C3b binding polypeptides

Abstract

The present invention relates to isolated nucleic acids encoding polypeptides capable of binding to C3b, vectors and pharmaceutical compositions. The nucleic acid comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35.

Description

[0001] This application is a divisional application of an application with an application date of January 15, 2019, application number “201980016721.2” (international application number “PCT / EP2019 / 050949”), and invention name “C3b binding polypeptide”. Technical Field

[0002] The present invention relates to the fields of molecular biology, immunology and medicine. More specifically, the present invention relates to a polypeptide that binds to C3b. Background Art

[0003] In developed countries, age-related macular degeneration (AMD) is the main cause of blindness. AMD currently accounts for 8.7% of all global blind registrations, and it is estimated that 196 million people will be affected by 2020 (Wong et al. Lancet Glob Heal (2014) 2: e106-16). AMD manifests as progressive destruction of the macula in the central part of the retina at the back of the eye, resulting in loss of central visual acuity. Early detection of the disease shows morphological changes in the macula, first including the loss of blood vessels in the choriocapillaris (Whitmore et al., Prog Retin Eye Res (2015) 45: 1-29), which is a reticular (fenestrated) blood vessel present in the choroid (a highly vascularized layer that supplies oxygen and nutrition to the outer retina).

[0004] AMD is primarily a genetic disease. Genetic mutations in the complement system, i.e., part of our immune system, are highly associated with an increased risk of AMD. Indeed, it has become clear that overactivation of complement is a major driver of disease pathogenesis, and many examples of overactivation of complement can be seen in the choriocapillaris layer. For example, Zipfel et al., in Lambris and Adamis (eds.), Inflammation and retinal diseases: Complement biology and pathology, Experimental Medicine and Biology Advances 703, Springer Science + Business Media LLC (2010), reviewed the role of complement in AMD in Chapter 2, the entire contents of which are incorporated herein by reference. Complement is activated by deposition on the surface of protein C3b, a pro-inflammatory breakdown product of an immune system protein C3. C3b associates with other proteins to form a convertase complex that activates and amplifies the complement response and initiates the amplification loop of the complement cascade, ultimately leading to cell / tissue destruction and local inflammatory responses, all hallmarks of AMD.

[0005] The choriocapillaris layer is separated from the metabolically active retinal pigment epithelium (RPE) by a thin (2-4 μm), non-cellular, five-layer extracellular matrix Bruch's membrane (BrM). The BrM has two main functions: the substratum of the RPE and the vascular wall. For example, the structure and function of the BrM are reviewed in Curcio and Johnson, Structure, Function and Pathology of Bruch's Membrane, In: Ryan et al. (2013), Retina, Vol. 1, Part 2: Basic Science and Translation to Therapy. 5th ed. London: Elsevier, pp466-481, the entire contents of which are incorporated herein by reference.

[0006] C3b activation of complement on non-cellular structures such as BrM and intercapillary septa (extracellular matrix filling the space between capillaries in the choriocapillaris layer) is regulated by the proteins "complement factor H" (FH) and "complement factor I" (FI). FI prevents complement activation by cleaving C3b into a proteolytically-inactive form, specifically iC3b, which cannot participate in convertase assembly. However, iC3b is an opsonin and is therefore a mediator of leukocyte recruitment with subsequent inflammatory responses, while further breakdown products of C3b, iC3dg and C3d, are weak opsonins. In order to cleave C3b, FI requires the presence of cofactors, examples of which include the blood-borne FH protein and the membrane-bound surface cofactor "complement factor 1" (CR1; CD35). CR1 is a membrane receptor expressed on a wide range of cells and is involved in immune complex clearance, phagocytosis and complement regulation. In addition to acting as a cofactor in the FI-mediated cleavage of C3b, CR1 also acts as a regulator of complement by accelerating the C3 and C5 convertases. The structure and function of CR1 are reviewed, for example, in Khera and Das, Mol Immunol (2009) 46(5):761-772 and Jacquet et al., J Immunol (2013) 190(7):3721-3731, the entire contents of which are incorporated herein by reference.

[0007] The hallmark lesions of early AMD, called drusen, develop in the BrM adjacent to the RPE layer (Bird et al, Surv Ophthalmol (1995) 39 (5): 367-374). Drusen are formed from the accumulation of lipids and cell debris, and include a series of complement activation products (Anderson et al., Prog Retin Eye Res (2009) 29: 95–112; Whitcupet al., Int J Inflam (2013) 1-10). The presence of drusen in BrM destroys the flow of nutrients from the choroid through the extracellular matrix to the RPE cells, which causes cell dysfunction and eventually death. Because the RPE cell monolayer supports the rods and cones of the retinal neurosensory layer by providing nutrients and removing waste, their cell death causes the dysfunction of photoreceptor cells (photoreceptor cell) and subsequently causes the loss of visual acuity.

[0008] This represents one of the late stages of AMD, known as "dry" AMD, and also known as geographic atrophy, which represents about 90% of AMD cases. In the remaining proportion of late AMD cases, the presence of drusen promotes choroidal neovascularization (CNV), in which the synthesis of vascular endothelial growth factor (VEGF) increased by RPE cells promotes the growth of new blood vessels from the choroid / choriocapillaris layer, which damages the growth and enters the retina through BRM. These new blood vessels leak and eventually form scar tissue; this is called "wet" AMD.

[0009] "Wet" AMD, which represents only 10% of cases, is the most lethal form of late-stage AMD and has a different disease profile than "dry" AMD. There are treatments for wet AMD, where, for example, injection of anti-VEGF agents into the vitreous of the eye can slow or reverse the growth of these blood vessels, although it cannot prevent the formation of these blood vessels in the first place. Geographic atrophy ("dry" AMD) remains untreatable. Summary of the invention

[0010] The present invention provides a polypeptide that binds to C3b, which can be used to treat or prevent complement-related diseases or disorders.

[0011] In one aspect, the present invention provides a polypeptide capable of binding to C3b, comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and wherein the polypeptide has a total length of less than 450 amino acids, and the polypeptide is used in a method for treating or preventing a complement-related disease or disorder.

[0012] Also provided is a nucleic acid encoding a polypeptide capable of binding to C3b, the polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and wherein the polypeptide has a total length of less than 450 amino acids, the polypeptide being used in a method for treating or preventing a complement-related disease or disorder.

[0013] In another aspect, the present invention provides a use of a polypeptide capable of binding to C3b in the manufacture of a medicament for treating or preventing a complement-related disease or disorder, wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 4, and wherein the polypeptide has a total length of less than 450 amino acids.

[0014] Also provided is a use of a nucleic acid in the manufacture of a medicament for treating or preventing a complement-related disease or disorder, wherein the nucleic acid encodes a polypeptide capable of binding to C3b, the polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and wherein the polypeptide has a total length of 450 or less amino acids.

[0015] In another aspect, a method of treating or preventing a complement-related disease or disorder is provided, the method comprising administering to a subject a polypeptide capable of binding to C3b, the polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and wherein the polypeptide has a total length of 450 or less amino acids.

[0016] In yet another aspect, a method of treating or preventing a complement-associated disease or disorder in a subject is provided, the method comprising modifying at least one cell of the subject to express or include a polypeptide capable of binding to C3b, the polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and wherein the polypeptide has a total length of 450 amino acids or less.

[0017] In some embodiments, the complement-associated disease or disorder is an ocular disease or disorder.

[0018] In some embodiments, the treatment or prevention of an ocular disease or condition includes modifying at least one ocular cell of a subject to express or include the polypeptide. In some embodiments, the treatment or prevention of an ocular disease or condition includes modifying at least one ocular cell of a subject to express or include a nucleic acid encoding the polypeptide. In some embodiments, the treatment or prevention of an ocular disease or condition includes administering a vector to at least one ocular cell of a subject, the vector including a nucleic acid encoding the polypeptide. In some embodiments, the at least one ocular cell is a retinal pigment epithelium (RPE) cell.

[0019] In some embodiments, the disease or disorder is one in which C3b or a C3b-containing complex, an activity / response associated with C3b or a C3b-containing complex, or a product of an activity / response associated with C3b or a C3b-containing complex is pathologically implicated.

[0020] In some embodiments, the disease or condition is macular degeneration. In some embodiments, the disease or condition is selected from one or more of: age-related macular degeneration (AMD), early AMD, mid-term AMD, late AMD, geographic atrophy ("dry" AMD), "wet" (neovascular) AMD, choroidal neovascularization (CNV), glaucoma, autoimmune uveitis, diabetic retinopathy, and early-onset macular degeneration (EOMD).

[0021] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, X of SEQ ID NO: 4 1 A or T, X 2 P or L, and / or X 3 It is G or R.

[0022] In some embodiments, the polypeptide has a total length of 50-250 amino acids.In some embodiments, the polypeptide comprises or consists of SEQ ID NO:2 or SEQ ID NO:3.

[0023] In some embodiments, the polypeptide comprises or consists of SEQ ID NO:13.

[0024] In some embodiments, the polypeptide is capable of acting as a co-factor of complement factor I. In some embodiments, the polypeptide is capable of diffusing across Bruch's membrane (BrM). In some embodiments, the polypeptide binds to C3b in a region bound by a co-factor of complement factor I. In some embodiments, the polypeptide binds to C3b in a region bound by complement receptor 1 (CR1).

[0025] In some embodiments, the polypeptide comprises a secretory pathway sequence. In some embodiments, the secretory pathway sequence comprises or consists of SEQ ID NO: 7. In some embodiments, the polypeptide comprises or consists of SEQ ID NO: 47, 49, or 51. In some embodiments, the polypeptide comprises a cleavage site for removal of the secretory pathway sequence.

[0026] In one aspect, the invention provides a polypeptide having at least 80% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a length of less than 700 amino acids.

[0027] In some embodiments, the polypeptide has a length of 50-700 amino acids. In some embodiments, the polypeptide has at least 80% sequence identity to SEQ ID NO: 4, wherein X 1 A or T, X 2 P or L, and / or X 3It is G or R.

[0028] Also provided is a polypeptide capable of binding to C3b, the polypeptide comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 4, and wherein the polypeptide has a total length of 450 amino acids or less.

[0029] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. 1 A or T, X 2 P or L, and / or X 3 It is G or R.

[0030] In some embodiments, the polypeptide has a total length of 50-250 amino acids.

[0031] In some embodiments, the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 13.

[0032] In some embodiments, the polypeptide is capable of binding to C3b. In some embodiments, the polypeptide binds to C3b in a region bound by a cofactor of complement factor I. In some embodiments, the polypeptide binds to C3b in a region bound by complement receptor 1 (CR1). In some embodiments, the polypeptide acts as a cofactor of complement factor I.

[0033] In some embodiments, the polypeptide is capable of diffusing across the Bruch's membrane (BrM). In some embodiments, the polypeptide is non-glycosylated or partially glycosylated. In some embodiments, the polypeptide comprises at least one amino acid substitution, for example, one, two, three or four substitutions at positions 509, 578, 959 and / or 1028 (numbered according to the Universal Protein Resource Database (UniProt): P17927 (SEQ ID NO: 1)). In some embodiments, at least one amino acid substitution is one or more of N509Q, N578Q, N959Q and / or N1028Q (numbered according to UniProt: P17927 (SEQ ID NO: 1)). In some embodiments, the polypeptide comprises SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 15, or consists of SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 15.

[0034] In some embodiments, the polypeptide additionally comprises a secretory pathway sequence. In some embodiments, the secretory pathway sequence comprises or consists of SEQ ID NO: 7. In some embodiments, the polypeptide additionally comprises a cleavage site for removal of the secretory pathway sequence. In some embodiments, the polypeptide comprises or consists of SEQ ID NO: 47, 49, or 51.

[0035] In another aspect, the present invention provides a nucleic acid encoding a polypeptide according to the present invention.

[0036] In yet another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.

[0037] In yet another aspect, the present invention provides a cell comprising the polypeptide, nucleic acid or vector according to the present invention.

[0038] In yet another aspect, the present invention provides a method for producing a polypeptide, the method comprising introducing a nucleic acid or a vector according to the present invention into a cell, and culturing the cell under conditions suitable for the expression of the polypeptide.

[0039] In a further aspect, the present invention provides a cell obtained or obtainable by the method for producing a polypeptide according to the present invention.

[0040] In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide, nucleic acid, vector or cell according to the present invention. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0041] In a further aspect, the present invention provides a polypeptide, a nucleic acid, a vector, a cell or a pharmaceutical composition according to the present invention for use in a method of treating or preventing a disease or disorder.

[0042] In yet another aspect, the present invention provides a use of a polypeptide, nucleic acid, vector, cell or pharmaceutical composition according to the present invention in the manufacture of a medicament for treating or preventing a disease or disorder.

[0043] In yet another aspect, the present invention provides a method for treating or preventing a disease or disorder, the method comprising administering to a subject a polypeptide, a nucleic acid, a vector, a cell or a pharmaceutical composition according to the present invention.

[0044] In yet another aspect, the invention provides a method of treating or preventing a disease or disorder in a subject, the method comprising modifying at least one cell of the subject to express or include a nucleic acid, vector or polypeptide according to the invention.

[0045] In some embodiments according to various aspects of the present invention, the disease or condition is a disease or condition wherein pathologically involves C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b. In some embodiments, the disease or condition is macular degeneration. In some embodiments, the disease or condition is age-related macular degeneration (AMD). In some embodiments, the method for treating or preventing a disease or condition includes modifying at least one retinal pigment epithelium (RPE) cell of a subject to express or include a nucleic acid, a vector or a polypeptide according to the present invention.

[0046] In yet another aspect, the present invention provides a kit comprising a plurality of parts, wherein the plurality of parts comprises a predetermined amount of a polypeptide, a nucleic acid, a vector, a cell or a pharmaceutical composition according to the present invention.

[0047] describe

[0048] To date, complement-based therapies for AMD have focused on injecting complement-modulating antibodies into the eye. Such therapies have provided little or no therapeutic benefit because these proteins simply fail to reach the target area, i.e., the BrM and its underlying vasculature, the choriocapillaris, or reach effective concentrations.

[0049] Complement factor I (FI)-mediated complement regulation, i.e., cleavage of C3b to iC3b (proteolytically inactivated C3b), requires a cofactor, such as membrane-anchored CR1. However, the inventors have discovered that for successful cofactor activity, it is not necessary to provide full-length membrane-bound CR1, or even a soluble form of CR1 lacking only the transmembrane domain. Instead, the inventors have discovered that a short CR1 fragment including the CR1C3b binding domain is sufficient to achieve efficient FI-mediated C3b cleavage.

[0050] Therefore, the present invention relates to a soluble truncated polypeptide derived from FI cofactor CR1. The polypeptide includes a domain that can be bound to C3b so that these domains can serve as necessary FI cofactors for regulating complement activation. The key advantage of the soluble truncated polypeptide is their ability to pass through BrM, so they can reach all regions associated with AMD, all regions associated with AMD, i.e., RPE / BrM interface, BrM and choroid, including capillary septa (extracellular matrix between blood vessels of the choriocapillaris layer). The present invention also provides a non-glycosylated polypeptide derived from CR1, which can assist the polypeptide to pass through BrM. The polypeptide is easy to express and secrete, so that it can be expressed in situ by cells local to the affected site, and the polypeptide is targeted to the region affected by complement overactivation. The in situ expression of the polypeptide can be achieved using gene therapy technology. In situ expression provides targeted therapy for the region in need without destroying the functional complement regulation in other regions of the body.

[0051] The present invention is able to supplement a degraded complement regulatory system without replacing the endogenous complement regulation currently in place, or interfering with the rest of the complement cascade.

[0052] Peptides

[0053] The polypeptide according to the invention may comprise or consist of one or more C3b binding regions.

[0054] The polypeptide according to the present invention has at least 80% sequence identity with SEQ ID NO: 4, wherein the polypeptide has a length of less than 700 amino acids. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4, wherein the polypeptide has a length of less than 700 amino acids.

[0055] The polypeptide according to the present invention may comprise or consist of an amino acid sequence of less than 700 amino acids having at least 80% sequence identity with SEQ ID NO:4.

[0056] In some embodiments, the polypeptide comprises an amino acid sequence of 650, 600, 550, 500, 450, 400, 350, 300, 250 or 200 amino acids or less. In some embodiments, the polypeptide comprises an amino acid sequence of 1-200 amino acids, 1-250 amino acids, 1-300 amino acids, 1-350 amino acids, 1-400 amino acids, 1-450 amino acids, 1-500 amino acids, 1-550 amino acids, 1-600 amino acids, 1-650 amino acids or 1-700 amino acids or consists of it. In some embodiments, the polypeptide has a length of 50-700 amino acids. In some embodiments, the polypeptide has a length of 100-650 amino acids. In some embodiments, the polypeptide has a length of 100-550 amino acids. In some embodiments, the polypeptide has a length of 150-450 amino acids. In some embodiments, the polypeptide has a length of 400-700 amino acids. In some embodiments, the polypeptide has a length of 700-1000, or greater than 1000 amino acids.

[0057] As used herein, "length" refers to the total length of a polypeptide; that is, "length" refers to the measurement or range of the entire polypeptide from end to end, i.e., from the N-terminus to the C-terminus. "Length" as used herein is measured by the number of amino acid residues in a polypeptide.

[0058] In some embodiments, polypeptide is a detached / discrete / separate / individual molecule. In some embodiments, polypeptide is a single continuous amino acid sequence that is not connected, i.e., not joined, not fused, or not attached to another amino acid sequence. In some embodiments, polypeptide is not attached to another polypeptide or amino acid sequence by an amino acid joint or a non-amino acid joint. In some embodiments, polypeptide is not a section, part or region of a longer amino acid sequence, i.e., polypeptide is not a part of an amino acid sequence that exceeds the maximum, specified polypeptide length. In some embodiments, polypeptide is not a part of a fusion protein or does not form a section of a fusion protein. In some embodiments, polypeptide may include a sequence provided herein, and one or more additional amino acids, as long as the maximum length of the polypeptide is not exceeded. The short length of the polypeptide described herein enables the polypeptide to pass through BrM and reach the site of complement activation.

[0059] In some embodiments, the polypeptide has a total length of 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, or 200 amino acids or less. In some embodiments, the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, or 70 amino acids or less. In some embodiments, the polypeptide has 1-70 amino acids, 1-80 amino acids, 1-90 amino acids, 1-100 amino acids, 1-110 amino acids, 1-120 amino acids, 1-130 amino acids, 1-140 amino acids, 1-150 amino acids, 1-160 amino acids, 1-170 amino acids, 1-180 amino acids, 1-190 amino acids, 1-200 amino acids, 1-210 amino acids, 1-220 amino acids, 1-230 amino acids, 1-240 amino acids, 1-250 amino acids, 1-260 1-260 amino acids, 1-270 amino acids, 1-280 amino acids, 1-290 amino acids, 1-300 amino acids, 1-310 amino acids, 1-320 amino acids, 1-330 amino acids, 1-340 amino acids, 1-350 amino acids, 1-360 amino acids, 1-370 amino acids, 1-380 amino acids, 1-390 amino acids, 1-400 amino acids, 1-410 amino acids, 1-420 amino acids, 1-430 amino acids, 1-440 amino acids, or a total length of 1-450 amino acids. In some embodiments, the polypeptide has a total length of 50-450 amino acids, 50-400 amino acids, 50-350 amino acids, 50-300 amino acids, 50-250 amino acids, 50-200 amino acids, 100-250 amino acids, 100-200 amino acids, 150-250 amino acids, or 150-200 amino acids. In some embodiments, the polypeptide has a total length of one of 61, 72, 194, 212, 231, 388, 406, or 644 amino acids.

[0060] In some embodiments, the polypeptides of the invention have a maximum molecular weight of 80 kDa, regardless of whether the polypeptide is covalently / non-covalently bound to a larger complex, part of a larger complex, or not part of a larger complex. In some embodiments, the polypeptides of the invention have a molecular weight of less than 75 kDa, less than 70 kDa, less than 65 kDa, less than 60 kDa, less than 55 kDa, less than 50 kDa, less than 45 kDa, less than 40 kDa, less than 35 kDa, less than 30 kDa, less than 29 kDa, less than 28 kDa, less than 27 kDa, less than 26 kDa, less than 25 kDa, less than 24 kDa, less than 23 kDa, less than 22 kDa, less than 21 kDa, less than 20 kDa, less than 19 kDa, less than 18 kDa, less than 17 kDa, less than 16 kDa, less than 15 kDa, less than 14 kDa, less than 13 kDa, less than 12 kDa, less than 11 kDa, or less than 10 kDa. In some embodiments, the polypeptide has a maximum molecular weight of 50 kDa, i.e., less than 50 kDa. In some embodiments, the polypeptide has a maximum molecular weight of 26 kDa, i.e., less than 26 kDa. In some embodiments, the polypeptide has a maximum molecular weight of 24 kDa, i.e., less than 24 kDa. In some embodiments, the polypeptide has a maximum molecular weight of 22 kDa, i.e., less than 22 kDa. In some embodiments, the polypeptide has a maximum molecular weight of 20 kDa, i.e., less than 20 kDa.

[0061] In some embodiments, a polypeptide of the invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, X 1 A or T, X 2 P or L, and / or X 3 is G or R. In some embodiments, X 1 A, X 2 is P, and / or X 3 is G. In some embodiments, X 1 A, X 2 is L, and / or X 3 is R. In some embodiments, X 1 A, X 2 is P, and / or X 3 is R. In some embodiments, X 1 A, X 2 is L, and / or X 3is G. In some embodiments, X 1 T, X 2 is L, and / or X 3 is R. In some embodiments, X 1 T, X 2 is P, and / or X 3 is G. In some embodiments, X 1 T, X 2 is L, and / or X 3 is G. In some embodiments, X 1 T, X 2 is P, and / or X 3 For R.

[0062] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4, wherein the polypeptide has a length provided herein. For example, in some embodiments, the polypeptide comprises or consists of an amino acid sequence having 85% sequence identity to SEQ ID NO:4, wherein the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, or less amino acids; in some embodiments, the polypeptide comprises an amino acid sequence having 85% sequence identity to SEQ ID NO:4, wherein the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210 NO:4 has a sequence identity of 90%, or consists of an amino acid sequence having a sequence identity of 90%, wherein the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190 or less amino acids; in some embodiments, the polypeptide comprises an amino acid sequence having a sequence identity of 90%, or consists of an amino acid sequence having a sequence identity of 90%, NO:4 has 95% sequence identity, or consists of an amino acid sequence, wherein the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190 or less amino acids; in some embodiments, the polypeptide comprises an amino acid sequence with SEQ ID NO:4 having 95% sequence identity, wherein the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190 or less amino acids. NO:4 has an amino acid sequence with 98% sequence identity, or consists of it, wherein the polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210 or less amino acids.

[0063] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 450 amino acids or less. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 50-450 amino acids. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 250 amino acids or less. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 50-250 amino acids. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 450 amino acids or less. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 50-450 amino acids. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of less than 250 amino acids. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein the polypeptide has a total length of 50-250 amino acids.

[0064] Human CR1 (UniProt: P17927 (entry version 181 (October 25, 2017), sequence version 3 (March 2, 2010)); SEQ ID NO: 1) has a sequence of 2039 amino acids (including the N-terminus, a 41-amino acid signal peptide), and includes 30 complement control protein (CCP) domains (also known as sushi domains or short consensus repeats (SCR)), and the N-terminal 28 CCPs are organized into four long homologous repeat (LHR) domains, each of which includes 7 CCPs: LHR-A, LHR-B, LHR-C and LHR-D. The C3b binding region of CR1 is present in CCP 8-10 of LHR-B (UniProt: P17927 positions 491-684; SEQ ID NO: 2) and CCP 15-17 of LHR-C (UniProt: P17927 positions 941-1134; SEQ ID NO: 3). CCP 8-10 and 15-17 differ in sequence by three amino acid residues, as shown in the consensus sequence SEQ ID NO: 4.

[0065] The polypeptide according to the present invention may include or consist of an amino acid sequence corresponding to CCP 8-10 (SEQ ID NO: 2) and / or CCP 15-17 (SEQ ID NO: 3). In some embodiments, the polypeptide of the present invention includes or consists of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2 and / or SEQ ID NO: 3. Such a polypeptide may have any length provided herein.

[0066] The polypeptide according to the invention may comprise or consist of an amino acid sequence corresponding to CCP 8-10 and 15-17. The polypeptide may comprise or consist of the native CR1 sequence (SEQ ID NO: 30) of CCP 8-10 and 15-17. The polypeptide may comprise or consist of CCP 8-10 joined to CCP 15-17. This may be a consensus sequence (SEQ ID NO: 13), or may be achieved by a linker between CCP 8-10 and 15-17 (e.g., SEQ ID NO: 14). In some embodiments, polypeptides of the invention comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14 and / or SEQ ID NO: 30. Such polypeptides can have any length provided herein.

[0067] The polypeptide according to the present invention may include or consist of an amino acid sequence corresponding to one or more of sequence "A" (SEQ ID NO: 8), sequence "B" (SEQ ID NO: 16) and / or sequence "C" (SEQ ID NO: 17). In some embodiments, the polypeptide consists of a sequence selected from sequence "A" (SEQ ID NO: 8), sequence "B" (SEQ ID NO: 16) and sequence "C" (SEQ ID NO: 17). In some embodiments, a polypeptide of the invention comprises, or consists of, an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of sequence "A" (SEQ ID NO: 8), sequence "B" (SEQ ID NO: 16) and / or sequence "C" (SEQ ID NO: 17).

[0068] In some embodiments, when a polypeptide according to the invention comprises or consists of sequence "B" (SEQ ID NO: 16), or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence "B" (SEQ ID NO: 16), X1 is A or T. In some embodiments, X 1 is A. In some embodiments, X 1 is T.

[0069] In some embodiments, when a polypeptide according to the invention comprises or consists of sequence "C" (SEQ ID NO: 17), or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence "C" (SEQ ID NO: 17), X 2 P or L, and / or X 3 is G or R. In some embodiments, X 2 For P and / or X 3 is G. In some embodiments, X 2 For P and / or X 3 is R. In some embodiments, X 2 L and / or X 3 is G. In some embodiments, X 2 L and / or X 3 For R.

[0070] In some embodiments, sequence "B" corresponds to SEQ ID NO:9 or SEQ ID NO: 11. In some embodiments, sequence "C" corresponds to SEQ ID NO:10 or SEQ ID NO:12.

[0071] The present invention includes polypeptides comprising the sequences "A", "B" and / or "C" as described herein, and combinations thereof, thereby comprising at least the following binding (organized from N-terminus to C-terminus):

[0072] A+B

[0073] ·B+C

[0074] ·A+C

[0075] ·C+A

[0076] A+B+C

[0077] ·B+C+A

[0078] C+A+B

[0079] A+B+C+A

[0080] B+C+A+B

[0081] C+A+B+C

[0082] A+B+C+A+B

[0083] B+C+A+B+C

[0084] A+B+C+A+B+C

[0085] A+B+C+A+B+C+Y (where Y=one or more of A, B and / or C).

[0086] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any combination of CCP domains or any combination of sequences "A", "B", and / or "C" described herein.

[0087] In some embodiments, the combination of CCP domains is the combination present in native CR1. In some embodiments, the combination of CCP domains is not the combination present in native CR1.

[0088] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having multiple copies of sequence "A", multiple copies of sequence "B", and / or multiple copies of sequence "C". In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8 or more copies of one or more of sequence "A", "B", and / or "C". In some embodiments, the polypeptide comprises: 1, 2, 3, 4, 5, 6, 7, 8 or more copies of sequence "A"; 1, 2, 3, 4, 5, 6, 7, 8 or more copies of sequence "B"; and / or 1, 2, 3, 4, 5, 6, 7, 8 or more copies of sequence "C". In some embodiments, the polypeptide comprises 9, 10 or more copies of one or more of sequence "A", "B", and / or "C".

[0089] In some embodiments, the polypeptides according to the invention lack substantial sequence identity with one or more of the amino acid sequences 1-490, 685-940 and / or 1135-2039 of human CR1 (SEQ ID NO: 1). The polypeptides lacking substantial sequence identity as described herein may have less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% sequence identity with one or more of the amino acid sequences 1-490, 685-940 and / or 1135-2039 of human CR1 (SEQ ID NO: 1). In some embodiments, the polypeptides according to the invention lack amino acid sequences having substantial sequence identity with the CR1 long homology repeat (LHR) domains LHR-A and / or LHR-D. In some embodiments, the polypeptides according to the invention lack an amino acid sequence having substantial sequence identity to CR1 CCP domains 1-7, 11-14 and / or 18-30. The amino acid residues of SEQ ID NO: 1 are numbered according to Uniprot P17927; entry version 181 (October 25, 2017), sequence version 3 (March 2, 2010).

[0090] The polypeptides according to the invention, and / or described herein may be isolated and / or substantially purified.

[0091] Other characteristics of peptides

[0092] The polypeptide according to the present invention may include modified and / or additional amino acid sequences. The modified and / or additional amino acid sequences may be included in the length limits of the polypeptides provided herein, so as not to exceed the length limits of the polypeptides.

[0093] In some embodiments, the additional amino acid sequence comprises no more than 25, 50, 100, 150 or 200 amino acids, or consists of no more than 25, 50, 100, 150 or 200 amino acids, i.e., the additional amino acid sequence comprises 1-25, 1-50, 1-100, 1-150 or 1-200 amino acids, or consists of 1-25, 1-50, 1-100, 1-150 or 1-200 amino acids. In some embodiments, the additional amino acid sequence comprises more than 200 amino acids. In some embodiments, the additional amino acid sequence comprises no more than 100 amino acids at the C-terminus of the polypeptide according to the invention, and / or comprises no more than 100 amino acids at the N-terminus of the polypeptide according to the invention.

[0094] In some embodiments, the additional amino acid sequence results in a polypeptide longer than 700 amino acids. In some embodiments, the polypeptide according to the invention comprises or consists of more than 700 amino acids. For example, the polypeptide may comprise or consist of 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000 or more than 1000 amino acids.

[0095] In some embodiments, the additional amino acid sequences described herein lack substantial sequence identity to one or more of amino acid sequences 1-490, 685-940, and / or 1135-2039 of human CR1 (SEQ ID NO: 1, numbered according to Uniprot P17927; entry version 181 (October 25, 2017), sequence version 3 (March 2, 2010)). In some embodiments, the additional amino acid sequences lack substantial sequence identity to CR1 CCP domains 1-7, 11-14, and / or 18-30. In some embodiments, the additional amino acid sequence has less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity to one or more of the amino acid sequences 1-490, 685-940, and / or 1135-2039 of human CR1 (SEQ ID NO: 1). In some embodiments, the additional amino acid sequence lacks substantial sequence identity to the CR1 long homology repeat (LHR) domains LHR-A and / or LHR-D.

[0096] In some embodiments, the polypeptide may lack an amino acid sequence that has substantial sequence identity to a region of a cofactor of complement factor I (e.g., CR1) except in the C3b binding region. For example, the polypeptide may lack an amino acid sequence that has substantial sequence identity to CR1 except in CR1 CCP domains 8-10 and / or 15-17 (residues 491-684 and / or 941-1134 of SEQ ID NO: 1, respectively). In some embodiments, the polypeptide may lack an amino acid sequence that has substantial sequence identity to CR1 CCP domains 1-7, 11-14, and / or 18-30. A polypeptide lacking an amino acid sequence having substantial sequence identity as described herein may have less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% sequence identity to one or more of the amino acid sequences 1-490, 685-940 and / or 1135-2039 of human CR1 (SEQ ID NO: 1).

[0097] In some embodiments, polypeptides according to the present invention may include secretory pathway sequences. As used herein, secretory pathway sequences are amino acid sequences that direct the secretion of polypeptides. Once the exit of the polypeptide chain across the rough endoplasmic reticulum is initiated, the secretory pathway sequences can be cleaved from the mature protein. Polypeptides secreted by mammalian cells generally have a signal peptide fused to the N-terminal of the polypeptide, which is cleaved from the translated polypeptide to produce a "mature" form of the polypeptide.

[0098] In some embodiments, the secretory pathway sequence may include a leader sequence (also referred to as a signal peptide or or signal sequence), or be composed thereof. The leader sequence is generally composed of a sequence of 5-30 hydrophobic amino acids forming a single alpha helix. Secreted proteins and proteins expressed on the cell surface generally include a leader sequence. The leader sequence may be present in a newly translated polypeptide (e.g., before processing to remove the leader sequence). The leader sequences of many proteins are known and recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

[0099] In some embodiments, the secretory pathway sequence is derived from complement factor H (FH). In some embodiments, the secretory pathway sequence comprises, or consists of, SEQ ID NO: 7. In some embodiments, the secretory pathway sequence of a polypeptide of the invention comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7.

[0100] In some embodiments, a polypeptide according to the invention comprises or consists of an amino acid sequence corresponding to SEQ ID NO: 47, 49 and / or 51. In some embodiments, a polypeptide comprises or consists of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 47, 49 and / or 51. Such a polypeptide can have any length provided herein.

[0101] In some embodiments, polypeptide according to the present invention can additionally include a cleavage site for removing secretory pathway sequences from polypeptide. In some embodiments, the cleavage site for removing secretory pathway sequences from polypeptide is a cleavage site for endoproteinase (endoprotease). In some embodiments, the cleavage site is for an endoproteinase expressed by the cell expressed therein by the polypeptide. In some embodiments, the cleavage site is a signal peptidase (signalpeptidase) cleavage site. In some embodiments, the cleavage site is a protease cleavage site, for example, for the cleavage site of an endoproteinase expressed by the cell expressing the polypeptide. In some embodiments, the cleavage site is for the cleavage site of an endoproteinase expressed by RPE cells.

[0102] The polypeptide according to the present invention may include one or more linker sequences between the amino acid sequences. The linker sequence may be arranged between any two or more of the sequences "A", "B" and / or "C". In some embodiments, the polypeptide according to the present invention comprises the amino acid sequence A+B+C-[linker]-A+B+C, or consists of it. In some embodiments, the polypeptide comprises SEQ ID NO: 14, or consists of it.

[0103] The linker sequence is known to those skilled in the art and is described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence can be a flexible linker sequence. The flexible linker sequence allows relative movement of the amino acid sequence linked by the linker sequence. Flexible linkers are known to the technician and some flexible linkers are identified in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369. Flexible linker sequences typically include a high proportion of glycine and / or serine residues.

[0104] In some embodiments, the joint sequence includes at least one glycine residue and / or at least one serine residue. In some embodiments, the joint sequence is composed of a glycine residue and a serine residue. In some embodiments, the joint sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 amino acid.

[0105] In some embodiments, polypeptides according to the present invention include non-amino acid linkers. In some embodiments, polypeptides according to the present invention may include two or more polypeptides linked by conjugation, such as by nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0106] In some embodiments, polypeptides according to the invention include a cleavable linker.

[0107] It may be desirable for a polypeptide according to the invention to lack certain properties of CR1. For example, it may be desirable for a polypeptide to lack a region that would otherwise inhibit diffusion through Bruch's membrane (BrM) or would interfere with the activity of a native cofactor family protein.

[0108] The polypeptide according to the invention lacks the CR1 transmembrane domain (SEQ ID NO: 32). The polypeptide according to the invention may lack the CR1 cytoplasmic tail (SEQ ID NO: 33). In a preferred embodiment, the polypeptide according to the invention is soluble.

[0109] The polypeptides according to the invention may lack regions that could otherwise be used by pathogenic bacteria to subvert the host immune system. Bacteria have developed molecules on their surfaces that can bind to and recruit soluble complement factor H from the blood. This allows the bacteria to effectively coat themselves in complement regulators and evade the host's immune response. The polypeptides according to the invention may lack bacterial binding sites, making them unusable by invading pathogens to evade the immune response.

[0110] In some embodiments, the polypeptides according to the present invention comprise one or more sites for glycosylation. In some embodiments, the polypeptides according to the present invention are glycosylated.

[0111] In some embodiments, the polypeptide according to the present invention is non-glycosylated. In some embodiments, the polypeptide according to the present invention lacks one or more glycosylation sites. In some embodiments, the polypeptide according to the present invention lacks one or more N-linked glycosylation sites. In some embodiments, the polypeptide according to the present invention lacks N-linked glycans. In some embodiments, the polypeptide according to the present invention is expressed and / or secreted by cells that cannot glycosylate polypeptides or cannot fully glycosylate polypeptides. For example, the cell may lack a functional glycosyltransferase enzyme. In some embodiments, the polypeptide is aglycosyl (i.e., non-glycosylated). In some embodiments, the polypeptide has been deglycosylated, for example, by treatment with a glycosidase (e.g., peptide N-glycosidase). Deglycosylation is preferably non-denatured. In some embodiments, the polypeptide according to the present invention is partially glycosylated, non-glycosylated, or deglycosylated.

[0112] In some embodiments, polypeptides according to the invention lack sequences conforming to the consensus sequence of SEQ ID NO: 27. In some embodiments, polypeptides according to the invention include one or more sequences conforming to the consensus sequence of SEQ ID NO: 27 that have been mutated to remove an N-glycosylation site. In some embodiments, an asparagine (Asn, N) residue in one or more consensus sequences according to SEQ ID NO: 27 is substituted with another amino acid residue, such as a residue selected from the group consisting of alanine (Ala, A), cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), phenylalanine (Phe, F), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), lysine (Lys, K), leucine (Leu, L), methionine (Met, M), proline (Pro, P), glutamine (Gln, Q), arginine (Arg, R), serine (Ser, S), threonine (Thr, T), valine (Val, V), tryptophan (Trp, W), or tyrosine (Tyr, Y). In some embodiments, an Asn (N) residue in one or more consensus sequences according to SEQ ID NO: 27 is substituted with a Gln (Q) residue. In some embodiments, residue X of SEQ ID NO: 27 2It is an amino acid other than Ser (S) or Thr (T), or is mutated to an amino acid other than Ser (S) or Thr (T).

[0113] In some embodiments, the polypeptide comprises one or more amino acid substitutions at position 509 and / or position 578 (numbered according to Uniprot: P17927), the polypeptide comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the one or more amino acid substitutions are selected from N509Q and / or N578Q. In some embodiments, the polypeptide comprises one or more amino acid substitutions at position 959 and / or position 1028 (numbered according to Uniprot: P17927), the polypeptide comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the one or more amino acid substitutions are selected from N959Q and / or N1028Q. In some embodiments, the polypeptide comprises one or more amino acid substitutions at positions 509, 578, 959 and / or 1028 (numbered according to Uniprot: P17927), comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the one or more amino acid substitutions are selected from N509Q, N578Q, N959Q and / or N1028Q. Such polypeptides can have any length provided herein.

[0114] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 15, and / or SEQ ID NO: 31. Such polypeptides can have any length provided herein.

[0115] In some embodiments, polypeptides according to the invention include secretory pathway sequences, and one or more sequences that conform to the consensus sequence of SEQ ID NO: 27 that has been mutated to remove an N-glycosylation site. In some embodiments, polypeptides according to the invention include or consist of an amino acid sequence corresponding to SEQ ID NO: 48, 50, 52, 53, and / or 54. In some embodiments, the polypeptide includes or consists of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 48, 50, 52, 53, and / or 54. Such polypeptides can have any length provided herein.

[0116] In some embodiments, the polypeptide according to the present invention may include an amino acid sequence to promote the expression, folding, transportation (trafficking), processing, purification or detection of the polypeptide. For example, the polypeptide may include a sequence encoding a protein tag (proteintag), optionally at the N or C terminus of the polypeptide, in a joint, or at the N or C terminus of the joint, a protein tag such as His (e.g., 6XHis), FLAG, Myc, GST, MBP, HA, E or a biotin tag. In some embodiments, the polypeptide includes a detectable portion, such as fluorescence, cold light, immunodetectable, radioactive, chemical, nucleic acid or enzyme label (label). In some embodiments, the detectable portion promotes the detection of the polypeptide in a sample obtained from a subject, for example, after applying a polypeptide, nucleic acid, carrier, cell or pharmaceutical composition according to the present invention to the subject. The sample can be any biological sample obtained from a subject. In some embodiments, the sample is a liquid biopsy, such as ocular fluid (tears, aqueous humour or vitreous fluid), blood, plasma, etc. In some embodiments, the sample is a cytological sample or a tissue sample, such as a surgical sample of an eye cell / tissue.

[0117] In some embodiments, polypeptides according to the invention can be detected and / or distinguished from endogenous CR1 by Western blotting, mass spectrometry and / or enzymatic digestion, for example, by specific peptidases. In some embodiments, the polypeptides can include point mutations to generate peptides by enzymatic digestion that are different from post-digestion peptides from endogenous CR1.

[0118] In some embodiments, the polypeptide according to the present invention may additionally include a cleavage site for removing a protein tag. For example, it may be necessary to remove the tag used to purify the polypeptide after purification. In some embodiments, the cleavage site may be, for example, a tobacco etch virus (Tobacco Etch Virus, TEV) protease cleavage site, for example, as shown in SEQ ID NO: 34.

[0119] In some embodiments, a polypeptide according to the invention comprises or consists of an amino acid sequence corresponding to SEQ ID NO: 40, 42, 44 and / or 46. In some embodiments, a polypeptide comprises or consists of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40, 42, 44 and / or 46. Such a polypeptide can have any length provided herein.

[0120] As used herein, "polypeptide" includes molecules that include more than one polypeptide chain that can be associated (e.g., covalently or non-covalently) into a complex. That is, "polypeptide" within the meaning of the present invention includes molecules that include one or more polypeptide chains. The polypeptides of the present invention can include, for example, signal peptides, protein tags, cleavage sites for removing protein tags, etc. in various embodiments and at different stages of expression / production in vitro or in vivo. Polypeptides of the invention may include any CR1 CCP sequence described herein, or any combination of CR1 CCP domains 8 (SEQ ID NO: 8), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), 15 (SEQ ID NO: 8), 16 (SEQ ID NO: 11) and / or 17 (SEQ ID NO: 12), or any combination of sequences "A", "B" and / or "C" described herein, optionally in combination with one or more of any other features of polypeptides of the invention described herein (e.g., signal peptide, linker, detection sequence, deletion of glycosylation site, substituted amino acid residues, protein tag, cleavage site for removal of protein tag, secretory pathway sequence, cleavage site for removal of secretory pathway sequence).

[0121] Sequence identity

[0122] As used herein, an amino acid sequence corresponding to a reference amino acid sequence can include at least 60% sequence identity to the reference sequence, such as at least one of 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0123] For the purpose of determining the percent identity between two or more amino acid or nucleic acid sequences, pairwise and multiple sequence alignments can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega ( J.2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780) software. When using such software, it is preferred to use default parameters such as gap penalty and extension penalty.

[0124] sequence

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] Functional properties of peptides

[0142] Polypeptides according to the invention may be characterized by reference to one or more functional properties.

[0143] In particular, the polypeptides according to the invention may have one or more of the following properties (as determined by analysis in appropriate assays for said properties):

[0144] Binds to C3b;

[0145] binds to C3b with a binding affinity similar to the binding affinity exhibited by the cofactor of complement factor I (or a fragment thereof) to C3b;

[0146] binds to C3b with a higher binding affinity than the binding affinity exhibited by the cofactor of complement factor I (or a fragment thereof) to C3b;

[0147] binds to C3b with a binding affinity similar to the binding affinity exhibited by complement receptor 1 (or a fragment thereof) to C3b;

[0148] binds to C3b in the region of C3b that is bound by a cofactor of complement factor I (or a fragment thereof);

[0149] binds to C3b in the region of C3b that is bound by complement receptor 1 (or a fragment thereof);

[0150] binds to C3b in the region of C3b bound by complement receptor 1 CCP domains 8-10 and / or 15-17 (or fragments thereof);

[0151] Acts as a cofactor to enable complement factor I-mediated inactivation of C3b;

[0152] Acts as a cofactor to achieve the decrease / prevention of complement factor I-mediated formation of functional C3bBb-type C3 convertase;

[0153] Acts as a cofactor to achieve complement factor I-mediated decrease / prevention of the formation of functional C3bBb3b-type C5 convertase;

[0154] Acts as a cofactor to achieve complement factor I-mediated reduction / prevention of the formation of functional C4b2a3b-type C5 convertase;

[0155] Acts as a cofactor to achieve complement factor I-mediated reduction of C3bBb-type C3 convertase activity;

[0156] Acts as a cofactor to achieve complement factor I-mediated reduction of C3bBb3b-type C5 convertase activity;

[0157] Acts as a cofactor to achieve complement factor I-mediated reduction of C4b2a3b-type C5 convertase activity;

[0158] Acts as a cofactor to achieve complement factor I-mediated reduction in the amount of C3bBb-type C3 convertase;

[0159] Acts as a cofactor to achieve complement factor I-mediated reduction in the amount of C3bBb3b-type C5 convertase;

[0160] Acts as a cofactor to achieve complement factor I-mediated reduction in the amount of C4b2a3b-type C5 convertase;

[0161] Reduction of the amount of C3b by complement factor I;

[0162] Increases the amount of iC3b through complement factor I;

[0163] Increases the amount of C3dg through complement factor I;

[0164] Increases the amount of C3d through complement factor I;

[0165] Increases the amount of C3f through complement factor I;

[0166] Reduction of the amount of C5b by complement factor I;

[0167] Reduction of the amount of C5a by complement factor I;

[0168] reducing the amount of iC3b by complement factor I compared to the amount of iC3b produced by FH and / or FHL-1 by complement factor I;

[0169] Increase the ratio of C3dg to iC3b via complement factor I;

[0170] Ability to inhibit complement activation;

[0171] Diffusion through Bruch's membrane (BrM);

[0172] Compared with complement factor I, it exhibits superior diffusion ability across BrM;

[0173] Compared to complement factor I cofactors (or fragments thereof), they exhibit superior diffusion through BrM;

[0174] Compared to the cofactor of complement factor I (or its fragments), it exhibits a similar ability to diffuse through BrM;

[0175] Compared with complement factor H, it exhibits superior ability to diffuse through BrM;

[0176] Compared with complement factor H isoform FHL-1, it exhibits a similar ability to diffuse through BrM;

[0177] Compared with complement factor H isoform FHL-1, it exhibits superior ability to diffuse through BrM;

[0178] Compared with soluble complement receptor 1, it exhibits a similar ability to diffuse across BrM;

[0179] Compared to soluble complement receptor 1, it exhibits superior diffusion ability across BrM.

[0180] For example, as described herein, a given polypeptide can be analyzed for its functional properties as mentioned in the preceding paragraph.

[0181] The polypeptides according to the invention may be capable of binding to C3b. In some embodiments, the polypeptides according to the invention may include or consist of a C3b binding region. In some embodiments, the C3b binding region of the polypeptides according to the invention includes or consists of a C3b binding region of CR1, such as CCP domains 8-10 and / or 15-17.

[0182] As used herein, "C3b binding region" refers to a region capable of binding to C3b. In some embodiments, the C3b binding region is capable of specifically binding to C3b. Binding to C3b can be mediated by non-covalent interactions, such as van der Waals forces, electrostatic interactions, hydrogen bonding, and hydrophobic interactions formed between the C3b binding region and C3b. In some embodiments, the C3b binding region binds to C3b with greater affinity, and / or for a greater duration, than it binds to molecules other than C3b.

[0183] The ability of a polypeptide or putative C3b binding region according to the invention to bind to C3b can be analyzed using techniques known to those skilled in the art, including ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442; or Rich et al., Anal Biochem. 2008 Feb 1; 373(1): 112-20), Bio-Layer Interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507; or Concepcion et al., Comb Chem High Throughput Screen. 2009 Sep; 12(8): 791-800), microscale thermophoresis (MST) analysis (see, e.g., Jerabek-Willemsen et al., Assay Drug Dev Technol. 2011 Aug; 9(4): 342–353) or by radiolabeled antigen binding assay (RIA). By such analysis, binding to a given target can be determined and quantified. In some embodiments, the binding can be a response detected in a given assay.

[0184] In some embodiments, polypeptides according to the invention exhibit binding to C3b in such assays that is greater than 1-fold, e.g., >1.01, >1.02, >1.03, >1.04, >1.05, >1.06, >1.07, >1.08, >1.09, >1.1, >1.2, >1.3, >1.4, >1.5, >1.6, >1.7, >1.8, >1.9, >2, >3, >4, >5, >6, >7, >8, >9, >10, >15, >20, >25, >30, >35, >40, >45, >50, >60, >70, >80, >90, or >100-fold, compared to the level of binding signal detected in such assays to a negative control molecule that does not bind to the C3b binding region.

[0185] In some embodiments, a polypeptide according to the invention is capable of binding to C3b with a binding affinity similar to the binding affinity exhibited by CR1 or other cofactor of complement factor I (or a fragment thereof) to C3b in a given assay. A binding affinity similar to a reference binding affinity can be, for example, ±40%, such as one of ±35%, ±30%, ±25%, ±20%, ±15%, ±10%, or ±5% of the level of binding to C3b exhibited by a reference CR1 or a reference cofactor of complement factor I in a comparable assay.

[0186] In some embodiments, the polypeptides according to the invention are capable of binding to C3b with a higher binding affinity than the binding affinity exhibited by a cofactor of complement factor I (or a fragment thereof) to C3b in a given assay. In some embodiments, the polypeptides according to the invention are capable of binding to C3b with a higher binding affinity than the binding affinity exhibited by a cofactor of complement factor I (or a fragment thereof) to C3b in a given assay. In some embodiments, the polypeptides according to the invention are capable of binding to C3b with a higher binding affinity than the binding affinity exhibited by a cofactor of complement factor I (or a fragment thereof) to C3b in a given assay. In some embodiments, the polypeptides according to the invention can bind to C3b with a binding affinity that is 10,000 times, 100,000 times, or 1,000,000 times greater than the binding affinity exhibited by a cofactor of complement factor I (or a fragment thereof) to C3b in a given assay. In some embodiments, a polypeptide according to the invention is capable of binding to C3b with a binding affinity that is 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude greater than the binding affinity exhibited by a cofactor of complement factor I (or a fragment thereof) to C3b in a given assay. In some embodiments, the cofactor of complement factor I is complement factor H or a truncated FH isoform FHL-1. The cofactor of complement factor I may be CR1.

[0187] In some embodiments, polypeptides according to the invention are capable of binding to C3b in a region of C3b that is bound by a cofactor of complement factor I (i.e., to the same region or an overlapping region). In some embodiments, the polypeptides are capable of binding to C3b in a region that is bound by CR1 (or a fragment thereof). In some embodiments, the polypeptides are capable of binding to C3b in a region that is bound by CR1 CCP domains 8-10 and / or 15-17. In some embodiments, the polypeptides are capable of binding to C3b in a region that is bound by one or more of the complement factor I cofactors complement factor H, CD46, CD55, C4BP, SPICE, VCP, or MOPICE (or a fragment thereof).

[0188] Whether a polypeptide according to the invention binds to C3b in the region of C3b bound by a given cofactor of complement factor I (or a fragment thereof) can be determined by various methods known to those skilled in the art, including ELISA and surface plasmon resonance (SPR) analysis. An example of a suitable assay for determining whether a C3b binding region binds to C3b in the region bound by a given cofactor of complement factor I (or a fragment thereof) is an ELISA assay.

[0189] For example, whether a polypeptide according to the invention binds to C3b in the region of C3b bound by a given complement factor I cofactor (or fragment thereof) can be determined by analyzing the interaction of the cofactor / fragment with C3b in the presence of a polypeptide according to the invention or after incubation of one or both of the cofactor / fragment and C3b with a polypeptide according to the invention. A C3b binding region that binds to C3b in the region of C3b bound by a given cofactor / fragment is identified by observing a decrease / reduction in the level of interaction between the cofactor / fragment and C3b in the presence of a polypeptide according to the invention or after incubation of one or both of the cofactor / fragment and C3b with a polypeptide according to the invention, compared to the level of interaction in the absence of a polypeptide according to the invention (or in the presence of an appropriate control peptide / polypeptide). Suitable analyses can be performed in vitro, for example using recombinant interaction partners. For the purposes of such assays, one or both of the interaction partners and / or the polypeptide according to the invention can be labeled or used in conjunction with a detectable entity for the purpose of detecting and / or measuring the level of interaction.

[0190] In some embodiments, the polypeptide according to the invention acts as a cofactor for complement factor I. For example, the polypeptide can enhance the cleavage of C3b by complement factor I, and / or present the proteolytic cleavage of C3b by complement factor I in a favorable direction. The polypeptide preferably does not inhibit the proteolytic cleavage of C3b by complement factor I. In some embodiments, complement factor I is endogenous. In some embodiments, complement factor I is exogenous.

[0191] As used herein, an "endogenous" protein / peptide refers to a protein / peptide encoded / expressed by a relevant cell type, tissue, or subject (before treatment with a polypeptide, nucleic acid, vector, cell, or pharmaceutical composition according to the invention). A "non-endogenous" or "exogenous" protein / peptide refers to a protein / peptide that is not encoded / expressed by a relevant cell type, tissue, or subject (before treatment with a polypeptide, nucleic acid, vector, cell, or pharmaceutical composition according to the invention).

[0192] A polypeptide according to the invention that acts as a cofactor of complement factor I can be determined, for example, by analyzing the following: the level or rate of proteolytic cleavage of C3b by complement factor I in the presence of the polypeptide according to the invention (or after incubation with the polypeptide according to the invention) in a suitable assay compared to the level or rate of proteolytic cleavage of C3b by complement factor I in the absence of the polypeptide according to the invention (or in the presence of an appropriate control peptide / polypeptide). A C3b binding region that acts as a cofactor of complement factor I is identified by detecting an increase in the level or rate of proteolytic cleavage of C3b by complement factor I in the presence of the polypeptide according to the invention (or after incubation with the polypeptide according to the invention) compared to the level or rate of proteolytic cleavage of C3b by complement factor I in the absence of the polypeptide according to the invention (or in the presence of an appropriate control peptide / polypeptide). The level or rate of proteolytic cleavage of C3b by complement factor I can be determined, for example, by detecting one or more products of cleavage of C3b by complement factor I, such as iC3b, C3dg, C3d or C3f. The level or rate of proteolytic cleavage of C3b by complement factor I can be determined, for example, by detecting a decrease in the presence of C3b. In some embodiments, a polypeptide according to the invention that acts as a cofactor for complement factor I produces less iC3b overall than the amount of iC3b produced by FH / FHL-1 by complement factor I. In some embodiments, a polypeptide according to the invention that acts as a cofactor for complement factor I increases the ratio of C3dg to iC3b by complement factor I compared to the ratio of C3dg to iC3b produced by FH / FHL-1 by complement factor I. For example, a polypeptide may not increase the overall amount of iC3b, but instead may increase the amount of C3dg, C3f, and / or C3d by complement factor I.

[0193] In some embodiments, polypeptides according to the present invention can inhibit or reduce complement activation. Polypeptides can inhibit or reduce complement overactivation. The level of complement activation / overactivation can be determined by tests described herein, such as abnormal levels of complement components, or by tests / tests known to those skilled in the art, such as those described in Shih and Murali Am. J. Hematol. 2015, 90: 1180-1186, Kirschfink and Mollnes, Clin Diagn Lab Immunol. 2003, 10 (6): 982–989, Nilsson and Ekdahl, Clinical and Developmental Immunology, 2012, Article ID 962702, the entire contents of which are incorporated herein by reference.

[0194] In some embodiments, a polypeptide according to the invention has the ability to diffuse through, ie, cross, a Bruch's membrane (BrM) as determined by analysis in an appropriate assay for said property.

[0195] The ability of a given polypeptide to diffuse through the BrM can be analyzed, for example, in vitro, for example, as described in Clark et al J. Immunol (2014) 193, 4962-4970. Briefly, the BrM can be isolated from the eye of the donor, and the macular region can be placed in an Ussing chamber, as described in McHarg et al., J Vis Exp (2015) 1-7. Once placed, the 5 mm diameter macular region is the only barrier between the two identical compartments. Both sides of the BrM can be washed with PBS, and human serum can be diluted 1:1 with PBS and added to the Ussing chamber (sample chamber) on one side of the BrM. The polypeptide to be analyzed can be added to the sample chamber in PBS, and PBS can be added separately to the chamber on the other side of the BrM (diffusion chamber), and the Ussing chamber can be incubated for 24 hours at room temperature with gentle stirring in the sample chamber and the diffusion chamber. Subsequently, the presence of polypeptides in samples from each chamber can be analyzed, for example, using antibody-based detection methods, such as ELISA analysis or Western blot. Detection of polypeptides in the diffusion chamber indicates that the polypeptides are able to diffuse through the BrM. Suitable positive and negative control proteins known to be able / incapable of diffusing through the BrM can be included in such experiments.

[0196] In some embodiments, the polypeptides according to the present invention exhibit a superior ability to diffuse through BrM than complement factor I. In some embodiments, the polypeptides according to the present invention exhibit a superior ability to diffuse through BrM than complement factor H. FH, which consists of 20 CCP domains, is a large molecule and does not pass through BrM. In some embodiments, the polypeptides according to the present invention exhibit a similar ability to diffuse through BrM compared to the truncated complement factor H isoform FHL-1 (UniProt: P08603-2; SEQ ID NO: 28). In some embodiments, the polypeptides according to the present invention exhibit a superior ability to diffuse through BrM compared to the complement factor H isoform FHL-1. In some embodiments, the polypeptides according to the present invention exhibit a similar ability to diffuse through BrM compared to the full-length soluble CR1 (30 CCP domains; SEQ ID NO: 1 lacking SEQ ID NOs: 32 and 33). In some embodiments, the polypeptides according to the present invention exhibit a superior ability to diffuse through BrM compared to the full-length soluble CR1. A polypeptide according to the invention which is able to diffuse through BrM preferably remains functionally active, ie acts as a cofactor for complement factor I, after diffusing through BrM.

[0197] A polypeptide of the invention that exhibits superior ability to diffuse through BrM compared to a given reference polypeptide can be identified by analyzing diffusion through BrM as described above. Diffusion through BrM can be detected by measuring the diffusion rate through a diffusion chamber and / or detecting the percentage of polypeptides present in the diffusion chamber at the end of the experiment. A polypeptide of the invention that exhibits similar ability to diffuse through BrM compared to a given reference polypeptide can be identified by analyzing diffusion through BrM as described above. Similar ability to diffuse through BrM can be indicated by detecting that the diffusion rate through the diffusion chamber is within 30% of the diffusion rate of the reference polypeptide, such as within one of 25%, 20%, 15% or 10%, and / or by detecting that the percentage of the polypeptide of the invention present in the diffusion chamber at the end of the experiment is within 30% of the percentage of the reference polypeptide present in the diffusion chamber, such as within one of 25%, 20%, 15% or 10%.

[0198] Due to the ability of polypeptides to diffuse through BrM, if / once the polypeptides of the invention perform their cofactor role with FI, the polypeptides of the invention may also be able to diffuse away from the site of C3b inactivation. In other words, the polypeptides of the invention may be able to transiently reside at areas of complement activation. This is advantageous because accumulation of complement-associated debris is undesirable, particularly in the case of macular degeneration where accumulation of cellular debris can lead to the formation of drusen.

[0199] The polypeptide of the present invention may be capable of being expressed in a cell, such as a cell as described herein. The polypeptide of the present invention may be capable of being secreted by a cell, such as a cell as described herein. In some embodiments, the cell is an eye cell, such as a RPE cell as described herein.

[0200] Nucleic acids, cells, compositions and kits

[0201] The invention provides a nucleic acid encoding a polypeptide according to the invention. In some embodiments, the nucleic acid is purified or separated, for example, from other nucleic acids or naturally occurring biological materials. In some embodiments, the nucleic acid comprises DNA and / or RNA, or is composed of DNA and / or RNA.

[0202] Provided herein are nucleic acid sequences encoding polypeptides comprising or consisting of SEQ ID NO: 2, 3, 5, 6, 13, 14, 15, 30, 31, 40, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, or 54. The encoded polypeptides can be produced with or without a leader sequence, such as a secretory pathway sequence. The encoded polypeptides can be produced with a leader sequence that is then subsequently removed from the polypeptide.

[0203] In some embodiments, the nucleic acid according to the present invention comprises or consists of one or more of SEQ ID NOs: 35, 36, 37, 38, 39, 41, 43 and / or 45, or equivalent nucleic acid sequences thereof that are translated into the same corresponding polypeptides due to codon degeneracy.

[0204] The present invention also provides a vector comprising a nucleic acid encoding the polypeptide according to the present invention.

[0205] The nucleotide sequence can be contained in a vector, such as an expression vector. "Vector" as used herein is a nucleic acid molecule used as a vehicle for transporting exogenous nucleic acid into a cell. A vector can be a vector for expressing nucleic acid in a cell. Such a vector can include a promoter sequence that is operably linked to a nucleotide sequence encoding a sequence to be expressed. The vector can also include a stop codon and an expression enhancer. The vector can include regulatory elements, such as a polyadenylation site. Any suitable vector, promoter, enhancer and stop codon known in the art can be used to express peptides or polypeptides from a vector according to the present invention. The nucleic acid sequences described herein can be codon optimized to optimize expression in desired cells or organisms.

[0206] The term "operably linked" may include situations in which a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked (forming an expression cassette) in a manner such that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of affecting transcription of the nucleic acid sequence. The resulting transcript may then be translated into a desired peptide / polypeptide.

[0207] Preferably, nucleic acid and / or vector according to the present invention are provided for introduction into cells, such as human cells. Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., vectors derived from murine leukemia virus (MLV)), lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus (adeno-associated virus, AAV) vectors, vaccinia virus vectors and herpes virus vectors such as herpes simplex virus vectors (Herpes Simplex Virus)), transposon-based vectors and artificial chromosomes (e.g., yeast artificial chromosomes), for example, as described in Maus et al., Annu Rev Immunol (2014) 32: 189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, the entire contents of which are incorporated herein by reference. In some embodiments, the lentiviral vector may be pELNS, or may be derived from pELNS. In some embodiments, the vector may be a vector encoding CRISPR / Cas9. In some embodiments, the adeno-associated virus (AAV) vector is selected from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a hybrid and / or mutant thereof. In some embodiments, the AAV vector is an AAV serotype 2 (AAV-2) vector, or a hybrid and / or mutant thereof. Viral and non-viral delivery systems for introducing genetic material into cells are reviewed in, for example, Nayerossadat et al., Adv Biomed Res. 2012; 1:27, MacLaren et al. Ophthalmology. 2016, 123(10 Suppl): S98–S106, Petit and Punzo, Discov Med. 2016, 22(121): 221–229, Aguirre, Invest Ophthalmol Vis Sci. 2017, 58(12): 5399–5411, Lundstrom, Diseases. 2018, 6(2): 42, the entire contents of which are incorporated herein by reference. In the context of the present invention, any suitable nucleotide or vector delivery method may be used.

[0208] In some embodiments, according to the present invention, expression of a nucleic acid or a nucleic acid contained in a vector is driven by a promoter that drives expression in a specific retinal cell type, such as rods, cones, RPE or ganglion cells, as described in, for example, Beltran WA, et al. Gene Ther. 2010; 17: 1162–74 and Boye SE, et al. Hum Gene Ther. 2012; 23: 1101–15, the entire contents of which are incorporated herein by reference.

[0209] In some embodiments, according to the present invention, the expression of nucleic acid or the nucleic acid included in the vector is driven by a promoter, which drives the expression of the nucleic acid in retinal pigment epithelium (RPE) cells. In some embodiments, the promoter is RPE65 or VMD2 promoter, or a modified form thereof. In some embodiments, the promoter is chicken beta actin promoter (chicken beta cactin promoter).

[0210] In some embodiments, the vector can be a eukaryotic vector, such as a vector comprising elements necessary for expression of a protein from the vector in a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, such as a vector comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0211] In some embodiments, the vector includes an inducible promoter, i.e., gene expression is activated by the promoter only in the presence or absence of a specific molecule. Suitable inducible promoters will be known to the skilled person. Examples of inducible promoters are described in, for example, Le at al. Invest Ophthalmol Vis Sci. 2008, 49 (3): 1248–1253 and McGee Sanftner et al. Mol Ther. 2001. 3 (5): 688-696, the entire contents of which are incorporated herein by reference.

[0212] In some embodiments, the nucleic acid comprises, or consists of, a nucleic acid sequence encoding a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 30, 31, 40, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or any combination of sequences A, B, and / or C as described herein.

[0213] The present invention also provides a cell comprising or expressing a polypeptide according to the present invention. A cell comprising or expressing a nucleic acid or vector according to the present invention is also provided. A cell comprising or expressing a polypeptide, nucleic acid or vector according to the present invention can secrete a polypeptide according to the present invention. That is, expression of a polypeptide, nucleic acid or vector by a cell can result in soluble production of a polypeptide according to the present invention from the cell.

[0214] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a human or non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows or any animal in the order Bos), horse (including any animal in the order Equidae), donkey and non-human primate). In some embodiments, the cell can be from a human subject or can have been obtained from a human subject.

[0215] In some embodiments, the cell is a cell of the eye. In some embodiments, the cell is a cell of the retinal neurosensory layer (neurosensory retina), retinal pigment epithelium (RPE), choroid or macula. In some embodiments, the cell is a retinal cell. In some embodiments, the cell is a retinal pigment epithelial cell. In some embodiments, the cell is a human retinal pigment epithelial cell (RPE). In some embodiments, the cell is a photoreceptor cell.

[0216] The present invention also provides a method for producing a cell comprising nucleic acid according to the present invention or a carrier, the method comprising introducing nucleic acid according to the present invention or a carrier into the cell. In some embodiments, introducing nucleic acid according to the present invention or a carrier into the cell comprises conversion, transfection, electroporation or transduction (e.g., retroviral transduction). The method for producing a cell according to the present invention can be performed according to the method for producing a cell comprising nucleic acid / carrier known to those skilled in the art.

[0217] The present invention also provides a method for producing a cell comprising or expressing a polypeptide according to the present invention, the method comprising introducing a nucleic acid or a vector according to the present invention into a cell. In some embodiments, the method additionally comprises culturing cells under conditions suitable for expression of the nucleic acid or the vector by the cell. In some embodiments, the method is performed in vitro or ex vivo. In some embodiments, the method is performed in vivo.

[0218] The invention also provides a cell obtained or obtainable by a method according to the invention.

[0219] The present invention also provides a composition comprising the polypeptide, nucleic acid, vector or cell according to the present invention.

[0220] The polypeptides, nucleic acids, vectors and cells according to the present invention can be formulated into pharmaceutical compositions for clinical use, and may include pharmaceutically acceptable vehicles, diluents, excipients or adjuvants.

[0221] According to the present invention, a method for producing a pharmaceutically useful composition is also provided, which production method may include one or more steps selected from the following: isolating the polypeptide, cell, nucleic acid or vector as described herein; and / or mixing the polypeptide, cell, nucleic acid or vector as described herein with a pharmaceutically acceptable vehicle, adjuvant, excipient or diluent.

[0222] A kit of parts is also provided. In some embodiments, the kit may have at least one container having a predetermined amount of a polypeptide, nucleic acid, vector, cell and / or composition according to the invention.

[0223] The kit can provide polypeptide, nucleic acid, carrier, cell or composition to the subject together with the application instructions, so as to treat specific disease / illness. Polypeptide, nucleic acid, carrier, cell or composition can be formulated to be suitable for injection or infusion. In some embodiments, polypeptide, nucleic acid, carrier, cell or composition can be formulated to be suitable for intravenous, intraocular, subretinal, suprachoroidal or intraconjunctival injection, as the use (i.e. ophthalmic use) or oral administration of eye drops.

[0224] In some embodiments, the kit may include materials for producing cells according to the present invention. For example, the kit may include materials for modifying cells to express or include a polypeptide, nucleic acid or vector according to the present invention, or materials for introducing a nucleic acid or vector according to the present invention into cells.

[0225] In some embodiments, the test kit may further include at least one container having a predetermined amount of another therapeutic agent (e.g., a therapeutic agent for treating AMD). In such embodiments, the test kit may also include a second medicament or pharmaceutical composition so that two medicaments or pharmaceutical compositions can be administered simultaneously or separately, thereby providing a combined therapy for a specific disease or condition. In some embodiments, the second medicament or pharmaceutical composition includes complement factor I.

[0226] Producing peptides

[0227] The present invention also provides a method for producing a polypeptide according to the present invention, the method comprising: introducing a nucleic acid or a vector according to the present invention into a cell; and culturing the cell under conditions suitable for expression of the polypeptide. The polypeptide may be a fusion protein. The polypeptide may then be isolated and / or substantially purified.

[0228] The polypeptide according to the present invention can be prepared according to methods known to those skilled in the art for producing polypeptides.

[0229] The polypeptide can be prepared by chemical synthesis, such as liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using methods such as those described in Chandrudu et al., Molecules (2013), 18:4373-4388, the entire contents of which are incorporated herein by reference.

[0230] Alternatively, the polypeptide can be produced by recombinant expression. Molecular biological techniques suitable for recombinant production of polypeptides are well known in the art, such as those proposed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012 and Nat Methods. (2008); 5 (2): 135-146, the entire contents of both of which are incorporated herein by reference.

[0231] For recombinant production according to the present invention, any cell suitable for expression of the polypeptide can be used. The cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell, such as a cell of an archaea or a bacterium. In some embodiments, the bacterium can be a gram-negative bacterium, such as a bacterium of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, for example a CHO, HEK (e.g., HEK293), HeLa, or COS cell.

[0232] In some cases, the cell is not a prokaryotic cell, as some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotic cells, and proteins can be more easily purified from eukaryotic cells using appropriate tags. Specific plasmids that enhance secretion of the protein into the culture medium can also be used.

[0233] In some embodiments, the polypeptide can be prepared by cell-free-protein synthesis (CFPS), for example using the system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431, the entire contents of which are incorporated herein by reference.

[0234] Production can involve the cultivation or fermentation of eukaryotic cells modified to express the target (of interest) polypeptide. Cultivation or fermentation can be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by distributing culture medium / fermentation broth from cells, extracting protein content, and separating individual proteins to separate secreted polypeptides. Cultivation, fermentation and separation techniques are well known to those skilled in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, which is incorporated by reference above).

[0235] A bioreactor includes one or more containers in which cells can be cultured. Cultivation in a bioreactor can occur continuously, with reactants flowing continuously into the reactor and cultured cells flowing continuously out of the reactor. Alternatively, cultivation can occur in batches. A bioreactor monitors and controls environmental conditions, such as pH, oxygen, flow rates in and out of the container, and agitation within the container, to provide optimal conditions for the cultured cells.

[0236] After culturing cells expressing antigen binding molecules / polypeptides, the target polypeptide can be separated. Any suitable method known in the art for separating proteins from cells can be used. In order to separate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide is secreted from the cells, the cells can be separated from the culture medium containing the secreted target polypeptide by centrifugation. If the target polypeptide is aggregated in the cell, protein separation can include: centrifugation to separate the cells from the cell culture medium; treatment of the cell pellet with a lysis buffer; and cell destruction, for example, by ultrasonic treatment, rapid freeze-thaw or osmotic lysis.

[0237] Then it may be necessary to separate the target polypeptide from the supernatant or culture medium, which may contain other proteins and non-protein components. A common method for separating protein components from supernatants or culture medium is by precipitation. Proteins of different solubility are precipitated under different concentrations of precipitation agents, such as ammonium sulfate. For example, under low concentrations of precipitation agents, water-soluble proteins are extracted. Therefore, by adding different increasing concentrations of precipitation agents, proteins of different solubility can be distinguished. Dialysis can then be used to remove ammonium sulfate from the separated protein.

[0238] Other methods for distinguishing different proteins are known in the art, such as ion exchange chromatography and size chromatography. The polypeptides can also be affinity-purified using binding partners for appropriate molecular tags on the polypeptides (e.g., His, FLAG, Myc, GST, MBP, HA, E or biotin tags). These can be used as an alternative to precipitation, or can be performed after precipitation.

[0239] In some cases, it may be desirable to further process the polypeptide, for example to remove sequences of amino acids, molecular tags, moieties, etc.

[0240] In some embodiments, the amino acid sequence is cleaved and removed by treatment with an appropriate endopeptidase.

[0241] In some embodiments, an enzyme is used to remove the target moiety. In some embodiments, the polypeptide is treated to remove glycans (ie, the polypeptide is deglycosylated), for example, by treatment with a glycosidase, such as with peptide:N-glycosidase (PNGase).

[0242] Once the polypeptide of interest has been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide. Many methods of concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.

[0243] In some embodiments, the generation of polypeptide occurs in vivo, for example, after the cell comprising nucleic acid encoding polypeptide of the present invention or vector is introduced into host, or after nucleic acid encoding polypeptide of the present invention or vector is introduced into host cell.In such embodiments, polypeptide is transcribed, translated and post-translation processed into mature polypeptide.In some embodiments, polypeptide is produced in situ at desired position in host.In some embodiments, desired position is eyes, for example, in the cell of retina, choroid, retinal pigment epithelium (RPE) or macula lutea.In some embodiments, desired position is at retinal cell or in retinal cell.In some embodiments, desired position is at RPE cell or in RPE cell.

[0244] Therapeutic applications

[0245] Any of the polypeptides, nucleic acids, vectors, cells and pharmaceutical compositions according to the invention are used in a method of treatment or prevention.

[0246] The present invention provides a polypeptide, nucleic acid, vector, cell or pharmaceutical composition according to the present invention for use in medical treatment or prophylaxis. The present invention also provides the use of a polypeptide, nucleic acid, vector, cell or pharmaceutical composition according to the present invention in the manufacture of a medicament for treating or preventing a disease or condition. The present invention also provides a method for treating or preventing a disease or condition, the method comprising administering to a subject a therapeutically or prophylactically effective amount of a polypeptide, nucleic acid, vector, cell or pharmaceutical composition according to the present invention.

[0247] In particular, the polypeptides, nucleic acids, vectors, cells and pharmaceutical compositions according to the present invention are used to treat or prevent diseases / disorders associated with complement dysregulation, particularly an overactive complement response. In some embodiments, an overactive complement response is associated with the presence of C3b. In some embodiments, the disease / disorder to be treated or prevented is a complement-related disease. In some embodiments, the disease / disorder to be treated or prevented is pathologically associated with complement activation. In some embodiments, the disease / disorder to be treated or prevented is pathologically associated with complement overactivation. In some embodiments, the disease / disorder to be treated or prevented is driven by complement activation or overactivation. In some embodiments, the disease / disorder is complement activation or overactivation.

[0248] The polypeptides, nucleic acids, vectors, cells and pharmaceutical compositions are used to treat or prevent diseases / disorders that would benefit from one or more of the following: decreased levels or activity of a C3bBb-type C3 convertase, a C3bBb3b-type C5 convertase, or a C4b2a3b-type C5 convertase; decreased levels of C3b, C5b, or C5a; increased levels of iC3b, C3f, C3dg, or C3d; or decreased levels or activity of iC3b; and increased levels of C3f, C3dg, or C3d.

[0249] For example, "treatment" can be a decrease in the development or progression of a disease / disorder, a reduction in the symptoms of a disease / disorder, or a decrease in the pathology of a disease / disorder. Treatment or relief of a disease / disorder can effectively prevent the progression of a disease / disorder, such as preventing the deterioration of the disorder or slowing the rate of development. In some embodiments, treatment or relief can lead to an improvement in the disease / disorder, such as a decrease in the symptoms of the disease / disorder or a decrease in some other correlation of the severity / activity of the disease / disorder. Prevention / prophylaxis of a disease / disorder can refer to preventing the deterioration of a disorder or preventing the development of a disease / disorder, such as preventing an early disease / disorder from developing into a late, chronic phase.

[0250] In some embodiments, the disease or condition to be treated or prevented can be a disease / condition associated with C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b. That is, in some embodiments, the disease or condition to be treated or prevented is a disease / condition in which C3b, a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of said activity / response is pathologically implicated. In some embodiments, the disease / condition can be associated with an increased level of C3b or a complex containing C3b, an increased level of an activity / response associated with C3b or a complex containing C3b, or an increased level of a product of an activity / response associated with C3b or a complex containing C3b, as compared to a control state.

[0251] Treatment may be directed to reducing the level of C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b. In some embodiments, treatment is directed to: reducing the level or activity of a C3bBb-type C3 convertase, a C3bBb3b-type C5 convertase, or a C4b2a3b-type C5 convertase; reducing the level of C3b, C5b, or C5a; increasing the level of iC3b, C3f, C3dg, or C3d, or reducing the level of iC3b and increasing the level of C3f, C3dg, or C3d.

[0252] Administration of the polypeptides, nucleic acids, vectors, cells and compositions of the invention may result in a decrease in the level of C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b, through cleavage of C3b.

[0253] In some embodiments, treatment may be intended to reduce the level of C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b, in, for example, a specific location, in a specific organ, tissue, structure, or cell type of a subject. In some embodiments, treatment may be intended to reduce the level of C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b, in, for example, the retina, choroid, RPE, macula, and / or at the BrM / RPE interface in the eye.

[0254] In some embodiments, treatment can include modified cells or cell groups to include / express polypeptides, nucleic acids or carriers of the present invention. In some embodiments, treatment can include modified cells / groups in vivo to produce polypeptides of the present invention in situ. In some embodiments, cells / cell groups are eye cells. In some embodiments, cells / cell groups are RPE cells and / or RPE cell groups. In some embodiments, cells / cell groups are photoreceptor cells and / or photoreceptor cell groups.

[0255] In some embodiments, the present invention provides nucleic acid or vector of the present invention used in gene therapy. In some embodiments, treatment includes administering nucleic acid and / or vector to a subject. In some embodiments, treatment includes introducing nucleic acid and / or vector into a subject's cell using techniques described herein or known in the art, and techniques known in the art are described, for example, in MacLaren et al. Ophthalmology. 2016, 123 (10 Suppl): S98–S106, Aguirre, Invest Ophthalmol Vis Sci. 2017, 58 (12): 5399–5411, Lundstrom, Diseases. 2018, 6 (2): 42, all of which are incorporated herein by reference. In some embodiments, the cell is one or more ocular cells. In some embodiments, the cell is one or more RPE cells. In some embodiments, the cell is one or more photoreceptor cells.

[0256] In some embodiments, treatment may include administering to a subject a cell or cell population modified to include / express a polypeptide, nucleic acid or vector of the invention. In some embodiments, treatment may include modifying a cell / population ex vivo or in vitro.

[0257] In some embodiments, treatment involves providing the subject with a cell or cell population that produces and / or will produce a polypeptide of the invention, for example by administering a cell according to the invention, or by generating a cell according to the invention.

[0258] In some embodiments, the cell mentioned herein is a cell of the eye, i.e. an eye cell. In some embodiments, the cell is a cell of the retina, choroid, retinal pigment epithelium (RPE) or macula lutea. In some embodiments, the cell is a retinal cell. In some embodiments, the cell is an RPE cell. In some embodiments, the cell is a photoreceptor cell.

[0259] The invention provides a method for treating or preventing a disease or condition in a subject, the method comprising modifying at least one cell to express or include a polypeptide, nucleic acid or carrier according to the present invention. In some embodiments, the at least one cell is an eye cell. In some embodiments, the at least one cell is a RPE cell.

[0260] At least one cell modified according to the present invention can be modified according to methods known to technicians. Modification can include nucleic acid transfer, for permanent or transient expression of the nucleic acid transferred. Any suitable genetic engineering platform can be used to modify the cell according to the present invention. Suitable methods for modifying cells include using genetic engineering platforms, such as gamma retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, DNA transfection, transposon-based gene delivery and RNA transfection, such as Maus et al., Annu Rev Immunol (2014) 32: described in 189-225, which is incorporated by reference above.

[0261] The subject to be treated can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be a non-human mammal, but more preferably a human. The subject can be male or female. The subject can be a patient. The subject may have been diagnosed with a disease or condition that requires treatment, or is suspected of having such a disease or condition.

[0262] The subject to be treated can exhibit elevated levels of C3b or a C3b-containing complex, an activity / response associated with C3b or a C3b-containing complex, or a product of an activity / reaction associated with C3b or a C3b-containing complex, e.g., as determined by analyzing the subject, or a sample (e.g., a cell, tissue, blood sample) obtained from the subject, using an appropriate assay.

[0263] The subject may have increased levels of expression or activity of a positive modulator / effector of C3b or a complex containing C3b, or an activity / response associated with C3b or a complex containing C3b, or may have increased levels of expression or activity of a product of an activity / response associated with C3b or a complex containing C3b. The subject may have increased levels of an activity upregulated by C3b or a complex containing C3b.

[0264] The subject may have a reduced level of expression or activity of a negative regulator of C3b or a complex containing C3b, or an activity / response associated with C3b or a complex containing C3b, or may have a reduced level of expression or activity of a factor that is down-regulated by C3b or a complex containing C3b. The subject may have a reduced level of an activity that is down-regulated by C3b or a complex containing C3b.

[0265] The increase / decrease may be related to the level of expression / activity in the absence of the relevant disease / disorder, such as the level of expression / activity in a healthy control subject or a sample obtained from a healthy control subject.

[0266] In some embodiments, the subject may be at risk of developing / infecting a disease or condition. In some embodiments, the subject may have one or more pathogenic factors that increase the risk of developing / infecting a disease or condition.

[0267] In some embodiments, the subject may have one or more risk factors for age-related macular degeneration (AMD). In some embodiments, the subject may have one or more AMD-associated genetic variants. AMD-associated genetic variants are described in, for example, Clark et al., J Clin Med (2015) 4 (1): 18-31, the entire contents of which are incorporated herein by reference. In some embodiments, the subject may have one or more of the following AMD-associated genetic variants (or have LD = r 2 ≥0.8, variants with this variant): Y402H in CFH (i.e., rs1061170 C )、rs1410996 C , I62V in CFH, R53C in CFH, D90G in CFH, R1210C in CFH, rs6685931 in CFHR4T .

[0268] In some embodiments, the subject may have one or more risk factors for early-onset macular degeneration (EOMD). EOMD is believed to be caused by monogenic inheritance of rare variants of the CFH gene (see, e.g., Boon CJ et al. AmJ Hum Genet 2008; 82(2): 516-23, van de Ven JP, et al. Arch Ophthalmol 2012; 130(8): 1038-47, Yu Y et al. Hum Mol Genet 2014; 23(19): 5283-93, Duvvari MR, et al. Mol Vis 2015; 21: 285-92 Hughes AE, et al. Acta Ophthalmol 2016; 94(3): e247-8, Wagner et al. Sci Rep 2016; 6: 31531). In some embodiments, the subject may have one or more EOMD-associated genetic variants. EOMD-associated genetic variants are described, for example, in Servais A et al. Kidney Int, 2012; 82(4):454-64 and Dragon-Durey MA, et al. J Am Soc Nephrol 2004; 15(3):787-95, the entire contents of which are incorporated herein by reference. In some embodiments, the subject may have one or more of the following EOMD-associated genetic variants: CFH c.1243del, p.(Ala415Profs*39)het; CFH c.350+1G>T het; CFH c.619+1G>Ahet; CFH c.380G>A, p.(Arg127His); CFH c.694C>T, p.(Arg232Ter); or CFH c.1291T>A, p.(Cys431Ser).

[0269] In some embodiments, based on determining that the subject has one or more risk factors for AMD and / or EOMD, for example one or more AMD / EOMD associated genetic variants, select the subject to use the treatment or preventive treatment of polypeptide, nucleic acid, carrier, cell or composition of the present invention.In some embodiments, it has been determined that the subject suffers from one or more such risk factors.In some embodiments, method of the present invention relates to determining whether the subject has one or more such risk factors.

[0270] In some embodiments, the disease or condition to be treated or prevented can be an ocular disease / disorder. In some embodiments, the disease or condition to be treated or prevented is a complement-related ocular disease. In some embodiments, the disease or condition to be treated or prevented is macular degeneration. In some embodiments, the disease or condition to be treated or prevented is age-related macular degeneration (AMD). AMD is generally defined as causing a loss of vision in subjects over 50 years old.

[0271] In some embodiments, the disease or condition to be treated or prevented is selected from: age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, geographic atrophy ("dry" (i.e., non-exudative) AMD), "wet" (neovascular or exudative) AMD, choroidal neovascularization (CNV), glaucoma, autoimmune uveitis, and diabetic retinopathy. In some embodiments, the disease or condition to be treated or prevented is AMD. In some embodiments, the disease or condition to be treated or prevented is geographic atrophy ("dry" AMD). In some embodiments, the disease or condition to be treated or prevented is "wet" AMD. In some embodiments, the disease or condition to be treated or prevented is a combination of the above-mentioned diseases / conditions, such as "dry" and "wet" AMD. In some embodiments, the disease or condition to be treated or prevented is not "wet" AMD or choroidal neovascularization. In some embodiments, the subject to be treated is over 50 years old, i.e., at least 50 years old.

[0272] As used herein, "early AMD" refers to the stage of an AMD, characterized in that there are medium-sized drusen in the Bruch's membrane adjacent to the RPE layer, usually with a width of ~200 μm. Subjects suffering from early AMD usually do not experience significant vision loss. As used herein, "mid-term AMD" refers to the stage of an AMD, characterized in that large drusen and / or pigment changes in the retina. Mid-term AMD may be accompanied by some vision loss. As used herein, "late AMD" refers to the stage of an AMD, characterized in that drusen and vision loss exist due to damage to the macula. In all stages of AMD, there may be "reticular pseudodrusen (reticular pseudodrusen, RPD)" or "reticular drusen", which refers to the accumulation of extracellular material in the subretinal space (subretinalspace) between the retinal neurosensory layer and the RPE. "Late AMD" covers "dry" and "wet" AMD. In "dry" AMD (also referred to as map-like atrophy), the photosensitive cells that transmit visual information to the brain in the macula gradually decompose, and the supporting tissue below the macula gradually decomposes. In "wet" AMD (also called choroidal neovascularization and exudative AMD), abnormal blood vessels grow beneath the retina and into the retina. These blood vessels leak fluid and blood, which can cause swelling and damage to the macula, and subsequent scarring. This damage can be rapid and severe.

[0273] In some embodiments, the disease or condition to be treated or prevented is early-onset macular degeneration (EOMD). As used herein, "EOMD" refers to a subtype of macular degeneration that is phenotypically severe, which exhibits a much earlier age of onset than classic AMD and leads to more years of substantial vision loss. The EOMD subset is described in, for example, Boon CJ et al. Am J Hum Genet 2008; 82 (2): 516-23 and van de Ven JP, et al. Arch Ophthalmol 2012; 130 (8): 1038-47. In some embodiments, the subject to be treated is under 49 years old. In some embodiments, the subject to be treated is between 15 and 49 years old, i.e., between 15 and 49 years old.

[0274] In some embodiments, the disease or condition to be treated or prevented is a disease / condition driven by excessive complement activation. In some embodiments, the disease or condition to be treated or prevented can be selected from: atypical hemolytic uremic syndrome (aHUS), Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, and paroxysmal nocturnal hemoglobinuria (PNH).

[0275] The medical treatment method may also involve in vivo, ex vivo and adoptive immunotherapy, including immunotherapy using syngeneic and / or allogeneic cells or immortalized cell lines.

[0276] Administration of the polypeptides described herein is preferably carried out in a "therapeutically effective amount", which is sufficient to show benefit to an individual. The actual amount administered, as well as the rate and time course of administration will depend on the nature and severity of the disease being treated. The prescription of treatment, such as the determination of dosage, etc., is within the responsibility of general practitioners and other physicians, and generally takes into account the condition to be treated, the condition of the individual patient, the delivery site, the method of administration, and other factors known to practitioners. Examples of the above-mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0277] According to the polypeptide of the present invention, nucleic acid, carrier and cell can be formulated into pharmaceutical composition or medicament for clinical use, and can include pharmaceutically acceptable vehicle, diluent, excipient or adjuvant.Said composition can be formulated into local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral or transdermal route of administration, and route of administration can include injection or infusion, or as the application (i.e. ophthalmic application) of eye drops.Suitable preparation can include polypeptide, nucleic acid, carrier or cell in sterile or isotonic culture medium.Medicament and pharmaceutical composition can be formulated into liquid form including gel.Liquid preparation can be formulated into selected organ or region applied to human or animal body by injection or infusion (e.g., via catheter).In some embodiments, polypeptide, nucleic acid, carrier, cell and composition of the present invention are formulated into intravitreal route of administration for example by intravitreal injection. In some embodiments, the polypeptides, nucleic acids, vectors, cells and compositions of the invention are formulated for submacular delivery, ie, direct contact of the therapeutic molecule with the target cell layer.

[0278] The position of C3b inactivation can be selected as required for specific mode of administration and / or site. In some embodiments, polypeptide, nucleic acid, carrier or pharmaceutical composition of the present invention are formulated for use and / or are administered to the subretinal space between the photoreceptor cells in the eye and the retinal pigment epithelium (RPE). In some embodiments, polypeptide, nucleic acid, carrier or pharmaceutical composition of the present invention are formulated for use and / or are administered to the retinal pigment epithelium (RPE).

[0279] According to the present invention, a method for producing a pharmaceutically useful composition is also provided, which production method may include one or more steps selected from the following: isolating the polypeptide, nucleic acid, vector or cell described herein; and / or mixing the polypeptide, nucleic acid, vector or cell described herein with a pharmaceutically acceptable vehicle, adjuvant, excipient or diluent.

[0280] For example, another aspect of the invention relates to a method of formulating or producing a medicament or pharmaceutical composition for use in a medical method, the method comprising formulating a pharmaceutical composition or medicament by mixing a polypeptide, nucleic acid, vector or cell described herein with a pharmaceutically acceptable vehicle, adjuvant, excipient or diluent.

[0281] Can be administered alone or in combination with other treatments (eg other therapeutic interventions or preventive interventions), either simultaneously or sequentially depending on the condition to be treated. The polypeptides, nucleic acids, vectors, cells or compositions and therapeutic agents according to the invention can be administered simultaneously or sequentially.

[0282] Administration simultaneously refers to the administration of a polypeptide, nucleic acid, vector, cell or composition and a therapeutic agent together, for example, as a pharmaceutical composition (combined preparation) comprising two agents, or immediately after each other and optionally by the same route of administration, for example, to the same tissue, artery, vein or other blood vessel. Sequential administration refers to the administration of one of a polypeptide, nucleic acid, vector, cell or composition or therapeutic agent, and then the administration of another agent separately after a given time interval. Although this is the case in some embodiments, it is not necessary to administer two agents by the same route. The time interval can be any time interval. In some embodiments, a polypeptide, nucleic acid, vector, cell or composition and a therapeutic agent are separately, simultaneously or sequentially applied to the eye.

[0283] In some embodiments, another treatment / therapeutic agent is a therapeutically effective amount of complement factor I. In some embodiments, complement factor I is administered to a subject simultaneously or sequentially with the administration of a polypeptide, nucleic acid, vector, cell or pharmaceutical composition according to the present invention. In some embodiments, treatment may include modifying a cell or cell group in vitro, ex vivo or in vivo to express and / or secrete complement factor I. The cell or cell group may be the same cell or cell group as the cell or cell group modified to include / express a polypeptide, nucleic acid or vector according to the present invention, for example, treatment may include modifying a cell or cell group in vitro, ex vivo or in vivo to express and / or secrete a polypeptide, nucleic acid or vector according to the present invention, and complement factor I. In some embodiments, complement factor I is administered to a subject, wherein the subject includes a cell or cell group modified to include / express a polypeptide, nucleic acid or vector according to the present invention. In some embodiments, complement factor I is administered to a subject, wherein the subject has expressed or is expressing a polypeptide, nucleic acid or vector of the present invention in situ.

[0284] Complement factor I or a composition comprising complement factor I can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intravitreal, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration, which may include injection or infusion, or administration as eye drops (i.e., ophthalmic administration). Suitable preparations may comprise sterile or isotonic culture media. Medicaments and pharmaceutical compositions may be formulated into liquid forms including gels. Liquid preparations may be formulated to be administered to selected organs or regions of the human or animal body by injection or infusion (e.g., via a catheter).

[0285] In some embodiments, the other treatment / therapeutic agent is a therapeutically effective amount of an anti-VEGF therapy (e.g., ranibizumab (Lucentis; Genentech / Novartis); bevacizumab (off label Avastin; Genentech); aflibercept (Eylea / VEGF Trap-Eye; Regeneron / Bayer); pegaptanib Laser photocoagulation; or, for example, using Visudyne TM (verteporfin) photodynamic therapy (PDT).

[0286] Multiple doses of polypeptides, nucleic acids, vectors, cells or compositions may be provided. One or more doses or each dose may be administered simultaneously or sequentially with another therapeutic agent.

[0287] Multiple doses can be separated by predetermined time intervals, which can be selected as one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days, or 1, 2, 3, 4, 5 or 6 months. For example, a dose can be given once every 7, 14, 21 or 28 days (plus or minus 3, 2 or 1 day).

[0288] Can be formulated in sustained-release delivery system according to polypeptide, nucleic acid, carrier or composition of the present invention, so that discharge polypeptide, nucleic acid, carrier or composition with predetermined rate.Sustained-release delivery system can keep constant drug / treatment concentration in the specified time period.In some embodiments, polypeptide, nucleic acid, carrier or composition of the present invention is formulated into liposome, gel, implant, device or drug-polymer conjugate, for example hydrogel.

[0289] Genetic Factors for Age-Related Macular Degeneration (AMD)

[0290] Complement factor H (encoded by the CHF gene) is a cofactor of another complement factor I. The structure and function of complement factor H are reviewed in Wu et al., Nat Immunol (2009) 10 (7): 728-733, the entire contents of which are incorporated herein by reference. Human complement factor H (UniProt: P08603; SEQ ID NO: 29) has a 1233 amino acid sequence (including a signal peptide of 18 amino acids at the N-terminus) and includes 20 complement control protein (CCP) domains. The first four CCP domains of complement factor H (i.e., CCP1 to CCP4) are essential for the activity of complement factor I cofactor to cleave C3b into iC3b. CCPs 19 to 20 have also been shown to bind to C3b and C3d (Morgan et al., Nat Struct Mol Biol (2011) 18(4):463-470), while CCP7 and CCP19-20 bind to glycosaminoglycan (GAG) and sialic acid and are involved in the discrimination between self and non-self (Schmidt et al., J Immunol (2008) 181(4):2610-2619, Kajander et al., PNAS (2011) 108(7):2897-2902).

[0291] One of the major SNPs associated with the genetic risk of developing AMD is present in the CFH gene and results in the Y402H polymorphism in complement factor H (e.g., see Haines et al., Science (2005) 308: 419-21), and it is an alternative indirect variant factor H-like protein 1 (FHL-1). Approximately 30% of European white genetic individuals have at least one copy of this polymorphism, and being a heterozygote increases the risk of AMD by ~3 times (Sofat et al., Int J Epidemiol (2012) 41: 250-262). The Y402H polymorphism embodied in the seventh complement control protein (CCP) domain reduces the binding of FH / FHL-1 to BrM, resulting in the binding of these blood-borne complement regulators and the disturbance in the apparently inhibited complement regulation (Clark et al., J Biol Chem (2010) 285: 30192-202).

[0292] The binding of FH / FHL-1 to BrM is mediated by sulfated sugars, including glycosaminoglucosan (GAG), heparan sulfate (HS) and dermatansulphate (DS). The GAG ​​sequence family present in BrM appears to have greater tissue specificity than previously thought, as they are able to recruit FH / FHL-1 through its CCP7 domain rather than the secondary anchoring site of FH present in CCP19-20 (Clark et al., J Immunol (2013) 190: 2049-2057). This may be an evolutionary twist, as the major regulator of complement in BrM has been found to be a truncated FHL-1 protein (Clark et al J. Immunol (2014) 193, 4962-4970), which has only one surface anchoring site in CCP7 and lacks CCP19-20. In contrast, the Y402H polymorphism is not associated with renal disease, where the CCP19-20 domain of FH is known to be the major GAG-mediated anchoring site (Clark et al., J Immunol (2013) 190: 2049-2057). Age-related changes in BrM expression levels in HS and DS, which themselves are also considered part of the normal aging process, are also associated with AMD and may explain, to some extent, the age-related nature of genetically driven AMD.

[0293] Rare mutation (R1210C) in the C-terminal CCP19-20 region of FH not combined with BrM, has a very high association level with AMD, and finds that the FH protein carrying this mutation is covalently bound to albumin (Sánchez-Corral et al., Am J Hum Genet (2002) 71: 1285-1295), preventing FH protein from leaving the circulatory system and entering eye tissue. Some studies show that the associated pigment changes in the large fusion drusen and RPE before the geographic atrophy show that dry AMD is first caused by the RPE dysfunction with secondary effect in the choroid (Bhutto and Lutty Mol Aspects Med (2012) 33: 295-317). On the contrary, Whitmore et al. reported the changes of choriocapillaris layer before all forms of late AMD including the deposition of terminal complement membrane attack complex (MAC), and believed that excessive complement activation in choriocapillaris layer is the main event (Whitmore et al., Prog Retin Eye Res (2015) 45: 1-29) of RPE atrophy secondary. These data show that the genetic susceptibility given by the change of complement genes is tolerant, until the change of BrM and lower choriocapillaris layer emerges. Whether these changes are age-related, driven by oxidative stress or caused by RPE cell dysfunction, it remains to be seen, but it is known that the changes occurring naturally in these structures are age-related.

[0294] C3 and C3b

[0295] Complement component 3 (C3) is an immune system protein that plays a central role in the innate immune and complement systems. The processing of C3 is described, for example, in Foley et al. J Thromb Haemostasis (2015) 13: 610-618, the entire contents of which are incorporated herein by reference. Human C3 (UniProt: P01024; SEQ ID NO: 18) includes a sequence of 1663 amino acids (including an N-terminal, 22 amino acid signal peptide). Amino acids 23 to 667 encode the C3 β chain (SEQ ID NO: 19), and amino acids 749 to 1663 encode the C3 α′ chain (SEQ ID NO: 20). The C3 β chain and the C3 α′ chain associate to form C3b via an interchain disulfide bond (formed between cysteine ​​559 of the C3 β chain and cysteine ​​816 of the C3 α′ chain). C3a is a 77 amino acid fragment corresponding to amino acid positions 672 to 748 of C3 (SEQ ID NO: 21) that is generated by proteolytic cleavage of C3 following activation by both the classical complement pathway and the lectin pathway.

[0296] C3b is an effective opsonin that targets pathogens, antibody-antigen immune complexes, and apoptotic cells for phagocytosis by phagocytes and NK cells. C3b also participates in the formation of convertase complexes to activate and amplify complement responses. C3b associates with factor B to form a C3bBb-type C3 convertase (alternative complement pathway), and can associate with C4b and C2a to form a C4b2a3b-type C5 convertase (classical pathway), or associate with C3bBb to form a C3bBb3b-type C5 convertase (alternative pathway).

[0297] Processing of C3b to form iC3b, which is proteolytically inactivated and cannot itself promote further complement expansion, involves proteolytic cleavage of the C3b α' chain at amino acid positions 1303 and 1320 to form α' chain fragment 1 (corresponding to amino acid positions 672-748 of C3; SEQ ID NO: 22), α' chain fragment 2 (corresponding to amino acid positions 1321-1663 of C3; SEQ ID NO: 23). Thus, iC3b includes the C3 β chain, C3 α' chain fragment 1, and C3 α' chain fragment 2 (associated by a disulfide bond). Cleavage of the α chain also releases C3f, which corresponds to amino acid positions 1304-1320 of C3 (SEQ ID NO: 24).

[0298] As used herein, "C3" refers to C3 from any species, and includes isoforms, fragments, variants or homologs of C3 from any species. In some embodiments, C3 is mammalian C3 (e.g., cynomolgus monkey, human and / or rodent (e.g., rat and / or mouse) C3). An isoform, fragment, variant or homolog of C3 can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of immature or mature C3 from a given species, such as human C3 (SEQ ID NO: 18).

[0299] As used herein, "C3b" refers to and includes isoforms, fragments, variants, or homologs of C3b from any species. In some embodiments, C3b is mammalian C3b (e.g., cynomolgus monkey, human, and / or rodent (e.g., rat and / or mouse) C3b).

[0300] An isoform, fragment, variant or homolog of C3b can optionally be characterized as including C3α' chain fragment 1, C3α' chain fragment 2 and C3β, which have at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of the individual polypeptides from a given species, such as human. That is, C3b may include: C3 α3 chain fragment 1, which has at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 22; C3 α' chain fragment 2, which has at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 23; and C3 β chain, which has at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 24. NO:19 has at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0301] An isoform, fragment, variant or homologue of C3b may optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of a reference C3b, as determined by analysis of a suitable assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of C3b may be characterized by the ability to act as an opsonin, and / or to form a functional C3 / C5 convertase.

[0302] Complement Factor I

[0303] Processing of C3b to iC3b is performed by complement factor I (encoded in humans by the gene CFI). Human complement factor I (UniProt: P05156; SEQ ID NO: 25) has a sequence of 583 amino acids (including an N-terminal, 18 amino acid signal peptide). The precursor polypeptide is cleaved by furin to produce mature complement factor I, which includes a heavy chain (amino acids 19 to 335) and a light chain (amino acids 340 to 583) linked by interchain disulfide bonds. Amino acids 340 to 574 of the light chain encode the proteolytic domain of complement factor I (SEQ ID NO: 26), which is a serine protease containing the catalytic triad responsible for cleaving C3b to produce iC3b (Ekdahl et al., J Immunol (1990) 144 (11): 4269-74).

[0304] As used herein, "complement factor I (FI)" refers to complement factor I from any species, and includes isoforms, fragments, variants, or homologs of complement factor I from any species. In some embodiments, complement factor I is mammalian complement factor I (e.g., cynomolgus monkey, human, and / or rodent (e.g., rat and / or murine) complement factor I).

[0305] An isoform, fragment, variant or homologue of complement factor I may optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of immature or mature complement factor I from a given species, such as human complement factor I (SEQ ID NO: 25). An isoform, fragment, variant or homologue of complement factor I may optionally be a functional isoform, fragment, variant or homologue, such as having a functional property / activity of a reference complement factor I (e.g., full-length human complement factor I), as determined by analysis of an appropriate assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of complement factor I may exhibit serine protease activity and / or may be capable of inactivating C3b.

[0306] Proteolytic cleavage of C3b by complement factor I to generate iC3b is facilitated by cofactors of complement factor I. Cofactors of complement factor I typically bind to C3b and / or complement factor I and enhance processing of C3b to iC3b by complement factor I.

[0307] Complement receptor 1

[0308] Complement receptor 1 (CR1) serves as a cofactor for complement factor I, which is able to cleave C3b into iC3b and downstream products.

[0309] iC3b does not amplify or activate the complement system, but can still act as an opsonin to target pathogens for phagocytosis. Therefore, the production of iC3b leads to local immune system activation and inflammatory effects. This may negatively impact patients with complement-associated diseases and may contribute to the development or exacerbation of existing complement-associated diseases / disorders.

[0310] Factor H (FH) and the truncated FH isoform FHL-1 act as cofactors for FI to generate iC3b, but they are unable to promote further degradation of iC3b, which can lead to undesirable iC3b accumulation. In addition, accumulation of iC3b may promote further debris in the affected area, leading to the (further) development of drusen in macular degeneration, for example.

[0311] In contrast, see e.g. Figure 2 , Figure 3B , Fig. 7A , CR1 and polypeptides according to the present invention can act in combination with FI to promote further breakdown of iC3b into favorable downstream products, such as C3c, C3dg and C3b. These molecules are not opsonins and therefore avoid recruiting immune system components. Their presence in the affected area is better than iC3b accumulation.

[0312] As used herein, "complement receptor 1 (CR1)" refers to CR1 from any species, and includes isoforms, fragments, variants or homologs of CR1 from any species. In some embodiments, CR1 is mammalian CR1 (e.g., cynomolgus monkey, human and / or rodent (e.g., rat and / or mouse) CR1).

[0313] ***

[0314] Aspects and embodiments of the present invention will now be described by way of example and with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0315] The present invention includes combinations of the described aspects and preferred features unless such combinations are expressly impermissible or explicitly avoided.

[0316] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0317] The features disclosed in the preceding description, or in the following claims or in the accompanying drawings, expressed in their specific form, or in terms of means for performing the disclosed functions or methods or processes for obtaining the disclosed results, may be utilized separately or in any combination of these features to implement the invention in a variety of forms, as the case may be.

[0318] Although the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when the present invention is given. Therefore, the exemplary embodiments of the present invention described above are considered to be illustrative, rather than restrictive. Various changes may be made to the embodiments without departing from the spirit and scope of the present invention.

[0319] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0320] Throughout the specification, including the following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0321] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about," it will be understood that the particular value forms another embodiment.

[0322] Where a nucleic acid sequence is disclosed, its reverse complement is also expressly contemplated.

[0323] The following numbered paragraphs (paras) describe specific aspects and embodiments of the invention:

[0324] 1. A polypeptide having at least 80% sequence identity with SEQ ID NO: 4, wherein the polypeptide has a total length of less than 700 amino acids.

[0325] 2. The polypeptide according to paragraph 1, wherein the polypeptide has a length of 50-700 amino acids.

[0326] 3. A polypeptide according to paragraph 1 or 2, wherein X 1 A or T, X 2 P or L, and / or X 3 It is G or R.

[0327] 4. A polypeptide according to any one of paragraphs 1-3, comprising SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 13.

[0328] 5. The polypeptide according to any one of paragraphs 1-4, which consists of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 13.

[0329] 6. A polypeptide according to any one of paragraphs 1-5, which is capable of binding to C3b.

[0330] 7. A polypeptide according to any of paragraphs 1-6, which binds to C3b in the region bound by a cofactor of complement factor I.

[0331] 8. A polypeptide according to any of paragraphs 1-7, which binds to C3b in the region that binds through complement receptor 1 (CR1).

[0332] 9. A polypeptide according to any one of paragraphs 1-8, which acts as a cofactor for complement factor I.

[0333] 10. A polypeptide according to any one of paragraphs 1-9, which is capable of diffusing across Bruch's membrane (BrM).

[0334] 11. The polypeptide according to any one of paragraphs 1-10, which is non-glycosylated or partially glycosylated.

[0335] 12. A polypeptide according to any of paragraphs 1-11, wherein the amino acid sequence comprises one or more amino acid substitutions at positions 509, 578, 959 and / or 1028 (numbered according to Uniprot: P17927).

[0336] 13. A polypeptide according to paragraph 12, wherein the one or more amino acid substitutions are selected from N509Q, N578Q, N959Q and / or N1028Q (numbered according to Uniprot: P17927).

[0337] 14. A polypeptide according to any one of paragraphs 1 to 13, comprising or consisting of SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 15.

[0338] 15. A polypeptide according to any of paragraphs 1-14, which additionally comprises a secretory pathway sequence.

[0339] 16. The polypeptide of paragraph 15, wherein the secretory pathway sequence comprises SEQ ID NO: 7.

[0340] 17. A polypeptide according to paragraph 15 or paragraph 16, wherein the polypeptide additionally comprises a cleavage site for removing the secretory pathway sequence.

[0341] 18. A nucleic acid encoding a polypeptide according to any one of paragraphs 1-17.

[0342] 19. A vector comprising the nucleic acid according to paragraph 18.

[0343] 20. A cell comprising a polypeptide according to any of paragraphs 1-17, a nucleic acid according to paragraph 18, or a vector according to paragraph 19.

[0344] 21. A method for producing a polypeptide, comprising: introducing the nucleic acid according to paragraph 18 or the vector according to paragraph 19 into a cell; and culturing the cell under conditions suitable for expression of the polypeptide.

[0345] 22. A cell obtained or obtainable by the method according to paragraph 21.

[0346] 23. A pharmaceutical composition comprising a polypeptide according to any one of paragraphs 1-17, a nucleic acid according to paragraph 18, a vector according to paragraph 19, or a cell according to paragraph 20 or 22, optionally comprising a pharmaceutically acceptable vehicle, adjuvant, excipient, or diluent.

[0347] 24. A polypeptide according to any one of paragraphs 1-17, a nucleic acid according to paragraph 18, a vector according to paragraph 19, a cell according to paragraph 20 or 22, or a pharmaceutical composition according to paragraph 23, for use in a method of treating or preventing a disease or condition.

[0348] 25. Use of a polypeptide according to any one of paragraphs 1-17, a nucleic acid according to paragraph 18, a vector according to paragraph 19, a cell according to paragraph 20 or 22, or a pharmaceutical composition according to paragraph 23 in the manufacture of a medicament for treating or preventing a disease or condition.

[0349] 26. A method of treating or preventing a disease or condition, the method comprising administering to a subject a polypeptide according to any one of paragraphs 1-17, a nucleic acid according to paragraph 18, a vector according to paragraph 19, a cell according to paragraph 20 or 22, or a pharmaceutical composition according to paragraph 23.

[0350] 27. A method of treating or preventing a disease or condition in a subject, comprising modifying at least one cell of the subject to express or include a polypeptide according to any of paragraphs 1-17, a nucleic acid according to paragraph 18, or a vector according to paragraph 19.

[0351] 28. The polypeptide, nucleic acid, vector, cell or pharmaceutical composition for use according to paragraph 24, or the use according to paragraph 25, or the method according to paragraph 26 or paragraph 27, wherein the disease or condition is a disease or condition in which C3b or a complex containing C3b, an activity / response associated with C3b or a complex containing C3b, or a product of an activity / response associated with C3b or a complex containing C3b is pathologically involved.

[0352] 29. The polypeptide, nucleic acid, vector, cell or pharmaceutical composition, use or method according to any one of paragraphs 24-28, wherein the disease or condition is age-related macular degeneration (AMD).

[0353] 30. A polypeptide, nucleic acid, vector, cell or pharmaceutical composition, use or method for use according to any one of paragraphs 24-29, wherein the method for treating or preventing a disease or condition comprises modifying at least one retinal pigment epithelial (RPE) cell of a subject to express or include a polypeptide according to any one of paragraphs 1-17, a nucleic acid according to paragraph 18 or a vector according to paragraph 19.

[0354] 31. A kit comprising a plurality of parts comprising predetermined amounts of a polypeptide according to any one of paragraphs 1-17, a nucleic acid according to paragraph 18, a vector according to paragraph 19, a cell according to paragraph 20 or paragraph 22, or a pharmaceutical composition according to paragraph 23. BRIEF DESCRIPTION OF THE DRAWINGS

[0355] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings.

[0356] Figure 1A and Figure 1B .CR1a and nCR1a protein expression from human HEK293 cells. ( Figure 1A )CR1a is expressed as two glycoforms, which are reduced to a lower MW band after deglycosylation treatment with peptide N-glycosidase. Figure 1B )nCR1a is expressed as a single form with the same MW as deglycosylated CR1a.

[0357] Figure 2 . Capacity of CR1a and nCR1a expressed and secreted by human cells to act as cofactors for Factor I-mediated C3b cleavage. The CR1a / nCR1a+FI+C3b reaction produced iC3b and C3dg (lanes 7 and 11).

[0358] FIG. 3A to FIG. 3C .( Figure 3A ) After 24 hours, CR1a was present in the diffusion chamber of the Ussing chamber, demonstrating the ability to diffuse through the Bruch membrane (BrM). Figure 3B and Figure 3C ) After the polypeptide diffuses through the Bruch's membrane (BrM), ( Figure 3B )CR1a and ( Figure 3C )nCR1a's ability to act as a cofactor for Factor I to cleave C3b.

[0359] Figure 4 .Schematic diagram of the Ussing chamber used in the diffusion experiment, where (a) is the enriched Bruch's membrane from a human donor eye, which covers the 5 mm pore size and represents the only passage of liquid from one chamber to the other; (b) is the sampling access point; and (c) is the magnetic stirring bar.

[0360] Figure 5 . Binding kinetics of CR1a, FH and FHL-1 to C3b measured by biolayer interferometry (BLI).

[0361] Figure 6 Secreted CR1a was detected using anti-CR1a antibody from tissue culture medium of ARPE-19 cells transduced with AAV-CR1a (AAV-CR1a culture medium). Recombinant CR1a protein was used as a positive control. Culture medium alone and culture medium from ARPE-19 cells transduced with AAV-GFP were used as negative controls.

[0362] Fig. 7A and Figure 7B .The ability of CR1a secreted from human ARPE-19 cells to act as a cofactor for factor I-mediated C3b cleavage. ( Fig. 7A ) Secreted CR1a acts as a cofactor for FI to produce iC3b (product e) and C3dg (product f). CR1a secreted from human HEK293 cells was provided as a control. ( Figure 7B ) Compared to culture medium containing non-transduced cells, cultured AAV-CR1a-transduced RPE cells showed an increased ability to cleave C3b (product a) into iC3b (product b).

[0363] Figure 8 . Schematic diagram of the macular region of the eye showing the photoreceptors, retinal pigment epithelium, Bruch's membrane, and the choriocapillaris layer and capillary septa in the choroid.

[0364] Fig. 9 Representative fluorescence microscopy images of retinal tissue from Forest et al. (2015) Dis. Mod. Mech. 8, 421-427 (Figure 1). Shown are degraded RPE cells and drusen deposits beneath areas of complement activation. Scale bar 20 μm.

[0365] Fig.10 . Schematic diagram of the macular region of the eye showing key stages of local expression of effective complement therapy.

[0366] Fig.11 . An embodiment of an expression vector comprising a nucleic acid encoding a polypeptide according to the invention (eg, CR1), a promoter element, a replication element, and a selection element. Example

[0367] In the following examples, the inventors describe the design of recombinant CR1 proteins that include the C3b-binding cofactor region of complement receptor 1. It is also described that these proteins are expressed by human cells, diffuse through Bruch's membrane enriched from human donor eyes, and confer regulatory activity, namely the ability to promote FI-mediated cleavage of C3b to iC3b and further cleavage products.

[0368] Example 1

[0369] DNA inserts encoding the amino acid sequences shown in SEQ ID NOs: 2 and 5 were prepared by recombinant DNA technology and cloned into vectors to generate constructs for recombinant expression of CR1 peptides. The amino acid sequences and their characteristics are shown below:

[0370] CR1a (SEQ ID NO: 47)

[0371] MRLLAKIICLMLWAICVA GHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII

[0372] Signal peptide (SEQ ID NO: 7) ; CCP 8-10 of CR1 (SEQ ID NO: 2) (UniProt: P17927, residues 491-685)

[0373] nCR1a (SEQ ID NO: 48)

[0374] MRLLAKIICLMLWAICVA GHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII

[0375] Signal peptide (SEQ ID NO: 7); CCP 8-10 (SEQ ID NO: 5) of CR1 including substitutions N509Q and N578Q (UniProt: P17927, residues 491-685)

[0376] An 18 amino acid signal peptide was designed to be cleaved from the polypeptide upon secretion.

[0377] In some embodiments, HIS-tagged CR1a and nCR1a are used, for example as shown in SEQ ID NOs: 40 and 21, respectively.

[0378] Protein expression from human cells

[0379] HEK 293T cells (7×10 6 Cells / plate) were incubated at 37°C and 5% CO 2 The cells were grown overnight in a 15 cm culture plate in 17 mL of high glucose Dulbecco's modified Eagle's medium (DMEM, Sigma, catalog number D469) supplemented with 10% fetal bovine serum (FBS, Sigma, catalog number F9665) in an incubator at 4 °C. Once the cells reached 60% confluence, the cells were transiently transfected with 86.4 μg of 7.5 mM polyethyleneimine (PEI, Polysciences, catalog number 24765-2), 150 mM NaCl (Fisher Scientific UK Ltd, catalog number 10739592) and 14.4 μg of a plasmid expressing either CR1a (SEQ ID NO: 2) or nCR1a (SEQ ID NO: 5) linked to a signal peptide (SEQ ID NO: 7). For negative control, 14.4 μg of Tris-EDTA buffer was used instead of plasmid DNA. Five hours after transfection, 17.5 ml of fresh high-glucose DMEM was used to replace the transfection medium containing 10% FBS, which was supplemented with 2% FBS (hereinafter referred to as expression medium). Expression medium was collected at 24, 48, 72 and 140 hours after transfection. 80 μl of 0.5M phenylmethylsulfonyl fluoride (Phenylmethanesulfonyl fluoride, PMSF, Sigma, catalog number P7626) was added to each 100 ml expression medium collected and stored at 4 ° C. The expression medium collected after 24 hours was used for diffusion and functional studies described below.

[0380] Characterization of secreted proteins from human cells

[0381] Protein expression from human cells Figure 1A and Figure 1BAs shown. Recombinantly expressed and purified CR1a protein was found to be secreted from cells in two glycoforms. Treatment with the deglycosylation enzyme, peptide N-glycosidase F, reduced the two bands to a single band of lower apparent molecular weight (1A). The non-glycosylated form of the protein (nCR1a) was expressed. Western blotting showed that nCR1a produced a single band that migrated to the same position as the enzymatically deglycosylated protein ( Figure 1B ).

[0382] C3b cleavage activity

[0383] CR1a and nCR1a expressed and secreted from human HEK293 cells were tested for their ability to act as cofactors for Factor I-mediated C3b cleavage.

[0384] 1 μg of recombinant CR1a or nCR1a protein was mixed with 2 μg of pure C3b protein and 0.04 μg of pure complement factor I (FI; VWR International, catalog number 341280) at 37°C for 15 minutes. FHL-1 was provided as a cofactor control for FI. CR1a / nCR1a, CR1a / nCR1a+FI, CR1a / nCR1a+C3b and C3b alone were also provided as controls. The reaction was terminated by adding 4× SDS-loading buffer and heated to 100°C for 5 minutes. The samples were then run on a 4-12% NuPAGE Bis-Tris gel at 200 V for 60 minutes. The samples were transferred to a nitrocellulose membrane at 80 mA for 1.5 hours in transfer buffer (25 mM Tris, 192 mM glycine, 10% (v / v) methanol) using a semi-dry transfer device. The membrane was blocked in PBS, 10% (w / v) milk, 0.2% (w / v) BSA for 16 hours at 4°C before adding 100 μg / ml anti-C3b antibody (Hycult Biotech, catalog number HM2287) in PBS, 0.2% (v / v) Tween-20 (PBS-T) at room temperature. The membrane was washed 2×30 min in PBS-T, protected from light, before adding a 1:2500 dilution of HRP-conjugated goat anti-mouse for 1 hour at room temperature. The membrane was washed 2×30 min in PBS-T before adding SuperSignal WestPico chemiluminescent substrate (Thermo Fisher Scientific, catalog number 34080) for 3 min at room temperature. Reactive bands were detected by exposing Super RX-N X-ray film (FujiFilm, catalog number PPB5080) to the treated membrane for 2 min at room temperature and developing on an automatic X-ray film developer.

[0385] The results are Figure 2 Both secreted proteins were found to be able to act as cofactors for factor I, leading to the breakdown of C3b first into iC3b (a 1 ), and further decomposed into C3dg(a 1-1 C3b cleavage using CR1a / nCR1a was further extended than the normal native cleavage of C3b observed using FI+FHL-1 (second lane), which produces only iC3b (a 1 ).

[0386] Ussing chamber diffusion test and C3b degradation activity

[0387] As described in McHarg et al., J Vis Exp (2015) 1-7, the macular region of the enriched Bruch's membrane isolated from the donor eye was mounted in a Ussing chamber (Harvard Apparatus, Hamden, CT). Once mounted, the 5 mm diameter macular region was the only barrier between the two identical compartments, i.e., fluid had to pass through the Bruch's membrane ( Figure 4 ). The two sides of the Bruch membrane were washed with 2 ml PBS for 5 minutes at room temperature. Prior to the experiment, the structural integrity of the Bruch membrane was tested by the ability to retain 2 ml of liquid in one chamber without leaking into the next chamber. 2 ml of expression medium containing recombinant protein (CR1a and / or nCR1a, see above) was added to one chamber (hereinafter referred to as the sample chamber), and 2 ml of fresh PBS was added to the other chamber (hereinafter referred to as the diffusion chamber / diffusion chamber). The Ussing chamber was placed at room temperature for 24 hours and gently stirred with a magnetic stirring bar in each compartment to avoid the generation of a gradient of diffused protein.

[0388] CR1a was added to the sample chamber. After 24 hours, the samples in each chamber (original sample chamber and diffusion chamber) were analyzed for the presence of CR1a.

[0389] The results are Figure 3A It is shown in Figure 2. CR1a was found to be present in the diffusion chamber after 24 hours.

[0390] CR1a and nCR1a were added separately to the sample chambers, and the samples in both chambers were analyzed for C3b-cleaving activity. After 24 hours, 18.6 μg of sample was removed from each chamber and mixed with 1 μg (1 μg) of purified C3b protein and 0.4 μg of purified complement factor I (0.04 μg, VWR International, catalog number 341280) at 371 for 15, 30, or 60 minutes. One of the four additions was added. The reaction was terminated with the above loading buffer and heated to 100°C for 4 minutes. The samples were then electrophoresed on a 4-12% NuPAGE Bis-Tris gel at 200V for 60 minutes.

[0391] The results are Figure 3B and Figure 3C The decomposition of C3b in the diffusion chamber shows that glycosylated CR1a ( Figure 3B ) and non-glycosylated nCR1a( Figure 3C ) were able to cross the Bruch's membrane from the sample chamber and remain functionally active. It was found that CR1a and nCR1a in both chambers successfully acted as cofactors for FI, thereby breaking down C3b into proteolytically inactivated C3b (iC3b) and further products, as evidenced by the presence of bands representing C3b breakdown products iC3b and C3dg. Therefore, in the presence of Factor I, CR1a and nCR1a are able to diffuse through the BrM and retain the ability to confer C3b breakdown.

[0392] Example 2

[0393] The expression levels of polypeptides according to the invention are compared to assess whether there is an optimal preparation, ie whether the glycosylated polypeptide is expressed at a higher level than the non-glycosylated polypeptide. It is expected that the glycosylation state of the polypeptide will have minimal effect on expression levels.

[0394] CR1a was found to be well expressed by human cells. nCR1a was found to be expressed at lower levels than CR1a, but both polypeptides were found to be functionally active, see e.g. Figure 2 .

[0395] Example 3

[0396] The binding kinetics of the purified polypeptide according to the invention to C3b were tested using surface plasmon resonance (SPR), whereby C3b was immobilized on an SPR chip and the polypeptide was used in liquid phase. The association and dissociation constants were directly measured and the kD value of the interaction was inferred.

[0397] Although no differences in binding of CR1a (CCP 8-10) or CR1b (CCP 15-17) to C3b were observed, glycosylated peptides bound more strongly to C3b than non-glycosylated peptides.

[0398] Binding kinetics measured by biolayer interferometry (BLI)

[0399] The affinity of CR1a for C3b protein was measured using the OctetRed96 system (ForteBio, Pall Corporation, USA). The biotinylated C3b protein was diluted to a final concentration of 0.4 μg / mL in 0.2% PBST and loaded onto a high-precision streptavidin (SAX) biosensor (ForteBio, Pall Corporation, USA) for 600 s, which was pre-hydrated with the same buffer for 20 minutes. Then, the C3b-loaded sensor was washed with 0.2% PBST for 150 s (baseline) and immersed in a well containing CR1a ranging from 30.0 μg / mL to 2.6 μg / mL for 600 s (association), and then washed with 0.2% PBST for 600 s (dissociation). The association and dissociation profiles were recorded and analyzed with Forte Biodata Analysis v9 (ForteBio, Pall Corporation, USA). Negative controls, wells containing 0.2% PBST, were used in parallel to subtract binding due to nonspecific interactions with the sensor. Experiments were performed at 25°C using kinetic mode and the sample plate was pre-stirred for 3 minutes. Binding profiles were globally fitted to 1:1 (one analyte in solution to one binding site on the surface). KD was determined by steady-state analysis using data from the association phase (from 0s to 600s) and dissociation phase (from 0s to 100s) at the four lowest available analyte concentrations. The binding affinity of native soluble C3b binding to complement regulatory factor H (FH) and FHL-1 to immobilised C3b was also determined.

[0400] The results are Figure 5 . The binding affinity of CR1a to C3b was found to be 21 nM. For comparison, the binding affinity of FH to C3b is 580 nM and the binding affinity of FHL-1 to C3b is 1.2 mM. Therefore, CR1a binds to C3b significantly more strongly than any of the natural soluble complement regulators. The strong binding affinity of CR1a to C3b means that CR1a is a more effective agent for promoting C3b degradation than agents based on FH or FHL-1.

[0401] The strong binding affinity also enables CR1a to promote the degradation of iC3b into further desired downstream products, such as C3dg (see Figure 2 ). In contrast, FH and FHL-1 do not cause degradation of C3b beyond iC3b. iC3b is a pro-inflammatory molecule that acts to recruit immune cells to complement activation sites, which in turn causes negative inflammatory effects. Therefore, further degradation of iC3b to C3dg by CR1a is advantageous and avoids further damage caused by the immune system.

[0402] Example 4

[0403] The diffusion rates of polypeptides according to the invention were compared using an Ussing chamber assay (described above) to see if any differences were due to the formulation of the proteins.

[0404] The experiment involved using Bruch's membrane from a donor with AMD to determine whether substances deposited in the Bruch's membrane, including drusen and basal linear deposits, impaired the ability of the polypeptide to cross the Bruch's membrane in this case. It is expected that the best polypeptides will effectively cross the Bruch's membrane even in the presence of AMD changes.

[0405] Example 5

[0406] The nucleic acid according to the present invention, optionally encoding a preferred signal peptide and a stop codon, is inserted into an AAV vector, the preferred signal peptide being a polypeptide having at least 80% sequence identity with SEQ ID NO: 4. The resulting expression vector is used to transfect cultured RPE cells. The expression and secretion of the encoded polypeptide are evaluated. It is expected that the polypeptide having at least 80% sequence identity with SEQ ID NO: 4 is secreted by RPE cells, and the signal peptide has been cleaved from the secreted polypeptide.

[0407] Adeno-associated virus (AAV) transduction The nucleic acid encoding the CR1a polypeptide described in Example 1 was transfected into human APRE-19 cells (ATCC, USA) derived from the retinal pigment epithelium.

[0408] AAV2 serotype virus particles were prepackaged with CR1a plasmid. ARPE-19 cells were seeded at a density of 300,000 cells per well in 6-well cell culture plates (Corning) in 2 ml of DMEM / F12 growth medium (ATCC, USA) supplemented with 10% (v / v) fetal bovine serum (ATCC, USA). The cells were then incubated at 5% CO 2 Incubate at 37°C in a humidified atmosphere for 24 hours. After incubation, wash the cells twice with 2 ml of serum free DMEM / F12 growth medium. Add AAV2-CR1a at a multiplicity of infection (MOI) of 100,000 in serum free DMEM / F12 growth medium in a total volume of 1 ml. Incubate the medium containing AAV2-CR1a with the cells for 24 hours (37°C, 5% CO 2), followed by 2 ml of fresh serum-free DMEM / F12 growth medium the next day. Control cells transduced with AAV-GFP were grown in parallel. Transduction efficiency was assessed 14 days after infection. Secretion of CR1a by ARPE-19 cells was detected by dot blot: Conditioned medium from CR1a-transduced RPE cells was contacted with in-house polyclonal anti-CR1a antibodies.

[0409] The results are Figure 6 As shown in . Compared with the culture medium of RPE cells transduced with AAV-GFP (control (Ctrl) culture medium) used as a negative control, immunoreactivity with anti-CR1a antibodies was found in both test samples (AAV-CR1a culture medium). Purified recombinant CR1a protein was included as a positive control.

[0410] Recombinant CR1a polypeptides secreted from human APRE-19 and HEK293 cells were evaluated for their ability to cleave C3b into iC3b and C3dg.

[0411] The results are Fig. 7A It was found that the functional CR1a polypeptide secreted from human APRE-19 cells acts as a cofactor for Factor I, resulting in the breakdown of C3b into iC3b (product e) and C3dg (product f). Reactions containing FHL-1+FI+C3b provide MW control for C3b and product iC3b.

[0412] The degradation of C3b was evaluated in tissue culture medium of human RPE cells (ARPE-19) transduced with CR1a delivered by AAV. RPE cells transduced with AAV-GFP were used as negative controls. 14 days after transduction, transduced cells were supplemented with purified C3b and factor I. C3b and iC3b levels in tissue culture medium were detected by Western blotting.

[0413] The results are Figure 7B RPE cells produce their own complement components and were found to have a low native turnover of C3b (product a) to iC3b (product b; lane 2). However, RPE cells expressing AAV-delivered CR1a were found to have an increased ability to resolve C3b to iC3b (product b; lane 3) compared to the native turnover.

[0414] This suggests that CR1a is successfully secreted from ocular cells, such as retinal pigment epithelial cells, is functionally active as a FI cofactor, enhances C3b breakdown to downstream products, is capable of complement regulation, and will provide therapeutic benefit for conditions involving complement hyperactivation, such as an excess of C3b.

[0415] Example 6

[0416] The polypeptides of the invention find use in methods of treating or preventing a complement-associated disorder.An example of a complement-associated disorder is macular degeneration in the eye, such as AMD.

[0417] Figure 8 A schematic diagram of the macular region of the eye is provided. The retinal pigment epithelium (RPE) is a continuous single layer of cuboidal / columnar epithelial cells between the neurosensory layer of the retina and the vascular choroid. The cells have physical, optical, metabolic / biochemical and transport functions and play a vital role in normal visual processes. The RPE is separated from the choroid by a thin (2-4μ-), non-cellular, five-layered, extracellular matrix of Bruch's membrane (BrM). The BrM has two main functions: the basis of the RPE and the vascular wall. Immediately adjacent to the BrM and within the choroid is a layer of capillaries, called the choriocapillaris. Complement activation is concentrated in the extracellular matrix of the choriocapillaris, called the capillary septum.

[0418] Drusen, the hallmark lesion of AMD, are formed from the accumulation of lipids and cellular debris, including many complement activation products. Drusen develop within the BrM adjacent to the RPE layer and disrupt the flow of nutrients from the choroid to the RPE, leading to cell dysfunction and death. The death of RPE cells also leads to dysfunction of photoreceptor cells and subsequent loss of visual acuity.

[0419] Fig. 9 Representative fluorescence microscopy images of drusen deposits are provided and are taken from Forest et al. (2015) Dis. Mod. Mech. 8, 421-427 (Figure 1). Fig. 9 Retinal tissue from an 82-year-old woman with AMD is shown. Cell membrane markers show degenerated RPE cells covering drusen. The terminal complement complex marker C5b-9 indicates areas of complement activation within drusen and around blood vessels. Cell nuclei are stained. Scale bar 20 μm.

[0420] The systemic administration of complement regulatory molecules will require high doses and bring substantial risks of harmful off-target effects (off-target effect) to the operation of the complement system. Local administration, such as expression from RPE cells, is a safer and more effective delivery method. However, to make the molecules expressed by RPE effective in the capillary compartment of the treatment / prevention choriocapillaris layer, i.e., complement-related disorders in the site of complement overactivation, the molecules will have to pass through Bruch's membrane (see Fig.10 ). That is, effective complement inhibition therapy must meet Fig.10 Requirements for all three stages.

[0421] As shown in this article, CR1a is:

[0422] 1. Secreted from human RPE cells (see, for example Figure 6 );

[0423] 2. Capable of passive diffusion across human BrM (see, e.g., FIG. 3 ); and

[0424] 3. In the presence of complement factor I, it is able to mediate the breakdown of C3b into iC3b and further desired breakdown products (see, for example, Figure 2 , 3, 7).

[0425] Therefore, CR1a can be expressed by RPE cells, can reach areas of complement activation that require C3b regulation, and may serve as an effective therapeutic agent for treating complement hyperactivation in the eye, such as AMD.

[0426] Example 7

[0427] Laser-induced choroidal neovascularization model

[0428] This model applies laser burns to the retina of mice or rats to induce choroidal neovascularization, for example, as described in Schnabolk et al, Mol Ther Methods Clin Dev. 2018; 9: 1-11. The size of the choroidal neovascular complex can be measured by fluorescein angiography or histology.

[0429] Rodents are given subretinal injections of AAV vectors containing CR1a cDNA, such as AAV2, or blank AAV vectors as controls. Rodents receiving AAV vectors containing CR1a cDNA secrete CR1a protein from their retinal pigment epithelial cells. When maximum CR1a protein excretion is reached, laser burns are applied to the rodent retina, and the size of choroidal neovascularization complexes is measured at a predetermined time after the laser burn. It was found that the complement inhibition of CR1a polypeptides reduced the size of choroidal neovascularization complexes compared to rodents administered blank AAV vectors.

[0430] Sodium iodate-induced retinal degeneration

[0431] Mice were injected intravenously with sodium iodate, which induces degeneration of the retinal pigment epithelium, which is partly dependent on complement activation, for example, as described in Katschke et al., Sci Rep. 2018; 8(1):7348. CR1a was delivered as a recombinant protein by intravitreal injection or by subretinal delivery using an AAV vector containing CR1a cDNA, such as AAV2. A blank AAV vector was administered as a control. It was found that CR1a-treated mice had less retinal degeneration than control mice that did not receive CR1a treatment.

Claims

1. An isolated nucleic acid encoding a polypeptide capable of binding to C3b, wherein: The nucleic acid comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

35.

2. The isolated nucleic acid according to claim 1, wherein The encoded polypeptide comprises an amino acid sequence comprising or consisting of SEQ ID NO:2 or SEQ ID NO:

3.

3. The isolated nucleic acid according to claim 1 or 2, wherein The nucleic acid comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:

35.

4. The isolated nucleic acid according to any one of claims 1 to 3, wherein The encoded polypeptide has a total length of less than 450 amino acids.

5. The isolated nucleic acid according to any one of claims 1 to 4, wherein The nucleic acid includes a nucleotide sequence encoding a secretory pathway sequence.

6. The isolated nucleic acid according to claim 5, wherein The amino acid sequence of the secretory pathway sequence comprises SEQ ID NO:7, or consists of SEQ ID NO:

7.

7. The isolated nucleic acid according to claim 5 or 6, wherein The polypeptide or the amino acid sequence comprises SEQ ID NO: 47 or SEQ ID NO: 49, or consists of SEQ ID NO: 47 or SEQ ID NO:

49.

8. The isolated nucleic acid according to any one of claims 1 to 7, wherein The nucleic acid includes nucleotides 67-648 of SEQ ID NO:

35.

9. The isolated nucleic acid according to any one of claims 1 to 7, wherein The nucleic acid comprises nucleotides 13-648 of SEQ ID NO:

35.

10. The isolated nucleic acid according to any one of claims 1 to 7, wherein The nucleic acid includes nucleotides 7-648 of SEQ ID NO:

35.

11. A vector comprising the isolated nucleic acid according to any one of claims 1 to 10.

12. The carrier according to claim 11, wherein The vector is a viral vector.

13. The vector according to claim 11 or 12, wherein The vector is a lentiviral vector, a retroviral vector, a gamma-retroviral vector, an adenoviral vector, an adeno-associated virus AAV vector, a vaccinia virus vector and a herpes virus vector.

14. The vector according to any one of claims 11 to 13, wherein The vector is an AAV vector, and the AAV vector is selected from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a hybrid and / or mutant thereof.

15. The vector according to any one of claims 11 to 14, wherein The vector is an AAV2 vector, or a hybrid and / or mutant thereof.

16. The vector according to any one of claims 11 to 15, wherein The vector may also include a promoter and / or a polyadenylation sequence.

17. A mammalian cell comprising or expressing the nucleic acid according to any one of claims 1 to 10, or the vector according to any one of claims 11 to 16.

18. The mammalian cell according to claim 17, wherein The mammalian cell is a cell of the eye.

19. The mammalian cell according to claim 17 or 18, wherein The cells are retinal pigment epithelial RPE cells, photoreceptor cells, retinal cells, or cells of the choroid or macula.

20. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 10, the vector according to any one of claims 11 to 16, and / or the cell according to any one of claims 17 to 19.

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