C3b-bonded polypeptide

Abstract

To provide polypeptides that bind to C3b which are useful for treating or preventing complement-related diseases or conditions.SOLUTION: Polypeptides comprising a C3b binding region are disclosed, as well as nucleic acids and vectors encoding such polypeptides, and cells and compositions comprising such polypeptides. Also disclosed are uses and methods using the polypeptides for treating and preventing diseases and conditions.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims priority to GB1800620.5, filed on January 15, 2018, the content and elements of which are incorporated herein by reference for all purposes. Field of the Invention The present invention relates to the fields of molecular biology, immunology, and medicine. More specifically, the present invention relates to polypeptides that bind to C3b.

Background Art

[0002] Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries; AMD currently accounts for 8.7% of all blindness registrations worldwide and is estimated to affect 196 million people by 2020 (Wong et al Lancet Glob Heal (2014) 2:e106-16). AMD presents as a progressive destruction of the macula, the central part of the retina on the back of the eye, leading to loss of central vision. In the early stages of the disease, morphological changes in the macula are seen, which first include the loss of blood vessels in the choroid capillaries, which are fenestrated blood vessels found in the choroid (a highly vascularized layer that supplies oxygen and nutrients to the outer retina) (Whitmore et al, Prog Retin Eye Res (2015) 45:1-29).

[0003] AMD is primarily a genetic disease. Mutations in genes of the complement system, which is part of the immune system, are strongly associated with an increased risk of AMD. In fact, it has become clear that complement hyperactivation is a major driving factor in disease pathogenesis, and many examples of complement hyperactivation can be found in choroidal capillaries. The role of complement in AMD is described, for example, in Zipfel et al., Chapter 2, Lambris and Adamis (eds.), Inflammation and Retinal Disease: Complement Biology and Pathology, Advances in Experimental Medicine and Biology 703, Springer Science+Business Media, LLC (2010), which is incorporated in its entirety herein. Complement is activated by the deposition of protein C3b, a pro-inflammatory degradation product of the immune system protein C3, on the surface. C3b associates with other proteins to form convertase enzyme complexes that activate and amplify the complement response, initiating an amplification loop of the complement cascade, ultimately leading to cell / tissue destruction and local inflammatory responses (all characteristic of AMD).

[0004] Choroidal capillaries are separated from the metabolically active retinal pigment epithelium (RPE) by Bruch's membrane (BrM), a thin (2-4 μm) cell-free, five-layer extracellular matrix. BrM performs two main functions: acting as the basal layer and vascular wall of the RPE. The structure and function of BrM are outlined in Curcio and Johnson, Structure, Function and Pathology of Bruch's Membrane, Ryan et al. (2013), Retina, Vol. 1, Part 2: Basic Science and Translation to Therapy. 5th edition, London: Elsevier, pp. 466-481, which are incorporated herein by reference.

[0005] Complement C3b activation in cell-free structures, such as BrM and intercapillary septa (the extracellular matrix filling the space between capillaries in choroidal capillaries), is regulated by the proteins "complement factor H" (FH) and "complement factor I" (FI). FI activates complement by cleaving C3b into a proteolytically inactive form called iC3b. To prevent this, iC3b is unable to participate in the assembly of the convertase. However, iC3b is an opsonin and therefore a mediator of leukocyte replacement, which subsequently triggers an inflammatory response, whereas further degradation products of C3b, iC3dg and C3d, are inferior opsonins. To cleave C3b, FI requires the presence of cofactors, examples of which include the vascular-derived 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. Acting as a cofactor in FI-mediated cleavage of C3b, CR1 also acts as a complement regulator by accelerating the breakdown of C3 and C5 convertases. The structure and function of CR1 are outlined, for example, in Khera and Das, Mol Immunol (2009) 46(5):761-772, both of which are incorporated herein in their entirety.

[0006] A characteristic lesion of early AMD, called a drusen, develops within the BrM adjacent to the RPE layer (Bird et al., Surv Ophthalmol (1995) 39(5):367-374). The drusen is formed from the accumulation of lipids and cellular debris, and it contains a series of complement activators (Anderson et al., Prog Retin). Eye Res (2009) 29:95-112; Whitcup et al., Int J Inflam (2013) 1-10). The presence of drusen within the BrM hinders the flow of nutrients from the choroid to RPE cells across the extracellular matrix, leading to cellular dysfunction and ultimately death. The monolayer of RPE cells supports the rod and cone cells of the sensory retina by providing nutrients and removing waste; therefore, the death of these cells leads to photoreceptor cell dysfunction and subsequent loss of vision.

[0007] This corresponds to one of the later stages of AMD, known as "dry" AMD and also as geographic atrophy, which accounts for nearly 90% of AMD cases. In the remaining proportion of late-stage AMD cases, the presence of drusen promotes choroidal neovascularization (CNV), in which case increased synthesis of vascular endothelial growth factor (VEGF) by RPE cells promotes the proliferation of new blood vessels from the choroid / choroidal capillaries, which then destroy the retina through the BrM. These new blood vessels leak out and eventually form scar tissue; this is referred to as "wet" AMD.

[0008] "Wet" AMD accounts for only 10% of cases, but it is the most aggressive form of late-stage AMD and has different disease characteristics from "dry" AMD. For example, there is a treatment for wet AMD, which involves injecting anti-VEGF agents into the vitreous cavity of the eye, which can delay or reverse the proliferation of these blood vessels, but this does not prevent their formation in the first place. Geographic atrophy ("dry" AMD) remains untreatable. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Wong et al. Lancet Glob Heal (2014) 2:e106-16 [Non-Patent Document 2] Whitmore et al., Prog Retin Eye Res (2015) 45:1-29 [Non-Patent Document 3] Zipfel et al., Chapter 2; Lambris and Adamis (eds.), Inflammation and Retinal Disease: Complement Biology and Pathology, Advances in Experimental Medicine and Biology 703, Springer Science+Business Media, LLC (2010). [Non-Patent Document 4] Curcio and Johnson, Structure, Function and Pathology of Bruch's Membrane, : Ryan et al. (2013), Retina, Vol. 1, Part 2: Basic Science and Translation to Therapy. 5th edition, London: Elsevier, pp. 466-481. [Non-Patent Document 5] Khera and Das, Mol Immunol (2009) 46(5):761-772 [Non-Patent Document 6] Jacquet et al., J Immunol (2013) 190(7):3721-3731 [Non-Patent Document 7] Bird et al., Surv Ophthalmol (1995) 39(5):367-374 [Non-Patent Document 8] Anderson et al., Prog Retin Eye Res (2009) 29:95-112 [Non-Patent Document 9] Whitcup et al., Int J Inflam (2013) 1-10 [Overview of the Initiative]

[0010] The present invention provides a C3b-binding polypeptide that is useful for treating or preventing complement-related diseases or conditions. In one aspect, the present invention provides a C3b-binding polypeptide for use in methods of treating or preventing complement-related diseases or conditions, comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 4, and having a total length of 450 amino acids or less.

[0011] The provision is a nucleic acid encoding a C3b-binding polypeptide for use in methods of treating or preventing complement-related diseases or conditions, wherein the polypeptide comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4 and having a total length of 450 amino acids or less.

[0012] In another aspect, the present invention provides the use of a C3b-binding polypeptide in the manufacture of a drug for treating or preventing a complement-related disease or condition, wherein the polypeptide comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4 and having a total length of 450 amino acids or less.

[0013] What is provided is the use of a nucleic acid encoding a C3b-binding polypeptide in the manufacture of a drug for treating or preventing a complement-related disease or condition, wherein the nucleic acid encoding the polypeptide comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4 and having a total length of 450 amino acids or less.

[0014] In another aspect, the present invention provides a method for treating or preventing a complement-related disorder or condition, comprising the step of administering to a subject a polypeptide that is C3b-binding, comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4, and has a total length of 450 amino acids or less.

[0015] In another aspect, there is provided a method of treating or preventing a complement-related disease or condition in a subject, the method comprising modifying at least one cell of the subject to express or contain a polypeptide that is a polypeptide capable of binding to C3b, has an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and has a full length of 450 amino acids or less.

[0016] In some embodiments, the complement-related disease or condition is an eye disease or condition. In some embodiments, treating or preventing an eye disease or condition comprises modifying at least one eye cell of the subject to express or contain the polypeptide. In some embodiments, treating or preventing an eye disease or condition comprises modifying at least one eye cell of the subject to express or contain a nucleic acid encoding the polypeptide. In some embodiments, treating or preventing an eye disease or condition comprises administering to at least one eye cell of the subject a vector comprising a nucleic acid encoding the polypeptide. In some embodiments, the at least one eye cell is a retinal pigment epithelial (RPE) cell.

[0017] In some embodiments, the disease or condition is a disease or condition in which C3b or a C3b-containing complex, an activity / reactivity associated with C3b or a C3b-containing complex, or a product of an activity / reactivity associated with C3b or a C3b-containing complex is pathologically involved.

[0018] In some embodiments, the disease or condition is age-related macular degeneration. In some embodiments, the disease or condition is selected from one or more of: age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, geographic atrophy (“dry” AMD), “wet” (neovascular) AMD, choroidal neovascularization (CNV), glaucoma, autoimmune uveitis, diabetic retinopathy, and early-onset macular degeneration (EOMD).

[0019] In some embodiments, the polypeptide contains an amino acid sequence having at least 95% identity with SEQ ID NO: 4. In some embodiments, in SEQ ID NO: 4, X1 is A or T, X2 is P or L, and / or X3 is G or R.

[0020] In some embodiments, the polypeptide has a total length of 50 to 250 amino acids. In some embodiments, the polypeptide includes or consists of sequences of SEQ ID NO: 2 or SEQ ID NO: 3.

[0021] In some embodiments, the polypeptide comprises or consists of sequence number 13. In some embodiments, the polypeptide can act as a cofactor for complement factor I. In some embodiments, the polypeptide can diffuse across the Bruch membrane (BrM). In some embodiments, the polypeptide binds to C3b in a region bound by a cofactor for complement factor I. In some embodiments, the polypeptide binds to C3b in a region bound by complement receptor 1 (CR1).

[0022] In some embodiments, the polypeptide includes a secretory pathway sequence. In some embodiments, the secretory pathway sequence includes or consists of SEQ ID NO: 7. In some embodiments, the polypeptide includes or consists of SEQ ID NOs: 47, 49, or 51. In some embodiments, the polypeptide includes a cleavage site for removing the secretory pathway sequence.

[0023] In one aspect, the present invention provides a polypeptide having at least 80% sequence identity to SEQ ID NO: 4 and having a length of 700 amino acids or less. In some embodiments, the polypeptide has a length of 50 to 700 amino acids. In some embodiments, the polypeptide has at least 80% sequence identity to SEQ ID NO: 4, where X1 is A or T, X2 is P or L, and / or X3 is G or R.

[0024] What is provided is a polypeptide that can bind to C3b, and less of the number in Sequence ID No. 4. Both have 85% sequence identity and are polypeptides containing amino acid sequences with a length of 450 amino acids or less.

[0025] In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 4. In some embodiments, X1 is A or T, X2 is P or L, and / or X3 is G or R.

[0026] In some embodiments, the polypeptide has a total length of 50 to 250 amino acids. In some embodiments, the polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 3. In some embodiments, the polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 13.

[0027] 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 for 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 for complement factor I.

[0028] In some embodiments, the polypeptide is diffusible across the Bruch membrane (BrM). In some embodiments, the polypeptide is not glycosylated or is partially glycosylated. In some embodiments, the polypeptide contains at least one amino acid substitution, e.g., one, two, three, or four substitutions, at positions 509, 578, 959, and / or 1028 (numbered according to Uniprot:P17927 (SEQ ID NO: 1)). In some embodiments, the 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 contains or consists of sequences of SEQ ID NO: 5, SEQ ID NO: 6, and / or SEQ ID NO: 15.

[0029] In some embodiments, the polypeptide further 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 further comprises a cleavage site for removing the secretory pathway sequence. In some embodiments, the polypeptide comprises or consists of SEQ ID NOs: 47, 49, or 51.

[0030] In another aspect, the present invention provides nucleic acids that encode the polypeptide described in the present invention. In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.

[0031] In another aspect, the present invention provides cells comprising the polypeptide, nucleic acid, or vector described in the present invention. In another aspect, the present invention provides a method for producing a polypeptide, comprising the steps of introducing a nucleic acid or vector described in the present invention into cells and culturing the cells under conditions suitable for polypeptide expression.

[0032] In another aspect, the present invention provides cells that can be obtained or obtained by a method for producing polypeptides described in the present invention. In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide, nucleic acid, vector, or cell described in the present invention. In some embodiments, the pharmaceutical composition is pharmaceutically Includes acceptable carriers, adjuvants, excipients, or diluents.

[0033] In another aspect, the present invention provides polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described herein for use in methods of treating or preventing a disease or condition.

[0034] In another aspect, the present invention provides the use of polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described in the present invention in the manufacture of agents for treating or preventing diseases or conditions.

[0035] In another aspect, the present invention provides a method for treating or preventing a disease or condition, comprising the step of administering to a subject a polypeptide, nucleic acid, vector, cell, or pharmaceutical composition described in the present invention.

[0036] In another aspect, the present invention provides a method for treating or preventing a disease or condition in a subject, comprising the step of modifying at least one cell of the subject to express or contain a polypeptide, nucleic acid, vector, or polypeptide according to the present invention.

[0037] In some embodiments according to the diverse aspects of the present invention, the disease or condition is a disease or condition in which C3b or a C3b-containing complex, an activity / reaction associated with C3b or a C3b-containing complex, or a product of an activity / reaction associated with C3b or a C3b-containing complex is pathologically involved. 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, a method for treating or preventing the disease or condition comprises the step of modifying at least one retinal pigment epithelial (RPE) cell of a subject to express or contain a nucleic acid, vector, or polypeptide described in the present invention.

[0038] In another aspect, the present invention provides a partial kit comprising a predetermined amount of the polypeptide, nucleic acid, vector, cell, or pharmaceutical composition described in the present invention. [Brief explanation of the drawing]

[0039] Embodiments and experiments illustrating the principle of the present invention will be discussed here with reference to the accompanying figures. [Figure 1] Figures 1A and 1B. Expression of CR1a and nCR1a proteins from human HEK293 cells. (1A) CR1a is expressed as two glycosylated forms, which are reduced to a single lower molecular weight band after deglycosylation with PNGase. (1B) nCR1a is expressed as a single form with the same molecular weight as deglycosylated CR1a. [Figure 2] The ability of CR1a and nCR1a expressed and secreted by human cells to act as cofactors in the factor I-mediated degradation of C3b. The CR1a / nCR1a+FI+C3b reaction produced iC3b and C3dg (lanes 7 and 11). [Figure 3-1] (3A) Presence of CR1a in the diffusion chamber of the Ussing chamber after 24 hours, demonstrating its ability to diffuse through Bruch's membrane (BrM). [Figure 3-2]The ability of (3B)(3B)CR1a and (3C)nCR1a to act as cofactors for factor I, by disrupting C3b after polypeptide diffusion through Bruch's membrane (BrM). [Figure 3-3] The ability of (3C)(3B)CR1a and (3C)nCR1a to act as cofactors for factor I, by disrupting C3b after polypeptide diffusion through Bruch's membrane (BrM). [Figure 4] (a) is a concentrated Bruch membrane derived from a human donor eye, covering a 5 mm opening and representing the sole passage of liquid from one chamber to the other; (b) is the sampling access point; and (c) is a magnetic stirrer bar; this is a schematic diagram of a Ussing chamber used for a diffusion experiment. [Figure 5-1] Coupling dynamics of CR1a, FH, and FHL-1 relative to C3b, as measured by Biolayer interferometry (BLI). [Figure 5-2] Coupling dynamics of CR1a, FH, and FHL-1 relative to C3b, as measured by Biolayer interferometry (BLI). [Figure 5-3] Coupling dynamics of CR1a, FH, and FHL-1 relative to C3b, as measured by Biolayer interferometry (BLI). [Figure 6] Detection of secreted CR1a (AAV-CR1a medium) from tissue culture medium of AAV-CR1a-transduced ARPE-19 cells using anti-CR1a antibodies. Recombinant CR1a protein was used as a positive control. Culture medium alone and culture medium derived from AAV-GFP-transduced ARPE-19 cells were used as negative controls. [Figure 7-1] The ability of CR1a secreted from human ARPE-19 cells to act as a cofactor for factor I-mediated degradation of C3b. (7A) Secreted CR1a acts as an FI cofactor to produce iC3b (product e) and C3dg (product f). CR1a secreted from human HEK293 cells was provided as a control. [Figure 7-2]The ability of CR1a secreted from human ARPE-19 cells to act as a cofactor for factor I-mediated degradation of C3b. (7B) Cultured AAV-CR1a transduced RPE cells showed increased ability to degrade C3b (product a) to iC3b (product b) compared to culture medium containing non-transduced cells. [Figure 8] A schematic diagram of the macula of the eye, showing photoreceptors, retinal pigment epithelium, Bruch's membrane, and choroidal capillaries and intercapillary septa within the choroid. [Figure 9] Representative fluorescence microscopy images of retinal tissue from Forest et al. (2015) Dis. Mod. Mech. 8, 421-427 (Figure 1). The images show drusen deposition underlying destroyed RPE cells and complement-activated regions. Scale bar: 20 μm. [Figure 10] A schematic diagram of the macula of the eye illustrating a crucial stage in the localization of effective complement therapy. [Figure 11-1] An example of an expression vector comprising nucleic acids encoding a polypeptide (e.g., CR1a), a promoter element, a replication element, and a selection element as described in the present invention. [Figure 11-2] An example of an expression vector comprising nucleic acids encoding a polypeptide (e.g., CR1a), a promoter element, a replication element, and a selection element as described in the present invention. [Modes for carrying out the invention]

[0040] Complement-based therapies for AMD have so far focused on injecting complement-regulating antibodies into the eye. Because these proteins cannot reach the target region, namely the BrM and its underlying vascular system and choroidal capillaries, at any or no effective concentrations, these therapies have provided little or no therapeutic benefit.

[0041] The complement factor I (FI)-mediated regulation of complement, i.e., the cleavage of C3b to iC3b (protein-inactive C3b), requires a cofactor such as membrane-bound CR1. However, we have found that it is not necessary to provide full-length membrane-bound CR1, or even a soluble form of CR1 lacking only the transmembrane domain, for successful cofactor activity. Instead, we have found that a short CR1 fragment containing the CR1 C3b-binding domain is sufficient to enable efficient FI-mediated C3b cleavage.

[0042] Therefore, the present invention relates to a soluble truncated polypeptide derived from the FI cofactor CR1. The polypeptide contains a domain that can bind to C3b, such that these domains can act as essential FI cofactors for regulating complement activation. An important advantage of the soluble truncated polypeptide is its ability to pass through BrM, and Therefore, these can reach all regions associated with AMD, namely the choroid, including the RPE / BrM interface, BrM, and intercapillary septa (the extracellular matrix between blood vessels in choroidal capillaries). The present invention also provides CR1-derived non-glycosylated polypeptides that can assist polypeptide passage through BrM. These polypeptides are readily expressed and secreted, enabling in situ expression by cells localized to the affected site, and targeting of polypeptides to regions affected by complement hyperactivation. In situ expression of polypeptides may be achieved using gene therapy techniques. In situ expression provides targeted therapy to the desired region without interfering with complement regulation functioning elsewhere in the body.

[0043] The present invention enables the supplementation of a degraded complement regulatory system without replacing the currently performed endogenous complement regulation or interfering with the remaining complement cascade.

[0044] Polypeptide The polypeptide described in the present invention may contain one or more C3b binding regions, or may consist of such regions.

[0045] The polypeptide described in the present invention has at least 80% sequence identity with SEQ ID NO: 4, wherein the polypeptide has a length of 700 amino acids or less. 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 700 amino acids or less.

[0046] The polypeptide described in the present invention may contain, or consist of, an amino acid sequence of 700 amino acids or less, having at least 80% sequence identity with SEQ ID NO: 4.

[0047] In some embodiments, the polypeptide contains or consists of 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 contains or consists of an amino acid sequence having 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. 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, polypeptides have a length of 700 to 1000, or more than 1000 amino acids.

[0048] In this specification, “length” refers to the total length of the polypeptide; that is, “length” refers to the measurement or degree of the entire polypeptide from end to end, i.e., from the N-terminus to the C-terminus. In this specification, “length” is measured by the number of amino acid residues in the polypeptide.

[0049] In some embodiments, polypeptides are isolated / separate / individual / separate molecules. In some embodiments, polypeptides are unlinked, i.e., unbonded, unfused or unattached, single continuous amino acid sequence with respect to another amino acid sequence. In some embodiments, polypeptides are not attached to another polypeptide or amino acid sequence by amino acid linkers or non-amino acid linkers. In some embodiments, the polypeptide is not a portion, part, or region of a longer amino acid sequence; that is, the polypeptide is not a portion of an amino acid sequence exceeding the maximum specified polypeptide length. In some embodiments, the polypeptide is not a portion of, or does not form a part of, a fusion protein. In some embodiments, the polypeptide may contain the sequence provided herein and one or more additional amino acids, as long as it does not exceed the maximum polypeptide length. Because the polypeptides described herein are short in length, they are able to pass through the BrM and reach the complement activation site.

[0050] 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 ways, polypeptides are composed of 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, and 1 The polypeptide has a total length of ~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 1~450 amino acids. In some aspects, 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 one full-length sequence of 61, 72, 194, 212, 231, 388, 406, or 644 amino acids.

[0051] In some embodiments, the polypeptides of the present invention have a maximum molecular weight of 80 kDa, and the polypeptides are covalently / non-covalently bonded to a larger complex, are part of a larger complex, or are not part of a larger complex. In some embodiments, the polypeptides of the present invention have a maximum molecular weight of 75 kDa or less, 70 kDa or less, 65 kDa or less, 60 kDa or less, 55 kDa or less, 50 kDa or less, 45 kDa or less, 40 kDa or less, 35 kDa or less, 30 kDa or less, 29 kDa or less, 28 kDa or less, 27 kDa or less, 26 kDa or less, 25 kDa or less. The polypeptide has a molecular weight of 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., a molecular weight of 50 kDa or less. In some embodiments, the polypeptide has a maximum molecular weight of 26 kDa, i.e., a molecular weight of 26 kDa or less. In some embodiments, the polypeptide has a maximum molecular weight of 24 kDa, i.e., a molecular weight of 24 kDa or less. In some embodiments, the polypeptide The thiosulfate has a maximum molecular weight of 22 kDa, i.e., a molecular weight of 22 kDa or less. In some embodiments, the polypeptide has a maximum molecular weight of 20 kDa, i.e., a molecular weight of 20 kDa or less.

[0052] In some embodiments, the polypeptide of the present 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 in SEQ ID NO: 4. In some embodiments, X1 is A or T, X2 is P or L, and / or X3 is G or R. In some embodiments, X1 is A, X2 is P, and / or X3 is G. In some embodiments, X1 is A, X2 is L, and / or X3 is R. In some embodiments, X1 is A, X2 is P, and / or X3 is R. In some embodiments, X1 is A, X2 is L, and / or X3 is G. In some embodiments, X1 is T, X2 is L, and / or X3 is R. In some embodiments, X1 is T, X2 is P, and / or X3 is G. In some embodiments, X1 is T, X2 is L, and / or X3 is G. In some embodiments, X1 is T, X2 is P, and / or X3 is R.

[0053] 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 the length provided herein. For example, in some embodiments, the polypeptide contains or consists of an amino acid sequence having 85% sequence identity with SEQ ID NO: 4, where the polypeptide comprises 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 160 amino acids or Having a total length less than that; in some embodiments, the polypeptide comprises or consists of an amino acid sequence having 90% sequence identity to SEQ ID NO: 4, where the polypeptide comprises 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 180 amino acids or so Having a total length less than ; in some embodiments, the polypeptide comprises or consists of an amino acid sequence having 95% sequence identity to SEQ ID NO: 4, where the polypeptide comprises 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 180 amino acids or so Having a total length of less than 200 amino acids; in some embodiments, the polypeptide comprises or consists of an amino acid sequence having 98% sequence identity with 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, or 200 amino acids or less.

[0054] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity with 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 with SEQ ID NO: 4 The polypeptide consists of, where the polypeptide has a total length of 50 to 450 amino acids. In some embodiments, the polypeptide contains or consists of an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 4, where the polypeptide has a total length of 250 amino acids or less. In some embodiments, the polypeptide contains or consists of an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 4, where the polypeptide has a total length of 50 to 250 amino acids. In some embodiments, the polypeptide contains or consists of an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4, where the polypeptide has a total length of 450 amino acids or less. In some embodiments, the polypeptide contains or consists of an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4, where the polypeptide has a total length of 50 to 450 amino acids. In some embodiments, the polypeptide contains or consists of an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4, where 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 95% sequence identity with SEQ ID NO: 4, wherein the polypeptide has a total length of 50 to 250 amino acids.

[0055] Human CR1 (UniProt:P17927 (Entry Version 181 (October 25, 2017), Sequence Version 3 (March 2, 2020)); Sequence ID 1) has a 2,039 amino acid sequence (including a 41-amino acid signal peptide at the N-terminus) and contains 30 complement regulatory protein (CCP) domains (also known as sushi domains or small molecule consensus repeats (SCRs)). The 28 CCPs at the N-terminus are organized into four long-chain homologous repeat (LHR) domains, each containing seven CCPs: LHR-A, LHR-B, LHR-C, and LHR-D. The C3b binding region of CR1 is found in CCPs 8-10 in LHR-B (UniProt: P17927, positions 491-684; SEQ ID NO: 2) and CCPs 15-17 in LHR-C (UniProt: P17927, positions 941-1134; SEQ ID NO: 3). CCPs 8-10 and 15-17 differ in sequence by three amino acid residues, as shown in the consensus sequence, SEQ ID NO: 4.

[0056] The polypeptides described in this invention include CCP 8-10 (SEQ ID NO: 2) and / or CCP The polypeptide may contain or consist of an amino acid sequence corresponding to 15-17 (SEQ ID NO: 3). In some embodiments, the polypeptide of the present invention contains 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 polypeptides may have any length provided herein.

[0057] The polypeptide described in the present invention may contain or consist of amino acid sequences corresponding to CCPs 8-10 and 15-17. The polypeptide may contain or consist of CCPs 8-10 and 15-17 in its natural CR1 sequence (SEQ ID NO: 30). The polypeptide may contain CCPs linked to CCPs 15-17. The polypeptide may include or consist of 8-10. This may be a continuous sequence (SEQ ID NO: 13) or may be achieved by linkers between CCPs 8-10 and 15-17 (e.g., SEQ ID NO: 14). 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: 13, SEQ ID NO: 14 and / or SEQ ID NO: 30. Such polypeptides may have any length provided herein.

[0058] The polypeptides described in the present invention may contain or consist of amino acid sequences 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 sequences selected from sequence "A" (SEQ ID NO: 8), sequence "B" (SEQ ID NO: 16), and sequence "C" (SEQ ID NO: 17). In some embodiments, the polypeptides of the present invention contain or consist of amino acid sequences 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).

[0059] In some embodiments, the polypeptide described in the present invention contains 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 sequence "B" (SEQ ID NO: 16), where X1 is A or T. In some embodiments, X1 is A. In some embodiments, X1 is T.

[0060] In some embodiments, the polypeptide described in the present 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 sequence "C" (SEQ ID NO: 17), where X2 is P or L and / or X3 is G or R. In some embodiments, X2 is P and / or X3 is G. In some embodiments, X2 is P and / or X3 is R. In some embodiments, X2 is L and / or X3 is G. In some embodiments, X2 is L and / or X3 is R.

[0061] In some embodiments, sequence "B" corresponds to sequence number 9 or sequence number 11. In some embodiments, sequence "C" corresponds to sequence number 10 or sequence number 12. The present invention includes polypeptides comprising sequences "A", "B", and / or "C", as described herein, and combinations thereof, including at least the following combinations (organized from the N-terminus to the C-terminus): A+B B+C A+C ·C+A A+B+C B+C+A ·C+A+B ·A+B+C+A B+C+A+B ·C+A+B+C · A+B+C+A+B ·B+C+A+B+C ·A+B+C+A+B+C · A + B + C + A + B + C + Y (where Y = one of A, B and / or C) (More).

[0062] In some embodiments, the polypeptide comprises or consists of amino acid sequences 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 any combination of the CCP domains described herein, or any combination of sequences "A", "B", and / or "C".

[0063] In some embodiments, the combination of CCP domains is the same as that found in natural CR1. In some embodiments, the combination of CCP domains is not the same as that found in natural CR1.

[0064] In some embodiments, the polypeptide comprises or consists of amino acid sequences having numerous copies of sequence "A", numerous copies of sequence "B", and / or numerous copies of sequence "C". In some embodiments, the polypeptide comprises one or more copies of sequences "A", "B", and / or "C", namely one, two, three, four, five, six, seven, eight, or more. In some embodiments, the polypeptide comprises one, two, three, four, five, six, seven, eight, or more copies of sequence "A"; one, two, three, four, five, six, seven, eight, or more copies of sequence "B"; and / or one, two, three, four, five, six, seven, eight, or more copies of sequence "C". In some embodiments, the polypeptide comprises one or more copies of sequences "A", "B", and / or "C", namely nine, ten, or more.

[0065] In some embodiments, the polypeptides described herein lack substantial 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). 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% 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 described in the present invention lack amino acid sequences having substantial sequence identity to the CR1 long-chain homologous repeat (LHR) domain, LHR-A and / or LHR-D. In some embodiments, the polypeptides described in the present invention lack amino acid sequences having substantial sequence identity to the CR1 CCP domains 1-7, 11-14 and / or 18-30. The amino acid residues of Sequence ID No. 1 are numbered according to Uniprot:P17927; entry version 181 (October 25, 2017), sequence version 3 (March 2, 2010).

[0066] Polypeptides prepared in accordance with the present invention and / or described herein may be isolated and / or substantially purified.

[0067] Further characteristics of polypeptides The polypeptides described in the present invention may include modifications and / or further amino acid sequences. These modifications and / or further amino acid sequences may be included within the length limits of the polypeptides provided herein, so as not to exceed the polypeptide length limits.

[0068] In some aspects, further amino acid sequences include 25, 50, 100, 150, and or comprising not more than 200 amino acids, i.e., further amino acid sequences comprising 1-25, 1-50, 1-100, 1-150, or 1-200 amino acids, or comprising such amino acids. In some embodiments, further amino acid sequences comprising more than 200 amino acids. In some embodiments, further amino acid sequences comprising not more than 100 amino acids at the C-terminus of the polypeptide described in the present invention, and / or not more than 100 amino acids at the N-terminus of the polypeptide described in the present invention.

[0069] In some embodiments, further amino acid sequences result in polypeptides longer than 700 amino acids. In some embodiments, the polypeptides described in the present invention contain or consist of 700 or more amino acids. For example, the polypeptide may contain or consist of 700-750, 750-800, 800-850, 850-900, 900-950, 950-100, or more than 1000 amino acids.

[0070] In some embodiments, the further amino acid sequences described herein lack substantial sequence identity with one or more amino acid sequences 1-490, 685-940 and / or 1135-2039 of human CR1 (SEQ ID NO: 1; Uniprot: P17927; numbered according to entry version 181 (October 25, 2017), sequence version 3 (March 2, 2020)). In some embodiments, the further amino acid sequences lack substantial sequence identity with CR1 CCP domains 1-7, 11-14 and / or 18-30. In some embodiments, the further amino acid sequences 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). In some embodiments, the further amino acid sequences lack substantial sequence identity to the CR1 long homologous repeat (LHR) domains, LHR-A, and / or LHR-D.

[0071] In some embodiments, the polypeptide may lack amino acid sequences that have substantial sequence identity in the cofactor region relating to complement factor I (e.g., CR1) other than the C3b binding region. For example, the polypeptide may lack amino acid sequences that have substantial sequence identity to CR1 other than CR1 CCP domains 8-10 and / or 15-17 (residues 491-684 and / or 941-1134, respectively, of SEQ ID NO: 1). In some embodiments, the polypeptide may lack amino acid sequences that have substantial sequence identity to CR1 CCP domains 1-7, 11-14 and / or 18-30. Polypeptides 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).

[0072] In some embodiments, the polypeptides described in the present invention may include a secretory pathway sequence. In this specification, the secretory pathway sequence is an amino acid sequence that directs the secretion of the polypeptide. The secretory pathway sequence may be cleaved from the mature protein once the efflux of the polypeptide chain beyond the rough endoplasmic reticulum is initiated. Polypeptides secreted by mammalian cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the “mature” form of the polypeptide.

[0073] In some embodiments, the secretory pathway sequence is a leader sequence (signal peptide or sy The leader sequence may contain or consist of a sequence (also known as a signal sequence). The leader sequence typically consists of a sequence of 5 to 30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed on the cell surface often contain the leader sequence. The leader sequence may be present in a newly translated polypeptide (e.g., before processing to remove the leader sequence). Leader sequences are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensemble, and InterPro, and / or may 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).

[0074] In some embodiments, the secretory pathway sequence is derived from complement factor H (FH). In some embodiments, the secretory pathway sequence includes or consists of sequence number 7. In some embodiments, the secretory pathway sequence of the polypeptide of the present invention includes 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 with the amino acid sequence of sequence number 7.

[0075] In some embodiments, the polypeptides described in the present invention include or consist of amino acid sequences corresponding to SEQ ID NOs. 47, 49, and / or 51. In some embodiments, the polypeptides include or consist of amino acid sequences 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 NOs. 47, 49, and / or 51. Such polypeptides may have any length provided herein.

[0076] In some embodiments, the polypeptide described in the present invention may further include cleavage sites for removing secretory pathway sequences from the polypeptide. In some embodiments, the cleavage site for removing secretory pathway sequences from the polypeptide is an endoprotease-related cleavage site. In some embodiments, the cleavage site is related to an endoprotease expressed by the cell expressing the polypeptide. In some embodiments, the cleavage site is a signal peptidase cleavage site. In some embodiments, the cleavage site is a protease cleavage site, for example, a cleavage site related to an endoprotease expressed by the cell expressing the polypeptide. In some embodiments, the cleavage site is related to an endoprotease expressed by RPE cells.

[0077] The polypeptides described in the present invention may include one or more linker sequences between amino acid sequences. The linker sequences may be provided between any two or more of sequences "A", "B", and / or "C". In some embodiments, the polypeptides described in the present invention include or consist of the amino acid sequence A+B+C-[linker]-A+B+C. In some embodiments, the polypeptide includes or consists of Sequence ID No. 14.

[0078] Linker sequences are known to those skilled in the art and, for example, are described in their entirety in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which are incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. The flexible linker sequence is linked by the linker sequence. This allows for the relative movement of the minoic acid sequence. Flexible linker sequences are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0079] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of a glycine and a serine residue. In some embodiments, the linker 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 acids.

[0080] In some embodiments, the polypeptides described in the present invention include a non-amino acid linker. In some embodiments, the polypeptides described in the present invention may include two or more polypeptides linked by conjugate, for example, by nucleophilic substitution (e.g., reaction of acyl halides, active esters with amines and alcohols), electrophilic substitution (e.g., enamine reaction), and addition to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder reaction). These and other useful reactions are discussed, for example, in March, Advanced Organic Chemistry, 3rd edition, 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.

[0081] In some embodiments, the polypeptides described in the present invention include cleavable linkers. It may be desirable for the polypeptide described in this invention to lack certain properties of CR1. For example, it may be desirable for the polypeptide to lack regions that would otherwise inhibit diffusion through the Bruch membrane (BrM) or regions that would interfere with the action of innate cofactor family proteins.

[0082] The polypeptide described in the present invention lacks a CR1 transmembrane domain (SEQ ID NO: 32). The polypeptide described in the present invention may also lack a CR1 cytoplasmic tail (SEQ ID NO: 33). In a preferred embodiment, the polypeptide described in the present invention is soluble.

[0083] The polypeptides described in this invention may lack a region that could otherwise be used by pathogenic bacteria to disrupt the host immune system. Bacteria develop molecules on their surface that can bind to and replenish soluble complement factor H derived from blood. This allows bacteria to effectively coat themselves with complement regulatory factors and evade the host immune response. The polypeptides described in this invention may lack a bacterial binding site so that invasive pathogens cannot use it to evade the immune response.

[0084] In some embodiments, the polypeptides described in the present invention contain one or more glycosylation sites. In some embodiments, the polypeptides described in the present invention are glycosylated.

[0085] In some embodiments, the polypeptides described in the present invention are not glycosylated. In some embodiments, the polypeptides described in the present invention lack one or more glycosylation sites. In some embodiments, the polypeptides described in the present invention lack one or more glycosylation sites. They lack a large number of N-linked glycosylation sites. In some embodiments, the polypeptides described in the present invention lack N-linked glycans. In some embodiments, the polypeptides described in the present invention are expressed and / or secreted by cells that are unable to glycosylate the polypeptide or are unable to glycosylate it completely. For example, the cells may lack a functional glycosyltransferase enzyme. In some embodiments, the polypeptide is aglycosyl (i.e., not glycosylated). In some embodiments, the polypeptide is deglycosylated, for example, by treatment with a glycosidase (e.g., peptide N-glycosidase). The deglycosylation is preferably non-denaturing. In some embodiments, the polypeptides described in the present invention are partially glycosylated, unglycosylated, or deglycosylated.

[0086] In some embodiments, the polypeptides described in the present invention lack a sequence matching the consensus sequence of SEQ ID NO: 27. In some embodiments, the polypeptides described in the present invention include one or more sequences matching the consensus sequence of SEQ ID NO: 27, which have been mutated to remove a site for N-glycosylation. In some embodiments, the Asn(N) residue in the one or more consensus sequences described in SEQ ID NO: 27 is replaced with another amino acid residue, for example: Ala(A), Cys(C), Asp(D), Glu(E), Phe(F), Gly(G), His(H), Ile(I), Lys(K), Leu(L), Met(M), Pro(P), Gln(Q), Arg(R), Ser(S), Thr(T), Val(V), Trp(W), or Tyr(Y). In some embodiments, one or more Asn(N) residues in the consensus sequence described in SEQ ID NO: 27 are replaced with Gln(Q) residues. In some embodiments, residue X2 in SEQ ID NO: 27 is an amino acid that is not Ser(S) or Thr(T), or is replaced with such amino acids.

[0087] In some embodiments, a 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 in SEQ ID NO: 2 or SEQ ID NO: 4 contains one or more amino acid substitutions at positions 509 and / or 578 (numbered according to Uniprot:P17927). In some embodiments, the one or more amino acid substitutions are selected from N509Q and / or N578Q. In some embodiments, a 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 in SEQ ID NO: 3 contains one or more amino acid substitutions at positions 959 and / or 1028 (numbered according to Uniprot:P17927). In some embodiments, the one or more amino acid substitutions are selected from N959Q and / or N1028Q. In some embodiments, a 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 in SEQ ID NO: 13 contains one or more amino acid substitutions at positions 509, 578, 959, and / or 1028 (numbered according to Uniprot:P17927). In some embodiments, the one or more amino acid substitutions are selected from N509Q, N578Q, N959Q, and / or N1028Q. Such polypeptides may have any length provided herein.

[0088] In some embodiments, the polypeptide is represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 15, and / or Sequence ID No. 31 contains or comprises 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. Such polypeptides may have any length provided herein.

[0089] In some embodiments, the polypeptides described in the present invention have one or more sequences that match the secretory pathway sequence and the consensus sequence of SEQ ID NO: 27, which has been mutated to remove a site for N-glycosylation. In some embodiments, the polypeptides described in the present invention include or consist of amino acid sequences corresponding to SEQ ID NOs: 48, 50, 52, 53, and / or 54. In some embodiments, the polypeptides include or consist of amino acid sequences 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 NOs: 48, 50, 52, 53, and / or 54. Such polypeptides may have any length provided herein.

[0090] In some embodiments, the polypeptides described in the present invention may include one or more amino acid sequences that facilitate polypeptide expression, folding, transport, processing, purification, or detection. For example, the polypeptide may optionally include a sequence encoding a protein tag, such as His (e.g., 6XHis), FLAG, Myc, GST, MBP, HA, E, or biotin tag: at the N or C terminus of the polypeptide; in the linker; or at the N or C terminus of the linker. In some embodiments, the polypeptide includes a detectable portion, such as fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzyme-labeled. In some embodiments, the detectable portion facilitates the detection of the polypeptide in a sample obtained from a subject after administration of the polypeptide, nucleic acid, vector, cells, or pharmaceutical composition described in the present invention to the subject. The sample may be any biological sample obtained from the subject. In some embodiments, the sample is a liquid biopsy, such as ocular fluid (tears, aqueous humor, or vitreous fluid), blood, plasma, etc. In some embodiments, the sample is a cytological or tissue sample, such as a cell / tissue sample of the eye, or a surgical sample.

[0091] In some embodiments, the polypeptides described in the present invention may be detected and / or distinguished from endogenous CR1 by Western blotting, mass spectrometry, and / or enzymatic digestion, for example, by digestion with a specific peptidase. In some embodiments, the polypeptides may contain point mutations that, upon enzymatic digestion, produce peptides different from the post-digestion peptide derived from endogenous CR1.

[0092] In some embodiments, the polypeptide described in the present invention may further include cleavage sites for removing protein tags. For example, it may be desirable to remove the tags used for the purification of the polypeptide after purification. In some embodiments, the cleavage sites may be, for example, tobacco etch virus (TEV) protease cleavage sites, such as those shown in SEQ ID NO: 34.

[0093] In some embodiments, the polypeptides described in the present invention include or consist of amino acid sequences corresponding to SEQ ID NOs. 40, 42, 44, and / or 46. In some embodiments, the polypeptides include or consist of amino acid sequences 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 NOs. Such polypeptides may have any length provided herein.

[0094] In this specification, “polypeptide” includes molecules comprising one or more polypeptide chains that may be associated in a complex (e.g., covalently or non-covalently). That is, “polypeptide” within the meaning of the present invention includes molecules comprising one or more polypeptide chains. The polypeptides of the present invention may comprise, in a variety of different embodiments and at different in vitro or in vivo expression / production stages, for example, signal peptides, protein tags, cleavage sites for their removal, etc. The polypeptide of the present invention may optionally contain 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 sequences "A", "B", and / or "C", in combination with one or more of the further features of the polypeptide of the present invention described herein (e.g., signal peptides, linkers, detection sequences, absence of glycosylation sites, substituted amino acid residues, protein tags, cleavage sites for removing protein tags, secretory pathway sequences, cleavage sites for removing secretory pathway sequences).

[0095] Sequence identity In this specification, an amino acid sequence corresponding to a reference amino acid sequence may contain at least 60% of one sequence identity to the reference sequence, for example, at least 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%.

[0096] Paired and multi-sequence sequence alignments for the purpose of determining the percentage identity between two or more amino acid or nucleic acid sequences, e.g., ClustalOmega (Soeding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol.) This may be achieved in various ways known to those skilled in the art using publicly available computer software such as (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). When using such software, it is preferable to use default parameters, for example, regarding gap penalties and extension penalties.

[0097] array

[0098] [Table 1-1]

[0099] [Table 1-2]

[0100] [Table 1-3]

[0101] Table 1-4

[0102] Table 1-5

[0103] Table 1-6

[0104] Table 1-7

[0105] Table 1-8

[0106] Table 1-9

[0107] Table 1-10

[0108] Table 1-11

[0109] Table 1-12

[0110] Table 1-13

[0111] Functional properties of polypeptides The polypeptides described in the present invention may be characterized by referring to one or more functional properties.

[0112] In particular, the polypeptides described in the present invention may possess one or more of the following properties (as determined by analysis in a suitable assay relating to the said properties): Join to C3b; It binds to C3b with a binding affinity similar to the affinity for binding to C3b shown by the cofactor (or fragment thereof) related to complement factor I; It binds to C3b with a binding affinity higher than the binding affinity to C3b indicated by the cofactor (or fragment thereof) related to complement factor I; It binds to C3b with a binding affinity similar to the affinity shown by complement receptor 1 (or a fragment thereof); Binds to C3b in a region of C3b that is bound by a cofactor (or fragment thereof) related to complement factor I; Binds to C3b in the region of C3b that is bound by complement receptor 1 (or a fragment thereof); Complement receptor 1 binds to C3b in the region of C3b, which is mediated by CCP domains 8-10 and / or 15-17 (or fragments thereof); It acts as a cofactor that enables complement factor I-mediated inactivation of C3b; It acts as a cofactor that enables the mediated reduction / prevention of the formation of functional C3bBb type C3 convertase by complement factor I; It acts as a cofactor that enables the mediated reduction / prevention of the formation of functional C3bBb3b type C5 convertase by complement factor I; It acts as a cofactor that enables the mediated reduction / prevention of the formation of functional C4b2a3b type C5 convertases via complement factor I; It acts as a cofactor that enables complement factor I-mediated reduction of C3bBb type C3 convertase activity; It acts as a cofactor that enables complement factor I-mediated reduction of C3bBb3b type C5 convertase activity; A cofactor that enables complement factor I-mediated reduction of C4b2a3b type C5 convertase activity To act; It acts as a cofactor that enables a complement factor I-mediated decrease in the amount of C3bBb type C3 convertase; It acts as a cofactor that enables a complement factor I-mediated decrease in the amount of C3bBb3b type C5 convertase; It acts as a cofactor that enables a complement factor I-mediated reduction in the amount of C4b2a3b type C5 convertase; Reduces the amount of C3b through complement factor I; Increases the amount of iC3b through complement factor I; Increases the amount of C3dg through complement factor I; Increases the amount of C3d through complement factor I; Increases the amount of C3f through complement factor I; Reduces the amount of C5b through complement factor I; Reduces the amount of C5a through complement factor I; To reduce the amount of iC3b produced via complement factor I compared to the amount of iC3b produced by FH and / or FHL-1 via complement factor I; Increase the ratio of C3dg to iC3b via complement factor I; It is possible to inhibit complement activation; Diffuses through Bruch's membrane (BrM) It exhibits superior ability to diffuse through BrM compared to complement factor I; It exhibits superior ability to diffuse through BrM compared to cofactors (or fragments thereof) related to complement factor I; It exhibits a similar ability to diffuse through BrM compared to the cofactor (or fragment thereof) related to complement factor I; It exhibits superior ability to diffuse through BrM compared to complement factor H; It exhibits a similar ability to diffuse through BrM compared to the complement factor H isoform FHL-1; It exhibits superior ability to diffuse through BrM compared to the complement factor H isoform FHL-1; It exhibits a similar ability to diffuse through BrM compared to soluble complement receptor 1; Compared to soluble complement receptor 1, it exhibits superior ability to diffuse through BrM.

[0113] Whether a given polypeptide possesses the functional properties described in the preceding paragraph may be analyzed, for example, as described herein. The polypeptides described in the present invention may be capable of binding to C3b. In some embodiments, the polypeptides described in the present invention may include or consist of a C3b binding region. In some embodiments, the C3b binding region of the polypeptides described in the present invention includes or consists of the C3b binding region of CR1, for example, CCP domains 8-10 and / or 15-17.

[0114] In this specification, “C3b-binding region” refers to a region capable of binding to C3b. In some embodiments, the C3b-binding region is specifically capable of binding to C3b. Binding to C3b may be mediated by non-covalent interactions, such as van der Waals forces, electrostatic interactions, hydrogen bonds, and hydrophobic interactions, formed between the C3b-binding region and C3b. In some embodiments, the C3b-binding region binds to C3b with higher affinity and / or for a longer duration than it would to other molecules.

[0115] ELISA, surface plasmon resonance (SPR); 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 The ability of the polypeptide or putative C3b-binding region described in the present invention to bind to C3b may be analyzed using techniques well known to those skilled in the art, including (see reference), Bio-Layer interferometry (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 (e.g., Jerabek-Willemsen et al., Assay Drug Dev Technol. 2011 Aug; 9(4): 342-353), or by radiolabeled antigen-binding assay (RIA). Through such analysis, binding to a given target may be determined and quantified. In some embodiments, binding may be a reaction detected by a given assay.

[0116] In some embodiments, the polypeptides described in the present invention exhibit a binding to C3b in such assays that is greater than 1 times the level of the binding signal detected in such assays compared to a negative control molecule to which the C3b binding domain does not bind, for example, >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 times.

[0117] In some embodiments, the polypeptides described in the present invention are capable of binding to C3b in a given assay with a binding affinity similar to the binding affinity to C3b shown by another cofactor (or fragment thereof) relating to CR1 or complement factor I. The binding affinity similar to the reference binding affinity may be, for example, ±40% of the binding level to C3b shown by the reference cofactor relating to reference CR1 or complement factor I in a comparative assay, for example, ±35%, ±30%, ±25%, ±20%, ±15%, ±10%, or ±5% of the binding level.

[0118] In some embodiments, the polypeptides described in the present invention are capable of binding to C3b with a higher binding affinity in a given assay than the binding affinity to C3b indicated by the cofactor (or fragment thereof) for complement factor I. In some embodiments, the polypeptides described in the present invention are capable of binding to C3b with a higher binding affinity in a given assay than the binding affinity to C3b indicated by the cofactor (or fragment thereof) for complement factor I, such as 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, and 1 The polypeptides are capable of binding to C3b with binding affinity of 5x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 75x, 100x, 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, or 1000x in some embodiments. In some embodiments, the polypeptides described in the present invention are capable of binding to C3b with binding affinity of 10000x, 100000x, or 1000000x in a given assay compared to the binding affinity to C3b indicated by a cofactor (or fragment thereof) for complement factor I. In some embodiments, the polypeptides described in the present invention are capable of binding to C3b in a given assay with a binding affinity 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude higher than the binding affinity to C3b shown by the cofactor (or fragment thereof) for complement factor I. In some embodiments, the cofactor for complement factor I is complement factor H or the partially excised FH isoform FHL-1. The cofactor for complement factor I may also be CR1.

[0119] In some embodiments, the polypeptides described in the present invention are cofactors related to complement factor I. Therefore, the polypeptide is bindable to C3b within the region of C3b to which it is bound (i.e., it binds to the same or overlapping region). In some embodiments, the polypeptide is bindable to C3b within the region to which it is bound by CR1 (or a fragment thereof). In some embodiments, the polypeptide is bindable to C3b within the region to which it is bound by CR1 CCP domains 8-10 and / or 15-17. In some embodiments, the polypeptide is bindable to C3b within the region to which it is bound by one or more of the following: complement factor I cofactor, complement factor H, CD46, CD55, C4BP, SPICE, VCP, or MOPICE (or a fragment thereof).

[0120] Whether the polypeptide described in the present invention binds to C3b in a region of C3b to which a predetermined cofactor (or fragment thereof) of complement factor I is bound may be determined by a variety of 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 the C3b binding region binds to C3b in a region to which a predetermined cofactor (or fragment thereof) of complement factor I is bound is a competitive ELISA assay.

[0121] For example, whether the polypeptide described in the present invention binds to C3b in a region of C3b to which a predetermined cofactor (or fragment thereof) related to complement factor I is bound may be determined by analyzing the interaction between C3b and the cofactor / fragment in the presence of the polypeptide described in the present invention, or after incubation of the polypeptide described in the present invention with one or both of the cofactor / fragment and C3b. The C3b-binding region that binds to C3b in a region of C3b to which a predetermined cofactor / fragment is bound is identified by observing a decrease in the level of interaction between the cofactor / fragment and C3b in the presence of the polypeptide described in the present invention, or after incubation of the polypeptide described in the present invention with one or both interaction partners, compared to the level of interaction in the absence of the polypeptide described in the present invention (or in the presence of a suitable control peptide / polypeptide). Appropriate analysis may be performed in vitro, for example, using a recombinant interaction partner. For the purposes of such assays, one or both of the interaction partners and / or the polypeptide described in the present invention may be labeled or used in combination with a detectable entity for the purpose of detecting and / or measuring the level of interaction.

[0122] In some embodiments, the polypeptides described in the present invention act as cofactors for complement factor I. For example, the polypeptides may enhance the cleavage of C3b by complement factor I and / or present C3b in a preferred orientation for proteolytic cleavage by complement factor I. Preferably, the polypeptides do 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.

[0123] In this specification, “endogenous” protein / peptide means a protein / peptide encoded / expressed by a suitable cell type, tissue, or subject (prior to treatment with the polypeptide, nucleic acid, vector, cell, or pharmaceutical composition described in the present invention). “Non-endogenous” or “exogenous” protein / peptide means a protein / peptide not encoded / expressed by a suitable cell type, tissue, or subject (prior to treatment with the polypeptide, nucleic acid, vector, cell, or pharmaceutical composition described in the present invention).

[0124] For example, by analyzing the level or rate of proteolytic cleavage of C3b by complement factor I in a suitable assay in the presence of the polypeptide described in the present invention (or after incubation with the polypeptide), compared to the level or rate of proteolytic cleavage of C3b by complement factor I in the absence of the polypeptide described in the present invention (or in the presence of a suitable control peptide / polypeptide), the polypeptide can be used as a cofactor for complement factor I. The polypeptide to be used according to the present invention may be determined. The C3b binding region acting as a cofactor for 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 present invention (or after incubation with the polypeptide), compared to the level or rate of proteolytic cleavage of C3b by complement factor I in the absence of the polypeptide according to the present invention (or in the presence of a suitable control peptide / polypeptide). For example, the level or rate of proteolytic cleavage of C3b by complement factor I may be determined by detecting one or more products of C3b cleavage by complement factor I, e.g., iC3b, C3dg, C3d, or C3f. For example, the level or rate of proteolytic cleavage of C3b by complement factor I may be determined by detecting a decrease in the presence of C3b. In some embodiments, the polypeptides described in the present invention, acting as cofactors for complement factor I, produce a smaller overall amount of iC3b compared to the amount of iC3b produced by FH / FHL-1 through complement factor I. In some embodiments, the polypeptides described in the present invention, acting as cofactors for complement factor I, increase the ratio of C3dg to iC3b through complement factor I compared to the ratio of C3dg to iC3b produced by FH / FHL-1 through complement factor I. For example, while the polypeptides cannot increase the overall amount of iC3b, they can instead increase the amounts of C3dg, C3f, and / or C3d through complement factor I.

[0125] In some embodiments, the polypeptides described in the present invention can inhibit or reduce complement activation. Polypeptides may also inhibit or reduce complement hyperactivation. The level of complement activation / hyperactivation may be determined by assays described herein, for example, by abnormal levels of complement components, or by tests / assays known to those skilled in the art, such as those described herein, in whole, Shih and Murali Am. J. Hematol. 2015, 90: 1180-1186; Kirschfink and Molnes, Clin Diagn Lab Immunol. 2003, 10(6): 982-989; Nilsson and Ekdahl, Clinical and Developmental Immunology, 2012, citation ID 962702.

[0126] In some embodiments, the polypeptides described in the present invention possess the ability to diffuse through a Bruch membrane (BrM), i.e., to pass through a Bruch membrane, as determined by analysis in a suitable assay relating to these properties.

[0127] The ability of a given polypeptide to diffuse through BrM may be analyzed, for example, in vitro, as described, for example, in Clark et al. J. Immunol (2014) 193, 4962-4970. Briefly, BrM may be isolated from a donor eye, as described in McHarg et al., J Vis Exp (2015) 1-7, and the macular region may be placed in a Ussing chamber. Once placed, the 5 mm diameter macular region is the only barrier between the two identical compartments. Both sides of the BrM may be washed with PBS, and human serum diluted 1:1 with PBS may be added to the Ussing compartment (sample chamber) on one side of the BrM. The polypeptide to be analyzed may be added to the sample chamber in PBS, and PBS alone may be added to the compartment on the other side of the BrM (diffuser chamber), and the Ussing chamber may be incubated at room temperature for 24 hours with gentle agitation of both the sample and diffuser chambers. Next, samples from each chamber may be analyzed for the presence of polypeptides using antibody-based detection methods, such as ELISA analysis or Western blotting. If polypeptides are detected in the diffusion chamber, it indicates that the polypeptides are diffusible through BrM. Appropriate positive and negative control proteins known to be diffusible / imfusible through BrM may be included in these experiments.

[0128] In some embodiments, the polypeptides described in the present invention exhibit a superior ability to diffuse through BrM compared to complement factor I. In some embodiments, the polypeptides described in the present invention exhibit a superior ability to diffuse through BrM compared to complement factor H. FH, consisting of 20 CCP domains, is a macromolecule and does not pass through BrM. In some embodiments, the polypeptides described in the present invention exhibit a similar ability to diffuse through BrM compared to partially excised complement factor H isoform FHL-1 (UniProt: P08603-2; SEQ ID NO: 28). In some embodiments, the polypeptides described in the present invention exhibit a superior ability to diffuse through BrM compared to complement factor H isoform FHL-1. In some embodiments, the polypeptides described in the present invention exhibit a similar ability to diffuse through BrM compared to full-length soluble CR1 (30 CCP domains; SEQ ID NO: 1 lacking SEQ ID NOs. 32 and 33). In some embodiments, the polypeptides described in the present invention exhibit a superior ability to diffuse through BrM compared to full-length soluble CR1. The polypeptides described in the present invention, which are diffusible through BrM, preferably remain functionally active after diffusion through BrM, i.e., act as cofactors for complement factor I.

[0129] Polypeptides of the present invention that exhibit superior ability to diffuse through BrM compared to a given reference polypeptide may be identified by analyzing their diffusion through BrM, as described above. Diffusion through BrM may be detected by measuring the rate of diffusion into the diffusion chamber and / or by detecting the proportion of polypeptide present in the diffusion chamber at the end of the experiment. Polypeptides of the present invention that exhibit similar ability to diffuse through BrM compared to a given reference polypeptide may be identified by analyzing their diffusion through BrM, as described above. Similar ability to diffuse through BrM may be demonstrated by detecting the diffusion rate into the diffusion chamber which is within 30% of the diffusion rate of the reference polypeptide, for example, one of 25%, 20%, 15%, or 10%, and / or by detecting the proportion of polypeptides of the present invention present in the diffusion chamber at the end of the experiment which is within 30% of the proportion of the reference polypeptide present in the diffusion chamber, for example, one of 25%, 20%, 15%, or 10%.

[0130] As a result of the polypeptide's ability to diffuse through BrM, the polypeptide of the present invention can also diffuse from the C3b inactivation site if, once, it has performed its cofactor role with FI. In other words, the polypeptide of the present invention may be transiently present in the complement activation region. This is preferable because the accumulation of complement-related debris is undesirable, particularly in the context of macular degeneration, where the accumulation of cellular debris can lead to drusen formation.

[0131] The polypeptide of the present invention may be expressed in cells, for example, cells as described herein. The polypeptide of the present invention may be secreted by cells, for example, cells as described herein. In some embodiments, the cells are eye cells, for example, RPE cells, as described herein.

[0132] Nucleic acids, cells, compositions and kits The present invention provides nucleic acids encoding polypeptides described in the present invention. In some embodiments, the nucleic acids are purified or isolated, for example, from other nucleic acids or from naturally occurring biological materials. In some embodiments, the nucleic acids (one or more) include or consist of DNA and / or RNA.

[0133] Provided herein are sequence numbers 2, 3, 5, 6, 13, 14, 15, 30, 31, 40, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, or 54. or a nucleic acid sequence encoding a polypeptide consisting of these sequences. The encoded polypeptide may be produced with or without a leader sequence, such as a secretory pathway sequence. The encoded polypeptide may be produced together with the leader sequence, and the leader sequence is subsequently removed from the polypeptide.

[0134] In some embodiments, the nucleic acids described in the present invention include or consist of one or more of SEQ ID NOs: 35, 36, 37, 38, 39, 41, 43, and / or 45, or their equivalent nucleic acid sequences which, by codon degeneracy, would be translated into the same respective polypeptides.

[0135] The present invention also provides a vector comprising a nucleic acid encoding the polypeptide described in the present invention. The nucleotide sequence may be contained in a vector, for example, an expression vector. "Vector" as used herein refers to a nucleic acid molecule used as a vehicle for transporting exogenous nucleic acids into a cell. The vector may be a vector for the expression of nucleic acids in a cell. Such a vector may include a promoter sequence ligated to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a termination codon and an expression enhancer. The vector may also include regulatory elements, such as a polyadenylation site. A peptide or polypeptide may be expressed from the vector described herein using any suitable vector, promoter, enhancer, and termination codon known in the art. The nucleic acid sequences described herein may be codon-optimized for optimized expression in a desired cell or organism.

[0136] The term “functionally linked” may include situations in which a selected nucleic acid sequence and a control nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked such that the expression of the nucleic acid sequence is under the influence or regulation of the control sequence (thereby forming an expression cassette). Thus, the control sequence is functionally linked to the selected nucleic acid sequence if transcription of the nucleic acid sequence is achievable. The resulting transcript(s) may then be translated into the desired peptide(s) / polypeptides(s)(s).

[0137] The nucleic acids and / or vectors described in the present invention are preferably provided for introduction into cells, for example, human cells. Suitable vectors include, for example, plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, and herpesvirus vectors, e.g., herpes simplex virus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, both of which are incorporated herein in their entirety. 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 its hybrids and / or mutants. In some embodiments, the AAV vector is an AAV serotype 2 (AAV-2) vector, or its hybrids and / or mutants. Viral and nonviral delivery systems for introducing genetic material into cells are, for example, as whole incorporated herein by 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. This is outlined in 2017, 58(12): 5399-5411; Lundstrom, Diseases. 2018, 6(2): 42. Any suitable nucleotide or vector delivery method may be used in the background of this invention.

[0138] In some embodiments, the expression of nucleic acids contained in the nucleic acids or vectors described in the present invention is driven by a promoter that drives expression in specific retinal cell types, such as rods, cones, RPEs, or ganglion cells, as described, for example, in Beltran WA et al. Gene Ther. 2010; 17:1162-74 and Boye SE et al. Hum Gene Ther. 2012; 23:1101-15, which are incorporated herein by reference in their entirety.

[0139] In some embodiments, the expression of nucleic acids contained in the nucleic acids or vectors described in the present invention is driven in retinal pigment epithelial (RPE) cells by a promoter that drives the expression of the nucleic acids. In some embodiments, the promoter is the RPE65 or VMD2 promoter, or a modified version thereof. In some embodiments, the promoter is the chicken β-actin promoter.

[0140] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing the elements necessary for expressing a protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, for example, containing a cytomegalovirus (CMV) or SV40 promoter that drives protein expression.

[0141] In some embodiments, the vector includes an inductive promoter, i.e., gene expression is activated by the promoter only in the presence or absence of a specific molecule. Suitable inductive promoters will be known to those skilled in the art. An example of an inductive promoter is, for example, Le et al., Invest Ophthalmol, which is incorporated herein by reference in its entirety. Vis Sci. 2008, 49(3): 1248-1253 and McGee This is described by Sanftner et al. in Mol Ther. 2001. 3(5):688-696.

[0142] 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%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence A, B, and / or C as described above herein.

[0143] The present invention also provides cells that contain or express the polypeptide described in the present invention. Also provided are cells that contain or express the nucleic acid or vector described in the present invention. Cells containing or expressing the polypeptide, nucleic acid or vector described in the present invention may secrete the polypeptide described in the present invention. That is, the expression of polypeptide, nucleic acid or vector by cells may result in soluble production of the polypeptide described in the present invention from the cells.

[0144] The cells may be eukaryotic cells, such as mammalian cells. The mammals may be humans or non-human mammals (e.g., rabbits, guinea pigs, rats, mice or other rodents (including any animals of the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows, such as dairy cows, or any animals of the order Artiodactyla), horses (including any animals of the order Perissodactyla), donkeys, and non-human primates). In some embodiments, the cells may be derived from or obtained from human subjects.

[0145] In some embodiments, the cells are eye cells. In some embodiments, the cells are cells of the neurosensory retina, retinal pigment epithelium (RPE), choroid, or macula. In some embodiments, the cells are retinal cells. In some embodiments, the cells are retinal pigment epithelial cells. In some embodiments, the cells are human retinal pigment epithelial cells (RPE). In some embodiments, the cells are photoreceptor cells.

[0146] The present invention also provides a method for producing cells comprising the nucleic acid or vector described herein, the method comprising the step of introducing the nucleic acid or vector described herein into cells. In some embodiments, the step of introducing the isolated nucleic acid(s) or vector(s) described herein into cells comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction). For the production of cells comprising nucleic acid / vectors, the method for producing cells described herein may be carried out according to methods known to those skilled in the art.

[0147] The present invention also provides a method for producing cells comprising or expressing a polypeptide described in the present invention, the method comprising the step of introducing a nucleic acid or vector described in the present invention into cells. In some embodiments, the method further comprises the step of culturing cells under conditions suitable for the expression of nucleic acid or vector by cells. In some embodiments, the method is performed in vitro or ex vivo. In some embodiments, the method is performed in vivo.

[0148] The present invention also provides cells that can be obtained or obtained by the methods described in the present invention. The present invention also provides compositions comprising polypeptides, nucleic acids, vectors, or cells as described in the present invention.

[0149] The polypeptides, nucleic acids, vectors, and cells described in the present invention may be formulated as pharmaceutical compositions for clinical use, and these may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

[0150] Methods for producing pharmaceutically useful compositions are also provided in accordance with the present invention, and such production methods may comprise one or more steps selected from: isolating a 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 carrier, adjuvant, excipient or diluent.

[0151] Partial kits are also provided. In some embodiments, a kit may have at least one container containing a predetermined amount of the polypeptides, nucleic acids, vectors, cells, and / or compositions described in the present invention.

[0152] The kit may provide polypeptides, nucleic acids, vectors, cells, or compositions, along with instructions for use to administer them to a subject to treat a specified disease / condition. The polypeptides, nucleic acids, vectors, cells, or compositions may be formulated to be suitable for injection or infusion. Polypeptides, nucleic acids, vectors, cells, or compositions may be formulated to be suitable for intravenous, intraocular, subretinal, parachoroidal or intraconjunctival injection, administration as eye drops (i.e., ocular application), or oral administration.

[0153] In some embodiments, the kit may include materials for producing the cells described in the present invention. For example, the kit may include materials for modifying cells to express or contain the polypeptide, nucleic acid, or vector described in the present invention, or materials for introducing the nucleic acid or vector described in the present invention into cells.

[0154] In some embodiments, the kit may further include at least one container containing a predetermined amount of another therapeutic agent (e.g., a therapeutic agent for the treatment of AMD). In such embodiments, the kit may also include a second drug or pharmaceutical composition so that the two drugs or pharmaceutical compositions may be administered simultaneously or separately so that they provide a combined treatment for a particular disease or condition. In some embodiments, the second drug or pharmaceutical composition comprises complement factor I.

[0155] Polypeptide production The present invention also provides a method for producing a polypeptide described in the present invention, comprising the steps of introducing a nucleic acid or vector described in the present invention into cells and culturing the cells under conditions suitable for the expression of the polypeptide. The polypeptide may be a fusion protein. The polypeptide may subsequently be isolated and / or substantially purified.

[0156] The polypeptides described in the present invention may be prepared according to methods for the production of polypeptides known to those skilled in the art. Polypeptides may be prepared by chemical synthesis, for example, by liquid-phase or solid-phase synthesis. For example, Chandrudu et al., Molecules, whose entire work is incorporated herein. Peptides / polypeptides may be synthesized using the method described in (2013), 18: 4373-4388.

[0157] Alternatively, polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant polypeptide production are well known in the art, for example, those shown in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, both of which are incorporated herein in their entirety, and in Nat Methods. (2008); 5(2): 135-146.

[0158] For recombinant production as described in the present invention, any cell suitable for polypeptide expression may be used. The cell may be a prokaryotic or eukaryotic cell. In some embodiments, the cell may be a prokaryotic cell, such as an archaeon or bacterial cell. In some embodiments, the bacterium may be a Gram-negative bacterium, such as an Enterobacteriaceae, such as Escherichia coli. In some embodiments, the cell may be a eukaryotic cell, such as a yeast cell, plant cell, insect cell, or mammalian cell, such as a CHO, HEK (e.g., HEK293), HeLa, or COS cell.

[0159] In some cases, cells are not prokaryotic because prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. Furthermore, very high expression levels are possible in eukaryotic cells, and with appropriate tags, proteins may be more easily purified from eukaryotic cells. Specific plasmids that enhance the secretion of proteins into the culture medium may also be utilized.

[0160] In some embodiments, polypeptides may be prepared by cell-free protein synthesis (CFPS) using, for example, the system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431, which is incorporated herein by reference in whole.

[0161] Production may involve culturing or fermenting eukaryotic cells modified to express the polypeptide(s) of interest. Culturing or fermentation may be carried out in a bioreactor, with adequate supply of nutrients, air / oxygen, and / or growth factors. Secreted proteins may be collected by distributing the medium / fermentation broth from the cells, extracting the protein contents, and isolating the secreted polypeptide(s). Culturing, 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; hereafter incorporated herein).

[0162] A bioreactor may contain one or more vessels in which cells may be cultured. Culture in the bioreactor may be carried out continuously, with a continuous inflow of reactants into the reactor and a continuous outflow of cultured cells from the reactor. Alternatively, culture may be carried out in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, inflow and outflow rates, and agitation within the vessels to provide optimal conditions for the cultured cells.

[0163] After culturing cells expressing an antigen-binding molecule / polypeptide(s)(s)(s), the polypeptide(s)(s)(s) of interest may be isolated. Any method known in the art suitable for isolating proteins from cells may be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide(s)

[0164] Next, it may be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from the supernatant or culture medium is by precipitation. Proteins with different solubility levels precipitate at different concentrations of precipitating agents, such as ammonium sulfate. For example, at low concentrations of precipitating agents, water-soluble proteins are extracted. Therefore, it is also possible to distinguish proteins with different solubility levels by adding increasing concentrations of precipitating agents. Subsequently, ammonium sulfate may be removed from the separated proteins using dialysis.

[0165] Other methods for distinguishing different proteins, such as ion-exchange chromatography and size chromatography, are known in the art. Furthermore, polypeptides may be affinity-purified using appropriate binding partners for molecular tags on the polypeptide (e.g., His, FLAG, Myc, GST, MBP, HA, E, or biotin tags). These may be used as an alternative to precipitation or performed following precipitation.

[0166] In some cases, processing the polypeptide may be even more desirable, for example, to remove amino acid sequences, molecular tags, or sub-parts. In some embodiments, the process involves appropriate envelopes for cutting and removing amino acid sequences. This involves treatment with dopeptidase.

[0167] In some embodiments, the treatment involves processing with an enzyme that removes the portion of interest. In some embodiments, the polypeptide is treated with a glycosidase, such as peptide:N-glycosidase (PNGase), to remove the glycan (i.e., deglycosylate the polypeptide).

[0168] Once the polypeptide(s) of interest have been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide(s). Several methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0169] In some embodiments, polypeptide production occurs in vivo after introducing cells containing, for example, nucleic acids or vectors encoding the polypeptide of the present invention into a host, or after introducing nucleic acids or vectors encoding the polypeptide of the present invention into host cells. In these embodiments, the polypeptide is transcribed, translated, and post-processed into a mature polypeptide. In some embodiments, the polypeptide is produced in situ at a desired site in the host. In some embodiments, the desired site is in the cells of the eye, for example, the retina, choroid, retinal pigment epithelium (RPE), or macula. In some embodiments, the desired site is in retinal cells or within retinal cells. In some embodiments, the desired site is in RPE cells or within RPE cells.

[0170] Therapeutic application Any of the polypeptides, nucleic acids, vectors, cells, and pharmaceutical compositions described in this invention may be found to have use in therapeutic and prophylactic methods.

[0171] The present invention provides polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described herein for use in methods of medical treatment or prevention. The present invention also provides the use of polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described herein in the manufacture of drugs for treating or preventing diseases or conditions. The present invention also provides methods for treating or preventing diseases or conditions, comprising the step of administering a therapeutically or prophylactically effective amount of polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described herein to a subject.

[0172] In particular, the polypeptides, nucleic acids, vectors, cells, and pharmaceutical compositions described in the present invention find use to treat or prevent diseases / conditions associated with complement dysfunction, particularly hyperactive complement reactions. In some embodiments, hyperactive complement reactions are associated with the presence of C3b. In some embodiments, the disease / condition to be treated or prevented is a complement-related disease. In some embodiments, the disease / condition to be treated or prevented is pathologically associated with complement activation. In some embodiments, the disease / condition to be treated or prevented is pathologically associated with complement hyperactivation. In some embodiments, the disease / condition to be treated or prevented is driven by complement activation or hyperactivation. In some embodiments, the disease / condition is complement activation or hyperactivation.

[0173] Polypeptides, nucleic acids, vectors, cells, and pharmaceutical compositions find use to treat or prevent diseases / conditions that would benefit from one or more of the following: a decrease in the levels of C3bBb type C3 convertase, C3bBb3B type C5 convertase, or C4b2a3b type C5 convertase; a decrease in the levels of C3b, C5b, or C5a; an increase in the levels of iC3b, C3f, C3dg, or C3d; or a decrease in the level or activity of iC3b and an increase in the levels of C3f, C3dg, or C3d.

[0174] "Treatment" may, for example, be a reduction in the development or progression of a disease / condition, a reduction in the symptoms of a disease / condition, or a reduction in the pathology of a disease / condition. Treatment or mitigation of a disease / condition may be effective in preventing the progression of a disease / condition, for example, preventing worsening of the condition or slowing the rate of development. In some embodiments, treatment or mitigation may lead to improvement of a disease / condition, for example, a reduction in the symptoms of a disease / condition or a reduction in some other correlate of the severity / activity of a disease / condition. Prevention / prevention of a disease / condition may refer to preventing worsening of the condition or preventing the development of a disease / condition, for example, preventing an early-stage disease / condition from progressing to a later chronic stage.

[0175] In some embodiments, the disease or condition to be treated or prevented may be a disease / condition related to C3b or a C3b-containing complex, an activity / reaction related to C3b or a C3b-containing complex, or a product of an activity / reaction related to C3b or a C3b-containing complex. That is, in some embodiments, the disease or condition to be treated or prevented is a disease / condition pathologically related to C3b, a C3b-containing complex, an activity / reaction related to C3b or a C3b-containing complex, or a product of said activity / reaction. In some embodiments, the disease / condition may be related to an increase in the level of C3b or a C3b-containing complex, an increase in the level of an activity / reaction related to C3b or a C3b-containing complex, or an increase in the level of a product of an activity / reaction related to C3b or a C3b-containing complex, compared to a control state.

[0176] The treatment may aim to reduce the level of C3b or C3b-containing complexes, the activity / reaction associated with C3b or C3b-containing complexes, or the level of the product of the activity / reaction associated with C3b or C3b-containing complexes. In some embodiments, the treatment aims to: reduce the level or activity of C3bBb-type C3 convertase, C3bBb3b-type C5 convertase, or C4b2a3b-type C5 convertase; reduce the level of C3b, C5b, or C5a; increase the level of iC3b, C3f, C3dg, or C3d, or reduce the level of iC3b and increase the levels of C3f, C3dg, or C3d.

[0177] Administration of the polypeptides, nucleic acids, vectors, cells, and compositions of the present invention may cause a decrease in the levels of C3b or C3b-containing complexes, activity / reactions associated with C3b or C3b-containing complexes, or products of activity / reactions associated with C3b or C3b-containing complexes, through cleavage of C3b.

[0178] In some embodiments, the treatment may aim to reduce the levels of C3b or C3b-containing complexes, activity / reactions associated with C3b or C3b-containing complexes, or products of activity / reactions associated with C3b or C3b-containing complexes in the subject, for example, at a specific location, in a specific organ, tissue, structure, or cell type. In some embodiments, the treatment may aim to reduce the levels of C3b or C3b-containing complexes, activity / reactions associated with C3b or C3b-containing complexes, or products of activity / reactions associated with C3b or C3b-containing complexes in the eye, for example, in the retina, choroid, RPE, macula, and / or at the BrM / RPE interface.

[0179] In some embodiments, the treatment may include a step of modifying cells or cell populations to contain / express the polypeptide, nucleic acid, or vector of the present invention. In some embodiments, the treatment may include in vivo modification of cells / populations for in situ production of the polypeptide of the present invention. In some embodiments, the cells / cell populations are single / multiple cells of an eye. In some embodiments, the cells / cell populations are RPE cells and / or RPE cell populations. In some embodiments, the cells / cell populations are photoreceptor cells and / or photoreceptor cell populations.

[0180] In some embodiments, the present invention provides nucleic acids or vectors for use in gene therapy. In some embodiments, the therapy includes the step of administering the nucleic acid and / or vector to a subject. In some embodiments, the therapy includes the step of introducing the nucleic acid and / or vector into the cells of the subject using techniques described herein or known in the art. For example, MacLaren et al., Ophthalmology. 2016, 123(10 Suppl): S98-S106; Aguirre, Invest Ophthalmol Vis, which is incorporated herein in its entirety. See Sci. 2017, 58(12): 5399-5411; Lundstrom, Diseases. 2018, 6(2): 42. In some embodiments, the cells are one or more eye cells. In some embodiments, the cells are one or more RPE cells. In some embodiments, the cells are one or more photoreceptor cells.

[0181] In some embodiments, the treatment may include administering cells or cell populations modified to express / contain the polypeptides, nucleic acids, or vectors of the present invention to a subject. In some embodiments, the treatment may include modification of cells / populations ex vivo or in vitro.

[0182] In some embodiments, the treatment aims to provide a subject with cells or cell populations that produce and / or will produce polypeptides of the present invention, for example, by administering the cells described in the present invention or by generating the cells described in the present invention.

[0183] In some embodiments, the cells referred to herein are cells of the eye, i.e., ophthalmos. In some embodiments, the cells are cells of the retina, choroid, retinal pigment epithelium (RPE), or macula. In some embodiments, the cells are retinal cells. In some embodiments, the cells are RPE cells. In some embodiments, the cells are photoreceptor cells.

[0184] The present invention provides a method for treating or preventing a disease or condition in a subject, comprising the step of modifying at least one cell to express or contain a polypeptide, nucleic acid, or vector described in the present invention. In some embodiments, the at least one cell is an ophthalmic cell. In some embodiments, the at least one cell is an RPE cell.

[0185] At least one cell modified according to the present invention may be modified according to methods well known to those skilled in the art. The modification may include nucleic acid transfer for permanent or transient expression of the transferred nucleic acid. Cells may be modified according to the present invention using any suitable genetic engineering platform. Suitable methods for modifying cells include the use of genetic engineering platforms, e.g., gamma retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225, as incorporated herein by reference.

[0186] The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient. The subject may have been diagnosed with, or suspected to have, a disease or condition requiring treatment.

[0187] Subjects to be treated may, for example, be determined by analysis of the subject or a sample obtained from the subject (e.g., cell, tissue, blood sample) using an appropriate assay, and may show elevated levels of C3b or C3b-containing complexes, activity / reactions related to C3b or C3b-containing complexes, or products of activity / reactions related to C3b or C3b-containing complexes.

[0188] The subject may have increased levels of expression or activity of C3b or a C3b-containing complex, or positive regulators / effectors of activity / reactions associated with C3b or a C3b-containing complex, or increased levels of expression or activity of products of activity / reactions associated with C3b or a C3b-containing complex. The subject may have increased levels of activity upregulated by C3b or a C3b-containing complex.

[0189] The subject may have reduced levels of expression or activity of C3b or C3b-containing complexes, or negative regulatory factors of activity / reaction associated with C3b or C3b-containing complexes, or reduced levels of expression or activity of factors downregulated by C3b or C3b-containing complexes. The subject may have reduced levels of activity downregulated by C3b or C3b-containing complexes.

[0190] The increase / decrease may be relative to the level of expression / activity in the absence of the associated disease / condition, for example, the level of expression / activity in a healthy control subject or in a sample obtained from a healthy control subject.

[0191] In some embodiments, the subject may have a risk of developing / contracting a disease or condition. In some embodiments, the subject may have one or more predisposing factors that increase the risk of developing / contracting a disease or condition.

[0192] In some embodiments, subjects may possess one or more risk factors for age-related macular degeneration (AMD). In some embodiments, subjects may possess one or more AMD-related gene variants. AMD-related gene variants are described, for example, in Clark et al., J Clin Med (2015) 4(1):18-31, which is incorporated herein by reference in whole. In some embodiments, subjects may possess the following AMD-related gene variants (or such variants and LD=r 2May possess one or more mutants having ≥0.8: Y402H in CFH (i.e., rs1061170) C ), rs1410996 C , I62V in CFH, R53C in CFH, D90G in CFH, R1210C in CFH, or rs6685931 in CFHR4 T .

[0193] In some embodiments, subjects may possess one or more risk factors for early-onset macular degeneration (EOMD). EOMD is thought to be caused by monogenic inheritance of rare variants of the CFH gene (e.g., Boon CJ et al. Am See J 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, subjects may possess one or more EOMD-related gene variants. EOMD-related gene 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; 1 As described in 5(3):787-95; the literature is incorporated herein by reference in its entirety. In some embodiments, a subject may possess one or more of the following EOMD-related gene variants: CFH c.1243del, p.(Ala415Profs*39) het; CFH c.350+1G>T het; CFH c.619+1G>A het; CFH c.380G>A, p.(Arg127His); CFH c.694C>T, p.(Arg232Ter); or CFH c.1291T>A, p.(Cys431Ser).

[0194] In some embodiments, subjects are selected for therapeutic or prophylactic treatment with the polypeptides, nucleic acids, vectors, cells, or compositions of the present invention based on the determination that they possess one or more risk factors related to AMD and / or EOMD, for example, one or more AMD / EOMD-related gene variants. In some embodiments, subjects are determined to have one or more such risk factors. In some embodiments, the method of the present invention includes the step of determining whether a subject possesses one or more such risk factors.

[0195] In some embodiments, the disease or condition to be treated or prevented may be an eye disease / condition. In some embodiments, the disease or condition to be treated or prevented may be a complement-related eye disease. In some embodiments, the disease or condition to be treated or prevented may be macular degeneration. In some embodiments, the disease or condition to be treated or prevented may be age-related macular degeneration (AMD). AMD is generally defined as causing blindness in subjects aged 50 years or older.

[0196] In some embodiments, the disease or condition to be treated or prevented may be 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 diseases / conditions, e.g., "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 50 years of age or older, i.e., at least 50 years of age.

[0197] In this specification, “early AMD” refers to a stage of AMD characterized by the presence of moderately sized drusen, typically up to ~200 μm in width, within the Bruch’s membrane adjacent to the RPE layer. Subjects with early AMD typically do not exhibit significant visual acuity loss. In this specification, “intermediate AMD” refers to a stage of AMD characterized by large drusen and / or pigment changes in the retina. Intermediate AMD may involve some degree of visual acuity loss. In this specification, “late AMD” refers to a stage of AMD characterized by the presence of drusen and visual acuity loss due to damage to the macula. In all stages of AMD, “reticular pseudodrusen” (RPD) or “reticular drusen,” which refer to the accumulation of extracellular material in the subretinal space between the neurosensory retina and the RPE, may also be present. “Late AMD” includes “dry” and “wet” AMD. In "dry" AMD (also known as geographic atrophy), there is progressive destruction of photosensitive cells in the macula that carry visual information to the brain, and of the supporting tissues beneath the macula. In "wet" AMD (also known as choroidal neovascularization and exudative AMD), abnormal blood vessels proliferate beneath and within the retina. These vessels contain fluid and It may cause blood to leak, which can lead to macula swelling and damage, and subsequently scarring. The damage can be rapid and severe.

[0198] In some embodiments, the disease or condition to be treated or prevented is early-onset macular degeneration (EOMD). In this specification, “EOMD” refers to a phenotypically severe subtype of macular degeneration that occurs at a much younger age than classical AMD and results in substantial vision loss many years later. EOMD subsets are described, for example, in 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 subjects to be treated are 49 years of age or younger. In some embodiments, the subjects to be treated are between 15 and 49 years of age, i.e., between 15 and 49 years of age.

[0199] In some embodiments, the disease or condition to be treated or prevented is a disease / condition driven by complement hyperactivation. In some embodiments, the disease or condition to be treated or prevented may be selected from atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis type II (MPGN II), sepsis, and paroxysmal nocturnal hemoglobinuria (PNH).

[0200] Medical treatments may also involve in vivo, ex vivo, and adoptive immunotherapy, including those using autologous and / or xenolytic or immortalized cell lines. The administration of polypeptides described herein is preferably a “therapeutic effective dose,” which is sufficient to demonstrate benefit to the 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, is the responsibility of the practicing physician and other physicians, and typically takes into account the condition to be treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to the physician. Examples of the techniques and protocols described above are from Remington’s This can be found in Pharmaceutical Sciences, 20th edition, 2000, published by Lippincott, Williams & Wilkins.

[0201] The polypeptides, nucleic acids, vectors, and cells described in the present invention may be formulated as pharmaceutical compositions or agents for clinical use and may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The compositions may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, subarachnoid, intraocular, intraconjunctival, subretinal, superchoroidal, subcutaneous, intradermal, subarachnoid, oral, or transdermal administration routes, including injection or infusion, or administration as eye drops (i.e., ocular application). Suitable formulations may contain polypeptides, nucleic acids, vectors, or cells in a sterile or isotonic medium. The agents and pharmaceutical compositions may be formulated in liquid form, including gels. Liquid formulations may be formulated for administration by injection or infusion (e.g., via catheter) into selected organs or regions of the human or animal body. In some embodiments, the polypeptides, nucleic acids, vectors, cells, and compositions of the present invention are formulated for intravitreous administration routes, for example, by intravitreous injection. In some embodiments, the polypeptides, nucleic acids, vectors, cells, and compositions of the present invention are formulated to deliver the therapeutic molecule submacularly, i.e., to direct contact with the target cell layer.

[0202] The specific mode and / or site of administration may be selected according to the location where C3b inactivation is desired. In some embodiments, the polypeptides, nucleic acids, vectors, or pharmaceutical compositions of the present invention are formulated for administration in the eye, into the subretinal space between photoreceptor cells and retinal pigment epithelium (RPE), and / or administered into such space. In some embodiments, the polypeptides, nucleic acids, vectors, or pharmaceutical compositions of the present invention are used in a network Formulated for and / or administered into the membrane pigment epithelium (RPE).

[0203] Methods for producing pharmaceutically useful compositions are also provided in accordance with the present invention, such production methods may comprise one or more steps selected from: isolation of polypeptides, nucleic acids, vectors, or cells as described herein; and / or mixing of polypeptides, nucleic acids, vectors, or cells as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0204] For example, a further aspect of the present invention relates to a method for formulating or producing a drug or pharmaceutical composition for use in a method of medical treatment, the method comprising the step of formulating the pharmaceutical composition or drug by mixing a polypeptide, nucleic acid, vector, or cell, as described herein, with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0205] The administration may be alone or, depending on the condition to be treated, may be simultaneous or sequential, in combination with other treatments (e.g., other therapeutic or prophylactic interventions). The polypeptides, nucleic acids, vectors, cells or compositions and therapeutic agents described in the present invention may be administered simultaneously or sequentially.

[0206] Simultaneous administration refers to the administration of polypeptides, nucleic acids, vectors, cells or compositions, and therapeutic agents together, for example, as a pharmaceutical composition (combination preparation) containing both agents, or immediately after each other, and optionally, through the same route of administration, for example, to the same tissue, artery, vein or other blood vessel. Sequential administration refers to the separate administration of one polypeptide, nucleic acid, vector, cell or composition, or therapeutic agent, after a predetermined time interval following the administration of the other agent. The two agents do not need to be administered by the same route, although this is the case in some embodiments. The time interval may be any time interval. In some embodiments, polypeptides, nucleic acids, vectors, cells or compositions, and therapeutic agents are administered to the eye separately, simultaneously, or sequentially.

[0207] In some embodiments, the other therapeutic agent is a therapeutically effective amount of complement factor I. In some embodiments, complement factor I is administered to a subject simultaneously with or consecutively with the administration of the polypeptide, nucleic acid, vector, cells, or pharmaceutical composition described in the present invention. In some embodiments, the treatment may include a step of modifying cells or a population of cells in vitro, ex vivo, or in vivo so that the cells or population of cells express and / or secrete complement factor I. The cells or population of cells may be the same cells or population of cells modified to contain / express the polypeptide, nucleic acid, or vector described in the present invention, for example, the treatment may include a step of modifying cells or a population of cells in vitro, ex vivo, or in vivo so that the cells or population of cells express and / or secrete the polypeptide, nucleic acid, or vector described in the present invention and complement factor I. In some embodiments, complement factor I is administered to a subject, wherein the subject contains cells or a population of cells modified to contain / express the polypeptide, nucleic acid, or vector described in the present invention. In some embodiments, complement factor I is administered to a subject, where the subject was expressing or in the process of expressing the polypeptide, nucleic acid, or vector of the present invention in situ.

[0208] Complement factor I or compositions containing complement factor I may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intravitreous, intraarterial, intramuscular, subarachnoid, intraocular, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, subarachnoid, oral, or transdermal administration routes, including injection or infusion, or administration as eye drops (i.e., ocular application). Suitable formulations may contain sterile or isotonic media. The drugs and pharmaceutical compositions are formulated in liquid form, including gels. The liquid formulation may be formulated for administration by injection or infusion (e.g., via catheter) into a selected organ or area of ​​the human or animal body.

[0209] In some embodiments, other therapeutic agents include therapeutically effective amounts of anti-VEGF therapy (e.g., ranibizumab (Lucentis; Genentech / Novartis), bevacizumab (unapproved Avastin; Genentech), afilbercept (Eylea / VEGF Trap-Eye; Regeneron / Bayer)), pegaptanib (Macugen®), laser photoaggregation, or photodynamic therapy (PDT), e.g., Visudyne TM This is the therapeutically effective dose when using (verteporfin).

[0210] Multiple doses of polypeptides, nucleic acids, vectors, cells, or compositions may be provided. One or more doses, or each dose, may be accompanied by the simultaneous or sequential administration of another therapeutic agent.

[0211] Multiple doses may be separated by predetermined time intervals, which may be selected from 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 one of 1, 2, 3, 4, 5, or 6 months. For example, doses may be administered once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0212] The polypeptides, nucleic acids, vectors, or compositions described in the present invention may be incorporated into a sustained-release delivery system to release the polypeptides, nucleic acids, vectors, or compositions at a predetermined rate. The sustained-release delivery system may maintain a constant drug / therapeutic concentration for a specified period. In some embodiments, the polypeptides, nucleic acids, vectors, or compositions described in the present invention may be incorporated into liposomes, gels, implants, devices, or drug-polymer conjugates, such as hydrogels.

[0213] Genetic factors in age-related macular degeneration (AMD) Complement factor H (encoded by the CFH gene) is another cofactor related to complement factor I. The structure and function of complement factor H are outlined, for example, in Wu et al., Nat Immunol (2009) 10(7): 728-733, the entire text of which is incorporated herein. Human complement factor H (UniProt:P08603; SEQ ID NO: 29) has a 1,233 amino acid sequence (including an 18-amino acid signal peptide at the N-terminus) and contains 20 complement regulatory protein (CCP) domains. The first four CCP domains of complement factor H (i.e., CCP1-CCP4) are required for complement factor I cofactor activity to cleave C3b to iC3b. CCPs 19-20 have also been shown to associate with C3b and C3d (Morgan et al., Nat Struct Mol Biol (2011) 18(4):463-470), while CCP7 and CCPs 19-20 bind to glycosaminoglycans (GAGs) and sialic acid and are involved in the distinction between self and non-self (Schmidt et al., J Immunol (2008) 181(4):2610-2619; Kajander et al., PNAS (2011) 108(7):2897-2902).

[0214] One of the main SNPs associated with the genetic risk of developing AMD is found in the CFH gene and leads to the Y402H polymorphism in complement factor H (see, e.g., Haines et al., Science (2005) 308:419-21) and its alternative splice variant factor H-like protein 1 (FHL-1). Approximately 30% of individuals with the Caucasian European genetic trait have at least one copy of this polymorphism, while heterozygotes have AM The risk of D increases by approximately 3 times (Sofat et al., Int J Epidemiol (2012) 41:250-262). The Y402H polymorphism appearing in the seventh complement regulatory protein (CCP) domain reduces the binding of FH / FHL-1 to BrM, leading to interference with the binding of these blood-derived complement regulatory factors and a reduction in complement regulation on this surface (Clark et al., J Biol Chem (2010) 285:30192-202).

[0215] The binding of FH / FHL-1 to BrM is mediated by sulfated sugars, including glycosaminoglycans (GAGs), heparan sulfate (HS), and dermatan sulfate (DS). The family of GAG sequences found in BrM appears to have higher tissue specificity than previously thought, as it can replenish FH / FHL-1 through its CCP7 domain and bypass the secondary FH anchoring sites on CCPs 19-20 (Clark et al., J Immunol (2013) 190:2049-2057). The main complement regulator within BrM is the partially excised FHL-1 protein (Clark et al., J. Immunol (2014) 193, 4962-4970), which has been found to have only one surface anchoring site on CCP7 and lacks CCPs 19-20, suggesting that this may be an evolutionary twist. In contrast, the Y402H polymorphism is not associated with renal disease in which the CCP 19-20 domains of FH are known to be the main 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 are themselves considered part of the normal aging process, have also been associated with AMD and may explain to some extent the age-related nature of genetically driven AMD.

[0216] A rare mutation (R1210C) in the C-terminal CCP 19-20 region of FH that does not bind to BrM has a very high level of association with AMD, and it has been found that FH proteins possessing this mutation covalently bind to albumin (Sanchez-Corral et al., Am J Hum Genet (2002) 71:1285-1295), preventing the FH protein from leaving the circulation and entering ocular tissue. Several studies suggest that large fused drusen preceding geographic atrophy and associated pigment changes in RPE indicate that dry AMD first arises from RPE dysfunction, followed by secondary effects within the choroid (Bhutto and Lutty Mol Aspects). Med (2012) 33:295-317). In contrast, Whitmore et al. reported capillary changes preceding all forms of late-stage AMD, including deposition of the terminal complement membrane attack complex (MAC), and argued that excessive complement activation in the choroid capillaries is the primary event and RPE atrophy is the secondary event (Whitmore et al., Prog Retin Eye Res (2015) 45:1-29). These data imply that the genetic predisposition conferred by complement gene modification is tolerated until changes in both the BrM and underlying choroid capillaries surface. Whether these changes are age-related, driven by oxidative stress, or a result of RPE cell dysfunction remains unclear, but natural-occurring changes in these structures are known to be age-related.

[0217] C3 and C3b Complement component 3 (C3) is an immune system protein that plays a central role in innate immunity and the complement system. The processing of C3 is described, for example, in Foley et al. J Thromb Haemostasis (2015) 13:610-618, which is hereby incorporated by reference in its entirety. Human C3 (UniProt: P01024; SEQ ID NO: 18) contains a 1,663 amino acid sequence (including a 22 amino acid signal peptide at the N-terminus). Amino acids 23-667 encode the C3 β-chain (SEQ ID NO: 19), and amino acids 749-1,663 encode the C3 α'-chain (SEQ ID NO: 20). The C3 β-chain and C3 α'-chain associate through an interchain disulfide bond (formed between cysteine 559 of the C3 β-chain and cysteine 816 of the C3 α'-chain) to form C3b. C3a is a 77 amino acid fragment (SEQ ID NO: 21) corresponding to amino acids 672-748 of C3 and is generated by proteolytic cleavage of C3 after activation through the classical complement pathway and the lectin pathway.

[0218] C3b is a potent opsonin that targets pathogens, antibody-antigen immune complexes, and apoptotic cells for phagocytosis by phagocytes and NK cells. C3b is also involved in the formation of convertase enzyme complexes for activating and amplifying the complement reaction. C3b associates with factor B to form the C3bBb type C3 convertase (alternative complement pathway), and associates with C4b and C2a to form the C4b2a3b type C5 convertase (classical pathway), or it is also possible to associate with C3bBb to form the C3bBb3b type C5 convertase (alternative pathway).

[0219] Processing of C3b to iC3b, which is proteolytically inactive and unable to promote further complement amplification itself, involves proteolytic cleavage of the C3b α’ chain at amino acids 1303 and 1320, forming α’ fragment 1 (corresponding to amino acids 672 - 748 of C3; SEQ ID NO: 22) and α’ fragment 2 (corresponding to amino acids 1321 - 1,663 of C3; SEQ ID NO: 23). Thus, iC3b contains the C3 β chain, C3 α’ fragment 1, and C3 α’ fragment 2 (associated through disulfide bonds). Cleavage of the α’ chain also releases C3f, which corresponds to amino acids 1304 - 1320 of C3 (SEQ ID NO: 24).

[0220] 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., cynomolgous, human, and / or rodent (e.g., rat and / or mouse) C3). Isoforms, fragments, variants, or homologs of C3 may optionally be characterized as having at least 70%, preferably 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 C3 from a given species, e.g., human C3 (SEQ ID NO: 18).

[0221] In this specification, "C3b" means, and includes, isoforms, fragments, variants or homologs of C3b from any species. In some embodiments, C3b is mammalian C3b (e.g., cynomolgus macaque, human and / or rodent (e.g., rat and / or mouse) C3b).

[0222] C3b isoforms, fragments, variants, or homologs may optionally be characterized by containing C3 α' chain fragment 1, C3 α' chain fragment 2, and C3 β having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a single amino acid sequence identity in the amino acid sequence of their respective polypeptides derived from a given species, such as human. That is, C3b is: C3 α' chain fragment 1 having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity in SEQ ID NO: 22; C3 α' chain fragment 2 having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity in SEQ ID NO: 23; and C3 having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity in SEQ ID NO: 19 It may also contain β-chains.

[0223] C3b isoforms, fragments, mutants, or homologs may optionally be functional isoforms, fragments, mutants, or homologs that possess the functional properties / activities of reference C3b, as determined, for example, by analysis using appropriate assays for functional properties / activity. For example, C3b isoforms, fragments, mutants, or homologs may be characterized by their ability to act as opsonins and / or to form functional C3 / C5 convertases.

[0224] Complement Factor I The processing of C3b to iC3b is carried out by complement factor I (encoded by the gene CFI in humans). Human complement factor I (UniProt: P05156; SEQ ID NO: 25) has a 583-amino acid sequence (including an 18-amino acid signal peptide at the N-terminus). The precursor polypeptide is cleaved by furin to produce mature complement factor I, which contains a heavy chain (amino acids 19-335) and a light chain (amino acids 340-583) linked by interchain disulfide bonds. Amino acids 340-574 of the light chain encode the proteolytic domain of complement factor I (SEQ ID NO: 26), which is a serine protease containing a catalytic triple structure involved in the cleavage of C3b to produce iC3b (Ekdahl et al., J Immunol (1990) 144 (11):4269-74).

[0225] In this specification, “complement factor I (FI)” refers to complement factor I of any species and includes isoforms, fragments, variants or homologs of complement factor I of 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 mouse) complement factor I).

[0226] Isoforms, fragments, mutants, or homologs of complement factor I may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identity of immature or mature complement factor I from a given species, such as human complement factor I (SEQ ID NO: 25). Isoforms, fragments, mutants, or homologs of complement factor I may optionally be functional isoforms, fragments, mutants, or homologs that have the functional properties / activities of reference complement factor I (e.g., full-length human complement factor I) as determined, for example, by analysis by an appropriate assay for functional properties / activity. For example, isoforms, fragments, mutants, or homologs of complement factor I may exhibit serine protease activity and / or be capable of inactivating C3b.

[0227] The proteolytic cleavage of C3b by complement factor I, which yields iC3b, is facilitated by cofactors related to complement factor I. Cofactors related to complement factor I typically bind to C3b and / or complement factor I, and enhance the processing of C3b to iC3b by complement factor I.

[0228] Complement receptor 1 Complement receptor 1 (CR1) acts as a cofactor for complement factor I, enabling cleavage from C3b to iC3b and its downstream products.

[0229] iC3b does not amplify or activate the complement system, but it can still act as an opsonin that targets pathogens for phagocytic purposes. iC3b production, therefore, results in local immune system activation and inflammatory effects. This may have negative consequences for patients with complement-related disorders and may contribute to the development or exacerbation of existing complement-related disorders / conditions.

[0230] Factor H (FH) and the partially resected FH isoform FHL-1 are cofactors related to FI. These cells act to produce iC3b, but they are unable to promote further degradation of iC3b, which can lead to undesirable iC3b accumulation. Furthermore, iC3b accumulation may contribute to further fragmentation in the affected area, for example, leading to the (further) development of drusen in macular degeneration.

[0231] In contrast, CR1 and the polypeptides described in this invention may act in combination with FI, as seen, for example in Figures 2, 3B, and 7A, to promote further degradation of iC3b to preferred downstream products, such as C3c, C3dg, and C3b. These molecules are not opsonins and therefore avoid replenishment of immune system components. Their presence in the affected area is favorable for iC3b accumulation.

[0232] In this specification, “complement receptor 1 (CR1)” refers to CR1 of any species, and this includes isoforms, fragments, variants or homologs of CR1 of any species. In some embodiments, CR1 is mammalian CR1 (e.g., cynomolgus monkey, human and / or rodent (e.g., rat and / or mouse) CR1).

[0233] Aspects and aspects of the present invention will be illustrated herein by reference with reference to the accompanying figures. Further aspects and aspects will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0234] The present invention includes combinations of the aspects and preferred features described, unless such combinations are clearly unacceptable or obviously to be avoided. Section headings used in this specification are for structural purposes only and should not be considered to limit the subject matter described herein.

[0235] To the extent appropriate, features disclosed in the foregoing description, or in the following claims or accompanying figures, expressed in a particular type or with respect to means for performing the disclosed function, or methods or processes for obtaining the disclosed results, may be used individually or in any combination of such features, in a variety of types, to realize the present invention.

[0236] While the present invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is provided. Therefore, the exemplary embodiments of the invention described above are considered illustrative and not limiting. Various modifications to the embodiments described may be made without departing from the spirit and scope of the invention.

[0237] To avoid any doubt, any theoretical explanations provided herein are provided solely for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0238] Throughout this specification, 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, including the following claims.

[0239] It should be noted that when used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In this specification, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from one particular value and / or to the other particular value. Similarly, when values are expressed by use of the antecedent "about", it will be understood that the particular value forms another aspect. When a value is expressed as approximate by use of the antecedent "about", it will be understood that the particular value forms another aspect.

[0240] When a nucleic acid sequence is disclosed, its reverse complement is also clearly intended. The following numbered paragraphs describe particular aspects and embodiments of the invention: 1. A polypeptide having at least 80% sequence identity to SEQ ID NO: 4 and having a length of 700 amino acids or shorter.

[0241] 2. The polypeptide according to paragraph 1, having a length of 50 to 700 amino acids. 3. The polypeptide according to paragraph 1 or 2, wherein X1 is A or T, X2 is P or L, and / or X3 is G or R.

[0242] 4. The polypeptide according to any one of paragraphs 1 to 3, comprising SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 13. 5. A polypeptide according to any one of paragraphs 1 to 4, comprising sequence number 2, sequence number 3, or sequence number 13.

[0243] 6. A polypeptide described in any one of paragraphs 1 to 5, which can be bound to C3b. 7. A polypeptide according to any one of paragraphs 1 to 6, which binds to C3b in a region bound by a cofactor related to complement factor I.

[0244] 8. A polypeptide according to any one of paragraphs 1 to 7, which binds to C3b in the region bound by complement receptor 1 (CR1). 9. A polypeptide described in any one of paragraphs 1 to 8, which acts as a cofactor for complement factor I.

[0245] 10. A polypeptide described in any one of paragraphs 1 to 9, which is diffusible beyond Bruch's membrane (BrM). 11. A polypeptide according to any one of paragraphs 1 to 10, which is not glycosylated or is partially glycosylated.

[0246] 12. A polypeptide according to any one of paragraphs 1 to 11, wherein the amino acid sequence contains one or more amino acid substitutions at positions 509, 578, 959 and / or 1028 (numbered according to UniProt:P17927).

[0247] 13. The polypeptide described in paragraph 12, wherein one or more amino acid substitutions are selected from N509Q, N578Q, N959Q and / or N1028Q (numbered according to UniProt:P17927).

[0248] 14. A polypeptide according to any one of paragraphs 1 to 13, comprising or consisting of sequence number 5, sequence number 6, and / or sequence number 15. 15. A polypeptide according to any one of paragraphs 1 to 14, further comprising a secretory pathway sequence.

[0249] 16. The polypeptide described in paragraph 15, wherein the secretory pathway sequence includes sequence number 7. 17. The polypeptide according to paragraph 15 or paragraph 16, further comprising a cleavage site for removing a secretory pathway sequence.

[0250] 18. A nucleic acid encoding a polypeptide as described in any one of paragraphs 1 to 17. 19. A vector containing the nucleic acid from paragraph 18. 20. A cell comprising a polypeptide as described in any one of paragraphs 1 to 17, a nucleic acid as described in paragraph 18, or a vector as described in paragraph 19.

[0251] 21. A method for producing a polypeptide, comprising the steps of introducing a nucleic acid described in paragraph 18 or a vector described in paragraph 19 into cells, and culturing the cells under conditions suitable for polypeptide expression.

[0252] 22. Cells obtained or obtainable by the method described in paragraph 21. 23. A pharmaceutical composition comprising a polypeptide according to any one of paragraphs 1 to 17, a nucleic acid according to paragraph 18, a vector according to paragraph 19, or a cell according to paragraph 20 or 22, and optionally comprising a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0253] 24. A polypeptide according to any one of paragraphs 1 to 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.

[0254] 25. Use of a polypeptide according to any one of paragraphs 1 to 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 drug for treating or preventing a disease or condition.

[0255] 26. A method for treating or preventing a disease or condition, comprising the step of administering to a subject a polypeptide described in any one of paragraphs 1 to 17, a nucleic acid described in paragraph 18, a vector described in paragraph 19, cells described in paragraph 20 or 22, or a pharmaceutical composition described in paragraph 23.

[0256] 27. A method for treating or preventing a disease or condition in a subject, comprising the step of modifying at least one cell of the subject to express or contain a polypeptide described in any one of paragraphs 1 to 17, a nucleic acid described in paragraph 18, or a vector described in paragraph 19.

[0257] 28. A polypeptide, nucleic acid, vector, cell, or pharmaceutical composition for use as described in paragraph 24, the use as described in paragraph 25, or the method as described in paragraph 26 or paragraph 27, wherein the disease or condition is a disease or condition in which C3b or a C3b-containing complex, an activity / reaction related to C3b or a C3b-containing complex, or a product of an activity / reaction related to C3b or a C3b-containing complex is pathologically involved.

[0258] 29. A polypeptide, nucleic acid, vector, cell, or pharmaceutical composition, use, or method for use as described in any one of paragraphs 24-28, wherein the disease or condition is age-related macular degeneration (AMD).

[0259] 30. A polypeptide, nucleic acid, vector, cell, or pharmaceutical composition, use, or method for use according to any one of paragraphs 24 to 29, comprising the step of modifying at least one retinal pigment epithelial (RPE) cell of a subject to express or contain a polypeptide according to any one of paragraphs 1 to 17, a nucleic acid according to paragraph 18, or a vector according to paragraph 19, for the treatment or prevention of a disease or condition.

[0260] 31. A partial kit comprising a predetermined amount of a polypeptide according to any one of paragraphs 1 to 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. [Examples]

[0261] In the following examples (one or more), we describe the design of a recombinant CR1 protein comprising the C3b-binding cofactor region of complement receptor 1. Also described is the ability of these proteins to be expressed by human cells, diffuse through a concentrated Bruch membrane from the eye of a human donor, and confer regulatory activity, namely the ability to promote FI-mediated degradation from C3b to iC3b and further degradation products.

[0262] Example 1 DNA insertions encoding the amino acid sequences shown in SEQ ID NOs: 2 and 5 were prepared using recombinant DNA technology and cloned into a vector to generate constructs for recombinant expression of the CR1 peptide. The amino acid sequences and their characteristics are shown below:

[0263] [ka]

[0264] The 18-amino acid signal peptide is designed to be cleaved from the polypeptide during secretion. In several experiments, HIS-tagged CR1a and nCR1a were used, as shown in sequence numbers 40 and 21, respectively.

[0265] Protein expression from human cells HEK 293T cells (7x10 per plate) 6Cells were grown overnight in 15 cm culture plates in 17 ml of high-glucose Dulbecco's modified Eagle medium (DMEM, Sigma, catalog number D469) supplemented with 10% fetal bovine serum (FBS, Sigma, catalog number F9665) in a 5% CO2 incubator at 37°C. Once the cells reached 60% density, they were transiently transfected with a 14.4 μg plasmid expressing either CR1a (sequence number 2) or nCR1a (sequence number 5) linked to a signal peptide (sequence number 7) using 86.4 μl of 7.5 mM polyethyleneimine (PEI, Polysciences, catalog number 24765-2) and 150 mM NaCl (Fisher Scientific UK Ltd, catalog number 1073592). For a negative control, 14.4 μl of Tris-EDTA buffer was used instead of plasmid DNA. Five hours after transfection, 10% F Transfection medium containing BS was replaced with 17.5 ml of fresh DMEM (hereinafter referred to as expression medium) containing high glucose supplemented with 2% FBS. Expression medium was collected 24, 48, 72, and 140 hours after transfection. 80 μl of 0.5 M phenylmethanesulfonyl fluoride (PMSF, Sigma, catalog number P7626) was added to each 100 ml of collected expression medium and stored at 4°C. Expression medium collected after 24 hours was used for the diffusion and functional studies described below.

[0266] Characterization of proteins secreted from human cells Protein expression from human cells is shown in Figures 1A and 1B. Recombinantly expressed and purified CR1a protein was found to be secreted from cells in two glycosylation forms. Treatment with PNGase F, an enzyme that removes glycosylation, reduced the two bands to a single band with a smaller apparent molecular weight (1A). The unglycosylated form of the protein (nCR1a) was expressed. Western blotting demonstrated that nCR1a produced a single band that moved to the same position as the enzymatically deglycosylated protein (1B).

[0267] C3b degrading activity We tested the ability of CR1a and nCR1a expressed and secreted from human HEK293 cells to act as cofactors for factor I-mediated degradation of C3b.

[0268] 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 no. 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 stopped by adding 4xSDS-loaded buffer and heating at 100°C for 5 minutes. The samples were then electrophoresed on 4–12% NuPAGE Bis-Tris gels at 200 V for 60 minutes. The samples were transferred to a nitrocellulose membrane at 80 mA for 1.5 hours using a semi-dry transfer apparatus in transfer buffer (25 mM Tris, 192 mM glycine, 10% (v / v) methanol). The membranes were blocked in PBS, 10% (w / v) milk, and 0.2% (w / v) BSA at 4°C for 16 hours. Then, 100 μg / ml of anti-C3b antibody (Hycult Biotech, catalog number HM2287) was added to PBS and 0.2% (v / v) Tween-20 (PBS-T) at room temperature for 1 hour. After washing the membranes in PBS-T for 2 x 30 minutes, a 1:2500 dilution of HRP-conjugated goat anti-mouse was added at room temperature for 1 hour, shielded from light. After washing the membranes in PBS-T for 2 x 30 minutes, SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, catalog number 34080) was added at room temperature for 3 minutes. The treated membranes were exposed to Super RX-N X-ray film (FujiFilm, catalog number PPB5080) for 2 minutes at room temperature, and reaction bands were detected by development on an automated X-ray film developer.

[0269] The results are shown in Figure 2. Both secreted proteins act as cofactors for complement factor I, first breaking down C3b to iC3b(a1), and then further breaking down C3dg(a 1-1 This led to the degradation of ) into ). The degradation of C3b using CR1a / nCR1a continued further than the normal natural degradation of C3b observed using FI+FHL-1 (second lane), which yields only iC3b(a1).

[0270] Ussing chamber diffusion experiment and C3b degradation activity McHarg et al., J Vis Exp (2015) 1-7, as described above. First, the macular region of the concentrated Bruch membrane isolated from the donor eye was placed in a Ussing chamber (Harvard Apparatus, Hampden, USA). Once placed, the 5 mm diameter macular region was the only barrier between two identical compartments, meaning that the liquid had to pass through the Bruch membrane (Figure 4). Both sides of the Bruch membrane were washed with 2 ml of PBS at room temperature for 5 minutes. Before the experiment, the structural integrity of the Bruch membrane was tested by its ability to hold 2 ml of liquid in one chamber without leakage to 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 diffuser / diffusion chamber). The Ussing chamber was left at room temperature for 24 hours, with each compartment gently agitated using a magnetic stirrer bar to avoid creating a diffusion gradient of the protein.

[0271] CR1a was added to the sample chamber. After 24 hours, samples from each chamber (the original sample chamber and the diffusion chamber) were tested for the presence of CR1a. The results are shown in Figure 3A. CR1a was found to be present in the diffusion chamber after 24 hours.

[0272] CR1a and nCR1a were added separately to sample chambers, and samples from both chambers were analyzed for C3b degradation activity. After 24 hours, 18.6 μl of sample was taken from each chamber and mixed with 1 μl (1 μg) of pure C3b protein and 0.4 μl of pure complement factor I (0.04 μg, VWR International, catalog no. 341280) at 37°C for 15, 30, or 60 minutes. 4xSDS-loaded buffer was added, and the reaction was stopped by heating at 100°C for 5 minutes. The samples were then electrophoresed on 4–12% NuPAGE Bis-Tris gels at 200 V for 60 minutes.

[0273] The results are shown in Figures 3B and 3C. The degradation of C3b in the diffusion chamber demonstrates that both glycosylated CR1a (3B) and non-glycosylated nCR1a (3C) were able to traverse the Bruch membrane from the sample chamber and remained functionally active. CR1a and nCR1a from both chambers were found to successfully act as cofactors for FI, degrading C3b into proteolytically inactive C3b (iC3b) and further products, as evidenced by the presence of bands corresponding to the C3b degradation products iC3b and C3dg. Thus, CR1a and nCR1a are capable of diffusing through BrM and retaining the ability to contribute to C3b degradation in the presence of factor I.

[0274] Example 2 The expression levels of the polypeptides described in this invention are compared to evaluate whether there is an optimal formulation, i.e., whether the glycosylated polypeptide is expressed at a higher level than the non-glycosylated polypeptide. It is expected that the glycosylation status of the polypeptide will have a minimal effect on the expression level.

[0275] CR1a was found to be well expressed in human cells. nCR1a was found to be expressed at lower levels than CR1a, but both polypeptides were found to be functionally active. See, for example, Figure 2.

[0276] Example 3 Using surface plasmon resonance (SPR), C3b is immobilized on an SPR tip, and the binding kinetics of the purified polypeptide described in this invention to C3b are tested using the polypeptide in liquid phase. Association and dissociation constants are measured directly, and the kD value for the interaction is estimated.

[0277] No difference is observed in the binding of CR1a (CCP 8-10) or CR1b (CCP 15-17) species to C3b, but glycosylated polypeptides bind more strongly to C3b than non-glycosylated polypeptides.

[0278] Couple dynamics measured by bio-layer interferometry (BLI) The affinity of CR1a for the C3b protein was measured using the OctetRed96 system (ForteBio, Pall Corp, USA). Biotinylated C3b protein was diluted to a final concentration of 0.4 μg / mL with 0.2% PBST and loaded onto a high-precision streptavidin (SAX) biosensor (ForteBio, Pall Corp, USA), which had been pre-hydrated in the same buffer for 20 minutes, for 600 seconds. The C3b-loaded sensor was then washed with 0.2% PBST for 150 seconds (baseline), dipped for 600 seconds into wells containing CR1a at different concentrations ranging from 30.0 μg / mL to 2.6 μg / mL (association), and then washed with 0.2% PBST for 600 seconds (dissociation). The association and dissociation profiles were recorded and analyzed using ForteBio Data Analysis v9 (ForteBio, Pall Corp, USA). A negative control, i.e., a well containing 0.2% PBST, was used in parallel to reduce binding resulting from nonspecific interactions with the sensor. Experiments were performed at 25°C using dynamic mode, and the sample plate was agitated for 3 minutes beforehand. The binding profile was fitted to a 1:1 ratio overall (one analyte in solution for one binding site on the surface). By steady-state analysis, the KD was determined using association (0 sec to 600 sec) and dissociation (0 sec to 100 sec) data from four of the lowest available analyte concentrations. Binding affinity for fixed C3b to the naturally soluble C3b-binding complement regulators, factor H (FH) and FHL-1, was also determined.

[0279] The results are shown in Figure 5. The binding affinity of CR1a to C3b was found to be 21 nM. For comparison, the binding affinity of FH to C3b was 580 nM, and that of FHL-1 was 1.2 mM. Therefore, CR1a binds to C3b significantly more strongly than either of the innate 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.

[0280] Strong binding affinity also allows CR1a to promote the degradation of iC3b to a desirable further downstream product, such as C3dg (see Figure 2). In contrast, FH and FHL-1 are unable to induce degradation of C3b beyond iC3b. iC3b is a pro-inflammatory molecule that acts to replenish immune cells at the site of complement activation, which in turn causes a negative inflammatory effect. Therefore, further degradation of iC3b to C3dg by CR1a is preferable and avoids further damage caused by the immune system.

[0281] Example 4 Using the Wushing chamber experiment (described above), the diffusion rates of the polypeptides described in the present invention were compared to see if any differences arose due to the protein composition.

[0282] The experiment involves using donor-derived Bruch membranes in this state to determine whether substances deposited in the Bruch membrane in AMD, including drusen and basal-backed (linear) deposits, impair the ability of polypeptides to traverse the Bruch membrane. Optimal polypeptides are expected to traverse the Bruch membrane even in the presence of AMD changes.

[0283] Example 5 Optionally, a preferred signal peptide having at least 80% sequence identity with SEQ ID NO: 4. The nucleic acid described in the present invention, which encodes a polypeptide and a termination codon, is inserted into an AAV vector. Cultured RPE cells are transfected with the resulting expression vector. The expression and secretion of the encoded polypeptide are evaluated. A polypeptide having at least 80% sequence identity to SEQ ID NO: 4 is expected to be secreted by RPE cells, and the signal peptide is expected to be cleaved from the secreted polypeptide.

[0284] 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 retinal pigment epithelium.

[0285] AAV2 serotype virus particles were prepackaged with a CR1a plasmid. ARPE-19 cells were inoculated at a density of 300,000 cells per well into 2 ml of DMEM / F12 growth medium (ATCC, USA) supplemented with 10% (v / v) fetal bovine serum (ATCC, USA) in a 6-well cell culture plate (Corning). The cells were then incubated at 37°C for 24 hours in humidified air with 5% CO2. After incubation, the cells were washed twice with 2 ml of serum-free DMEM / F12 growth medium. AAV2-CR1a with an infection efficiency (MOI) of 100,000 was added to 1 ml of serum-free DMEM / F12 growth medium. The AAV2-CR1a-containing medium was incubated with the cells for 24 hours (37°C, 5% CO2), and the following day it was replaced with 2 ml of fresh serum-free DMEM / F12 growth medium. Control cells transduced with AAV-GFP were grown in parallel. Transduction efficiency was evaluated 14 days after infection. CR1a secretion by ARPE-19 cells was detected by dot blotting; and conditioned medium derived from CR1a-transduced RPE cells was contacted with in-house polyclonal anti-CR1a antibody.

[0286] The results are shown in Figure 6. Compared to the medium derived from RPE cells transduced with AAV-GFP (control medium), which was used as a negative control, immunoreactivity with anti-CR1a antibody was observed in both tested samples (AAV-CR1a medium). Purified recombinant CR1a protein was included as a positive control.

[0287] We evaluated the ability of recombinant CR1a polypeptides secreted from human APRE-19 and HEK293 cells to degrade from C3b to iC3b and C3dg. The results are shown in Figure 7A. Functional CR1a polypeptide secreted from human APRE-19 cells was found to act as a cofactor for factor I, leading to the degradation of C3b to iC3b (product e) and C3dg (product f). Reactions containing FHL-1 + FI + C3b provided a MW control for C3b and its product iC3b.

[0288] C3b degradation was evaluated in tissue culture medium of human RPE cells (ARPE-19) transduced with AAV-delivered CR1a. AAV-GFP transduced RPE cells were used as a negative control. Fourteen days after transduction, the transduced cells were supplemented with purified C3b and factor I. C3b and iC3b levels in tissue culture medium were detected by Western blotting.

[0289] The results are shown in Figure 7B. RPE cells were found to produce their own complement components and to have a low intrinsic turnover of C3b (product a) to iC3b (product b; lane 2). However, RPE cells expressing AAV-delivered CR1a were found to have increased C3b-degrading ability to iC3b (product b; lane 3) compared to the intrinsic turnover rate.

[0290] This means that CR1a is successfully secreted from ocular cells, such as retinal pigment epithelial cells, and is functionally active as an FI cofactor, promoting C3b degradation to downstream products, enabling complement regulation, and offering therapeutic benefits against complement hyperactivation, such as conditions involving excessive C3b. They will provide it.

[0291] Example 6 The polypeptide of the present invention finds use in methods for treating or preventing complement-related disorders. An example of a complement-related disorder is macular degeneration of the eye, e.g., AMD.

[0292] Figure 8 provides a schematic diagram of the macula of the eye. The retinal pigment epithelium (RPE) is a continuous monolayer of cuboidal / columnar epithelial cells between the neurosensory retina and the vascular choroid. The cells possess physical, optical, metabolic / biochemical, and transport functions and play an essential role in normal visual processes. The RPE is separated from the choroid by Bruch's membrane (BrM): a thin (2-4 μm) cell-free, five-layer extracellular matrix. The BrM performs two main functions: acting as the basal layer of the RPE and as the vascular wall. Immediately adjacent to the BrM, and within the choroid, is a layer of capillaries called choroidal capillaries. Complement activation is centered on the extracellular matrix of the choroidal capillaries, called the intercapillary septum.

[0293] The characteristic lesions of AMD, drusen, form from accumulations of lipids and cellular debris containing numerous complement activators. Drusens develop within the BrM adjacent to the RPE layer and disrupt the flow of nutrients from the choroid to the RPE, leading to cellular dysfunction and cell death. RPE cell death also causes dysfunction of photoreceptor cells and subsequent vision loss.

[0294] Representative fluorescence microscopy images of drusen deposition taken from Forest et al. (2015) Dis. Mod. Mech. 8, 421-427 (Figure 1) are provided in Figure 9. Figure 9 shows retinal tissue from an 82-year-old woman with AMD. Cell membrane markers indicate degraded RPE cells layered on drusen. Complement-activated regions within and around drusen are indicated by the terminal complement complex marker C5b-9. Nuclei are stained. Scale bar 20 μm.

[0295] Systemic administration of complement regulatory molecules requires high doses and carries a substantial risk of harmful off-target effects on the functional complement system. Local administration, such as expression from RPE cells, is a safer and more effective delivery method. However, since RPE-expressed molecules are effective in treating / preventing complement-related disorders in the intercapillary septum of choroidal capillaries, i.e., at the site of complement hyperactivation, the molecules must cross Bruch's membrane (see Figure 10). In other words, effective complement inhibition therapy must meet the requirements of all three stages in Figure 10.

[0296] As shown herein, CR1a is: 1. Secreted from human RPE cells (see, for example, Figure 6); 2. Passive diffusion beyond human BrM is possible (see, for example, Figure 3); and 3. In the presence of complement factor I, the degradation of C3b to iC3b and, moreover, to more desirable degradation products can be mediated (see, for example, Figures 2, 3, and 7).

[0297] Therefore, CR1a can be expressed by RPE cells, reach the complement activation domain when C3b regulation is needed, and can act as an effective therapeutic agent for treating complement hyperactivation in the eye, such as in AMD.

[0298] Example 7 Laser-induced choroidal angiogenesis model This model involves applying laser burns to the retina of a mouse or rat, for example, Schnabolk et al. Mol Ther Methods Clin Dev. 2018; 9 As described in 1-11, it causes choroidal angiogenesis. The size of the choroidal angiogenic complex may be measured by fluorescein angiography or histology.

[0299] Rodents are administered subretinal injections of AAV vectors, such as AAV2 containing CR1a cDNA, or empty AAV vectors as a control. Rodents administered with the CR1a cDNA-containing AAV vector secrete CR1a protein from their retinal pigment epithelial cells. Once maximal CR1a protein efflux is achieved, a laser burn is applied to the rodent retina, and the size of the choroidal angiogenic complex is measured at a predetermined time after the laser burn. The complement inhibitory effect of the CR1a polypeptide is observed by reducing the size of the choroidal angiogenic complex compared to rodents administered with empty AAV vectors.

[0300] Sodium iodide-induced retinal degeneration For example, as described in Katschke et al., Sci Rep. 2018; 8(1):7348, mice are intravenously injected with sodium iodide, which induces retinal pigment epithelial degeneration partially dependent on complement activation. CR1a is delivered either by intravitreous injection as a recombinant protein or by subretinal delivery using an AAV vector containing CR1a cDNA, such as AAV2. CR1a-treated mice are found to have less retinal degeneration than control mice that have not received CR1a treatment. Non-limitingly, the present invention includes the following embodiments. [Aspect 1] A C3b-binding polypeptide for use in methods of treating or preventing complement-related diseases or conditions, comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 4, and having a total length of 450 amino acids or less. [Aspect 2] A nucleic acid encoding a C3b-binding polypeptide for use in methods of treating or preventing complement-related disorders or conditions, wherein the polypeptide comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4 and having a total length of 450 amino acids or less. [Aspect 3] The use of a C3b-binding polypeptide in the manufacture of a drug for treating or preventing a complement-related disease or condition, wherein the polypeptide comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4 and having a total length of 450 amino acids or less. [Aspect 4] The use of a nucleic acid encoding a C3b-binding polypeptide in the manufacture of a drug for treating or preventing a complement-related disorder or condition, wherein the nucleic acid encoding the polypeptide comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4 and having a total length of 450 amino acids or less. [Aspect 5] Use of the polypeptide according to Embodiment 1 or Embodiment 2, or according to Embodiment 3 or Embodiment 4, wherein the complement-related disease or condition is an eye disease or condition. [Aspect 6] The polypeptide or use according to embodiment 5, comprising the step of modifying at least one eye cell of a subject to express or contain the polypeptide, for the treatment or prevention of an eye disease or condition. [Aspect 7] The polypeptide or use according to embodiment 5 or 6, comprising the step of modifying at least one eye cell of a subject to express or contain the nucleic acid encoding the polypeptide, for the treatment or prevention of an eye disease or condition. [Aspect 8] The polypeptide or use according to any one of embodiments 5 to 7, wherein the treatment or prevention of an eye disease or condition comprises the step of administering a vector containing a nucleic acid encoding the polypeptide to at least one eye cell of a subject. [Aspect 9] A polypeptide or use according to any one of embodiments 6 to 8, wherein at least one cell of the eye is a retinal pigment epithelial (RPE) cell. [Aspect 10] The polypeptide or use according to any one of embodiments 1 to 9, wherein the disease or condition is a disease or condition in which C3b or a C3b-containing complex, an activity / reaction related to C3b or a C3b-containing complex, or a product of an activity / reaction related to C3b or a C3b-containing complex is pathologically involved. [Aspect 11] The polypeptide or use according to any one of the embodiments 1 to 10, wherein the disease or condition is macular degeneration. [Aspect 12] The polypeptide or use according to any one of embodiments 1 to 11, wherein the disease or condition is: selected from one or more of the following: age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, geographic atrophy ("dry" AMD), "wet" (neovascular) AMD, choroidal neovascularization (CNV), glaucoma, autoimmune uveitis, diabetic retinopathy, and early-onset macular degeneration (EOMD). [Aspect 13] The polypeptide or use according to any one of embodiments 1 to 12, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 4. [Aspect 14] A polypeptide or use according to any one of embodiments 1 to 13, wherein X1 is A or T, X2 is P or L, and / or X3 is G or R. [Aspect 15] The polypeptide according to any one of embodiments 1 to 14, wherein the polypeptide has a total length of 50 to 250 amino acids, or the use thereof. [Aspect 16] The polypeptide according to any one of embodiments 1 to 15, wherein the polypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 3 or consists of a sequence thereof. [Aspect 17] The polypeptide or use according to any one of embodiments 1 to 14, wherein the polypeptide comprises or consists of sequence number 13. [Aspect 18] A polypeptide or use according to any one of embodiments 1 to 17, wherein the polypeptide is capable of acting as a cofactor for complement factor I. [Aspect 19] The polypeptide or use according to any one of embodiments 1 to 18, wherein the polypeptide is diffusible beyond the Bruch membrane (BrM). [Aspect 20] The polypeptide or use according to any one of embodiments 1 to 19, wherein the polypeptide binds to C3b in a region bound by a cofactor relating to complement factor I. [Aspect 21] The polypeptide according to any one of embodiments 1 to 20, wherein the polypeptide binds to C3b in the region bound by complement receptor 1 (CR1), or the use thereof. [Aspect 22] The polypeptide according to any one of embodiments 1 to 21, wherein the polypeptide includes a secretory pathway sequence, or the use thereof. [Aspect 23] The polypeptide or use according to embodiment 22, wherein the secretory pathway sequence includes or consists of sequence number 7. [Aspect 24] The polypeptide or use according to embodiment 22 or embodiment 23, wherein the polypeptide comprises or consists of sequence numbers 47, 49, or 51. [Pattern 25] The polypeptide according to any one of embodiments 22 to 24, wherein the polypeptide includes a cleavage site for removing a secretory pathway sequence, or the use thereof. [Aspect 26] A C3b-binding polypeptide comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 4, and having a total length of 450 amino acids or less. [Aspect 27] The polypeptide according to embodiment 26, comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 4. [Aspect 28] A polypeptide according to either embodiment 26 or embodiment 27, wherein X1 is A or T, X2 is P or L, and / or X3 is G or R. [Aspect 29] The polypeptide according to any one of embodiments 26 to 28, wherein the polypeptide has a total length of 50 to 250 amino acids. [Aspect 30] A polypeptide according to any one of embodiments 26 to 29, comprising or consisting of sequence number 2 or sequence number 3. [Aspect 31] A polypeptide according to any one of embodiments 26 to 28, comprising or consisting of sequence number 13. [Aspect 32] The polypeptide according to any one of embodiments 26 to 31, wherein the polypeptide can act as a cofactor for complement factor I. [Aspect 33] A polypeptide according to any one of embodiments 26 to 32, wherein the polypeptide is diffusible beyond Bruch's membrane (BrM). [Aspect 34] A polypeptide according to any one of embodiments 26 to 33, which binds to C3b in a region bound by a cofactor related to complement factor I. [Aspect 35] A polypeptide according to any one of embodiments 26 to 34, which binds to C3b in a region bound by complement receptor 1 (CR1). [Aspect 36] A polypeptide according to any one of embodiments 26 to 35, wherein the polypeptide includes a secretory pathway sequence. [Aspect 37] The polypeptide according to embodiment 36, wherein the secretory pathway sequence includes or consists of sequence number 7. [Aspect 38] The polypeptide according to embodiment 36 or embodiment 37, wherein the polypeptide comprises or consists of sequences of sequence numbers 47, 49, or 51. [Aspect 39] The polypeptide according to any one of embodiments 36 to 38, wherein the polypeptide includes a cleavage site for removing a secretory pathway sequence. [Aspect 40] A nucleic acid encoding a polypeptide according to any one of embodiments 26 to 39. [Aspect 41] A vector containing nucleic acid according to embodiment 40. [Aspect 42] A cell comprising a polypeptide according to any one of embodiments 26 to 39, a nucleic acid according to embodiment 40, or a vector according to embodiment 41. [Aspect 43] A method for producing a polypeptide, comprising the steps of introducing a nucleic acid according to embodiment 40 or a vector according to embodiment 41 into cells, and culturing the cells under conditions suitable for polypeptide expression. [Aspect 44] Cells obtained or obtainable by the method described in aspect 43. [Aspect 45] A pharmaceutical composition comprising a polypeptide according to any one of embodiments 26 to 39, a nucleic acid according to embodiment 40, a vector according to embodiment 41, or a cell according to embodiment 42 or 44, and optionally comprising a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent. [Aspect 46] A polypeptide according to any one of embodiments 26 to 39, a nucleic acid according to embodiment 40, a vector according to embodiment 41, a cell according to embodiment 42 or 44, or a pharmaceutical composition according to embodiment 45 for use in a method of treating or preventing a disease or condition. [Aspect 47] Use of a polypeptide according to any one of embodiments 26 to 39, a nucleic acid according to embodiment 40, a vector according to embodiment 41, a cell according to embodiment 42 or 44, or a pharmaceutical composition according to embodiment 45 in the manufacture of a drug for treating or preventing a disease or condition. [Aspect 48] A partial kit comprising a predetermined amount of a polypeptide according to any one of embodiments 26 to 39, a nucleic acid according to embodiment 40, a vector according to embodiment 41, cells according to embodiment 42 or embodiment 44, or a pharmaceutical composition according to embodiment 45.

Claims

1. An isolated nucleic acid encoding a polypeptide capable of binding to C3b, comprising a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 35, wherein the encoded polypeptide has a full length of 180-300 amino acid residues.

2. The isolated nucleic acid according to claim 1, wherein the encoded polypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 3, or comprises an amino acid sequence 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 SEQ ID NO: 7 or a nucleic acid sequence encoding a secretory pathway sequence having an amino acid sequence consisting of SEQ ID NO:

7.

4. The isolated nucleic acid according to claim 3, wherein the polypeptide comprises SEQ ID NO: 47 or 49, or has an amino acid sequence consisting of SEQ ID NO: 47 or 49.

5. The isolated nucleic acid according to any one of claims 1 to 4, wherein the nucleic acid comprises nucleotides 13-648 of sequence number 35.

6. The isolated nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid comprises nucleotides 7-648 of sequence number 35.

7. A vector comprising an isolated nucleic acid according to any one of claims 1 to 6.

8. The vector according to claim 7, which is a viral vector.

9. The vector according to claim 7 or 8, which is a lentiviral vector, a retroviral vector, a gamma-retroviral vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a vaccinia virus vector, or a herpesvirus vector.

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

11. The vector according to any one of claims 7 to 10, which is an AAV-2 vector, or a hybrid and / or mutant thereof.

12. The vector according to any one of claims 7 to 11, further comprising a promoter and / or a polyadenylated sequence.

13. A mammalian cell comprising or expressing a nucleic acid according to any one of claims 1 to 6, or a vector according to any one of claims 7 to 12.

14. A mammalian cell according to claim 13, which is an eye cell.

15. The mammalian cell according to claim 13 or 14, wherein the cell is a retinal pigment epithelial cell (RPE), a photoreceptor cell, a retinal cell, or a choroidal or macular cell.

16. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 6, a vector according to any one of claims 7 to 12, or a mammalian cell according to any one of claims 13 to 15.

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