Renal Activating Fusion Protein and Therapeutic Method Using the Same

Abstract

Described herein is a fusion protein that includes a factor H catalytic domain and may include a VHH domain, a factor H-related protein 5 domain, or an integrin binding domain, and the use of such a fusion protein in a method for treating a disease mediated by alternative complement pathway activation or dysregulation, such as a renal disease. The fusion protein may be used to treat a patient with a disease associated with alternative complement pathway activation or dysregulation, such as a renal disease. Provided herein is a fusion protein having the structure, from N-terminus to C-terminus, D1-L1-D2-L2-D3, where D1 includes a fragment of complement factor H (FH); L1 is absent, covalently linked, or an amino acid sequence of at least one amino acid; D2 includes 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.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, prepared on 16 May 2022, is named 50694-083WO2_Sequence_Listing_5_16_22_ST25 and has a size of 140,215 bytes. [Background technology]

[0002] The complement system plays a central role in the elimination of immune complexes and in immune responses to 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 accessory pathway. The accessory pathway of complement is in a constant state of low-level activation. Unregulated activation or inadequate regulation of the accessory pathway of complement (CAP) can lead to inflammation, cytotoxicity, and tissue damage. Local accessory pathway activation within the kidney is a contributing factor to renal pathology and loss of function. Therefore, the accessory pathway of complement is involved in the pathogenesis of many kidney diseases. Inhibition or regulation of accessory pathway activity in the absence of lectin and classical pathway initiation is recognized as a promising therapeutic strategy. For example, the accessory pathway plays a role in amplifying complement activation initiated from all three pathways. The number of available therapeutic options for diseases associated with accessory pathway of complement is limited. Therefore, developing innovative strategies to treat diseases associated with activation or dysregulation of accessory pathways of complement, such as kidney diseases affecting an estimated 37 million people in the United States alone, is a critical unmet need. [Overview of the project] [Means for solving the problem]

[0003] This specification discloses fusion polypeptides comprising an H factor catalytic domain. The fusion proteins may be used to treat patients with diseases associated with complement accessory pathway activation or dysregulation, such as renal disease.

[0004] This specification provides fusion proteins having a D1-L1-D2-L2-D3 structure from the N-terminus to the C-terminus, where D1 comprises a fragment of complement factor H (FH); L1 is absent, covalently bonded, or an amino acid sequence of at least one amino acid; D2 comprises or is absent VHH; L2 is absent, covalently bonded, 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, and optionally, one or more SCR domains are selected from the group consisting of SCR 1, 2, 3, 4, 5, 6, 19, and 20. In some embodiments, the FH SCR domains are selected from the group consisting of SCR 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 embodiment, the VHH of D2 comprises a single-domain antibody of a camel. In one embodiment, the integrin-recognizing domain of D3 comprises an integrin-recognizing domain containing an arginylglycylaspartate (RGD) peptide motif. In another embodiment, the integrin-recognizing domain of D3 comprises a cyclo(RGD)4 peptide motif.

[0006] In one embodiment, L1 and L2 have the same amino acid sequence. In another embodiment, L1 and L2 have different amino acid sequences. In some embodiments, L1 and / or L2 are (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGGAGGGGAGGGGS, GGGGSGGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GTSSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G12 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, 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 contains FH SCR domains 1-5; L1 contains G4A; D2 is absent; L2 is absent; and D3 contains cyclo(RGD)4; D1 contains FH SCR domains 1-5; L1 is absent; D2 contains VHH; L2 contains G4A; and D3 contains cyclo(RGD)4; D1 contains FH SCR domains 1-5; L1 contains G4A; D2 is absent; L2 contains G4A; and D3 contains cyclo(RGD)4; D1 contains FH SCR domains 1-5; L1 is absent; D2 contains VHH; L2 contains G4A; and D3 contains cyclo(RGD)4; D1 contains FH Includes SCR domains 1-5; L1 is absent; D2 includes VHH; L2 includes G4A; and D3 includes cyclo(RGD)4 or; D1 includes FH SCR domains 1-6; L1 is absent; D2 includes VHH; L2 includes G4A; and D3 includes cyclo(RGD)4 or; D1 includes FH SCR domains 1-5; L1 includes G4A; D2 includes VHH; L2 includes G4A; and D3 includes 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., 1, 2, 3, 4, 5, 6, 7, 8, and up to 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., 1, 2, 3, 4, 5, 6, 7, 8, and up to 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., 1, 2, 3, 4, 5, 6, 7, 8, and up to 9 amino acids); or the amino acid sequence of SEQ ID NO: 9 or up to 10 amino acids (e.g., 1 It has a variant having substitutions, additions, or deletions of up to 2, 3, 4, 5, 6, 7, 8, and 9 amino acids; it has the amino acid sequence of SEQ ID NO: 13 or a variant having substitutions, additions, or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); or it has the amino acid sequence of SEQ ID NO: 14 or a variant having substitutions, additions, or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and 9 amino acids); it has the amino acid sequence of SEQ ID NO: 15 or a variant having substitutions, additions, or deletions of up to 10 amino acids (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 with SEQ ID NO: 4; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 5; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 8; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 9; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 13; an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 14; and an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 15.

[0010] In another embodiment, the Disclosure provides a fusion protein comprising a D1-L1-D2 structure from the N-terminus to the C-terminus, wherein D1 comprises FH fragments, e.g., FH1-5; L1 comprises a linker, or is absent; and D2 comprises H factor-associated protein 5 (FHRP5) domains, e.g., FHRP domains 7 and 8. In one embodiment, L1 is G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGGAGGGGAGGGGS, GGGGSGGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GTSSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12Selected 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, for example, 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., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10); 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., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10). 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 with SEQ ID NO: 6; or an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 10.

[0012] In another embodiment, the disclosure provides a fusion protein comprising a D1-L1-D2-L2-D3 structure from the N-terminus to the C-terminus, where D1 comprises an integrin-recognizing 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 have the same amino acid sequence. In another embodiment, L1 and L2 have different amino acid sequences. In some embodiments, L1 and / or L2 are G4A, (G4A)3, (G4A)2G3AG4S, G4SDAA, (G4A)2G4S, G4AG3AG4S, GGGGAGGGGAGGGGS, GGGGSGGGGSGGGGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (EAAAK)3, PAPAP, G4SPAPAP, PAPAPG4S, GTSSGKSSEGKG, (GGGDS)2, (GGGES)2, GGGDSGGGGS, GGGASGGGGS, GGGESGGGGS, ASTKGP, ASTKGPSVFPLAP, G3P, G7P, PAPNLLGGP, G6, G 12 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, for example, 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 acids (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, and 9) substituted, added, or deleted; or the amino acid sequence of SEQ ID NO: 3 or a variant having up to 10 amino acids (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, and 9) substituted, added, or deleted. 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 with SEQ ID NO: 2; or an amino acid sequence having at least 85% (e.g., at least 90%, 95%, and 99%) sequence identity with SEQ ID NO: 3.

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

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

[0020] In another aspect, the present disclosure provides a method for 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 present disclosure provides a method for treating a disease mediated by complement alternative pathway activation or dysregulation, the method comprising administering to a subject in need thereof a composition comprising an effective amount of any one of the fusion proteins described herein, the pharmaceutical compositions described herein, the polynucleotides described herein, the vectors described herein or the 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 carrier is saline. In some embodiments, the composition is formulated for daily, weekly or monthly administration.

[0022] In some embodiments, the composition is formulated for intravenous, subcutaneous, intramuscular, oral, nasal, sublingual, intrathecal, and intradermal administration. In some embodiments, the composition is formulated for administration in doses of about 0.1 mg / kg to about 150 mg / kg (e.g., about 0.5 to 150 mg / kg, 1 to 150 mg / kg, 10 to 150 mg / kg, 25 to 150 mg / kg, 50 to 150 mg / kg, 100 to 150 mg / kg, 125 to 150 mg / kg, 0.1 to 125 mg / kg, 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, 0.1 to 25 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, and 0.1 to 1 mg / kg). In some embodiments, the composition is formulated for administration in combination with additional therapeutic agents.

[0023] In some embodiments, diseases mediated by complement accessory pathway activation or dysregulation include renal impairment, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, minimal change disease (MCD), diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3 nephropathy (C3G), high-density deposition disease, membranoproliferative glomerulonephritis, glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, atypical hemolytic uremic syndrome (aHUS), ischemia-reperfusion injury, or rejection of transplanted organs such as kidneys. In some embodiments, the subjects are mammals. In some embodiments, the mammals are humans.

[0024] In another embodiment, the Disclosure provides a kit comprising a composition selected from one of the fusion proteins described herein, the pharmaceutical compositions described herein, the polynucleotides described herein, the vectors described herein, or the host cells described herein. In some embodiments, the kit further includes instructions for administering an effective amount of the composition to a subject requiring it. [Brief explanation of the drawing]

[0025] [Figure 1]This is a schematic diagram showing complement factor H (FH) fusion proteins of formulas I and III containing an integrin recognition domain (Figure 1A), and a factor H fusion protein of formula II containing a fragment of FHRP5 (Figure 1B). [Figure 2A] This graph shows the assay results for comparative inhibition of CAP-mediated hemolysis by compound A and H factors SCR1-5. [Figure 2B] This graph shows the assay results for the comparative inhibition of CAP-mediated hemolysis by compounds D, H, E, and I. [Figure 2C] This graph shows the assay results for comparative inhibition of CAP-mediated hemolysis by compounds E and I, and reference protein 6, an anti-HSA H factor-VHH fusion protein used as a positive control. [Figure 2D] This graph shows the assay results for the comparative inhibition of CAP-mediated hemolysis by compounds E, M, N, and O. [Figure 3] This is a set of in vivo whole-body and kidney images of wild-type mice treated with compound B. The images were prepared using a LI-COR Odyssey microscope. [Figure 4] This graph shows the serum levels of the fusion protein in ng / mL 1 hour and 24 hours after administration to wild-type mice. [Figure 5] This graph shows the proteinuria levels in wild-type Balb / c mice in an FSGS-induced adriamycin nephropathy model, after intravenous administration of the fusion protein on day 0 and subcutaneous administration on days 7, 9, 11, and 13. Data are shown as the mean + or - of the standard error of the mean (n=4~9). Statistical significance compared to the vehicle is indicated by *p<0.05 and ***p<0.001, and statistical significance compared to the vehicle and adriamycin is indicated by †p<0.5. [Figure 6]This graph shows albuminuria levels in a wild-type Balb / c mouse model of FSGS (Adriamycin Nephropathy) after intravenous administration of the fusion protein on day 0 and subcutaneous administration on days 7, 9, 11, and 13. Data are shown as the mean + or - of the standard error of the mean (n=4~9). Statistical significance compared to the vehicle is indicated by *p<0.05 and ***p<0.001, and statistical significance compared to the vehicle and Adriamycin is indicated by †p<0.5. [Figure 7] This graph shows blinded tubular protein scoring of Masson trichrome-stained Adriamycin-induced nephropathy mouse kidneys, assigned scores from 1 to 5 according to an accepted method. N=8-10. Statistical significance compared to the vehicle using multiple t-tests is indicated by **p<0.01**, and results using ANOVA with multiple comparisons are indicated by #p<0.5. [Figure 8A] This is a set of images showing exemplary immunofluorescence evaluations of kidney sections for C3 deposition in a model of adriamycin nephropathy in wild-type Balb / c mice 7 days after administration of compound E. Other molecules gave similar results or were closer to vehicle-negative controls. [Figure 8B] Figure 9 is a graph showing the average pixel intensity of the results, where the C3 pixel average intensity value represents the average signal intensity within the selected target region / renal medulla on day 14 (day 7 post-treatment). [Figure 9A] This graph shows the urinary protein / creatinine ratio in male Balb / c mice with adriamycin-induced renal disease after treatment with a fusion protein, administered intravenously on day 0 and subcutaneously on days 7, 9, 11, and 13. Data are shown as the mean + or - of the standard error of the mean (n=4-9). Statistical significance compared to the vehicle is indicated by ***p<0.001. No statistically significant differences were observed compared to the vehicle and adriamycin. A positive trend was observed. [Figure 9B]This graph shows the urinary albumin / creatinine ratio after treatment with the fusion protein, administered intravenously on day 0 and subcutaneously on days 7, 9, 11, and 13. Data are shown as the mean + or - of the standard error of the mean (n=4-9). Statistical significance compared to the vehicle is indicated by ***p<0.001. There was no statistically significant difference compared to the vehicle and adriamycin. [Figure 10A] This is a Western blot showing the SDS-PAGE gels of purified compounds D and E. [Figure 10B] This graph shows the hydrophobic interaction chromatogram of compound E. [Figure 11] This graph shows the mass spectrometry results for compound E, indicating that its molecular weight is approximately 50 kDa. A slight peak at +162 Da is observed, which is likely due to glycation. [Figure 12] This graph shows the melting curve of compound E using dynamic light scattering. [Figure 13A] This graph shows the retention time of compound E at 37°C for day 0, measured using size exclusion chromatography to determine the relative stability of the compound. [Figure 13B] This graph shows the retention time of compound E after 14 days at 37°C, measured using size exclusion chromatography to determine the relative stability of the compound. [Figure 14A] This graph shows the retention time of compound E after 0 days at 37°C, measured using hydrophobic interaction chromatography to determine the relative stability of the compound. [Figure 14B] This graph shows the retention time of compound E after 14 days at 37°C, measured using hydrophobic interaction chromatography to determine the relative stability of the compound. [Figure 15A] This graph shows the alignment time of the non-reduced compound E after 0 and 14 days at 37°C, using capillary electrophoresis-SDS chromatography to measure the relative stability of the compound. [Figure 15B]This graph shows 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 compound. [Figure 16] This graph shows the chromatogram signature of compound E obtained using isoelectric focusing capillary electrophoresis (iCE). [Figure 17] This graph shows the mass spectra of compound E measured at 37°C after 0, 3, 7, and 14 days to characterize the compound's stability at room temperature. [Figure 18A] This graph shows the binding curves of compounds E and K to C3b, compared to the H factor (fH), over a period of 0 to 2500 seconds. [Figure 18B] This graph shows an enlarged view of the coupling curve in Figure 18A at 40 seconds, and the time point t=0 seconds in Figure 18B corresponds to the time point t=720 seconds in Figure 18A. [Figure 19] This graph shows the assay results of comparative inhibition of liquid-phase CAP activation by compound E in the Complement system Alternative Pathway WIESLAB® across two lots of normal human serum (NHS). [Figure 20] This graph shows the combined single-dose serum pharmacokinetic (PK) data of compound E after subcutaneous (SC) administration to wild-type male C57Bl / 6 mice across two separate studies. [Figure 21A] This graph shows the serum pharmacokinetics of compound E after intravenous (IV) or seroconcentrated (SC) administration to female cynomolgus monkeys. Each graph includes comparative PK profiles across a series of dose levels after both the initial dose given on day 0 of the study and the fourth dose administered on day 12 of the study. [Figure 21B] Includes data from Figure 21A, replotted to compare equivalent dose levels given via IV or SC administration routes. [Modes for carrying out the invention]

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

[0027] As used herein, "administering" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. The fusion protein can be administered by any method known to those of skill in the art. Suitable methods for administering the fusion protein can include, for example, oral, injection (e.g., intravenous, intraperitoneal, intramuscular, intravitreal, and subcutaneous), infusion, inhalation, intranasal, and the like. In some embodiments, administration is via intravenous and / or subcutaneous infusion. The fusion proteins prepared as described herein can be administered in a variety of forms depending on the disorder being treated and the age, condition, and weight of the subject, as is known in the art. The formulation 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 non-covalent 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 that reflects the specific interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be represented 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 readily from their binding partners, while high-affinity complexes generally contain molecules that tend to remain bound to their binding partners for a longer duration. "Specifically binds" means at least 1×10 -6 M or less (e.g., 1×10 -6 M to 1×10 -12 M range, e.g., 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, and 1×10 -12This refers to molecules and binding partner pairs that possess Kd (M).

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

[0030] As used herein, the term “complemental pathway” refers to one of the three pathways of complement activation (the other being the classical pathway and the lectin pathway).

[0031] As used herein, the term “complement accessory pathway activation or dysregulation” refers to any abnormality in the complement accessory pathway’s ability to provide host defense against pathogens and to eliminate immune complexes and damaged cells for immunomodulation. Complement accessory pathway activation or dysregulation can occur in the fluid phase and on the cell surface. Complement accessory pathway activation or dysregulation can result in excessive complement activation or insufficient complement regulation, both of which can cause tissue damage.

[0032] As used herein, the term “disease” means an interruption, cessation, or impairment of a bodily function, system, or organ. Diseases or disorders of interest include those that would benefit from treatment with fusion proteins or treatment by the methods described herein. Non-exclusive examples of diseases or disorders treated herein are those mediated by complement accessory pathway activation or dysregulation, including, but not limited to, renal impairment, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, minimal change disease (MCD), diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3 nephropathy (C3G), high-density 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. In some embodiments, the disease is FSGS.

[0033] As used herein, “Factor H” refers to the protein component of the complement subpathway encoded by the complement factor H gene (“FH;” NM000186; GeneID: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 processed by the removal of an 18-amino acid signal peptide, resulting in the mature factor H protein (amino acids 19-1231). Factor H consists of 20 short complement regulator (SCR) domains. Amino acids 1-18 contain the signal peptide, residues 21-80 contain SCR1 (SEQ ID NO: 24), residues 85-141 contain SCR2 (SEQ ID NO: 25), residues 146-205 contain SCR3 (SEQ ID NO: 26), residues 201-262 contain SCR4 (SEQ ID NO: 27), residues 267-320 contain SCR5 (SEQ ID NO: 28), and residues 326-384 contain 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 disintegration-promoting activity for the secondary 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 formation of enzyme complexes that amplify the complement cascade. On the cell surface, C3b is rapidly converted to inactive iC3b when deposited on a host surface (i.e., most host tissues) containing complement activation regulators. Even in the absence of membrane-bound complement regulators, considerable levels of iC3b are formed due to the action of serum factor H and serum factor I. iC3b is then digested into the membrane-bound fragment C3dg, and subsequently digested into C3d by factor I and other proteases and cofactors, although this process is relatively slow.

[0035] As used herein, the terms “Factor H-related protein 5” or “FHRP5” refer to the protein component of the complement subpathway 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, regions within SCR5-7 have heparin-binding and C-reactive protein-binding properties, and the two C-terminal SCRs are analogous to the complement component 3b (C3b) binding domain. FHRP5 co-localizes with C3 and binds to C3b in a dose-dependent manner, and is recruited to tissue damaged by C-reactive proteins.

[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.), but containing, for example, 5, 10, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350 or more amino acids. The fragment may be long enough to maintain the desired function of the full-length protein. For example, regulation of a complement sub-pathway in the liquid phase by a fragment (e.g., a fragment of factor H) is maintained. Such a fragment is a “biologically active fragment.”

[0037] As used herein, “functional fragment” or “biologically active fragment” refers to a fragment or portion of a protein that has some or all of the activity of a full-length protein. For example, a functional or biologically active fragment of factor H refers to any fragment of the factor H protein that has some or all of the activity of factor H, e.g., some or all of the complement accessory pathway regulatory activity of the full-length factor H protein. Examples include, but are not limited to, N-terminal 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 “biologically active fragment” of the FHRP5 protein is one that has some or all of the activity of FHRP5, e.g., some or all of the complement accessory pathway regulatory activity of the full-length FHRP5 protein. Examples include, but are not limited to, N-terminal to C-terminal FHRP5 fragments containing SCR:[7-8]. As used herein, the terms “fused” or “joined” refer to the combination or attachment of two or more elements, components, or protein domains, such as polypeptides, by means of chemical conjugation, recombination, and chemical bonding, such as disulfide bonds and amide bonds. For example, two single polypeptides can be joined to form a single continuous protein structure by means of recombinant expression, chemical conjugation, chemical bonding, peptide linkers, or any other covalent bonding.

[0038] As used herein, the term “fusion protein” refers to a complex polypeptide composed of two or more different heterologous polypeptides. These heterologous polypeptides may be full-length proteins or fragments of full-length proteins. The fusion proteins described herein may 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 manipulated 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 transformant 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 arginylglycylaspartate moieties, e.g., (RGD) 1-4 (Sequence number 21) and other (RGD) 1-8 This refers to [the specified part]. In some embodiments, the arginylglycylaspartate moiety can be cyclized.

[0041] As used herein, the term “intrarenal residence time” refers to the period during which compounds such as compounds A-O described herein are present in the extravascular compartment, e.g., along the renal epithelium or within Bowman’s dorsal pouch within the kidney. Intrarenal residence time can be measured using longitudinal in vivo imaging. For example, in animal studies, the 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 automated 2D epi-illumination exposure settings, field of view (FOV) C, F / Stop 2, moderate binning, and an 800nm ​​emission / 750nm excitation filter, where the subject receives, for example, 1 mg / kg of AlexaFluor 750-labeled test material via intravenous injection. In clinical settings, longitudinal in vivo imaging can be achieved, for example, using radiolabeled test material 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 may be a covalent bond. A linker may also be a molecule of any length that can be used to link, for example, H factor fragments and / or VHH and / or integrin recognition motifs. A linker may also refer to a portion (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence, 1-150 amino acid sequence, 1-100 amino acid sequence, 5-50 amino acid sequence, or 1-10 amino acid sequence, e.g., amino acids with smaller side chains and / or flexible amino acid sequences) that exists between two polypeptides or polypeptide domains to provide space and / or flexibility between them. An amino acid linker may be part of the primary sequence of a polypeptide (e.g., linked to polypeptides or polypeptide domains linked via a polypeptide backbone). Non-restrictive examples include (G4A)2G4S, G4A, (G4A)3, and (G4A)2G3AG4S (Sequence IDs 32, 80, 81, and 30).

[0043] As used herein, the terms “patient in need of it” or “subject in need of it” refer 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). A subject may be identified as having a need for treatment of a disease or disorder (e.g., renal impairment, 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, high-density deposition 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 the administration of treatment. In some embodiments, the disease is FSGS, and the need for treatment is based on an early diagnosis by a person skilled in the art (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. These terms also include amino acid polymers modified, in particular, by disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with labeling components.

[0045] The “sequence identity percentage (%)” for 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 the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after the sequences have been aligned and, if necessary, gaps have been introduced to achieve the maximum possible sequence identity percentage. Alignment for the purpose of determining the nucleic acid or amino acid sequence identity percentage can be achieved in various ways within the capabilities of a person skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum possible alignment over the entire length of the sequences being compared. For example, a sequence identity percentage value can be generated using the sequence comparison computer program BLAST. As an example, the sequence identity percentage of a given nucleic acid or amino acid sequence A to or with 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 specific sequence identity percentage to or with a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) In the formula, X is the number of nucleotides or amino acids scored as identical in the alignment of A and B by a sequence alignment program (e.g., BLAST), 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, the sequence identity percentage of A to B will not be equal to the sequence identity percentage of B to A.

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

[0047] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition and / or dosage form suitable for contact with the tissues of a subject, such as a mammal (e.g., human), without excessive toxicity, irritation, allergic reactions and other problematic complications commensurate with a reasonable benefit / risk ratio.

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

[0049] As used herein, the terms “short consensus repeat,” “sushi domain,” or “complement regulatory protein,” or “CCP,” “short complement regulator,” or “SCR,” describe domains found in all regulatory factors (RCAs) of the complement activation gene cluster that contribute to their ability to regulate complement activation in the blood or on the cell surface to which they specifically bind. SCRs typically consist of about 60 amino acids, with four cysteine ​​disulfide residues linked in 1-3 and 2-4 sequences, and a hydrophobic core constructed around a nearly immutable tryptophan residue. SCRs are found in proteins including, but are not limited to, factor H and FHRP5.

[0050] As used herein, the terms “single-domain antibody” and “VHH” define molecules formed by a single immunoglobulin domain. A single-domain antibody includes an antibody whose complementarity-determining region (“CDR”) is part of a single-domain polypeptide. A single-domain antibody often includes an antibody or its antigen-binding fragment that specifically binds to a single antigen (for example, a VHH antibody may have a capacity of 1 × 10⁻⁶). -6 K below M D For example, 1 × 10 -6 M~1×10 -12 K in the range of M D For example, 1 × 10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M and 1×10 -12 M's K D(It binds to the antigen). Generally, the antigen-binding site of an immunoglobulin monovariate domain is formed by three or fewer CDRs. The monovariate domain may include, for example, 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 VHH sequence) or a suitable fragment thereof. Such antibodies may be derived from antibodies produced in, for example, camels, dromedary camels, llamas, alpacas, or guanacos, for example, in species of the Camelidae family. Further antibodies include, for example, immunoglobulin neoantigen receptors (IgNARs) of cartilaginous fish (e.g., sharks, e.g., nurse sharks). Other species other than Camelidae and cartilaginous fish may produce antibodies in which their CDRs are part of a monopolypeptide. Antibodies can 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., that is a recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein with respect to human subjects.

[0052] "Therapeutic dose" means the amount of composition administered to improve, inhibit, or alleviate the symptoms of a disorder or disease in a clinically relevant manner or condition of the subject. Any improvement in the subject is considered sufficient to achieve treatment. In some embodiments, a therapeutic dose is an amount that reduces, inhibits, or prevents the onset of a disease or disorder (e.g., a disease or disorder mediated by complement accessory pathway activation or dysregulation) or one or more of its symptoms, or an amount that reduces the severity of any disease or disorder mediated by CAP activation or dysregulation, or the duration of time the subject suffers from one or more of its symptoms (e.g., at least about 10%, about 20%, or about 30% compared to a control subject not treated with the composition described herein, e.g., at least about 50%, about 60%, or about 70%, and e.g., at least about 80%, about 90%, about 95%, about 99%, or more). The therapeutic dose of the pharmaceutical composition used to carry out the methods described herein (e.g., treatment of renal disease) may vary depending on the mode of administration and the age, weight, and overall health of the subject being treated. A physician or researcher can determine the appropriate dosage and administration plan. Dosages vary and can be administered in one or more doses, once daily, once weekly, once monthly, or once a year, or over several days.

[0053] As used herein, the terms “treatment,” “treating,” or “to treat” refer to therapeutic actions whose purpose is to inhibit or reduce undesirable physiological changes or impairments or to promote beneficial phenotypes in a patient. For example, “treatment,” “treating,” or “to treat” refer to clinical interventions in an attempt to alter the natural course of an individual’s suffering, disease, or disability. These terms include, for example, prevention before or during the course of a clinicopathological condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing of disease progression, remission or mitigation of the disease state, and improvement of prognosis. In some embodiments, the fusion protein is used to control the cellular and clinical manifestations of renal impairment, 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney. In some embodiments, the disease is FSGS.

[0054] A “mutant” refers to a polynucleotide or polypeptide that is substantially homologous to a natural or reference polynucleotide or polypeptide. For example, a mutant polynucleotide is substantially homologous to a natural or reference polynucleotide but has a different polynucleotide sequence due to one or more deletions, insertions, and / or substitutions. In another example, a mutant polypeptide is substantially homologous to a natural or reference polypeptide but has a different amino acid sequence due to one or more deletions, insertions, and / or substitutions. A mutant polypeptide sequence encoding a polynucleotide sequence includes sequences that encode an active mutant protein or fragment thereof, and which involve one or more additions, deletions, or substitutions of nucleotides compared to the natural or reference polynucleotide sequence. A wide variety of mutagenesis approaches are known in the art and can be applied by those skilled in the art. A mutant polynucleotide or polypeptide sequence may be identical to the natural or reference sequence by 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. The degree of homology (percent identity) between the natural and mutant sequences can be determined, for example, by comparing the two sequences using a freely available computer program commonly used on the World Wide Web for this purpose (e.g., BLASTp or BLASTn with default settings).

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

[0056] Complement accessory pathway-specific C3 and C5 convertase inhibitors that modulate complement accessory pathway activity are described herein. A series of low molecular weight complement accessory pathway (CAP) activation and amplification loop inhibitors designed to have a mechanism for binding to renal epithelial cells are described herein. The compositions and methods described herein are characterized by a fusion protein comprising a fragment of complement factor H (FH), a fragment of factor H-related protein 5 (FHRP5), and / or one or more kidney target 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 disintegration-promoting function via C3b binding. Additional factor H SCRs (e.g., SCR5 and SCR6) may be included to increase activity, stability, or structural flexibility. According to the disclosure herein, fusion proteins containing FH catalytic domain SCRs may also include short amino acid sequence motifs or complement molecule domains that recognize integrins or damage markers present on the surface of damaged renal epithelial and tubulointerstitial cells. Fusion proteins containing only single-domain variable heavy chain (VHH) camel antibodies are described herein to enable deposition in renal epithelial cells, improve expression, facilitate purification, and provide an exogenous probe for detection. In summary, the use of these targeting residues, coupled with the intrinsic exclusion 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 complement accessory pathway activation or dysregulation include, for example, renal impairment, 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney. In some embodiments, the disease is FSGS.

[0059] The fusion proteins disclosed herein modulate complement subpathway activity by irreversibly inactivating C3b and attenuating C3 and C5 convertase activity. The constructs target the complement subpathway while preserving activation (protection) via the classical and lectin pathways.

[0060] Fusion protein As described herein, the fusion proteins of this disclosure may comprise a fragment of factor H and may comprise an integrin recognition motif or a fragment of FHRP5. The constructs may be used as therapeutic agents for treating diseases mediated by complement accessory 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 complement activation (RCA) gene cluster. Proteins within the RCA family differ in size but share similarities in their 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 exist in multiple copies within the protein. Each SCR consists of approximately 60-70 highly conserved amino acids, each containing four cysteine ​​residues.

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

[0063] In certain embodiments, the portion of the fusion protein suitable for inhibiting the activity of the complement accessory 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 cofactor and disintegration-promoting 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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, the H factor fragment may contain 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 sequence number 18.

[0065] The fusion protein may include 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 containing 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: 21.

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

[0067] The fusion protein may contain a fragment of factor H in addition to a fragment of factor H-related protein 5 (FHRP5). In some embodiments, the fusion protein may contain a fragment of the FHRP5 domain containing 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 contain a fragment of the FHRP5 domain containing the polypeptide sequence of SEQ ID NO: 22. The fragment of factor H in the fusion protein may contain 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 contain at least the seventh and / or eighth N-terminal SCR domain of FHRP5.

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

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

[0070] In some embodiments, the FHRP5 portion of the fusion protein is a functional fragment of wild-type FHRP5. In some embodiments, the FHRP5 or its fragment portion of the fusion protein composition is derived from substituted (e.g., conservatively substituted) FHRP5 or manipulated FHRP5 (e.g., FHRP5 manipulated to increase protein stability, activity and / or other desirable properties, as determined by predictive models or assays known to those skilled in the art, e.g., the assay 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, and the amino acid substitutions increase the binding affinity of the fragment of factor H, an integrin-binding domain, or a fragment of FHRP5 to 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 may be fused with other compounds, such as compounds that increase the half-life of the polypeptide and / or compounds that reduce the potential immunogenicity of the fusion protein (e.g., polyethylene glycol (PEG)). PEG can be used to improve water solubility, reduce renal clearance rate, and reduce the immunogenicity of the fusion protein (see, for example, U.S. Patent No. 6,214,966, the disclosure of which is incorporated herein by reference). The fusion proteins described herein may be PEGylated by any means known to those skilled in the art.

[0073] H factor fragments can be prepared by many synthetic methods of peptide synthesis involving the 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, the integrin-binding domain, and / or FHRP5 can be produced by expression in a suitable prokaryotic or eukaryotic system. In some embodiments, the DNA construct can be inserted into a plasmid vector suitable for expression in a suitable host cell (e.g., Escherichia 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 cells (HEK) (e.g., HEK 293), Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, or COS-7 cells. A suitable expression vector contains the necessary and sufficient regulatory elements for DNA expression in the host cell. In some embodiments, a leader or secretion sequence or a sequence used for the purification of the fusion protein (e.g., a histidine tag) may be included in the fusion protein. Fragments of factor H, integrin-binding domains, and / or FHRP5 produced by gene expression in recombinant prokaryotes or eukaryotes can be purified according to methods known in the art (see, for example, Structural Genomics Consortium, Nat. Methods, 5:135-46, 2008).

[0075] In certain embodiments, the cyclized integrin-binding domain and the FHRP5 fragment are also produced by the same method described for the expression and purification of the H factor fragment.

[0076] In some embodiments, the fusion protein has the structure of formula I from the N-terminus to the C-terminus: D1-L1-D2-L2-D3 Equation I During the ceremony, D1 is a fragment of FH (for example, a fragment of FH having any one amino acid sequence from sequence numbers 16-18, or a variant thereof having 85% or more sequence identity); L1 is either absent (for example, L1 is a covalent bond between D1 and D2 or between D1 and D3) or a linker having an amino acid sequence of at least one amino acid between D1 and D2 or between D1 and D3 (for example, the linker may have one of the amino acid sequences of SEQ ID NOs. 30-122 or a variant thereof with 85% or more sequence identity); D2 is either absent (for example, D2 is a covalent bond between L1 and D3, between D1 and L2, or between L1 and L2), or it is a VHH domain, such as a single-domain antibody (for example, a camel single-domain antibody VHH having any one amino acid sequence of SEQ ID NOs. 19-20 and 23, or a variant thereof having 85% or more sequence identity); L2 is either absent (for example, L2 is a covalent bond between D2 and D3) or a linker having an amino acid sequence of at least one amino acid between D2 and D3 (for example, the linker may have one of the amino acid sequences of SEQ ID NOs. 30-122 or a variant thereof with 85% or more sequence identity); D3 is an integrin recognition domain (for example, an arginylglycylaspartate (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).

[0077] In some embodiments, the FH fragment of D1 comprises one or more FH SCR domains, and optionally, one or more SCR domains are selected from the group consisting of variants thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity with any one of SCR 1, 2, 3, 4, 5, or 6 or SEQ ID NOs: 24-29. In some embodiments, the FH SCR domain is selected from the group consisting of variants thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity with any one of SCR[1-5] or SEQ ID NOs: 16 or 17, or variants thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity with any one of SCR[1-6] or SEQ ID NOs: 18.

[0078] L1 and L2 may be linkers of the same type and / or array, or different types and / or arrays.

[0079] In some embodiments, the composition of formula I comprises one amino acid sequence of sequence numbers 4, 5, 8, 9 and 13-15 and a variant 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 one nucleic acid sequence of sequence numbers 128, 129, 132, 133 and 137-139 and a variant thereof having at least 85%, 87%, 90%, 95%, 97%, or 99% sequence identity thereto.

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

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

[0082] In some embodiments, the composition of formula II comprises either sequence number 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 sequence number 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 of formula III from the N-terminus to the C-terminus: D1-L1-D2-L2-D3 Equation III During the ceremony, D1 is an integrin recognition domain (for example, an arginylglycylaspartate (RGD) peptide motif, e.g., cyclo(RGD)4 of SEQ ID NO: 21 or a variant thereof having 85% or more sequence identity); L1 is either absent (for example, L1 is a covalent bond between D1 and D2 or between D1 and D3) or a linker having an amino acid sequence of at least one amino acid between D1 and D2 or between D1 and D3 (for example, the linker may have one of the amino acid sequences of SEQ ID NOs. 30-122 or a variant thereof with 85% or more sequence identity); D2 is either absent (for example, D2 is a covalent bond between L1 and D3, between D1 and L2, or between L1 and L2), or it is a VHH domain, such as a single-domain antibody (for example, a camel single-domain antibody VHH having any one amino acid sequence of SEQ ID NOs. 19-20 and 23, or a variant thereof having 85% or more sequence identity); L2 is either absent between D2 and D3 (for example, L2 is a covalent bond between D2 and D3) or is a linker having the amino acid sequence of at least one amino acid (for example, the linker may have any one amino acid sequence from SEQ ID NOs. 30 to 122 or a variant thereof having 85% or more sequence identity); and D3 is a fragment of FH (for example, a fragment of FH having one of the amino acid sequences of sequence numbers 16-18, or a variant thereof having 85% or more sequence identity).

[0084] In some embodiments, the FH fragment of D3 comprises one or more FH SCR domains, and optionally, one or more SCR domains are selected from the group consisting of variants thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity with any one of SCR 1, 2, 3, 4, 5, or 6 or SEQ ID NOs: 24-29. In some embodiments, the FH SCR domain is selected from the group consisting of variants thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity with any one of SCR[1-5] or SEQ ID NOs: 16 or 17, or variants thereof having at least 85% (e.g., 87%, 90%, 95%, 97%, or 99%) sequence identity with any one of SCR[1-6] or SEQ ID NOs: 18.

[0085] L1 and L2 may be linkers of the same type and / or array, or different types and / or arrays.

[0086] In some embodiments, the composition of formula III comprises either sequence number 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 either sequence number 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 of formula IV from the N-terminus to the C-terminus: D1-D2 or D2-D1 Formula IV During the ceremony, D1 is a VHH domain, for example, a single-domain antibody (e.g., a camel single-domain antibody VHH having one of the amino acid sequences of SEQ ID NOs. 19-20 and 23, or a variant thereof having 85% or more sequence identity); D2 is a fragment of FH (for example, a fragment of either FH sequence number 16 or 17, or a variant thereof having 85% or more sequence identity).

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

[0089] In some embodiments, the composition of formula IV comprises one of sequence numbers 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 one of sequence numbers 125, 131, 135, and 136 nucleic acid sequence 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 contain a single-chain VHH domain. Such antibodies are naturally present in camels and sharks (Saerens et al., Curr. Opin. Pharmacol., 8:600-608, 2008). Camel 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 full contents of which are incorporated herein by reference.

[0091] An example of a VHH domain is, QVQLVESGGGLVKPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAADLGDGSWVDYVNAEPYEYDYWGQGTLVTVSS (Sequence ID 19), EVQLVESGGGLVKPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAADLGDGSWVDYVNAEPYEYDYWGQGTLVTVSS (Sequence ID 20), or EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCAADLGDGSWVDYVNMEPYEYDYWGQGTQVTVSS(Sequence ID 23) Includes those having the following sequence.

[0092] In some embodiments, the fusion protein may include a VHH domain containing 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, the fusion protein may include a VHH domain containing 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 include a VHH domain containing 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 may contain D1-L1-D2-L2-D3 from the N-terminus to the C-terminus, where D1 contains a fragment of an FH protein, e.g., FH SCR 1-5 or FH SCR 1-6; L1 is absent or contains a linker; D2 contains a VHH domain; L2 is absent or contains a linker; and D3 contains an integrin recognition domain, e.g., cyclo(RGD)4.

[0094] In another example, a fusion protein may contain D1-L1-D2-L2-D3 from the N-terminus to the C-terminus, where D1 contains an integrin recognition domain such as cyclo(RGD)4, L1 is absent or contains a linker, D2 contains a VHH domain, L2 contains or does not contain a linker, and D3 contains a fragment of an FH protein such as FH SCR 1-5.

[0095] In another example, the fusion protein may contain D1-D2 or D2-D1 from the N-terminus to the C-terminus, where D1 contains a VHH domain and D2 contains a fragment of the FH protein. The fusion protein may have a variant thereof that has 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, H factor fusion proteins containing a VHH domain have increased intrarenal residence times along the renal epithelial surface compared to fusion proteins lacking a VHH domain. While not bound by any particular theory, the size of the fusion proteins described herein (e.g., about ≤60 kDa (e.g., <60 kDa)) is thought to allow the fusion proteins to acquire access to extravascular compartments within the kidney that are inaccessible by monoclonal antibody and albumin-binding bispecificity, and the use of the VHH domain in the fusion proteins described herein is thought to allow the fusion proteins to deposit on the apical membrane of proximal tubules and parietal epithelial cells, where naturally occurring low levels of membrane-related surface modulofactors confer sensitivity to CAP products and exhibit extended residence along the renal epithelium. In some embodiments, intrarenal residence times are 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 a VHH domain. In some embodiments, the intrarenal residence time is 24 to 96 hours (e.g., 36 to 96 hours, 48 ​​to 96 hours, 60 to 96 hours, 72 to 96 hours, and 60 to 84 hours).

[0097] Integrin-binding domain The fusion protein may also have an integrin-binding domain that can improve the pharmacokinetics of the fusion protein and act as a targeting motif that mediates renal cell-specific targeting at the site 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-terminus or C-terminus of the fusion protein. The RGD motifs bind 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). While not bound by any particular theory, it is expected that the inclusion of cyclic RGD motifs may restrict pro-TGF-β ligand binding and prevent pro-fibrotic signaling. Different variants of the integrin-binding motifs can be constructed and bound to the fusion protein. In some embodiments, the fusion protein may contain an integrin-binding domain containing an amino 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: 21.

[0099] The fusion protein may contain D1-L1-D2-L2-D3 from the N-terminus to the C-terminus, where D1 contains a fragment of an FH protein such as FH with SCR[1~5] or FH with SCR[1~6]. The fusion protein may have one 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 may also contain a fragment of an FH protein such as FH with SCR[1~5] or FH with SCR[1~6]. The fusion protein may have one 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] Linker of fusion protein The L1 and L2 domains of the fusion proteins described herein are linkers. Linkers are used, for example, to create binding or connections between polypeptide or protein domains. For example, a fragment of factor H may be directly bound to a VHH domain (e.g., a single-domain camel VHH domain) by one or more suitable linkers. Linkers can be any kind of binding produced from simple covalent bonds, e.g., peptide bonds, synthetic polymers, e.g., PEG polymers, or chemical reactions, e.g., chemical conjugations. A peptide linker may be, for example, a linker of one or more amino acid residues inserted into or included in the transition between two domains (e.g., a fragment of an FH protein and a VHH domain). The identity and sequence of amino acid residues in the linker may vary depending on the desired secondary structure. For example, glycine, serine, and alanine are useful as linkers given their flexibility. Depending on the desired length and / or properties, any amino acid residue can be considered as a linker in combination with one or more other amino acid residues that may be the same as or different from the first amino acid residue, in order to construct a larger peptide linker as needed.

[0101] Various linkers can be used to fuse two or more protein domains (e.g., a fragment of factor H and a VHH domain). Linkers may be flexible, rigid, or cleavable. Linkers may be structured or unstructured. Linker residues may be selected from naturally occurring amino acids, non-naturally occurring amino acids, and modified amino acids. Linkers may contain at least one, two, five, ten, fifteen, or twenty amino acid residues. Examples of peptide linkers include, but are not limited to, glycine linkers, glycine-rich linkers, and serine-glycine linkers. Glycine-rich linkers contain at least about 50% glycine.

[0102] In some embodiments, the linker used imparts one or more other desirable properties or functionalities to the polypeptide described herein and / or provides one or more sites for derivative formation and / or functional group attachment. For example, a linker containing one or more charged amino acid residues can provide improved hydrophilicity, while a linker forming or containing a small epitope or tag can be used for detection, identification and / or purification purposes. Those skilled in the art will be able to determine the optimal linker for use in a particular polypeptide.

[0103] When two or more linkers are used in a polypeptide, the linkers may be the same or different.

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

[0105] In another embodiment, the linker comprises the amino acid sequence GGGGA (SEQ ID NO: 80) or a repeat thereof. In a particular embodiment, the linker comprises two or more repeats of GGS or GGGGS (U.S. Patent No. 6,541,219, the full 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 also 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] Examples of linkers include G4S (sequence number 36), (G4A)2G4S (sequence number 34), (G4A)2G3AG4S (sequence number 30), G4AG3AG4S (sequence number 33), G4SDA (sequence number 79), G4SDAA (sequence number 31), G4S (sequence number 36), (G4S)2 (sequence number 37), (G4S)3 (sequence number 35), (G4S)4 (sequence number 39), (G4S)5 (sequence number 40), (G4S)6 (sequence number 41), EAAAK (sequence number 95), (EAAAK)3 (sequence number 42), and PAPAP (sequence number 4 3) G4SPAPAP (SEQ ID NO: 44), PAPAPG4S (SEQ ID NO: 45), GTSSGKSSEGKG (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) nExamples of rigid linkers include, but are not limited to, KESGSVSSEQLAQFRSLD (sequence number 82) and EGKSSGSGSESKST (sequence number 83), (Gly)8 (sequence number 84), GSAGSAAGSGEF (sequence number 87) and (Gly)6 (sequence number 57). Exemplary rigid linkers include A(EAAAK)A (sequence number 86), A(EAAAK) n A (where n can be any number) or (XP) nExamples of in vivo cleavable linkers include, but are not limited to, those shown in the formulas (wherein n can be any number and X represents any amino acid). Examples of in vivo cleavable linkers include, for example, LEAGCKNFFPRSFTSCGSLE (SEQ ID NO: 87), GSST (SEQ ID NO: 88), and CRRRRRREAEAC (SEQ ID NO: 89). In some embodiments, the linker may include 2 to 12 amino acids containing the GS motif, e.g., 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 may include 3 to 12 amino acids containing the GGS motif, e.g., GGS, GGSGGS (SEQ ID NO: 96), GGSGGSGGS (SEQ ID NO: 97), and GGSGGSGGSGGS (SEQ ID NO: 98). In yet another embodiment, the linker may include 4 to 12 amino acids containing the GGSG motif, for example, GGSG (SEQ ID NO: 99), GGSGGGSG (SEQ ID NO: 100), or GGSGGGSGGGSG (SEQ ID NO: 101). In yet another embodiment, the linker may include the GGGGS (SEQ ID NO: 36) motif. In other embodiments, the linker may also include amino acids other than glycine and serine, such as 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), GGGGAGGGGAGGGAGGGGS (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. Including a cleavable linker can facilitate the detection of fusion proteins. Enzymatically cleavable linkers may be cleavable by, for example, trypsin, human rhinovirus 3C protease (3C), enterokinase (Ekt), factor Xa (FXa), tobacco etch virus protease (TEV), or thrombin (Thr). The cleavage sequences of 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 on the carboxyl side of the cleavage site, cleavage does not occur. It has been shown that the rate of hydrolysis is slower when an acidic residue is on either side of the cleavage site. The following linker is an example of a linker that can be cleaved using trypsin: K(G4A)2G3AG4SK (Sequence ID 113) , R(G4A)2G3AG4SR (Sequence ID 114) , K(G4A)2G3AG4SR (Sequence ID 115) , R(G4A)2G3AG4SK (Sequence ID 140) , K(G4A)2G4SK (Sequence ID 117) , K(G4A)2G4SR (Sequence ID 118) , R(G4A)2G4SK (Sequence ID 119) and R(G4A)2G4SR (Sequence ID 120) .

[0108] Examples of protease cleavage sites that can be included in enzymatically cleavable linkers include the tobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS. (Sequence ID 141) The protease cleaves between glutamine and serine. Another example of a protease cleavage site that can be included in an enzymatically cleavable linker is the enterokinase cleavage site, e.g., DDDDK (Sequence ID 121) The cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in an enzymatically cleavable linker is the thrombin cleavage site, e.g., LVPR (Sequence ID 142) Regarding human rhinovirus 3C protease, the cleavage site is LEVLFQGP. (Sequence ID 122)The cleavage occurs between the glutamine and glycine residues. The cleavage site of factor Xa protease is IEDGR. (Sequence ID 143) Therefore, the cleavage occurs between the glutamate residue and the aspartate residue.

[0109] The inclusion of a cleavable linker is useful in that it has a different amino acid sequence from other peptides in the human proteome produced by the enzymes described above. Therefore, this excised linker can function as a unique identifying peptide of 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 polymer or oligomeric glycine linker, which may contain lysine at the N-terminus, C-terminus, or both the N-terminus and C-terminus.

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

[0112] Referring to Equation II above, the C-terminus of D1 may be bonded to the N-terminus of D2. In certain embodiments, the C-terminus of the FH fragment is bonded 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 This specification describes a method for producing fusion proteins using nucleic acid molecules encoding fusion proteins, such as the fusion proteins shown in Tables 1-5. The nucleic acid molecules can be operably bound to appropriate regulatory sequences to form protein-coding expression units. These expression units can be used to transform suitable host cells, which can then be cultured under conditions that allow for the production of recombinant proteins. Optionally, recombinant proteins can be isolated from the culture medium or cells, and protein recovery and purification may not be necessary if some impurities are acceptable. To facilitate purification, additional residues (e.g., histidine tags) may be included at the N-terminus or C-terminus of the protein-coding sequence and, if necessary, subsequently removed to form the final protein product.

[0119] A fusion protein can be expressed as a single polynucleotide encoding the entire fusion protein, or as multiple (e.g., two or more) polynucleotides that can be expressed by an appropriate expression system or co-expressed. Polypeptides encoded by 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 may be encoded by a polynucleotide separate from the heavy chain portion of the monoclonal antibody. When co-expressed in host cells, the heavy chain polypeptide associates with the light chain polypeptide to form a monoclonal antibody.

[0120] Typically, the nucleic acid encoding the desired fusion protein is generated using molecular cloning methods and generally placed within a vector, such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell suitable for the 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 selected because they can provide appropriate post-translational modifications. Host cells may 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 cells 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 a particular embodiment, a host cell comprising one or more fusion expression vectors is provided. The fusion protein can be produced by the expression of a nucleotide sequence in any suitable expression system known in the art. Any expression system can be used, including yeast, bacteria, animals, plants, eukaryotes, and prokaryotes. In some embodiments, a yeast system modified to reduce natural 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 protein described herein is 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 fusion protein expression. In some embodiments, the factor H fusion protein is obtained from a cell culture or culture medium.

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

[0123] Isolation / purification of fusion proteins 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, including high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, such as protein A affinity chromatography and size exclusion chromatography. The conditions used to purify specific proteins depend, as will be apparent to those skilled in the art, in part, on factors such as net charge, hydrophobicity, and hydrophilicity.

[0124] Assay of fusion protein activity Hemolysis assay The fusion proteins described herein were evaluated for activity using a complement pathway hemolysis assay that measures complement-mediated lysis of rabbit erythrocytes secondary to the activation of a sub-pathway on the cell surface. Rabbit erythrocytes generally activate complement-mediated lysis in mouse or human serum. When serum C3 is activated, C3 convertase, C3 activating fragments, and C5 convertase are deposited in rabbit RBCs. For example, serum complement sub-pathway activity in the presence of fusion proteins containing a fragment of factor H and a VHH domain, a fragment of factor H and a fragment of FHRP5, or a fragment of factor H, VHH, and an integrin-binding domain (e.g., fusion proteins in Tables 1-5) was evaluated in a concentration-dependent manner in human or mouse serum in which the complement activation sub-pathway was promoted by supplementing with Mg++ and EGTA as Ca scavengers. Incubation of rabbit erythrocytes in normal mouse or human serum induces cell lysis, while the addition of nanomolar amounts of fusion proteins, for example, a fragment of factor H and a VHH domain, or a fragment of factor H and a FHRP5 fragment, or a fragment of factor H, a VHH domain, and an integrin-binding domain, reduces the degree of lysis (see Figures 2A-2D). The fusion proteins of this disclosure can be used to achieve semi-maximal inhibitory concentrations (IC) of approximately 15 nM to 250 nM (e.g., approximately 15 nM to 240 nM, approximately 15 nM to 220 nM, approximately 200 nM to 150 nM, approximately 15 nM to 100 nM, approximately 15 nM to 40 nM, or approximately 15 nM to 50 nM).50 ) may exhibit. In some embodiments, the fusion protein has an IC of 19nM to 240nM (e.g., about 19nM to about 230nM, about 50nM to about 240nM, about 100nM to about 240nM, about 150nM to about 240nM, about 200nM to about 240nM, about 19nM to about 50nM, about 19nM to about 100nM, about 19nM to about 150nM, about 19nM to about 200nM and about 19nM to about 230nM). 50 This could demonstrate that.

[0125] [Table 6]

[0126] Complement activity assay The fusion proteins described herein (e.g., the fusion proteins in Tables 1-5) can be evaluated for complement alternative pathway activity in the liquid phase using a complement alternative pathway assay kit, e.g., Complement system Alternative Pathway WIESLAB®, Lund, Sweden. This method combines the principle of a hemolysis assay for complement activation with the use of a labeled antibody specific to the 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, the plate wells are coated with a specific activator for the alternative pathway. Serum is diluted in a diluent containing a specific blocker to ensure that only the alternative pathway is activated. For example, antiproperdin VHH can be mixed into the patient's blood in a concentration-dependent manner. 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 (optical density (OD)) at 405 nm. Addition of nanomolar amounts of factor H fusion protein according to this disclosure reduces the degree of activity, for example. Further exemplary assays for determining complement pathway activity are those described in Hebell et al., (Science (1991) 254(5028):102-105).

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

[0128] Compositions containing factor H fusion proteins can be formulated for either a single or multiple dosing regimen. Doses can be formulated for, for example, hourly, every two hours, daily, every two days, twice a week, three times a week, four times a week, five times a week, six times a week, weekly, every other week, monthly, every other month, or yearly. Alternatively, doses can be formulated for, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times a day.

[0129] Pharmaceutical compositions containing factor H fusion proteins can be formulated according to standard methods. Pharmaceutical formulation is a well-established technique and is further described, for example, in 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] A pharmaceutical composition may include a fusion protein and at least one pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. The term “pharmaceutically acceptable carrier” excludes tissue culture media containing bovine or equine serum. A pharmaceutically acceptable carrier or adjuvant does not, by itself, induce the production of antibodies harmful to the individual receiving the composition, nor does it induce protection. Therefore, 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 water, physiological saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and one or more combinations thereof. Some embodiments may include isotonic agents in the composition, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable substances may include small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, stabilizers, or buffers that enhance the shelf life or efficacy of the antibody.

[0131] The compositions described herein can be prepared in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and injectable 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, for example, 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 increased circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0132] Pharmaceutical compositions containing H factor fusion proteins can also be formulated with carriers that protect the composition (e.g., H factor fusion protein) from rapid release, such as controlled-release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. 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 depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or injectable solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. Compositions can be delivered, for example, by parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or topical administration (e.g., directly to the kidney).

[0134] Pharmaceutical compositions can be provided stably in sterile form under manufacturing and storage conditions. Compositions can be formulated into 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 into a suitable solvent, along with one or a combination of the components listed above as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the fusion protein into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods are vacuum drying and freeze-drying, yielding a powder of the active ingredient and any additional desired components from a pre-sterilized filtered solution. 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. Sustained absorption of injectable compositions can be achieved by including absorption-delaying reagents, such as monostearate and gelatin, in the composition. The chosen form depends in part on the intended mode of administration and therapeutic application. For example, compositions intended for systemic or local delivery may be in the form of injectable or injectable solutions. Compositions can be formulated, for example, as buffer solutions suitable for storage at 2–8°C (e.g., 4°C) at appropriate concentrations. Compositions can also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). Compositions can be further formulated for storage at 2–8°C (e.g., 4°C) for up to 2 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 and 1 / 2 years, or 2 years). Therefore, the compositions described herein may be stable for storage at 2–8°C (e.g., 4°C) for at least 1 year.

[0135] The fusion proteins described herein can be administered by various methods known in the art, but for many therapeutic applications, the chosen route / mode of administration is intravenous injection or infusion. The fusion proteins can also be administered by intramuscular or subcutaneous injection. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome.

[0136] In certain embodiments, the fusion protein may be prepared using a carrier that protects it from rapid release, such as a controlled-release formulation including an implant, a transdermal patch, and a microencapsulated delivery system.

[0137] Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate 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). Further methods applicable to the controlled or sustained release of the fusion proteins disclosed herein are described, for example, in International Publication No. 2016 / 081884, the entire contents of which are incorporated herein by reference.

[0138] Pharmaceutical compositions may have a pH of approximately 5.6–10.0, approximately 6.0–8.8, or approximately 6.5–8.0. For example, the pH may be approximately 6.2, 6.5, 6.75, 7.0, or 7.5, for example, pH 7.0. Pharmaceutical compositions may be formulated for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical, intravitreous, or parenteral administration. Parenteral administration may include intradermal, subcutaneous (SC, sq, subQ, Hypo), intramuscular (im), intravenous (IV), intraperitoneal (ip), intra-arterial, intramedullary, intracardiac, intravitreous (ocular), intra-articular (articular), intra-synovial (synovial fluid area), intracranial, intraspinal, and intrathecal (cerebrospinal fluid) injection or infusion. SC administration may include SC infusion or SC push. Any device suitable for parenteral injection or infusion of the drug formulation may be used for such administration. For example, the pharmaceutical composition may be contained in a sterile, pre-filled syringe.

[0139] Further active compounds may 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 may be co-formulated with the second drug or formulated separately from the second drug formulation. For example, each pharmaceutical composition may be mixed, for example, immediately before administration and administered together, or administered separately, for example, at the same or different times. In some embodiments, the fusion protein may be co-formulated and / or co-administered with one or more further antibodies that bind to other targets (e.g., antibodies that bind to regulators of the complement accessory pathway). Such combination therapies may utilize lower doses of the therapeutic agents administered and thus avoid potential toxicity or complications associated with various monotherapies. Furthermore, the compositions described herein may be co-formulated or co-administered with other therapeutic agents (e.g., therapeutic agents that minimize the risk of infection in an immunocompromised environment, e.g., antibacterial agents, antifungal agents and antiviral agents) to mitigate the side effects of administering the compositions described herein.

[0140] Preparations of compositions containing factor H fusion proteins can be provided to subjects in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions, or emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, fish oils, and organic esters for injection. Aqueous carriers include water, water-alcohol solutions, emulsions, or suspensions containing sodium chloride solution, ringer's dextrose solution, dextrose + sodium chloride solution, ringer's solution containing lactose, or non-volatile oils, as well as physiological saline and buffered parenteral vehicles for medical use.

[0141] Intravenous vehicles may include fluids and nutritional supplements, electrolyte supplements, such as those based on ringer's dextrose; pharmaceutically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may 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 contain a "therapeutic effective dose" or a "preventive effective dose" of the fusion protein. The "therapeutic effective dose" refers to an effective amount in a dosage and duration sufficient to achieve the desired therapeutic outcome. The therapeutic effective dose of the fusion protein may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the fusion protein's ability to induce the desired response in the individual. The "preventive effective dose" refers to an effective amount in a dosage and duration sufficient to achieve the desired preventive outcome. In some embodiments, the preventive dose is used in subjects in a pre-disease or early-stage stage where the preventive effective dose is less than the therapeutic effective dose.

[0143] The dosage regimen may be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and uniformity of dosage, it is advantageous to formulate parenteral compositions in dose units. As used herein, dose unit forms refer to physically distinct units suitable as unit doses for the mammalian subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. It should be noted that dose values ​​may vary depending on the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, a specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the administering clinician.

[0144] The efficacy of therapies with the fusion proteins described herein can be evaluated based on the improvement of one or more symptoms or indicators of the disease condition or disorder being treated (e.g., improvement of one or more symptoms of complement accessory pathway (CAP)-mediated diseases or disorders, e.g., renal diseases or disorders mediated by CAP dysregulation). An improvement of at least 10% in one or more clinical indicators (increase or decrease, depending on the indicator measured) is considered an “effective treatment,” but greater improvements such as 20%, 30%, 40%, 50%, 75%, 90%, or even 100% are possible, or exceed 100% depending on the indicator measured (e.g., 2x, 3x, 10x, etc., up to and including the achievement of disease-free status).

[0145] Therapeutic methods using fusion proteins The complement factor H fusion proteins described herein (see, for example, Tables 1-5) may be used to treat diseases mediated by complement accessory pathway activation or dysregulation in mammals (e.g., humans) by inhibiting complement accessory pathway activation. The fusion proteins described herein can be used to treat a variety of diseases or disorders mediated by complement accessory pathway activation or dysregulation. Such disorders include, but are not limited to, renal impairment, 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney.

[0146] A therapeutically effective dose of the complement factor H fusion proteins disclosed herein (e.g., fusion proteins having any one of SEQ ID NOs: 1-15 or their variants 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 mammalian subjects in need of such treatment. In some embodiments, the subject is a human patient. The dose administered should be sufficient to inhibit complement activation and / or restore normal complement accessory pathway regulation. Determining the therapeutically effective dose is within the scope of the expertise of physicians in this field. However, as an example, in embodiments of the methods described herein that utilize systemic administration of a fusion protein for diseases mediated by complement accessory pathway activation or dysregulation, the effective human dose is 0.01 mg / kg to 150 mg / kg ((e.g., 0.05 mg / kg to 500 mg / kg, 0.1 mg / kg to 20 mg / kg, 5 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 10 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.5 mg / kg to 25 mg / kg, 1.0 mg / kg to 10 mg / kg, 1.5 mg / kg to 5 mg / kg, or 2.0 mg / kg to 3.0 mg / kg) or 1 μg / kg to 1,000 μg / kg (e.g., 5 μg) The recommended dose will likely be within the range of g / kg~1,000 μg / kg, 1 μg / kg~750 μg / kg, 5 μg / kg~750 μg / kg, 10 μg / kg~750 μg / kg, 1 μg / kg~500 μg / kg, 5 μg / kg~500 μg / kg, 10 μg / kg~500 μg / kg, 1 μg / kg~100 μg / kg, 5 μg / kg~100 μg / kg, 10 μg / kg~100 μg / kg, 1 μg / kg~50 μg / kg, 5 μg / kg~50 μg / kg, or 10 μg / kg~50 μg / kg. The route of administration may affect the recommended dose. Repeated systemic doses are intended to maintain an effective level, 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 in the treatment of renal impairment mediated by dysregulation of the capillary arteries, such as FSGS. FSGS is characterized by occlusion of the glomerular capillary acceleroles 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 few 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) resulting from primary kidney 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 activation of a secondary pathway, 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 to patients with low serum C3 levels. Low serum C3 indicates complement activation. Therefore, complement system activation may play a significant role in the pathogenesis and outcomes of FSGS (Liu et al., Scientific Reports, 7:4095, 2017). In humans, tubulointerstitial deposition of complement membrane attack complex (C5b-9) correlates with interstitial myofibroblast accumulation and proteinuria. Experimentally, intratubular formation of C5b-9 cells was found to promote the accumulation of peritubular myofibroblasts in focal segmental glomerulosclerosis (FSGS). These myofibroblasts act as sentinel inflammatory cells and can deposit extracellular matrix. These cells can also constrict the tubules, leading to atrophic 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 exhibit 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 complement secondary pathway is activated in the glomeruli and tubulointerstitium of mice with adriamycin nephropathy (Turnberg et al., J Immunol.177(6):4094-4102, 2006). Furthermore, complement H-deficient mice show 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 of C3a in progressive proteinuria nephropathy. Therefore, clinical utility in FSGS can be achieved by using inhibitors of the complement activation side pathway.

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

[0149] The methods described herein may be useful in treating high-density deposit disease, a rare renal disorder resulting in persistent proteinuria, hematuria, and nephritis syndrome. Simultaneous dysfunction of factor H deficiency and high-density deposit disease has been reported in several cases. For example, mutations in factor H have been found in human patients with high-density deposit disease. Symptoms of high-density deposit disease include, for example, hematuria and / or proteinuria; acute nephritis syndrome; development of drusen and / or visual impairment; acquired partial lipodystrophy and its complications; and the presence of serum C3 nephritis factor (C3NeF), an autoantibody against C3bBb, a C3 convertase of the complement accessory pathway (Appel et al., J.Am.Soc.Nephrol., 16:1392-1404, 2005). Targeting factor H at complement activation sites has a therapeutic effect on individuals with high-density deposit disease. In some embodiments, administering compositions containing the fusion molecules described herein to an individual is effective in treating high-density deposition diseases. The route of administration may affect the recommended dose. Repeated systemic doses are intended to maintain an effective level, for example, to attenuate or inhibit complement activation in the patient's system, depending on the mode of administration employed.

[0150] The compositions and methods described herein may be useful in the treatment of nephritis 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 lesions, 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 serious complication that occurs in subpopulations of SLE patients. SLE is a proto-autoimmune disease resulting in multi-organ involvement. This anti-autoimmune response is characterized by autoantibodies against various nuclear and cytoplasmic cellular components. These autoantibodies bind to their respective antigens, circulate, and ultimately form immune complexes that deposit in tissues. The deposition of these immune complexes leads to chronic inflammation and tissue damage. The complement pathway (including the complement accessory pathway) is involved in the pathology of SLE, and therefore, the fusion proteins provided herein are useful for treating lupus nephritis.

[0151] The compositions and methods described herein may be useful in treating the most common causes of membranous nephropathy (MN), glomerular disease, and idiopathic nephrotic syndrome in non-diabetic Caucasian adults. If left untreated, approximately one-third of MN patients progress to end-stage renal disease within 10 years. The incidence of ESRD due to MN in the United States is approximately 1.9 per million per year. The majority of primary MN cases (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, suggesting the involvement of lectins and potentially complementary activation pathways. Over time, IgG4 and C5b-9 deposition leads to podocyte injury, proteinuria, and nephrotic syndrome (Couser, Clin J Am Soc Nephrol 12:983-997, 2017). Mice lacking factor B, an essential component of the complement activation secondary pathway, did not show C3 and C5B-9 deposition and did not develop albuminuria in the mouse model of MN (Wentian et al., Front Immunol. 9:1433, 2018). Therefore, this disease can be treated using complement inhibitors that reduce the amount of C3 and C5 convertases deposited in glomerular lesions, such as the fusion proteins described herein.

[0152] In some embodiments, the method involves treating a subject having a disease or disorder mediated by complement accessory pathway activation or dysregulation by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1-15 or a variant thereof having at least 85% sequence identity (or more)) 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.

[0153] In some embodiments, the method involves treating a subject with renal impairment by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0154] In some embodiments, the method involves treating a subject with membranous nephropathy by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0155] In some embodiments, the method involves treating a subject having FSGS by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0156] In some embodiments, the method involves treating a subject having glomerulonephritis by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0157] In some embodiments, the method involves treating a subject having membranoproliferative glomerulonephritis by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0158] In some embodiments, the method involves treating a subject with complement 3 nephropathy (C3G) by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0159] In some embodiments, the method involves treating a subject with IgA nephropathy by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0160] In some embodiments, the method involves treating a subject having MCD by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0161] In some embodiments, the method involves treating a subject with diabetic nephropathy by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0162] In some embodiments, the method involves treating a subject with Alport syndrome by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0163] In some embodiments, the method involves treating a subject having lupus nephritis by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0164] In some embodiments, the method involves treating a subject with acute kidney injury by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0165] In some embodiments, the method involves treating a subject having Goodpasture syndrome by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0166] In some embodiments, the method involves treating a subject with nephrotic syndrome by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0167] In some embodiments, the method involves treating a subject with chronic proteinuria by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0168] In some embodiments, the method involves treating a subject with chronic kidney disease by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0169] In some embodiments, the method involves treating a subject with a high-density deposition disease by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0170] In some embodiments, the method involves treating a subject with polycystic kidney disease by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0171] In some embodiments, the method involves treating a subject with hypertensive nephropathy by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0172] In some embodiments, the method involves treating a subject with nephrosclerosis by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0173] In some embodiments, the method involves treating a subject having aHUS by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having any one amino acid sequence from SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0174] In some embodiments, the method involves treating a subject with ischemia-reperfusion injury by administering a therapeutically effective amount of a fusion protein (e.g., a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0175] In some embodiments, the method involves treating a subject with transplanted organ rejection by administering a therapeutically effective amount of a fusion protein (for example, a fusion protein having one of the amino acid sequences of SEQ ID NOs: 1 to 15) or a variant thereof, 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.

[0176] This disclosure further relates to compositions comprising the fusion protein provided above for use in the treatment of diseases or disorders mediated by CAP activation or dysregulation. In some embodiments, the disease or disorder is selected from the group consisting of renal impairment, 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney.

[0177] This disclosure further relates to compositions for use in the treatment of renal impairment, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0178] This disclosure further relates to compositions for use in the treatment of FSGS, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0179] This disclosure further relates to compositions for use in the treatment of membranous nephropathy, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0180] This disclosure further relates to compositions for use in the treatment of IgA nephropathy, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0181] This disclosure further relates to compositions for use in the treatment of MCD, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0182] This disclosure further relates to compositions for use in the treatment of diabetic nephropathy, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0183] This disclosure further relates to compositions for use in the treatment of Alport syndrome, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0184] This disclosure further relates to compositions for use in the treatment of lupus nephritis, 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15).

[0185] This disclosure further relates to compositions for use in the treatment of acute kidney injury, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0186] This disclosure further relates to compositions for use in the treatment of Goodpasture syndrome, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0187] This disclosure further relates to compositions for use in the treatment of nephrotic syndrome, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0188] This disclosure further relates to compositions for use in the treatment of chronic proteinuria, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0189] This disclosure further relates to compositions for use in the treatment of chronic kidney disease, 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15).

[0190] This disclosure further relates to compositions for use in the treatment of C3G, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0191] This disclosure further relates to compositions 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for use in the treatment of high-density deposition diseases.

[0192] This disclosure further relates to compositions for use in the treatment of glomerulonephritis, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0193] This disclosure further relates to compositions for use in the treatment of membranoproliferative glomerulonephritis, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0194] This disclosure further relates to compositions for use in the treatment of polycystic kidney disease, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0195] This disclosure further relates to compositions for use in the treatment of hypertensive nephropathy, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0196] This disclosure further relates to compositions for use in the treatment of nephrosclerosis, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0197] This disclosure further relates to compositions for use in the treatment of aHUS, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0198] This disclosure further relates to compositions for use in the treatment of ischemia-reperfusion injury, 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15).

[0199] This disclosure further relates to compositions 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for use in treating rejection of transplanted organs such as kidneys.

[0200] In some embodiments, the present disclosure relates to pharmaceutical compositions for treating diseases or disorders mediated by CAP activation or dysregulation. In some embodiments, the diseases are renal impairment, 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney.

[0201] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating renal impairment, 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15).

[0208] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating rejection of transplanted organs such as kidneys, 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15).

[0215] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating Goodpasture 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15).

[0218] In some embodiments, the present disclosure relates to a pharmaceutical composition for treating high-density deposition diseases, 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15).

[0224] In some embodiments, the present disclosure relates to the use of compositions comprising the fusion proteins provided above for the manufacture of pharmaceuticals for treating diseases or disorders mediated by CAP activation or dysregulation. In some embodiments, the diseases are selected from the group consisting of renal impairment, 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney.

[0225] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for renal impairment.

[0226] In some embodiments, the present disclosure relates to the 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 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for FSGS.

[0227] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for IgA nephropathy.

[0228] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for diabetic nephropathy.

[0229] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for acute kidney injury.

[0230] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for chronic kidney disease.

[0231] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for membranous nephropathy.

[0232] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for the rejection of transplanted organs such as kidneys.

[0233] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for MCD.

[0234] In some embodiments, the present disclosure relates to the use of a composition comprising a fusion protein (e.g., a fusion protein having an 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) 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 Alport syndrome.

[0235] In some embodiments, the present disclosure relates to the use of a composition comprising a fusion protein (e.g., a fusion protein having an 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) 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 nephrotic syndrome.

[0236] In some embodiments, the present disclosure relates to the use of a composition comprising a fusion protein (e.g., a fusion protein having an 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) 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 chronic proteinuria.

[0237] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for lupus nephritis.

[0238] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for membranoproliferative glomerulonephritis.

[0239] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for glomerulonephritis.

[0240] In some embodiments, the present disclosure relates to the 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 to 15) or a variant thereof (e.g., a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a medicament for Goodpasture syndrome.

[0241] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for C3 nephropathy.

[0242] In some embodiments, the present disclosure relates to the 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 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 the manufacture of a pharmaceutical for high-density deposition diseases.

[0243] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for polycystic kidney disease.

[0244] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 15) for the manufacture of a pharmaceutical for hypertensive nephropathy.

[0245] In some embodiments, the present disclosure relates to the 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 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 the manufacture of a pharmaceutical for nephrosclerosis.

[0246] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for aHUS.

[0247] In some embodiments, the present disclosure relates to the 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 (for example, a fusion protein having the amino acid sequence of any one of sequence numbers 1 to 15) or a variant thereof (for example, a fusion protein having at least 85% sequence identity with any one of sequence numbers 1 to 15) for the manufacture of a pharmaceutical for ischemia-reperfusion injury. [Examples]

[0248] The following examples are provided to those skilled in the art to disclose and describe how the methods and compounds claimed herein are carried out and prepared. They are intended to be purely illustrative and not to limit the scope of this disclosure.

[0249] Example 1. In silico design and construction of a factor H fusion protein localized in the kidney. Constructs containing various combinations of the H factor N-terminal functional domain, VHH domain, and polyarginylglycylaspartate (RGD) domain 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 provided to GeneArt (ThermoFisher) for codon optimization and gene synthesis. The nucleotide sequences were cloned into a proprietary vector for expression in mammalian cells. Plasmid DNA was then transiently transfected into HEK 293 and CHO cells. The supernatant was collected after 4-5 days. The concentration of the fusion protein was determined by SDS-PAGE and densitometry.

[0251] Example 2. Inhibition of a secondary pathway using a fusion protein. The compounds disclosed herein were tested for their ability to inhibit the secondary pathway using a CAP-specific hemolysis assay. The results are shown in Figures 2A, 2B, 2C, and 2D. In short, rabbit red blood cells were washed and Mg 2+ The inhibitors were added to 10% human serum containing EGTA. Serial dilutions of the 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. Inhibition of the secondary pathway lysis was shown for all molecules tested, confirming that the molecules functioned 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 non-inbred nude mice (Jackson Laboratories, Bar Harbor, ME). Figure 3 shows representative longitudinal in vivo images of the same subjects acquired over four days. For image acquisition, subjects were maintained under 2-3% isoflurane anesthesia on a heated imaging platform in 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, moderate binning, and an 800nm ​​emission / 750nm excitation filter. Subjects received 1 mg / kg of AlexaFluor 750-labeled test material via volume-standardized 100 μL tail vein intravenous 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 IACUC under Alexion Pharmaceuticals, Inc., New Haven, CT, Protocol #917103.

[0253] Figure 3 also shows kidneys from the same mice, imaged via fluorescence microscopy. The signals indicate the in vivo distribution of the test compound and exhibit selectivity for parietal and apical tubular epithelial cells. Renal residence time was prolonged by the presence of VHH and RGD-containing motifs.

[0254] Example 4. Determination of the pharmacokinetics of the selected fusion protein In female J:NU non-inbred nude mice (Jackson Laboratories, Bar Harbor, ME), single-dose pharmacokinetic exposures to the selected compounds were determined. Serum exposures were measured by an electrochemiluminescence detection assay developed on the MSD (Meso Scale Discovery, Rockville, Maryland) platform. Figure 4 shows the PK of the compound 1 hour and 24 hours after IV bolus administration. In contrast to the renal retention kinetics shown in Figure 3, in serum, the compound fell below the lower limit of detection by 24 hours after dosing, confirming the target specificity of the compound.

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

[0256] Example 6. Determination of Therapeutic Efficacy in the 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 effect on urinary albumin. Factor H 1~5 Alone, it was insufficient to provide a significant benefit. The efficacy was enhanced by the presence of the VHH and RGD-containing motifs.

[0257] Example 7. Determination of Therapeutic Efficacy in the 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 effect on tubular protein. Factor H 1~5 Alone, it was insufficient to provide a significant benefit.

[0258] Example 8. Visualization of the kidney in an FSGS model after administration of a fusion protein. Immunofluorescence C3 activation product staining was performed on mouse-derived kidney sections from an FSGS-induced adriamycin-induced nephropathy mouse model 7 days after therapeutic administration of compound E (day 14 of the study). Figure 8A illustrates a region of interest (ROI) manually applied to approximate the renal medulla to assess local complement accessory pathway (CAP) activation, using area-normalized C3 fragment mean pixel intensity analysis. Figure 8B qualitatively shows medullary CAP regulation occurring after treatment with compound E.

[0259] Example 9. Determination of therapeutic efficacy in the FSGS model. The therapeutic efficacy of compounds D, E, and G in a mouse model of FSGS-induced adriamycin-induced nephropathy was evaluated. On day 13 of the study, animals were individually placed in metabolic cages for 16 hours. The collected urine was analyzed for albumin, protein, and creatinine using a Cobas analyzer. The calculated urinary protein / creatinine (Figure 9A) and albumin / creatinine (Figure 9B) ratios showed a positive, but not statistically significant, benefit trend after treatment with compound E.

[0260] Example 10. Characterization of fusion protein purity The protein was evaluated using a non-reducing SDS-PAGE gel to confirm its purity and molecular weight. Most fusion proteins were readily purified to high purity by anion exchange chromatography followed by hydrophobic interaction chromatography (HIC). Anion exchange was performed in 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. Unreduced SDS-PAGE showed a single band at a loading of 2 μg per well after two-step purification. Selected proteins were readily purified to high purity by protein A chromatography. The concentration of the purified fusion proteins was determined by UV spectroscopic absorbance at 280 nm, corrected for molar extinction. Purity was evaluated by SDS-PAGE and size exclusion chromatography (SEC) HPLC. Exemplary recovered cell culture supernatants evaluated via SDS-PAGE and proteins purified 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, and their intact molecular weights were determined. Figure 11 shows the confirmation of the predicted theoretical molecular weight for a representative compound E.

[0262] Example 12. Characterization of fusion proteins using dynamic light scattering The melting point was determined by dynamic light scattering (DLS). First, the target compound was diluted to 1 mg / mL in PBS (pH 7.4) and evaluated at a scanning rate of 60°C-hours. Figure 12 shows an exemplary scan, illustrating the satisfactory melting point measured by DLS for compound E. Similar results were obtained for other compounds.

[0263] Example 13. Characterization of fusion proteins using size exclusion chromatography. Size exclusion chromatograms of compound E were obtained at 0 and 14 days of incubation at 37°C. The relative percentages of aggregated protein and intact fusion protein were calculated from the chromatogram measured at 0 days (Figure 13A) (1.1% aggregates and 98.4% fusion protein) and the chromatogram measured at 14 days (Figure 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 time period.

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

[0265] Example 15. Characterization of fusion protein stability using capillary electrophoresis The time-dependent stability of the compound (14 days at 37°C) was evaluated by CE-SDS. Figure 15A shows the unreduced CE-SDS chromatograms of compound E at time zero and after 14 days. Figure 15B shows the reduced CE-SDS chromatograms at time zero and after 14 days. The profiles at day 0 and day 14 were similar, confirming the stability of the compound. No significant differences were observed under reduced vs. unreduced 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 according to standard techniques to determine stability and lack of charge heterogeneity. Figure 16 shows the ICE results for a representative compound E. A total of six iterations 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 evaluated by MS using standard techniques. Figure 17 shows compound E at time 0, day 3, day 7, and day 14. The profiles were all similar, confirming the stability of compound E. Similar results were obtained for compounds D and G.

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

[0269] Example 19. Measurement of the effect of fusion proteins on complement accessory pathway regulation. The modulation of the complement accessory pathway (CAP) in normal human serum (NHS) by compound E was evaluated in the complement accessory pathway WIESLAB® assay according to the manufacturer's instructions. Equivalent dose-dependent inhibition of the C5b-9 neoantigen expressed during MAC formation was observed across 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 for compound E was determined across a series of subcutaneous (SC) doses in wild-type male C57Bl / 6J mice (Jackson Laboratories, Bar Harbor, ME). Figure 20 shows composite data from two separate studies illustrating 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 across a series of intravenous (IV) and subcutaneous (SC) doses was determined in female cynomolgus monkeys (Macaca fascicularis) (Charles River Laboratories, Inc., Mattawan, MI). Figure 21A shows the serum pharmacokinetics of compound E after both the initial dose given on day 0 of the study and the fourth dose administered on day 12 of the study. Figure 21B includes 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 referenced herein are incorporated by reference to the same extent that each individual publication or patent application is specifically and individually indicated as being incorporated by reference. While specific embodiments are described herein, those skilled in the art will understand that further modifications and embodiments are encompassed, including variations, uses, or adaptations that generally follow the principles described herein, are within the scope of publicly known or customary practices in the art, and include such deviations from this disclosure that may be applied to the essential features described above and below in the claims. In embodiments of the present invention, for example, the following items are provided. (Item 1) Structure from the N-terminus to the C-terminus: D1-L1-D2-L2-D3 (In the formula, D1 contains a fragment of complement factor H (FH); L1 is either absent, covalently bonded, or an amino acid sequence of at least one amino acid; D2 may or may not contain VHH; L2 is either absent, covalently bonded, or an amino acid sequence of at least one amino acid; and D3 is the integrin recognition domain. A fusion protein having the following properties. (Item 2) D1 is the fusion protein described in item 1, comprising one or more FH short consensus repeat (SCR) domains, wherein the one or more SCR domains are optionally selected from the group consisting of SCR 1, 2, 3, 4, 5, and 6. (Item 3) The FH SCR domain is a fusion protein as described in item 2, selected from the group consisting of SCR 1-4; 1-5; and 1-6. (Item 4) The VHH of D2 is a fusion protein according to any one of items 1 to 3, comprising a single-domain antibody. (Item 5) The VHH of D2 is a fusion protein according to any one of items 1 to 4, comprising a camel single-domain antibody. (Item 6) The integrin-recognizing domain of D3 comprises an integrin-recognizing domain containing an arginylglycylaspartate (RGD) peptide motif, as described in any one of items 1 to 5. (Item 7) The integrin recognition domain of D3 is a fusion protein according to any one of items 1 to 6, comprising a cyclo(RGD)4 peptide motif. (Item 8) L1 and L2 are fusion proteins described in any one of items 1 to 7, containing the same amino acid sequence. (Item 9) L1 and L2 are fusion proteins described in any one of items 1-7, containing different amino acid sequences. (Item 10) L1 and / or L2 are (G 4 A) 2 G 3 AG 4 S sociG 4 SDAA, (G 4 A) 2 G 4 S sociG 4 AG 3 AG 4 S,GGGGAGGGGAGGGS,GGGGSGGGGSGGGS,G 4 S, (G 4 S) 2 、(G 4 S) 3 、(G 4 S) 4 、(G 4 S) 5 、(G 4 S) 6 , (EAAAK) 3 PAPAP, G 4 SPAPAP, PAPAPG 4 S, GSTSGKSSEGKG, (GGGDS) 2 (GGGES) 2 ,GGGDSGGGGS,GGGASGGGGS,GGGESGGGGS,ASTKGP,ASTKGPSVFPLAP,G 3 P-Sensing 7 P, PAPNLLGGP, G 6 、G 12 APELPGGP, SEPQPQPG, (G 3 S2) 3 , GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS) 3 , (GS 4 ) 3 、G 4 A(G 4 S) 2 、G 4 SG 4 AG 4 S sociG 3 AS(G 4 S) 2 、G 4 SG 3 ASG 4 S sociG 4 SAG 3 SG 4 S, (G 4 S) 2 AG 3 S sociG 4 SAG 3 SAG 3 S sociG 4 D(G 4 S) 2 、G 4 SG 4 DG 4 S, (G 4 D) 2 G 4 S sociG 4 E(G 4 S) 2 、G 4 SG 4 EG 4 S, (G 4 E) 2 G 4 S sociG 4 SDA, G 4 A and (G 4 A) 3 A fusion protein selected from the group consisting of items 8 or 9. (Item 11) L1 and / or L2 are (G 4 A) 2 G 3 AG 4 S sociG 4 SDAA, (G 4 A) 2 G 4 S sociG 4 SDAA, (G 4 S) 4 、G 4 AG 3 AG 4 S sociG 4 A and (G 4A) 3 A fusion protein selected from the group consisting of the following, as described in item 10. (Item 12) (a) D1 contains FH SCR domains 1-5; L1 contains G 4 A is included; D2 is not present; L2 is not present; and D3 is cyclo(RGD) 4 Does it include; (b) D1 contains FH SCR domains 1-5; L1 is absent; D2 contains the aforementioned VHH; L2 contains G4A; and D3 is cyclo(RGD) 4 Does it include; (c) D1 contains FH SCR domains 1-5; L1 contains G 4 A is included; D2 does not exist; L2 is G 4 A includes; and D3 is cyclo(RGD) 4 Does it include; (d) D1 contains FH SCR domains 1-5; L1 is absent; D2 contains VHH; L2 is G 4 A includes; and D3 is cyclo(RGD) 4 Does it include; (f) D1 contains FH SCR domains 1-5; L1 is absent; D2 contains VHH; L2 is G 4 A includes; and D3 is cyclo(RGD) 4 Does it include; (g) D1 contains FH SCR domains 1-6; L1 is absent; D2 contains VHH; L2 is G 4 A includes; and D3 is cyclo(RGD) 4 including; or (h)D1 contains FH SCR domains 1-5; L1 contains G 4 A is included; D2 is VHH; L2 is G 4 A includes; and D3 is cyclo(RGD) 4 A fusion protein, including the one described in item 1. (Item 13) (a) Does it have the amino acid sequence of SEQ ID NO: 4 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions? (b) Having the amino acid sequence of SEQ ID NO: 5 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions; (c) Whether it 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) Whether it 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) Having 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) Does it have the amino acid sequence of SEQ ID NO: 14 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions? (g) A fusion protein as described in item 1, having the amino acid sequence of Sequence ID No. 15 or a variant thereof having up to 10 amino acid substitutions, additions, or deletions. (Item 14) (a) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 4; (b) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 5; (c) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 8; (d) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 9; (e) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 13; (f) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 14; (g) A fusion protein as described in item 1, having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 15. (Item 15) Structure from the N-terminus to the C-terminus: D1-L1-D2 (In the formula, D1 contains FH fragments such as FH1-5; L1 may or may not include a linker; and D2 contains H factor-related protein 5 (FHRP5) domains such as FHRP domains 7 and 8. A fusion protein containing the above. (Item 16) L1 is G 4 A, (G 4 A) 3 、(G 4 A) 2 G 3 AG 4 S sociG 4 SDAA, (G 4 A) 2 G 4 S sociG 4 AG 3 AG 4 S,GGGGAGGGGAGGGS,GGGGSGGGGSGGGS,G 4 S, (G 4 S) 2 、(G 4 S) 3 、(G 4 S) 4 、(G 4 S) 5 、(G 4 S) 6 , (EAAAK) 3 PAPAP, G 4 SPAPAP, PAPAPG 4 S, GSTSGKSSEGKG, (GGGDS) 2 (GGGES) 2 ,GGGDSGGGGS,GGGASGGGGS,GGGESGGGGS,ASTKGP,ASTKGPSVFPLAP,G 3 P-Sensing 7 P, PAPNLLGGP, G 6 、G 12 APELPGGP, SEPQPQPG, (G 3 S2) 3 , GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS) 3 , (GS 4 ) 3 、G 4 A(G 4 S) 2 、G 4 SG 4 AG 4 S sociG 3 AS(G 4 S) 2 、G 4 SG 3 ASG 4 S sociG 4 SAG 3 SG 4 S, (G 4 S) 2 AG 3 S sociG 4 SAG 3 SAG 3 S sociG 4 D(G 4 S) 2、G 4 SG 4 DG 4 S, (G 4 D) 2 G 4 S sociG 4 E(G 4 S) 2 、G 4 SG 4 EG 4 S, (G 4 E) 2 G 4 S and G 4 A fusion protein selected from the group consisting of SDAs, as described in item 15. (Item 17) L1 is G 4 A and (G 4 A) 3 、(G 4 A) 2 G 3 AG 4 S sociG 4 SDAA, (G 4 A) 2 G 4 S sociG 4 SDAA, (G 4 S) 4 and G 4 AG 3 AG 4 A fusion protein selected from the group consisting of S, as described in item 16. (Item 18) (a) Having the amino acid sequence of SEQ ID NO: 6 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) A fusion protein as described in 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) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 6; or (b) A fusion protein according to any one of items 15 to 17, having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 10. (Item 20) Structure from the N-terminus to the C-terminus: D1-L1-D2-L2-D3 (In the formula, D1 is Cyclo (RGD) 4 Includes integrin recognition domains such as; L1 may or may not include a linker; D2 is a VHH such as a single-domain antibody. L2 is either a linker or does not exist; and D3 is an FH fragment such as FH1-5. A fusion protein containing the above. (Item 21) A fusion protein described in item 20, having a C-terminal His tag. (Item 22) L1 and L2 are fusion proteins as described in item 20 or 21, containing the same amino acid sequence. (Item 23) L1 and L2 are fusion proteins described in any one of items 20-22, containing different amino acid sequences. (Item 24) L1 and / or L2 are G 4 A, (G 4 A) 3 、(G 4 A) 2 G 3 AG 4 S sociG 4 SDAA, (G 4 A) 2 G 4 S sociG 4 AG 3 AG 4 S,GGGGAGGGGAGGGS,GGGGSGGGGSGGGS,G 4 S, (G 4 S) 2 、(G 4 S) 3 、(G 4 S) 4 、(G 4 S) 5 、(G 4 S) 6 , (EAAAK) 3 PAPAP, G 4 SPAPAP, PAPAPG 4 S, GSTSGKSSEGKG, (GGGDS) 2 (GGGES) 2 ,GGGDSGGGGS,GGGASGGGGS,GGGESGGGGS,ASTKGP,ASTKGPSVFPLAP,G 3 P-Sensing 7 P, PAPNLLGGP, G 6 、G 12 APELPGGP, SEPQPQPG, (G 3 S2) 3 , GGGGGGGGGSGGGS, GGGGSGGGGGGGGGS, (GGSSS) 3 , (GS 4 ) 3 、G 4 A(G 4 S) 2 、G 4 SG 4 AG 4 S sociG 3 AS(G 4 S) 2 、G 4 SG 3 ASG 4 S sociG 4 SAG 3 SG 4 S, (G 4 S) 2 AG 3 S sociG 4 SAG 3 SAG 3 S sociG 4 D(G 4 S) 2 、G 4 SG 4 DG 4 S, (G 4 D) 2 G 4 S sociG 4 E(G 4 S) 2 、G 4 SG 4 EG 4 S, (G 4 E) 2 G 4 S and G 4 A fusion protein as described in item 22 or 23, selected from the group consisting of SDAs. (Item 25) L1 and / or L2 are G 4 A, (G 4 A) 3 、(G 4 A) 2 G 3 AG 4 S sociG 4 SDAA, (G 4 A) 2 G 4 S sociG 4 SDAA, (G 4 S) 4 and G 4 AG 3 AG 4 A fusion protein selected from the group consisting of S, as described in item 24. (Item 26) (a) Having the amino acid sequence of SEQ ID NO: 2 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) A fusion protein as described in 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) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 2; or (b) A fusion protein according to any one of items 20 to 26, having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 3. (Item 28) Structure from the N-terminus to the C-terminus: 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 containing the above. (Item 29) A fusion protein described in item 28, having a C-terminal His tag. (Item 30) (a) Having the amino acid sequence of SEQ ID NO: 1 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) A fusion protein as described in 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) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 1; or (b) A fusion protein according to item 28 or 29, having an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 7. (Item 32) (a) Having the amino acid sequence of SEQ ID NO: 11 or a variant having up to 10 amino acid substitutions, additions, or deletions; or (b) A fusion protein as described in 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) Having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 11; or (b) A fusion protein according to item 28 or 29, having an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 12. (Item 34) A fusion protein according to any one of items 1 to 33, having an increased intrarenal residence time compared to the fusion protein lacking the VHH domain. (Item 35) A pharmaceutical composition comprising a fusion protein described in any one of items 1 to 34 and a pharmaceutically acceptable carrier. (Item 36) A polynucleotide encoding a fusion protein as described in any one of items 1 through 35. (Item 37) A vector containing the polynucleotides described in item 36. (Item 38) A host cell containing the polynucleotide described in item 36 or the vector described in item 37. (Item 39) A method for producing a fusion protein as described in any one of items 1 to 34, comprising the step of culturing one or more host cells containing one or more nucleic acid molecules capable of expressing the fusion protein under conditions suitable for the expression of the fusion protein. (Item 40) The method according to item 39, further comprising the step of obtaining the fusion protein from a cell culture or culture medium. (Item 41) A method for treating a disease mediated by activation or dysregulation of an accessory pathway of complement, comprising administering an effective amount of a composition comprising any one of items 1 to 34, a pharmaceutical composition as described in item 35, a polynucleotide as described in item 36, a vector as described in item 37, or a host cell as described in item 38, to a subject in need thereof. (Item 42) The method according to item 41, wherein the fusion protein is formulated as a pharmaceutical composition with at least one pharmaceutically acceptable carrier. (Item 43) The composition is freeze-dried according to the method described in item 42. (Item 44) The composition is rehydrated before administration, according to the method described in item 43. (Item 45) The method according to item 42, wherein the at least one pharmaceutically acceptable carrier is physiological saline. (Item 46) The composition described above is formulated for daily, weekly, or monthly administration, according to any one of items 41 to 45. (Item 47) The composition according to any one of items 41 to 46, formulated for intravenous, subcutaneous, intramuscular, oral, nasal, sublingual, intrathecal, and intradermal administration. (Item 48) The method according to any one of items 41 to 47, wherein the composition is formulated for administration at a dose of about 0.1 mg / kg to about 150 mg / kg. (Item 49) The composition described above is formulated for administration in combination with an additional therapeutic agent, according to any one of items 41 to 48. (Item 50) The aforementioned disease is a kidney disorder such as focal glomerulosclerosis (FSGS), IgA nephropathy, minimal change disease (MCD), diabetic nephropathy, Alport syndrome, lupus nephritis, membranous nephropathy, acute kidney injury, Goodpasture syndrome, nephrotic syndrome, chronic proteinuria, chronic kidney disease, C3 nephropathy (C3G), high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, atypical hemolytic uremic syndrome (aHUS), ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney, as described in any one of items 41 to 49. (Item 51) The disease is FSGS, as described in any one of items 41 to 50. (Item 52) The subject is a mammal, and the method described in any one of items 41 to 51. (Item 53) The mammal is a human, as described in item 52. (Item 54) A kit comprising a fusion protein as described in any one of items 1 to 34, a pharmaceutical composition as described in item 35, a polynucleotide as described in item 36, a vector as described in item 37, or a composition selected from host cells as described in item 38. (Item 55) The kit according to item 54, further comprising instructions for administering an effective amount of the composition to a subject in need thereof. (Item 56) Use of a composition comprising a fusion protein as described in any one of items 1 to 34 for the manufacture of a pharmaceutical product for a disease mediated by activation or dysregulation of the complement accessory pathway. (Item 57) The aforementioned diseases are kidney disorders such as 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, high-density deposition disease, glomerulonephritis, membranoproliferative glomerulonephritis, polycystic kidney disease, hypertensive nephropathy, nephrosclerosis, aHUS, ischemia-reperfusion injury, or rejection of transplanted organs such as the kidney, as described in item 56.

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