Covarcin for use in the treatment of rheumatic diseases

Abstract

The present invention relates to a method for treating or preventing rheumatic diseases.

Description

[Technical field]

[0001] The present invention relates to methods for treating and preventing rheumatic diseases, particularly rheumatoid arthritis.

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

[0003] complement The complement system is an essential part of the body's natural defense mechanism against foreign invaders and is also involved in inflammatory processes. More than 30 proteins in serum and on the cell surface are involved in the function and regulation of the complement system. Recently, it has become clear that the complement system itself interacts with at least 85 biological pathways with functions as diverse as angiogenesis, platelet activation and hemostasis, glucose metabolism, and spermatogenesis, as well as about 35 known components of the complement system that may be involved in both beneficial and pathological processes.

[0004] The complement system is activated by the presence of substances recognized by the immune system as non-self. There are three activation pathways: (1) the classical pathway, activated by IgM and IgG complexes or by recognition of carbohydrates; (2) the alternative pathway, activated by non-self surfaces (lacking specific regulatory molecules) and bacterial endotoxins; and (3) the lectin pathway, activated by binding of mannan-binding lectin (MBL) to mannose residues on the surface of pathogens. These three pathways bind to similar C3 1 The classical pathway involves a parallel cascade of events leading to the production of complement activation through the formation of C1q convertase and C5 convertase, resulting in the release of acute phase inflammatory mediators (C3a and C5a) and the formation of the membrane attack complex (MAC). The parallel cascades involved in the classical pathway (here defined as classical via C1q and lectin via MBL) and the alternative pathway are shown in Figure 1. 1It is conventional to designate the components of the complement pathway with the letter "C" followed by a number, such as "3" so that "C3" designates complement protein C3. Some of these components are cleaved upon activation of the complement system, and the cleavage products are designated by a lower case letter after the number. Thus, C5 is cleaved into fragments conventionally called C5a and C5b. Because complement proteins do not necessarily act in numerical order, the numbers do not necessarily indicate an order of action. This designation convention is used in this application.)

[0005] Classical, alternative and lectin complement pathways are collectively referred to herein as complement pathways. C5b initiates the "late" or "final" events of complement activation. These include a series of polymerization reactions in which the final complement components interact to form MAC, which opens holes in the cell membranes of some pathogens, which can lead to pathogen death or activate body cells without causing lysis. The final complement components include C5b (which initiates the assembly of the membrane attack system), C6, C7, C8 and C9.

[0006] LTB4 Leukotriene B4 (LTB4) is the most potent chemotactic and chemokinetic eicosanoid described, promoting neutrophil adhesion to vascular endothelium through upregulation of integrins [1]. It is also a complete secretagogue for neutrophils, inducing their aggregation and increasing microvascular permeability. LTB4 recruits and activates natural killer cells, monocytes and eosinophils. It increases the formation of superoxide radicals [2] and modulates gene expression, including the production of several proinflammatory cytokines and mediators that may enhance and prolong tissue inflammation [3,4]. LTB4 also plays a role in the induction and management of adaptive immune responses, such as regulating dendritic cell trafficking to draining lymph nodes [5,6], production of the Th2 cytokine IL-13 from pulmonary T cells [7], recruitment of antigen-specific effector CD8+ T cells [8] and activation and proliferation of human B lymphocytes [9].

[0007] LTB4 and hydroxyeicosanoids mediate their effects through the BLT1 and BLT2 G protein-coupled receptors [10,11]. Human BLT1 is a high-affinity receptor specific for LTB4 (Kd 0.39-1.5 nM;

[12] ), and only 20-hydroxyLTB4 and 12-epiLTB4 can displace LTB4 in competitive binding studies

[13] . Human BLT2 has 20-fold lower affinity for LTB4 (Kd 23 nM) than BLT1, and is activated by binding a broader range of eicosanoids, including 12-epiLTB4, 20-hydroxyLTB4, 12(S)-HETE, 15(S)-HETE, 12(S)-HPETE, and 15(S)-HPETE

[13] . Human BLT2 shares 45.2 and 44.6% amino acid identity with human and mouse BLT1, while human and mouse BLT2 share 92.7% identity

[11] .

[0008] Human BLT1 is expressed primarily on the surface of leukocytes, although it has recently been described to be expressed on endothelial cells and vascular smooth muscle cells. Human BLT2 is expressed in a wider range of tissues and cell types. Several specific antagonists of BLT1 and BLT2 have been described that inhibit human neutrophil activation, extravasation and apoptosis

[14] and attenuate symptoms caused by neutrophil infiltration in mouse models of inflammatory arthritis

[15] and renal ischemia-reperfusion

[16] . Although both BLT1 and BLT2 can mediate pathological effects via LTB4 and hydroxyeicosanoids

[17] , an increasing number of studies have shown that BLT1 undoubtedly plays a dominant role in some pathologies, such as collagen-induced arthritis in mice

[18] . BLT1- / - deficient mice also highlight the importance of BLT1 in directing neutrophil migration in inflammatory responses. Notably, using a 5LO-deficient mouse strain, it was shown that autocrine activation of BLT1 on neutrophils is required for neutrophil recruitment to arthritic joints

[19] .

[0009] Several marketed drugs target eicosanoids. These include glucocorticoids

[20] , which modulate phospholipase A2 (PLA2) and thereby inhibit the release of arachidonic acid (AA), an eicosanoid precursor. Nonsteroidal anti-inflammatory drugs (NSAIDs) and other COX2 inhibitors prevent the synthesis of prostaglandins and thromboxanes

[21] . There are also several leukotriene (LK) modulators that either inhibit the 5-LOX enzyme required for LTB4 synthesis and other leukotrienes (zileuton;

[22] ) or antagonize the CysLT1 receptor, which mediates the effects of cysteinyl leukotrienes (zafirlukast and montelukast)

[23] . LK modulators are orally available and have been approved by the FDA for use in the treatment of asthma, for example. No drugs are yet on the market that act specifically on LTB4 or its receptor.

[0010] Rheumatic diseases background The term rheumatic disease includes chronic, often intermittent painful conditions that affect the joints and / or connective tissue. Of the at least 200 different conditions that can be described as rheumatic disease, rheumatoid arthritis (RA) is a particularly well-known example.

[0011] Certain rheumatic diseases are caused by injury or infection, but most are autoimmune-driven conditions, including ankylosing spondylitis, relapsing polychondritis, systemic lupus erythematosus, rheumatoid arthritis, gout, inflammatory arthritis, pseudogout, juvenile arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Behcet's disease, and psoriatic arthritis.

[0012] severity Symptoms of an established arthritic condition include pain and limited joint function, such as joint stiffness, swelling, redness, and warmth. Tenderness of the inflamed joints may be present, in addition to symptoms from fever, malaise, and organ abnormalities such as the lungs, heart, or kidneys, caused by the systemic nature of the disease.

[0013] Inflammation is therefore one of the key symptoms of chronic musculoskeletal diseases, and the resulting joint and muscle pain can be particularly debilitating. Many of these diseases are chronic and progressive, requiring long-term drug therapy. An example of a rheumatic disease that affects the joints is arthritis, which comes in many forms. Rheumatoid arthritis (RA) is a particular example associated with inflammation due to autoimmunity.

[0014] Being highly prevalent chronic diseases with long-term effects and no cure, the burden on healthcare systems to treat these conditions is incredible. For example, RA affected approximately 24.5 million people as of 2015, or 0.5-1% of adults in developed countries.

[24] It was responsible for 38,000 deaths in 2013, up from 28,000 in 1990.

[25]

[0015] Rheumatoid arthritis (RA) is a chronic systemic disease characterized by inflammatory and erosive synovitis. Its pathological diagnosis is synovial cell hyperplasia, hyperemia, vascular wall thickening, inflammatory cell infiltration, and fibrous tissue hyperplasia, clearing, and degeneration. Synovial changes are characterized by the formation of new blood vessels (called angiogenesis), which plays a key role in the formation and maintenance of the inflammatory vascular tissue pannus. This pannus covers and erodes the articular cartilage, ultimately leading to joint destruction.

[0016] Although the pathogenesis of RA is not fully understood, it is thought to be an autoimmune disease associated with pathological angiogenesis and inflammation in affected tissues.

[0017] The symptom that distinguishes rheumatoid arthritis from other forms of arthritis is the simultaneous inflammation of many joints and swelling of soft tissues (polyarthritis). Rheumatoid arthritis is a painful, disabling inflammatory condition that can cause pain and joint destruction resulting in significant loss of mobility. RA is a systemic disease that frequently affects extra-articular tissues throughout the body, including the skin, blood vessels, heart, lungs, and muscles.

[0018] For rheumatoid arthritis, the basic goals of treatment are to reduce pain and inflammation; to prevent deformation of bone, cartilage, and soft tissue; and to maintain normal function of the joints; thereby enabling the patient to maintain normal daily activities for as long as possible.

[0019] Pharmacological treatments for RA can be divided into disease-modifying antirheumatic drugs (DMARDs), anti-inflammatory agents, and analgesics. The goals of treatment are to minimize symptoms such as pain and swelling, prevent bone deformation (e.g. bone erosion visible on X-ray), and maintain daily function.

[0020] DMARDs, for example, have been found to improve symptoms, reduce joint damage, and improve overall functional ability. Anti-inflammatory and analgesic drugs improve pain and stiffness, but do not prevent joint damage or slow disease progression.

[0021] Examples of DMARDs include traditional small molecules (cyclosporine, cyclophosphamide, hydroxychloroquine, gold salt methotrexate, leflunomide, methotrexate (MTX), mycophenolate, and sulfasalazine) as well as recombinantly produced, for example, biological agents with immunomodulatory properties, such as: tumor necrosis factor alpha (TNFa) blockers (etanercept, certolizumab pegol, golimumab, infliximab, and adalimumab), interleukin-1 blockers (anakinra), anti-B cell (CD20) antibodies (rituximab), and blockers of T cell activation (abatacept).

[0022] Therapeutic anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs, most of which also act as analgesics) and glucocorticoids.

[0023] NSAIDs reduce inflammation and pain in the early stages of the disease by inhibiting cyclooxygenase (COX) and therefore the production of prostaglandins (PGs), which play a central role in inflammation and pain. NSAIDs can treat the symptoms of arthritis, but do little to prevent the progression of the disease. Examples include acemetacin, diclofenac, ibuprofen, indomethacin, meloxicam, ketoprofen, sulindac, auranofin, naproxen, nabumetone, piroxicam, mecolfenamic acid, clofenamic acid, mefenamic acid, pirprofen, fenbufen, tolmetin, flufenamic acid, fenoprofen, methocarbamol, and nimesulide. NSAIDs can cause adverse effects due to inhibition of production of COX-I and COX-2 products (e.g., complex gastrointestinal syndrome, upset stomach, abdominal pain, ulcers, gastrointestinal bleeding, and damage to the kidneys, liver, and blood systems).

[0024] Corticosteroids (glucocorticoids such as cortisone and prednisolone) are effective immunosuppressants but also cannot cure arthritis. Their side effects (including increased infections, osteoporosis, and adrenal insufficiency) increase with increasing dosage and length of treatment, so they are not recommended for use as long-term drug therapy.

[0025] Painkillers are commonly used to treat pain due to arthritis. They include paracetamol, combination analgesics, and opioid analgesics.

[0026] Although DMARDs can improve the symptoms of RA, this class of drugs has many serious side effects that may be intolerable to patients. Lack of efficacy, intolerance, and recurrent secondary infections in some patients are factors that have prompted the need for the development of new therapies. Treatment of rheumatoid arthritis remains an unmet clinical need, with approximately 20-40% of patients with rheumatoid arthritis not responding adequately to any of the currently available therapies.

[0027] Complement inhibitors WO2004 / 106369 (Evolutec Limited

[26] ) relates to complement inhibitors. A particular subset of the disclosed complement inhibitors is directed to C5 and prevents it from being cleaved into C5a and C5b by any of the complement activation pathways. A particular example of such a C5 cleavage inhibitor is a protein produced by ticks of the species Ornithdoros moubata, the mature form of which consists of amino acids 19 to 168 of the amino acid sequence shown in FIG. 4 of WO2004 / 106369 (SEQ ID NO: 2). The amino acid sequence of the mature form is shown in SEQ ID NO: 4. In WO2004 / 106369, this protein is known under the name "EV576" and "OmCI protein" and, more recently, as "Coversin"

[27] . In 2019, Covasin was renamed "Nomacopan" after being approved as an international non-proprietary name by the World Health Organization. The protein is referred to herein as "Covasin."

[0028] In ticks, coversin is expressed as a preprotein with a leader sequence comprising amino acids 1 to 18 of the amino acid sequence shown in Figure 4 of WO2004 / 106369 at the N-terminus of the mature coversin protein (SEQ ID NO:2). The leader sequence is cleaved off after expression. The mature protein has a sequence consisting of amino acids 19 to 168 of the amino acid sequence shown in Figure 4 of WO2004 / 106369 and in Figure 2A of the present application (SEQ ID NO:2). The amino acid sequence of the mature form is shown in SEQ ID NO:4.

[0029] Coversin also has the ability to inhibit leukotriene B4 (LTB4) activity. The ability to bind LTB4 may be demonstrated by standard in vitro assays known in the art, for example, by competitive ELISA between Coversin and anti-LTB4 antibodies competing for binding to labeled LTB4, by isothermal titration calorimetry, or by fluorescence titration.

[0030] There are several further patent applications relating to the use of Covarcin or its functional equivalents in various applications, such as WO2007 / 028968, WO2008 / 029167, WO2008 / 029169, WO2011 / 083317 and WO2016 / 198133, for which there is no experimental evidence confirming the effectiveness of Covarcin or its functional equivalents in the treatment of rheumatic diseases such as RA.

[0031] In the studies leading to the present invention, Coversin, a molecule that binds LTB4 and also inhibits the complement pathway by binding C5, has been shown to prevent the onset of arthritis in a mouse model of RA and ameliorate established arthritic disease in the same model, as discussed above. Coversin has the ability to inhibit both complement (by inhibiting C5) and LTB4, making it particularly advantageous in the prevention and treatment of rheumatic diseases such as RA, either alone or in combination with other therapies. It has long been known that inhibition of either LTB4 or C5a alone in the K / BxN serum transfer mouse model can completely prevent the onset of arthritis. In the same model, inhibition of LTB4 exerts the ability to ameliorate established arthritic disease. Thus, prior to this study, it was expected that combined inhibition of C5 activation and LTB4 would only be as effective as LTB4 inhibition alone in a therapeutic model. However, unexpectedly, combined inhibition of C5 and LTB4 by Coversin has proven to be much more effective in a therapeutic model than LTB4 inhibition alone. Summary of the Invention

[0032] Coversin has been shown to prevent the development of arthritis in a mouse model of RA and improve established arthritic disease in the same model. Coversin has the ability to inhibit both complement (by inhibiting C5) and LTB4, which makes it particularly advantageous in the prevention and treatment of rheumatic diseases such as RA, either alone or in combination with other therapies. In Example 1, administration of Coversin with induction of disease was shown to completely prevent the development of arthritis. A greater effect can be observed with administration of Coversin compared to zileuton, since zileuton only partially improves the development of arthritis in this model. Thus, Coversin appears to be more effective than LTB4 inhibitors (zileuton) in preventing the development of arthritis in this mouse model. As noted in the background of the invention, zileuton inhibits the synthesis of LTB4 and other LKs, some of which may be anti-inflammatory.

[0033] It has also been shown that administration of Covarcin after disease induction can ameliorate established arthritis disease (Example 2). The results of this experiment are particularly interesting because they show that the dual inhibitory activity of Covarcin, which targets both C5 and LTB4, appears to be particularly advantageous in the treatment of arthritis.

[0034] In these experiments, a modified covarcin polypeptide with reduced or absent C5 binding activity but retaining LTB4 binding ability was compared to covarcin. This molecule (designated L-covarcin) was more effective than zileuton but less effective than covarcin in ameliorating established arthritic disease, highlighting the advantage of covarcin in treating rheumatic diseases such as RA.

[0035] Thus, the inventors have demonstrated that administration of the tick protein covarcin (also referred to in the art and herein as EV576 and OmCI

[26] ) can be used to treat or prevent rheumatic diseases.

[0036] Thus, the present invention provides a method for treating or preventing a rheumatic disease, comprising administering a therapeutically or prophylactically effective amount of a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO: 2), or an agent which is a functional equivalent of this protein.

[0037] The present invention also provides an agent which is a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or a functional equivalent of this protein, for use in a method for treating or preventing a rheumatic disease.

[0038] The present invention also provides a method for treating or preventing a rheumatic disease, comprising administering a therapeutically or prophylactically effective amount of an agent which is a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or a nucleic acid molecule encoding a functional equivalent of this protein.

[0039] The present invention also provides an agent which is a nucleic acid molecule encoding a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or a functional equivalent of this protein, for use in a method of treating or preventing a rheumatic disease.

[0040] The present invention also provides a method of treating or preventing a rheumatic disease, comprising administering (a) a therapeutically or prophylactically effective amount of a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or an agent that is a functional equivalent of this protein, and (b) a second rheumatic disease treatment.

[0041] The invention also provides (a) an agent which is a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or a functional equivalent of this protein, for use in a method of treating or preventing a rheumatic disease, and (b) a second rheumatic disease treatment.

[0042] The present invention also provides a method of treating or preventing a rheumatic disease, comprising administering (a) a therapeutically or prophylactically effective amount of an agent that is a nucleic acid molecule encoding a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or a functional equivalent of this protein, and (b) a second rheumatic disease treatment.

[0043] The invention also provides (a) an agent that is a nucleic acid molecule encoding a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or a functional equivalent of this protein, for use in a method of treating or preventing a rheumatic disease, and (b) a second rheumatic disease treatment.

[0044] The present invention also provides a method for reducing the amount of a second rheumatic disease treatment required to treat or prevent a rheumatic disease, or for reducing the duration of treatment with a second rheumatic disease treatment required to treat or prevent a rheumatic disease, the method comprising administering a therapeutically or prophylactically effective amount of a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or an agent that is a functional equivalent of this protein, or a nucleic acid molecule encoding said agent, and the second rheumatic disease treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] disease The subject may be suffering from, suspected to suffer from, or at risk of developing a rheumatic disease. In one embodiment, the rheumatic disease is arthritis, including its subtypes. In one embodiment, the rheumatic disease is an autoimmune rheumatic disease. Examples of autoimmune rheumatic diseases include ankylosing spondylitis, relapsing polychondritis, systemic lupus erythematosus, rheumatoid arthritis, gout, inflammatory arthritis, pseudogout, juvenile arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Behcet's disease, and psoriatic arthritis. In certain embodiments, the rheumatic disease is RA. In other embodiments, the RA is associated with vasculitis.

[0046] Subtypes of arthritis include osteoarthritis, gout (or gouty arthritis), RA, psoriatic arthritis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, and polymyalgia rheumatica. Here is a list of arthritis types according to the Arthritis Foundation:

[0047] [Table 1] JPEG0007629394000002.jpg74164

[0048] RA is the preferred disease.

[0049] The presence of these diseases can be determined by routine diagnostics well understood in the art. The severity of certain conditions can also be scored to help evaluate whether a particular treatment will be effective.

[0050] For example, for RA, clinical scoring may be performed using clinically accepted criteria, such as those established by the American College of Rheumatology (ACR). One example is the ACR score, which allows for the measurement of the amount of improvement following treatment (https: / / www.rheumatoidarthritis.org / treatment / acr-score / ). The ACR score considers various factors to create a score that evaluates the amount of improvement that has occurred in a patient's rheumatoid arthritis. The ACR criteria also evaluate and establish improvements in tender and painful joint counts, as well as improvements in three out of five of the following parameters: (1) Inflammation - A laboratory test that measures either erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) determines the amount of inflammation in the joints. (2) Patient's assessment - The patient describes and determines their own assessment of progress. (3) Physician's assessment - The physician describes and determines the patient's progress. (4) Pain scale - How much pain the patient feels in their joints on a daily basis. (5) Disability / functionality questionnaire - How easily the patient can use their joints to complete daily activities.

[0051] An ACR20 score means that the patient experienced a 20% improvement in tender and painful joint counts and a 20% improvement in three of the five parameters. ACR20 is measured on a 28-point scale, representing a 20% improvement. ACR50 and ACR70 represent a 50% and 70% improvement.

[0052] An alternative is the Disease Activity Score 28-joint count (DAS28). In one embodiment, a DAS28 score above 5.1 is considered severe, 3.2 to 5.1 is considered moderate, and patients with a score below 2.6 are defined as in remission. Furthermore, a reduction in DAS28 score of 0.6 or less after treatment is considered to indicate a poor response, and a reduction of more than 1.2 points indicates a moderate or good response, depending on whether the individual's DAS28 score at endpoint is above or below 3.2, respectively.

[0053] A further alternative is the Clinical Disease Activity Index (CDAI). A CDAI score of more than 10 and less than 22 is considered moderate, a CDAI score of more than 22 is considered severe, a CDAI score of more than 2.8 and less than 10 is considered low disease activity, and a CDAI score of less than 2.8 is considered remission. A reduction in CDAI of 6.5 represents a moderate improvement. In a further alternative embodiment, a RAPID3

[28] score of 1 to 3 indicates near remission, a score of 4 to 6 indicates low severity, a score of 7 to 12 is considered moderate, and a RAPID3 score of 13 to 30 is considered severe.

[0054] Subjects at risk of developing rheumatic disease may benefit from administration of the agents referred to herein to prevent rheumatic disease or its symptoms. Risk factors for certain rheumatic diseases are listed in Table 2 below and discussed in more detail in

[29] .

[0055] [Table 2]

[0056] In terms of treating or preventing rheumatic disease, subjects with one or more of these risk factors are preferred.

[0057] In some embodiments, a subject may have one or more of these risk factors, but may not exhibit clinical symptoms. In some embodiments, a subject may be in the effector phase of a condition, such as RA. In such a subject, one or more autoantibodies associated with RA may be detected in said subject at a level that indicates, for example, risk of disease progression. The K / BxN mouse model of RA is a model of the effector phase of RA, in which autoantibodies are involved. Without being bound by theory, Coversin may be particularly effective in treating cases where autoantibodies are present or where rheumatic disease continues to be caused by autoantibodies. In certain embodiments, any method of treatment may further include a step of selecting a subject for treatment based on the presence of autoantibodies.

[0058] RA In a particular embodiment, the rheumatic disease is RA. RA subjects can be seropositive or seronegative (depending on the presence or absence of detectable rheumatoid factor (RF), e.g., antibodies against the Fc portion of human IgG, which can be determined by agglutination or nephrometry). Other antibodies with specificity for RA are also known, including antibodies against certain peptides that contain citrullinated arginine residues (anti-citrullinated protein antibodies (ACPA)). RA subjects may additionally or alternatively have autoantibodies against cyclic citrullinated peptides (CCPs), which can be detected using commercially available tests. ACPAs can diagnose rheumatoid arthritis (RA) at a very early stage, and ACPA tests form part of the 2010 ACR-EULAR classification criteria for rheumatoid arthritis.

[0059] Thus, RA may be described as autoantibody-driven RA, and in particular, the subject may be RF-positive and / or anti-CCP (or ACPA)-positive. In certain embodiments, any method of treatment may further include selecting a subject for treatment based on the presence of RF and / or anti-CCP autoantibodies (or ACPA).

[0060] vasculitis In further embodiments, the RA subject may have vasculitis associated with RA, such as rheumatic vasculitis, such as systemic vasculitis. The term "vasculitis" indicates that blood vessels are in an inflammatory state. The outcome of vasculitis depends on the size, location, and number of blood vessels involved. Infarction of the tissues that the blood vessels supply can occur when small or medium-sized arteries are involved (e.g., coronary artery vasculitis can cause heart attacks), but the impact is less severe when very small blood vessels, such as capillaries, are involved. The exception is widespread local vasculitis, such as that which occurs in the kidney and can cause glomerulonephritis.

[0061] Vasculitis can occur as a complication of rheumatoid arthritis, for example small vasculitis (e.g., involving small arteries and arterioles). Vasculitis is also associated with most of the extra-articular (meaning "outside the joint") symptoms described in rheumatoid arthritis. These include inflammation of the eye (iritis), inflammation of the lining of the heart and lungs (pericarditis and pleuritis), and other pulmonary and cardiac symptoms, including inflammation of the base of the lungs (fibrosing alveolitis) and arrhythmias, including heart block. Subjects with systemic vasculitis usually have high levels of RF in their blood.

[0062] Systemic vasculitis is a rare but serious complication of rheumatoid arthritis and may be considered one of the most serious extra-articular events of the disease. As an example, rheumatoid vasculitis (RV), characterized by inflammation of medium-sized arteries and capillaries, is severe and up to 40% of patients die within 5 years due to damage caused by vasculitis and / or consequences of immunosuppressive therapy. RV remains difficult to treat and is associated with a high mortality rate.

[0063] The subject may have been previously treated with one or more other rheumatic disease treatments (e.g., one or more treatments or DMARDs discussed above). In some embodiments, the previous treatment was not effective, became ineffective, or was discontinued due to one or more adverse events. Discontinuing one rheumatic disease treatment is relatively common, especially with TNF inhibitors. Thus, in certain embodiments, the subject has been previously treated with one or more TNF inhibitors. In certain embodiments, such treatment was discontinued due to lack of efficacy or adverse effects. Lack of efficacy can be evaluated as discussed elsewhere herein. Examples of adverse effects include cough, headache, heartburn, nausea or vomiting, abdominal pain, weakness, severe allergic reactions, infections, cancer, such as tumor development, and the like.

[0064] timing It may be advantageous to initiate treatment early after diagnosis or onset of the disease. In a preferred embodiment of the invention, treatment of a rheumatic disease in a subject according to the invention is initiated within about 6 months of initial diagnosis, within about 12 months of initial diagnosis, within about 18 months of initial diagnosis, within about 2 years of initial diagnosis, within about 3 years of initial diagnosis, within about 4 years of initial diagnosis, within about 5 years of initial diagnosis.

[0065] In a preferred embodiment of the invention, the treatment of a rheumatic disease in a subject according to the invention is in a subject having disease duration of about 6 months or less, disease duration of about 12 months or less, disease duration of about 18 months or less, disease duration of about 2 years or less, disease duration of about 3 years or less, disease duration of about 4 years or less, disease duration of about 5 years or less.

[0066] Usually, the treatment of rheumatic disease is in the subject of the acute phase of the disease.The term "acute phase" in the context of rheumatoid arthritis is to be interpreted as meaning that the patient experiences significant inflammation of one or more joints, as opposed to only mild or moderate inflammation.Acute phase is also referred to in the art as "flare".

[0067] Administration results As a result of treatment, the subject may have a reduction in the incidence of symptoms, relief of symptoms, inhibition or delay of the occurrence or reoccurrence of symptoms, or a combination thereof.Preferably, treatment results in relief of symptoms of typical disease states.For example, this may be manifested in a reduction in the amount of inflammation or swelling, the size of joints, the number of affected joints, or the amount of pain, or a prolonged interval to recurrence.Also, the fever and / or severity and / or the number of painful areas may be reduced.Some subjects may experience complete resolution of symptoms and no further recurrence.

[0068] As a result of treatment, the subject may show improvement in clinical score, for example using one of the methods cited above. For example, the subject may be considered in remission after treatment, or may have a score that shows improvement of symptoms after a certain treatment period. If the subject has a rheumatic disease, such as RA, that is scored by DAS28, CDAI or RAPID3, treatment may, in one embodiment, result in improvement of DAS28 score, CDAI or RAPID3. The subject may have a reduction in DAS28 of at least 0.8 or 1.0 or 1.2 points after a course of treatment (preferably, the subject's DAS28 score at endpoint is less than 3.2). The subject may have a reduction in CDAI of at least 6.5 after a course of treatment. In a further alternative embodiment, the subject may have a RAPID3 score of less than 6 after a course of treatment.

[0069] In other words, a subject may have an ACR20, ACR50 or ACR70 score after a course of treatment.

[0070] Methods for diagnosing acute rheumatoid arthritis are known in the art and may include analysis of erythrocyte sedimentation rate (ESR) and / or serum C-reactive protein (CRP) levels, where, for example, an ESR of 50 or greater is considered acute. If a subject has acute RA, treatment may result in a decrease in erythrocyte sedimentation rate (ESR) and / or serum C-reactive protein (CRP) levels.

[0071] If the subject has RV, treatment may result in improvement as described above and / or improvement in clinical symptoms of RV (including skin ulcers, peripheral gangrene, vascular neuropathy, inflammatory eye disease and visceral infarction, i.e., one or more of these clinical symptoms may be reduced and / or their severity reduced).

[0072] Treatment may also result in a reduction in the amount or duration of second rheumatic disease treatment required.

[0073] Thus, in a further embodiment of the invention, there is provided a method of reducing clinical scores in a subject with a rheumatic disease, or reducing joint inflammation in a subject with a rheumatic disease, or reducing joint stiffness in a subject with a rheumatic disease, comprising administering a therapeutically or prophylactically effective amount of an agent which is a protein comprising amino acids 19 to 168 of the amino acid sequence of Figure 2 (SEQ ID NO:2) or a functional equivalent of this protein, or a nucleic acid molecule encoding said agent, either alone or in conjunction with the treatment of a second rheumatic disease.

[0074] The present invention also provides a protein comprising amino acids 19 to 168 of the amino acid sequence of Figure 2 (SEQ ID NO:2), or an agent which is a functional equivalent of this protein, or a nucleic acid molecule encoding said agent, for use in a method of reducing the clinical score of a subject with a rheumatic disease, or a method of reducing joint inflammation in a subject with a rheumatic disease, or a method of reducing joint stiffness in a subject with a rheumatic disease, either alone or in conjunction with the treatment of a second rheumatic disease.

[0075] The agent of the present invention can be used in combination with other rheumatic disease treatments as described above. The combination of the agent of the present invention with other (herein referred to as "second") rheumatic disease treatments can be such that the amount of the second rheumatic disease drug is reduced compared to the amount used in the absence of treatment with the agent of the present invention, or the duration of treatment with the second rheumatic disease drug is reduced compared to the treatment duration used in the absence of treatment with the agent of the present invention. This is advantageous in view of the side effects of certain known therapies. Thus, a method of reducing the amount of the second rheumatic disease treatment used in treatment or a method of reducing the duration of treatment with the second rheumatic disease treatment are also provided.

[0076] Preferably, the second rheumatic disease treatment is selected from a DMARD (e.g., one or more of cyclosporine, cyclophosphamide, hydroxychloroquine, gold salts, methotrexate, leflunomide, mycophenolate, sulfasalazine, etanercept, certolizumab pegol, golimumab, infliximab, and adalimumab, anakinra, rituximab, and abatacept), an anti-inflammatory agent (e.g., an NSAID, or a glucocorticoid), and an analgesic (e.g., selected from paracetamol, a combination analgesic, and an opioid analgesic).

[0077] When an agent of the invention and a second rheumatic disease treatment are used, they may be administered together or separately. The agent of the invention may be administered first and the second rheumatic disease treatment may be administered second, or vice versa.

[0078] Thus, for example in the above methods, when an agent of the invention is used in combination with one or more other rheumatic disease treatments, it can be described as a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or an agent that is a functional equivalent of this protein, as a second rheumatic disease treatment for use in a method of treating or preventing a rheumatic disease with a second rheumatic disease treatment, or for use in a method of treating or preventing a rheumatic disease with a protein comprising amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2), or an agent that is a functional equivalent of this protein.

[0079] Where the treatment results in a reduction in the amount or duration of a second rheumatic disease treatment, the reduction may be up to or at least 10, 20, 30, 40, 50, 60, 70, 80% compared to the amount of the second treatment used in the absence of the agent of the invention.

[0080] subject Preferred subjects, agents, dosages and the like are as disclosed herein.

[0081] Any reference to any decrease or increase is the decrease or increase in disease parameter compared to said subject in the absence of treatment.Preferably, the parameter can be quantified, and in this case, the increase or decrease is preferably statistically significant.For example, the increase or decrease can be at least 3, 5, 10, 15, 20, 30, 40, 50% or more compared to the parameter in the absence of treatment (e.g., before said treatment is started).

[0082] The subject to whom the agent is administered in the practice of the present invention is preferably a mammal, preferably a human being. The subject to whom the agent is administered is a subject at risk of or has a rheumatic disease.

[0083] The methods of the invention may also include one or more additional steps of (i) determining whether the subject is at risk for or has a rheumatic disease; (ii) determining the severity of the rheumatic disease, which may be performed before and / or after administration of Covarcin.

[0084] Drugs to be used in the present invention According to one embodiment of the invention, the agent is Coversin itself or a functional equivalent thereof. In the following, the term "Coversin-type protein" is used as shorthand for "a protein comprising amino acids 19 to 168 of the amino acid sequence shown in Figure 2 (SEQ ID NO: 2), or a functional equivalent thereof."

[0085] Conversen was isolated from the salivary glands of the tick Ornithodoros moubata. Conversen is a distant member of the lipocalin family and was the first member of the lipocalin family shown to inhibit complement activation. Conversen inhibits the classical, alternative, and lectin complement pathways by binding to C5 and preventing cleavage of C5 by C5 convertase into C5a and C5b, thereby inhibiting both the production of the active (e.g., pro-inflammatory) peptide C5a and the formation of MAC. Conversen has been demonstrated to bind to C5 and prevent cleavage of C5 by C5 convertase with an IC50 of approximately 0.02 mg / ml in rat, mouse, and human serum.

[0086] Thus, a covercin-type protein may comprise or consist of amino acids 19 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2) or amino acids 1 to 168 of the amino acid sequence in Figure 2 (SEQ ID NO:2). As the first 18 amino acids of the protein sequence shown in Figure 2 form a signal sequence that is not required for C5 binding or LTB4 binding activity, this may be omitted if desired, for example for efficiency of recombinant protein production.

[0087] The coversin protein has been demonstrated to bind to C5 with a Kd of 1 nM as determined using surface plasmon resonance (SPR)

[30] . Coversin-type peptides (e.g. functional equivalents of the coversin protein) preferably retain the ability to bind to C5 with a Kd of advantageously less than 360 nM, more advantageously less than 300 nM, most advantageously less than 250 nM, preferably less than 200 nM, more advantageously less than 150 nM, most advantageously less than 100 nM, even more advantageously less than 50, 40, 30, 20, or 10 nM, advantageously less than 5 nM, wherein said Kd is preferably determined using surface plasmon resonance by the methods described in

[30] .

[0088] Conversin inhibits the classical, alternative and lectin complement pathways. Preferably, the conversin-type protein binds to C5 in such a way that it stabilizes the overall conformation of C5 but does not directly block the C5 cleavage site targeted by the C5 convertases of the three activation pathways. Conversin binding to C5 leads to the stabilization of the overall conformation of C5 but does not block the convertase cleavage site. Functional equivalents of conversin also preferably share these properties.

[0089] C5 is cleaved by the enzyme C5 convertase (Figure 1). The products of this cleavage include the anaphylatoxin C5a and the lytic complex C5b, which promotes the formation of a complex of C5b, C6, C7, C8 and C9, also known as the membrane attack complex (MAC). C5a is a highly pro-inflammatory peptide that has been implicated in many pathological inflammatory processes, including neutrophil and eosinophil chemotaxis, neutrophil activation, increased capillary permeability and inhibition of neutrophil apoptosis

[31] .

[0090] Monoclonal antibodies and small molecules that bind and inhibit C5 have been developed to treat various diseases, particularly PNH, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and transplant rejection

[32] . However, some of these monoclonal antibodies do not bind to certain C5 proteins from subjects with C5 polymorphisms, and are therefore ineffective in these subjects

[33] . Preferably, the covasin protein binds to and inhibits the cleavage of C5 from subjects with C5 polymorphisms (e.g., C5 polymorphisms that render eculizumab treatment ineffective or reduce the efficacy of eculizumab treatment), as well as wild-type C5. The term "C5 polymorphism" includes any version of C5 that has been altered by insertion, deletion, amino acid substitution, frameshift, truncation, or one or more combinations of these changes, any of which may be one or more, compared to wild-type C5. In human subjects, wild-type C5 is considered to be the C5 protein with the accession number NP_001726.2; version GI:38016947. Examples of C5 polymorphisms include polymorphisms at amino acid position 885, such as Arg885Cys (encoded by c.2653C>T), p.Arg885His (encoded by c.2654G>A) and Arg885Ser, which reduce the efficacy of the mAb eculizumab

[33] .

[0091] The ability of an agent to bind to C5, including C5 from a subject having a C5 polymorphism, e.g., a C5 polymorphism that renders treatment with eculizumab ineffective or reduces the effectiveness of treatment with eculizumab, may be determined by standard in vitro assays known in the art, e.g., surface plasmon resonance or western blot after incubating the protein on a gel with labeled C5. Preferably, the covasin type protein binds to C5, either wild type C5 and / or C5 from a subject having a C5 polymorphism, e.g. a C5 polymorphism that renders treatment with eculizumab ineffective or reduces the efficacy of treatment with eculizumab, with a Kd of less than 360 nM, more advantageously less than 300 nM, most advantageously less than 250 nM, preferably less than 200 nM, more advantageously less than 150 nM, most advantageously less than 100 nM, even more advantageously less than 50, 40, 30, 20 or 10 nM, advantageously less than 5 nM, wherein said Kd is determined using surface plasmon resonance, preferably by the method described in

[30] .

[0092] This may show a higher, lower or the same affinity for wild-type C5 and C5 from subjects having a C5 polymorphism, e.g., a C5 polymorphism that renders treatment with eculizumab ineffective or reduces the effectiveness of treatment with eculizumab.

[0093] The ability of a covercin-type protein to inhibit complement activation may also be determined by measuring the ability of the agent to inhibit complement activation in serum. For example, the activity of complement in serum can be measured by any means known in the art or described herein.

[0094] Conversin-type proteins may also be defined as having the function of inhibiting eicosanoid activity. Conversin has also been demonstrated to bind to LTB4. Functional equivalents of the conversin protein may also retain the ability to bind to LTB4 with an affinity similar to that of the conversin protein.

[0095] The ability of the coversin type protein to bind LTB4 may be determined by standard in vitro assays known in the art, such as competitive ELISA between coversin and anti-LTB4 antibodies competing for binding to labeled LTB4, isothermal titration calorimetry or fluorescence titration. Data obtained using fluorescence titration shows that coversin binds to LTB4 with a Kd between 200 and 300 pM. For example, the binding activity to LTB4 (Caymen Chemicals, Ann Arbor, MI, USA) in phosphate buffered saline (PBS) can be quantified with a spectrofluorometer, such as an LS 50 B spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA). This can be performed as follows:

[0096] A 100 nM solution of purified covercin in 2 mL of PBS was added into a quartz cuvette (10 mm path length; Hellma, Mühlheim, Germany) equipped with a magnetic stirrer. After temperature adjustment to 20 °C and equilibration, the fluorescence of the protein Tyr / Trp was excited at 280 nm (slit width: 15 nm). The fluorescence emission corresponding to the emission maximum was measured at 340 nm (slit width: 16 nm). A 30 μM LTB4 ligand solution in PBS was added stepwise up to a maximum volume of 20 μL (1% of the total sample volume) and the steady-state fluorescence was measured after 30 s of incubation. For calculation of KD values, data were normalized to 100% initial fluorescence intensity, the inner filter effect was corrected using a titration of 3 μM N-acetyl-tryptophanamide solution, and the data were plotted against the corresponding ligand concentration. The data were then fitted using nonlinear least-squares regression based on the law of mass action for the formation of bimolecular complexes using published equations ( Breustedt et al., 2006 ) [ 34 ] with Origin software version 8.5 (OriginLab, Northampton, MA, USA).

[0097] Coversin may bind to LTB4 with a Kd of less than 1 nM, more advantageously less than 0.9 nM, most advantageously less than 0.8 nM, preferably less than 0.7 nM, more advantageously less than 0.6 nM, most advantageously less than 0.5 nM, even more advantageously less than 0.4 nM, advantageously less than 0.3 nM, said Kd being determined using fluorescence titration, preferably by the methods described above. Coversin type proteins preferably share these properties.

[0098] According to one embodiment of the invention, the covasin-type protein may bind to both C5 and LTB4, e.g., wild-type C5 and C5 from a subject having a C5 polymorphism, e.g., a C5 polymorphism that renders treatment with eculizumab ineffective or reduces the effectiveness of treatment with eculizumab, and LTB4.

[0099] Thus, the covasin-type protein may act to prevent the cleavage of complement C5 into complement C5a and complement C5b by C5 convertase, and also to inhibit LTB4 activity. It is particularly advantageous to use an agent that binds to both C5 and LTB4. Both C5 and eicosanoid pathways may contribute to pathology observed in rheumatic diseases. Thus, by using a single agent that inhibits multiple pathways involved in rheumatic diseases, enhanced efficacy can be achieved compared to using an agent that inhibits only a single pathway involved in the inflammatory effects of complement-mediated diseases and disorders (see, for example, Example 2, which compares covasin and L-covasin). Furthermore, there are practical advantages associated with administering a single molecule.

[0100] Preferably, the agent of the present invention is derived from a blood-sucking arthropod. The term "blood-sucking arthropod" includes all arthropods, such as insects, ticks, lice, fleas and mites, that feed by sucking blood from a suitable host. Preferably, the agent is derived from a tick, preferably the tick Ornithodoros moubata.

[0101] A functional equivalent of covercin may be a homolog or fragment of covercin that retains the ability to bind C5, either wild-type C5 or C5 from a subject with a C5 polymorphism (e.g., the Arg885Cys, Arg885His, or Arg885Ser polymorphisms described above), and to prevent cleavage of C5 into C5a and C5b by C5 convertase. The homolog or fragment may also retain the ability to bind LTB4.

[0102] Homologues include, for example, Rhipicephalus appendiculatus, R. sanguineus, R. bursa, A. americanum, A. cajennense, A. hebraeum, Boophilus microplus, B. annulatus, B. decoloratus, Dermacentor reticulatus, D. andersoni, D. marginatus, D. variabilis, Haemaphysalis inermis, Ha.leachii, Ha.punctata, Hyalomma anatolicum anatolicum, Hy.dromedarii, Hy.marginatum marginatum, Ixodes ricinus, I.persulcatus, I.scapularis, I.hexagonus, Argas persicus, A.reflexus, Ornithodoros erraticus, O.moubata The present disclosure includes paralogs and orthologs of the covercin sequence explicitly identified in FIG. 2 (SEQ ID NO:2), including covercin protein sequences from other tick species, including Ixodes moubata, Omporcinus, and O. savignyi.

[0103] The term "homologue" is also meant to include equivalent covercin protein sequences from mosquito species including Culex, Anopheles and Aedes species, particularly Culex quinquefasciatus, Aedes aegypti and Anopheles gambiae; flea species such as Ctenocephalides felis (cat flea); horsefly; sandfly; blackfly; tsetse fly; louse; tick; leech; and flatworm. The native covercin protein is believed to exist in three alternative forms of approximately 18 kDa in O. moubata, and the term "homologue" is meant to include these alternative forms of covercin.

[0104] Methods for identifying homologues of the covercin sequence shown in Figure 2 will be apparent to those skilled in the art. For example, homologues may be identified by homology searches of both public and non-public sequence databases. Conveniently, publicly available databases may be used. However, non-public or commercially available databases are equally useful, especially if they contain data not represented in the public databases. Primary databases are depository sites for primary nucleotide or primary amino acid sequence data and may be publicly or commercially available.Examples of publicly available primary databases include the GenBank database (http: / / www.ncbi.nlm.nih.gov / ), the EMBL database (http: / / www.ebi.ac.uk / ), the DDBJ database (http: / / www.ddbj.nig.ac.jp / ), the SWISS-PROT protein database (http: / / expasy.hcuge.ch / ), PIR (http: / / pir.georgetown.edu / ), TrEMBL (http: / / www.ebi.ac.uk / ), the TIGR database (see http: / / www.tigr.org / tdb / index.html), the NRL-3D database (http: / / www.nbrfa.georgetown.edu), and the Protein Data These include the Base (http: / / www.rcsb.org / pdb), the NRDB database (ftp: / / ncbi.nlm.nih.gov / pub / nrdb / README), the OWL database (http: / / www.biochem.ucl.ac.uk / bsm / dbbrowser / OWL / ) and the secondary databases PROSITE (http: / / expasy.hcuge.ch / sprot / prosite.html), PRINTS (http: / / iupab.leeds.ac.uk / bmb5dp / prints.html), Profiles (http: / / ulrec3.unil.ch / software / PFSCAN_form.html), Pfam (http: / / www.sanger.ac.uk / software / pfam), Identify (http: / / dna.stanford.edu / identify / ) and Blocks (http: / / www.blocks.fhcrc.org) databases. Examples of commercially available or non-public databases include PathoGenome (Genome Therapeutics Inc.) and PathoSeq (formerly of Incyte Pharmaceuticals Inc.).

[0105] Typically, greater than 30% identity between two polypeptides (preferably over a particular region such as the active site) is considered to be an indication of functional equivalence and therefore that the two proteins are homologous. Preferably, a protein that is a homolog has a level of sequence identity of greater than 60% with the coversin protein sequence (SEQ ID NO:2) identified in Figure 2. More preferred homologs have a level of identity of greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively, with the coversin protein sequence (SEQ ID NO:2) shown in Figure 2. A homolog may have a level of identity of greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%, respectively, with the coversin protein sequence (SEQ ID NO:2) shown in Figure 2. In some embodiments, a homolog has a level of identity of greater than 90% with the coversin protein sequence (SEQ ID NO:2) shown in Figure 2. Percentages of identity referred to herein are determined using BLAST version 2.1.3 using the default parameters [Blosum 62 matrix; gap opening penalty = 11 and gap extension penalty = 1] as specified by NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ). The % identity may be over the full length of the relevant reference sequence (e.g., amino acids 1-168 of SEQ ID NO:2 or amino acids 19-168 of SEQ ID NO:2).

[0106] Thus, a coversin type protein can be described by reference to a certain % amino acid sequence identity to a reference sequence, e.g., amino acids 19-168 of FIG. 2, SEQ ID NO:2, or amino acids 1-168 of FIG. 2, SEQ ID NO:2, as a protein comprising or consisting of a sequence having at least 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to amino acids 19-168 of FIG. 2, SEQ ID NO:2, or amino acids 1-168 of FIG. 2, SEQ ID NO:2. In some embodiments, the coversin type protein comprises or consists of a sequence having at least 90% identity to the coversin protein sequence of SEQ ID NO:4. When the coversin type protein comprises said sequence, the coversin type protein can be a fusion protein (e.g., with a second protein, e.g., a heterologous protein). Suitable second proteins are described below.

[0107] In various aspects and embodiments of the disclosure, modified coversin polypeptides (e.g., coversin-type proteins) may differ from unmodified coversin polypeptides in SEQ ID NO:2 and SEQ ID NO:4 by 1 to 50, 2 to 45, 3 to 40, 4 to 35, 5 to 30, 6 to 25, 7 to 20, 8 to 25, 9 to 20, 10 to 15 amino acids, up to 1, 2, 3, 4, 5, 7, 8, 9, 10, 20, 30, 40, 50 amino acids. These may be substitutions, insertions, or deletions, but are preferably substitutions. If deleted, these are preferably deletions of up to 1, 2, 3, 4, 5, 7 or 10 amino acids (e.g., deletions from the N- or C-terminus). Thus, variants include proteins that contain amino acid substitutions, such as conservative amino acid substitutions that do not adversely affect the function or activity of the protein. The term is also intended to include natural biological variants (e.g., allelic variants or geographic variations within the species from which the covercin protein is derived). Mutants with improved ability to bind C5 and / or LTB4 from subjects with wild-type C5 and / or C5 polymorphisms (e.g., C5 polymorphisms that render treatment with eculizumab ineffective or reduce the effectiveness of treatment with eculizumab) may also be engineered by systematic or directed mutation of specific residues in the protein sequence.

[0108] These modifications may be made to the coversin polypeptide as set forth in SEQ ID NO:2 and SEQ ID NO:4 and will be considered to be functional variants, provided that the molecule remains useful and the resulting modified coversin polypeptide retains LTB4 binding activity and C5 binding comparable to the coversin polypeptide as set forth in SEQ ID NO:2 and SEQ ID NO:4, as can be determined, for example, using the tests mentioned elsewhere herein (e.g., binding to each of these is at least 80, 85, 90, 95% binding compared to the unmodified coversin polypeptide).

[0109] Given the requirement for a functional variant that binds to C5 and LTB4, certain residues should be excluded from modification, if any, and these include conserved cysteine ​​residues. Other residues should be excluded from modification or, if substituted, should only be subjected to conservative modifications. These are the LTB4-binding and C5-binding residues defined below. Given that the binding of LTB4 and C5 is relatively well understood, it is possible to design a molecule that may have a percentage of identity of about 65% to covercin, but where changes are limited to residues that are not involved in the binding of C5 and LTB4.

[0110] In some embodiments, each of the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of the mature covercin molecule (e.g., corresponding to residues 19 to 168 of the full-length protein including the signal sequence as set forth in SEQ ID NO:4) are retained, and at least 5, 10, or 15, or each of the LTB4 binding residues and at least 5, 10, or 15, or 20, or each of the C5 binding residues set forth below are retained or conservatively modified.

[0111] In some embodiments, each of the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 are retained, and at least 5, 10 or 15 or each of the LTB4 binding residues and at least 5, 10 or 15 or 20 or each of the C5 binding residues set forth below are retained or subjected to conservative modification, and up to 2, 3, 4, 5, 10, 15, 20 of the LTB4 and C5 binding residues are conservatively modified.

[0112] In some embodiments, each of the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 are retained, and at least 5, 10 or 15 or each of the LTB4 binding residues and at least 5, 10, or 15, or 20, or each of the C5 binding residues set forth below are retained.

[0113] In some embodiments, each of the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 are retained, and each of the LTB4 binding residues and each of the C5 binding residues described below are retained or conservatively modified.

[0114] In some embodiments, each of the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 are retained, and each of the LTB4 binding residues and each of the C5 binding residues described below are retained or subjected to conservative modifications, with up to 2, 3, 4, 5, 10, 15, 20 of the C5 and / or LTB4 binding residues being conservatively modified.

[0115] In some embodiments, each of the six cysteine ​​amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO:4 are retained, and each of the LTB4 binding residues and each of the C5 binding residues described below are retained.

[0116] Modifications made outside of these regions may be conservative or non-conservative.

[0117] In each of these embodiments, the spacing between these six cysteine ​​amino acid residues is preferably maintained to preserve the overall structure of the molecule (e.g., the molecule contains six cysteine ​​residues spaced from one another at distances of 32 amino acids apart, 62 amino acids apart, 28 amino acids apart, 1 amino acid apart, and 21 amino acids apart when arranged from the amino terminus to the carboxyl terminus in a sequence according to amino acids 1-168 of the amino acid sequence of Figure 2).

[0118] LTB4 binding residue Residues believed to be involved in binding to LTB4 and which are preferably retained in unmodified form or undergo conservative changes only in the sequence of any molecule modified relative to SEQ ID NO:2 or SEQ ID NO:4 are Phe18, Tyr25, Arg36, Leu39, Gly41, Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71, Gln87, Arg89, His99, His101, Asp103, and Trp115 (numbered according to SEQ ID NO:4).

[0119] C5 binding residue Residues believed to be involved in binding to LTB4 are preferably retained in unmodified form in the sequence of SEQ ID NO:2 or any molecule modified relative to SEQ ID NO:4 and are: Val26, Val28, Arg29, Ala44, Gly45, Gly61, Thr62, Ser97, His99, His101, Met114, Met116, Leu117, Asp118, Ala119, Gly120, Gly121, Leu122, Glu123, Val124, Glu125, Glu127, His146, Leu147, and Asp149 (numbering according to SEQ ID NO:4).

[0120] LTB4 and / or C5 binding residues There are two histidine residues involved in both LTB4 and C5 binding, His99 and His101. Thus, the list of residues involved in LTB4 and / or C5 binding is: Phe18, Tyr25, Val26, Val28, Arg29, Arg36, Leu39, Gly41, Pro43, Ala44, Gly45, Leu52, Val54, Met56, Phe58, Gly61, Thr62, Thr67, Trp69, Phe71, Gln87 ... g89, Ser97, His99, His101, Asp103, Met114, Trp115, Met116, Leu117, Asp118, Ala119, Gly120, Gly121, Leu122, Glu123, Val124, Glu125, Glu127, His146, Leu147, and Asp149 (numbering according to SEQ ID NO: 4).

[0121] Functional equivalents of coversin include fragments of the coversin protein, provided that such fragments retain the ability to bind C5 and / or LTB4 from subjects with wild-type C5 and / or C5 polymorphisms (e.g., C5 polymorphisms that render treatment with eculizumab ineffective or reduce the effectiveness of treatment with eculizumab). Fragments may include, for example, polypeptides derived from the coversin protein sequence (or homologs) that are less than 150 amino acids, less than 145 amino acids, provided that such fragments retain the ability to bind C5 and / or LTB4 from subjects with complement wild-type C5 and / or C5 polymorphisms (e.g., C5 polymorphisms that render treatment with eculizumab ineffective or reduce the effectiveness of treatment with eculizumab). Fragments may include, for example, polypeptides derived from the covercin protein sequence (or homologues) that are at least 150 amino acids, at least 145 amino acids, provided that these fragments retain the ability to bind to complement wild-type C5 and / or C5 polymorphisms (e.g., C5 polymorphisms that render treatment with eculizumab ineffective or reduce the effectiveness of treatment with eculizumab).

[0122] Any functional equivalent or fragment thereof preferably retains the pattern of cysteine ​​residues found in covercin. For example, said functional equivalent comprises six cysteine ​​residues spaced from each other at distances of 32 amino acids apart, 62 amino acids apart, 28 amino acids apart, 1 amino acid apart and 21 amino acids apart when arranged from the amino terminus to the carboxyl terminus of the sequence according to amino acids 1 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO:2). Exemplary fragments of covercin protein are disclosed in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14. DNA encoding the corresponding fragments is disclosed in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13.

[0123] Included as such fragments are fragments of the O. moubata coversin protein explicitly identified in FIG. 2 herein, as well as fragments of homologues of this protein as described above. Such fragments of homologues typically have greater than 60% identity with the fragments of the coversin protein sequence in FIG. 2, while more preferred fragments of homologues show levels of identity greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively, with the fragments of the coversin protein sequence in FIG. 2. Preferably, such fragments retain the cysteine ​​spacings mentioned above. Fragments with improved properties may of course be rationally designed by systematic mutation or fragmentation of the wild-type sequence followed by appropriate activity assays. The fragments may show similar or greater affinity than coversin for either or both wild-type or polymorphic variants of C5 and / or LTB4. These fragments may be of the sizes described above for fragments of the coversin protein.

[0124] As described above, the covasin-type protein preferably binds to both C5 and LTB4 from subjects with wild-type C5 and / or C5 polymorphisms (e.g., C5 polymorphisms that render treatment with eculizumab ineffective or reduce the effectiveness of treatment with eculizumab).

[0125] Any substitutions 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 may be substituted for each other: [Table 3]

[0126] The functional equivalents used according to the invention may be, for example, fusion proteins obtained by cloning a polynucleotide encoding the covercin protein or a functional equivalent in frame with a coding sequence for a heterologous protein sequence. The term "heterologous" as used herein is intended to refer to any polypeptide other than the covercin protein or its functional equivalent. Examples of heterologous sequences that may be included at either the N- or C-terminus of the soluble fusion protein are: extracellular domains of membrane-bound proteins, immunoglobulin constant regions (Fc regions), PAS or XTEN or similar unstructured polypeptides, multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences that allow purification by affinity chromatography. Many of these heterologous sequences are commercially available in expression plasmids, since they are generally included in fusion proteins to provide additional properties without significantly impairing the specific biological activity of the protein fused with them

[35] . Examples of such additional properties are a longer half-life in body fluids (e.g., as a result of the addition of an Fc region or PASylation

[36] ), extracellular localization, or easier purification procedures, such as those enabled by tags such as histidine, GST, FLAG, avidin or HA tags. Fusion proteins may additionally contain a linker sequence (e.g., 1-50 amino acids in length), whereby the components are separated by the linker.

[0127] Thus, fusion proteins are examples of proteins that contain covercin-like proteins, including proteins that contain PAS sequences and covercin-type protein sequences as specific examples. PAS sequences are described, for example, in

[36] , and in EP2173890, and PASylated covercin molecules are described in Kuhn et al.

[37] . PASylation describes the genetic fusion of a protein with a conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser. This is a technique developed by XL Protein (http: / / xl-protein.com / ) and provides a simple method of attaching solvated random chains with large hydrodynamic volumes to the proteins to which it is fused. The polypeptide sequence adopts a random coil conformation. Thus, the apparent molecular weight of the resulting fusion protein is much larger than the actual molecular weight of the fusion protein. This significantly reduces the rate of clearance by renal filtration in biological systems. Suitable PAS sequences are described in EP2173890 as well as in

[36] . Any suitable PAS sequence may be used for the fusion protein. Examples include amino acid sequences of at least about 100 amino acid residues that form a random coil conformation and consist of or consist essentially of alanine, serine and proline residues (or consist of or consist essentially of proline and alanine residues). It may include multiple amino acid repeats, where the repeats consist of or consist essentially of Ala, Ser and Pro residues (or proline and alanine residues) and no more than six consecutive identical amino acid residues. Proline residues may constitute more than 4% and less than 40% of the amino acids of the sequence. The sequence may be 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 circularly permuted versions or multimers of these sequences as the whole or part of these sequences. For example, there may be 5-40, 10-30, 15-25, 18-20, preferably 20-30 or 30 copies of one of the repeats present in the PAS sequence, i.e. one, preferably 15, of SEQ ID NOs: 15-21. Preferably, the PAS sequence comprises or consists of 30 copies of SEQ ID NO: 15. Preferably, the PAS sequence is fused (directly or via a linker sequence) to the N-terminus of the covercin-type protein, and in certain preferred embodiments, the covercin-type protein may comprise or consist of amino acids 19-168 of SEQ ID NO: 2 (e.g., a fusion protein comprises (a) a PAS sequence consisting of 30 copies of SEQ ID NO: 15 and (b) amino acids 19-168 of SEQ ID NO: 2, where (a) is fused directly or via a linker sequence to the N-terminus of (b). Exemplary sequences are provided in FIG. 5 and SEQ ID NO: 22.

[0128] The fusion protein may further comprise a linker sequence (e.g., 1-50, 2-30, 3-20, 5-10, 2-4, 3-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids in length), whereby the components are separated by the linker. In one embodiment, the linker sequence may be a single alanine residue.

[0129] Currently, "PAS-coversin" is intended to refer to the functional equivalent of PASylated coversin, e.g., as described above. The exact sequence of the PAS-coversin molecule tested in Examples 1 and 2 is shown in FIG. 5 and SEQ ID NO: 22. PAS-coversin has the advantage that it has a longer half-life, which allows for less frequent administration and is more convenient for patients. Thus, PAS-coversin combines the advantages of coversin in that it inhibits both C5 and LTB4-dependent pathways, but has administration advantages due to less frequent administration than coversin.

[0130] The protein and its functional equivalents may be prepared in recombinant form by expression in a host cell. Such expression methods are known to those skilled in the art and are described in detail by

[38] and

[39] . The recombinant form of the covercin protein and its functional equivalents is preferably non-glycosylated. Preferably, the host cell is E. coli.

[0131] The coversin protein and its functional equivalents are preferably in isolated form, 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 coversin protein or its functional equivalent is purified to a purity of at least 90%, 95%, or 99%, e.g., as determined by electrophoresis or chromatography. The proteins and fragments of the invention can also be prepared using routine techniques of protein chemistry. For example, protein fragments may be prepared by chemical synthesis. Methods for producing fusion proteins are standard in the art and known to the skilled reader. For example, most general molecular biology, microbiology, recombinant DNA techniques and immunological techniques can be found in

[38] or

[40] .

[0132] According to a further embodiment of the present invention, the agent may be a nucleic acid molecule encoding a covercin-type protein. For example, gene therapy may be employed to induce endogenous production of covercin-type proteins by relevant cells in a subject, either in vivo or ex vivo. Another approach is the administration of "naked DNA", where a therapeutic gene is injected directly into the bloodstream or muscle tissue.

[0133] Preferably, such a nucleic acid molecule comprises or consists of bases 55 to 507 of the nucleotide sequence in Figure 2 (SEQ ID NO:1). This nucleotide sequence encodes the covercin protein in Figure 2 without a signal sequence. The first 54 bases of the nucleotide sequence in Figure 2 encode a signal sequence that is not required for complement inhibitory activity or LTB4 binding activity. Alternatively, the nucleic acid molecule may comprise or consist of bases 1 to 507 of the nucleic acid sequence in Figure 2, which encodes a protein with a signal sequence.

[0134] Mode of administration Conversin proteins do not require a medical professional to administer, and these molecules are rapidly absorbed. In contrast, many recombinant antibodies are absorbed very slowly or cannot be administered by subcutaneous injection or other administration routes, and as a result must be infused (e.g., intravenously) over a long period of time. Thus, administration of such molecules also requires a medical professional. Conversin proteins therefore have the advantage of being easier to administer than other drugs that require injection.

[0135] The agent is administered in a therapeutically or prophylactically effective amount. The term "therapeutically effective amount" refers to the amount of agent required to treat a rheumatic disease. In this context, "treating" includes reducing the severity of the disorder.

[0136] The term "prophylactically effective amount" as used herein refers to the amount of drug required to prevent the relevant condition, e.g., rheumatic disease. In this context, "preventing" includes reducing the severity of the disorder, e.g., where the presence of the disorder is not detected before administration of the drug begins. Reducing the severity of the disorder can be, for example, reducing the level of pain, inflammation, joint stiffness, or fever.

[0137] The reduction or improvement is relative to the results in the absence of administration and the agent described herein. The results are evaluated by standard criteria used to evaluate such patients. To the extent that this can be quantified, there is at least a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction or improvement in relative criteria (e.g., pain, inflammation, joint stiffness, or fever levels).

[0138] Preferably, the dose calculated based on the Coversin molecule is 0.1 mg / kg / day to 10 mg / kg / day (mass of drug compared to patient's mass), e.g., 0.2-5, 0.25-2, or 0.1-1 mg / kg / day. Fusion proteins (e.g., as discussed herein) are larger than the Coversin molecule, so equimolar amounts can be used for such proteins. Thus, for functional equivalents of Coversin, equimolar amounts of the doses mentioned above can be used. For example, for a fusion protein comprising Coversin and a PAS moiety of about 600 amino acids, or a PAS moiety as defined herein, e.g., PAS-Coversin) an equimolar amount of 0.1 mg / kg / day is 0.4 mg / kg / day, so the dose can be 0.4 mg / kg / day to 40 mg / kg / day (mass of drug compared to patient's mass), e.g., 0.8-20, 1-8, or 0.4-4 mg / kg / day. Alternatively, to provide a longer half-life of these fusion proteins, more amounts can be given per dose and doses can be administered less frequently, e.g., 40 mg to 2 g, 50 mg to 1.5 g, 75 mg to 1 g, for example, administered once or twice a week, over the course of a week.

[0139] A therapeutically or prophylactically effective amount can be further defined in terms of inhibition of terminal complement, e.g., an amount that means that terminal complement activity (TCA) is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% compared to terminal complement activity in the absence of treatment. Dosage and frequency can be adjusted to maintain terminal complement activity at a desired level, which can be, e.g., 10% or less, e.g., 9, 8, 7, 6, 5, 4, 3, 2, 1% or less compared to terminal complement activity in the absence of treatment.

[0140] A therapeutically or prophylactically effective amount can be further defined in terms of a reduction in plasma LTB4 levels, e.g., an amount meaning that the plasma LTB4 level is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% compared to the plasma LTB4 level in the absence of treatment, or an amount such that the LTB4 level is within a particular range of normal levels (e.g., 90-110% of normal, 85-115% of normal). Dosage and frequency can be adjusted to maintain plasma LTB4 levels at a desired level, which may be, for example, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less, e.g., 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, compared to plasma LTB4 levels in the absence of treatment, or within a certain range of normal levels (e.g., 90-110% of normal, 85-115% of normal). LTB4 levels can be determined by routine methods (e.g., immunoassays, see commercially available R&D Systems assays based on sequential competitive binding technology

[41] ).

[0141] When a dose is given, this is with respect to the dose of the agent that is a protein or its functional equivalent. These levels may occur using doses appropriate for agents that are nucleic acid molecules. The dose can be varied to effect the presence of non-active protein present (e.g., PAS-coversin, which has a PAS portion of 600 amino acids, has about four times the molecular weight of coversin, so an equimolar amount would be about four times the amount of coversin). Equimolar amounts of any dose provided for coversin can be used for any coversin functional equivalent thereof, including additional sequences. Equimolar amounts can be calculated using routine methods.

[0142] Terminal complement activity can be measured by standard assays known in the art, for example, Quidel CH 50 It can be measured using the hemolysis assay and the sheep red blood cell lysis CH50 assay.

[0143] The frequency with which a dose needs to be administered depends on the half-life of the agent involved. Coversen protein or its functional equivalent may be administered, for example, twice a day, daily, or every 2, 3, 4, 5, 6, or 7 days or more, e.g., twice a day, daily. Extended half-life versions, e.g., PAS-ylated Coversen molecules, can be administered less frequently (e.g., every 2, 3, 4 days, every 5, 6, 7, 10, 15, or 20 days or more, e.g., once a day or every 2 days or more, or weekly).

[0144] The exact dosage and frequency of the dose may also depend on the condition of the patient at the time of administration.Factors that may be considered when determining the dosage include the need for treatment or prevention, the severity of the disease in the patient, the patient's overall health condition, age, body weight, sex, diet, time and frequency of administration, drug combination, reaction sensitivity and the patient's resistance or response to treatment.The exact amount may be determined by routine experimentation, but may ultimately be left to the discretion of the physician.

[0145] The dosing regimen was also initially (ablating regimen) " followed by one or more subsequent doses (e.g., maintenance doses). Generally, the resolution regimen will be higher than the subsequent doses. As an example for Covarcin, this may be a resolution regimen of 0.6-1.2 mg / kg, followed 8-18, 10-14, or 11-13 hours (e.g., about 12 hours) later by 0.3-0.6 mg / kg, followed by a maintenance dose of 0.45-0.9 mg / kg, which may be administered, for example, once daily.

[0146] For PASified versions (e.g. PAS-Covarcin, e.g. as described elsewhere herein), a suitable regimen may be a resolution regimen of 6-12 mg / kg (e.g. 600 mg), followed by 6-12 mg / kg (e.g. 600 mg) for 3-10, 4-8, 5-7, e.g. about 7 days later, followed by a maintenance dose of 4-8 mg / kg (e.g. 400 mg), which may be administered, for example, once daily.

[0147] One or more dissipative doses (ablating dose) may be at least 1.5, 2, or 5 times the maintenance dose. The dissipation dose may be administered as a single dose or as one or more doses (e.g., two doses) within a particular time frame. Typically, the loading dose will be 1, 2, 3, 4, or 5 doses administered during a single 24-hour period (or one week for extended half-life versions). The maintenance dose may be a lower dose repeated at regular intervals. The maintenance dose may be repeated at intervals such as every 12, 24, or 48 hours (or every week, or every two weeks for extended half-life versions). The exact regimen may be determined by routine experimentation, but may ultimately be left to the discretion of the clinician. The maintenance dose can be at least 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the initial dissipated dose, or up to 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the initial dissipated dose.

[0148] In further embodiments, the same dose is used over the course of treatment (eg, daily or twice daily or weekly).

[0149] Agents are generally administered in combination with or in a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is generally a liquid, but may include other agents, provided that the carrier itself does not induce toxic effects or cause the production of antibodies harmful to the individual receiving the pharmaceutical composition. Pharmaceutically acceptable carriers may contain, for example, liquids such as water, saline, glycerol, ethanol, or auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like. Thus, the pharmaceutical carrier employed will vary depending on the route of administration. A detailed discussion of pharma- ceutically acceptable carriers is available in

[42] . In a preferred embodiment, the agent is administered in a liquid, e.g., a solution of water or PBS.

[0150] Drugs may optionally be delivered using colloidal delivery systems (e.g., liposomes, nanoparticles, or microparticles (e.g., as discussed in

[43] ). Advantages of these carrier systems include protection of sensitive proteins, sustained release, reduced dosing frequency, patient compliance, and controlled plasma levels.

[0151] Liposomes (e.g., comprising phospholipids of synthetic and / or natural origin) may be, for example, 20 nm to 100 or 200 micrometers, e.g., small unilamellar vesicles (25-50 nm), large unilamellar vesicles (100-200 nm), giant unilamellar vesicles (1-2 μm) or multilamellar vesicles (MLV; 1 μm-2 μm).

[0152] Nanoparticles (colloidal carriers with sizes between 10 and 1000 nm) can be manufactured from lipids, polymers, or metals. Polymeric nanoparticles can be made from natural or synthetic polymers (e.g., chitosan, alginate, PCL, polylactic acid (PLA), poly(glycolide), PLGA) and may be produced as nanospheres (where molecules are uniformly distributed within a polymer matrix) or nanocapsules (carrying drug molecules entrapped within a polymeric membrane).

[0153] Microparticles, such as those made from starch, alginate, collagen, poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL), can also be used.

[0154] A hydrogel may alternatively or additionally be present.

[0155] For larger molecular weight molecules, such as fusion proteins, additional excipients such as hyaluronidase can also be used, for example to allow for the administration of larger volumes (e.g., 2-20 ml).

[0156] The drug is preferably delivered by subcutaneous injection or injection into synovial fluid.Subcutaneous injection is preferred from the viewpoint of subject's ease of administration.In some embodiments, this is via subcutaneous injection once or twice a day.

[0157] Preferably, the course of treatment lasts for at least 1, 2, 3, 4, 5 or 6 weeks, or at least 1, 2, 3, 4, 5 or 6 months, or at least 1, 2, 3, 4, 5 or 6 years. The course of treatment preferably lasts at least until the subject's symptoms are alleviated. Thus, the course of treatment can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 weeks of administration of the agent (e.g., daily, every other day or weekly).

[0158] The maintenance dose (e.g., a single daily or weekly maintenance dose) may remain constant throughout the course of treatment) or the maintenance dose (e.g., a daily maintenance dose) may be modified (e.g., increased or decreased) during the course of treatment. The maintenance dose may be modified to maintain terminal complement activity and plasma LTB4 levels at a desired level, e.g., 10% or less terminal complement activity compared to serum from said patient in the absence of treatment or compared to normal control serum, and / or 90% or less plasma LTB4 levels compared to plasma from said patient in the absence of treatment, or to achieve plasma LTB4 levels within a certain range of normal levels (e.g., 90-110% of normal, 85-115% of normal). Alternatively, each maintenance dose may be continued daily for 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, 10 weeks. As the subject's symptoms improve, the maintenance dose may be reduced. As the subject's symptoms improve, the amount of the drug or the frequency of administration of the drug can be reduced.

[0159] Thus, there may be an initial dissipation dose or regimen, followed by an initial maintenance dose (e.g., an initial daily or weekly maintenance dose), which may be a maintenance dose as defined above, and one or more further maintenance doses (e.g., a further daily or weekly maintenance dose), for example at least 2, 3, 4, 5 further maintenance doses.

[0160] Thus, the present invention further includes a method of treating or preventing a rheumatic disease in a subject comprising administering to the subject an initial resolution dose or regimen of an agent defined above, followed by administration of a maintenance dose (e.g. a daily or weekly maintenance dose) of an agent defined above, wherein there is the initial maintenance dose and one or more further maintenance doses.

[0161] Thus, the present invention relates to an agent as defined above for use in a method for treating or preventing a rheumatic disease in a subject, the method further comprising administering to a subject an initial resolution dose or regimen of an agent as defined above, followed by administration of a maintenance dose (e.g. a daily or weekly maintenance dose) of an agent as defined above, wherein there is an initial maintenance dose and one or more further maintenance doses.

[0162] One or more further maintenance doses can be determined by testing the terminal complement activity or plasma LTB4 level in the subject (e.g., a biological sample from the subject), determining the further maintenance dose based on the level of terminal complement activity and / or plasma LTB4 level, and / or testing the subject for symptoms, and determining the further maintenance dose based on symptoms.The method can optionally further comprise administering the further maintenance dose.The further dose can be calculated to be a level that maintains terminal complement activity at a desired level.

[0163] When a biological sample is taken, it can be blood, for example, whole blood, plasma or serum sample. Optionally, the method further comprises taking a sample, and further optionally comprises determining the TCA of the sample and / or determining the plasma LTB4 level.

[0164] One or more further maintenance doses can be determined by testing the terminal complement activity and / or plasma LTB4 level in a subject (e.g., a biological sample), determining the further maintenance dose based on the level of terminal complement activity and / or plasma LTB4 level, and / or testing the subject for symptoms, and determining the further maintenance dose based on symptoms. The method can optionally further include administering the further maintenance dose. The further dose can be calculated to be a level that maintains the terminal complement activity and / or plasma LTB4 level at a desired level.

[0165] In certain embodiments, the desired complement activity level is 10% or less compared to serum from the subject in the absence of treatment or compared to normal control serum, and / or the plasma LTB4 level is 90% or less compared to serum from the subject in the absence of treatment, and / or the plasma LTB4 level is within a particular range of normal levels (e.g., 90-110% of normal, 85-115% of normal).

[0166] In certain embodiments, the maintenance dose is increased if TCA and / or plasma LTB4 are higher than desired levels, and optionally, the dose is maintained or decreased if TCA is less than 5, 4, 3, 2, 1% and / or LTB4 plasma levels are 90% or less compared to plasma from said patient in the absence of treatment (or if plasma LTB4 levels are within a certain range of normal levels (e.g., 90-110% of normal, 85-115% of normal)).

[0167] In certain embodiments, if symptoms worsen, the maintenance dose is increased, and optionally, if symptoms improve, the dose is maintained or decreased.

[0168] In some embodiments, the subject is examined within one month of initiating treatment, within two weeks of initiating treatment, within one week of initiating treatment, in other embodiments, the subject is examined once a day or at least once a day, once a week or at least once a week, once every two weeks or at least once every two weeks, once a month or once every two months.

[0169] The dosing regimen may also take the form of a fixed dose that is independent of the body weight of the subject being treated. The fixed dose may be administered as a single dose or as one or more doses within a particular time frame. The fixed dose may be 1 mg to 100 mg of coversin (e.g., SEQ ID NO: 4) for a typical human patient (e.g., a patient weighing between 50 kg and 100 kg). The molecular weight of the coversin type protein may be used to calculate the equivalent fixed dose of the functionally equivalent drug. In some embodiments, the fixed dose is between 1 mg to 80 mg, 1 mg to 50 mg, 5 mg to 80 mg, 5 mg to 50 mg, 10 mg to 60 mg, 10 mg to 50 mg, 20 mg to 50 mg, 20 mg to 40 mg, or 25 mg to 35 mg of coversin (e.g., SEQ ID NO: 4) or coversin type protein in molar equivalent. Preferably, the fixed dose is 30 mg, or 45 mg of coversin (SEQ ID NO: 4) or coversin type protein in molar equivalent. Typically, a fixed dose is 1, 2, 3, 4 or 5 doses administered during a single 24-hour period. Fixed doses may be repeated at intervals such as every 3, 4, 6, 8, 12, 24, or 48 hours. The exact regimen can be determined by routine experimentation, but may ultimately be left to the discretion of the physician. [Brief description of the drawings]

[0170] [Figure 1] FIG. 1 shows a schematic diagram of the classical and alternative pathways of complement activation, with anaphylatoxins 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 numbers are shown to the right. The nucleotide sequence is SEQ ID NO:1 and the amino acid sequence is SEQ ID NO:2. [Figure 2B] FIG. 1 shows examples of coversen variants. [Diagram 3] FIG. 1 shows experimental RA clinical scores and paw width in vehicle versus mice treated with prophylactic Covarcin, PAS-Covarcin or Zileuton. [Figure 4]FIG. 1 shows that mice treated with PAS-coversin show improved disease in experimental RA clinical scores in PAS-coversin vs. vehicle treated mice compared to vehicle control, zileuton, and PAS-L-coversin which does not bind C5. [Diagram 5] FIG. 2 shows the PAS-conversin sequence. Example 1

[0171] Effect of Covarcin (prophylactic) in the K / BxN serum transfer model We used the K / BxN serum transfer arthritis model to test whether Covarcin could prevent the development of arthritis. This model is a well-known mouse model in which immunological mechanisms occurring in rheumatoid arthritis (RA) are induced by transferring serum from arthritic transgenic K / BxN mice to naive mice. In the absence of treatment, arthritic symptoms develop after a few days. This model is recognized to be highly relevant in RA, especially for preclinical screening of new therapeutic targets for RA and possibly other forms of inflammatory arthritis

[44] .

[0172] Experimental RA was induced using the protocol described in

[45] . Five mice were studied in each treatment group.

[0173] On day 0, clinical scores and paw thickness of C57BL / 6 mice were assessed using standard protocols

[45] . All mice received injections of K / BxN serum on days 0 and 2. Each treatment was administered daily from day 0 to day 14. On days when serum was also injected (days 0 and 2), drugs were administered immediately after serum injection. Clinical scores and paw thickness were measured every other day.

[0174] The treatment groups were as follows: Group 1: PBS (vehicle, subcutaneous, q24h) Group 2: Treatment (Covarcin 5 mg / kg subcutaneously, q12h) Group 3: Treatment (zileuton 50 mg / kg orally once daily) Group 4: Treatment (PAS-Covarcin 20 mg / kg subcutaneously, once daily)

[0175] The results of experiment 1 are shown in Figure 3. It can be seen that covercin (q12h) and PAS-covercin (q24h) completely suppress the development of arthritis in this model. Zileuton (5-LOX inhibitor) partially ameliorates the development of arthritis in this model.

[0176] Covarcin and PAS-Covarcin reduced the development of arthritis in this model to a greater extent than N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea (zileuton), an oral inhibitor of 5-lipoxygenase (5-LOX), a key enzyme in the arachidonic acid cascade involved in the formation of bioactive leukotrienes (LTs).

[0177] The difference between PAS-coversin and coversin is that PAS-coversin is "PASylated", which is described as a genetic fusion of a protein with a structurally disordered polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser. This technology was developed by XL Protein (http: / / xl-protein.com / ) and provides a simple method to attach solvated random chains with a large hydrodynamic volume to the protein to which it is fused. The polypeptide sequence adopts a random coil conformation, which greatly increases the apparent molecular weight and decreases the rate of clearance by renal filtration. The sequence of PAS-coversin used in this experiment is shown in Figure 5 and SEQ ID NO:22.

[0178] Due to the large molecular weight of PAS-Cov, 20mg / kg of PAS-Cov is equivalent to 5mg / kg of Covarcin, so in this experiment, an equal dose of active Covarcin agent is administered daily, but Covarcin is administered twice as frequently (every 12 hours). In this experiment, PAS-Covarcin was as effective as Covarcin, but administered less frequently. Therefore, there may be advantages to using a longer half-life version of Covarcin therapeutically, such as PAS-Covarcin. Example 2

[0179] Effect of Covarcin on K / BxN serum transfer model (treatment) The K / BxN serum transfer arthritis (STA) model was used to test whether Covarcin could ameliorate established arthritic disease.

[0180] Experimental RA was induced using the protocol described in

[45] . Five mice were studied in each treatment group.

[0181] On day 0, clinical scores and paw thickness of C57BL / 6 mice were assessed using standard protocols

[45] . All mice received injections of K / BxN serum on days 0 and 2. Treatment was administered daily from day 4 to day 14. On days when serum was also injected (days 0 and 2), drugs were administered immediately after serum injection. Clinical scores and paw thickness were measured every other day.

[0182] The treatment groups were as follows: Group 1: PBS (vehicle) Group 2: Treatment (PAS-L Covarcin 20 mg / kg subcutaneously once daily) Group 3: Treatment (zileuton 50 mg / kg orally once daily) Group 4: Treatment (PAS-Covarcin 20 mg / kg subcutaneously once daily)

[0183] The clinical score results from the second experiment are shown in Figure 4. It can be seen that the mice developed arthritis after serum injection. In the vehicle and zileuton groups, the maximum clinical scores were seen on the sixth day of the experiment. After administration of PAS-L-Covarcin (q24h) and PAS-Covarcin (q24h), the clinical scores of the PAS-Covarcin and PAS-L-Covarcin groups decreased. For the PAS-L-Covarcin group, the clinical scores were significantly lower than the vehicle and zileuton treatment groups.

[0184] The difference between PAS-coversin and PAS-L-coversin is that the L-coversin molecule has a mutation in the coversin sequence that allows it to bind to LTB4 but not to C5 (referred to as "L-coversin"). The sequence of the L-convertin sequence is a variant of the mature convertin sequence (SEQ ID NO: 4) in which the following residues are modified: Ala44 to Asn, Met116 to Gln, Leu117 to Ser, Gly121 to Ala, Leu122 to Asp, Glu123 to Ala, and Asp149 to Gly (referred to as variant 2, the sequence is dsesdctgse pvdafqafse gkeayvlvrs tdpkardclk gepNgekqdn tlpvmmtfkn gtdwastdwt ftldgakvta tlgnltqnre vvydsqshhc hvdkvekevp dyemwQSdag ADAveveccr qkleelasgr nqmyphlkGc (SEQ ID NO: 23), where changes compared to the native convertin sequence of SEQ ID NO: 4 are shown in uppercase).

[0185] PAS-L-coversin was not as effective as PAS-coversin in improving RA, but was more effective than zileuton. This suggests that the dual inhibitory activity of coversin (C5 and LTB4 inhibition) provides improved therapeutic efficacy in this model. It was expected that simultaneous inhibition of C5 activation and LTB4 would only be as effective as LTB4 inhibition alone in the therapeutic model. However, unexpectedly, simultaneous inhibition of C5 and LTB4 by coversin was found to be much more effective than LTB4 inhibition alone in the therapeutic model, as shown by the improved efficacy of coversin compared to L-coversin.

[0186] References [1] Hoover et al, 1984, Proc. Nat. Acad. Sci. USA 81, 2191-2193 [2] Harrison and Murphy, 1995, J. Biol. Chem. 270, 17273-17276 [3] Ford-Hutchinson, 1990, Crit. Rev. Immunol. 10, 1-12 [4] Showell et al., 1995, J. Pharm. Exp. Ther. 273, 176-184 [5] Klaas et al, 2005 J. Exp. Med. 201, 1281-1292 [6] Del Prete et al, 2007 Blood, 109, 626-631 [7] Miyahara et al, 2006 A llergol Int. 55, 91-7 [8] Taube et al, 2006 J. Immunol. 176, 3157-3164 [9] Yamaoka et al, 1989 J. Immunol. 143, 1996-2000

[10] Yokomizo et al, 1997 Nature 387, 620-624

[11] Yokomizo et al, 2000 J. Exp. Med. 192, 421-432

[12] Tager and Luster, 2003 Prostaglandins Leukot. Essent. Fatty Acids 69, 123-134

[13] Yokomizo et al., 2001, J. Biol. Chem. 276, 12454-12459

[14] Kim, N. D. and Luster, A.D. (2007) The Scientific World Journal 7, 1307-1328.

[15] Kim et al., 2006. J. Exp. Med. 203, 829-835

[16] Noiri et al., 2000 Proc Nat Acad Sci USA 97, 823-828

[17] Lundeen et al., 2006 . J. Immunol. 177, 3439-3447

[18] Shao et al, 2006 . J. Immunol. 176, 6254-6261

[19] Chen et al., 2006 . J. Exp. Med. 203, 837-842

[20] Sebaldt et al., 1990 Proc Natl Acad Sd. U.S.A. 8, 6974-6978

[21] Curry et al., 2005 Journal of the American Animal Hospital Association 41 , 298- 309

[22] Dube et al., 1998. Zileuton: the first leukotriene inhibitor for use in the management of chronic asthma. In: Drazen JM, Dahlen S, Lee TH, eds. Five-lipoxygenase Products in Asthma. New York, NY: Marcel Dekkar, Inc

[23] Sharma and Mohammed, 2006 Immunopharmacology 14, 10–16

[24] GBD 2015 Disease and Injury Incidence and Prevalence, Collaborators. The Lancet. 388(10053):1545–1602. (2016)

[25] GBD 2013 Mortality and Causes of Death, Collaborators (17 December 2014). The Lancet. 385(9963): 117–71.

[26] WO2004 / 106369

[27] Jore, MM et al, Nature Structural & Molecular Biology 2016 volume 23, pages 378-386

[28] Pincus et al Rheumatology 2008;47:345–349

[29] Oliver J et al Arthritis Res Ther. 2009; 11(3):

[30] Roversi, P et al Journal of Biological Chemistry 2013, 288(26) 18789-18802

[31] Guo, RF and PA Ward, Annu Rev Immunol, 2005, 23: p. 821-52

[32] Ricklin D & Lambris J, Nature Biotechnology, 25: 1265-1275 (2007)

[33] Nishimura, J et al., New Engl J. Med., 30;7:632-639 (2014)

[34] Breustedt DA, Schonfeld DL, Skerra A. (2006) Comparative ligand-binding analysis of ten human lipocalins. Biochim Biophys Acta 1764(2):161-173.

[35] Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003

[36] Schlapschy M, et al Protein Eng Des Sel. 2013 Aug;26(8):489-501

[37] Kuhn et al Bioconjugate Chem., 2016, 27 (10), pp 2359-2371

[38] Sambrook et al (2000)

[39] Fernandez & Hoeffler (1998)

[40] Ausubel et al. (1991)

[41] https: / / resources.rndsystems.com / pdfs / datasheets / kge006b.pdf

[42] Remington's Pharmaceutical Sciences; Mack Pub. Co., NJ 1991

[43] Patel et al Ther. Delivery. (2014) 5(3), 337-365

[44] Christensen, A. et al. Front Immunol. 2016; 7:213. <h2 style=";text-align:left;direction:ltr">

[45] Kim et al JEM 2006; 203(4) 829-835

Claims

1. A medicament for treating or preventing rheumatoid arthritis (RA) in a subject, the medicament comprising a fusion protein or a nucleic acid molecule encoding the fusion protein, The fusion protein comprises: a) (i) a protein comprising a sequence having at least 90% sequence identity with amino acids 19 to 168 of SEQ ID NO:2, or (ii) a fragment of a protein having an amino acid sequence that has at least 90% sequence identity with the sequence of amino acids 19 to 168 of SEQ ID NO:2; wherein the protein or fragment binds C5 to prevent cleavage of complement C5 by C5 convertase into complement C5a and complement C5b, and binds LTB4; and b) PAS sequence Including, The fusion protein binds to C5 to prevent cleavage of complement C5 by C5 convertase into complement C5a and complement C5b, and binds to LTB4.

2. The drug according to claim 1, wherein the RA is RF and / or anti-CCP positive RA.

3. The method of claim 2, wherein the RA is accompanied by vasculitis.

4. The agent according to any one of claims 1 to 3, wherein the fusion protein comprises a protein comprising a sequence having at least 95% sequence identity with the sequence of amino acids 19 to 168 of SEQ ID NO:

2.

5. The method according to any one of claims 1 to 4, wherein the fusion protein comprises a protein comprising or consisting of the sequence of amino acids 19 to 168 of SEQ ID NO:

2.

6. The fusion protein comprises a fragment of a protein, The protein has an amino acid sequence that is at least 90%, or at least 95%, identical to the sequence of amino acids 19 to 168 of SEQ ID NO:2; or The protein comprises or consists of the sequence of amino acids 19 to 168 of SEQ ID NO:2, 2. The agent of claim 1, wherein the fragment binds to C5 to prevent cleavage of complement C5 by C5 convertase into complement C5a and complement C5b, and binds to LTB4.

7. The method according to any one of claims 1 to 6, wherein the agent is administered subcutaneously or intrasynovially.

8. 8. The method of claim 1, wherein the agent is administered to the subject as an initial ablating regimen of the agent, followed by a maintenance dose of the agent, optionally with an initial maintenance dose and one or more further maintenance doses.

9. The medicament of any one of claims 1 to 8, wherein the medicament is administered together with a second rheumatic disease treatment.

10. 10. The method of claim 9, wherein the second rheumatic disease treatment is selected from a disease-modifying antirheumatic drug (DMARD), an anti-inflammatory agent, and an analgesic agent.

11. (a) the DMARD is selected from cyclosporine, cyclophosphamide, hydroxychloroquine, gold salts, methotrexate, leflunomide, mycophenolate, sulfasalazine, etanercept, certolizumab pegol, golimumab, infliximab, and adalimumab, anakinra, rituximab, and abatacept, optionally selected from etanercept, certolizumab pegol, golimumab, infliximab, and adalimumab, anakinra, rituximab, and abatacept; (b) the anti-inflammatory agent is an NSAID or a glucocorticoid, and / or 11. The method of claim 10, wherein (c) the analgesic is selected from paracetamol, combined analgesics, and opioid analgesics.

12. 12. The agent of any one of claims 1 to 11, wherein the fusion protein comprises multiple copies of one of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 15); AAPASPAPAPSAPAAPS (SEQ ID NO: 16); APSSPSPSSAPSSPSPAASPSS (SEQ ID NO: 17), SAPSSPSPSSAPSSPSPASPS (SEQ ID NO: 18), SSPSAPSPSPSPSPSPSP (SEQ ID NO: 19), AASPAAPSAPPAAASAPAAPSAPPA (SEQ ID NO: 20) and ASAAAPAAAASAAAAASAPSAAA (SEQ ID NO: 21), optionally 20 to 30 or 30 copies of one of SEQ ID NOs: 15 to 21.

13. The agent according to any one of claims 1 to 12, wherein the fusion protein comprises (a) a PAS sequence consisting of 30 copies of SEQ ID NO: 15 and (b) amino acids 19 to 168 of SEQ ID NO: 2, wherein (a) is fused to the N-terminus of (b).

14. The agent according to any one of claims 1 to 13, wherein the fusion protein comprises the sequence of SEQ ID NO:22.

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