Therapeutic Compositions and Methods for Age-Related Macular Degeneration

Abstract

Engineered polypeptides for use in the treatment of age-related macular degeneration (AMD) comprising engineered variant peptides of FHL-1, compositions comprising these engineered polypeptides, and methods of using them. Further, the polypeptide comprises a linker domain separating a first peptide sequence from a second peptide sequence, a first junction region between the first peptide sequence and the linker domain, and a second junction region between the second peptide sequence and the linker domain.

Description

Technical Field

[0001] Claim of Priority

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 389,355, filed on July 14, 2022, entitled "THERAPEUTIC COMPOSITIONS AND METHODS FOR AGE-RELATED MACULAR DEGENERATION", which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference

[0002] All publications and patent applications mentioned in this specification are hereby incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Background Art

[0003]

[0003] Age-related macular degeneration (AMD) is a chronic metabolic inflammatory disease of the eye. AMD is the leading cause of blindness in people over 55 years old and has a relatively high prevalence in the United States (e.g., 8.7%) and worldwide. Furthermore, this problem is expected to increase with the aging of the world population. AMD is categorized into various types (e.g., early, intermediate, exudative, and atrophic), but the majority of AMD cases are considered "atrophic" AMD, and there is only one approved treatment for this.

[0004]

[0004] There are many associated factors that can contribute to AMD. For example, extracellular deposits of lipids (drusen) are the first pathological signs of AMD. Drusen damage and compress retinal pigment epithelium (RPE) cells, and the loss of RPE cells leads to photoreceptor degeneration and severe disease late stages including geographic atrophy (GA) and neovascular AMD (nvAMD). There is a need for treatments that can treat both atrophic and exudative AMD.

Summary of the Invention

[0005] The engineered polypeptide is described herein. In particular, an engineered therapeutic polypeptide (e.g., a "CFH-FHL" polypeptide) is provided that includes a short consensus repeat (SCR) region of complement factor H (or CFH) and an SCR region of factor H-like protein 1 (or FHL) peptide, which can provide one or more therapeutic benefits as described herein.

[0006] Generally, the engineered polypeptide can be used for the treatment of age-related macular degeneration (AMD). The engineered polypeptide can have a first peptide sequence with at least 80% homology to SEQ ID NO: 3 (CFH SCR1-7 N-terminal domain), a second peptide sequence with at least 80% homology to SEQ ID NO: 17 (FHL-1 SCR6-7 C-terminal domain), and a linker domain separating the first peptide sequence from the second peptide sequence.

[0007] In some examples, the linker domain includes an FHL-1 SCR7 junction linked to a first peptide sequence, and the FHL-1 SCR7 junction may have a peptide sequence of SEQ ID NO: 4 to SEQ ID NO: 10. The linker domain includes a CFH SCR6 junction linked to a second peptide sequence, and the CFH SCR6 junction may have a peptide sequence of SEQ ID NO: 13 to SEQ ID NO: 16. The linker domain may include a peptide sequence of SEQ ID NO: 11 or SEQ ID NO: 12. The engineered polypeptide may have an amino acid sequence with 90% or more homology to SEQ ID NO: 18 to SEQ ID NO: 132. The first peptide sequence may have 95% or more homology with the CFH SCR1-7 peptide sequence of SEQ ID NO: 3, while the second peptide sequence may have 95% or more homology with the FHL-1 SCR6-7 peptide sequence of SEQ ID NO: 17. The first peptide sequence may be the CFH SCR1-7 peptide sequence of SEQ ID NO: 3, while the second peptide sequence may be the FHL-1 SCR6-7 peptide sequence of SEQ ID NO: 17. The linker domain may include a Gly / Ser linker, a poly-Gly linker, or a poly-Ala linker. The linker domain may include GGGS, GGGSGGGS, GGGGSGGGS, GGGGSGGGSGGGS, or GGGGSGGGGSGGGGS. The linker domain may include EAAAK, EAAAKEAAAK, or EAAAAKEAAAKEAAAK.

[0008] In some examples, the engineered polypeptide may include a linker domain having a peptide sequence with 90% or more homology to SEQ ID NO: 11 or SEQ ID NO: 12. The linker domain may be a Gly / Ser linker, a poly-Gly linker, or a poly-Ala linker. The linker domain may include a first peptide sequence comprising SEQ ID NO: 4 to SEQ ID NO: 10. The linker domain may also include a second peptide sequence comprising SEQ ID NO: 13 to SEQ ID NO: 16.

[0009] Generally, an engineered polypeptide for use in treating AMD can have a first peptide sequence of SEQ ID NO: 3, which is linked to a second peptide sequence having at least 80% homology to the FHL-1 SCR6-7 peptide sequence of SEQ ID NO: 17, where the second peptide sequence can be separated from the second peptide sequence by a peptide linker comprising the peptide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0010] Generally, an engineered polypeptide for use in treating AMD can have a first region of a peptide sequence having at least 90% homology to SEQ ID NO: 3, which is peptide-linked to a second peptide sequence having at least 90% homology to any of SEQ ID NO: 17 by a peptide linker region.

[0011] For example, an engineered polypeptide (which may be for use in treating age-related macular degeneration) described herein can generally have any of the sequences of SEQ ID NO: 125, SEQ ID NO: 131, or SEQ ID NO: 132.

[0012] In some examples, an engineered polypeptide comprises one first peptide sequence of SEQ ID NO: 3, SEQ ID NO: 139, or SEQ ID NO: 140, a second peptide sequence of SEQ ID NO: 17, and a linker domain between the one first peptide sequence and the second peptide sequence of SEQ ID NO: 138. Any of these polypeptides can include a first junction region between the first peptide sequence and the linker domain, where the first junction region has one of the sequences of SEQ ID NOs: 4-10, and can also include a second junction region between the linker domain and the second peptide sequence, where the second junction region has one of the sequences of SEQ ID NOs: 13-16. For example, the first junction region can have the sequence of SEQ ID NO: 7 and the second junction region can have the sequence of SEQ ID NO: 13.

[0013] An engineered polypeptide for use in treating age-related macular degeneration (AMD) can have a first peptide sequence of SEQ ID NO: 3, a second peptide sequence of SEQ ID NO: 17, and a linker domain separating the first peptide sequence from the second peptide sequence. As mentioned, the engineered polypeptide can include a first peptide sequence having at least 80% homology to SEQ ID NO: 3, a second peptide sequence having at least 80% homology to SEQ ID NO: 17, and a linker domain separating the first peptide sequence and the second peptide sequence. The linker domain can include the FHL-1 SCR7 junction linked to the first peptide sequence, and the FHL-1 SCR7 junction has the peptide sequences of SEQ ID NOs: 4 to SEQ ID NO: 10. In some examples, the linker domain includes the CFH SCR6 junction linked to the second peptide sequence, and the CFH SCR6 junction has the peptide sequences of SEQ ID NOs: 13 to SEQ ID NO: 16. The linker domain can generally include the peptide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0014] For example, engineered polypeptides having any of the amino acid sequences of SEQ ID NOs: 18 to 97, SEQ ID NOs: 99 to 106, SEQ ID NOs: 122 to 125, or SEQ ID NOs: 128 to 132 are described herein. The first peptide sequence can be homologous (e.g., at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 99% homologous) to the CFH SCR1-7 peptide sequence of SEQ ID NO: 3, and the second peptide sequence can be homologous (e.g., at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 99% homologous) to the FHL-1 SCR6-7 peptide sequence of SEQ ID NO: 17. The linker domain can include a Gly / Ser linker, a poly-Gly linker, or a poly-Ala linker, and in some examples, the linker domain includes one of GGGS, GGGSGGGS, GGGGSGGGS, GGGGSGGGSGGGS, or GGGGSGGGGSGGGGS, EAAAK, EAAAKEAAAK, or EAAAAKEAAAKEAAAK.

[0015]

[0015] For example, an engineered polypeptide for use in treating age-related macular degeneration (AMD) is described herein, which comprises a first peptide sequence having at least 80% homology with SEQ ID NO: 3, a second peptide sequence having at least 80% homology with SEQ ID NO: 17, a linker domain separating the first peptide sequence from the second peptide sequence, a first junction region between the first peptide sequence and the linker domain, and a second junction region between the second peptide sequence and the linker domain. The first junction region can have the sequence of SEQ ID NO: 7, and the second junction region can have the sequence of SEQ ID NO: 13 or SEQ ID NO: 14. The linker domain can include the peptide sequence of SEQ ID NO: 11.

[0016]

[0016] For example, an engineered polypeptide for use in treating age-related macular degeneration (AMD) can have a sequence having at least 80% homology with SEQ ID NO: 131. For example, an engineered polypeptide for use in treating age-related macular degeneration (AMD) can have a sequence having at least 90% homology with SEQ ID NO: 131. In some examples, an engineered polypeptide for use in treating age-related macular degeneration (AMD) has the sequence of SEQ ID NO: 131.

[0017]

[0017] For example, an engineered polypeptide for use in treating age-related macular degeneration (AMD) can include the sequence of SEQ ID NO: 125. An engineered polypeptide for use in treating age-related macular degeneration (AMD) can include the sequence of SEQ ID NO: 131. An engineered polypeptide for use in treating age-related macular degeneration (AMD) can include the sequence of SEQ ID NO: 132.

[0018]

[0018] In some examples, an engineered polypeptide for use in treating age-related macular degeneration (AMD) can comprise a first peptide sequence of SEQ ID NO: 3 linked to a second peptide sequence having at least 80% homology to the FHL-1 SCR6-7 peptide sequence of SEQ ID NO: 17, where the second peptide sequence is separated from the second peptide sequence by a peptide linker comprising the peptide sequence of SEQ ID NO: 11 or SEQ ID NO: 12. An engineered polypeptide for use in treating age-related macular degeneration (AMD) can have a first region of a peptide sequence having at least 90% homology to SEQ ID NO: 3 linked to a second peptide sequence having at least 90% homology to any of SEQ ID NO: 17 by a peptide linker region.

[0019]

[0019] An engineered polypeptide for use in treating age-related macular degeneration (AMD) can have a peptide sequence having at least 80% homology to any one of SEQ ID NOs: 18-137. For example, an engineered polypeptide for use in treating age-related macular degeneration (AMD) can have a peptide sequence having at least 90% homology to any one of SEQ ID NOs: 18-137. An engineered polypeptide for use in treating age-related macular degeneration (AMD) can have the sequence of any one of SEQ ID NOs: 18-137.

[0020]

[0020] Also described herein are pharmaceutical compositions using any of these engineered polypeptides. For example, a pharmaceutical composition for use in the prevention or treatment of age-related macular degeneration (AMD) in a patient can comprise any of the polypeptides described above and a pharmaceutically acceptable excipient. In some examples, the composition can be formulated, adapted, and / or compounded for administration by intravitreal injection. In some examples, a plurality of different engineered polypeptides described herein can be used. For example, a pharmaceutical composition can comprise two or more of the engineered polypeptides described herein.

[0021]

[0021] Any of these engineered polypeptides can be glycosylated at one or more sites. For example, all of the polypeptides of SEQ ID NO: 125, 131, or 132 contain engineered glycosylation sites. Any of the compositions described herein can be fully glycosylated or partially glycosylated (e.g., some or all of the engineered polypeptides can be glycosylated). For example, more than 40% of the engineered polypeptides in the composition can be glycosylated (more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially all of the engineered polypeptides in the composition can be glycosylated).

[0022]

[0022] Also described herein are methods of treating a patient using any of these engineered polypeptides. For example, methods of treating or preventing age-related macular degeneration (AMD) in a patient using an engineered polypeptide or a pharmaceutical composition comprising any of these engineered polypeptides are described herein.

[0023]

[0023] For example, a method of treating or preventing age-related macular degeneration (AMD) in a patient using an engineered polypeptide or a pharmaceutical composition of any of these engineered polypeptides (e.g., when the prevention or treatment is the prevention of said AMD) may include administering the composition to a patient diagnosed as having a tendency to develop AMD. The engineered polypeptide (or composition comprising the engineered polypeptide) can be delivered in any suitable manner, including orally, systemically, by injection, etc. For example, a method of treating or preventing age-related macular degeneration (AMD) in a patient using a pharmaceutical composition comprising any of the engineered polypeptides or engineered polypeptides described herein may include administering one or more doses of the engineered polypeptide or composition comprising the engineered polypeptide to the patient. The patient may exhibit signs or symptoms of AMD. These methods may include the prevention or treatment of early-stage AMD. Any of these methods may include delivering an engineered polypeptide or a composition of any of these engineered polypeptides to the patient's eye. For example, any of these methods may include delivering an engineered polypeptide or a composition of the engineered polypeptide by intravitreal injection. In some examples, delivering includes delivering two or more of an engineered polypeptide or a composition of any of these engineered polypeptides.

[0024]

[0024] All of the methods and apparatuses described herein can be used in any combination, are contemplated herein, and are used to achieve the benefits described herein.

[0025]

[0025] A better understanding of the features and benefits of the methods and apparatuses described herein can be obtained by reference to the following detailed description, which illustrates exemplary embodiments, and the following accompanying drawings.

Brief Description of the Drawings

[0026]

Figure 1

[0026] Figure 1A schematically illustrates the proteoglycan binding activity of CFH protein in the complement pathway under normal conditions. Figure 1B schematically illustrates the disruption of proteoglycan binding activity when the CFH protein fails to bind properly. Figure 1C schematically illustrates an example of a engineered variant of FHL-1 described herein.

Figure 2

[0027] Figure 2 schematically illustrates an example of the arrangement of a therapeutic polypeptide described herein (the therapeutic polypeptide shown in Figure 2 may be referred to herein as an engineered variant of FHL-1).

Figure 3

[0028] Figure 3 schematically illustrates an example of an engineered variant of FHL-1 described herein that binds to a surface via interaction with two adjacent proteoglycans or via interaction with a single proteoglycan.

Figure 4

[0029] Figure 4 shows the alignment of the amino acid sequences between human CFH and FHL-1.

Figure 5

[0030] Figure 5 schematically illustrates an example of a library of engineered variants of FHL-1 described herein. The variants include an N-terminal constant region comprising the CFH SCR1-7 region, followed by eight different variants of the FHL-1 SCR7 junction sequence, followed by three different variants of a linker sequence, followed by five different variants of the CFH SCR6 junction sequence, followed by a C-terminal constant region comprising the FHL-1 SCR6-7 region.

Figure 6

[0031] Figure 6 is a bar graph showing the amount of full-length protein expressed from a library of engineered variants of FHL-1 as measured by Western blot analysis (anti-CFH antibody).

Figure 7

[0032] Figures 7A and 7B are examples of a representative set of Western blots showing FHL-1 and engineered FHL-1 variants expressed using a CHO expression system.

Figure 8

[0033] Figures 8A and 8B are bar graphs showing the amount of full-length protein expressed on day 7 after transfection for a subset of engineered variants of FHL-1 containing the [GGGGS]3 linker, as measured by Western blot analysis (anti-CFH antibody). Figure 8A compares a set of variants containing the same FHL-1 SCR7 junction. Figure 8B compares a set of variants containing the same CFH SCR6 junction.

Figure 9

[0034] Figure 9 is a bar graph showing the heparin-binding activity of proteins expressed from a library of engineered variants of FHL-1, as measured by ELISA.

Figure 10

[0035] Figure 10A is a bar graph showing the amount of full-length protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing the [GGGGS]3 linker, as measured by Western blot analysis (anti-CFH antibody). A set of variants containing the same FHL-1 SCR7 junction sequence is compared.

[0036] Figure 10B is a bar graph showing the total amount of protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing the [GGGGS]3 linker, as measured by interpolation of Western blot band intensity relative to a reference standard of purified FHL-1 protein. A set of variants containing the same FHL-1 SCR7 junction sequence is compared.

Figure 11

[0037] Figure 11A is a bar graph showing the amount of full-length protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing the [GGGGS]3 linker, as measured by Western blot analysis (anti-CFH antibody). A set of variants containing the same CFH SCR6 junction sequence is compared.

[0038] Figure 11B is a bar graph showing the total amount of protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing the [GGGGS]3 linker, as measured by interpolation of Western blot band intensity relative to a reference standard of purified FHL-1 protein. A set of variants containing the same CFH SCR6 junction sequence is compared.

Figure 12

[0039] Figure 12 is a table showing an example of a summary of results by measuring protein expression of engineered variants of FHL-1 containing the [GGGGS]3 linker. The optimal junction sequence is a sequence that confers significantly improved properties compared to non-optimal junction sequences, as shown in the source data.

Figure 13

[0040] Figures 13A and 13B illustrate the heparin-binding activity of engineered variants of FHL-1 (e.g., Var004, Var020) and control proteins (e.g., FHL-1, CFH) as measured by ELISA. The graph shows mean absorbance values from multiple independent heparin-binding assays. The table summarizes the EC50 values across multiple experiments.

Figure 14

[0041] Figures 14A and 14B illustrate the C3b-binding activity of engineered variants of FHL-1 (e.g., Var004) and control proteins (e.g., FHL-1, CFH) as measured by ELISA. The graph shows mean absorbance values from multiple independent C3b-binding assays. The table summarizes the EC50 values across multiple experiments.

Figure 15

[0042] Figure 15 is an example of a representative Western blot (anti-C3b antibody) showing surface-dependent complement inhibitory activity of engineered variants of FHL-1 (e.g., Var004) and control proteins (e.g., FHL-1, CFH). Bands labeled a’ and β represent intact C3b bands of 130 kDa and 70 kDa, respectively. Bands labeled a’68 and a’43 represent cleaved forms of C3b (iC3b) at 68 kDa and 43 kDa, respectively. Lanes labeled CFH, FHL-1 supe, and Var004 supe contain decreasing amounts of each protein in each set (333 nM, 111 nM, 37 nM, 12.3 nM). Control lane 1 contains cells, conditioned media, C3b, and CFI. Control lane 2 contains C3b and CFI. Control lane 3 contains C3b, CFI, and CFH.

Figure 16

[0043] Figure 16 illustrates an example of surface-dependent complement inhibitory activity of engineered variants of FHL-1 (e.g., Var004) and control proteins (FHL-1, CFH) as measured by Western blot analysis (anti-C3b antibody). Activity is represented as the ratio of iC3b to total C3b generated at each protein concentration tested, measured by the intensity of the bands representing intact C3b and cleaved C3b: [a’68 + a’43] / [a’68 + a’43 + a’ + β].

Figure 17

[0044] Figures 17A and 17B schematically illustrate an example of a library of glycoengineered variants of FHL-1 described herein. Each amino acid substitution introduces an N-linked glycosylation motif (NxS / T).

Figure 18

[0045] Figures 18A and 18B show an example of a representative Western blot (anti-CFH antibody) showing the degree of glycosylation of single amino acid substitutions in the FHL-1 framework (showing three independent transfections for one construct). Arrows indicate the mobility shift between glycosylated (+Gly) and non-glycosylated (-Gly) proteins. Control lane 1 contains purified FHL-1 protein.

Figure 19

[0046] Figure 19 shows an example of a representative Western blot (anti-CFH antibody) showing the degree of glycosylation of single amino acid substitutions in the Var004 framework (showing three independent transfections for one construct). Arrows indicate the mobility shift between glycosylated (+Gly) and non-glycosylated (-Gly) proteins. Control lane 1 contains purified FHL-1.

Figure 20

[0047] Figure 20 illustrates a table and examples of amino acid substitutions and the degree of their glycosylation in the FHL-1 variants described herein. Asterisks indicate variants with glycosylation exceeding 90%.

Figure 21

[0048] Figures 21A and 21B show examples of representative Western blots (anti-CFH antibody) showing the degree of glycosylation for complex amino acid substitutions in the FHL-1 variants described herein. The mobility shift is evident between the multiply glycosylated variants and the singly glycosylated parental construct. The lane labeled FHL-1 contains CHO expression supernatant from the native FHL-1 sequence. The table summarizes the amino acid substitutions present in each variant.

Figure 22

[0049] Figure 22A is a bar graph showing the amount of full-length protein measured from Western blot analysis using an anti-CFH antibody for the multiple glycosylated variants described herein. The % full-length monomer was quantified by measuring the concentration of the full-length monomer and dividing it by the concentration measurement of the entire lane from the monomer to 25 kDa.

[0050] Figure 22B is a bar graph showing the relative protein expression levels of the multiple glycosylated variants compared to the FHL-1 variants described herein. The relative protein expression level was determined by dividing the band intensity of each variant by the band intensity of Var004. [[ID=2

[0051] Figure 23 shows an example of data in a summary table quantifying the protein integrity and relative protein expression levels measured from Western blot analysis using an anti-CFH antibody. ​

[0052] Figures 24A and 24B illustrate the heparin-binding activities of engineered variants of FHL-1 (e.g., Var108, Var114, Var115) and control proteins (e.g., FHL-1, CFH) as measured by ELISA. The graph in Figure 24A shows the mean absorbance values from multiple independent heparin-binding assays. The table in Figure 24B is a summary of the EC50 values over multiple experiments. ​

[0053] Figures 25A and 25B illustrate the C3b-binding activities of engineered variants of FHL-1 (e.g., Var108, Var114) and control proteins (e.g., FHL-1, CFH) as measured by ELISA. The graph in Figure 25A shows the mean absorbance values from multiple independent C3b-binding assays. The table in Figure 25B is a summary of the EC50 values over multiple experiments. ​

[0054] Figures 26A and 26B illustrate the binding activities of engineered variants of FHL-1 (e.g., Var108, Var114, Var115) and control proteins (e.g., FHL-1, CFH) to cultured human RPE cells (ARPE-19 cell line) as measured by anti-CFH antibody immunostaining. The table in Figure 26B summarizes the EC50 values and maximum binding signals over multiple experiments. ​

[0055] Figures 27A and 27B illustrate examples of surface-dependent complement inhibitory activities of engineered variants of FHL-1 (e.g., Var108, Var114) and control proteins (e.g., FHL-1, CFH) in cultured human RPE cells (ARPE-19 cell line) as measured by anti-C5b9 (MAC) antibody immunostaining. The table in Figure 27B summarizes the IC50 values and maximum complement inhibition over multiple experiments. ​

[0056] Figure 28 is a graph showing the binding-functional relationships for engineered variants of FHL-1 (e.g., (Var108, Var114)) and control proteins (FHL-1, CFH) in the surface-dependent complement inhibition assay from Figures 27A - 27B and the binding assay from Figure 26. The correlation between cell surface binding and complement inhibition was tested by linear regression. ​

[0057] Figures 29A and 29B illustrate the surface-independent complement inhibitory activity of an engineered FHL-1 variant (e.g., Var114) compared to control proteins (e.g., FHL-1, CFH) in cultured human RPE cells (ARPE-19 cell line), as measured by anti-C5b9 (MAC) antibody immunostaining. The table in Figure 29B summarizes the IC50 values for complement inhibition across multiple experiments (e.g., Figure 29A). ​

[0058] Figures 30A and 30B illustrate the complement inhibitory activity of an engineered variant of FHL-1 (e.g., Var114) and a control protein (CFH) in cultured human RPE cells (ARPE-19 cells), as measured by C5a ELISA. Figure 30B is a table summarizing the IC50 values from multiple experiments. ​

[0059] Figures 31A and 31B illustrate the binding activity of an engineered variant of FHL-1 (e.g., Var114) and control proteins (e.g., FHL-1, CFH) in cultured human iPS-RPE cells, as measured by anti-CFH antibody immunostaining. The table in Figure 31B summarizes the EC50 values across multiple experiments. ​

[0060] Figure 32 is a graph showing the inhibition of C3a in rat aqueous humor samples after IVT delivery of engineered variants of FHL-1 (e.g., Var108, Var114) and control proteins (e.g., FHL-1, CFH) in a laser-induced CNV model of complement activation. ​

[0061] Figure 33 is a graph showing the inhibition of MAC deposition in rat RPE after IVT delivery of engineered variants of FHL-1 (e.g., Var108, Var114) and control proteins (e.g., FHL-1, CFH) in a laser-induced CNV model of complement activation. ​

[0062] Figure 34 is a graph showing the inhibition of macrophage recruitment in rat RPE after IVT delivery of engineered variants of FHL-1 (e.g., Var108, Var114) and control proteins (e.g., FHL-1, CFH) in a laser-induced CNV model of complement activation.

Mode for Carrying Out the Invention

[0027]

[0063] The compositions and methods described herein can be used to treat AMD, particularly AMD patients in whom the disease is primarily driven by complement dysfunction. In some examples, including (but not limited to) diseases in which the main contributing factor is the complement pathway, the patient can be treated with one or more engineered variants of FHL-1. In particular, these one or more engineered variants of FHL-1 (also referred to herein as CFH-FHL peptides or CFH-FHL peptide variants) are variants that improve complement inhibition, reduce lipid accumulation in Bruch's membrane, and prevent or reduce AMD-related effects. For example, the methods described herein can replace complement and lipid regulatory defects in Bruch's membrane by intravitreal injection of recombinant FHL-1 variants having improved proteoglycan binding activity, such as those having the protein sequences shown in SEQ ID NOs: 18 to 137.

[0028]

[0064] CFH is a negative regulator of complement activation and acts upstream of angiogenesis and retinal cell death. The compositions and methods described herein may provide therapeutics for AMD that modulate and / or restore complement and lipid homeostasis. These compositions are engineered to have improved proteoglycan binding activity, which may improve complement inhibition and reduce apolipoprotein binding.

[0029]

[0065] These compositions, such as engineered variants of FHL-1 described herein, may include one or more repeats of SCR6, SCR7, SCR8 modules from human CFH or FHL-1 separated by one or more linker regions.

[0030]

[0066] The present disclosure herein provides compositions and methods for treating, preventing, or inhibiting eye diseases. For example, the present disclosure herein provides recombinant factor H-like protein 1 (FHL-1) proteins and engineered variant proteins of FHL-1. The present disclosure provides methods for treating, preventing, or inhibiting eye diseases by administering to the eye (e.g., intravitreally) an effective amount of these compositions of the present disclosure for treating or preventing eye diseases using the methods provided herein. Eye diseases that can be treated or prevented using these methods include, but are not limited to, glaucoma, macular degeneration (e.g., age-related macular degeneration, AMD), diabetic retinopathy, hereditary retinal degenerations such as retinitis pigmentosa, retinal detachment or injury, and retinopathy (whether hereditary; surgical; traumatic; underlying etiology such as severe anemia, SLE, hypertension, blood disorders, systemic infections, or underlying carotid artery disease; induced by toxic compounds or drugs; or induced by light).

[0031]

[0067] Unless otherwise defined herein, scientific and technical terms used in this application have the meanings commonly understood by one of ordinary skill in the art.

[0068] Generally, the nomenclature and techniques used in connection with pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics, and protein and nucleic acid chemistry as described herein are those nomenclature and techniques well known and generally used in the art. In cases where there is a conflict, this specification, including definitions, will prevail.

[0032]

[0069] The practice of the present disclosure uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art unless otherwise indicated. Such techniques are described in Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait (ed.), 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis (ed.), 1998) Academic Press; Animal Cell Culture (R.I. Freshney (ed.), 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell (eds.), 1993 - 1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos (eds.), 1987); Current Protocols in Molecular Biology (F.M.Fully described in the literature such as Ausubel et al. (eds.), 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (eds.), 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0033]

[0070] Enzyme reactions and purification techniques are performed according to the manufacturer's instructions as generally accomplished in the art or as described herein. The nomenclature used in connection with the analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as their laboratory procedures and techniques, are well known in the art and are the generally used nomenclature and procedures and techniques. Standard techniques are used for chemical synthesis and chemical analysis.

[0034]

[0071] When aspects or examples of the present disclosure are described with respect to a Markush group or other group of alternatives, the present disclosure encompasses not only the entire recited group as a whole, but also each individual member of the group and all possible subgroups of the main group, as well as main groups in which one or more of the members of the group are absent. The present disclosure also contemplates any one or more explicit exclusions of members of the groups in the disclosed examples. Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0035] ​

[0072] The following terms are to be understood to have the following meanings unless otherwise indicated.

[0073] As used herein, "residue" refers to a position in a protein and its attendant amino acid identity. As is known in the art, "polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substitutions that can be incorporated into the chain by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation to a labeling component. Other types of modifications include, for example, "cap" substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications, such as modifications by uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.); modifications containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); modifications by intercalators (e.g., acridine, psoralen, etc.); modifications containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.); modifications containing alkylating agents; modifications having modified linkages (e.g., α-anomeric nucleic acids, etc.); and polynucleotides in unmodified form. Further, any of the hydroxyl groups normally present on the sugar can be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to provide additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OHs can be phosphorylated or replaced by an amine or an organic capping group moiety of 1 to 20 carbon atoms. Also, other hydroxyls can be derivatized with standard protecting groups.In addition, the polynucleotide may contain analogs of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomer sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include those in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NRi ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Examples include, but are not limited to, these. Not all linkages in the polynucleotide need to be the same. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0036]

[0074] The terms "polypeptide", "oligopeptide", "peptide", and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. The chain may be linear or branched, and it may contain modified amino acids and / or be interrupted by non-amino acids. Also, the term includes amino acid chains that are modified naturally or by intervention, such as disulfide bond formation, sugar chain addition, lipid addition, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeling component. Also, polypeptides containing, for example, one or more analogs of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are included in this definition. It is understood that the polypeptide may occur as a single chain or as associated chains.

[0037]

[0075] "Identical" refers to the relationship between two proteins that possess a common sequence, including protein sequences from the same superfamily in the same species of organism and homologous proteins from different species of organism, in all of their grammatical forms and spelling variations. Such proteins (and their coding nucleic acids) have sequence homology that is reflected by their sequence similarity, either in terms of % identity or by the presence and conserved positions of specific residues or motifs. However, in general usage and in this application, the term "identical" may, particularly (but not exclusively) when modified by a percentage, refer to sequence similarity, and may or may not be related to common evolutionary origin.

[0038]

[0076] The term "sequence similarity" refers to the degree of identity or correspondence between nucleic acid or amino acid sequences, in all of their grammatical forms, which may or may not share a common evolutionary origin. "Percent sequence identity" or "identical to %" for a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences to achieve the maximum % sequence identity and introducing gaps if necessary, where any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity % can be achieved in a variety of ways within the scope of the art, using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0039]

[0077] As used herein, an "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or fragment thereof) is one that, due to its source of origin or derivation, (1) is not associated with one or more of the naturally associated components that accompany it in its native state, (2) does not substantially contain one or more other molecules from the same species, (3) is expressed by cells from a different species, or (4) is a molecule that does not occur naturally.

[0040]

[0078] As used herein, "purify" and its grammatical variants, whether complete or partial, refer to the removal of at least one impurity from a mixture containing a polypeptide and one or more impurities, and the removal thereby improves the level of purity of the polypeptide in the composition (i.e., by reducing the amount (ppm) of impurities in the composition).

[0041]

[0079] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminating substances), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0042]

[0080] The terms "patient", "subject", or "individual" are used interchangeably herein and refer to either a human or a non-human animal. These terms include mammals such as humans, non-human primates, research animals, livestock animals (including cows, pigs, camels, etc.), companion animals (e.g., dogs, cats, other domesticated animals, etc.), and rodents (e.g., mice and rats). In some examples, the subject is a human who is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old.

[0043]

[0081] In one example, the subject has or is at risk of developing an eye disease. Eye diseases include, but are not limited to, retinitis pigmentosa, rod-cone dystrophy, Leber congenital amaurosis, Usher syndrome, Bardet-Biedl syndrome, Best disease, retinal detachment, Stargardt disease (autosomal dominant or autosomal recessive), untreated retinal detachment, pattern dystrophy, cone-rod dystrophy, color vision abnormalities, albinism, S cone opsin excess syndrome, diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy, congenital stationary night blindness, glaucoma, or retinal vein occlusion. In another example, the subject has or is at risk of developing glaucoma, Leber hereditary optic neuropathy, lysosomal storage disease, or peroxisomal disease. In some examples, the subject exhibits clinical signs of an eye disease.

[0044]

[0082] In some examples, the subject has or is at risk of developing a kidney disease or complication. In some examples, the kidney disease or complication is associated with AMD or aHUS. In some examples, the subject has or is at risk of developing AMD or aHUS.

[0045]

[0083] Clinical signs of an eye disease include, but are not limited to, reduced peripheral vision, reduced central (reading) vision, reduced night vision, loss of color vision, reduced visual acuity, reduced photoreceptor function, and pigmentary changes. In one example, the subject exhibits degeneration of the outer nuclear layer (ONL). In another example, the subject is diagnosed with an eye disease. In yet another example, the subject has not yet exhibited clinical signs of an eye disease.

[0046]

[0084] As used herein, the terms "prevent", "preventing", and "prevention" refer to the prevention of recurrence or onset of a disease or condition (e.g., an eye disease) in a subject, or reduction in one or more symptoms of the disease or condition, as a result of administration of a treatment (e.g., a prophylactic or therapeutic agent). For example, with respect to administration of a treatment to a subject for an infectious disease, "prevent", "preventing", and "prevention" refer to inhibition or reduction in the onset or initiation of a disease or condition (e.g., an eye disease) in the subject, or prevention of recurrence, initiation, or onset of one or more symptoms of the disease or condition (e.g., an eye disease), resulting from administration of the treatment (e.g., a prophylactic or therapeutic agent) or a combination of treatments (e.g., a combination of prophylactic or therapeutic agents). In some examples, prevention may refer to the result of administering a polypeptide described herein to a patient who does not have a disease or condition, or who does not exhibit signs or symptoms of a disease or condition.

[0047]

[0085] "Treating" a condition or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. For a disease or condition (e.g., an eye disease), treatment refers to reduction or improvement in the progression, severity, and / or duration of an infectious disease (e.g., an eye disease or symptoms associated therewith), or improvement in one or more symptoms resulting from administration of one or more treatments (including, without limitation, administration of one or more prophylactic or therapeutic agents).

[0048]

[0086] "Administering" a substance, compound or agent to a subject, or the "administration" thereof, can be effected using one of a variety of methods known to those skilled in the art. For example, a compound or agent can be administered intravitreally or subretinally. In certain instances, a compound or agent is administered intravitreally. In some examples, administration can be local. In other examples, administration can be systemic. Also, administration can be effected, for example, once, a plurality of times, and / or over one or more extended periods. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who teaches a patient to self-administer a drug, or to have another person administer a drug to the patient, and / or provides a prescription for a drug to the patient, is administering the drug to the patient.

[0049]

[0087] Each example described herein can be used individually or in combination with any other example described herein.

[0088] The therapeutic compositions described herein can include one or more therapeutic peptides having a proteoglycan-binding activity equal to or greater than that of the proteoglycan-binding activity of CFH or FHL-1, and providing improvement in complement inhibition and reduction in apolipoprotein binding in the eye. For example, the therapeutic compositions described herein can include one or more therapeutic peptides comprising the human FHL-1 protein (e.g., SEQ ID NO: 2), and / or one or more engineered variants of FHL-1, such as any of those described in SEQ ID NOs: 3-137 (e.g., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17... SEQ ID NO: 137). Accordingly, in some examples, therapeutic compositions of recombinant FHL-1 having an amino acid sequence similar or identical to native FHL-1 (e.g., SEQ ID NO: 2), and methods of using recombinant FHL-1 to treat a patient described herein are described herein.

[0050]

[0089] Figures 1A-1C schematically illustrate the therapeutic activity of engineered variants described herein and suggest a mechanism by which CFH or an engineered variant thereof regulates complement activation and lipid deposition in disease. In Figure 1A (“normal” case), the proteoglycan-binding activity of the CFH protein localizes CFH-mediated complement inhibition (CFH SCR 7 domain binding to proteoglycan) to the ocular surface and inhibits apolipoprotein binding (HDL). In some cases, e.g., some forms of AMD, functional problems with the patient's CFH protein and / or defects in CFH expression or other functions impair the binding of the CFH protein to proteoglycan, reduce complement inhibition at the ocular surface, and accumulate apolipoproteins. Without being bound by a particular theory, the methods and devices described herein may include a therapeutic protein in which engineered FHL-1 variant activity (engineered to improve proteoglycan binding) results in improved complement inhibition and reduced apolipoprotein binding at the ocular surface. This may result in alleviation of AMD symptoms and / or reversal of the adverse effects of AMD.

[0051]

[0090] The engineered variants of FHL-1 described herein can be configured to enhance proteoglycan-binding affinity / activity without significantly reducing the permeability of the engineered variant of FHL-1 to Bruch's membrane in the eye, e.g., by restricting the molecular weight. The engineered variants of FHL-1 described herein can also be configured to reduce the risk of immunogenicity of the engineered variant of FHL-1.

[0052]

[0091] The engineered variants of FHL-1 can include one or more duplications of the first, second, third, fourth, fifth, sixth, and seventh short consensus repeats (SCRs) of CFH (referred to as SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, and SCR7) or the short consensus repeats of FHL-1 (referred to as SCR6 and SCR7). Collectively, these regions can be referred to as CFH SCR1-7 (or SCR1-7) or FHL-1 SCR6-7 (or SCR6-7). All of these regions can contribute to GAG binding activity and can be used as SCR1-7 units as a whole. For example, in some variant forms, the therapeutic polypeptides described herein can include a protein sequence that substantially corresponds to native FHL-1 (e.g., having 85% or more identity, 90% or more identity, 95% or more identity, etc.) and is linked to SCR1-7 or SCR6-7 units via a linker region (e.g., a Gly-Ser linker region or a poly-Ala linker region), as shown in FIG. 2. Alternatively, as shown in FIG. 2, the engineered FHL-1 variant can include a protein sequence that substantially corresponds to CFH (e.g., 85% or more identity, 90% or more identity, 95% or more identity, etc.) and is linked to SCR1-7 units via a linker region (e.g., a Gly-Ser linker region). FIG. 2 shows an engineered variant of FHL-1 that includes FHL-1 (e.g., native FLH-1), but other engineered variants of FHL-1 can include other CFH or other splice variants of CFH. The therapeutic polypeptides shown in FIG. 2 can be referred to herein as engineered variants of FHL-1.

[0053]

[0092] The linker region of the FHL-1 variant described herein may include a linker to which one or more junctions are linked. The junction of the linker region may be linked to a linker (e.g., a Gly-Ser linker or a poly-Ala linker) between the linker and the CFH SCR1-7 unit. The junction of the linker region may be linked to a linker (e.g., a Gly-Ser linker or a poly-Ala linker) between the linker and the FHL-1 SCR6-7 unit. As shown in Figure 2, the engineered variant of FHL-1 may include a linker region having a linker between two junctions. In some examples, the junction may include an FHL-1 SCR7 junction linked to the linker and the CFH SCR1-7 unit. Alternatively, the junction may include a CFH SCR6 junction linked to the linker and the FHL-1 SCR6-7 unit. The linker region may include a peptide such as the peptides described in SEQ ID NO: 4 to SEQ ID NO: 16. In some examples, the linker region includes a combination of one or more peptides described in SEQ ID NO: 4 to 16. For example, the linker may include a junction peptide described in SEQ ID NO: 4 to SEQ ID NO: 10 that is linked to the linker and the N-terminal CFH SCR1-7 unit. The linker may further or alternatively include a junction peptide described in SEQ ID NO: 13 to SEQ ID NO: 16 that is linked to the linker and the C-terminal FHL-1 SCR6-7 unit. The linker may also include a first junction peptide described in SEQ ID NO: 4 to SEQ ID NO: 10 that is linked to the linker and the N-terminal CFH SCR1-7 unit, and a junction peptide described in SEQ ID NO: 13 to SEQ ID NO: 16 that binds to the linker and the C-terminal FHL-1 SCR6-7 unit.

[0054]

[0093] Any suitable linker region can be used as part of an engineered variant of FHL-1. For example, poly-Ala (e.g., AAA, [EAAAK]3) or poly-GlySer linkers (e.g., [GGGGS]3GGGGSGGGGSGGGGS, GGSGGSGGSGGS, GGGGSGGGGS, etc.) or poly-Gly linkers (e.g., GGG) can be used. The length of the linker can, as described herein, allow for the simultaneous binding of multiple SCR domains to cell surface glycosaminoglycan (GAG). This is schematically illustrated in Figure 3, which shows how an engineered variant of FHL-1 can bind to multiple GAGs simultaneously, either by binding to two different GAGs or by binding to the same GAG. As shown, the engineered variant of FHL-1 also includes a binding region that binds to C3b. In some examples, only a single binding region for C3b is included. In some examples, multiple C3b binding regions can be included (e.g., by including multiple repeats of SCR). Figure 3 illustrates two potential ways in which a CFH variant with overlapping surface binding domains can function.

[0055]

[0094] The engineered variants of FHL-1 described herein can generally have two or more tandem repeats of the SCR6-7 unit separated by linkers (e.g., GGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc.). The molecular weight of the engineered variants of FHL-1 can be between about 64 kDa and about 100.0 kDa.

[0056]

[0095] Figure 4 shows the sequence comparison of CFH (see SEQ ID NO: 1 and SEQ ID NO: 133) and FHL-1 (see SEQ ID NO: 2), and illustrates the short consensus repeats (SCRs) and the SCR junction sequences between each short consensus repeat. The alignment of the CFH sequence and the FHL-1 sequence in Figure 4 includes examples from the SCR1 domain to the SCR9 domain (designated as SCR1-SCR9). The sequences shown in Figure 4 illustrate the SCR junctions (shown as enclosed regions) in the CFH protein and the FHL-1 protein, and the unique C-terminus (SFTL) of FHL-1, which were used in the construction of the variant sequences described herein. The signal sequence (MRLLAKIICLMLWAICVA) common to both CFH and FHL-1 is highlighted.

[0057]

[0096] Figure 5 schematically illustrates other examples of engineered variants of FHL-1 described herein. The variant can include an N-terminal constant region comprising the CFH SCR1-7 region, followed by a variant of the FHL-1 SCR7 junction sequence, followed by a variant of the linker sequence, followed by a different variant of the CFH SCR6 junction sequence, followed by a C-terminal constant region comprising the FHL-1 SCR6-7 region. In some examples, as illustrated in Figure 5, the therapeutic polypeptides described herein can include a linker. Alternatively, a subset of the peptide combinations described herein do not include a linker. The polypeptide can include a linker having junctions (e.g., the FHL-1 SCR7 junction and the CFH SCR6 junction). The schematic shown in Figure 5 summarizes a matrix of variants tested to identify ways to add additional surface-binding domains to FHL-1.

[0058]

[0097] In some examples, the engineered variant of FHL-1 comprises the full-length CFH SCR1-7 sequence (SCR1-7 or CFH), to which one additional FHL-1 SCR7 unit is linked (e.g., SEQ ID NO: 98). For example, SEQ ID NO: 21 comprises CFH SCR1-7 linked via a linker region that includes an FHL-1 SCR7 junction (e.g., SEQ ID NO: 7), linked to a CFH SCR6 junction (e.g., SEQ ID NO: 13), and then to a GlySer linker (e.g., SEQ ID NO: 11) that is linked to an FHL-1 SCR6-7 unit (e.g., SEQ ID NO: 17). SEQ ID NO: 37 comprises CFH SCR1-7 linked via a linker region that includes an FHL-1 SCR7 junction (e.g., SEQ ID NO: 7), linked to a CFH SCR6 junction (e.g., SEQ ID NO: 14 LKP), and then to a GlySer linker (e.g., SEQ ID NO: 11) that is linked to an FHL-1 SCR6-7 unit (e.g., SEQ ID NO: 17). The example illustrated in FIG. 5 provides a library of peptide combinations that can be included in the therapeutic polypeptides described herein. As described in SEQ ID NOS: 1-137, the polypeptide can include all of the peptide regions from the N-terminus to the C-terminus. Alternatively, the therapeutic polypeptides described herein can include fewer peptide regions than all of the peptide regions described herein, from the N-terminus to the C-terminus.

[0059]

[0098] All of these engineered variants can be expressed in a soluble form, such as transient CHO expression, in a cell-based expression system (e.g., bacteria, insect, mammalian, etc.). The therapeutic peptides described herein (e.g., engineered variants of FHL-1) can be produced in any suitable protein expression system, including cell-based or in vitro expression systems.

[0060]

[0099] Engineered variants of FHL-1 can be engineered to include a linker, as described herein, based on the integrity of the resulting protein. Analysis of protein integrity was considered, and in FIG. 6, examples of engineered variants of FHL-1 and their relative expression of full-length protein versus cleaved protein as measured by Western blot analysis are shown. Here, the engineered variant sequences of FHL-1 were cloned into the pcDNA3.1(+) vector and transiently expressed using the expi-CHO expression system at a 0.8 mL scale in a 96-well culture format. The culture supernatant was collected on day 7 after transfection. Protein expression and integrity were analyzed by Western blot using an anti-CFH antibody. The amount of full-length protein was represented by the intensity of the 50 kDa band, and the cleaved protein was represented by combining the intensities of the 38 kDa and 35 kDa bands. The % of full-length protein was expressed as the intensity of the 50 kDa band divided by the sum of the 50 kDa, 38 kDa, and 35 kDa bands, multiplied by 100. In some examples, engineered variants of FHL-1 can have different protein integrity. For example, variants containing a poly-Ala linker (e.g., [EAAAK]3 linker) can have lower protein integrity compared to variants containing a GlySer linker (e.g., [GGGGS]3 linker) or variants without a linker. FIG. 6 illustrates an example of a functional screen to identify variant sequences that result in high levels of intact protein expression. In this example, constructs containing a GlySer linker produced higher levels of full protein than constructs containing an EAAAK linker or constructs without a linker.

[0061]

[0100] Figures 7A and 7B illustrate the transient expression of representative engineered variants of FHL-1 described herein. This data shows that the GlySer linker construct (underlined) results in the expression of nearly intact proteins. Here, the variant sequences of FHL-1 were cloned into the pcDNA3.1(+) vector and transiently expressed using the expi-CHO expression system standard protocol at a 0.8 mL scale in a 96-well culture format. The culture supernatant was collected on day 7 after transfection. The culture supernatant was analyzed by non-reducing SDS-PAGE using a 4-12% Bis-Tris gel. Protein expression and integrity were analyzed by Western blot using an anti-CFH antibody (e.g., clone OX-23). The amount of full-length protein was represented by the intensity of the 50 kDa band, and the cleaved protein was represented by combining the intensities of the 38 kDa and 35 kDa bands. Figure 7A shows a subset of variants containing GlySer and polyAla linkers (e.g., SEQ ID NO: 97, SEQ ID NO: 93, SEQ ID NO: 90, SEQ ID NO: 89, SEQ ID NO: 87, SEQ ID NO: 86, SEQ ID NO: 83, SEQ ID NO: 81, SEQ ID NO: 78, SEQ ID NO: 76, SEQ ID NO: 45, SEQ ID NO: 42, SEQ ID NO: 41, SEQ ID NO: 38, SEQ ID NO: 34, SEQ ID NO: 32, SEQ ID NO: 30, SEQ ID NO: 28, SEQ ID NO: 25, SEQ ID NO: 21, and SEQ ID NO: 18). Variants containing the GlySer linker are underlined and variants containing the poly-Ala linker are shown in italics. Figure 7B shows the expression of representative engineered variants described herein that do not contain linker sequences (e.g., SEQ ID NOs: 106-99).

[0062]

[0101] The junctions of the linker region (e.g., FHL-1 SCR7 and CFH SCR6) were examined for their effects on protein integrity. Figures 8A and 8B illustrate the results showing the amount of full-length protein expressed on day 7 after transfection for a subset of engineered variants of FHL-1 containing a GlySer linker, as measured by Western blot analysis (anti-CFH antibody). In particular, a representative FHL-1 construct containing a GlySer linker was transiently expressed using an expi-CHO expression system using a standard protocol on a 0.8 mL scale in a 96-well culture format. The culture supernatant was collected on day 7 after transfection. Protein integrity was analyzed by Western blot using an anti-CFH antibody (clone OX-23). The amount of full-length protein was represented by the intensity of the 50 kDa band, and the cleaved protein was represented by combining the intensities of the 38 kDa and 35 kDa bands. The % of full-length protein was represented as the intensity of the 50 kDa band divided by the sum of the 50 kDa, 38 kDa, and 35 kDa bands, multiplied by 100. Figure 8A illustrates a comparison of examples of engineered variants of FHL-1 containing the same FHL-1 SCR7 junction sequence. Figure 8B illustrates a comparison of examples of engineered variants of FHL-1 containing the same CFH SCR6 junction sequence. Both graphs in Figures 8A and 8B show the mean ± SEM of a set of variants containing the same FHL-1 SCR7 or CFH SCR6 junction sequence (n = 5 per group except for IRVSF = 4). As shown, sequence analysis identified which SCR7 junction sequences improved the protein integrity of the expression construct more. All SCR6 junction sequences tested had similar expression integrity. Differences between groups were analyzed by one-way ANOVA and Tukey's multiple comparison test. Of note, engineered FHL-1 variants containing an FHL-1 SCR7 junction comprising I, IR, IRV, IRVS, IRVSF, or IRVSFT may have significantly higher protein integrity compared to variants containing an FHL-1 SCR7 junction comprising IRVSFTL or variants without an SCR7 junction sequence.No significant difference in protein integrity was observed in any of the tested CFH SCR6 junction arrays.

[0063]

[0102] The linkers of the engineered variants of FHL-1 described herein can affect heparin binding activity. Figure 9 illustrates the heparin binding activity of representative engineered variants of FHL-1. Many constructs containing GlySer linkers showed a more than two-fold improvement in heparin binding activity compared to constructs containing poly-Ala linkers or constructs without linkers. Some EAAAK linker constructs also showed improved heparin binding activity, but none of the constructs without linkers performed well in this assay. For example, the FHL-1 variant sequences were cloned into the pcDNA3.1(+) vector and transiently expressed using the expi-CHO expression system standard protocol at a 0.8 mL scale in a 96-well culture format. The culture supernatant was collected on day 7 after transfection and tested in a heparin binding ELISA. For each construct, 50 μL of the expression culture was incubated on plates coated with heparin together with a reference standard of purified FHL-1 protein at 0 - 2000 nM. Heparin binding activity was measured with a mouse anti-CFH antibody (e.g., OX23), followed by an HRP-conjugated anti-mouse secondary antibody, and read via a TMB substrate. The graph in Figure 9 depicts the average FHL-1 molar equivalent of the protein bound for each expression construct. The dashed lines indicate 100% and 200% values for the FHL-1 native sequence construct. It should be noted that some examples of engineered variants of FHL-1 containing GlySer linkers can have increased heparin binding activity compared to variants containing poly-Ala linkers or variants without linkers.

[0064]

[0103] The engineered variants of FHL-1 described herein may show differences in protein integrity and expression levels. Figures 10A and 10B are graphs representing data showing the integrity and expression levels of full-length proteins of representative examples of engineered variants of FHL-1 containing GlySer linkers and different FHL-1 SCR7 junction sequences. The graphs of Figures 10A and 10B illustrate data for FHL-1 variant sequences for seven constructs transiently expressed using the 0.8 mL scale expi-CHO expression system high-titer protocol in a 96-well culture format. The culture supernatants were collected on day 13 post-transfection. This is the same analysis as shown in Figure 8, but using replicates of a selected number of top hits from the initial screening and expressing them for a longer period of time. This test shows that the IRVS sequence of the SCR7 junction is better than other junctions in optimizing expression levels and construct integrity.

[0065]

[0104] Figure 10A is a bar graph showing the amount of full-length protein expressed 13 days after transfection for a subset of engineered variants of FHL-1 containing GlySer linkers and different FHL-1 SCR7 junction sequences. For example, the variants represented by Figure 10A include a comparison of sets of variants containing the same FHL-1 SCR7 junction sequence (e.g., SEQ ID NO: 71 vs. SEQ ID NO: 28 / SEQ ID NO: 54 vs. SEQ ID NO: 21 / SEQ ID NO: 37 vs. SEQ ID NO: 20 / SEQ ID NO: 84). Protein integrity was analyzed by Western blot using an anti-CFH antibody (e.g., clone OX-23). The amount of full-length protein was represented by the intensity of the 50 kDa band, and the cleaved protein was represented by combining the intensities of the 38 kDa and 35 kDa bands. The % of full-length protein was represented as the intensity of the 50 kDa band divided by the sum of the 50 kDa, 38 kDa, and 35 kDa bands, multiplied by 100. The graph in Figure 10A shows the mean ± SEM for groups of constructs containing a common FHL-1 SCR7 junction sequence. Of note, variants containing an FHL-1 SCR7 junction comprising IR, IRV, or IRVS may have significantly higher protein integrity compared to variants containing an FHL-1 SCR7 junction comprising the IRVSF sequence.

[0066]

[0105] Figure 10B is a bar graph showing the amount of protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing GlySer linkers and different FHL-1 SCR7 junction sequences, as measured by interpolation of Western blot band intensity against a reference standard of purified FHL-1 protein. In Figure 10B, the expression levels of different constructs were measured by Western blot (e.g., OX-23 antibody). The intensity of the monomer band was interpolated against a reference standard of purified FHL-1 protein. It should be noted that variants containing an FHL-1 SCR7 junction comprising IRVS may have significantly higher protein expression levels compared to variants containing an FHL-1 SCR7 junction comprising IR, IRV, or IRVSF.

[0067]

[0106] Figures 11A and 11B are graphs representing data showing the integrity and expression levels of full-length proteins of representative examples of engineered variants of FHL-1 containing GlySer linkers and different CFH SCR6 junction sequences. Variant sequences of FHL-1 for seven constructs containing GlySer linkers were transiently expressed using the expi-CHO expression system high-titer protocol at a 0.8 mL scale in a 96-well culture format (n = 3 per construct). Culture supernatants were collected on day 13 after transfection for both variants in Figures 11A and 11B. This is the same analysis as shown in Figure 8, but using replicates of a selected number of top hits from the initial screening and expressing them for a longer period. This study revealed that the sequences of LKP and TLKP of the SCR6 junction result in more optimal levels of expression of the full-length construct.

[0068]

[0107] Figure 11A is a bar graph showing the amount of full-length protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing CFH SCR6 junction sequences different from the GlySer linker. For example, the variants represented by Figure 11A include a comparison of a set of variants containing the same CFH SCR6 junction sequence (e.g., SEQ ID NO: 84 vs. SEQ ID NO: 71 vs. SEQ ID NO: 54 vs. SEQ ID NO: 37 / SEQ ID NO: 38 vs. SEQ ID NO: 20 / SEQ ID NO: 21). Protein integrity was analyzed by Western blot using an anti-CFH antibody (e.g., clone OX-23). The amount of full-length protein was represented by the intensity of the 50 kDa band, and the cleaved protein was represented by combining the intensities of the 38 kDa and 35 kDa bands. The % of full-length protein was represented as the intensity of the 50 kDa band divided by the sum of the 50 kDa, 38 kDa, and 35 kDa bands, multiplied by 100. The graph shows the mean ± SEM of the groups. Differences between groups were analyzed by one-way ANOVA and Tukey's multiple comparison test. Of note, variants containing a CFH SCR6 junction comprising P, KP, LKP, or TLKP had significantly higher protein integrity compared to variants containing a CFH SCR6 junction without a sequence.

[0069]

[0108] Figure 11B is a bar graph showing the amount of protein expressed on day 13 after transfection for a subset of engineered variants of FHL-1 containing CFH SCR6 junction sequences different from the GlySer linker, as measured by interpolation of WB band intensity against a reference standard of purified FHL-1 protein. A set of variants containing the same CFH SCR6 junction sequence was compared. Of note, variants containing a CFH SCR6 junction sequence comprising LKP or TLKP had significantly higher protein expression levels compared to variants containing the junction sequences P, KP, or variants without a sequence.

[0070]

[0109] The table of FIG. 12 summarizes the results of an example of protein expression of representative engineered variants of FHL-1 containing a GlySer linker related to the junction of the linker region. For example, an optimal junction can confer significantly improved properties. From a matrix of 8 SCR7 junction sequences and 5 SCR6 junction sequences tested (40 variants), two combinations, IRVS+LKP (Var020) and IRVS+TLKP (Var004), were identified as more optimal sequences. As shown in FIG. 12, the FHL-1 SCR7 junction can be optimal in an engineered construct of FHL-1 having SEQ ID NO: 7 (e.g., IRVS). Engineered constructs of FHL-1 containing the CFH SCR6 junctions of SEQ ID NO: 13 and SEQ ID NO: 14 can be optimal as linker junctions of the polypeptide, as described above, at SEQ ID NO: 21 (e.g., Var004) and SEQ ID NO: 37 (e.g., Var020) in FIGS. 8, 10A, 10B, 11A, and 11B. In some examples, a linker junction can include a combination of a linker and one or more junctions, based on significantly improved properties. In some examples, an optimal linker junction can include a combination of the peptide of SEQ ID NO: 11 linked to the peptides of SEQ ID NO: 7 and SEQ ID NO: 13. In some examples, an optimal linker junction can include a combination of the peptide of SEQ ID NO: 11 linked to the peptides of SEQ ID NO: 7 and SEQ ID NO: 14. In some examples, an optimal linker junction can include any combination of the peptide sequences described herein.

[0071]

[0110] Any of the therapeutic polypeptides described herein can be assayed and / or functionally characterized to show that it has either or both of the GAG and C3b binding properties sufficient to localize complement inhibition to the ocular surface and inhibit apolipoprotein binding (HDL). For example, the therapeutic polypeptides described herein can be shown to bind to either or both of C3b and GAG by one or more assays, including, for example, showing the C3b and GAG binding kinetics by ELISA. Generally, such assays can be used to confirm that the binding kinetics of the therapeutic polypeptide (e.g., recombinant FHL-1 or engineered variants of FHL-1) to C3b and GAG are equivalent to or better than those of native CFH and / or FHL-1.

[0072]

[0111] In FIGS. 13A and 13B, heparin binding affinity was measured by ELISA on culture supernatants from CHO cells transfected with representative examples of purified CFH, purified FHL-1, and FHL-1 or engineered variant constructs collected after 8 days of expression at 30 mL scale. Analytes were incubated on plates coated with heparin, and FH / variant binding was measured with an anti-CFH (clone OX-23) antibody and an HRP-conjugated anti-mouse secondary antibody. Signals were detected by incubation with TMB substrate. EC 50 values were measured by 4PL non-linear regression. FIG. 13A shows the mean regression fit and 95% confidence intervals of assay absorbance values from 25 independent assays. The table in FIG. 13B summarizes the EC 50 values over multiple experiments. Notably, the engineered variants of FHL-1 of SEQ ID NOs: 21 and 37 were found to have improved heparin binding activity compared to FHL-1 or CFH, and the polypeptide of SEQ ID NO: 21 was found to have increased heparin binding activity compared to the polypeptide of SEQ ID NO: 37. The results of the assays shown in FIGS. 13A-13B suggest that the Var004 sequence has superior heparin binding activity compared to Var020.

[0073]

[0112] Representative examples of engineered variants of FHL-1 (e.g., the polypeptide of SEQ ID NO: 21) were compared for C3b binding activity with CFH and FHL-1, as shown in FIGS. 14A and 14B. The graph in FIG. 14A shows the average absorbance values from multiple independent C3b binding assays, and the table in FIG. 14B summarizes the EC 50 values over multiple experiments. Generally, the data were obtained by measuring binding on C3b-coated plates by ELISA for the test protein (either as the purified protein or as the culture supernatant of CHO cells transfected with an expression vector encoding the test protein). C3b binding activity was measured by absorbance at 450 nM. The mean ± SEM absorbance values are shown with a non-linear sigmoid 4PL fit. Notably, the engineered variants of FHL-1 (e.g., Var004, SEQ ID NO: 21) have increased C3b binding activity compared to native FHL-1. FIGS. 14A - 14B show that the Var004 construct did not impair C3b binding activity compared to the control protein.

[0074]

[0113] As shown in Figure 15, Western blot analysis was performed using an anti-C3b antibody that shows surface-dependent complement inhibitory activity of engineered variants of FHL-1 (e.g., Var004, SEQ ID NO: 21) and control proteins (FHL-1 and CFH). 2 million expi-CHO cells / test were resuspended in PBS containing purified CFH, FHL-1 culture supernatant, or engineered variant of FHL-1 (e.g., Var004) culture supernatant at [12.3 nM, 37 nM, 111 nM, or 333 nM]. Control lane 1 contains cells, conditioned medium, C3b, and CFI. Control lane 2 contains C3b and CFI. Control lane 3 contains C3b, CFI, and CFH. FH / variant binding was performed at 37°C for 30 minutes. The cells were then washed thoroughly and resuspended in PBS containing 0.5 μg of C3b and 1.5 μg of CFI per test. The cell / C3b / CFI mixture was incubated at 37°C for 15 minutes. The reaction supernatant was then analyzed by reducing SDS-PAGE and the cleavage of C3b was monitored by anti-C3b Western. Four C3b bands were quantified: C3b a’ (˜130 kDa), C3b β (70 kDa), iC3b a’ (68 kDa), and iC3b a’ (43 kDa). Representative engineered variants of FHL-1 have high surface-dependent complement inhibitory activity compared to FHL-1 and CFH, as demonstrated by an increase in the accumulation of C3b cleavage products.

[0075]

[0114] Figure 16 illustrates the surface-dependent complement inhibitory activities of representative engineered variants of FHL-1 (e.g., Var004 / SEQ ID NO: 21) and the control proteins FHL-1 and CFH, measured by Western blot analysis using anti-C3b antibody. The activity is represented as the ratio of iC3b to total C3b produced at each tested protein concentration, measured by the intensity of the bands representing intact C3b and cleaved C3b: [a’68 + a’43] / [a’68 + a’43 + a’ + β]. Two million expi-CHO cells / test were resuspended in PBS containing purified CFH, FHL-1 CHO culture supernatant, or engineered variant of FHL-1 (e.g., Var004) CHO culture supernatant at [0, 12.3 nM, 37 nM, 111 nM, or 333 nM]. Conditioned CHO medium was used as a negative control for the engineered variant of FHL-1 and FHL-1 culture supernatant. FH / variant binding was performed at 37°C for 30 minutes. Cells were then washed thoroughly and resuspended in PBS containing 0.5 μg of C3b and 1.5 μg of CFI / test. The cell / C3b / CFI mixture was incubated at 37°C for 15 minutes. The reaction supernatant was then analyzed by reducing SDS-PAGE and C3b cleavage was monitored by anti-C3b Western. Four C3b bands were quantified: C3b a’ (≈130 kDa), C3b β (70 kDa), iC3b a’ (68 kDa), and iC3b a’ (43 kDa). The graph shows the mean ± SEM from four independent experiments. Differences between groups were analyzed by two-way ANOVA using Tukey's multiple comparisons. The iC3b / total C3b ratio was calculated by dividing the total intensity of the a’68 + a’43 bands by the total intensity of the a’68, a’43, a’ (≈130 kDa), and β (70 kDa) bands. Representative engineered variants of FHL-1 have high surface-dependent complement inhibitory activity compared to FHL-1 and CFH, as demonstrated by an increase in the production of C3b cleavage products. Figures 15 and 16 demonstrate the improved surface-dependent complement inhibitory activity by Var004. This was an important design feature intended in the screening campaign. Complement inhibition is measured by the ability of constructs to act as cofactors for CFI, which promotes the cleavage of C3b to iC3b.

[0076]

[0115] The therapeutic polypeptides tested in FIGS. 13A - 16 are examples of engineered variant polypeptides of FHL-1 for use as described herein, and according to these assays, may exhibit similar or superior properties.

[0077]

[0116] The therapeutic polypeptides described herein (e.g., engineered variants of FHL-1) can be glycosylation-engineered variants of FHL-1 having one or more amino acid substitutions. Some examples of representative amino acid substitutions are shown in FIGS. 17A and 17B, which illustrate libraries of amino acid substitutions within the CFH SCR1 - 7 region, the FHL-1 SCR7 junction region, and the GlySer linker region. Thus, FIGS. 17A - 17B illustrate a glycoengineering strategy for identifying point mutations that result in effective glycosylation addition either within FHL-1 or within the GlySer linker sequence. For example, 15 variants of the FHL-1 SCR1 - 7 framework contain single amino acid substitutions at positions L6S, L6T, T16N, D21N, D258N, G286N, A289S, A289T, Y334N, H354N, Y380S, Y380T, A407N, R426N, and S428N. As a representative example, four variants of the engineered variant of FHL-1 of SEQ ID NO: 21 framework contain single amino acid substitutions at positions G431N, G436N, G441N, and G442N. Each amino acid substitution introduces an N-linked glycosylation addition motif (NxS / T). Amino acid substitutions are numbered from the first amino acid of the mature secreted protein (E) after cleavage of the signal transduction sequence (e.g., MGWSCIILFLVATATGVHS).

[0078]

[0117] The Western blot analysis shown in FIGS. 18A and 18B indicates the degree of glycosylation of single amino acid substitutions in the FHL-1 framework after three independent transfections per construct. The data in FIGS. 18A-18B show which point mutations resulted in complete glycosylation, partial glycosylation, or no glycosylation of the parental framework. Arrows indicate the mobility shift between glycosylated (+Gly) and non-glycosylated (-Gly) proteins. Control lane 1 contains purified FHL-1 protein. Examples of glycosylation-engineered variants of FHL-1 were cloned into the pcDNA3.1(+) vector and expressed using the expi-CHO expression system (standard protocol) at a 0.8 mL scale in a 96-well culture format. The culture supernatant was collected on day 6 after transfection. Protein expression and integrity were analyzed by non-reducing Western blot using an anti-CFH antibody (A237). Glycosylation was detected by the shift of the full-length expression band on the gel. Arrows indicate the shift for glycosylated and non-glycosylated bands.

[0079]

[0118] A similar analysis is shown in Figure 19, presenting a representative Western blot (anti-CFH antibody A237) indicating the degree of glycosylation of single amino acid substitutions in the engineered variant Var004 framework of FHL-1. In this example, the substitution at G442N (Var108) is an effective substitution for introducing strong glycosylation within the GlySer linker sequence of the construct. The arrow indicates the mobility shift between the glycosylated (+Gly) and non-glycosylated (-Gly) proteins. Control lane 1 contains purified FHL-1. Again, the glycosylation-engineered variants of the Var004 framework were cloned into the pcDNA3.1(+) vector and expressed using the expi-CHO expression system (standard protocol) at a 0.8 mL scale in a 96-well culture format. The culture supernatant was collected on day 6 post-transfection. Protein expression and integrity were analyzed by non-reducing Western blot using the anti-CFH antibody (A237). Glycosylation was detected by the shift of the full-length expression band on the gel. The arrow indicates the shift of the glycosylated and non-glycosylated bands. The table in Figure 20 represents the percentage of glycosylation of the exemplified engineered variants of FHL-1 assayed as described above. The percentage of glycosylation was calculated by dividing the signal intensity of the glycosylated monomer band by the signal intensities of both the glycosylated and non-glycosylated monomer bands. " * " indicates variant sequences where the glycosylated monomer exceeds 90%. Figure 20 shows the average percentage of glycosylation and the standard deviation (SD) for n = 3 independent transfections per construct. Of note, the representative engineered variants of FHL-1, Var093, Var099, Var100, Var102, and Var108, have monomer glycosylation exceeding 90%. The various substitutions tested to introduce the NxS / T glycosylation motif differed in their effectiveness in promoting glycosylation of the construct.

[0080]

[0119] Additional glycosylation analysis is shown in FIGS. 21A and 21B, and a representative Western blot (anti-CFH antibody A237) reveals the degree of glycosylation for the complex amino acid substitutions in the FHL-1 and Var004 frameworks. This data shows single, double, and triple glycosylation variants on the frameworks of FHL-1 and Var004. Each sequence included represents a construct in which the introduced NxS / T motif is fully glycosylated when the construct is expressed in CHO culture. A mobility shift is evident between the multiply glycosylated variants and the singly glycosylated parental construct. The table in FIG. 21B summarizes the amino acid substitutions present in each representative variant, and the lane labeled "purified FHL-1" contains the expression supernatant from the native FHL-1 sequence as a reference standard. The constructs were cloned into the pcDNA3.1(+) vector and expressed using the expi-CHO expression system (standard protocol) at a 0.8 mL scale in a 96 well culture format. The culture supernatant was collected on day 7 post-transfection. Protein expression and integrity were analyzed by non-reducing Tris-glycine SDS-PAGE followed by Western blot using anti-CFH antibody (A237). Glycosylation was detected by the shift of the full-length expression band on the gel.

[0081]

[0120] Figures 22A and 22B are bar graphs showing the quantification of protein integrity and expression levels measured from Western blot analysis using anti-CFH antibody (A237) for a plurality of glycosylated engineered variants of FHL-1 (e.g., engineered variants of FHL-1 having one or more amino acid substitutions) described herein. This data shows how glycosylation improves the stability and titer of the expression construct. Figure 22A shows the % of full-length monomer quantified by measuring the concentration of the full-length monomer by dividing the concentration measurement of the full-length monomer by the concentration measurement of the entire lane from the monomer to 25 kDa. Figure 22B is a bar graph showing the relative protein expression levels of a plurality of glycosylated variants compared to the engineered variant Var004 of FHL-1. The relative protein expression levels were determined by dividing the band intensity of each variant by the band intensity of Var004 from non-reducing Western blot analysis using anti-CFH antibody A237. The graph shows the mean ± SEM for the D7 supernatant from three transfections on a 0.8 mL scale. A representative Western blot from this experiment is shown in Figure 21A. The differences between the variants and Var004 were analyzed by one-way ANOVA using Dunnett's multiple comparison. Of note, all glycosylated variants (e.g., engineered variants of FHL1 containing one or more amino acid substitutions described herein) have improved protein integrity compared to the representative engineered variant Var004 of FHL-1. Also of note is that Var108 has a significantly improved expression titer compared to Var004.

[0082]

[0121] The table in Figure 23 shows a summary of the quantification of protein integrity and expression levels measured from Western blot analysis using the anti-CFH antibody (A237), as detailed in Figures 21A through 22B, and illustrates which glycosylation sites improve the titer and integrity of the expressed constructs. The % expression of the full-length monomer was quantified by measuring the concentration of the full-length monomer and dividing it by the concentration measurement of the entire lane from the monomer of the A237 Western blot up to 25 kDa. The relative protein expression levels were determined by dividing the band intensity of each variant by the band intensity of Var004. It should be noted that Var108, Var114, and Var115 have protein expression levels more than twice as high as that of Var004.

[0083]

[0122] Compared to CFH and FHL-1, engineered variants such as Var108, Var114, and Var115 can bind to heparin with significantly higher affinity. For example, Figures 24A and 24B show the results of experiments testing the binding of these variants (Var108, Var114, and Var115) of FHL-1 compared to CFH and FHL-1. Figure 24A is a graph showing the average absorbance values of multiple heparin binding assays comparing CFH and FHL-1 with each of Var108, Var114, and Var115. Figure 24B is a table summarizing this data, showing significantly lower EC 50 values for Var108, Var114, and Var115 compared to CFH and FHL-1. As shown in the figure, Var108, Var114, and Var115 each have a heparin binding activity improved by more than 500-fold compared to CFH and FHL-1. This data suggests that these variants may have the ability to bind to surface ligands recognized by the significantly improved CFH SCR7 domain.

[0084]

[0123] As shown in FIGS. 25A and 25B, the engineered glycan variant proteins described herein can have improved C3b binding activity compared to control proteins, indicating that the C3b regulatory activity of CFH / FHL-1 is conserved (and may be slightly better compared to CFH and FHL-1). C3b binding activity is measured by incubating the glycan variant protein on an ELISA plate functionalized with human C3b and detecting the bound protein with an anti-CFH antibody.

[0085]

[0124] The engineered FHL-1 variants can bind to human RPE cells at least as well as the controls (e.g., FHL-1 and CFH). For example, as shown in FIGS. 26A and 26B, variants Var108 and Var114 show improved binding to RPE cells compared to CFH and FHL-1, demonstrating the improved ability of constructs that bind to cells via the SCR7 domain. FIG. 26A is a graph showing the binding activity of test proteins over a range of protein concentrations. FIG. 26B is a table summarizing the results of multiple assays measuring EC 50 and Emax. In this example, ARPE-19 cells were grown to confluence on 96-well tissue culture plates. The cells were serum-starved for 24 hours and incubated for 20 minutes at 37 °C in serum-free medium containing 125 - 4000 nM of CFH, FHL-1, Var108, Var114, or Var115. After washing away unbound protein, the cells were fixed in 2% PFA, blocked, stained overnight with an anti-FH antibody (OX23), followed by Texas Red secondary staining and DAPI nuclear staining. Protein binding was measured by a high-content imager and the signal area was normalized to the DAPI nuclear count. The graph shows the mean ± SEM from multiple experiments. EC 50 and Emax values were calculated by 4PL sigmoid non-linear regression.

[0086]

[0125] Among the variants described herein, in some cases (e.g., Var114), the engineered variants of FHL-1 described herein can inhibit complement activation in a surface-dependent manner. Surprisingly, in some cases, the variants can be better surface-dependent regulators of complement than any of the control proteins, as shown in FIGS. 27A-27B. In FIGS. 27A-27B, ARPE-19 cells were grown to confluence, serum-starved overnight, and then incubated at 37° C. for 20 minutes in serum-free medium containing 62.5-4000 nM CFH, FHL-1, Var108, Var114, or medium alone. Unbound protein was removed by washing the cells with PBS, and the cells were subsequently challenged with FH-depleted human serum (5% serum volume) for 2 hours. After complement challenge, the cells were washed, fixed with 2% PFA, stained with anti-C5b9 antibody (clone AE11) to visualize MAC deposition, and DAPI was used to visualize the cell nuclei. The MAC signal area was normalized to the number of DAPI nuclei. The graph shows the mean ± SEM from multiple experiments. IC 50 and Imax values were calculated by 4PL sigmoid non-linear regression. Note that the Var108 regression was performed at concentrations of 0-2000 nM because activity was lost at a concentration of 4000 nM ( ** ).

[0087]

[0126] Generally, as shown in Figure 28, there is a very strong linear correlation between the improvement of cell binding by Var114 and its ability to regulate complement activation. This supports the design concept behind the FHL-1 variant campaign described above. As shown, the duplication of the SCR7 domain achieves an improvement in complement regulation by giving an increase in cell surface binding activity. Note that Var108 demonstrated an increase in cell binding that was not associated with an increase in complement regulatory activity at concentrations above 2000 nM. Figure 28 shows the binding and complement inhibitory potencies for the measured variants (0 - 4000 nM concentration) of Figures 26A - 26B and 27A - 27B, such as engineered variants of FHL-1 (e.g., Varl08, Var114), and for control proteins (FHL-1, CFH). For each protein tested, the average FH binding signal quantification value was plotted against the average MAC deposition. The correlation was measured by linear regression.

[0088]

[0127] The complement regulatory activities of Var114 and the control proteins were examined in experimental systems that included both liquid-phase and surface-dependent complement activation. The results are shown in Figures 29A - 29B. The engineered variant Va114 has an improved ability to regulate complement activation on RPE cells in this system. ARPE-19 cells were grown to confluence, serum-starved overnight, and then incubated at 37 °C for 2 hours in serum-free medium containing FH-depleted serum (5% serum) and 62.5 - 4000 nM of CFH, FHL-1, or Var114. After complement challenge, the cells were washed, fixed with 2% PFA, stained with anti-C5b9 antibody (clone AE11) to visualize MAC deposition, and with DAPI to visualize cell nuclei. The MAC signal area was normalized to the number of DAPI nuclei. The graph shows the mean ± SEM from multiple experiments. IC 50 values were calculated by 4PL sigmoid non-linear regression.

[0089]

[0128] In one example, Var114 (or related variants) demonstrated excellent ability to inhibit C5a generation (compared to CFH control). This is illustrated in FIGS. 30A - 30B. For example, in FIG. 30A, ARPE - 19 cells were grown to confluence and serum - starved overnight, then incubated at 37°C for 2 hours in serum - free medium containing FH - depleted serum (5% serum) and 62.5 - 4000 nM of CFH, FHL - 1, or Var114. After complement challenge, assay supernatants were collected and C5a levels were analyzed by ELISA. The graph shows mean ± SEM from multiple experiments. IC 50 values were calculated by 4 - parameter logistic (4PL) sigmoid non - linear regression. FIG. 30B illustrates an example of summary data showing the IC 50 values for the inhibition of C5a production by Var114 and CFH as measured by ELISA in the ARPE - 19 cell culture supernatant after complement challenge.

[0090]

[0129] FIGS. 31A - 31B illustrate another example showing the improved cell - surface binding activity of Var114. In this example, iPS - cell - derived RPE cultures were used to support the conclusions shown in FIG. 26. In particular, Var114 has an improved ability to bind to human iPS - derived RPE cells compared to control proteins. In this experiment, human iPS - RPE cells were matured in transwell plates for 4 - 6 weeks. The cells were serum - starved for 24 hours and incubated at 37°C for 20 minutes in serum - free medium containing 62.5 - 4000 nM of CFH, FHL - 1, or Var114. After washing away unbound proteins, the cells were fixed with 2% PFA, blocked, and stained overnight with anti - FH antibody (OX23) and DAPI nuclear stain. The binding signal area was measured. The graph shows mean ± SEM from multiple experiments. EC 50 values were calculated by 4 - parameter logistic (4PL) sigmoid non - linear regression.

[0091]

[0130] Figures 32-34 show the in vivo efficacy of Var108 and Var114 using a rat laser-induced retinal injury model known to induce complement pathway activation. For example, in the experiment summarized by FIG. 32, adult male Brown Norway rats were given laser-induced retinal injury (4 injuries / eye), and immediately thereafter, an engineered variant of 170 pM FHL-1 (e.g., Var108, Var114) or a control protein (e.g., CFH, FHL-1) or vehicle was intravitreally injected. Vitreous humor samples were collected on day 3 after laser irradiation, and C3a levels were measured by ELISA. The graph depicts the mean ± SEM values of C3a normalized to the vehicle control. Differences between groups were measured by one-way ANOVA + Tukey's multiple comparison test. This experimental system uses an established model of complement-induced retinal injury to compare the efficacy of engineered variants of FHL-1 to control proteins. In this system, the engineered variants are effective in reducing the C3a level in the eye compared to the vehicle-treated control.

[0092]

[0131] Figure 33 illustrates the inhibition of MAC deposition by some engineered variants of FHL-1 (e.g., (Var108, Var114)) in a laser-induced retinal injury model that is known to induce complement pathway activation. In this example, adult male Brown Norway rats were given laser-induced retinal injury (4 injuries / eye), and immediately afterwards, 170 pM of an engineered variant of FHL-1 (e.g., Var108, Var114) or a control protein (e.g., CFH, FHL-1) or vehicle was injected intravitreally. Three days after laser irradiation, the eyes of the rats were dissected, and RPE flat mounts were immunostained for MAC deposition with an anti-C5b9 antibody (clone AE11), and lesions were visualized by phalloidin staining. The graph depicts the mean ± SEM MAC signal area within the phalloidin lesion area. Differences between groups were measured by one-way ANOVA + Tukey's multiple comparison test. The established model of laser-induced retinal injury can be used to compare the efficacy of engineered variants of FHL-1 with control proteins. In this system, the modified variants (e.g., Var108 and Var114) are effective in reducing MAC deposition on the RPE (a readout of terminal complement activation).

[0093]

[0132] Figure 34 shows the inhibition of macrophage recruitment by engineered variants of FHL-1 (e.g., Var108 and Var114) and control proteins (e.g., FHL-1 and CFH) in rat RPE of a laser-induced retinal injury model, which is known to induce complement pathway activation. Adult male Brown Norway rats were given laser-induced retinal injury (4 lesions / eye), and immediately afterwards, 170 pM of an engineered variant of FHL-1 (e.g., Var108, Var114) or a control protein (e.g., CFH, FHL-1) or vehicle was injected intravitreally. Three days after laser irradiation, the eyes of the rats were dissected, RPE flat mounts were immunostained for macrophage infiltration with an anti-CD68 antibody, and lesions were visualized by phalloidin staining. The graph depicts the mean ± SEM macrophage signal area within the phalloidin lesion area. Differences between groups were measured by one-way ANOVA + Tukey's multiple comparison test. Thus, the efficacy of the engineered variants of FHL-1 was compared to control proteins using an established model of laser-induced retinal injury. The engineered variants were more effective in suppressing macrophage recruitment (measured by CD68) compared to the control proteins. This demonstrates the immunomodulatory activity of the variants in the context of complement activation in the eye.

[0094] ​

[0133] Any of the engineered variants of FHL-1 described herein can be used as part of a pharmaceutical composition. A pharmaceutical composition comprising an engineered variant of FHL-1 described herein can include one or more pharmaceutically acceptable carriers. The pharmaceutical composition can be suitable for any mode of administration, for example, a mode of administration by intravitreal injection.

[0095]

[0134] In some examples, the composition comprises the polypeptides of SEQ ID NOs: 18 to 137. For example, in some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 3. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 4. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 5. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 6. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 7. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 8. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 9. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 10. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 11. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 12. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 13. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 14. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 15. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 16. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 17. In some examples, the pharmaceutical composition comprises a combination of any two or more peptides having any two sequences of SEQ ID NOs: 3 to 137. In some examples, the pharmaceutical composition comprises three or more peptides having any three or more sequences of SEQ ID NOs: 3 to 137.

[0096]

[0135] In some examples, a pharmaceutical composition comprising any one or more of the peptides of SEQ ID NOs: 3 to 137 described herein and a pharmaceutically acceptable carrier is suitable for administration to a human subject. Such carriers are well known in the art (see, e.g., Remington’s Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). In some examples, a pharmaceutical composition comprising any one or more of the peptides of SEQ ID NOs: 3 to 137 described herein and a pharmaceutically acceptable carrier is suitable for intravitreal injection. In some examples, the pharmaceutical composition is suitable for subretinal delivery. Such pharmaceutically acceptable carriers may be sterile liquids such as water and oils, and the oils include oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Also, saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions can be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may further comprise additional ingredients such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity increasing agents, and the like. The pharmaceutical compositions described herein may be packaged in single unit dosages or in multiple dosage forms. The compositions are generally formulated as sterile and substantially isotonic solutions.

[0136] In one example, a peptide having any one or more of the sequences of SEQ ID NOs: 8 to 137 described herein is formulated into a pharmaceutical composition intended for subretinal or intravitreal injection. Such formulations include a pharmaceutically and / or physiologically acceptable medium or carrier, particularly a medium or carrier suitable for administration to the eye, for example by subretinal injection, such as buffered saline or other buffer solutions, for example HEPES, to maintain the pH at an appropriate physiological level, and optionally the use with other pharmaceutical agents, formulations, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier is typically liquid. Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate buffered saline. In one example, the carrier is an isotonic sodium chloride solution. In another example, the carrier is a balanced salt solution. In one example, the carrier contains Tween. If the product is intended for long-term storage, it can be frozen in the presence of glycerol or Tween 20. In another example, the pharmaceutically acceptable carrier includes a surfactant such as perfluorooctane (Perfluoron solution).

[0097]

[0137] In certain examples of the methods described herein, the pharmaceutical composition described above is administered to a subject by subretinal injection. In other examples, the pharmaceutical composition is administered by intravitreal injection. Other forms of administration that may be useful in the methods described herein include direct delivery to the desired organ (e.g., the eye), oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration, but are not limited thereto. The routes of administration can be combined if desired. In certain examples, the pharmaceutical composition of the present disclosure is administered after administration of an initial loading dose of a complement system protein.

[0098]

[0138] In some examples, the route of administration is selected such that the route of administration reduces the risk of retinal detachment in the patient (e.g., intravitreal or suprachoroidal rather than subretinal). In some examples, when the composition is intended to be administered to an elderly person (e.g., at least 60 years old), intravitreal administration is selected. In certain examples, any of the pharmaceutical compositions disclosed herein is administered intravitreally to a subject. The technique of intravitreal injection is known in the art (see, e.g., Peyman, G.A. et al. (2009) Retina 29(7):875-912; and Fagan, X.J. and Al-Qureshi, S. (2013) Clin. Experiment. Ophthalmol. 41(5):500-7). Briefly, for intravitreal injection, the subject can be prepared for the procedure by dilation of the pupil, disinfection of the eye, and administration of an anesthetic. Any suitable mydriatic known in the art can be used for pupil dilation. Adequate pupil dilation can be confirmed prior to the procedure. Disinfection can be achieved by applying an eye disinfection procedure, such as an iodide-containing solution such as povidone iodine (BETADINE®). Also, a similar solution can be used to clean the eyelids, eyelashes, and any other adjacent tissues (e.g., skin). Any suitable anesthetic, such as lidocaine or proparacaine, can be used at any suitable concentration. The anesthetic can be administered by any method known in the art, including, but not limited to, topical eye drops, gels or jellies, and subconjunctival application of the anesthetic. Prior to injection, a sterilized eyelid speculum can be used to remove the eyelashes from the area. The site of injection can be marked with a syringe. The site of injection can be selected based on the patient's lens. For example, the injection site can be 3-3.5 mm from the limbus in pseudophakic or aphakic patients and 3.5-4 mm from the limbus in phakic patients. The patient can look in the direction opposite to the injection site. During injection, the needle can be inserted perpendicular to the sclera and directed towards the center of the eye. The needle can be inserted such that the tip stops intravitreally rather than in the subretinal space. Any suitable volume known in the art for injection can be used. After injection, the eye can be treated with a disinfectant such as an antibiotic.Also, the eye may be rinsed to remove excess disinfectant.

[0099]

[0139] The composition can be delivered in a volume of from about 0.1 μL to about 1 mL, including all numbers within the range, depending on the size of the area to be treated, the route of administration, and the desired effect of the method. In one example, the volume is about 50 μL. In another example, the volume is about 70 μL. In one example, the volume is about 100 μL. In another example, the volume is about 125 μL. In another example, the volume is about 150 μL. In another example, the volume is about 175 μL. In yet another example, the volume is about 200 μL. In another example, the volume is about 250 μL. In another example, the volume is about 300 μL. In another example, the volume is about 450 μL. In another example, the volume is about 500 μL. In another example, the volume is about 600 μL. In another example, the volume is about 750 μL. In another example, the volume is about 850 μL. In another example, the volume is about 1000 μL.

[0100]

[0140] For example, the dosage can be from about 100 ng / eye to about 10 mg / eye (e.g., about 100 ng / eye, about 150 ng / eye, about 200 ng / eye, about 250 ng / eye, about 300 ng / eye, about 400 ng / eye, about 500 ng / eye, about 600 ng / eye, about 700 ng / eye, about 800 ng / eye, about 900 ng / eye, about 1 μg / eye, about 2 μg / eye, about 3 μg / eye, about 5 μg / eye, about 10 μg / eye, about 15 μg / eye, about 20 μg / eye, about 25 μg / eye, about 30 μg / eye, about 35 μg / eye, about 40 μg / eye, about 50 μg / eye, about 60 μg / eye, about 70 μg / eye, about 80 μg / eye, about 90 μg / eye, about 100 μg / eye, about 120 μg / eye, about 150 μg / eye, about 175 μg / eye, about 200 μg / eye, about 250 μg / eye, about 300 μg / eye, about 350 μg / eye, about 400 μg / eye, about 500 μg / eye, about 750 μg / eye, about 1 mg / eye, about 1.5 mg / eye, about 2 mg / eye, about 2.5 mg / eye, about 3 mg / eye, about 3.5 mg / eye, about 4 mg / eye, about 4.5 mg / eye, about 5 mg / eye, about 5.5 mg / eye, about 6.0 mg / eye, about 6.5 mg / eye, about 7.0 mg / eye, about 7.5 mg / eye, about 8.0 mg / eye, about 8.5 mg / eye, about 9.0 mg / eye, about 9.5 mg / eye, about 10 mg / eye or any range thereof).

[0101]

[0141] Still other dosages and volumes within these ranges can be selected by the attending physician taking into account the subject to be treated, preferably the general condition of a human, the age of the subject, the particular eye disorder, and the extent to which the disorder has progressed if it is progressive. For extraocular delivery, the dosage can be increased according to the scale-up from the retina.

[0102] ​

[0142] Various methods for preventing, treating, arresting, or improving eye disorders and the associated retinal changes are described herein. Any of these methods can include identifying patients who may benefit from one or more of these treatments and / or identifying which one or more of the treatments described herein may be most beneficial for a particular patient. Any of these methods can include determining the dosage to be delivered, the route of delivery, and / or the schedule for delivering one or more doses.

[0103]

[0143] Generally, the method includes administering to a mammalian subject in need of administration of a composition, an effective amount of any of the compositions described herein. For example, treatment of age-related macular degeneration can include localized delivery of a therapeutic composition described herein to the patient's retina. The cells that are the treatment targets in these diseases can include photoreceptor cells in the retina or cells of the RPE under the neurosensory retina. In certain embodiments, the present disclosure provides a method of treating a subject having age-related macular degeneration (AMD), the method comprising administering to the subject any of the compositions described herein.

[0104]

[0144] In certain examples, methods are provided for preventing, arresting, or improving vision loss associated with an eye disorder in a subject. Vision loss associated with an eye disorder refers to any decrease in peripheral vision, central (reading) vision, night vision, daytime vision; loss of color vision; loss of contrast sensitivity; or reduction in visual acuity. The methods and compositions described herein can be directed to increasing photoreceptor function. As used herein, "increasing photoreceptor function" means improving the function of photoreceptors, or increasing the number or proportion of functional photoreceptors, compared to the affected eye (having the same eye disease), the same eye at an earlier time point, the untreated portion of the same eye, or the contralateral eye of the same patient. Photoreceptor function can be evaluated using functional studies conventional in the art, such as ERG or visual field measurements.

[0105]

[0145] For each of the methods described, the treatment can be used to prevent the occurrence of retinal damage or to rescue an eye with a mild or advanced disease. As used herein, the term "rescue" means preventing the progression of the disease to complete blindness, preventing the spread of damage to the cells of an intact eye, improving the damage in the damaged cells of the eye, or providing an improvement in vision. In one example, the composition is administered before the disease becomes symptomatic or before photoreceptor loss. Symptomatic means any of the various retinal changes described above or the onset of vision loss. In another example, the composition is administered after the disease has become symptomatic. In yet another example, the composition is administered after the onset of photoreceptor loss. In yet another example, the composition is administered after the onset of outer nuclear layer (ONL) degeneration. In some examples, it is desirable for the composition to be administered while the bipolar cells that connect the ganglion cells and the optic nerve remain intact. In another example, the composition is administered after the onset of photoreceptor loss. In yet another example, the composition is administered when less than 90% of the photoreceptors are functional or remaining compared to an unaffected eye. In another example, the composition is administered when less than 80% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 70% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 60% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 50% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 40% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 30% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 20% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 10% of the photoreceptors are functional or remaining. In one example, the composition is administered only to one or more regions of the eye. In another example, the composition is administered to the entire eye. In another example, the method includes performing functional and imaging studies to determine the effectiveness of the treatment.These studies include ERG and in vivo retinal imaging as described in the following examples. In addition, visual field studies, perimetry and microperimetry, pupillometry, mobility tests, visual acuity, contrast sensitivity, and color vision tests may be performed.

[0106]

[0146] In another example, any of the methods described herein may be performed in combination with another or secondary treatment. The treatment may be any currently known or yet-to-be-discovered treatment that helps prevent, arrest, or improve any of the described retinal changes and / or vision loss.

[0107]

[0147] Any of the methods described herein (including user interfaces) may be implemented as software, hardware, or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet, smartphone, etc.), which instructions, when executed by the processor, cause the processor to perform any of the steps including, but not limited to, displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, warning, and others.

[0108]

[0148] When a feature or element is referred to in this specification as being "above" another feature or element, it can be directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly above" another feature or element, there are no intervening features or elements. Also, when a feature or element is referred to as being "connected to", "coupled to", or "linked to" another feature or element, it is understood that it may be directly connected, coupled, or linked to the other feature or element, or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected to", "directly coupled to", or "directly linked to" another feature or element, there are no intervening features or elements. Although an example is described or shown, the features and elements so described or shown can apply to other examples. Also, it is understood by those skilled in the art that a reference to a structure or feature being "adjacent to" another feature can have a portion that overlaps or is beneath the adjacent feature.

[0109]

[0149] The terms used in this specification are for the purpose of describing particular examples only and are not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprising" and / or "comprises" as used in this specification, when used, specify the presence of the stated feature, step, operation, element, and / or component, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0110]

[0150] Spatial relative terms, such as "under", "below", "lower", "over", "upper", etc., may be used herein for ease of explanation to describe the relationship of one element or feature to another element or feature as illustrated in the drawings. It is understood that spatial relative terms are intended to encompass various orientations of a device in use or operation in addition to the orientation depicted in the drawings. For example, if a device in the drawings is turned upside down, an element described as "under" or "beneath" another element or feature may be oriented "over" the other element or feature. Thus, the exemplary term "under" can encompass both upward and downward orientations. The device may be oriented in another manner (rotated 90 degrees or otherwise), and the spatial relative expressions used herein shall be construed accordingly. Similarly, terms such as "upwardly", "downwardly", "vertical", "horizontal", etc. are used herein only for purposes of explanation unless specifically indicated otherwise.

[0111]

[0151] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, without departing from the teachings of the present invention, the first feature / element described below may be referred to as the second feature / element, and similarly, the second feature / element described below may be referred to as the first feature / element.

[0112]

[0152] Throughout this specification and the following claims, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", are meant that the various components can be used together in a method and an article (e.g., a composition, an apparatus including a device, and a method). For example, the term "comprising" is understood to imply the inclusion of any indicated element or step, but not the exclusion of any other element or step.

[0113]

[0153] Generally, any of the apparatus and methods described in this specification should be understood to be inclusive, but all or subsets of components and / or steps may alternatively be exclusive, and various components, steps, sub-components or sub-steps may be expressed as "consisting of" or alternatively "consisting essentially of".

[0114]

[0154] In this specification and the claims, as used herein, including when used in examples and unless otherwise expressly specified, all numbers can be interpreted as if the term "about" or "approximately" were prefixed thereto even if the term is not expressly indicated. The phrases "about" or "approximately" can be used to describe a magnitude and / or a position to indicate that the value and / or position being described is within the reasonably expected range of the value and / or position. For example, a numerical value can have a value that is ±0.1% of the value shown (or range of values), ±1% of the value shown (or range of values), ±2% of the value shown (or range of values), ±5% of the value shown (or range of values), ±10% of the value shown (or range of values), etc. Also, any numerical value given herein should be understood to include about or approximately that value unless the context indicates otherwise. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. Also, as would be appropriately understood by one of ordinary skill in the art, when a value is disclosed, it is understood that the value "less than", "greater than", and the possible ranges between values are also disclosed. For example, if the value "X" is disclosed, "less than X" and "greater than X" (where, for example, X is a numerical value) are also disclosed. Also, throughout this application, it is understood that the data is provided in several different formats and that this data represents ranges of endpoints and starting points, as well as any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that points greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are disclosed along with the points between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0115]

[0155] Although various exemplary examples are described above, any of several variations can be made to the various examples without departing from the scope of the invention as described by the claims. For example, the order in which the various described method steps are performed may often vary in alternative examples, and in other alternative examples, one or more method steps may be entirely omitted. Optional features of the various device and system examples may be included in some examples but not in others. Therefore, the above description is provided primarily for illustrative purposes, and since the scope of the invention is set forth in the claims, the above description should not be construed as limiting the scope of the invention.

[0116]

[0156] The examples and illustrations included in this specification are for illustrative purposes, not for limitation, and show specific examples in which the subject matter may be practiced. As mentioned, other examples may be utilized and derived from, such that structural and logical substitutions and changes can be made without departing from the scope of this disclosure. Such examples of the subject matter of the invention may be referred to herein individually or collectively by the term "invention," which is for convenience only, and is not intended to spontaneously limit the scope of this application to any single invention or inventive concept. Thus, while specific examples are illustrated and described herein, any arrangement calculated to achieve the same purpose can replace the specific examples shown. This disclosure is intended to embrace any and all adaptations or variations of the various examples. Combinations of the above examples, and other examples not specifically described herein, will be apparent to those skilled in the art upon reading the above description.

Claims

1. A polypeptide comprising: a first region containing the amino acid sequence of complement factor H domain SCR1-SCR7 having at least 80% homology with SEQ ID NO: 3; a second region containing the amino acid sequence of factor H-like protein 1 domain SCR6-SCR7 having at least 80% homology with SEQ ID NO: 17; and a linker domain separating the first region from the second region.

2. The polypeptide according to claim 1, wherein the first region has the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 139, or SEQ ID NO: 140, the second region has the amino acid sequence of SEQ ID NO: 17, and the linker domain includes the amino acid sequence of SEQ ID NO:

138.

3. The polypeptide according to claim 1 or 2, comprising a first junction region between the first region and the linker domain, and a second junction region between the second region and the linker domain.

4. The polypeptide according to claim 3, wherein the first junction region has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 IRV, IR, or I.

5. The polypeptide according to claim 3, wherein the second junction region has the amino acid sequence of SEQ ID NO: 13, LKP, KP, or P.

6. The polypeptide according to claim 3, wherein the first junction region has the amino acid sequence of SEQ ID NO: 7, and the second junction region has the amino acid sequence of SEQ ID NO: 13 or LKP.

7. The polypeptide according to claim 1 or 2, wherein the linker domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

8. The polypeptide according to claim 1 or 2, wherein the first region comprises an amino acid sequence having at least 95% homology with SEQ ID NO: 3, and the second region comprises an amino acid sequence having at least 95% homology with SEQ ID NO:

17.

9. The polypeptide according to claim 1 or 2, wherein the first region comprises the amino acid sequence of SEQ ID NO: 3, and the second region comprises the amino acid sequence of SEQ ID NO:

17.

10. The polypeptide according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 21 having one or more amino acid substitutions selected from the group consisting of L6S, L6T, A289S, A289T, Y380S, Y380T, T16N, D258N, Y334N, D21N, G286N, H355N, A407N, R426N, S428N, G431N, G436N, G441N, and G442N.

11. The polypeptide according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 21 having one or more amino acid substitutions selected from the group consisting of L6S, L6T, A289S, A289T, Y380S, Y380T, T16N, D258N, Y334N, D21N, G286N, H355N, A407N, R426N, and S428N.

12. The polypeptide according to claim 1 or 2, having an amino acid sequence having at least 80% homology to SEQ ID NO:

131.

13. The polypeptide according to claim 1 or 2, having an amino acid sequence having at least 90% homology to SEQ ID NO:

131.

14. The polypeptide according to claim 1 or 2, having the amino acid sequence of SEQ ID NO:

131.

15. The polypeptide according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 125 or SEQ ID NO:

132.

16. The polypeptide according to claim 1 or 2, having any of the amino acid sequences of SEQ ID NOs: 18-97, 99-106, 122-124, or 128-130.

17. The polypeptide according to claim 1 or 2, wherein the linker domain comprises a Gly / Ser linker, a poly-Gly linker, or a poly-Ala linker.

18. The polypeptide according to claim 1 or 2, wherein the linker domain includes one of GGGS (SEQ ID NO: 141), GGGSGGGS (SEQ ID NO: 142), GGGGGSGGGS (SEQ ID NO: 143), GGGGGSGGGGGSGGGS (SEQ ID NO: 144), or GGGGGSGGGGGSGGGS (SEQ ID NO: 11).

19. A pharmaceutical composition for treating age-related macular degeneration (AMD) in a subject, comprising a polypeptide comprising: a first region comprising the amino acid sequence of complement factor H domain SCR1-SCR7 having at least 80% homology with SEQ ID NO: 3; a second region comprising the amino acid sequence of factor H-like protein 1 domain SCR6-SCR7 having at least 80% homology with SEQ ID NO: 17; and a linker domain separating the first region from the second region.

20. The pharmaceutical composition according to claim 19, wherein the first region has the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 139, or SEQ ID NO: 140, the second region has the amino acid sequence of SEQ ID NO: 17, and the linker domain includes the amino acid sequence of SEQ ID NO:

138.

21. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide comprises a first junction region between the first region and the linker domain, and a second junction region between the second region and the linker domain.

22. The pharmaceutical composition according to claim 21, wherein the first junction region has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 IRV, IR, or I.

23. The pharmaceutical composition according to claim 21, wherein the second junction region has the amino acid sequence of SEQ ID NO: 13, LKP, KP, or P.

24. The pharmaceutical composition according to claim 21, wherein the first junction region has the amino acid sequence of SEQ ID NO: 7, and the second junction region has the amino acid sequence of SEQ ID NO: 13 or LKP.

25. The pharmaceutical composition according to claim 19 or 20, wherein the linker domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

26. The pharmaceutical composition according to claim 19 or 20, wherein the first region comprises an amino acid sequence having at least 95% homology with SEQ ID NO: 3, and the second region comprises an amino acid sequence having at least 95% homology with SEQ ID NO:

17.

27. The pharmaceutical composition according to claim 19 or 20, wherein the first region comprises the amino acid sequence of SEQ ID NO: 3, and the second region comprises the amino acid sequence of SEQ ID NO:

17.

28. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21 having one or more amino acid substitutions selected from the group consisting of L6S, L6T, A289S, A289T, Y380S, Y380T, T16N, D258N, Y334N, D21N, G286N, H355N, A407N, R426N, S428N, G431N, G436N, G441N, and G442N.

29. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21 having one or more amino acid substitutions selected from the group consisting of L6S, L6T, A289S, A289T, Y380S, Y380T, T16N, D258N, Y334N, D21N, G286N, H355N, A407N, R426N, and S428N.

30. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide has an amino acid sequence having at least 80% homology to SEQ ID NO:

131.

31. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide has an amino acid sequence having at least 90% homology with SEQ ID NO:

131.

32. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide has the amino acid sequence of SEQ ID NO:

131.

33. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 125 or SEQ ID NO:

132.

34. The pharmaceutical composition according to claim 19 or 20, wherein the polypeptide has any of the amino acid sequences of SEQ ID NOs: 18-97, 99-106, 122-124, or 128-130.

35. The pharmaceutical composition according to claim 19 or 20, wherein the linker domain comprises a Gly / Ser linker, a poly-Gly linker, or a poly-Ala linker.

36. The pharmaceutical composition according to claim 19 or 20, wherein the linker domain comprises one of GGGS (SEQ ID NO: 141), GGGSGGGS (SEQ ID NO: 142), GGGGGSGGGS (SEQ ID NO: 143), GGGGGSGGGGGSGGGS (SEQ ID NO: 144), or GGGGGSGGGGGSGGGS (SEQ ID NO: 11).

37. The pharmaceutical composition according to claim 19 or 20, which is intended to be administered to a subject by intraocular injection.

38. The pharmaceutical composition according to claim 19 or 20, which is intended to be administered to a subject by intravascular (IV) injection.

39. The pharmaceutical composition according to claim 19 or 20, which is intended to be administered to a subject by subcutaneous (SC) injection.

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