Kidney active fusion proteins and methods of treatment using the same

Abstract

To provide fusion proteins which include factor H catalytic domains and may include VHH domains, factor H-related protein 5 domains, or integrin binding domains, and the use of such fusion proteins in methods of treatment of diseases mediated by complement alternative pathway activation or dysregulation, for example, kidney diseases.SOLUTION: A fusion protein having a structure, from N-terminus to C-terminus: D1-L1-D2-L2-D3, wherein D1 comprises a fragment of complement factor H (FH); L1 is absent, is a covalent bond, or is an amino acid sequence of at least one amino acid; D2 comprises a VHH or is absent; L2 is absent, is a covalent bond, or is an amino acid sequence of at least one amino acid; and D3 is an integrin recognition domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 16, 2022, is named 50694-083WO2_Sequence_Listing_5_16_22_ST25 and is 140,215 bytes in size. [Background technology]

[0002] The complement system plays a central role in the clearance of immune complexes and immune responses against infectious agents, foreign antigens, virus-infected cells, and tumor cells. Complement activation occurs primarily through three pathways: the classical pathway, the lectin pathway, and the alternative pathway. The alternative complement pathway is in a constant state of low-level activation. Uncontrolled activation or insufficient regulation of the alternative complement pathway (CAP) can lead to inflammation, cellular injury, and tissue damage. Local alternative pathway activation within the kidney contributes to renal pathology and loss of function. Therefore, the alternative complement pathway is involved in the pathogenesis of many kidney diseases. Inhibition or modulation of alternative complement pathway activity in the absence of lectin and classical pathway initiation has been recognized as a promising therapeutic strategy. For example, the alternative pathway plays a role in amplifying complement activation initiated by all three pathways. The number of treatment options available for diseases associated with the alternative complement pathway is limited. Therefore, there is a significant unmet need to develop innovative strategies to treat diseases associated with activation or dysregulation of the alternative complement pathway, such as kidney diseases, which affect an estimated 37 million people in the United States alone. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein are fusion polypeptides comprising the Factor H catalytic domain. The fusion proteins can be used to treat patients with diseases associated with alternative complement pathway activation or dysregulation, such as renal disease.

[0004] Provided herein are fusion proteins having the structure, from N-terminus to C-terminus, D1-L1-D2-L2-D3, where D1 comprises a fragment of complement factor H (FH); L1 is absent, covalently linked, or an amino acid sequence of at least one amino acid; D2 comprises a VHH or is absent; L2 is absent, covalently linked, or an amino acid sequence of at least one amino acid; and D3 is an integrin recognition domain. In some embodiments, D1 comprises one or more (e.g., two, three, four, five, or more) FH short consensus repeat (SCR) domains, optionally wherein the one or more SCR domains are selected from the group consisting of SCRs 1, 2, 3, 4, 5, 6, 19, and 20. In some embodiments, the FH SCR domains are selected from the group consisting of SCRs 1-4; 1-5; 1-6, 19, and 20; 1-5, 19, and 20; or 19 and 20.

[0005] In one embodiment, the VHH of D2 comprises a single domain antibody. In another aspect, the VHH of D2 comprises a camelid single domain antibody. In one embodiment, the integrin recognition domain of D3 comprises an integrin recognition domain comprising an arginylglycylaspartic acid (RGD) peptide motif. In another embodiment, the integrin recognition domain of D3 comprises a cyclo(RGD)4 peptide motif.

[0006] In one embodiment, L1 and L2 comprise the same amino acid sequence. In another embodiment, L1 and L2 comprise different amino acid sequences. In some embodiments, L1 and / or L2 are selected from the group consisting of (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGAGGGGAGGGGS, GGGGSGGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GSTSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G12 , APELPGGP, SEPQPQPG, (G3S2)3, GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS)3, (GS4)3, G4A(G4S)2, G4SG4AG4S, G3AS(G4S)2, G4SG3ASG4S, G4SAG3SG4S, (G4S)2AG3S, G4SAG3SAG3S, G4D(G4S)2, G4SG4DG4S, (G4D)2G4S, G4E(G4S)2, G4SG4EG4S, (G4E)2G4S, G4SDA, G4A and (G4A)3, for example G4A. In some embodiments, L1 and / or L2 are selected from the group consisting of (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4SDAA, (G4S)4, G4AG3AG4S, G4A, and (G4A)3.

[0007] In some embodiments, the fusion protein comprises FH SCR domains 1-5; L1 comprises G4A; D2 is absent; L2 is absent; and D3 comprises cyclo(RGD)4; or D1 comprises FH SCR domains 1-5; L1 is absent; D2 comprises a VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; or D1 comprises FH SCR domains 1-5; L1 comprises G4A; D2 is absent; L2 comprises G4A; and D3 comprises cyclo(RGD)4; or D1 comprises FH SCR domains 1-5; L1 is absent; D2 comprises a VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; or D1 comprises FH It comprises SCR domains 1 to 5; L1 is absent; D2 comprises a VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; D1 comprises FH SCR domains 1 to 6; L1 is absent; D2 comprises a VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; or D1 comprises FH SCR domains 1 to 5; L1 comprises G4A; D2 comprises a VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4.

[0008] In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 4 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); the amino acid sequence of SEQ ID NO: 5 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); the amino acid sequence of SEQ ID NO: 8 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); the amino acid sequence of SEQ ID NO: 9 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); or having the amino acid sequence of SEQ ID NO: 13 or a variant thereof having up to 10 amino acid substitutions, additions or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8 and 9 amino acids); or having the amino acid sequence of SEQ ID NO: 14 or a variant thereof having up to 10 amino acid substitutions, additions or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8 and 9 amino acids); or having the amino acid sequence of SEQ ID NO: 15 or a variant thereof having up to 10 amino acid substitutions, additions or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8 and 9 amino acids).

[0009] In some embodiments, the fusion protein has an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:4; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:5; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:8; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:9; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:13; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:14; and an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO:15.

[0010] In another aspect, the present disclosure provides a fusion protein comprising, from N-terminus to C-terminus, the structure D1-L1-D2, where D1 comprises an FH fragment, e.g., FH1-5, L1 comprises a linker or is absent, and D2 comprises a factor H-related protein 5 (FHRP5) domain, e.g., FHRP domains 7 and 8. In one embodiment, L1 is G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGAGGGGAGGGGS, GGGGSGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GSTSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12, APELPGGP, SEPQPQPG, (G3S2)3, GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS)3, (GS4)3, G4A(G4S)2, G4SG4AG4S, G3AS(G4S)2, G4SG3ASG4S, G4SAG3SG4S, (G4S)2AG3S, G4SAG3SAG3S, G4D(G4S)2, G4SG4DG4S, (G4D)2G4S, G4E(G4S)2, G4SG4EG4S, (G4E)2G4S and G4SDA, e.g., G4A. In some embodiments, L1 is selected from the group consisting of G4A and (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4SDAA, (G4S)4 and G4AG3AG4S.

[0011] In one embodiment, the fusion protein has the amino acid sequence of SEQ ID NO: 6 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acids); or the amino acid sequence of SEQ ID NO: 10 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acids). In some embodiments, the fusion protein has an amino acid sequence that has at least 85% (e.g., at least 90%, 95, and 99%) sequence identity to SEQ ID NO: 6; or an amino acid sequence that has at least 85% (e.g., at least 90%, 95, and 99%) sequence identity to SEQ ID NO: 10.

[0012] In another aspect, the present disclosure provides a fusion protein comprising, from N-terminus to C-terminus, the structure D1-L1-D2-L2-D3, where D1 comprises an integrin recognition domain such as cyclo(RGD)4, L1 may or may not contain a linker, D2 is a VHH such as a single-domain antibody, L2 may or may not contain a linker, and D3 is an FH fragment such as FH1-5. In some embodiments, the fusion protein has a C-terminal His tag. In one embodiment, L1 and L2 comprise the same amino acid sequence. In another embodiment, L1 and L2 comprise different amino acid sequences. In some embodiments, L1 and / or L2 are G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGAGGGGAGGGGS, GGGGSGGGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GSTSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12 , APELPGGP, SEPQPQPG, (G3S2)3, GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS)3, (GS4)3, G4A(G4S)2, G4SG4AG4S, G3AS(G4S)2, G4SG3ASG4S, G4SAG3SG4S, (G4S)2AG3S, G4SAG3SAG3S, G4D(G4S)2, G4SG4DG4S, (G4D)2G4S, G4E(G4S)2, G4SG4EG4S, (G4E)2G4S and G4SDA, e.g., G4A. In some embodiments, L1 and / or L2 are selected from the group consisting of G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4SDAA, (G4S)4, and G4AG3AG4S.

[0013] In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 2 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); or the amino acid sequence of SEQ ID NO: 3 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids). In some embodiments, the fusion protein has an amino acid sequence that has at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO: 2; or an amino acid sequence that has at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity to SEQ ID NO: 3.

[0014] In another aspect, the present disclosure provides fusion proteins comprising, from N-terminus to C-terminus, the structure D1-D2 or D2-D1, where D1 is a VHH, e.g., a single-domain antibody, and D2 is an FH fragment, e.g., FH1-5. In some embodiments, the fusion protein has a C-terminal His tag. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 1 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); or the amino acid sequence of SEQ ID NO: 7 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids). In some embodiments, the fusion protein has an amino acid sequence that has at least 85% (e.g., at least 90%, 95%, or 99%) sequence identity to SEQ ID NO: 1; or an amino acid sequence that has at least 85% (e.g., at least 90%, 95%, or 99%) sequence identity to SEQ ID NO: 7. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 11 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); or the amino acid sequence of SEQ ID NO: 12 or a variant having up to 10 amino acid substitutions, additions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids). In some embodiments, the fusion protein has an amino acid sequence that has at least 85% (e.g., at least 90%, 95%, or 99%) sequence identity to SEQ ID NO: 11; or an amino acid sequence that has at least 85% (e.g., at least 90%, 95%, or 99%) sequence identity to SEQ ID NO: 12.

[0015] In one embodiment, the fusion protein has an increased intrarenal residence time compared to a fusion protein lacking the VHH domain.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the fusion proteins described herein and a pharmaceutically acceptable carrier.

[0017] In another aspect, the disclosure provides a polynucleotide encoding any one of the fusion proteins described herein.

[0018] In another aspect, the present disclosure provides a host cell comprising a vector comprising a polynucleotide described herein.

[0019] In another aspect, the present disclosure provides a host cell comprising a polynucleotide described herein or a vector described herein.

[0020] In another aspect, the disclosure provides a method of producing any one of the fusion proteins described herein, the method comprising culturing one or more host cells comprising one or more nucleic acid molecules capable of expressing the fusion protein under conditions suitable for expression of the fusion protein. In some embodiments, the method further comprises obtaining the fusion protein from the cell culture or culture medium.

[0021] In another aspect, the disclosure provides a method of treating a disease mediated by alternative complement pathway activation or dysregulation, comprising administering to a subject in need thereof an effective amount of a composition comprising any one of the fusion proteins described herein, pharmaceutical compositions described herein, polynucleotides described herein, vectors described herein, or host cells described herein. In some embodiments, the fusion protein is formulated as a pharmaceutical composition with at least one (e.g., at least one, two, five, or ten) pharmaceutically acceptable carriers. In one embodiment, the composition is lyophilized. In another embodiment, the composition is rehydrated prior to administration. In another embodiment, the at least one (e.g., at least one, two, five, or ten) pharmaceutically acceptable carriers is saline. In some embodiments, the composition is formulated for daily, weekly, or monthly administration.

[0022] In some embodiments, the compositions are formulated for intravenous, subcutaneous, intramuscular, oral, nasal, sublingual, intrathecal, and intradermal administration. In some embodiments, the compositions are formulated for administration at a dosage of about 0.1 mg / kg to about 150 mg / kg (e.g., about 0.5-150 mg / kg, 1-150 mg / kg, 10-150 mg / kg, 25-150 mg / kg, 50-150 mg / kg, 100-150 mg / kg, 125-150 mg / kg, 0.1-125 mg / kg, 0.1-100 mg / kg, 0.1-50 mg / kg, 0.1-25 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, and 0.1-1 mg / kg). In some embodiments, the compositions are formulated for administration in combination with an additional therapeutic agent.

[0023] In some embodiments, the disease mediated by alternative complement pathway activation or dysregulation is nephropathy, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, minimal change disease (MCD), diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3 nephropathy (C3G), dense deposit disease, membranoproliferative glomerulonephritis, glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, atypical hemolytic uremic syndrome (aHUS), ischemia-reperfusion injury, or rejection of a transplanted organ such as a kidney. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0024] In another aspect, the present disclosure provides a kit comprising a composition selected from any one of a fusion protein described herein, a pharmaceutical composition described herein, a polynucleotide described herein, a vector described herein, or a host cell described herein. In some embodiments, the kit further comprises instructions for administering an effective amount of the composition to a subject in need thereof. [Brief explanation of the drawings]

[0025] [Figure 1]1A and 1B are schematic diagrams showing complement factor H (FH) fusion proteins of formula I and III containing an integrin recognition domain (FIG. 1A) and a factor H fusion protein of formula II containing a fragment of FHRP5 (FIG. 1B). [Figure 2A] 1 is a graph showing the results of an assay for the comparative inhibition of CAP-mediated hemolysis by Compound A and Factor H SCR1-5. [Figure 2B] 1 is a graph showing the results of an assay for the comparative inhibition of CAP-mediated hemolysis by compounds D, H, E, and I. [Figure 2C] 1 is a graph showing the results of an assay for the comparative inhibition of CAP-mediated hemolysis by compounds E, I and reference protein 6, an anti-HSA Factor H-VHH fusion protein used as a positive control. [Figure 2D] 1 is a graph showing the results of an assay for the comparative inhibition of CAP-mediated hemolysis by compounds E, M, N, and O. [Figure 3] 1 is a set of in vivo whole body and kidney images showing a wild-type mouse after treatment with Compound B. Images were generated using a LI-COR Odyssey microscope. [Figure 4] Graph showing serum levels in ng / mL for the indicated fusion proteins 1 and 24 hours after administration to wild-type mice. [Figure 5] Figure 1 shows graphs of proteinuria levels in wild-type Balb / c mice after intravenous administration of the fusion protein on day 0 and subcutaneous administration on days 7, 9, 11, and 13 in the adriamycin nephropathy model of FSGS. Data are presented as mean + or - standard error of the mean (n = 4-9). Statistically significant differences compared to vehicle are indicated by *p<0.05 and ***p<0.001, and statistically significant differences compared to vehicle and adriamycin are indicated by †p<0.5. [Figure 6]Figure 1 shows graphs depicting albuminuria levels in wild-type Balb / c mice in the adriamycin nephropathy model of FSGS after intravenous administration of the fusion protein on day 0 and subcutaneous administration on days 7, 9, 11, and 13. Data are presented as mean + or - standard error of the mean (n = 4-9). Statistically significant differences compared to vehicle are indicated by *p<0.05 and ***p<0.001, and statistically significant differences compared to vehicle and adriamycin are indicated by †p<0.5. [Figure 7] Graph showing blinded tubular protein scoring of Masson's trichrome stained adriamycin-nephrotic mouse kidneys, scored 1-5 according to accepted methods. N=8-10. Statistically significant differences compared to vehicle using multiple t-test are indicated by **p<0.01 and by ANOVA with multiple comparisons by #p<0.5. [Figure 8A] 1 is a set of images showing exemplary immunofluorescence evaluation of kidney sections for C3 deposition in an adriamycin nephropathy model in wild-type Balb / c mice 7 days after administration of Compound E. Other molecules gave similar results or were closer to the vehicle negative control. [Figure 8B] FIG. 10 is a graph showing the pixel mean intensity results from FIG. 9, where C3 pixel mean intensity values represent the mean signal intensity within the selected region of interest / renal medulla on day 14 (day 7 post-treatment). [Figure 9A] Figure 1 shows a graph showing urinary protein / creatinine ratios in male Balb / c mice with adriamycin-induced kidney disease after treatment with fusion protein administered intravenously on day 0 and subcutaneously on days 7, 9, 11, and 13. Data are shown as mean + or - standard error of the mean (n = 4-9). Statistically significant differences compared to vehicle are indicated by ***p<0.001. There were no statistically significant differences compared to vehicle and adriamycin. A positive trend was observed. [Figure 9B]Figure 1 shows a graph depicting urinary albumin / creatinine ratios after treatment with fusion protein, administered intravenously on day 0 and subcutaneously on days 7, 9, 11, and 13. Data are presented as mean + or - standard error of the mean (n = 4-9). Statistically significant differences compared to vehicle are indicated by ***p<0.001. There were no statistically significant differences compared to vehicle and adriamycin. [Figure 10A] 1 is a Western blot showing an SDS-PAGE gel of purified Compounds D and E. [Figure 10B] 1 is a graph showing a hydrophobic interaction chromatogram of Compound E. [Figure 11] 1 is a graph showing the mass spectrometry of Compound E, indicating that the molecular weight of Compound E is approximately 50 kDa. A slight peak at +162 Da is observed, which is likely due to glycation. [Figure 12] 1 is a graph showing the melting curve of Compound E using dynamic light scattering. [Figure 13A] 1 is a graph showing the retention time of Compound E at 37° C. at day 0 using size exclusion chromatography to measure the relative stability of the compounds. [Figure 13B] 1 is a graph showing the retention time of Compound E after 14 days at 37° C. using size exclusion chromatography to measure the relative stability of the compounds. [Figure 14A] 1 is a graph showing the retention time of Compound E after 0 days at 37° C. using hydrophobic interaction chromatography to measure the relative stability of the compound. [Figure 14B] 1 is a graph showing the retention time of Compound E after 14 days at 37° C. using hydrophobic interaction chromatography to measure the relative stability of the compound. [Figure 15A] 1 is a graph showing the alignment time of non-reduced Compound E after 0 and 14 days at 37° C. using capillary electrophoresis-SDS chromatography to measure the relative stability of the compounds. [Figure 15B]1 is a graph showing the alignment time of reduced Compound E after 0 and 14 days at 37° C. using capillary electrophoresis-SDS chromatography to measure the relative stability of the compounds. [Figure 16] 1 is a graph showing the chromatogram signature of Compound E obtained using isoelectric focusing capillary electrophoresis (iCE). [Figure 17] 1 is a graph showing the mass spectrum of Compound E measured after 0, 3, 7, and 14 days at 37° C. to characterize the stability of the compound at room temperature. [Figure 18A] 1 is a graph showing the binding curves of Compound E and Compound K to C3b compared to Factor H (fH) over the period 0-2500 seconds. [Figure 18B] 18B is a graph showing a 40 second zoom of the binding curve of FIG. 18A, where the t=0 second time point in FIG. 18B corresponds to the t=720 second time point in FIG. 18A. [Figure 19] 1 is a graph showing the assay results of comparative inhibition of fluid-phase CAP activation by Compound E in the Complement system Alternative Pathway WIESLAB® across two lots of normal human serum (NHS). [Figure 20] 1 is a graph showing combined single-dose serum pharmacokinetic (PK) data for Compound E following subcutaneous (SC) administration to wild-type male C57B1 / 6 mice across two separate studies. [Figure 21A] 1A-1C are graphs showing the serum pharmacokinetics of Compound E after intravenous (IV) or SC administration to female cynomolgus monkeys. Each graph includes comparative PK profiles across a range of dose levels after both the initial dose given on study day 0 and the fourth dose administered on study day 12. [Figure 21B] Includes data from Figure 21A replotted to compare equivalent dose levels given by IV or SC routes of administration. DETAILED DESCRIPTION OF THE INVENTION

[0026] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0027] As used herein, "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. The fusion protein can be administered by any method known to those skilled in the art. Suitable methods for administering the fusion protein can be, for example, orally, by injection (e.g., intravenous, intraperitoneal, intramuscular, intravitreal, and subcutaneous), infusion formulation, inhalation, intranasal, etc. In some embodiments, administration is via intravenous and / or subcutaneous injection. The fusion protein prepared as described herein can be administered in various forms depending on the disorder being treated and the age, condition, and weight of the subject, as is known in the art. The preparation can be administered prophylactically, i.e., to reduce the likelihood of developing a disease or condition.

[0028] As used herein, the terms "binding affinity," "specifically binds," and "affinity" refer to the strength of all noncovalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the specific interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain molecules that tend to dissociate easily from their binding partners, while high affinity complexes generally contain molecules that tend to remain bound to their binding partners for longer durations. "Specifically binds" refers to binding affinity of at least 1×10 -6 M or less (e.g., 1×10 -6 M~1×10 -12 M range, e.g., 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M and 1 x 10 -12M) refers to a molecule and binding partner pair that has a Kd of 0.05 M.

[0029] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically or substantially specifically binds to or is immunologically reactive with a particular antigen. Antibodies can be natural or artificial monovalent or multivalent antibodies, including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies. Antibodies can be genetically engineered or otherwise modified forms of antibodies, including, but not limited to, heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specific antibodies, diabodies, triabodies, and tetrabodies) and antigen-binding fragments of antibodies (e.g., single domain, VHH, Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments).

[0030] As used herein, the term "alternative complement pathway" refers to one of three pathways of complement activation (the others are the classical pathway and the lectin pathway).

[0031] As used herein, the term "alternative complement pathway activation or dysregulation" refers to any abnormality in the ability of the alternative complement pathway to provide host defense against pathogens and remove immune complexes and damaged cells for immunoregulation. Activation or dysregulation of the alternative complement pathway can occur in the fluid phase and on cell surfaces. Activation or dysregulation of the alternative complement pathway can result in excessive complement activation or insufficient regulation, both of which can cause tissue injury.

[0032] As used herein, the term "disease" refers to the interruption, cessation, or impairment of a bodily function, system, or organ. Diseases or disorders of interest include those that would benefit from treatment with a fusion protein or the methods described herein. Non-limiting examples of diseases or disorders treated herein are diseases or disorders mediated by alternative complement pathway activation or dysregulation, including, but not limited to, nephropathy, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, minimal change disease (MCD), diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3 nephropathy (C3G), dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, atypical hemolytic uremic syndrome (aHUS), ischemia-reperfusion injury, or rejection of a transplanted organ, such as a kidney. In some embodiments, the disease is FSGS.

[0033] As used herein, "Factor H" refers to a protein component of the alternative complement pathway encoded by the complement factor H gene ("FH;" NM000186; Gene ID: 3075; UniProt ID P08603; Ripoche, J. et al., Biochem. J., 249:593-602, 1988) (SEQ ID NO: 123). Factor H is translated as a 1,213 amino acid precursor polypeptide that is processed by removal of an 18 amino acid signal peptide to yield the mature factor H protein (amino acids 19-1231). Factor H consists of 20 short complement regulatory (SCR) domains. Amino acids 1-18 comprise the signal peptide, residues 21-80 comprise SCR1 (SEQ ID NO:24), residues 85-141 comprise SCR2 (SEQ ID NO:25), residues 146-205 comprise SCR3 (SEQ ID NO:26), residues 201-262 comprise SCR4 (SEQ ID NO:27), residues 267-320 comprise SCR5 (SEQ ID NO:28), and residues 326-384 comprise SCR6 (SEQ ID NO:29). Factor H regulates complement activation on autologous cells by possessing both cofactor activity for factor I-mediated C3b cleavage and decay-accelerating activity for the alternative pathway C3 convertase C3bBb.

[0034] Cleavage of C3 initially leads to the generation and deposition of C3b on the surface of activated cells. The C3b fragment is involved in the generation of an enzyme complex that amplifies the complement cascade. On the cell surface, C3b is rapidly converted to inactive iC3b when deposited, for example, on host surfaces containing regulators of complement activation (i.e., most host tissues). Even in the absence of membrane-bound complement regulators, significant levels of iC3b are formed due to the action of serum factor H and serum factor I. iC3b is subsequently digested into the membrane-bound fragment C3dg, which is then digested into C3d by factor I and other proteases and cofactors, but this process is relatively slow.

[0035] As used herein, the term "factor H-related protein 5" or "FHRP5" refers to a protein component of the alternative complement pathway encoded by the complement factor H-related protein 5 gene ("CFHR5"; NM_030787.3; Gene ID: 81494; UniProt ID: Q9BXR6) (SEQ ID NO: 124). FHRP5 has nine SCRs. The first two SCRs have heparin-binding properties, the region within SCRs 5-7 has heparin-binding and C-reactive protein-binding properties, and the two C-terminal SCRs resemble the complement component 3b (C3b)-binding domain. FHRP5 colocalizes with C3, binds C3b in a dose-dependent manner, and is recruited to tissues damaged by C-reactive protein.

[0036] As used herein, the term "fragment" refers to less than 100% of the amino acid sequence of a full-length reference protein (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc. of the full-length sequence), but including, for example, 5, 10, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, or more amino acids. A fragment may be of sufficient length such that a desired function of the full-length protein is maintained. For example, regulation by a fragment of the alternative complement pathway in fluid phase (e.g., a fragment of factor H) is maintained. Such a fragment is a "biologically active fragment."

[0037] As used herein, a "functional fragment" or a "biologically active fragment" refers to a fragment or portion of a protein that has some or all of the activity of the full-length protein. For example, a functional or biologically active fragment of Factor H refers to any fragment of Factor H protein that has some or all of the activity of Factor H, e.g., the alternative complement pathway regulatory activity of the full-length Factor H protein. Examples include, but are not limited to, N- to C-terminal Factor H fragments containing the following SCRs: [1-4], [1-5], [1-6], [1-7], [1-20], [19-20], [1-4 and 19-20], and [1-5] and [19-20]. A "functional fragment" or a "biologically active fragment" of the FHRP5 protein has some or all of the activity of FHRP5, e.g., the alternative complement pathway regulatory activity of the full-length FHRP5 protein. Examples include, but are not limited to, an N- to C-terminal FHRP5 fragment containing SCR: [7-8]. As used herein, the terms "fused" or "linked" refer to the combination or attachment of two or more elements, components, or protein domains, e.g., polypeptides, by chemical conjugation, recombinant means, and chemical bonds, including disulfide bonds and amide bonds. For example, two single polypeptides can be linked to form one continuous protein structure via recombinant expression, chemical conjugation, chemical bonds, peptide linkers, or any other covalent linking means.

[0038] As used herein, the term "fusion protein" refers to a composite polypeptide composed of two (or more) different heterologous polypeptides. The heterologous polypeptides can be either full-length proteins or fragments of full-length proteins. Fusion proteins herein can be prepared by either synthetic or recombinant techniques known in the art.

[0039] As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce the fusion proteins described herein. Non-limiting examples of host cells include Expi CHO-S, Expi 293 F, HEK, HEK 293, HT-1080, CHO, Pichia pastoris, Saccharomyces cerevisiae, and transformable insect cells such as High Five, Sf9, and Sf21 cells.

[0040] As used herein, the term "integrin recognition motif" refers to a polypeptide oligomer of repeating arginylglycylaspartic acid moieties, e.g., (RGD) 1-4 (RGD) such as (SEQ ID NO: 21) 1-8 In some embodiments, the arginylglycylaspartic acid moiety can be cyclized.

[0041] As used herein, the term "renal residence time" refers to the period during which a compound, such as Compounds A-O described herein, resides in the extravascular compartment, e.g., along the renal epithelium or within Bowman's dorsal pouch within the kidney. Renal residence time can be measured using longitudinal in vivo imaging. For example, in animal studies, an IVIS Spectrum Imaging System (PerkinElmer Inc., Waltham, MA) can be used for image acquisition. Fluorescence imaging analysis can be performed using Living Image 4.5.1 software (PerkinElmer Inc., Waltham, MA) with an automatic 2D epi-illumination exposure setting, field of view (FOV) C, F / Stop 2, medium binning, and an 800 nm emission / 750 nm excitation filter, and subjects receive, for example, 1 mg / kg of AlexaFluor 750-labeled test substance via intravenous injection. In clinical settings, longitudinal in vivo imaging can be achieved, for example, using a radiolabeled test substance and PET or SPECT imaging.

[0042] As used herein, the terms "linker," "L1," and "L2" refer to a bond between two elements, e.g., polypeptide or protein domains. A linker can be a covalent bond. A linker can also be a molecule of any length that can be used to connect, for example, factor H fragments and / or VHHs and / or integrin recognition motifs. A linker also refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids, 1 to 150 amino acids, 1 to 100 amino acids, 5 to 50 amino acids, or 1 to 10 amino acids, e.g., amino acids with smaller side chains and / or a flexible amino acid sequence) that is present between two polypeptides or polypeptide domains to provide space and / or flexibility between the two polypeptides or polypeptide domains. An amino acid linker can be part of the primary sequence of a polypeptide (e.g., attached to the connected polypeptides or polypeptide domains via the polypeptide backbone). Non-limiting examples include (G4A)2G4S, G4A, (G4A)3 and (G4A)2G3AG4S (SEQ ID NOs: 32, 80, 81 and 30).

[0043] As used herein, the term "patient in need thereof" or "subject in need thereof" refers to a subject in need of treatment, for example, based on the presence of a disease or disorder (e.g., one or more symptoms of a disease or disorder). The subject may be identified as having a need for treatment of a disease or disorder (e.g., kidney damage, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of a transplanted organ such as a kidney) prior to administration of treatment. In some embodiments, the disease is FSGS, and the need for treatment is based on early diagnosis by a skilled artisan (e.g., a physician). For example, the patient is a mammal, such as a human.

[0044] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. This term also encompasses amino acid polymers that have been modified by, among other things, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.

[0045] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST. As an example, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to or relative to a given nucleic acid or amino acid sequence, B (which can alternatively be expressed as a given nucleic acid or amino acid sequence, A, having a particular percent sequence identity to or relative to a given nucleic acid or amino acid sequence, B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids in A and B scored by a sequence alignment program (e.g., BLAST) as identical matches in the program's alignment, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0046] By "pharmaceutical composition" is meant any composition containing a therapeutically or biologically active agent (e.g., a fusion protein) suitable for administration to a subject. Any of these formulations can be prepared by methods well known and accepted in the art. See, e.g., Remington: The Science and Practice of See Pharmacy (21st ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2005 and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is incorporated herein by reference.

[0047] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit / risk ratio.

[0048] The terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides, or their analogs. A polynucleotide may contain modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure described herein that is a double-stranded polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0049] As used herein, the term "short complement regulator" or "SCR," also known as "short consensus repeat," "sushi domain," or "complement control protein" or "CCP," describes a domain found in all regulators of complement activation gene clusters (RCA) that specifically bind to and contribute to their ability to regulate complement activation in the blood or on cell surfaces. SCRs are typically composed of approximately 60 amino acids, with four cysteine disulfide residues linked in a 1-3, 2-4 sequence, and a hydrophobic core built around a nearly invariant tryptophan residue. SCRs are found in proteins including, but not limited to, Factor H and FHRP5.

[0050] As used herein, the terms "single domain antibody" and "VHH" define a molecule formed by a single immunoglobulin domain. Single domain antibodies include antibodies whose complementarity determining regions ("CDRs") are part of a single domain polypeptide. Single domain antibodies often include antibodies or antigen-binding fragments thereof that specifically bind to a single antigen (e.g., VHH antibodies can be up to 1x10 -6 K below M D , e.g., 1 x 10 -6 M~1×10 -12 K in the M range D , e.g., 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M and 1 x 10 -12 K of M D(which binds to antigens via the CDRs). Generally, the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. A single variable domain may, for example, comprise a light chain variable domain sequence (VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof. Such antibodies may be derived from antibodies produced in, for example, Camelidae species, e.g., in camels, dromedaries, llamas, alpacas, or guanacos. Further antibodies include, for example, the immunoglobulin neoantigen receptors (IgNARs) of cartilaginous fish (e.g., sharks, e.g., nurse sharks). Other species besides Camelidae and cartilaginous fish may produce antibodies whose CDRs are part of a single polypeptide. Antibodies may be prepared by either synthetic or recombinant techniques known in the art.

[0051] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, etc., who will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0052] A "therapeutically effective amount" refers to the amount of a composition administered to improve, inhibit, or ameliorate the condition of a subject or the symptoms of a disorder or disease in a clinically relevant manner. Any improvement in a subject is considered sufficient to achieve treatment. In some embodiments, an amount sufficient to treat is an amount that reduces, inhibits, or prevents the occurrence of a disease or disorder (e.g., a disease or disorder mediated by alternative complement pathway activation or dysregulation) or one or more symptoms thereof, or reduces the severity of a disease or disorder, e.g., any disease or disorder mediated by CAP activation or dysregulation, or the length of time a subject suffers from one or more symptoms of the disease or disorder (e.g., by at least about 10%, about 20%, or about 30%, e.g., by at least about 50%, about 60%, or about 70%, and e.g., by at least about 80%, about 90%, about 95%, about 99%, or more, compared to a control subject not treated with a composition described herein). Effective amounts of pharmaceutical compositions used to practice the methods described herein (e.g., treating renal disease) may vary depending on the mode of administration and the age, weight, and general health of the subject being treated. A physician or researcher can determine the appropriate amount and dosing regimen. Dosages can vary and can be administered daily, weekly, monthly, or yearly, or over several days in one or more dose administrations.

[0053] As used herein, the terms "treatment," "treating," or "treat" refer to therapeutic treatment, the purpose of which is to inhibit or reduce an undesirable physiological change or disorder or to promote a beneficial phenotype in a patient. For example, "treatment," "treating," or "treat" refers to clinical intervention in an attempt to alter the natural course of an individual's affliction, disease, or disorder. These terms include, for example, prevention before or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequence of disease, slowing the rate of disease progression, remission or alleviation of the disease state, and improved prognosis. In some embodiments, the fusion proteins are used to control the cellular and clinical symptoms of kidney damage, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as kidneys. In some embodiments, the disease is FSGS.

[0054] "Variant" refers to a polynucleotide or polypeptide that is substantially homologous to a native or reference polynucleotide or polypeptide. For example, a variant polynucleotide has a polynucleotide sequence that is substantially homologous to a native or reference polynucleotide but differs from the native or reference polynucleotide due to one or more deletions, insertions, and / or substitutions. In another example, a variant polypeptide has an amino acid sequence that is substantially homologous to a native or reference polypeptide but differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, and / or substitutions. A variant polypeptide sequence encoded by a polynucleotide sequence encompasses sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to a native or reference polynucleotide sequence that encode a variant protein or fragment thereof that retains activity. A wide variety of mutagenesis approaches are known in the art and can be applied by those skilled in the art. A variant polynucleotide or polypeptide sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0055] As used herein, a "vector" refers to a macromolecule or an association of macromolecules that contains or associates with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

[0056] Described herein are alternative pathway-specific C3 and C5 convertase inhibitors that modulate alternative pathway activity. Described herein are a series of low molecular weight alternative complement pathway (CAP) activation and amplification loop inhibitor molecules designed to possess mechanisms for binding to kidney epithelial cells. The compositions and methods described herein feature fusion proteins containing a fragment of complement factor H (FH), a fragment of factor H-related protein 5 (FHRP5), and / or one or more kidney-targeting motifs (e.g., one or more cyclic arginylglycylaspartate (RGD) motifs) that can be fused to a VHH domain.

[0057] These fusion molecules contain complement factor H (FH) catalytic domain short consensus repeats (SCRs) 1-4 to provide factor I-mediated cofactor activity and decay-accelerating function via C3b binding. Additional factor H SCRs (e.g., SCR5 and SCR6) can be included to increase activity, stability, or structural flexibility. According to the present disclosure, fusion proteins containing FH catalytic domain SCRs can also contain short amino acid sequence motifs or complement molecule domains that recognize integrins or injury markers present on the surface of damaged renal epithelial and tubulointerstitial cells. Described herein are fusion proteins that can include single-domain variable heavy chain-only (VHH) camelid antibodies to enable renal epithelial cell deposition, improve expression, facilitate purification, and provide exogenous probes for detection. Collectively, the use of these targeting residues, coupled with the inherent clearance kinetics of low molecular weight proteins, provides selective localization of CAP inhibitor fusion proteins to renal epithelial cells.

[0058] Diseases mediated by complement dysregulation are often the result of complement hyperactivity in both the fluid phase and on the cell surface. Compositions and methods for treating diseases mediated by complement dysregulation are described herein. Examples of disorders mediated by alternative complement pathway activation or dysregulation include, for example, nephropathy, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as kidneys. In some embodiments, the disease is FSGS.

[0059] Fusion proteins according to the present disclosure modulate alternative complement pathway activity, including by irreversibly inactivating C3b and attenuating C3 and C5 convertase activity. The constructs target the alternative complement pathway, leaving activation (protection) via the classical and lectin pathways intact.

[0060] fusion proteins As described herein, the fusion proteins of the present disclosure comprise a fragment of Factor H and may include an integrin recognition motif or a fragment of FHRP5. The constructs can be used as therapeutic agents to treat diseases mediated by alternative complement pathway activation or dysregulation (e.g., FSGS).

[0061] In humans, several regulatory proteins are encoded by a cluster of genes located on the long arm of chromosome 1. This region is called the regulator of complement activation (RCA) gene cluster. Although proteins within the RCA family vary in size, they share similarities in primary amino acid structure. The best-studied members of the RCA family are factor H, FHL-1, CR1, DAF, MCP, and C4b-binding protein (C4BP). Members of this family are organized into tandem structural units called short consensus repeats (SCRs), which are present in multiple copies in the protein. Each SCR consists of approximately 60-70 highly conserved amino acids, including four cysteine residues.

[0062] In some embodiments, the portion of the fusion protein suitable for inhibiting the activity of the alternative complement pathway is fused to a VHH to increase the duration of effect.

[0063] In certain embodiments, the portion of the fusion protein suitable for inhibiting the activity of the alternative complement pathway comprises a fragment of Factor H. The fragment of Factor H may comprise at least the first four N-terminal SCR domains of Factor H (e.g., SCR1, 2, 3, and 4). In certain embodiments, the fragment of Factor H comprises at least the first five N-terminal SCR domains of Factor H (e.g., SCR1, 2, 3, 4, and 5), also known as the cofactor and decay-accelerating domains. In certain embodiments, the fragment of Factor H comprises at least the first six N-terminal SCR domains of Factor H (e.g., SCR1, 2, 3, 4, 5, and 6).

[0064] In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:24. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:25. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:26. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:27. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:28. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:29. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 16. In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 17.In some embodiments, a fragment of Factor H may comprise a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99%) identical to SEQ ID NO:18.

[0065] The fusion protein may contain an integrin-binding domain in addition to a fragment of factor H. The fragment of factor H in the fusion protein may include at least the first four, five, or six N-terminal SCR domains of factor H, and the integrin-binding domain may include an arginylglycylaspartate (RGD) peptide motif. The arginylglycylaspartate peptide motif may include a cyclo(RGD)4 peptide (SEQ ID NO: 21). In some embodiments, the fusion protein may include an integrin-binding domain comprising a polypeptide sequence at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 21.

[0066] In certain embodiments, the fragment of factor H comprises at least the first five N-terminal SCR domains of factor H (e.g., SCR1, 2, 3, 4, and 5), and the integrin-binding domain comprises a cyclo(RGD)4 peptide. In certain embodiments, the fragment of factor H comprises at least six, five N-terminal SCR domains of factor H (e.g., SCR1, 2, 3, 4, 5, and 6), and the integrin-binding domain comprises a cyclo(RGD)4 peptide.

[0067] The fusion protein may comprise a fragment of factor H-related protein 5 (FHRP5) in addition to a fragment of factor H. In some embodiments, the fusion protein may comprise a fragment of an FHRP5 domain comprising a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 22. In some embodiments, the fusion protein may comprise a fragment of an FHRP5 domain comprising the polypeptide sequence of SEQ ID NO: 22. The fragment of factor H in the fusion protein may comprise at least the first four, five, or six N-terminal SCR domains of factor H, and the fragment of FHRP5 in the fusion protein may comprise at least the seventh and / or eighth N-terminal SCR domain of FHRP5.

[0068] In certain embodiments, the fragment of Factor H comprises at least the first five N-terminal SCR domains of Factor H (eg, SCR1, 2, 3, 4 and 5), and the fragment FHRP5 comprises at least the seventh and eighth N-terminal SCR domains of FHRP5.

[0069] In some embodiments, the fragment of the Factor H portion of the fusion protein is a functional fragment of wild-type Factor H. In some embodiments, the Factor H or fragment thereof portion of the fusion protein is derived from a substituted (e.g., conservatively substituted) or engineered Factor H (e.g., a Factor H engineered to increase protein stability, activity, and / or other desirable properties as determined by predictive models or assays known to those of skill in the art, such as those described herein).

[0070] In some embodiments, the fragment of the FHRP5 portion of the fusion protein is a functional fragment of wild-type FHRP5. In some embodiments, the FHRP5 or fragment thereof portion of the fusion protein composition is derived from a substituted (e.g., conservatively substituted) FHRP5 or an engineered FHRP5 (e.g., an FHRP5 engineered to increase protein stability, activity, and / or other desirable properties, as determined by predictive models or assays known to those of skill in the art, such as the assays described herein).

[0071] Amino acid substitutions can be introduced into the fusion proteins described herein to improve functionality. For example, amino acid substitutions can be introduced into a fragment of factor H, an integrin-binding domain, or a fragment of FHRP5, where the amino acid substitutions increase the binding affinity of the fragment of factor H, the integrin-binding domain, or the fragment of FHRP5 for its ligand. Similarly, amino acid substitutions can be introduced into factor H or a fragment thereof to increase functionality and / or improve the pharmacokinetics of the fusion protein.

[0072] In certain embodiments, the fusion proteins described herein can be fused to another compound, such as a compound that increases the half-life of the polypeptide and / or a compound that reduces the potential immunogenicity of the fusion protein (e.g., polyethylene glycol (PEG)). PEG can be used to improve water solubility, decrease the renal clearance rate, and reduce the immunogenicity of the fusion protein (see, e.g., U.S. Pat. No. 6,214,966, the disclosure of which is incorporated herein by reference). The fusion proteins described herein can be PEGylated by any means known to those of skill in the art.

[0073] Fragments of factor H can be prepared by a number of synthetic methods for peptide synthesis by fragment condensation of one or more amino acid residues according to conventional peptide synthesis methods known in the art (Amblard, M. et al., Mol. Biotechnol., 33:239-54, 2006).

[0074] Alternatively, fragments of factor H, integrin-binding domains, and / or fragments of FHRP5 can be produced by expression in suitable prokaryotic or eukaryotic systems. In some embodiments, the DNA construct can be inserted into a plasmid vector adapted for expression in a suitable host cell (e.g., E. coli) or yeast cell (e.g., S. cerevisiae or P. pastoris), or into a baculovirus vector for expression in insect cells, or into a viral vector for expression in mammalian cells. Examples of mammalian cells suitable for recombinant expression include, for example, human embryonic kidney (HEK) cells (e.g., HEK 293), Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, or COS-7 cells. Suitable expression vectors contain the necessary and sufficient regulatory elements for expression of DNA in the host cell. In some embodiments, a leader or secretory sequence or a sequence used for purification of the fusion protein (e.g., a histidine tag) can be included in the fusion protein. Fragments of factor H, integrin-binding domains, and / or FHRP5 produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified according to methods known in the art (e.g., Structural (See Genomics Consortium, Nat. Methods, 5:135-46, 2008).

[0075] In certain embodiments, the cyclized integrin binding domain and fragments of FHRP5 are also produced by the same methods described for expression and purification of fragments of Factor H.

[0076] In some embodiments, the fusion protein has the structure of Formula I, from N-terminus to C-terminus: D1-L1-D2-L2-D3 Formula I During the ceremony, D1 is a fragment of FH (e.g., a fragment of FH having the amino acid sequence of any one of SEQ ID NOs: 16 to 18, or a variant thereof having 85% or more sequence identity thereto); L1 is absent (e.g., L1 is a covalent bond between D1 and D2 or between D1 and D3) or is a linker having an amino acid sequence of at least one amino acid between D1 and D2 or between D1 and D3 (e.g., the linker may have the amino acid sequence of any one of SEQ ID NOs: 30-122 or a variant thereof having 85% or more sequence identity thereto); D2 is absent (e.g., D2 is a covalent bond between L1 and D3, between D1 and L2, or between L1 and L2) or is a VHH domain, e.g., a single-domain antibody (e.g., a camelid single-domain antibody VHH having the amino acid sequence of any one of SEQ ID NOs: 19 to 20 and 23, or a variant thereof having 85% or more sequence identity thereto); L2 is absent (e.g., L2 is a covalent bond between D2 and D3) or is a linker having an amino acid sequence of at least one amino acid between D2 and D3 (e.g., the linker can have the amino acid sequence of any one of SEQ ID NOs: 30-122 or a variant thereof having 85% or more sequence identity thereto); D3 is an integrin recognition domain (eg, an arginylglycylaspartic acid (RGD) peptide motif, such as cyclo(RGD)4 having the amino acid sequence of SEQ ID NO: 21, or a variant thereof having 85% or more sequence identity thereto).

[0077] In some embodiments, the fragment of FH of D1 comprises one or more FH SCR domains, and optionally the one or more SCR domains are selected from the group consisting of SCR 1, 2, 3, 4, 5, or 6, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any one of SEQ ID NOs: 24-29. In some embodiments, the FH SCR domain is selected from the group consisting of a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any of SCR[1-5] or SEQ ID NO: 16 or 17, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SCR[1-6] or SEQ ID NO: 18.

[0078] L1 and L2 can be linkers of the same type and / or sequence or different types and / or sequences.

[0079] In some embodiments, the composition of Formula I comprises the amino acid sequence of any one of SEQ ID NOs: 4, 5, 8, 9, and 13-15, and variants thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto. In some embodiments, the composition of Formula I is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 128, 129, 132, 133, and 137-139, and variants thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto.

[0080] In some embodiments, the fusion protein has the structure, from N-terminus to C-terminus, of Formula II: D1-L1-D2 Formula II During the ceremony, D1 is a fragment of FH (for example, a fragment of FH having any one of the amino acid sequences of SEQ ID NOs: 16 to 18, or a variant thereof having 85% or more sequence identity thereto). L1 is absent (e.g., L1 is a covalent bond between D1 and D2) or is a linker having an amino acid sequence of at least one amino acid between D1 and D2 (e.g., the linker can have the amino acid sequence of any one of SEQ ID NOs: 30-122 or a variant thereof having 85% or more sequence identity thereto); D2 is a fragment of factor H-related protein 5 (FHRP5) (eg, a fragment of FHRP5 having the amino acid sequence of SEQ ID NO: 22 or a variant thereof having 85% or more sequence identity thereto).

[0081] In some embodiments, the fragment of FH of D1 comprises one or more FH SCR domains, and optionally the one or more SCR domains are selected from the group consisting of SCR 1, 2, 3, 4, 5, or 6, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any one of SEQ ID NOs: 24-29. In some embodiments, the FH SCR domain is selected from the group consisting of a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any of SCR[1-5] or SEQ ID NO: 16 or 17, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SCR[1-6] or SEQ ID NO: 18. In some embodiments, the fragment of FHRP5 comprises one or more FHRP5 domains, optionally the domains are selected from domains 7 and 8 (e.g., the amino acid sequence of SEQ ID NO: 22). In some embodiments, the fragment of FHRP5 comprises domains 7-8 or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SEQ ID NO: 22.

[0082] In some embodiments, the composition of Formula II comprises either SEQ ID NO: 6 or 10, or a variant thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto. In some embodiments, the composition of Formula II is encoded by the nucleic acid sequence of SEQ ID NO: 130 or 134, or a variant thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto.

[0083] In some embodiments, the fusion protein has the structure, from N-terminus to C-terminus, of Formula III: D1-L1-D2-L2-D3 Formula III During the ceremony, D1 is an integrin recognition domain (e.g., an arginylglycylaspartic acid (RGD) peptide motif, e.g., cyclo(RGD)4 of SEQ ID NO: 21, or a variant thereof having 85% or greater sequence identity thereto); L1 is absent (e.g., L1 is a covalent bond between D1 and D2 or between D1 and D3) or is a linker having an amino acid sequence of at least one amino acid between D1 and D2 or between D1 and D3 (e.g., the linker may have the amino acid sequence of any one of SEQ ID NOs: 30-122 or a variant thereof having 85% or more sequence identity thereto); D2 is absent (e.g., D2 is a covalent bond between L1 and D3, between D1 and L2, or between L1 and L2) or is a VHH domain, e.g., a single-domain antibody (e.g., a camelid single-domain antibody VHH having the amino acid sequence of any one of SEQ ID NOs: 19 to 20 and 23, or a variant thereof having 85% or more sequence identity thereto); L2 is not present between D2 and D3 (e.g., L2 is a covalent bond between D2 and D3) or is a linker having an amino acid sequence of at least one amino acid (e.g., the linker can have the amino acid sequence of any one of SEQ ID NOs: 30-122 or a variant thereof having 85% or more sequence identity thereto); and D3 is a fragment of FH (for example, a fragment of FH having any one of the amino acid sequences of SEQ ID NOs: 16 to 18, or a variant thereof having 85% or more sequence identity thereto).

[0084] In some embodiments, the fragment of FH of D3 comprises one or more FH SCR domains, and optionally the one or more SCR domains are selected from the group consisting of SCR 1, 2, 3, 4, 5, or 6, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any one of SEQ ID NOs: 24-29. In some embodiments, the FH SCR domain is selected from the group consisting of a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any of SCR[1-5] or SEQ ID NO: 16 or 17, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SCR[1-6] or SEQ ID NO: 18.

[0085] L1 and L2 can be linkers of the same type and / or sequence or different types and / or sequences.

[0086] In some embodiments, the composition of Formula III comprises either SEQ ID NO: 2 or 3, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity thereto. In some embodiments, the composition of Formula III is encoded by the nucleic acid sequence of either SEQ ID NO: 126 or 127, or a variant thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto.

[0087] In some embodiments, the fusion protein has the structure, from N-terminus to C-terminus, of Formula IV: D1-D2 or D2-D1 Formula IV During the ceremony, D1 is a VHH domain, e.g., a single-domain antibody (e.g., a camelid single-domain antibody VHH having the amino acid sequence of any one of SEQ ID NOs: 19 to 20 and 23, or a variant thereof having 85% or more sequence identity thereto); D2 is a fragment of FH (eg, a fragment of FH of either SEQ ID NO: 16 or 17 or a variant thereof having 85% or more sequence identity thereto).

[0088] In some embodiments, the fragment of FH in D3 comprises one or more FH SCR domains, and optionally the one or more SCR domains are selected from the group consisting of SCR 1, 2, 3, 4, 5, or 6, or a variant thereof having 85% or more sequence identity thereto. In some embodiments, the FH SCR domain is selected from the group consisting of SCR[1-5] or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SEQ ID NO: 16 or 17, or SCR[1-6] or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to SEQ ID NO: 18.

[0089] In some embodiments, the composition of Formula IV comprises any one of SEQ ID NOs: 1, 7, 11, and 12, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity thereto. In some embodiments, the composition of Formula IV is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 125, 131, 135, and 136, or a variant thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto.

[0090] Immunoglobulin Proteins and Domains The fusion proteins described herein may comprise a single VHH domain. Such antibodies naturally occur in camelids and sharks (Saerens et al., Curr. Opin. Pharmacol., 8:600-608, 2008). Camelid antibodies are described, for example, in U.S. Patent Nos. 5,759,808, 5,800,988, 5,840,526, 5,874,541, 6,005,079, and 6,015,695, the entire contents of each of which are incorporated herein by reference.

[0091] Exemplary VHH domains include: QVQLVESGGGLVKPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAADLGDGSWVDYVNAEPYEYDYWGQGTLVTVSS (SEQ ID NO: 19), EVQLVESGGGLVKPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAADLGDGSWVDYVNAEPYEYDYWGQGTLVTVSS (SEQ ID NO: 20), or EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAADLGDGSWVDYVNMEPYEYDYWGQGTQVTVSS (SEQ ID NO: 23) This includes those having the sequence:

[0092] In some embodiments, a fusion protein may comprise a VHH domain comprising a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 19. In some embodiments, a fusion protein may comprise a VHH domain comprising a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:20. In some embodiments, the fusion protein may comprise a VHH domain comprising a polypeptide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 23.

[0093] The fusion protein can comprise, from N-terminus to C-terminus, D1-L1-D2-L2-D3, where D1 is a fragment of an FH protein, such as FH SCR 1-5 or FH SCR 1 to 6, wherein L1 is absent or comprises a linker, D2 comprises a VHH domain, L2 is absent or comprises a linker, and D3 comprises an integrin recognition domain, for example, cyclo(RGD)4.

[0094] In another example, the fusion protein can comprise, from N-terminus to C-terminus, D1-L1-D2-L2-D3, where D1 comprises an integrin recognition domain such as cyclo(RGD)4, L1 is absent or comprises a linker, D2 comprises a VHH domain, L2 comprises a linker or is absent, and D3 comprises a fragment of an FH protein such as FH SCR 1-5.

[0095] In another example, the fusion protein can comprise, from N- to C-terminus, D1-D2 or D2-D1, where D1 comprises a VHH domain and D2 comprises a fragment of a FH protein. The fusion protein can have the amino acid sequence of any one of SEQ ID NOs: 1, 7, 11, and 12, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any one of SEQ ID NOs: 1, 7, 11, and 12.

[0096] In some embodiments, Factor H fusion proteins comprising a VHH domain have increased intrarenal residence time along the renal epithelial surface compared to fusion proteins lacking the VHH domain. Without being bound by theory, it is believed that the size of the fusion proteins described herein (e.g., about ≦60 kDa (e.g., less than 60 kDa)) enables the fusion proteins to gain entry into extravascular compartments within the kidney that are inaccessible by monoclonal antibodies and albumin-binding bispecifics, and the use of a VHH domain in the fusion proteins described herein enables the fusion proteins to deposit on the apical membrane of proximal tubules and parietal epithelial cells, where naturally low levels of membrane-associated surface regulators confer sensitivity to CAP products, resulting in extended residence along the renal epithelium. In some embodiments, intrarenal residence time is increased by at least 1-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold) compared to fusion proteins lacking the VHH domain. In some embodiments, the intrarenal residence time is between 24 hours and 96 hours (eg, between 36 hours and 96 hours, between 48 hours and 96 hours, between 60 hours and 96 hours, between 72 hours and 96 hours, and between 60 hours and 84 hours).

[0097] Integrin-binding domain The fusion protein may also have an integrin-binding domain, which may improve the pharmacokinetics of the fusion protein and act as a targeting motif to mediate kidney cell-specific targeting at sites of injury or remodeling. The integrin-binding domain may be added as an additional domain to any one of the fusion proteins described herein.

[0098] An exemplary integrin-binding domain comprises one or more cyclic arginylglycylaspartate (RGD) peptide motifs fused to either the N- or C-terminus of the fusion protein. The RGD motif binds to the extracellular domains of integrin α- and β-subunits on the cell surface, which can be upregulated in response to injury (e.g., renal fibrosis mediated by TGF-β signaling). Without being bound by theory, it is believed that the inclusion of a cyclic RGD motif can limit the binding of pro-TGF-β ligands and prevent profibrotic signaling. Different variants of the integrin-binding motif can be constructed and attached to the fusion protein. In some embodiments, the fusion protein can comprise an integrin-binding domain comprising an amino acid sequence at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 21.

[0099] The fusion protein can comprise, from N-terminus to C-terminus, D1-L1-D2-L2-D3, where D1 comprises a fragment of an FH protein, such as an FH having SCRs [1-5] or an FH having SCRs [1-6]. The fusion protein can have the amino acid sequence of any one of SEQ ID NOS: 4, 5, 8, 9, and 13-15, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any one of SEQ ID NOS: 4, 5, 8, 9, and 13-15. The fusion protein can also comprise a fragment of an FH protein, such as an FH having SCRs [1-5] or an FH having SCRs [1-6]. The fusion protein can have the amino acid sequence of any one of SEQ ID NOS: 2 or 3, or a variant thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity to any one of SEQ ID NOS: 2 or 3.

[0100] Fusion protein linker The L1 and L2 domains of the fusion proteins described herein are linkers. Linkers are used, for example, to create bonds or connections between polypeptide or protein domains. For example, a fragment of factor H can be directly linked to a VHH domain (e.g., a single-domain camelid VHH domain) by one or more appropriate linkers. The linker can be a simple covalent bond, such as a peptide bond, a synthetic polymer, such as a PEG polymer, or any type of bond created by a chemical reaction, such as chemical conjugation. The peptide linker can be, for example, a linker of one or more amino acid residues inserted or included at the transition between two domains (e.g., a fragment of an FH protein and a VHH domain). The identity and sequence of the amino acid residues in the linker can vary depending on the desired secondary structure. For example, glycine, serine, and alanine are useful linkers given their flexibility. Depending on the desired length and / or properties, any amino acid residue can be considered a linker in combination with one or more other amino acid residues, which can be the same as or different from the first amino acid residue, to construct larger peptide linkers as needed.

[0101] A variety of linkers can be used to fuse two or more protein domains (e.g., a fragment of factor H and a VHH domain) together. Linkers can be flexible, rigid, or cleavable. Linkers can be structured or unstructured. Residues of the linker can be selected from naturally occurring amino acids, non-naturally occurring amino acids, and modified amino acids. Linkers can contain at least 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more amino acid residues. Peptide linkers include, but are not limited to, glycine linkers, glycine-rich linkers, serine-glycine linkers, etc. A glycine-rich linker contains at least about 50% glycines.

[0102] In some embodiments, the linker used confers one or more other desirable properties or functionalities to the polypeptides described herein and / or provides one or more sites for derivatization and / or attachment of functional groups. For example, a linker containing one or more charged amino acid residues can provide improved hydrophilic properties, while a linker that forms or contains a small epitope or tag can be used for detection, identification, and / or purification purposes. One of skill in the art will be able to determine the optimal linker for use in a particular polypeptide.

[0103] If more than one linker is used in a polypeptide, the linkers may be the same or different.

[0104] The linker can include a motif, for example, multiple motifs or repeated motifs. In one embodiment, the linker has the amino acid sequence GS or repeats thereof (Huston, J. et al., Methods Enzymol., 203:46-88, 1991). In another embodiment, the linker includes the amino acid sequence EK or repeats thereof (Whitlow, M. et al., Protein Eng., 6:989-95, 1993). In another embodiment, the linker includes the amino acid sequence GGS or repeats thereof.

[0105] In another embodiment, the linker comprises the amino acid sequence GGGGA (SEQ ID NO: 80) or repeats thereof. In certain embodiments, the linker comprises two or more repeats of GGS or GGGGS (U.S. Patent No. 6,541,219, the entire contents of which are incorporated herein by reference). In one embodiment, the peptide linker may be rich in small or polar amino acids such as G and S, but may contain additional amino acids such as T and A to maintain flexibility and polar amino acids such as K and E to improve solubility.

[0106] Exemplary linkers include G4S (SEQ ID NO: 36), (G4A)2G4S (SEQ ID NO: 34), (G4A)2G3AG4S (SEQ ID NO: 30), G4AG3AG4S (SEQ ID NO: 33), G4SDA (SEQ ID NO: 79), G4SDAA (SEQ ID NO: 31), G4S (SEQ ID NO: 36), (G4S)2 (SEQ ID NO: 37), (G4S)3 (SEQ ID NO: 35), (G4S)4 (SEQ ID NO: 39), (G4S)5 (SEQ ID NO: 40), (G4S)6 (SEQ ID NO: 41), EAAAK (SEQ ID NO: 95), (EAAAK)3 (SEQ ID NO: 42), PAPAP (SEQ ID NO: 4 3), G4SPAPAP (SEQ ID NO: 44), PAPAPG4S (SEQ ID NO: 45), GSTSGKSSEGKG (SEQ ID NO: 46), (GGGDS)2 (SEQ ID NO: 47), (GGGES)2 (SEQ ID NO: 48), GGGDSGGGGS (SEQ ID NO: 49), GGGASGGGGS (SEQ ID NO: 50), GGGESGGGGS (SEQ ID NO: 51), ASTKGP (SEQ ID NO: 52), ASTKGPSVFPLAP (SEQ ID NO: 53), G3P (SEQ ID NO: 54), G7P (SEQ ID NO: 55), PAPNLLGGP (SEQ ID NO: 56), G6 (SEQ ID NO: 57), G 12 (SEQ ID NO: 58), APELPGGP (SEQ ID NO: 59), SEPQPQPG (SEQ ID NO: 60), (G3S2)3 (SEQ ID NO: 61), GGGGGGGGGSGGGS (SEQ ID NO: 62), GGGGSGGGGGGGGGS (SEQ ID NO: 63), (GGSSS)3 (SEQ ID NO: 64), (GS4)3 (SEQ ID NO: 65), G4A(G4S)2 (SEQ ID NO: 66), G4SG4AG4S (SEQ ID NO: 67), G3AS(G4S)2 (SEQ ID NO: 68), G4SG3ASG4S (SEQ ID NO: 69), G4SAG3SG4S (SEQ ID NO: 70), (G4S)2AG3S (SEQ ID NO: 71), G4SAG3SAG3S (SEQ ID NO: 72), G4D(G4S)2 (SEQ ID NO: 73), G4SG4DG4S (SEQ ID NO: 74), (G4D)2G4S (SEQ ID NO: 75), G4E(G4S)2 (SEQ ID NO: 76), G4SG4EG4S (SEQ ID NO: 77) and (G4E)2G4S (SEQ ID NO: 78), (GGGGS) n(wherein n can be any number), KESGSVSSEQLAQFRSLD (SEQ ID NO: 82), and EGKSSGSGSESKST (SEQ ID NO: 83), (Gly) (SEQ ID NO: 84), GSAGSAAGSGEF (SEQ ID NO: 87), and (Gly) (SEQ ID NO: 57). Exemplary rigid linkers include, but are not limited to, A(EAAAK)A (SEQ ID NO: 86), A(EAAAK) n A (wherein n can be any number) or (XP) n(wherein n can be any number and X represents any amino acid). Exemplary in vivo cleavable linkers include, but are not limited to, LEAGCKNFFPRSFTSCGSLE (SEQ ID NO: 87), GSST (SEQ ID NO: 88), and CRRRRRREAEAC (SEQ ID NO: 89). In some embodiments, the linker can comprise 2 to 12 amino acids containing a GS motif, e.g., GS, GSGS (SEQ ID NO: 90), GSGSGS (SEQ ID NO: 91), GSGSGSGS (SEQ ID NO: 92), GSGSGSGSGS (SEQ ID NO: 93), or GSGSGSGSGSGS (SEQ ID NO: 95). In certain other embodiments, the linker can comprise 3 to 12 amino acids containing a GGS motif, e.g., GGS, GGSGGS (SEQ ID NO: 96), GGSGGSGGS (SEQ ID NO: 97), and GGSGGSGGSGGS (SEQ ID NO: 98). In yet other embodiments, the linker can comprise 4 to 12 amino acids containing a GGSG motif, e.g., GGSG (SEQ ID NO: 99), GGSGGGSG (SEQ ID NO: 100), or GGSGGGSGGGSG (SEQ ID NO: 101). In other embodiments, the linker can comprise a GGGGS (SEQ ID NO: 36) motif. In other embodiments, the linker can also include amino acids other than glycine and serine, for example, GENLYFQSGG (SEQ ID NO: 102), SACYCELS (SEQ ID NO: 103), RSIAT (SEQ ID NO: 104), RPACKIPNDLKQKVMNH (SEQ ID NO: 105), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGT GSG (SEQ ID NO: 16), AAANSSIDLISVPVDSR (SEQ ID NO: 107), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 108), GGGGAGGGGAGGGGS (SEQ ID NO: 32), GGGGAGGGGAGGGGAGGGGS (SEQ ID NO: 110), DAAGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 111), GGGGAGGGGAGGGGA (SEQ ID NO: 81), GGGGAGGGGAGGGAGGGGGS (SEQ ID NO: 30), or GGSSRSSSSGGGGAGGGG (SEQ ID NO: 112).

[0107] In one embodiment, the linker is a cleavable linker, such as an enzymatically cleavable linker. The inclusion of a cleavable linker can facilitate detection of the fusion protein. The enzymatically cleavable linker can be cleavable, for example, by trypsin, human rhinovirus 3C protease (3C), enterokinase (Ekt), factor Xa (FXa), tobacco etch virus protease (TEV), or thrombin (Thr). The cleavage sequences for each of these enzymes are well known in the art. For example, trypsin cleaves peptides at the C-terminal side of lysine and arginine amino acid residues. If a proline residue is located on the carboxyl side of the cleavage site, cleavage does not occur. It has been shown that the rate of hydrolysis is slower when acidic residues are located on either side of the cleavage site. The following linkers are examples of linkers that can be cleaved using trypsin: K(G4A)2G3AG4SK, R(G4A)2G3AG4SR, K(G4A)2G3AG4SR, R(G4A)2G3AG4SK, K(G4A)2G4SK, K(G4A)2G4SR, R(G4A)2G4SK and R(G4A)2G4SR.

[0108] An example of a protease cleavage site that can be included in an enzymatically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS, where the protease cleaves between glutamine and serine. Another example of a protease cleavage site that can be included in an enzymatically cleavable linker is an enterokinase cleavage site, e.g., DDDDK, where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in an enzymatically cleavable linker is a thrombin cleavage site, e.g., LVPR. For human rhinovirus 3C protease, the cleavage site is LEVLFQGP, where cleavage occurs between glutamine and glycine residues. For factor Xa protease, the cleavage site is IEDGR, where cleavage occurs between glutamic acid and aspartic acid residues.

[0109] The inclusion of a cleavable linker is useful in that it has an amino acid sequence that is different from other peptides in the human proteome produced by the enzyme. Thus, the cleaved linker can serve as a unique identifying peptide for the fusion protein when administered to humans as a pharmaceutical formulation. In this way, the cleavable linker can be detected and quantified by mass spectrometry and used to monitor the pharmacokinetics of the fusion protein.

[0110] In another embodiment, the linker is a polymeric or oligomeric glycine linker and can contain lysines at the N-terminus, C-terminus, or both the N-terminus and C-terminus.

[0111] With reference to Formulas I and III above, the C-terminus of D1 may be linked to the N-terminus of D2. In certain embodiments, the C-terminus of the FH fragment is linked to the N-terminus of the VHH. In certain embodiments, the C-terminus of the integrin-binding domain is linked to the N-terminus of the VHH. In certain embodiments, the C-terminus of D2 may be linked to the N-terminus of D3. In certain embodiments, the C-terminus of the VHH may be linked to the N-terminus of the integrin-binding domain. In certain embodiments, the C-terminus of the VHH may be linked to the N-terminus of the FH fragment. In another example, the C-terminus of D1 may be linked to the N-terminus of D3. In certain embodiments, the C-terminus of the FH fragment is linked to the N-terminus of the integrin-binding domain. In certain embodiments, the C-terminus of the integrin-binding domain is linked to the N-terminus of the FH fragment. In another example, the C-terminus of D2 may be linked to the N-terminus of D3. In certain embodiments, the C-terminus of the VHH may be linked to the N-terminus of the integrin-binding domain. In certain embodiments, the C-terminus of the VHH may be linked to the N-terminus of the FH fragment.

[0112] Referring to Formula II above, the C-terminus of D1 can be linked to the N-terminus of D2. In certain embodiments, the C-terminus of the FH fragment is linked to the N-terminus of the FHRP5 fragment.

[0113] [Table 1]

[0114] [Table 2]

[0115] [Table 3]

[0116] [Table 4]

[0117] [Table 5]

[0118] Production of fusion proteins Described herein are methods for producing fusion proteins using nucleic acid molecules encoding fusion proteins, such as those shown in Tables 1-5. The nucleic acid molecule can be operably linked to appropriate control sequences to form an expression unit encoding the protein. The expression unit can be used to transform a suitable host cell, and the transformed host cell can be cultured under conditions that allow for the production of the recombinant protein. Optionally, the recombinant protein can be isolated from the medium or cells; recovery and purification of the protein may not be necessary if some impurities can be tolerated. To facilitate purification, additional residues (e.g., a histidine tag) can be included at the N- or C-terminus of the protein-coding sequence, which can then be removed, if necessary, to form the final protein product.

[0119] Fusion proteins can be expressed from a single polynucleotide encoding the entire fusion protein, or as multiple (e.g., two or more) polynucleotides that can be expressed or co-expressed by an appropriate expression system. Polypeptides encoded by the co-expressed polynucleotides can associate, for example, via disulfide bonds or other means, to form a functional fusion protein. For example, the light chain portion of a monoclonal antibody can be encoded by a polynucleotide separate from the heavy chain portion of the monoclonal antibody. When co-expressed in a host cell, the heavy chain polypeptides associate with the light chain polypeptides to form the monoclonal antibody.

[0120] Typically, a nucleic acid encoding the desired fusion protein is generated using molecular cloning methods and is generally placed into a vector, such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell suitable for expression of the fusion polypeptide. Representative methods are disclosed, for example, in Maniatis et al. (Cold Springs Harbor Laboratory, 1989). While many cell types can be used as suitable host cells, mammalian cells are often chosen because of their ability to provide appropriate post-translational modifications. Host cells can include, for example, human embryonic kidney (HEK) (e.g., HEK 293) cells, Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, or any other suitable host cell known in the art.

[0121] In one embodiment, a nucleic acid or polynucleotide encoding a fusion protein is provided. In one embodiment, a vector comprising a nucleic acid or polynucleotide encoding a fusion protein is provided. In one embodiment, a host cell comprising one or more polynucleotides encoding a fusion protein is provided. In certain embodiments, a host cell comprising one or more fusion expression vectors is provided. Fusion proteins can be produced by expression of a nucleotide sequence in any suitable expression system known in the art. Any expression system can be used, including yeast, bacterial, animal, plant, eukaryotic, and prokaryotic systems. In some embodiments, yeast systems modified to reduce native yeast glycosylation, hyperglycosylation, or proteolytic activity can be used. Furthermore, any in vivo expression system designed for high-level expression of recombinant proteins in organisms known in the art can be used to produce the fusion proteins identified herein. In some embodiments, the Factor H fusion proteins described herein are produced by culturing one or more host cells comprising one or more nucleic acid molecules capable of expressing the fusion protein under conditions suitable for expression of the fusion protein. In some embodiments, the Factor H fusion protein is obtained from the cell culture or culture medium.

[0122] Fusion proteins can also be produced using chemical methods to synthesize the desired amino acid sequence, in whole or in part. For example, polypeptides can be synthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative high-performance liquid chromatography (e.g., Creighton (1983) Proteins: Structures and Molecular Principles, W.H. Freeman and Co., New York, NY). The composition of the synthetic polypeptide can be confirmed by amino acid analysis or sequencing. Furthermore, the amino acid sequence of the fusion protein, or any portion thereof, can be modified during direct synthesis and / or combined with sequences from other subunits or any portion thereof using chemical methods to produce variant polypeptides.

[0123] Fusion protein isolation / purification The secreted biologically active fusion proteins described herein, such as those listed in Tables 1-5, can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography such as Protein A affinity chromatography, size exclusion chromatography, etc. The conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., as will be apparent to one of skill in the art.

[0124] Assay of fusion protein activity Hemolysis assay The fusion proteins described herein were evaluated for activity using a complement pathway hemolysis assay, which measures complement-mediated lysis of rabbit red blood cells following activation of the alternative pathway on the cell surface. Rabbit red blood cells typically undergo complement-mediated lysis in mouse or human serum. Upon serum C3 activation, C3 convertase, C3 activation fragments, and C5 convertase are deposited on rabbit RBCs. For example, serum alternative complement pathway activity in the presence of fusion proteins containing a factor H fragment and a VHH domain, a factor H fragment and an FHRP5 fragment, or a factor H fragment, a VHH, and an integrin-binding domain (e.g., the fusion proteins in Tables 1-5) was assessed in a concentration-dependent manner in human or mouse serum in which the alternative pathway of complement activation was stimulated by supplementing Mg++ and EGTA as Ca scavengers. While incubation of rabbit erythrocytes in normal mouse or human serum causes cell lysis, the addition of nanomolar amounts of a fusion protein comprising, for example, a fragment of Factor H and a VHH domain, or a fragment of Factor H and a fragment of FHRP5, or a fragment of Factor H, a VHH domain, and an integrin-binding domain reduces the extent of lysis (see Figures 2A-2D). Fusion proteins of the present disclosure exhibit half-maximal inhibitory concentrations (IC) of about 15 nM to about 250 nM (e.g., about 15 nM to about 240 nM, about 15 nM to about 220 nM, about 200 nM to about 150 nM, about 15 nM to about 100 nM, about 15 nM to about 40 nM, or about 15 nM to about 50 nM).50 In some embodiments, the fusion protein may exhibit an IC of 19 nM to 240 nM (e.g., about 19 nM to about 230 nM, about 50 nM to about 240 nM, about 100 nM to about 240 nM, about 150 nM to about 240 nM, about 200 nM to about 240 nM, about 19 nM to about 50 nM, about 19 nM to about 100 nM, about 19 nM to about 150 nM, about 19 nM to about 200 nM, and about 19 nM to about 230 nM). 50 can be shown.

[0125] [Table 6]

[0126] Complement activation assay Fusion proteins described herein (e.g., those in Tables 1-5) can be assessed for alternative pathway activity in solution using an alternative pathway assay kit, such as the Complement system Alternative Pathway kit (WIESLAB®, Lund, Sweden). This method combines the principles of a hemolytic assay for complement activation with the use of a labeled antibody specific for a neoantigen produced as a result of complement activation. The amount of neoantigen produced is proportional to the functional activity of the alternative pathway. In the Complement system Alternative Pathway kit, plate wells are coated with a specific activator of the alternative pathway. Serum is diluted in a diluent containing a specific blocker to ensure that only the alternative pathway is activated. For example, anti-properdin VHH can be mixed in a concentration-dependent manner into patient blood. During incubation of the diluted patient serum in the wells, complement is activated by the specific coating. The wells are then washed, and C5b-9 is detected using a specific alkaline phosphatase-labeled antibody against the neoantigen as a result of complement activation. The amount of complement activation correlates with color intensity and is measured in terms of absorbance at 405 nm (optical density (OD)). Addition of nanomolar amounts of Factor H fusion proteins according to the present disclosure, for example, reduces the degree of activity. Further exemplary assays for determining complement pathway activity include those described in Hebell et al., (Science (1991) 254(5028):102-105).

[0127] Pharmaceutical Compositions, Dosages and Administration The fusion proteins described herein (see, e.g., Tables 1-5, e.g., those described in Table 1) can be incorporated into pharmaceutical compositions suitable for administration to a subject. Pharmaceutical compositions containing the Factor H fusion proteins described herein can be formulated for administration at individual doses ranging, for example, from 0.01 mg / kg to 500 mg / kg. Pharmaceutical compositions can contain, for example, 0.1 μg / 0.5 mL to 1 g / 5 mL of fusion protein. In some embodiments, pharmaceutical compositions described herein contain about 1 to 200 mg / mL, e.g., about 30 to 100 mg / mL, e.g., about 50 mg / mL (e.g., 50 mg / mL) of fusion protein.

[0128] Compositions containing Factor H fusion proteins can also be formulated for either single or multiple administration regimens. Doses can be formulated for administration, for example, hourly, every 2 hours, daily, every other day, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, weekly, biweekly, monthly, bimonthly, or yearly. Alternatively, doses can be formulated for administration, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times per day.

[0129] Pharmaceutical compositions containing factor H fusion protein can be formulated according to standard methods.Pharmaceutical formulation is a well-established technology, and is further described in, for example, Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 3rd Edition, (ISBN: 091733096X).

[0130] Pharmaceutical compositions can include a fusion protein and at least one pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The term "pharmaceutically acceptable carrier" excludes tissue culture media containing bovine or horse serum. A pharmaceutically acceptable carrier or adjuvant does not itself induce the production of antibodies harmful to or confer protection in the individual receiving the composition. Thus, pharmaceutically acceptable carriers are inherently non-toxic and non-therapeutic and are known to those skilled in the art. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Some embodiments will include an isotonic agent, such as a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable substances include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, stabilizers or buffers, which enhance the shelf life or effectiveness of the antibody.

[0131] The compositions described herein can be prepared in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Such formulations can be prepared by methods known in the art, such as those described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0132] Pharmaceutical compositions containing Factor H fusion proteins can also be formulated with carriers that protect the composition (e.g., Factor H fusion proteins) from rapid release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978) Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York.

[0133] The final form will depend on the intended mode of administration and therapeutic application. Typical compositions are in the form of an injectable or infusible solution, such as compositions similar to those used for passive immunization of humans with other antibodies. The compositions can be delivered, for example, by parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or local administration (e.g., directly to the kidney).

[0134] The pharmaceutical compositions can be provided in a sterile form and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of fusion protein in an appropriate solvent, optionally with one or a combination of the ingredients listed above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the fusion protein into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as monostearate salts and gelatin, in the composition. The form chosen will depend, in part, on the intended mode of administration and therapeutic application. For example, compositions intended for systemic or local delivery can be in the form of an injectable or infusible solution. The compositions can be formulated, for example, as a buffer solution at an appropriate concentration suitable for storage at 2-8°C (e.g., 4°C). The compositions can also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). The compositions can further be formulated for storage at 2-8°C (e.g., 4°C) for up to two years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, the compositions described herein can be stable upon storage at 2-8°C (e.g., 4°C) for at least one year.

[0135] The fusion proteins described herein can be administered by a variety of methods known in the art, although for many therapeutic applications, the route / mode of administration of choice is intravenous injection or infusion. Fusion proteins can also be administered by intramuscular or subcutaneous injection. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results.

[0136] In certain embodiments, the fusion protein may be prepared with a carrier that will protect against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.

[0137] Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Prolonged absorption of the injectable composition can be achieved by including an absorption-delaying agent, such as monostearate salts and gelatin, in the composition. Many methods for preparing such formulations are known to those skilled in the art (e.g., "Sustained and Controlled Release Drug Delivery Systems," JR Robinson, ed., Marcel Dekker, Inc., New York, 1978). Additional methods applicable to the controlled or sustained release of the fusion proteins disclosed herein are described, for example, in International Publication No. WO 2016 / 081884, the entire contents of which are incorporated herein by reference.

[0138] The pharmaceutical composition may have a pH of about 5.6 to 10.0, about 6.0 to 8.8, or about 6.5 to 8.0. For example, the pH may be about 6.2, 6.5, 6.75, 7.0, or 7.5, e.g., pH 7.0. The pharmaceutical composition may be formulated for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical, intravitreal, or parenteral administration. Parenteral administration may include intradermal, subcutaneous (SC, sq, subQ, hypo), intramuscular (im), intravenous (IV), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intravitreal (eye), intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluid) injection or infusion. SC administration may include SC infusion or SC push. Any device suitable for parenteral injection or infusion of drug formulations may be used for such administration. For example, the pharmaceutical compositions may be packaged in sterile, pre-filled syringes.

[0139] Additional active compounds can also be incorporated into the composition. In certain embodiments, the fusion protein is co-formulated and / or co-administered with one or more additional therapeutic agents. When the composition is used in combination with a second active agent, the composition can be co-formulated with the second agent or can be formulated separately from the second agent. For example, the respective pharmaceutical compositions can be mixed, e.g., immediately prior to administration, and administered together, or can be administered separately, e.g., at the same or different times. In some embodiments, the fusion protein can be co-formulated and / or co-administered with one or more additional antibodies that bind to other targets (e.g., antibodies that bind to regulators of the alternative complement pathway). Such combination therapy can utilize lower doses of the administered therapeutic agent, thus avoiding potential toxicities or complications associated with various monotherapies. Furthermore, the compositions described herein can be co-formulated or co-administered with other therapeutic agents (e.g., therapeutic agents that minimize the risk of infection in immunocompromised settings, e.g., antibacterial, antifungal, and antiviral agents) to ameliorate side effects of administering the compositions described herein.

[0140] The composition containing the Factor H fusion protein can be administered to a subject in combination with a pharmaceutically acceptable sterile aqueous or non-aqueous solvent, suspension, or emulsion. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions, or suspensions, including saline and buffered medical parenteral vehicles, including sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride solution, Ringer's solution containing lactose, or fixed oils.

[0141] Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, etc. Pharmaceutically acceptable salts can be included therein, such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can be present in such vehicles. A complete discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0142] The pharmaceutical composition may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the fusion protein. A "therapeutically effective amount" refers to an amount effective, at a dosage and for a duration sufficient to achieve a desired therapeutic result. A therapeutically effective amount of a fusion protein may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the fusion protein to elicit a desired response in the individual. A "prophylactically effective amount" refers to an amount effective, at a dosage and for a duration sufficient to achieve a desired prophylactic result. In some embodiments, a prophylactic dose is used in subjects prior to or at an early stage of disease, where the prophylactically effective amount is less than the therapeutically effective amount.

[0143] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. It should be noted that dosage values can vary depending on the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the administering clinician.

[0144] The effectiveness of treatment with the fusion proteins described herein can be assessed based on improvement in one or more symptoms or indicators of the disease state or disorder being treated (e.g., improvement in one or more symptoms of an alternative complement pathway (CAP)-mediated disease or disorder, e.g., a renal disease or disorder mediated by dysregulation of CAP). An improvement of at least 10% (an increase or decrease, depending on the indicator measured) in one or more clinical indicators is considered "effective treatment," although greater improvements are possible, such as 20%, 30%, 40%, 50%, 75%, 90%, or even 100% improvement, or greater than 100% (e.g., 2-fold, 3-fold, 10-fold, etc., up to and including achievement of a disease-free state), depending on the indicator measured.

[0145] Therapeutic methods using fusion proteins The complement factor H fusion proteins described herein (see, e.g., Tables 1-5) can be used to treat diseases mediated by alternative complement pathway activation or dysregulation in mammals (e.g., humans) by inhibiting alternative complement pathway activation. The fusion proteins described herein can be used to treat various diseases or disorders mediated by alternative complement pathway activation or dysregulation. Such disorders include, but are not limited to, renal injury, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as kidneys.

[0146] A therapeutically effective amount of a complement factor H fusion protein disclosed herein (e.g., a fusion protein having any one of SEQ ID NOS: 1-15 or a variant thereof having at least 85% (e.g., at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto) can be administered to a mammalian subject in need of such treatment. In some embodiments, the subject is a human patient. The amount administered should be sufficient to inhibit complement activation and / or restore normal alternative complement pathway regulation. Determination of a therapeutically effective dose is within the capabilities of a physician skilled in the art. However, by way of example, in embodiments of the methods described herein utilizing systemic administration of the fusion protein for diseases mediated by alternative complement pathway activation or dysregulation, an effective human dose may be between 0.01 mg / kg and 150 mg / kg (e.g., 0.05 mg / kg and 500 mg / kg, 0.1 mg / kg and 20 mg / kg, 5 mg / kg and 500 mg / kg, 0.1 mg / kg and 100 mg / kg, 10 mg / kg and 100 mg / kg, 0.1 mg / kg and 50 mg / kg, 0.5 mg / kg and 25 mg / kg, 1.0 mg / kg and 10 mg / kg, 1.5 mg / kg and 5 mg / kg, or 2.0 mg / kg and 3.0 mg / kg) or between 1 μg / kg and 1,000 μg / kg (e.g., 5 μg / kg and 1,000 μg / kg). The recommended dose may be within the range of 1 μg / kg to 1,000 μg / kg, 1 μg / kg to 750 μg / kg, 5 μg / kg to 750 μg / kg, 10 μg / kg to 750 μg / kg, 1 μg / kg to 500 μg / kg, 5 μg / kg to 500 μg / kg, 10 μg / kg to 500 μg / kg, 1 μg / kg to 100 μg / kg, 5 μg / kg to 100 μg / kg, 10 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 5 μg / kg to 50 μg / kg, or 10 μg / kg to 50 μg / kg. The route of administration may affect the recommended dose. Repeated systemic doses are contemplated to maintain effective levels, for example, to attenuate or inhibit complement activation in the patient's system, depending on the mode of administration employed.

[0147] The compositions and methods described herein may be useful for treating renal disorders mediated by CAP dysregulation, such as FSGS. FSGS is characterized by obstruction of the glomerular capillary tuft with increased matrix deposition and scarring (D'Agati et al., Am J Kidney Dis. 43(2):368-382, 2004). The incidence of FSGS has increased over the past several decades and is one of the leading causes of nephrotic syndrome in adults (Korbet, J Am Soc Nephrol. 23(11):1769-1776, 2012). Spontaneous remission is rare (<5%), and the presence of persistent nephrotic syndrome indicates a poor prognosis, with 50% of patients progressing to end-stage renal disease (ESRD) 6-8 years after initial diagnosis (Korbet, Nephrol Dial Transplant. 14 Suppl 3:68-73, 1999). Primary FSGS accounts for 3.3% of all cases of end-stage renal disease (ESRD) due to primary renal disease in the United States. The complement system has been shown to be activated in patients with primary FSGS, and elevated plasma Ba levels, indicating alternative pathway activation, correlate with disease severity. Patients with low serum C3 levels had a higher rate of interstitial injury. Furthermore, patients with normal serum C3 levels were found to have a higher renal survival rate compared with patients with low serum C3 levels. Low serum C3 levels indicate complement activation. Therefore, activation of the complement system may play an important role in the pathogenesis and outcome of FSGS (Liu et al., Scientific Reports, 7:4095, 2017). In humans, tubulointerstitial deposition of the complement membrane attack complex (C5b-9) correlates with interstitial myofibroblast accumulation and proteinuria. Experimentally, in focal segmental glomerulosclerosis, intratubular formation of C5b-9 was found to promote the accumulation of peritubular myofibroblasts. Myofibroblasts act as sentinel inflammatory cells and can deposit extracellular matrix. These cells can also cause tubular contraction, resulting in atubular glomeruli. Through this mechanism, complement activation may contribute to tubulointerstitial damage and fibrosis in FSGS (Rangan et al., Kidney Int. 66:1838-1848, 2004).Factor B and factor D-deficient mice have lower proteinuria than wild-type controls in an adriamycin-induced FSGS model, suggesting that CAP activation plays a pathogenic role (Lenderink et al., Am. J. Physiol. Renal Physio. 293:F555-F564, 2007). The alternative complement pathway is activated in the glomeruli and tubulointerstitium of mice with adriamycin-induced nephropathy (Turnberg et al., J Immunol. 177(6):4094-4102, 2006). Furthermore, complement factor H-deficient mice exhibit higher C3b glomerular deposition and more severe renal injury than wild-type controls (Morigi et al., Sci Rep. 6:28445, 2016), confirming a previously unrecognized role for C3a in proteinuric progressive nephropathy. Therefore, inhibitors of the alternative pathway of complement activation may be used to achieve clinical benefit in FSGS.

[0148] The methods described herein may be useful for treating renal lesions characterized histologically by predominant C3 accumulation in glomeruli in the absence of significant immunoglobulin deposition due to aberrant regulation of the alternative complement pathway, also known as C3G (Nester and Smith, Curr. Opin. Nephrol. Hypertens., 22:231-237, 2013).

[0149] The methods described herein may be useful for treating dense deposition disease, a rare renal disease that results in persistent proteinuria, hematuria, and nephritic syndrome. Concomitant dysfunction in factor H deficiency and dense deposition disease has been reported in several cases. For example, mutations in factor H have been found in human patients with dense deposition disease. Symptoms of dense deposition disease include, for example, hematuria and / or proteinuria; acute nephritic syndrome; the development of drusen and / or visual impairment; acquired partial lipodystrophy and its complications; and the presence of serum C3 nephritic factor (C3NeF), an autoantibody against C3bBb, the C3 convertase of the alternative complement pathway (Appel et al., J. Am. Soc. Nephrol., 16:1392-1404, 2005). Targeting factor H to complement activation sites has therapeutic effects on individuals with dense deposition disease. In some embodiments, administering a composition comprising a fusion molecule described herein to an individual is effective in treating dense deposition disease. The route of administration may affect the recommended dose. Repeated systemic doses are contemplated to maintain effective levels, for example, to attenuate or inhibit complement activation in the patient's system, depending on the mode of administration used.

[0150] The compositions and methods described herein may be useful for treating renal inflammation caused by systemic lupus erythematosus (SLE), such as lupus nephritis. Lupus glomerulonephritis involves diverse and complex morphological lesions, depending on the degree of active or chronic disease, the degree of interstitial inflammation or fibrosis, and the proportion of glomeruli affected by vascular lesions (Weening et al., J. Am. Soc. Nephrol., 15:241-250, 2004). Lupus nephritis is a severe complication that occurs in a subset of SLE patients. SLE is a prototypical autoimmune disease resulting in multiorgan involvement. This anti-self response is characterized by autoantibodies directed against various nuclear and cytoplasmic cellular components. These autoantibodies bind to their respective antigens, forming immune complexes that circulate and ultimately deposit in tissues. The deposition of these immune complexes causes chronic inflammation and tissue damage. The complement pathway (including the alternative complement pathway) is involved in the pathology of SLE; therefore, the fusion proteins provided herein are useful for treating lupus nephritis.

[0151] The compositions and methods described herein may be useful for treating membranous nephropathy (MN), the most common cause of glomerular disease and idiopathic nephrotic syndrome in non-diabetic Caucasian adults. If left untreated, approximately one-third of patients with MN progress to end-stage renal disease over a 10-year period. The incidence of ESRD due to MN in the United States is approximately 1.9 per million per year. Most cases of primary MN (70%) have circulating pathogenic IgG4 autoantibodies against the podocyte membrane antigen PLA2R. Complement components including C3, C4d, and C5b-9 are also commonly present, but C1q is absent, indicating the involvement of lectins and potentially the alternative pathway of complement activation. Over time, IgG4 and C5b-9 deposition leads to podocyte injury, urinary protein excretion, and nephrotic syndrome (Couser, Clin J Am Soc Nephrol 12:983-997, 2017). Mice lacking factor B, an essential component of the alternative pathway of complement activation, did not exhibit C3 and C5B-9 deposition and did not develop albuminuria in a mouse model of MN (Wentian et al., Front Immunol. 9:1433, 2018). Therefore, complement inhibitors, such as the fusion proteins described herein, that reduce the amount of C3 and C5 convertases deposited in glomerular lesions can be used to treat this disease.

[0152] In some embodiments, the method includes treating a subject having a disease or disorder mediated by alternative complement pathway activation or dysregulation by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15, or a variant thereof having at least 85% sequence identity (or greater) thereto).

[0153] In some embodiments, the method includes treating a subject with a renal disorder by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0154] In some embodiments, the method includes treating a subject with membranous nephropathy by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0155] In some embodiments, the method includes treating a subject with FSGS by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0156] In some embodiments, the method includes treating a subject with glomerulonephritis by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0157] In some embodiments, the method includes treating a subject with membranoproliferative glomerulonephritis by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0158] In some embodiments, the method includes treating a subject with complement 3 nephropathy (C3G) by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0159] In some embodiments, the method includes treating a subject with IgA nephropathy by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0160] In some embodiments, the method includes treating a subject with MCD by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0161] In some embodiments, the method includes treating a subject with diabetic nephropathy by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0162] In some embodiments, the method includes treating a subject with Alport syndrome by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0163] In some embodiments, the method includes treating a subject with lupus nephritis by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0164] In some embodiments, the method includes treating a subject having acute kidney injury by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0165] In some embodiments, the method includes treating a subject with Goodpasture's syndrome by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0166] In some embodiments, the method includes treating a subject with nephrotic syndrome by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0167] In some embodiments, the method includes treating a subject with chronic proteinuria by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0168] In some embodiments, the method includes treating a subject with chronic kidney disease by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0169] In some embodiments, the method includes treating a subject with a dense deposition disease by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0170] In some embodiments, the method includes treating a subject with polycystic kidney disease by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0171] In some embodiments, the method includes treating a subject with hypertensive nephropathy by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0172] In some embodiments, the method includes treating a subject with nephrosclerosis by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0173] In some embodiments, the method includes treating a subject with aHUS by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0174] In some embodiments, the method includes treating a subject having ischemia-reperfusion injury by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0175] In some embodiments, the method includes treating a subject having rejection of a transplanted organ by administering to the subject a therapeutically effective amount of a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof.

[0176] The present disclosure further relates to a composition comprising the fusion protein provided above for use in treating a disease or disorder mediated by CAP activation or dysregulation. In some embodiments, the disease or disorder is selected from the group consisting of renal injury, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of a transplanted organ such as a kidney.

[0177] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating renal disorders.

[0178] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating FSGS.

[0179] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating membranous nephropathy.

[0180] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating IgA nephropathy.

[0181] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating MCD.

[0182] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating diabetic nephropathy.

[0183] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating Alport syndrome.

[0184] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating lupus nephritis.

[0185] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating acute kidney injury.

[0186] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating Goodpasture's syndrome.

[0187] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating nephrotic syndrome.

[0188] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating chronic proteinuria.

[0189] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating chronic kidney disease.

[0190] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating C3G.

[0191] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating dense deposition disease.

[0192] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating glomerulonephritis.

[0193] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating membranoproliferative glomerulonephritis.

[0194] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating polycystic kidney disease.

[0195] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating hypertensive nephropathy.

[0196] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating nephrosclerosis.

[0197] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for use in treating aHUS.

[0198] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating ischemia-reperfusion injury.

[0199] The present disclosure further relates to a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15) for use in treating rejection of a transplanted organ such as a kidney.

[0200] In some embodiments, the present disclosure relates to pharmaceutical compositions for treating a disease or disorder mediated by CAP activation or dysregulation, in some embodiments, the disease is renal injury, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of a transplanted organ such as a kidney.

[0201] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating renal disorders, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0202] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating FSGS, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0203] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating IgA nephropathy, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15).

[0204] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating diabetic nephropathy, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15).

[0205] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating acute kidney injury, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0206] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating chronic kidney disease, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0207] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating membranous nephropathy, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0208] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating rejection of a transplanted organ such as a kidney, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0209] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating MCD, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0210] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating Alport syndrome, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0211] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating nephrotic syndrome, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0212] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating lupus nephritis, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0213] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating glomerulonephritis, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0214] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating membranoproliferative glomerulonephritis, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0215] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating Goodpasture's syndrome, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0216] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating chronic proteinuria, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0217] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating C3G, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0218] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating dense deposition disease, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0219] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating polycystic kidney disease, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0220] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating hypertensive nephropathy, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15).

[0221] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating nephrosclerosis, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 15).

[0222] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating aHUS, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0223] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating ischemia-reperfusion injury, comprising as an active ingredient a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15).

[0224] In some embodiments, the present disclosure relates to the use of a composition comprising the fusion protein provided above for the manufacture of a medicament for treating a disease or disorder mediated by CAP activation or dysregulation, in some embodiments, the disease is selected from the group consisting of renal injury, FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of a transplanted organ such as a kidney.

[0225] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for renal disorders.

[0226] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O for the manufacture of a medicament for FSGS.

[0227] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for IgA nephropathy.

[0228] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for diabetic nephropathy.

[0229] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for acute kidney injury.

[0230] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for chronic kidney disease.

[0231] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for membranous nephropathy.

[0232] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for rejection of a transplanted organ, such as a kidney.

[0233] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for MCD.

[0234] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for Alport syndrome.

[0235] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for nephrotic syndrome.

[0236] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for chronic proteinuria.

[0237] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for lupus nephritis.

[0238] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for membranoproliferative glomerulonephritis.

[0239] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for glomerulonephritis.

[0240] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O, for the manufacture of a medicament for Goodpasture's syndrome.

[0241] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for C3 nephropathy.

[0242] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for a dense deposition disease.

[0243] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for polycystic kidney disease.

[0244] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for hypertensive nephropathy.

[0245] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for nephrosclerosis.

[0246] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for aHUS.

[0247] In some embodiments, the present disclosure relates to use of a composition comprising a fusion protein selected from the group consisting of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, Compound H, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N, and Compound O (e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1-15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity to any one of SEQ ID NOs: 1-15) for the manufacture of a medicament for ischemia-reperfusion injury. [Example]

[0248] The following examples are put forth to provide those of ordinary skill in the art with a disclosure and description of how to perform and make the methods and compounds claimed herein. They are intended to be purely illustrative and are not intended to limit the scope of the present disclosure.

[0249] Example 1. In silico design and construction of a kidney-localized Factor H fusion protein Constructs containing various combinations of Factor H N-terminal functional domains, VHH domains, and polyarginylglycylaspartic acid (RGD) domains were designed in silico. Exemplary constructs are shown in Figures 1A and 1B.

[0250] The amino acid sequences of the constructs shown in Figures 1A and 1B were submitted to GeneArt (ThermoFisher) for codon optimization and gene synthesis. The nucleotide sequences were cloned into a proprietary vector for expression in mammalian cells. The plasmid DNA was then transiently transfected into HEK 293 and CHO cells. After 4–5 days, the supernatants were harvested. The concentrations of the fusion proteins were determined by SDS-PAGE and densitometry.

[0251] Example 2. Inhibition of the alternative pathway using fusion proteins Compounds according to the present disclosure were tested for their ability to inhibit the alternative pathway using a CAP-specific hemolysis assay. The results are shown in Figures 2A, 2B, 2C, and 2D. Briefly, rabbit red blood cells were washed and treated with Mg 2+ The inhibitors were added to 10% human serum containing EGTA and 10% ATP. Serial dilutions of inhibitors were added, and the cells were incubated at 37°C for 30 minutes. The cells were removed by centrifugation, and the amount of cell lysis was determined by measuring the absorbance of the supernatant at 415 nm. All molecules tested demonstrated inhibition of alternative pathway lysis, confirming that the molecules were functional as expected.

[0252] Example 3. Visualization of fusion proteins in the kidney In vivo imaging of fluorescently labeled constructs was performed in female J:NU outbred nude mice (Jackson Laboratories, Bar Harbor, ME). Figure 3 shows representative longitudinal in vivo imaging in the same subject acquired over a 4-day period. For image acquisition, subjects were maintained under 2-3% isoflurane anesthesia on a heated imaging platform within an IVIS Spectrum Imaging System (PerkinElmer Inc., Waltham, MA). Fluorescence imaging analysis was performed using Living Image 4.5.1 software (PerkinElmer Inc., Waltham, MA) with automated 2D epi-illumination exposure settings: field of view (FOV) C, F / Stop 2, medium binning, and an 800 nm emission / 750 nm excitation filter. Subjects received 1 mg / kg of AlexaFluor 750-labeled test substance via a volume-standardized 100 μL intravenous tail vein injection. All animal studies were conducted in accordance with the provisions of the Animal Welfare Act and the principles of the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the IACUC of Alexion Pharmaceuticals, Inc., New Haven, CT, protocol #917103.

[0253] Figure 3 also shows a kidney from the same mouse imaged via fluorescence microscopy. The signal indicates biodistribution of the test compound, with selectivity for the parietal and apical sides of proximal tubular epithelial cells. Renal residence time was prolonged by the presence of VHH and RGD-containing motifs.

[0254] Example 4. Pharmacokinetic determination of selected fusion proteins Single-dose pharmacokinetic exposures for select compounds were determined in female J:NU outbred nude mice (Jackson Laboratories, Bar Harbor, ME). Serum exposure was measured by an electrochemiluminescence detection assay developed on an MSD (Meso Scale Discovery, Rockville, Maryland) platform. Figure 4 shows the PK of the compounds 1 and 24 hours after IV bolus administration. In contrast to the renal retention kinetics shown in Figure 3, the compounds were below the lower limit of detection in serum by 24 hours after administration, confirming the target specificity of the compounds.

[0255] Example 5. Determining Treatment Efficacy in an FSGS Model Compounds D, E, and G were evaluated for therapeutic efficacy in an adriamycin-induced nephropathy mouse model of focal segmental glomerulosclerosis (FSGS). Figure 5 shows the therapeutic effects on urinary protein. Factor H 1~5 Alone, it was insufficient to provide significant benefit. Efficacy was enhanced by the presence of VHH and RGD-containing motifs.

[0256] Example 6. Determining Treatment Efficacy in an FSGS Model Compounds D, E, and G were evaluated for their therapeutic efficacy in an adriamycin-induced nephropathy mouse model of FSGS. Figure 6 shows the therapeutic effects on urinary albumin and factor H. 1~5 Alone, it was insufficient to provide significant benefit. Efficacy was enhanced by the presence of VHH and RGD-containing motifs.

[0257] Example 7. Determining Treatment Efficacy in an FSGS Model Compounds D, E, and G were evaluated for their therapeutic efficacy in an adriamycin-induced nephropathy mouse model of FSGS. Figure 7 shows the therapeutic effects on renal tubular proteins. Factor H 1~5 Alone, it was insufficient to provide significant benefit.

[0258] Example 8. Kidney visualization in an FSGS model after fusion protein administration Immunofluorescent C3 activation product staining was performed on kidney sections from mice in the adriamycin-induced nephropathy mouse model of FSGS 7 days after therapeutic administration of Compound E (study day 14). Figure 8A illustrates a region of interest (ROI) manually applied to approximate the renal medulla to perform area-normalized C3 fragment mean pixel intensity analysis and assess local alternative complement pathway (CAP) activation. Figure 8B qualitatively demonstrates medullary CAP modulation occurring after treatment with Compound E.

[0259] Example 9. Determining Treatment Efficacy in an FSGS Model Compound D, Compound E, and Compound G were evaluated for therapeutic efficacy in the adriamycin-induced nephropathy mouse model of FSGS. On study day 13, animals were individually placed in metabolic cages for 16 hours. Collected urine was analyzed for albumin, protein, and creatinine using a Cobas analyzer. Calculated urinary protein / creatinine (Figure 9A) and albumin / creatinine (Figure 9B) ratios show a positive, but not statistically significant, trend toward benefit after treatment with Compound E.

[0260] Example 10. Characterization of fusion protein purity Proteins were assessed by non-reducing SDS-PAGE gels to confirm purity and molecular weight. Most fusion proteins were easily purified to high levels of purity by anion exchange chromatography followed by hydrophobic interaction chromatography (HIC). Anion exchange was performed on a 34 mL Capto Q Impres. HIC (HiTrap Phenyl FF (HS)) was equilibrated in buffer A (20 mM Tris-HCl, 3 M NaCl, pH 8.2) and eluted with buffer B (20 mM Tris-HCl, pH 8.2). Purity was greater than 95% for compound E. Non-reducing SDS-PAGE showed a single band at a load of 2 μg per well after two-step purification. Selected proteins were easily purified to high levels of purity by protein A chromatography. The concentration of the purified fusion proteins was determined by UV spectrophotometric absorbance at 280 nm corrected for molar extinction coefficient. Purity was assessed by SDS-PAGE and size-exclusion chromatography (SEC) HPLC. Exemplary recovered cell culture supernatants assessed via SDS-PAGE and purified proteins via SEC-HPLC are shown in Figures 10A and 10B, respectively.

[0261] Example 11. Characterization of fusion proteins using mass spectrometry Selected compounds were evaluated by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry to determine intact molecular weight. Figure 11 shows confirmation with the expected theoretical molecular weight for representative compound E.

[0262] Example 12. Characterization of fusion proteins using dynamic light scattering The melting point was determined by dynamic light scattering (DLS). The compound of interest was first diluted to 1 mg / mL in PBS (pH 7.4) and evaluated at a scan rate of 60 °C-h. Figure 12 shows an exemplary scan showing a satisfactory melting point measured by DLS for compound E. Other compounds gave similar results.

[0263] Example 13. Characterization of fusion proteins using size exclusion chromatography Size-exclusion chromatograms of Compound E were obtained after 0 and 14 days of incubation at 37° C. The relative percentage of aggregated protein and the relative percentage of intact fusion protein were calculated from the chromatogram measured after 0 days (FIG. 13A) (1.1% aggregates and 98.4% fusion protein) and after 14 days (FIG. 13B) (2.4% aggregates and 97.4% fusion protein). The negligible 1.3% increase in aggregated protein over 14 days indicates that the fusion protein is stable over this length of time.

[0264] Example 14. Characterization of fusion proteins by hydrophobic interactions Hydrophobic interaction chromatograms of compound E were obtained after 0 and 14 days of incubation at 37° C. The resulting chromatograms show that the retention time and area under the peak remain unchanged from day 0 to day 14, indicating the stability of the fusion protein over this time period, as shown in Figures 14A and 14B.

[0265] Example 15. Characterization of fusion protein stability using capillary electrophoresis The stability of the compound over time (14 days at 37°C) was assessed by CE-SDS. Figure 15A shows the non-reduced CE-SDS chromatograms of Compound E at time zero and after 14 days. Figure 15B shows the reduced CE-SDS chromatograms after 0 and 14 days. The profiles at day 0 and day 14 were similar, confirming the stability of the compound. No obvious differences were observed under reduced vs. non-reduced conditions, further confirming protein stability.

[0266] Example 16. Characterization of fusion protein stability using isoelectric focusing capillary electrophoresis Isoelectric focusing capillary electrophoresis (ICE) was performed to determine stability and lack of charge heterogeneity according to standard techniques. Figure 16 shows a representative ICE result for Compound E. A total of six replicates confirmed a consistent stability and manufacturability profile for Compound E.

[0267] Example 17. Characterization of fusion protein stability using mass spectrometry The stability of the intact molecular weight of the compound was measured at various time points over 14 days at 37°C and assessed by MS via standard techniques. Figure 17 shows Compound E at time 0, 3 days, 7 days, and 14 days. The profiles were all similar, confirming the stability of Compound E. Similar results were obtained for Compound D and Compound G.

[0268] Example 18. Characterization of fusion protein binding to C3b The binding of compounds to C3b was assessed by biolayer interferometry (BLI). Biotinylated C3b was immobilized on a streptavidin biosensor chip and exposed to molar equivalent concentrations of analyte diluted in kinetic buffer. Figure 18A shows the predicted binding profiles of Compound E and Compound K compared to non-binding controls (Reference Protein 11, an anti-C5 VHH reference protein used as a negative control, and Reference Protein 6, an anti-HSA Factor H-VHH fusion protein used as a positive control), as represented by the shift in optical thickness across the sensor chip. Figure 18B shows an expanded 40-second section of the binding curve in Figure 18A; the t = 0 second time point in Figure 18B corresponds to the t = 720 second time point in Figure 18A. These results confirm the binding of the compounds to C3b.

[0269] Example 19. Determining the effect of fusion proteins on alternative complement pathway regulation Modulation of the alternative complement pathway (CAP) in normal human serum (NHS) by Compound E was assessed in the alternative complement pathway WIESLAB® assay according to the manufacturer's instructions. Comparable dose-dependent inhibition of C5b-9 neoantigen expressed during MAC formation was observed across the two tested NHS lots under otherwise identical conditions, as shown in Figure 19.

[0270] Example 20. Pharmacokinetic evaluation of Compound E in wild-type mice Single-dose pharmacokinetic exposure was determined for Compound E across a range of subcutaneous (SC) doses in wild-type male C57B1 / 6J mice (Jackson Laboratories, Bar Harbor, ME). Figure 20 shows combined data from two separate studies demonstrating the dose-dependent pharmacokinetics of Compound E administered at 10, 30, and 100 mg / kg. Serum exposure was measured by LC-MS / MS.

[0271] Example 21. Pharmacokinetic evaluation of Compound E in cynomolgus monkeys The single-dose pharmacokinetic exposure of Compound E was determined across a range of intravenous (IV) and subcutaneous (SC) doses in female cynomolgus (Macaca fascicularis) monkeys (Charles River Laboratories, Inc., Mattawan, MI). Figure 21A shows the serum pharmacokinetics of Compound E after both the initial dose given on study day 0 and the fourth dose administered on study day 12. Figure 21B contains the data from Figure 21A replotted to compare equivalent dose levels given by the IV or SC administration route. Serum exposure was measured by LC-MS / MS.

[0272] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. While particular embodiments have been described herein, those skilled in the art will recognize that further modifications and embodiments are encompassed, including variations, uses, or adaptations that generally follow from the principles described herein, and that are within known or customary practice in the art, including such departures from the present disclosure as may be applicable to the essential features described above and in the claims below. In an embodiment of the present invention, for example, the following items are provided: (Item 1) From N-terminus to C-terminus, the structure: D1-L1-D2-L2-D3 (In the formula, D1 contains a fragment of complement factor H (FH); L1 is absent, a covalent bond, or an amino acid sequence of at least one amino acid; D2 contains or is absent a VHH; L2 is absent, a covalent bond, or an amino acid sequence of at least one amino acid; and D3 is the integrin recognition domain) A fusion protein comprising: (Item 2) 2. The fusion protein of item 1, wherein D1 comprises one or more FH short consensus repeat (SCR) domains, optionally wherein the one or more SCR domains are selected from the group consisting of SCR 1, 2, 3, 4, 5 and 6. (Item 3) 3. The fusion protein according to item 2, wherein the FH SCR domain is selected from the group consisting of SCRs 1 to 4; 1 to 5; and 1 to 6. (Item 4) 4. The fusion protein of any one of items 1 to 3, wherein the VHH of D2 comprises a single domain antibody. (Item 5) 5. The fusion protein according to any one of items 1 to 4, wherein the VHH of D2 comprises a camelid single domain antibody. (Item 6) 6. The fusion protein according to any one of items 1 to 5, wherein the integrin recognition domain of D3 comprises an integrin recognition domain containing an arginylglycylaspartic acid (RGD) peptide motif. (Item 7) 7. The fusion protein according to any one of items 1 to 6, wherein the integrin recognition domain of D3 comprises a cyclo(RGD)4 peptide motif. (Item 8) 8. The fusion protein according to any one of items 1 to 7, wherein L1 and L2 comprise the same amino acid sequence. (Item 9) 8. The fusion protein according to any one of items 1 to 7, wherein L1 and L2 comprise different amino acid sequences. (Item 10) L1 and / or L2 are (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGAGGGGAGGGGS, GGGSGGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GSTSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12 , APELPGGP, SEPQPQPG, (G3S2)3, GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS)3, (GS4)3, G4A(G4S)2, G4SG4AG4S, G3AS(G4S)2, G4SG3ASG4S, G4SAG3SG4S, (G4S)2AG3S, G4SAG3SAG3S, G4D(G4S)2, G4SG4DG4S, (G4D)2G4S, G4E(G4S)2, G4SG4EG4S, (G4E)2G4S, G4SDA, G4A and (G4A)3. (Item 11) 11. The fusion protein according to item 10, wherein L1 and / or L2 are selected from the group consisting of (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4SDAA, (G4S)4, G4AG3AG4S, G4A and (G4A)3. (Item 12) (a) D1 contains FH SCR domains 1-5; L1 contains G4A; D2 is absent; L2 is absent; and D3 contains cyclo(RGD)4; (b) D1 comprises FH SCR domains 1 to 5; L1 is absent; D2 comprises the VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; (c) D1 contains FH SCR domains 1-5; L1 contains G4A; D2 is absent; L2 contains G4A; and D3 contains cyclo(RGD)4; (d) D1 contains FH SCR domains 1-5; L1 is absent; D2 contains VHH; L2 contains G4A; and D3 contains cyclo(RGD)4; (f) D1 comprises FH SCR domains 1-5; L1 is absent; D2 comprises VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; (g) D1 comprises FH SCR domains 1-6; L1 is absent; D2 comprises a VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4; or (h) The fusion protein of item 1, wherein D1 comprises FH SCR domains 1 to 5; L1 comprises G4A; D2 comprises VHH; L2 comprises G4A; and D3 comprises cyclo(RGD)4. (Item 13) (a) has the amino acid sequence of SEQ ID NO: 4 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; (b) has the amino acid sequence of SEQ ID NO: 5 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; (c) has the amino acid sequence of SEQ ID NO: 8 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; (d) has the amino acid sequence of SEQ ID NO: 9 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; (e) has the amino acid sequence of SEQ ID NO: 13 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; or (f) has the amino acid sequence of SEQ ID NO: 14 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; (g) The fusion protein according to item 1, having the amino acid sequence of SEQ ID NO: 15 or a variant thereof having up to 10 amino acid substitutions, additions or deletions. (Item 14) (a) has an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:4; (b) has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:5; (c) having an amino acid sequence having at least 85% sequence identity to SEQ ID NO:8; (d) has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:9; (e) having an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13; (f) having an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 14; (g) The fusion protein of item 1, having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 15. (Item 15) From N-terminus to C-terminus, the structure: D1-L1-D2 (In the formula, D1 includes FH fragments such as FH1-5; L1 comprises a linker or is absent; and D2 contains factor H-related protein 5 (FHRP5) domains, including FHRP domains 7 and 8 A fusion protein comprising: (Item 16) L1 is G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGGAGGGAGGGS, GGGSGGGGSGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK) 3, PAPAP, G4SPAPAP, PAPAPG4S, GSTSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12, APELPGGP, SEPQPQPG, (G3S2)3, GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS)3, (GS4)3, G4A(G4S)2, G4SG4AG4S, G3AS(G4S)2, G4SG3ASG4S, G4SAG3SG4S, (G4S)2AG3S, G4SAG3SAG3S, G4D(G4S)2, G4SG4DG4S, (G4D)2G4S, G4E(G4S)2, G4SG4EG4S, (G4E)2G4S and G4SDA. (Item 17) 17. The fusion protein of item 16, wherein L1 is selected from the group consisting of G4A and (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4SDAA, (G4S)4 and G4AG3AG4S. (Item 18) (a) has the amino acid sequence of SEQ ID NO: 6 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) The fusion protein according to any one of items 15 to 17, having the amino acid sequence of SEQ ID NO: 10 or a variant having up to 10 amino acid substitutions, additions or deletions. (Item 19) (a) has an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:6; or (b) The fusion protein according to any one of items 15 to 17, having an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. (Item 20) From N-terminus to C-terminus, the structure: D1-L1-D2-L2-D3 (In the formula, D1 contains an integrin recognition domain such as cyclo(RGD)4; L1 comprises a linker or is absent; D2 is a VHH, such as a single domain antibody, L2 is a linker or is absent; and D3 is an FH fragment such as FH1-5) A fusion protein comprising: (Item 21) 21. The fusion protein of item 20, having a C-terminal His tag. (Item 22) 22. The fusion protein according to item 20 or 21, wherein L1 and L2 comprise the same amino acid sequence. (Item 23) 23. The fusion protein according to any one of items 20 to 22, wherein L1 and L2 comprise different amino acid sequences. (Item 24) L1 and / or L2 are G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGAGGGGAGGGGS, GGGGSGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GSTSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12 24. The fusion protein according to item 22 or 23, wherein the fusion protein is selected from the group consisting of: APELPGGP, SEPQPQPG, (G3S2)3, GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS)3, (GS4)3, G4A(G4S)2, G4SG4AG4S, G3AS(G4S)2, G4SG3ASG4S, G4SAG3SG4S, (G4S)2AG3S, G4SAG3SAG3S, G4D(G4S)2, G4SG4DG4S, (G4D)2G4S, G4E(G4S)2, G4SG4EG4S, (G4E)2G4S and G4SDA. (Item 25) 25. The fusion protein according to item 24, wherein L1 and / or L2 are selected from the group consisting of G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4SDAA, (G4S)4 and G4AG3AG4S. (Item 26) (a) has the amino acid sequence of SEQ ID NO: 2 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) The fusion protein according to item 18 or 19, having the amino acid sequence of SEQ ID NO: 3 or a variant having up to 10 amino acid substitutions, additions or deletions. (Item 27) (a) has an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:2; or (b) The fusion protein according to any one of Items 20 to 26, having an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3. (Item 28) From N-terminus to C-terminus, the structure: D1-D2 or D2-D1 (In the formula, D1 is a VHH, such as a single domain antibody, and D2 is an FH fragment such as FH1-5) A fusion protein comprising: (Item 29) 29. The fusion protein of item 28, having a C-terminal His tag. (Item 30) (a) has the amino acid sequence of SEQ ID NO: 1 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) The fusion protein according to item 28 or 29, having the amino acid sequence of SEQ ID NO: 7 or a variant having up to 10 amino acid substitutions, additions or deletions. (Item 31) (a) has an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:1; or (b) The fusion protein according to item 28 or 29, having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 7. (Item 32) (a) has the amino acid sequence of SEQ ID NO: 11 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) The fusion protein according to item 28 or 29, having the amino acid sequence of SEQ ID NO: 12 or a variant having up to 10 amino acid substitutions, additions or deletions. (Item 33) (a) has an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 11; or (b) the fusion protein according to item 28 or 29, having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 12. (Item 34) 34. The fusion protein of any one of items 1 to 33, having an increased intrarenal residence time compared to the fusion protein lacking the VHH domain. (Item 35) 35. A pharmaceutical composition comprising the fusion protein according to any one of items 1 to 34 and a pharmaceutically acceptable carrier. (Item 36) A polynucleotide encoding the fusion protein according to any one of items 1 to 35. (Item 37) 37. A vector comprising the polynucleotide of Item 36. (Item 38) 38. A host cell comprising the polynucleotide of item 36 or the vector of item 37. (Item 39) 35. A method for producing a fusion protein according to any one of items 1 to 34, comprising culturing one or more host cells comprising one or more nucleic acid molecules capable of expressing the fusion protein under conditions suitable for expression of the fusion protein. (Item 40) 40. The method of claim 39, further comprising obtaining the fusion protein from the cell culture or culture medium. (Item 41) A method for treating a disease mediated by alternative complement pathway activation or dysregulation, comprising administering to a subject in need thereof an effective amount of a composition comprising the fusion protein of any one of Items 1 to 34, the pharmaceutical composition of Item 35, the polynucleotide of Item 36, the vector of Item 37, or the host cell of Item 38. (Item 42) 42. The method of claim 41, wherein the fusion protein is formulated as a pharmaceutical composition together with at least one pharmaceutically acceptable carrier. (Item 43) 43. The method of claim 42, wherein the composition is lyophilized. (Item 44) 44. The method of claim 43, wherein the composition is rehydrated prior to administration. (Item 45) Item 43. The method of item 42, wherein the at least one pharmaceutically acceptable carrier is saline. (Item 46) 46. The method of any one of items 41 to 45, wherein the composition is formulated for daily, weekly or monthly administration. (Item 47) 47. The method according to any one of items 41 to 46, wherein the composition is formulated for intravenous, subcutaneous, intramuscular, oral, nasal, sublingual, intrathecal and intradermal administration. (Item 48) 48. The method of any one of items 41 to 47, wherein the composition is formulated for administration at a dosage of about 0.1 mg / kg to about 150 mg / kg. (Item 49) 49. The method of any one of items 41 to 48, wherein the composition is formulated for administration in combination with an additional therapeutic agent. (Item 50) 50. The method according to any one of Items 41 to 49, wherein the disease is a renal disorder such as focal glomerulosclerosis (FSGS), IgA nephropathy, minimal change disease (MCD), diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3 nephropathy (C3G), dense deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, atypical hemolytic uremic syndrome (aHUS), ischemia-reperfusion injury, or rejection of a transplanted organ such as a kidney. (Item 51) 51. The method according to any one of items 41 to 50, wherein the disease is FSGS. (Item 52) 52. The method according to any one of items 41 to 51, wherein the subject is a mammal. (Item 53) 53. The method of claim 52, wherein the mammal is a human. (Item 54) A kit comprising a composition selected from the fusion protein according to any one of Items 1 to 34, the pharmaceutical composition according to Item 35, the polynucleotide according to Item 36, the vector according to Item 37, or the host cell according to Item 38. (Item 55) 55. The kit of item 54, further comprising instructions for administering an effective amount of the composition to a subject in need thereof. (Item 56) 35. Use of a composition comprising the fusion protein of any one of items 1 to 34 for the manufacture of a medicament for a disease mediated by alternative complement pathway activation or dysregulation. (Item 57) 57. The use according to Item 56, wherein the disease is a renal disorder such as FSGS, IgA nephropathy, MCD, diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture's syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3G, dense deposit disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury or rejection of a transplanted organ such as a kidney.

Claims

[Claim 1] The invention described in this specification.

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