu, I Le, Phe, Tyr, Met, Pro, or His, preferably Ser or Asn has been replaced with; g. Gly121 is Ala, Asp, Asn, Glu, Arg, Lys, Leu, I Le, Phe, Tyr, Met, Pro, or His, preferably Ala or Asn has been replaced with; h. Leu122 is Asp, Glu, Asn, Ala, Gln, Arg, Lys, P ro, or His, preferably substituted with Asp or Ala; i. Glu123 is Asp, Ala, Gln, Asn, Arg, Lys, Gly, L eu, Ser, Ile, Phe, Tyr, Pro, His, or Thr, preferably A substituted with sp, Ala, Gln, or Asn; j. Val124 is Lys, Gln, Asn, Arg, Lys, Gly, Ala, P substituted with ro, His, or Thr, preferably Lys or Ala The invention has been modified so that one or more of the following is performed:

[0117] Reduced C5 binding activity or no C5 binding activity but LTB-4 binding ability In a modified coversin polypeptide comprising the formula: The covercin polypeptide comprises the following substitutions (a) to (j) at positions 114 to 124 of SEQ ID NO:4: : a. Met114 is replaced by Gln; b. Met116 is replaced by Gln; c. Leu117 is replaced by Ser; d. Asp118 is replaced by Asn; e. Ala119 is replaced with Gly; f. Gly120 is replaced by Ser; g. Gly121 is replaced by Ala; h. Leu122 is replaced by Asp; i. Glu123 is replaced with Asp, or Ala; j. Val124 is replaced by Lys The invention can be modified so that one or more of the following is achieved:

[0118] In the modified covercin polypeptide, 2, 3, 4, 5, 6 of the substitutions (a) to (j) , 7, 8, 9, or 10 are present. Preferably, two or more of the substitutions (a) to (j) are present. , there are five or more or eight or more.

[0119] Reduced C5 binding activity or no C5 binding activity but LTB-4 binding ability In a modified coversin polypeptide comprising the formula: The covercin polypeptide comprises the following substitutions at positions 114-124 of SEQ ID NO:4: a. Met114 is replaced by Gln; b. Met116 is replaced by Gln; c. Leu117 is replaced by Ser; d. Asp118 is replaced by Asn; e. Ala119 is replaced with Gly; f. Gly120 is replaced by Ser; g. Gly121 is replaced by Ala; h. Leu122 is replaced by Asp; i. Glu123 is replaced by Asp; j. Val124 is replaced by Lys can be modified so that

[0120] Optionally, in the modified covercin polypeptide described above, Trp115 is substituted. A preferred modified covercin polypeptide has the amino acid sequence at amino acid positions 114 to 116 of SEQ ID NO:4. Between beta H and alpha 2 of 124, the sequence Gln-Trp-Gln-Ser-Asn - a loop having Gly-Ser-Ala-Asp-Asp-Lys (SEQ ID NO: 27) Yes.

[0121] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In a modified covercin polypeptide that retains the function, the covercin polypeptide has SEQ ID NO: The following substitutions at positions 114-124 of issue 4: Met114 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A substituted with La, Pro, His, or Thr, preferably Gln; b. Leu117 is Ser, Asp, Asn, Glu, Arg, Lys, Gly, A la, or Pro, preferably substituted with Ser; c. Gly121 is Ala, Asp, Glu, Arg, Lys, Leu, Ile, P substituted with he, Tyr, Met, Pro, or His, preferably Ala; d. Leu122 is Asp, Glu, Asn, Gln, Arg, Lys, Pro, or or His, preferably Asp; e. Glu123 can be Asp, Ala, Gln, Asn, Arg, Lys, Gly, or L eu, Ser, Ile, Phe, Tyr, Pro, His, or Thr, preferably A Substituted with sp can be modified so that

[0122] In a more detailed embodiment; a. Met116 is replaced by Gln; b. Leu117 is replaced by Ser; c. Gly121 is replaced with Ala; d. Leu122 is replaced by Asp; e. Glu123 is substituted with Ala.

[0123] Optionally, in the modified covercin polypeptide described above, Trp115 is replaced. Optionally, in this embodiment, Met114, Trp115, Asp11 8, Ala119, Gly120 and Val124 are unsubstituted or Conservative substitutions have been made as noted elsewhere in the specification. or a preferred modified cover that does not have C5 binding activity but retains LTB-4 binding ability. The synthase polypeptide is a beta H and alpha 2 polypeptide at amino acid positions 114-124 of SEQ ID NO:4. Between It has a loop having sp-Ala-Val (SEQ ID NO:28).

[0124] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In a modified covercin polypeptide that retains the function, the covercin polypeptide has SEQ ID NO: In issue 4, positions 114-124, the following substitutions: Met116 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A substituted with La, Pro, His, or Thr, preferably Gln; b. Leu122 is Asp, Glu, Asn, Gln, Arg, Lys, Pro, or or His, preferably Asp; can be modified so that

[0125] In a more detailed embodiment; a. Met116 is replaced by Gln; b. Leu122 is replaced with Asp.

[0126] Optionally, in the modified covercin polypeptide described above, Trp115 is substituted Optionally, in this embodiment, Met114, Trp115, Le u117, Asp118, Ala119, Gly120, Gly121, Glu123 and and Val124 are not substituted. Preferred modified covercin polypeptides that have no binding activity but retain LTB-4 binding ability The sequence is Met-Trp-Gln-Leu-Asp-Ala-Gly-Gly-Asp -amino acid positions 114 to 124 of SEQ ID NO: 4 (SEQ ID NO: 29) containing Glu-Val It has a loop between beta H and alpha 2.

[0127] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In a modified covercin polypeptide that retains the function, the covercin polypeptide has SEQ ID NO: Ala44 in No. 4 (Ala62 in SEQ ID NO: 2) is Asn, Asp, Gln, Glu, Replace with Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His. It can be modified so that it can be converted.

[0128] In a preferred embodiment, Ala44 in SEQ ID NO:4 is replaced with Asn.

[0129] This substitution at position 44 of SEQ ID NO:4 (or position 62 of SEQ ID NO:2) is These substitutions may be made in combination with any of the other substitutions listed above.

[0130] Reduced C5 binding activity or no C5 binding activity but LTB-4 binding ability In another modified coversin polypeptide, the coversin polypeptide has the sequence The following substitutions (a) to (j) are made at positions 114 to 124 of number 4: Met114 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A la, Pro, His, or Thr, preferably Gln or Ala, e.g. Gln Has been replaced; b. Met116 is Gln, Asp, Asn, Glu, Arg, Lys, Gly, A la, Pro, His, or Thr, preferably Gln or Ala, e.g. Gln Has been replaced; c. Leu117 is Ser, Asp, Asn, Glu, Arg, Lys, Gly, A la, or Pro, preferably substituted with Ser or Ala, e.g., Ser; d. Asp118 is Asn, Gln, Arg, Lys, Gly, Ala, Leu, S Er, Ile, Phe, Tyr, Met, Pro, His, or Thr, preferably A Substituted by sn; e. Ala119 is Gly, Asp, Glu, Arg, Lys, Leu, Ile, P he, Tyr, Met, Pro, or His, preferably Gly or Asn, e.g. substituted with Gly; f. Gly120 is Ser, Asp, Glu, Arg, Lys, Leu, Ile, P he, Tyr, Met, Pro, or His, preferably Ser or Asn, e.g. substituted with Ser; g. Gly121 is Ala, Asp, Glu, Arg, Lys, Leu, Ile, P he, Tyr, Met, Pro, or His, preferably Ala or Asn, e.g. substituted with Ala; h. Leu122 is Asp, Glu, Asn, Gln, Arg, Lys, Pro, or or His, preferably Asp or Ala, e.g., Asp; i. Glu123 is Asp, Ala, Gln, Asn, Arg, Lys, Gly, L eu, Ser, Ile, Phe, Tyr, Pro, His, or Thr, preferably A substituted with sp, Ala, Gln or Asn, e.g. Asp or Ala; j. Val124 is Lys, Gln, Asn, Arg, Lys, Gly, Ala, P substituted with ro, His, or Thr, preferably Lys or Ala, e.g., Lys; Is and additionally, Ala44 in SEQ ID NO: 4 (SEQ ID NO: 2 Ala62 in the nucleus is Asn, Asp, Gln, Glu, Arg, Lys, Leu, Il e, Phe, Tyr, Met, Pro, or His, preferably Asn. It can be modified as follows.

[0131] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In some modified coversin polypeptides that retain the function, the coversin polypeptide is The following substitutions at positions 114-124 of SEQ ID NO:4: a. Met116 is replaced by Gln; b. Leu117 is replaced by Ser; c. Gly121 is replaced by Ala; d. Leu122 is replaced by Asp; e. Glu123 is replaced by Ala and Ala44 in SEQ ID NO:4 is modified to be substituted with Asn. can.

[0132] In a preferred embodiment of this embodiment, the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO:4 are The group is as shown in SEQ ID NO:28.

[0133] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In some modified coversin polypeptides that retain the function, the coversin polypeptide is The following substitutions at positions 114-124 of SEQ ID NO:4: a. Met116 is replaced by Gln; b. Leu122 is replaced by Asp is present, and has been modified so that Ala44 in SEQ ID NO:4 is replaced with Asn.

[0134] In a preferred embodiment of this embodiment, the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO:4 are The group is as shown in SEQ ID NO:29.

[0135] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In some modified coversin polypeptides that retain the function, the coversin polypeptide is Asp149 in SEQ ID NO: 4 is Gly, Gln, Asn, Ala, Met, Arg, L Substitute with ys, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr In some embodiments, the coversin polypeptide can be modified to: Asp149 of SEQ ID NO:4 is modified to be replaced with Gly. This substitution at position 167 of SEQ ID NO:2 is not required to be one of the other substitutions described herein. This may be done in combination with either

[0136] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In some modified coversin polypeptides that retain the function, the coversin polypeptide is The following substitutions at positions 114-124 of SEQ ID NO:4: a. Met116 is replaced by Gln; b. Leu117 is replaced by Ser; c. Gly121 is replaced by Ala; d. Leu122 is replaced by Asp; e. Glu123 is replaced by Ala Ala44 in SEQ ID NO:4 is replaced with Asn, and Asp149 in SEQ ID NO:4 is can be modified to be substituted with Gly149.

[0137] In a preferred embodiment of this embodiment, the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO:4 are The group is as shown in SEQ ID NO:28.

[0138] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity In some modified coversin polypeptides that retain the function, the coversin polypeptide is The following substitutions at positions 114-124 of SEQ ID NO:4: a. Met116 is replaced by Gln; b. Leu122 is replaced by Asp exists, Ala44 in SEQ ID NO:4 is replaced with Asn, and Asp149 in SEQ ID NO:4 is replaced with Gly It can be modified to be substituted with 149.

[0139] In a preferred embodiment of this embodiment, the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO:4 are The group is as shown in SEQ ID NO:29.

[0140] In various aspects and embodiments of the present disclosure, the C5 binding activity is reduced or A modified covasin polypeptide that does not have 5-binding activity but retains LTB-4 binding ability is The unmodified covercin polypeptide and amino acids 1 to 30 of SEQ ID NO: 2 and SEQ ID NO: 4 The amino acids are different. can be made, provided that the resulting modified covasin polypeptide is similar to the unmodified covasin polypeptide. exhibiting LK / E binding activity and reduced C5 binding activity compared to the syn polypeptide; does not exhibit C5 binding.

[0141] In some embodiments, the nucleotides at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 The six cysteine ​​amino acids are retained in the modified covercin polypeptide of the present invention. .

[0142] In some modified covercin polypeptides, Asn60 and Asn in SEQ ID NO:4 84 are each replaced with Gln. This modification allows the polypeptide to be expressed in yeast. To prevent N-linked hyperglycosylation when expressed, site-directed mutagenesis was performed. This can be done.

[0143] In some modified covercin polypeptides, the following amino acid in SEQ ID NO:4: Phe 18, Tyr25, Arg36, Leu39, Gly41, Pro43, Leu52, V al54, Met56, Phe58, Thr67, Trp69, Phe71, Gln87 , Arg89, His99, His101, Asp103, and Trp115 One or more of these are believed to be involved in binding to LTB4 and therefore are preserved in their unmodified form. In some modified covasin polypeptides, at least one of these amino acids may be present. At least 5, 10 or 15, or all, of the modified coversin polypeptides of the invention The C5 binding activity of the ribozyme is reduced, or the C5 binding activity of the ribozyme is reduced. In some modified covercin polypeptides, which do not have the same activity as the covercin polypeptides described above but retain the LTB-4 binding ability, In this case, one or more of these amino acids may be conservatively substituted. Some of these have C5 binding activity or no C5 binding activity but retain LTB-4 binding ability. In the modified covasin polypeptide of the present invention, at most 5, 10 or Fifteen or all of the amino acids are conservatively substituted in the modified covercin polypeptides of the present invention. is.

[0144] The following positions in SEQ ID NO: 4: 5, 6, 11, 13-15, 20-21, 24-27, 2 9~32, 35~41, 45, 47~48, 50, 52~60, 64, 66, 69~81 , 83, 84, 86, 90-94, 97-104, 112-113, 115, 125-1 Amino acids 29, 132–139, 145, 148, and 150 correspond to covercin and TS It is highly conserved between GP2 and TSGP3.

[0145] The following positions in SEQ ID NO: 4: 5, 6, 11, 13-15, 18, 20-21, 24-2 7, 29-32, 35-41, 43, 45, 47-48, 50, 52-60, 64, 66 , 67, 69-81, 83, 84, 86, 87, 89, 90-94, 97-104, 11 2-113, 115, 125-129, 132-139, 145, 148, and 150 The amino acids in are thought to be involved in binding to LTB4 and / or interact with covercin and T It is highly conserved between SGP2 and TSGP3.

[0146] The following positions in SEQ ID NO: 4: 5, 6, 11, 13-15, 18, 20-21, 24-2 5, 27, 30-32, 35-41, 43, 47-48, 50, 52-60, 64, 66 , 67, 69-81, 83, 84, 86, 87, 89, 90-94, 98, 100, 10 2-104, 112-113, 115, 126, 128-129, 132-139, 14 The amino acids 5, 148, and 150 are thought to be involved in binding to LTB4, and and / or is highly conserved between covercin and TSGP2 and TSGP3.

[0147] Thus, those with reduced or no C5 binding activity but L In some modified covercin polypeptides that retain TB-4 binding ability, the amino acids In some embodiments, at least one of these amino acids is retained in its unmodified form. At least 5, 10 or 15, or all, of the modified coversin polypeptides of the invention In some embodiments, one or two of these amino acids are retained in their unmodified form. or more may be conservatively substituted. In some embodiments, up to five of these amino acids may be conservatively substituted. , 10 or 15, 20, 25, 30, 40, 50 or all of the modified cover of the present invention. Conservative substitutions are made in the syn polypeptide.

[0148] The modified coversin polypeptides described herein typically have the sequence set forth in SEQ ID NO:2 or SEQ ID NO: 4 and 1 to 30, preferably 2 to 25, more preferably 3 to 20, and even more preferably or 4-15 amino acid differences. Typically, the differences are 5-12, or 6-10. Amino acid changes, e.g., 1-30, 2-25, 3-30, 4-15, 5-1 Two, or six to ten amino acid substitutions can be made in SEQ ID NO:2 or SEQ ID NO:4.

[0149] Amino acid positions 114 to 124 of SEQ ID NO: 4 (amino acid positions 132 to 142 of SEQ ID NO: 2 ) having a loop between beta H and alpha 2 as shown in SEQ ID NO:27 The bassin polypeptide has an additional 10 amino acids compared to SEQ ID NO:4 as a result of the presence of this loop. Thus, in some embodiments, the modified codons described herein have amino acid substitutions. The barsin polypeptide preferably has the same structure as SEQ ID NO:4, except for the substitutions shown in SEQ ID NO:22. Compare (e.g., in the loop of SEQ ID NO:27) 1 to 15, 2 to 10, 3 to 5, or up to and having 2, 3, 4 or 5 additional substitutions.

[0150] As shown in SEQ ID NO:28, amino acid positions 114 to 124 of SEQ ID NO:4 (SEQ ID NO: 2 (amino acid positions 132-142) with a loop between beta H and alpha 2 The covercin polypeptide has 5 amino acids as compared to SEQ ID NO:4 as a result of the presence of this loop. Thus, in some embodiments, the modified codons described herein have amino acid substitutions. The barsin polypeptide preferably has the same structure as SEQ ID NO:4, except for the substitutions shown in SEQ ID NO:23. Compare (e.g., in the loop of SEQ ID NO:28) 1 to 20, 2 to 15, 3 to 10, or With up to 2, 3, 4, 5, 6, 7, 8, 9, 10 additional substitutions. Preferably, the amino acid sequence includes substitutions at positions 44 and 149 as set forth elsewhere herein. Can be enjoyed.

[0151] As shown in SEQ ID NO:29, amino acid positions 114 to 124 of SEQ ID NO:4 (SEQ ID NO:2 A modified codon with a loop between beta H and alpha 2 at amino acid positions 132-142 The bassin polypeptide has two nucleotides as a result of the presence of this loop, compared to SEQ ID NO:4. Thus, in some embodiments, the modified coversin polypeptide has Preferably, in addition to the substitutions shown in SEQ ID NO:24, there are 1 to 25, 2 to 4, or more substitutions, compared to SEQ ID NO:4. 12, 3-15, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15 additional substitutions (e.g., substitutions in the loop of SEQ ID NO:29). Additional substitutions preferably include those at positions 44 and 149 as set forth elsewhere herein. Position substitution is included.

[0152] A modified covercytidine derivative having a substitution at position 44 of SEQ ID NO:4 as shown elsewhere herein. The polypeptide is preferably 1 to 25, 2 to 12, 3 to 15, or more amino acids longer than SEQ ID NO: 4. , or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 has the additional substitution:

[0153] Substitutions other than those explicitly mentioned above are preferably conservative substitutions, for example according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column are: may be substituted for one another.

[0154] [Table 1]

[0155] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity Preferred modified coversin polypeptides that retain the function are those represented by SEQ ID NOs: 22, 23, 24, 25 The amino acid sequence may comprise or consist of the amino acid sequence shown in one of:

[0156] The present invention also relates to antibodies having reduced or no C5 binding activity. A fragment of a modified covasin polypeptide that retains the above-mentioned LTB-4 binding ability, Use of a fragment of the covercin polypeptide with up to 5 amino acids deleted from the N-terminus The fragments include 1, 2, 3, 4 or 5 fragments from the N-terminus of the modified coversin polypeptide. 5 deletions. Other positions in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 Deletions from the IL-1 domain also enhance the LK / E binding activity of the resulting polypeptide. If the compound has a reduced or no complement inhibitor activity, it is considered to be a soluble form of the compound. are considered to form part of the present invention.

[0157] have reduced C5 binding activity or no C5 binding activity but LTB-4 binding activity When a modified covasin polypeptide that retains the ability to bind C5 is used, the C5 binding ability can be determined by, for example, Compared to the binding exhibited by the unmodified covercin polypeptide in columns 2 or 4. At least half, fifth, tenth, fifteenth, twentieth, fiftieth, C5 binding may be reduced or eliminated by 100-fold. At least 50%, 60%, 70%, 80% The modified covercin polypeptide may be reduced by 90% or 95%. 2013) J Biol Chem. 288, 18789-18802, or surface plasma resonance (SPR) according to the method shown in Example 2 of GB1706406.4. rface Plasma Resonance) is determined by It can bind C5 with a KD greater than molar.

[0158] Functional equivalents as used according to the invention include, for example, coding sequences for heterologous protein sequences. A polynucleotide encoding the covercin protein was cloned in frame with the covercin sequence. As used herein, a "heterologous" protein may be a fusion protein obtained by linking the The term "covasin" refers to any polypeptide other than the covasin protein or its functional equivalent. The term is intended to refer to a soluble fusion protein that is included at either the N-terminus or the C-terminus. Examples of heterologous sequences that may be present include the following: extracellular domains of membrane-bound proteins, immunoglobulin constants, A constant region (Fc region), a PAS or XTEN or a similar unstructured polypeptide, Multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or affinities These heterologous sequences allow purification by affinity chromatography. Many are commercially available inserted into expression plasmids that contain To provide additional properties without significantly compromising the specific biological activity of a protein because these sequences are commonly included in fusion proteins

[36] . Examples of additive properties include a longer sustained half-life in body fluids (e.g., the addition of an Fc region or P as a result of pasylation

[37] , extracellular localization, or histone This is made possible by tags such as sucrose, GST, FLAG, avidin or HA tags. The fusion protein may additionally contain a linker sequence (e.g., The linker may be, for example, 1 to 50 amino acids in length, whereby the components are separated by the linker. can be.

[0159] Thus, a fusion protein is an example of a protein that contains a covercin-like protein. These include, for example, proteins containing PAS sequences and covercin-type protein sequences. PAS sequences are described, for example, by Schlapschy M et al.

[37] and The PAS-modified covercin molecule is described in EP08773567.6 and Kuhn et al.

[0038] . PAS-modification is carried out by combining proteins with the amino acids Pro, Ala, and and / or a conformationally disordered polypeptide sequence composed of Ser This describes the genetic fusion of XL Protein (http: / / xl-protein.jp / ). This technique was developed by the group TEIN (http: / / www.tein.com / ) and involves the synthesis of solvated random chains in large flows. This provides a simple method for binding to proteins fused to it in a biodynamic volume. The peptide sequence adopts a bulky random coil structure. The size of the fusion protein is much larger than the protein to which it is fused. It has been shown that the PAS sequence reduces clearance from the body system. P08773567.6 and in the Schlapschy references cited above. Any suitable PAS sequence can be used in the fusion protein. Examples include random coil conformation and consists of alanine, serine and proline residues (or proline Amino acid sequences consisting of at least about 100 amino acid residues (consisting of amino acid residues of alpha-, alpha- and alanine). It may contain multiple amino acid repeats, where The repeats consist of Ala, Ser, and Pro residues (or proline and alanine residues). The sequence consists of no more than six identical amino acid residues. It may constitute greater than 4% but less than 40% of the acid. ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 15); AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 16); APSSPSPSAPSSPSPASPSS (SEQ ID NO: 17), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 18), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 19), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 20) and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 21) or circular permuted versions of these sequences as whole or part of these sequences. For example, the amino acid sequence may be selected from the group consisting of a nucleotide sequence, a nucleotide sequence, a nucleotide sequence, and a nucleotide sequence. Of the repeats, 5 to 40, 10 to 30, 15 to 25, 18 to 20, preferably , 20 to 30 or 30 copies, i.e., one of SEQ ID NOs: 15 to 21, preferably 1 5 may be present. Preferably, the PAS sequence contains 30 copies of SEQ ID NO: 15 or Preferably, the PAS sequence is directly linked to the N-terminus of the covercin-type protein. or via a linker sequence), and in certain preferred embodiments, The type protein comprises or consists of amino acids 19 to 168 of SEQ ID NO:2. (e.g., the fusion protein may comprise (a) a PAS consisting of 30 copies of SEQ ID NO:15) and (b) amino acids 19 to 168 of SEQ ID NO:2, wherein (a) comprises (either directly or via a linker sequence) to the N-terminus of

[0160] The fusion protein may additionally contain a linker sequence (e.g., 1-50, 2-30, 3-20, 5 10 amino acids in length), whereby the components are linked by this linker. In one embodiment, the linker sequence may be a single alanine residue.

[0161] The protein and its functional equivalents can be expressed in recombinant form by expression in a host cell. Such expression methods are known to those skilled in the art and are described in

[39] and

[40] . Recombinant forms of the covasin protein and its functional equivalents are described in detail herein. The form is preferably non-glycosylated. Preferably, the host cell is Escherichia coli (E. coli).

[0162] The covercin protein and its functional equivalents are preferably, e.g. Separated from at least one component of the host cell and / or cell growth medium in which it is expressed In some embodiments, the covasin protein or a functional equivalent thereof is in isolated form. is at least 90% as determined, for example, by electrophoresis or chromatography. , 95%, or 99% pure. The proteins and fragments of the invention are also They can be prepared using conventional techniques of protein chemistry. For example, protein fragments The fusion protein may be prepared by chemical synthesis. Methods for producing fusion proteins are known in the art. are standard and well known to the skilled reader. The techniques for the synthesis of genomic DNA, recombinant DNA and immunological methods can be found in

[39] or

[41] . Cut.

[0163] According to a further embodiment of the invention, the agent comprises a nucleic acid encoding a covercin-type protein. For example, the in vivo expression of a covercin-type protein by relevant cells in a subject may be Gene therapy to induce endogenous production either in vivo or ex vivo Another approach is to inject the therapeutic gene directly into the bloodstream or muscle tissue. This involves the administration of "naked DNA."

[0164] Preferably, such a nucleic acid molecule has the nucleotide sequence of This nucleotide sequence comprises or consists of bases 55 to 507. The nucleotide sequence in FIG. 2 is not included in the covasin protein. The first 54 bases of the sequence are not required for complement inhibitory or LTB4 binding activity. Alternatively, the nucleic acid molecule may encode a protein having a signal sequence. 2, which may include or consist of bases 1 to 507 of the nucleic acid sequence in FIG. good.

[0165] Mode of administration The covasin protein does not require a medical professional to administer, and these In contrast, most recombinant antibodies are absorbed very slowly. As a result, it must be infused (e.g., intravenously) over a long period of time. Administration of such molecules also requires medical personnel. It has the advantage of being more effective at inhibiting activation of the complement pathway in Covarsin-type proteins also have a higher potency than other drugs, such as antibodies like eculizumab. It also has the advantage of being easy to administer.

[0166] The agent is administered in a therapeutically or prophylactically effective amount. The term "therapeutically effective amount" refers to an amount In this context, "treating" refers to the amount of drug required to treat a disorder. Including reducing severity.

[0167] As used herein, the term "prophylactically effective amount" refers to a dose that is effective to treat or prevent a related condition, such as AIBD. It refers to the amount of drug required to prevent. In this context, "preventing" can refer to, for example, Reducing the severity of a disorder when its presence is not detected before administration of the drug is started Reducing the severity of a disorder can include, for example, the use of any of the methods described elsewhere herein. Thus, the purpose of the present invention may be to reduce the size or number of blisters or affected body surfaces.

[0168] The reduction or improvement is relative to results in the absence of administration and the agents described herein. The results were evaluated according to standard criteria used to evaluate such patients. To the extent that this can be quantified, it can be measured on a relative basis (e.g., by the size of the blister or the affected body surface). or number) at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 % decrease or improvement.

[0169] Preferably, the dose of the agent is adjusted to provide as much available C5 as possible to the subject, more preferably or is sufficient to bind all available C5. As much available LTB4 as possible in the subject, more preferably all available In some embodiments, the dose of the agent may be as low as possible. Many available C5 and LTB4, e.g., all available C5 and LTB4 The dose of drug delivered is sufficient to bind all available C 5 and / or at least 1x the molar dose required to bind LTB4 or 1. The dose of drug delivered may be, for example, 5-fold or 2-fold greater than the dose required to achieve full benefit in a subject. Approximately or less than the molar dose required to bind available C5 and / or LTB4 Both can be 1, 1.5, 2, 2.5, 3 or 4 times.

[0170] Preferably, the dose is between 0.0001 mg / kg (mass of drug compared to the mass of the patient) and 20mg / kg, 0.001mg / kg~10mg / kg, 0.01mg / kg~2mg / kg, 0.1mg / kg~1mg / kg, 0.2mg / kg~0.8mg / kg, 0. 3mg / kg~0.6mg / kg, 0.4mg / kg~0.6mg / kg (e.g. about 0. 57mg / kg).

[0171] A therapeutically or prophylactically effective amount may additionally be defined in terms of inhibition of terminal complement, e.g. For example, terminal complement activity (TCA) is at least 100% relative to terminal complement activity in the absence of treatment. 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, It can be an amount that means a 95, 96, 97, 98, 99, or 100% reduction. The amount and frequency may be adjusted to maintain terminal complement activity at a desired level. The level may be, for example, 10% or less, for example, 9, 8, or less, compared to terminal complement activity in the absence of treatment. , 7, 6, 5, 4, 3, 2, 1% or less.

[0172] When a dose is given, this refers to the dose of an agent that is a protein or its functional equivalent. These levels are achieved using appropriate doses of the nucleic acid molecule drug. obtain.

[0173] Terminal complement activity can be measured by standard assays known in the art, for example, Qu idel CH 50Measured using hemolysis assay and sheep red blood cell lysis CH50 assay It can be determined.

[0174] The frequency with which doses need to be administered depends on the half-life of the drug involved. The protein or functional equivalent may be administered, for example, on a twice-daily basis, on a daily basis, or on a few days. May be administered every 4, 5, 6, 7, 10, 15 or 20 days or more. .

[0175] Single or multiple doses can be administered. For example, at least 2, 3, 4, 5, 6, 7, or or 8 doses may be administered. A single dose is one embodiment. The exact dosage and The frequency may also depend on the condition of the patient at the time of administration. Factors that influence the outcome include the need for treatment or prevention, the severity of the patient's condition, and the patient's overall health. The following factors must be considered: morphology, age, weight, sex, diet, time and frequency of administration, drug combinations, sensitivity and The exact amount can be determined by routine experimentation and the patient's resistance or response to treatment. The decision may be made, but ultimately it is up to the physician to decide.

[0176] The dosing regimen also includes an initial "loading dose" followed by one or more subsequent "maintenance doses." Generally, the loading dose is greater than the maintenance dose. The loading dose may be administered as a single dose or in a dose that is 2, 5, 10 or more times the amount of the drug. The loading dose may be administered as one or more doses within a fixed time frame. Typically, the loading dose is administered as a single Maintenance doses are 1, 2, 3, 4 or 5 doses administered during a single 24-hour period. Maintenance doses may be at intervals of 3, 4, 6, 8, 12, 2 The exact regimen may be determined by routine experimentation. The maintenance dose can be determined by the physician's discretion. At least 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the amount or a maximum of 20, 30, 40, 50, 60, 70, 80, 90 or 10 loading doses It could be 0%.

[0177] In further embodiments, the same dose is used over the course of treatment (e.g., daily or once a day). twice a day).

[0178] The loading dose ranges from 0.0001 mg / kg (mass of drug compared to the patient's mass) to 20 mg / kg and the maintenance dose may be between 0.0001 mg / kg and 20 mg / kg Alternatively, the loading dose is 0.001 mg / kg to 10 mg / kg and the maintenance dose is 0.001 mg / kg to 10 mg / kg, or the loading dose is 0.01 mg / The administration dose is 0.01 mg / kg to 2 mg / kg, and the maintenance dose is 0.01 mg / kg to 2 mg / kg; Alternatively, the loading dose is 0.1 mg / kg to 1 mg / kg, and the maintenance dose is 0.1 mg / kg. / kg to 1 mg / kg; or the loading dose is 0.1 mg / kg to 1 mg / kg and the maintenance dose is 0.05 mg / kg to 0.5 mg / kg; or The dose is 0.2mg / kg to 0.8mg / kg, and the maintenance dose is 0.1mg / kg to 0 or the loading dose is 0.3mg / kg to 0.7mg / kg. and the maintenance dose is 0.1 mg / kg to 0.3 mg / kg; or the loading dose is The initial dose is 0.4mg / kg to 0.6mg / kg, and the maintenance dose is 0.1mg / kg to 0.2 For example, if the loading dose is 0.57 mg / kg, the maintenance dose is 0. For example, for a loading dose of 0.6mg / kg to 1.8mg / kg, A maintenance dose of 0.2 mg / kg to 0.6 mg / kg (e.g., 0.3 mg / kg) follows.

[0179] The loading dose ranges from 0.0001 mg / kg (mass of drug compared to the patient's mass) to 20 mg / kg and the maintenance dose may be between 0.0001 mg / kg and 20 mg / kg Alternatively, the maintenance dose may be from 0.001 mg / kg to 10 mg / kg, or The maintenance dose may be 0.01 mg / kg to 2 mg / kg; alternatively, the maintenance dose may be 0. 1mg / kg to 1mg / kg; alternatively the maintenance dose may be 0.1mg / kg to 0. or the maintenance dose may be 0.1 mg / kg to 0.6 mg / kg. Alternatively, the maintenance dose may be 0.1 mg / kg to 0.4 mg / kg; or The maintenance dose may be 0.1 mg / kg to 0.2 mg / kg.

[0180] The loading dose ranges from 0.0001 mg / kg (mass of drug compared to the patient's mass) to 20 mg / kg and the maintenance dose may be between 0.0001 mg / kg and 20 mg / kg Alternatively, the loading dose may be from 0.001 mg / kg to 10 mg / kg, or The loading dose can be 0.01 mg / kg to 2 mg / kg; alternatively, the loading dose can be 0. Alternatively, the loading dose may be from 0.1 mg / kg to 1 mg / kg. g / kg; alternatively, the loading dose may be 0.2 mg / kg to 0.8 mg / kg. Alternatively, the loading dose may be 0.3 mg / kg to 0.6 mg / kg; or The loading dose is 0.4mg / kg to 0.6mg / kg or 0.6mg / kg to 1.8mg / kg. It may be g / kg.

[0181] Agents are generally administered with a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any carrier that does not itself induce toxic effects and The gene, polypeptide, or other component of the pharmaceutical composition may be used without causing the production of antibodies harmful to the individual receiving the pharmaceutical composition. peptides, antibodies, liposomes, polysaccharides, polylactic acid, polyglycolic acid and inactive virus particles Pharmaceutically acceptable carriers may additionally include water, Liquids such as saline, glycerol, ethanol, or auxiliary substances such as wetting agents or emulsifying agents The pharmaceutical carrier employed may contain various additives, such as terpenes, pH buffering substances, and the like. The route will vary. To aid in ingestion by the patient, the carrier may be used to administer the pharmaceutical composition in the form of a tablet, It can be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries and suspensions. A thorough discussion of pharma- ceutically acceptable carriers is available in

[42] . In a preferred embodiment, the agent is administered in water or PBS.

[0182] The agent may be delivered by any known route of administration. The agent may be administered locally or systemically. The agent may be delivered by a parenteral route (e.g., subcutaneous, intraperitoneal, intravenous). The composition is delivered either intramuscularly or intramuscularly, or into the interstitial space of a tissue. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transdermal applications, needles, and hyposprays. , local administration, e.g., application to the affected area, e.g., to the skin. This is particularly true for mild AIB D, for example of particular use in mild BP.

[0183] Preferably, the agent is delivered via subcutaneous injection. In some embodiments, this is done via, for example, For example, from 0.0001 mg / kg (the mass of the drug compared to the mass of the patient) to 20 mg / kg followed by a loading dose of between 0.0001 mg / kg and 20 mg / kg once daily once or twice daily with a maintenance dose of 0.1 mg / kg or other doses disclosed elsewhere herein. Alternatively, the drug can be delivered by subcutaneous injection every other day.

[0184] In a preferred embodiment, the drug is administered at a dose of 0.6 mg / kg to 1.8 mg / kg (e.g., 0.5 7mg / kg) loading dose followed by 0.2mg / kg to 0.6mg / kg (e.g. 0 0.3mg / kg) once daily by subcutaneous injection or at a maintenance dose of 0.6mg / kg once daily An initial loading dose of 0.2 mg / kg to 3.6 mg / kg (e.g., 1.0 mg / kg) followed by 1 mg / kg to 1.2 mg / kg (e.g., 0.6 mg / kg) once daily at a maintenance dose It is delivered by subcutaneous injection once daily.

[0185] Preferably, the course of treatment lasts for at least 1, 2, 3, 4, 5 or 6 weeks.

[0186] The course of treatment is preferably continued until the subject's symptoms are alleviated. The period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 , 35, 40 weeks of administration (eg, daily) of the drug.

[0187] The maintenance dose (e.g., a single daily maintenance dose) should remain constant throughout the course of treatment. ) or maintenance dose (e.g., daily maintenance dose) may be altered (e.g., increased or decreased) during the course of treatment. A maintenance dose can be used to restore terminal complement activity to a desired level, e.g., in the absence of treatment. maintained at 10% or less compared to sera from the above patients or compared to normal control sera The or each maintenance dose may be modified to provide at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9 , may be continued daily for 10 weeks. The maintenance dose may be decreased as the subject's symptoms improve. The amount of the drug or the frequency with which the drug is administered may be decreased as the subject's condition improves. .

[0188] Thus, an initial loading dose is followed by an initial maintenance dose ( e.g., a daily initial maintenance dose) and one or more further maintenance doses (e.g., a further daily maintenance dose), for example at least 2, 3, 4, 5 further maintenance doses.

[0189] Thus, the present invention further relates to a method of treating or preventing AIBD in a subject. administering to the subject an initial loading dose of an agent as defined above, and then administering to the subject an initial loading dose of an agent as defined above. and administering a maintenance dose (e.g., a daily maintenance dose) of a drug to There is one maintenance dose and one or more further maintenance doses.

[0190] Thus, the present invention further provides a method for treating a subject comprising administering to the subject an initial loading dose of the agent defined above. and then administering a maintenance dose (e.g., a daily maintenance dose) of the agent defined above. as defined above for use in a method for treating or preventing AIBD in a subject, and wherein there is an initial maintenance dose and one or more further maintenance doses. do.

[0191] One or more further maintenance doses may be administered to the subject (e.g., to a biological sample from the subject). and determining whether or not to further maintain the sample based on the level of terminal complement activity. determining a sustained dose and / or examining the subject for symptoms and determining based on the symptoms; The method can be optionally carried out by determining a further maintenance dose. The further dose may further comprise administering a maintenance dose comprising: It may be calculated that this is the level that will maintain the desired level.

[0192] If a biological sample is taken, this may be a blood, e.g., a whole blood or serum sample. The method optionally further comprises the step of taking a sample, and optionally Further comprising the step of determining the TCA.

[0193] One or more further maintenance doses may be administered to a subject (e.g., in a biological sample) to determine terminal end-point concentration. Testing complement activity and determining further maintenance doses based on the level of terminal complement activity and / or examining the subject for symptoms and further maintaining the subject based on the symptoms. The method may be further modified by titrating the additional maintenance dose, if necessary. The additional dose may further comprise administering to the patient a dose of 100 mg / kg of terminal complement activity to the patient. It can be calculated that the level to be maintained.

[0194] In certain aspects, the desired complement activity level is determined by measuring serum complement activity from said subject in the absence of treatment. or less than 10% compared to normal control serum.

[0195] In certain embodiments, if the TCA is at a higher than desired level, the maintenance dose is increased and, if necessary, If necessary, maintain or reduce dose if TCA is less than 5, 4, 3, 2, or 1%. Can.

[0196] In certain embodiments, if symptoms worsen, the maintenance dose is increased, and as needed, If improvement occurs, maintain or reduce the dose.

[0197] In some embodiments, the subject is diagnosed with idiopathic pulmonary disease within one month of initiating treatment, within two weeks of initiating treatment, In another embodiment, the subject is examined once a day or once a week after the start of treatment. at least once a day, once a week, or at least once a week, once every two weeks The tests are conducted at least once every two weeks or once a month or once every two months.

[0198] Preferably, the loading dose is at or about 1.2 mg / kg protein or is functionally equivalent, and the maintenance dose is at least 0.6 mg / kg (e.g., at least 0 0.7mg / kg, 0.8-1.5, 0.9-1.2 or 1.0-1.25mg / kg ) or up to 0.3 mg / kg (e.g. up to 0.2 mg / kg, 0.15 ~0.4, 0.2-0.3 mg / kg), and if necessary, (i) the maintenance dose is a small and / or (ii) treatment continues for at least 6 and / or (iii) treatment is continued daily for at least 3, 4, 5, or 6 weeks. To be continued.

[0199] Preferably, the loading dose is 0.6 to 1.8 mg / kg protein or a functional equivalent. and the maintenance dose is 0.2 to 0.6 mg / kg, for example about 0.3 mg / kg, (i (ii) the maintenance dose is continued for at least 2, 3, 4, 5, or 6 weeks; and / or and / or (iii) treatment is continued for at least 3 consecutive days daily. , which will last for 4, 5 or 6 weeks.

[0200] The dosing regimen may also take the form of a fixed dose that is independent of the body weight of the subject being treated. Fixed doses are administered as a single dose or as one or more doses within a specific time frame. A fixed dose may be administered to a typical human patient (e.g., between 50 kg and 100 kg in weight). The dose may be 1 mg to 100 mg of Covasin (SEQ ID NO: 4) for each patient. The molecular weights of the covercin-type protein and the modified covercin polypeptide were used to identify functionally equivalent The equivalent fixed dose of the drug can be calculated. In some embodiments, the fixed dose is 1 ml g~80mg, 1mg~50mg, 5mg~80mg, 5mg~50mg, 10mg~6 0mg, 10mg-50mg, 20mg-50mg, 20mg-40mg or 25mg ~35mg. Preferably, the fixed dose is 30mg of sCoveSyn. rsin) (SEQ ID NO: 4) or a molar equivalent of a covercin-type protein or a modified covercin Typically, a fixed dose is one or more doses administered during a single 24-hour period. 2, 3, 4 or 5 doses. Fixed doses are 3, 4, 6, 8, 12, 24 or 48 The exact regimen can be determined by routine experimentation. However, the final decision is left to the doctor. [Brief description of the drawings]

[0201] [Figure 1] Schematic diagram of the classical and alternative pathways of complement activation. Enzymatic components are in dark grey. Anaphylatoxins are surrounded by a star. [Figure 2A] Figure 1 shows the primary sequence of covercin. The signal sequence is underlined. Cysteine ​​residues are in bold. Nucleotide and amino acid numbering is shown to the right. [Figure 2B] FIG. 1 shows examples of coversen variants. [Diagram 3] FIG. 1 shows Experiment 1: Clinical scores of experimental EBA in mice treated with prophylactic Covarcin compared to vehicle, demonstrating reversal of disease in mice treated with all doses of Covarcin compared to vehicle control and methylprednisolone groups. [Figure 4] Figure 2 shows Experiment 2, in which the clinical scores of experimental EBA in mice treated with prophylactic Covarcin compared to vehicle demonstrates disease reversal in mice treated with all doses of Covarcin compared to the vehicle control and methylprednisolone groups. Compared to Experiment 1, only the 2.5 mg / kg dose of Covarcin had a significant effect compared to the vehicle control group. Notably, in this experiment, some mice in the control group did not develop significant levels of disease, which may be the reason for this difference compared to Experiment 1. [Diagram 5] Figure 1 shows the combined results of experiments 1 and 2, where clinical scores of experimental EBA in mice treated with prophylactic Covarcin compared to vehicle demonstrate reversal of disease in mice treated with all doses of Covarcin compared to the vehicle control and methylprednisolone groups. Two-way ANOVA analysis reveals statistically significant differences between vehicle control and Covarcin at doses of 250 and 2500 μg / ml. [Figure 6A]FIG. 1 shows experimental EBA clinical scores compared to vehicle in mice treated with prophylactic Covarcin and prophylactic PAS-L-Covarcin, demonstrating disease reversal in mice treated with 2.5 mg / kg Covarcin (Example 4, Experiment 1). [Figure 6B] FIG. 1 shows experimental EBA clinical scores in mice treated with prophylactic Covarcin and prophylactic PAS-L-Covarcin compared to vehicle, demonstrating disease reversal in mice treated with 2.5 mg / kg Covarcin (Example 4, Experiment 2), where both the 10 mg / kg dose of PAS-L-Covarcin and 2.5 mg / kg Covarcin show statistically significant effects on ABSA. [Figure 6C] 1 shows experimental EBA clinical scores in mice treated with prophylactic Covarcin and prophylactic PAS-L-Covarcin compared to vehicle, showing the combined results of Experiments 1 and 2 of Example 4. [Figure 7] Figure 2 shows the effect of therapeutically administered Covarcin on the course of pemphigoid disease-like dermatitis in a passive EBA mouse model (Example 2, Experiment 1). Covarcin dose-dependently reversed the dermatitis. Data are presented as mean ± SEM; N = 5 mice / group; Two-way ANOVA test for statistical significance confirms significant differences between vehicle control and all doses of Covarcin used. Experiments were performed on females only. [Figure 8] Figure 2 shows the effect of therapeutically administered Covarcin on the course of pemphigoid disease-like dermatitis in a passive EBA mouse model (Example 2, Experiment 2). Covarcin dose-dependently reversed the dermatitis. Data are presented as mean ± SEM; N = 5 mice / group; Two-way ANOVA test for statistical significance confirms significant differences between vehicle control and all doses of Covarcin used. Experiments were performed on males only. [Figure 9A] FIG. 1 shows C5a levels in blister fluid of patients with bullous pemphigoid. [Figure 9B] FIG. 1 shows LTB4 levels in blister fluid of patients with bullous pemphigoid. [Figure 10] FIG. 1 shows the sequences of certain modified coversin polypeptides that have reduced or no C5 binding activity but retain LTB-4 binding ability. Example 1

[0202] Efficacy of Covarcin in an EBA induction model using prophylactically administered Covarcin A modified version of the protocol described by Sitaru et al., 2005

[43] was used. Antibody-transduced EBA was induced by injecting the mice with 5 mice per treatment group. Mice were injected subcutaneously with 50 μg of affinity purified anti-Col7 IgG on days 0, 2, and 4. Starting 4 days before the first injection of anti-Col7 IgG (day -2), three Mice were subcutaneously injected with different doses of Covaxin twice a day. In the control group, mice received either vehicle alone or 20 μg of g / kg methylprednisolone was administered once daily.

[0203] To generate antibodies against mouse type VII collagen ("anti-COL7"), Zeeland White rabbits were cultured using a 5'-dimer of the non-collagen 1 (NC1) domain of collagen VII. Proteins containing three different recombinant proteins ("Col7A, B, and C") from the past The mice were immunized with 200 μg of the protein mixture together with Freund's incomplete adjuvant. From the serum of the rabbits, IgG was isolated by using protein G, and then his- Col7 was used to affinity purify IgG, and rabbit anti-Col7 IgG was specifically The quality of the antibodies was checked by determining the titer and potency in a frozen section assay. I did.

[0204] Beginning on day 4, the severity of skin lesions in each individual mouse was scored every other day. Skin areas showing erythema, blisters, erosions, crusts, or alopecia were scored as "affected" by a trained observer. The total body surface area affected by skin lesions (ABSA) was classified as “affected” or “unaffected”

[43] . ) was calculated as a percentage.

[0205] Two independent experiments were performed.

[0206] The results of the first experiment are shown in Figure 4. In mice treated with Covaxin 25 μg / kg The percentage of ABSA was reduced at 2500 μg / kg and Treatment with 250 μg / kg of Covarcin further reduced the There was a 63% difference between the 0.25 mg / kg and 0.5 mg / kg doses. A two-tailed t-test confirmed this difference. The p-value for all was 0.0023. The mean ABSA scores for the two groups were 6.23+1.1 and 2.65+0.4. Values ​​after the mean are SD.

[0207] Covarcin reduces the percentage ABSA to a greater extent than previously tested molecules. For example, previous similar experiments showed that 5-lipoxygenase (a cascade enzyme that converts arachidonic acid) It is an important enzyme in the glycation process and is involved in the formation of bioactive leukotrienes (LTs) 5-LO) Oral Inhibitor N-[1-(1-benzothien-2-yl)ethyl]-N- Hydroxyurea (zileuton) was administered prophylactically to 8-week-old female C57Bl / 6J mice. In this initial study, mice were divided into two groups and administered 0.6 mg of riboflavin supplemented in their drinking water. Zileuton or vehicle control was administered at ~0.8 mg / mouse / day. Experimental EBA The %ABSA decreased from day 7 to the end of the study. Mice treated with zileuton in 2016 showed significantly reduced disease severity compared to the control group. (Clinical score on the 7th day; control group: X - = 9.2 ± 0.7; zileuton treatment group: X - =5 The study concluded that "the proportion of affected body surface area was 1.9 ± 0.5% (median 1.9 ± 0.5% of total body surface area)."

[0208] In the antibody induction model of EBA, 20 mg / kg / day of methylprednisolone was administered. In a similar previous study, control mice had a 7.5±0.0% increase in body surface area at the end of the experiment. 1% suffered from skin lesions, but the severity of the disease was significantly greater in methylprednisolone-treated mice. in the control group, the rate was significantly reduced to 4.7 ± 0.4%

[45] . Example 2

[0209] Efficacy of Covaxin in an EBA-introduced model The study included the following experimental groups with 5 mice per experimental group: 1. Vehicle (PBS) control group 2. Covaxin 125 μg / kg sc twice daily 3. 2500 μg / kg Covarcin, sc, twice daily

[0210] Antibody-induced EBA-like dermatitis was induced as described by Sezin et al.

[46] . C57Bl / 6J WT mice were inoculated with affinity-purified anti-CoA on days 0, 2, and 4. 50 μg of l7 IgG was injected subcutaneously (sc).

[0211] Treatment of mice with Covarcin began on day 5 of the experiment (day 0 = anti-COL7 antibody treatment). After that, mice were injected with either Covaxin or vehicle control in the shoulder region until the end of the experiment. Injections were given sc every 12 hours.

[0212] The severity of the disease was scored. To score the severity of the disease, erythema, blisters, Skin areas showing erosion, crusting, or alopecia were examined by a single subject blinded to treatment. A trained examiner classified them as "affected". Absolute body surface area affected by skin lesions (ABSA) To score disease, the percentage of mice with pulmonary edema was calculated. Mice were anesthetized with a mixture of ketamine / xylazine administered ip every other day.

[0213] This protocol was followed in two independent experiments.

[0214] Preparation of test items Convolvlin 18 mg was reconstituted in 0.6 ml of sterile water to achieve a concentration of 30 mg / ml. The solution was aliquoted and stored at -20°C until final dilution.

[0215] Before the start of the experiment, the average body weight of the mice was calculated and the doses were adjusted to 2.5 mg / kg and 0.125 mg / kg. 1 kg of drug was freshly prepared in PBS, pH 7.2 in a final volume of 100 μl. The solution was freshly prepared twice, 30 min before the final injection into the mice. Based on the above, the appropriate concentration was adjusted to achieve the required mg / kg dose in a final volume of 100 μl. Drugs were adjusted in PBS, pH 7.2. Drugs were administered twice daily, 30 minutes before the final application to the animals. As a vehicle control, 100 μl of PBS, pH 7.2 was administered to mice. .c was injected.

[0216] statistical analysis Treatments were assessed by two-way ANOVA performed using GraphPad Prism 7. ABSA between groups was evaluated.

[0217] result Clinical findings and treatment effectiveness In Study 1, Covarcin reduced dermatitis as measured by absolute affected body surface area (ABSA). Covaxin 2.5mg / kg reduced ABSA in a dose-dependent manner. The ABSA in the negative control (vehicle) group was approximately 7% in experiment 1. Although there were some differences, this is typical of what has been achieved in this model. showed a similar inflammatory response.

[0218] In experiment 2, 2.5 mg / kg Covaxin reduced dermatitis as measured by (ABSA). The negative control (vehicle) group was significantly (P<0.01) restored (Figure 7). The ABSA in the 100-mL 100-mL 15 ...50-mL 150-mL 150-mL slightly lower than the achieved value.

[0219] No mice died during treatment in these two experiments. Mice were sacrificed 12 days after initiation. Example 3

[0220] C5 and LTB4 in blister fluid from patients We examined levels of C5a and LTB4 in blister fluid from four patients with bullous pemphigoid. The results for each of the four patients are shown in Figures 9A (C5a) and 9B (LTB4). We aspirated blister fluid with a syringe from the blisters of four patients who were admitted to the inpatient clinic for BP. The samples were immediately frozen in liquid nitrogen. was used to measure LTB4 and C5a levels in the fluid. Example 4

[0221] Comparison of the efficacy of dual-acting Conversin and single-acting Conversin Further experiments evaluated the effect of PAS-L-Covarcin at 0.1 mg / kg, 1 0 mg / kg, 1 mg / kg PAS-L-Covarcin, or 2.5 mg / kg Covarcin Mice were treated with covacin and the experiments were performed as described above. The covercin sequence has a PAS sequence fused to the N-terminus of the covercin sequence, which is identical to LTB4. It was mutated to bind to C5 but not to C5 (called "L-covasin"). The sequence of the covercin sequence is the mature covercin sequence (SEQ ID NO: 4) variant: Ala44 to Asn, Met116 to Gln, Leu11 7 to Ser, Gly121 to Ala, Leu122 to Asp, Glu123 to Al a, and Asp149 to Gly (referred to as variant 2, the sequence is dsesdc tgse pvdafqafse gkeayvlvrs tdpkardclk ge pNgekqdn tlpvmmtfkn gtdwastdwt ftldgakvt a tlgnltqnre vvydsqshhc hvdkvekevp dyemw QSdag ADAveveccr qkleelasgr nqmyphlkGc(distribution In column number 23), changes in the sequence compared to the native covercin sequence of SEQ ID NO: 4 are indicated in capital letters. Since the molecular weight of PAS-L-Cov is higher, 10 mg / kg PAS-L- Cov corresponds to 2.5 mg / kg Covarcin. In the first experiment (Figure 6A), PAS -L-Covarcin at a dose of 1 mg / kg or 0.1 mg / kg, but not at a dose of 10 mg / kg In the second experiment (Fig. 6B), the PAS- L-Covarcin was administered at a dose of 1 mg / kg or 0.1 mg / kg, but not at a dose of 10 mg / kg. The 10 mg / kg dose of PAS-L-cova reduced ABSA compared to the control. Covaxin was not as effective as the molar equivalent 2.5 mg / kg dose of Covaxin (although However, the 10 mg / kg dose of PAS-L-Covarcin did not show any statistically significant effect in the second experiment. This indicates that the dual inhibitory activity of covercin (inhibition of C5 and LTB4) is essential in this model. These results suggest that improved therapeutic benefit may be achieved in patients with

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[46] Sezin T, Krajewski M, Wutkowski A, Mousavi S, Chakievska L, Bieber K, et al . The Leukotriene B4 and Its Receptor BLT1 Act as Critical Drivers of Neutrophil Recruitment in Murine Bullous Pemphigoid-Like Epidermolysis Bullosa Acquisita. J Invest Dermatol. 2017, 137(5):1104-13. [Sequence Listing] JPEG0007600278000002.jpg221164JPEG0007600278000003.jpg221165JPEG0007600278000004.jpg221164JPEG0007600278000005.jpg213164JPEG0007600278000006.jpg212162JPEG0007600278000007.jpg214164JPEG0007600278000008.jpg190164

Claims

1. 1. A medicament for treating or preventing bullous pemphigoid or epidermolysis bullosa acquisita in a subject, comprising: The agent comprises a modified coversin polypeptide that exhibits leukotriene or hydroxyeicosanoid binding activity and exhibits reduced or no C5 binding, the modified coversin polypeptide comprising a sequence having 1-30 amino acid substitutions in SEQ ID NO:4, with the proviso that a) the following substitutions at positions 114-124 of SEQ ID NO:4: Met116 is substituted with Gln, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; b. Leu117 is substituted with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro; c. Gly121 is substituted with Ala, Asp, Asn, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; d. Leu122 is substituted with Asp, Glu, Asn, Ala, Gln, Arg, Lys, Pro, or His; e. Glu123 is replaced with Asp, Ala, Gln, Asn, Arg, Lys, Gly, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr; or a fragment thereof in which up to 5 amino acids have been deleted from the N-terminus of said modified coversin polypeptide.

2. (i) Ala44 in SEQ ID NO:4 is substituted with Asn, Asp, Gln, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; and (ii) Asp149 in SEQ ID NO: 4 is substituted with Gly, Gln, Asn, Ala, Met, Arg, Lys, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr; The drug according to claim 1.

3. At positions 114 to 124 of SEQ ID NO:4: (i) Met114 is substituted with Gln, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; (ii) Asp118 is replaced with Asn, Gln, Arg, Lys, Gly, Ala, Leu, Ser, Ile, Phe, Tyr, Met, Pro, His, or Thr; (iii) Ala119 is substituted with Gly, Asp, Asn, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; (iv) Gly120 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; and (v) Val124 is substituted with Lys, Gln, Asn, Arg, Lys, Gly, Ala, Pro, His, or Thr; The drug according to claim 1.

4. At positions 114 to 124 of SEQ ID NO:4: a. Met116 is replaced with Gln; b. Leu117 is replaced with Ser; c. Gly121 is replaced with Ala; d. Leu122 is replaced with Asp; and e. Glu123 is replaced with Ala; The drug according to claim 1 or 2.

5. The method of claim 4, wherein Ala44 of SEQ ID NO:4 is replaced with Asn and Asp149 of SEQ ID NO:4 is replaced with Gly149.

6. 6. The method of claim 1, wherein Trp115 of SEQ ID NO:4 is not substituted.

7. The agent according to any one of claims 1, 2, or 4 to 6, wherein Met114, Trp115, Asp118, Ala119, Gly120 and Val124 of SEQ ID NO:4 are not substituted.

8. having a loop sequence as set forth in SEQ ID NO:28 between amino acid positions 114-124 of SEQ ID NO:4, and having from 1 to 20 additional substitutions compared to SEQ ID NO:4 other than the substitutions as set forth in SEQ ID NO:23; and optionally (i) having from 2 to 15 additional substitutions compared to SEQ ID NO:4 other than the substitutions set forth in SEQ ID NO:23; or (ii) other than the substitutions shown in SEQ ID NO:23, has 3 to 10 additional substitutions compared to SEQ ID NO:4; The drug according to any one of claims 1, 2, or 4 to 7.

9. (a) the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 are retained in their unmodified form; (b) Asn60 and Asn84 of SEQ ID NO:4 are each replaced with Gln; (c) one or more, or all of the following amino acids in SEQ ID NO: 4: Phe18, Tyr25, Arg36, Leu39, Gly41, Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71, Gln87, Arg89, His99, His101, Asp103, and Trp115 are not substituted; (d) none of the amino acids at positions 5, 6, 11, 13-15, 20-21, 24-27, 29-32, 35-41, 45, 47-48, 50, 52-60, 64, 66, 69-81, 83, 84, 86, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150 in SEQ ID NO:4 are substituted; (e) none of the amino acids at positions 5, 6, 11, 13-15, 18, 20-21, 24-27, 29-32, 35-41, 43, 45, 47-48, 50, 52-60, 64, 66, 67, 69-81, 83, 84, 86, 87, 89, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150 in SEQ ID NO:4 are substituted; and / or (f) none of the amino acids at positions 5, 6, 11, 13-15, 18, 20-21, 24-25, 27, 30-32, 35-41, 43, 47-48, 50, 52-60, 64, 66, 67, 69-81, 83, 84, 86, 87, 89, 90-94, 98, 100, 102-104, 112-113, 115, 126, 128-129, 132-139, 145, 148, and 150 in SEQ ID NO:4 are substituted; The drug according to any one of claims 1, 2, or 4 to 8.

10. The agent according to any one of claims 1, 2, or 4 to 8, comprising or consisting of the base sequence of SEQ ID NO:

23.

11. An agent according to any one of claims 1 to 10 which binds to LTB4.

12. (a) the medicament is one that is administered subcutaneously; and / or (b) the subject is a human The drug according to any one of claims 1 to 11.

13. (a) the agent is administered at a dose sufficient to bind as much available LTB4 as possible in the subject; and / or (b) The agent of any one of claims 1 to 12, wherein the agent is administered at a dose that is 1.5 times the molar dose required to bind all available LTB4 in the subject.

14. The method of any one of claims 1 to 13, wherein the medicament is administered to the subject with an initial loading dose of the medicament followed by a maintenance dose thereof.

15. 15. The method of claim 14, wherein there is an initial maintenance dose and one or more further maintenance doses.

16. The method of any one of claims 1 to 15, wherein the medicament is administered in conjunction with a second AIBD treatment.

17. 17. The method of claim 16, wherein the second AIBD treatment is selected from systemic corticosteroid therapy, topical corticosteroid therapy, immunosuppressive therapy, and immunosuppressive biologic therapy.

18. 18. The method of claim 17, wherein the corticosteroid is selected from prednisone and prednisolone, the immunosuppressive therapy is selected from methylprednisolone, mycophenolate, azathioprine, dapsone and cyclophosphamide, and the immunosuppressive biological therapy is selected from rituximab and intravenous immunoglobulin G (IVIG).

19. The medicament according to any one of claims 1 to 18, which is a medicament for treating bullous pemphigoid.

20. 1. A medicament for treating or preventing bullous pemphigoid or epidermolysis bullosa acquisita in a subject, comprising: The agent is a fusion protein comprising: (a) the modified coversin polypeptide of any one of claims 1 to 19; and (b) a second sequence.

21. The second sequence is (i) contains a PAS sequence; or (ii) a PAS sequence consisting of 30 copies of SEQ ID NO: 15, fused to the N-terminus of the modified covasin polypeptide; The drug according to claim 20.

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