Complement factor I related compositions and methods

Abstract

Provided herein are complement factor I (CFI) variants and CFI-containing fusion constructs that exhibit at least one improved characteristic compared to wild-type CFI. The CFI variants and fusion constructs of the present disclosure can exhibit tunable specificity and activity. The CFI variants and fusion constructs provided herein can be useful for treating diseases or conditions associated with dysregulation of the complement system or deficiencies in CFI.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 293,040, filed December 22, 2021, the entire contents of which are incorporated herein by reference.

[0002] Electronic sequence list reference The contents of the electronic sequence listing (VTEX_706_01WO_SeqList_ST26.xml; size: 53,828 bytes; and creation date: December 20, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] The complement system includes the classical, lectin and alternative pathways and is tightly controlled by several regulators. Complement factor I (CFI) is one such regulator, which acts to regulate the complement system by cleaving C4b and C3b proteins, thereby inactivating these proteins. Such cleavage results in the inhibition of the classical and lectin pathways (through cleavage of C4b), as well as the inhibition of the alternative pathway (through cleavage of C3b), thus ultimately preventing the assembly of C3 and C5 convertase enzymes.

[0004] CFI is encoded as a proenzyme and is activated by proteolytic cleavage into a heterodimeric glycoprotein, the heterodimeric protein having heavy and light chains connected by disulfide bonds. The light chain (also called B chain) contains a serine protease domain (SPD) involved in the cleavage of C3b and C4b, and contains a catalytic triad (His362, Asp411, and Ser507) in a region called the active site. The heavy chain (also called A chain) contains four domains: the FI membrane attack complex (FIMAC) domain, the scavenger receptor cysteine-rich domain SRCR (also called CD5 domain) domain, the low density lipoprotein receptor 1 domain (LDLr1), and the low density lipoprotein receptor 2 domain (LDLr2). CFI is post-translationally processed into its active form by the addition of six Asn-linked glycans and proteolytic activation by furin, which excises the RRKR linker (SEQ ID NO: 25) to generate a two-chain mature protein.

[0005] With regard to its ability to cleave C3b or C4b, CFI is proteolytically active when it forms a ternary complex with its cofactor; factor H (FH) or complement receptor 1 (CR1, also called CD35) and its physiological substrates, C3b and C4b. FH is an example of a soluble member of a group of proteins called regulators of complement activation (RCA). The formation of the complex made by CFI and FH, and the subsequent cleavage of C3b together act to regulate the alternative pathway of the complement system. The continuous regulation of C3b levels by CFI acts to maintain a balance between the classical and alternative pathways. For example, it has been shown that removal of CFI causes immediate activation of the alternative pathway, resulting in overactivity. CR1 is an example of a monomeric single-pass type I membrane glycoprotein and is also a member of the RCA group of proteins. The formation of the complex made between CFI and CR1, and the subsequent cleavage of C3b and C4b, act to regulate the alternative or classical and lectin pathways, respectively.

[0006] Dysregulation of CFI, mutations and dysfunction of CFI, and CFI deficiency are involved in diseases involving the complement system. There is a need for methods to regulate or inhibit specific regulatory points within the complement system. Provided herein are compositions and methods to address this need. Summary of the Invention

[0007] In one aspect, the present disclosure provides complement factor I (CFI) variants that include at least one modification relative to wild-type CFI, and fusion proteins that include such CFI variants. Exemplary CFI variants and fusion proteins are provided in Tables 2 and 3. The CFI variants of the present disclosure can regulate the complement system and can have at least one improved characteristic compared to wild-type CFI. Methods of making and using the CFI variants and fusion proteins of the present disclosure are also provided. [Brief description of the drawings]

[0008] [Figure 1] Schematic diagram of the domains of wild-type complement factor I (CFI), showing the heavy and light chains. The heavy chain (A chain) contains the FIMAC, SRCR, LDLr1, LDLr2 domains, and a linker. The light chain (B chain) contains the serine protease domain. Regions of the light chain include the activation loop, 37-loop, 60-loop, 70-loop, 99-loop, 110-loop, 150-autolysis loop, 180-loop oxyanion stabilization, and / or the 220-loop S1 entrance frame. [Figure 2A] 1 shows an exemplary model of a fusion construct of the present disclosure between albumin (eg, serum albumin, such as human serum albumin (HSA)) and CFI, including a CFI variant, where the CFI variant comprises an AB chain inversion. [Figure 2B] 1 shows an exemplary model of a fusion construct of the present disclosure between albumin (eg, serum albumin, such as human serum albumin (HSA)) and CFI, including a CFI variant, where the CFI variant comprises an AB chain inversion. [Figure 2C]1 shows an exemplary model of a fusion construct of the present disclosure between albumin (eg, serum albumin, such as human serum albumin (HSA)) and CFI, including a CFI variant, where the CFI variant comprises an AB chain inversion. [Figure 2D] 1 shows an exemplary model of a fusion construct of the present disclosure between albumin (eg, serum albumin, such as human serum albumin (HSA)) and CFI, including a CFI variant, where the CFI variant comprises an AB chain inversion. [Figure 3A] 1 shows a model of an exemplary CFI-albumin (e.g., serum albumin, such as human serum albumin (HSA)) fusion construct comprising serum albumin fused to CFI. [Figure 3B] 1 shows a model of an exemplary CFI-HSA fusion construct comprising HSA fused to the serine protease domain of CFI. [Figure 4A] 1 shows a schematic diagram of Factor H (FH) showing its 20 domains. [Figure 4B] A schematic diagram of mini-factor H showing domains 1-4 connected to domains 19-20 of FH is shown. [Diagram 5] 1 shows a model of an exemplary fusion construct containing portions of Factor H and CFI, including domains 1-8 of FH fused to CFI. [Figure 6] Schematic diagrams of three exemplary fusion constructs of the present disclosure are shown, each comprising HSA, at least one CFI domain, and various domains of Factor H, portions of which are connected by optional / exemplary linkers. [Figure 7] Schematic diagrams of exemplary CFI fusion constructs are shown, in which a "core" domain may comprise any one or more of the CFIs of the present disclosure and may be flanked at either or both the N-terminus and C-terminus by one or more domains. The depicted domains may optionally be linked by linkers. [Figure 8A] FIG. 1 is a graph showing the measured concentrations of wild-type CFI-HSA fusion construct compared to free plasma-purified CFI after a single subcutaneous dose of 1 mg / kg in monkeys. [Figure 8B]FIG. 1 is a graph showing the measured concentrations of wild-type CFI-HSA fusion construct compared to free plasma-purified CFI after a single subcutaneous dose of 1 mg / kg in monkeys. [Figure 9A] Graphs of C3b and C4b degradation by CFI-HSA and plasma-derived CFI, respectively, are shown. [Figure 9B] Graphs of C3b and C4b degradation by CFI-HSA and plasma-derived CFI, respectively, are shown. [Figure 9C] 1 shows graphs of hemolytic assays of CFI-HSA and plasma-derived CFI, where AP represents an assay focused on the alternative pathway and CP+AP represents an assay focused on the alternative and classical pathways. [Figure 9D] 1 shows graphs of hemolytic assays of CFI-HSA and plasma-derived CFI, where AP represents an assay focused on the alternative pathway and CP+AP represents an assay focused on the alternative and classical pathways. [Figure 10] FIG. 1 shows a plot depicting the amount of C3a detected in samples from vitreous humor following intravitreal injection (IVT) of CFI-HSA at doses of 250 μg or 500 μg in an African green monkey primate model. [Figure 11] FIG. 1 shows plots of Factor I levels detected in plasma at the indicated time points following intravenous injection of 3 mg / kg CFI-HSA (Factor I-HSA) or 1.3 mg / kg CFI (Factor I) in CD1 mice. [Figure 12] FIG. 1 shows plots of Factor I levels detected in plasma at the indicated time points following subcutaneous injection of 3 mg / kg CFI-HSA (Factor I-HSA) or 6.5 mg / kg CFI (Factor I) in CD1 mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure provides compositions and methods useful for regulating signaling and amplification of the complement system. By providing CFI variants and CFI-containing fusion constructs that are more or less active against one or more physiological substrates of complement factor I (CFI) and / or more stable than plasma-derived CFI, modulation of the complement system is observed. Such modulation includes increased amounts of C3b cleavage and / or C4b cleavage, thus reducing complement activation and reducing amplification of the complement pathway. For example, some CFI variants can alter the levels of regulators in the complement system. In some embodiments, the CFI variants and fusion constructs provided herein can act on the classical and lectin pathways of the complement system, the alternative pathway of the complement system, or both pathways. The present disclosure also provides methods of making and using such variants and constructs, for example, in the treatment of diseases or conditions associated with complement dysregulation, for example, in the treatment of an overactive complement response.

[0010] I. Complement Factor I Proteins Useful in Regulating the Complement System Complement factor I mutants Complement factor I variants (CFIs) are provided herein, such variants comprising one or more modifications relative to wild-type CFI, referred to herein as "CFI variants." As used herein, a "modification" relative to wild-type CFI includes a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion (i.e., addition) of one or more amino acid residues, an insertion (i.e., addition) of one or more domains, an inversion of one or more domains, and a substitution of one or more domains.

[0011] The CFI mutants of the disclosure do not act directly on C3, e.g., the mutants of the disclosure do not directly cleave C3, do not directly inhibit C3, do not directly inhibit the activation of C3, or do not directly reduce the activation of C3.

[0012] As used herein, "wild-type CFI" refers to any naturally occurring full-length CFI, with or without a signal sequence, and of any species, that is not a disease-causing CFI.

[0013] In some embodiments, the wild-type CFI is plasma-derived. In some embodiments, the wild-type CFI is human wild-type CFI. In some embodiments, the wild-type human CFI with a signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 1 (shown in Table 1 below). In some embodiments, the wild-type CFI is human CFI. In some embodiments, the wild-type human CFI does not comprise a signal sequence. In some embodiments, the wild-type CFI without a signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 5 (shown in Table 1 below).

[0014] Wild-type CFI comprises a heavy chain and a light chain, also referred to as A chain and B chain, respectively. Figure 1 shows a schematic diagram of CFI showing the two chains. The heavy chain (A chain) has four domains: a FI membrane attack complex (FIMAC) domain (residues 36-90 of SEQ ID NO:5), a SRCR domain further composed of multiple scavenger receptor cysteine ​​rich (SRCR) domains, a low density lipoprotein 1 domain (LDLr1), and a low density lipoprotein 2 domain (LDLr2). The light chain (B chain) consists of a serine protease domain (SPD). The interface between these chains is referred to as the A:B chain interface.

[0015] The CFI mutants of the present disclosure include one or more of the following: a deletion of one or more amino acid residues of wild-type CFI, a deletion of one or more CFI domains of wild-type CFI, a substitution of one or more amino acid residues of wild-type CFI, an insertion of one or more amino acid residues into wild-type CFI, an inversion of one or more CFI domains of wild-type CFI, and an insertion of one or more domains into wild-type CFI.

[0016] CFI variants of the present disclosure may be generated by introducing one or more modifications to a CFI base molecule, where the domains of the CFI base molecule correspond to those found in wild-type CFI, e.g., as described in FIG. 1. Thus, the CFI base molecule may be the wild-type CFI of any species, or the CFI base molecule may contain only a portion of a wild-type CFI having only some of the domains of the wild-type CFI of any species (e.g., already a CFI variant). In some embodiments, the CFI base molecule is wild-type mouse CFI. In some embodiments, the CFI base molecule is wild-type human CFI. In some embodiments, the CFI base molecule is wild-type non-human primate CFI. In some embodiments, the CFI base molecule contains only some of the domains of wild-type human CFI.

[0017] In some embodiments, the CFI variants provided herein modulate the activity of the complement system and have at least one improved characteristic compared to wild-type CFI. Such improved characteristics include, but are not limited to, an increase or decrease in any one or more of bioavailability, half-life, activity, potency, catalytic activity, cofactor affinity (e.g., affinity for factor H and / or CR1), substrate specificity, and substrate affinity (e.g., affinity for C3b and / or C4b). In some embodiments, the improved characteristic is an increase in half-life. In some embodiments, the improved characteristic is an increase in activity, which is discussed in more detail in the subsequent sections below. In other embodiments, the improved characteristic is an altered substrate specificity for C3b and / or C4b, allowing for tunability of the CFI variants.

[0018] Table 1 provides exemplary base molecules that may be used to generate CFI variants. The base molecules provided herein may be useful for regulating the complement system without further modification, or may be useful for regulating the complement system with further modification. For example, any one of the base molecules provided in Table 1 may be further modified to include one or more modifications, such as the deletion of one or more amino acid residues, the deletion of one or more CFI domains, the substitution of one or more amino acid residues, or the addition of one or more amino acid residues or CFI domains. The base molecules in Table 1 may be further moieties of fusion constructs, which are further described below.

[0019] [Table 1-1]

[0020] [Table 1-2]

[0021] [Table 1-3]

[0022] In some embodiments, the base molecule itself may be a CFI variant, for example, in some embodiments, a CFI variant containing only the serine protease domain (CFI-SPD) is itself a CFI variant. In some embodiments, a CFI variant is derived from any of the base molecules in Table 1 and includes modifications to loops that correspond to loops of unmodified CFI. In some embodiments, a CFI variant is derived from any of the base molecules in Table 1 and includes substitution mutations. In some embodiments, a CFI variant is derived from any of the base molecules in Table 1 and includes a deletion of one or more domains of CFI. In some embodiments, a CFI variant is derived from any of the base molecules in Table 1 and includes an inversion of strand A and strand B of CFI.

[0023] In some embodiments, provided herein are CFI variants comprising at least one CFI domain, where the at least one CFI domain corresponds to that of wild-type CFI of any species. For example, the amino acid sequence of the at least one CFI domain may comprise an amino acid sequence derived from wild-type human CFI as set forth in SEQ ID NO: 5. The CFI variants provided herein comprising an amino acid sequence derived from SEQ ID NO: 5 may comprise one or more modifications relative to the sequence set forth in SEQ ID NO: 5. For example, the one or more modifications may comprise a deletion of one or more amino acid residues, a substitution mutation of one or more amino acid residues, an addition of one or more amino acid residues, a deletion of one or more domains of CFI, a substitution of one or more domains of CFI, or an addition of one or more domains of CFI.

[0024] In some embodiments, provided herein are CFI variants comprising at least one CFI domain of any species, wherein the at least one CFI domain comprises any one or more CFI domains selected from a serine protease domain (SPD), a factor I membrane attack complex (FIMAC) domain, a scavenger receptor cysteine-rich domain (SRCR), a low density lipoprotein receptor 1 (LDLr1), and a low density lipoprotein receptor 2 (LDLr2) domain. In some embodiments, any one or more CFI domains are of human CFI. In some embodiments, any one or more CFI domains comprise an amino acid sequence derived from the sequence set forth in SEQ ID NO:5.

[0025] In some embodiments, the CFI mutant comprises all of the domains of wild-type CFI, i.e., one each of the SPD, FIMAC domain, SRCR domain, LDLr1 domain, and LDLr2 domain, and includes any one or more modifications of these domains relative to wild-type CFI.

[0026] In some embodiments, the CFI mutant does not contain all of the domains corresponding to those of wild-type CFI. In some embodiments, the CFI mutant contains an SPD. In some embodiments, the CFI mutant contains only the SPD and lacks the A strand of CFI, referred to herein as "CFI-SPD". In some embodiments, the CFI-SPD contains the amino acid sequence set forth in SEQ ID NO: 12 (shown in Table 1), which is the SPD of human CFI. In some embodiments, the CFI-SPD does not contain any further modifications to those of the wild-type CFI SPD. In some embodiments, the CFI-SPD contains one or more modifications to those of the wild-type CFI SPD. In some embodiments, the CFI-SPD contains at least one modification to the amino acid sequence set forth in SEQ ID NO: 12.

[0027] Exemplary mutants of CFI are described in more detail below. Exemplary CFI mutants contain one or more substitutions of amino acid residues relative to a CFI having an amino acid sequence set forth in SEQ ID NO: 5. For example, a CFI mutant containing substitutions at positions S499 and I500 has substitutions at positions S499 and I500 in the amino acid sequence set forth in SEQ ID NO: 5.

[0028] Exemplary CFI Mutants Provided herein are CFI variants that contain or consist of at least one modification to wild-type CFI, which can increase complement system inhibition and have at least one improved characteristic compared to wild-type CFI. Examples of improved characteristics include, but are not limited to, increased half-life, increased bioavailability, or an increase or decrease in any one or more of activity, substrate specificity, potency, substrate affinity, cofactor affinity, and catalytic ability. In an exemplary embodiment, the improved characteristic is increased half-life. In another exemplary embodiment, the improved characteristic is increased or altered substrate specificity.

[0029] Without being limiting, the present disclosure contemplates exemplary CFI mutants set forth in Table 3. The mutants in Table 3 include modified CFIs as described herein, as well as CFI fusion constructs. For the avoidance of doubt, unless otherwise indicated, when a residue number is given, it refers to SEQ ID NO: 5 (wild type human CFI) or a sequence corresponding thereto. For the avoidance of doubt, by way of example, a mutant whose description is P433A is a CFI mutant comprising a P433A substitution, e.g., a CFI mutant comprising a P433A substitution in SEQ ID NO: 5 (or a sequence corresponding thereto). The present disclosure also provides CFI mutants consisting of a P433A substitution, e.g., a CFI mutant in SEQ ID NO: 5 having a P433A substitution.

[0030] CFI variants of the disclosure are provided individually in Table 2. In some embodiments, a CFI variant of the disclosure comprises or consists of any one or more of the modifications presented in Table 2, where the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. Construct numbers refer to Table 3, which indicates the construct in which the indicated variant is present.

[0031] The CFI variants of the present disclosure may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or more modifications, such as substitutions, deletions, insertions, and fusions. Modifications, such as substitutions, for a given variant can be expressed in one of many ways recognized by those of skill in the art. For example, a hCFI variant having substitutions at D395A and E416A may be referred to as having the substitutions: "D395A and E416A", "D395A-E416A", "D395A+E416A", "D395A / E416A", or "D395A;E416A", which are used interchangeably herein. In some examples, a CFI variant having substitutions at D395A and E416A may be referred to as "hCFI;D395A;E416A" or CFI variant (D395A;E416A)". As described herein, variants having other modifications, such as deletions, or combinations of modifications, such as deletions, fusions and substitutions, can follow a similar nomenclature.

[0032] Table 3 provides exemplary CFI variants of the disclosure, each represented by a unique construct number (abbreviated in Table 3 as "Const. No."). This and other tables disclosing variants may include the following symbols and abbreviations and associated meanings: HSA = human serum albumin; CFI = complement factor I; Δ = deletion of the indicated amino acid range; → or > = deletion of the indicated sequence and substitution with the indicated amino acid; Cr1 = CR1 fusion; Fh = FH fusion; G(#) indicates a linker of glycine that is repeated the indicated number of instances; (GGSS) n represents a linker of GGSS repeated n times (SEQ ID NO: 26); (GGSS) n GG indicates a linker (SEQ ID NO: 27) consisting of n repeats of GGSS followed by GG.

[0033] 7 corresponds to the layout of Table 3 and shows a schematic diagram of an exemplary CFI fusion construct, in which a "core" domain may contain any one or more of the amino acid modifications of the present disclosure and may be flanked at either or both the N-terminus and C-terminus by one or more domains. The depicted domains may optionally be linked by linkers.

[0034] [Table 2]

[0035] Adjustment of activity and specificity The activity and specificity of the CFI variants provided herein can be tailored (tuned) for a particular use and therapeutic indication. For example, activity and specificity can be tailored by selection of a C3b degrading agent, or a C4b degrading agent, or a degrading agent for both C3b and C4b. As referred to herein, protease activity on a substrate refers to the ability of a CFI variant of the present disclosure to cleave its substrates C4b and C3b. This can be expressed in several ways, for example, as an increase in C4b degrading agent activity, protease activity on C4b, C3b degrading agent activity, protease activity on C3b, yield of cleavage products, etc.

[0036] As used herein, a C3b degrading agent is a CFI mutant capable of cleaving C3b. Similarly, a C4b degrading agent is a CFI mutant capable of cleaving C4b. The use of a C3b degrading agent does not mean that it does not degrade C4b. A CFI mutant may be both a C3b degrading agent and a C4b degrading agent, and may, but does not necessarily, show specificity for one over the other.

[0037] The CFI variants provided herein have altered characteristics, including increased or decreased protease activity towards a substrate, as well as increased or decreased substrate specificity.

[0038] As used herein and as demonstrated in the examples provided herein, specificity for a substrate, also referred to as substrate specificity, refers to the specificity that a CFI variant demonstrates for one over the other. If the substrate specificity of a CFI variant is about 1, then the specificity for both C4b and C3b is equal to wild-type CFI activity. If the specificity of a CFI variant is 2-fold higher for C4b, then it would be considered to indicate increased specificity of cleavage for C4b compared to C3b. A greater fold increase in protease activity for one substrate compared to another substrate is an example of increased specificity for that substrate.

[0039] In some embodiments, the amino acid modifications (e.g., substitutions) increase activity, confer specificity, or both. In some embodiments, increased C4b degrader activity includes increased cleavage of C4b (and generation of cleavage products such as C4c), and increased specificity for C4b includes increased cleavage of C4b and decreased cleavage of C3b (and generation of cleavage products such as iC3b). Generally, comparisons are provided relative to wild-type CFI or a fusion construct comprising wild-type CFI.

[0040] In some embodiments, the combination of two or more alterations (eg, substitutions) may confer unexpected increases in activity that are synergistic or additive.

[0041] In some embodiments, the combination of one or more modifications may confer an unexpected increase or decrease in activity that is synergistic when C4b is a substrate and additive or less than additive when C3b is a substrate.

[0042] In some embodiments, the combination of one or more modifications may confer an unexpected increase or decrease in activity that is synergistic when C3b is a substrate and additive or less than additive when C4b is a substrate.

[0043] In some embodiments, altered characteristics can be achieved by selection (e.g., adjustment) of one or more modifications that confer increased C3b degrading agent activity and decreased C4b degrading agent activity (increased C3b substrate specificity), or that confer increased C4b degrading agent activity and decreased C3b degrading agent activity (increased C4b substrate specificity), or that provide increased activity as a degrader of both C3b and C4b (specificity is unchanged, but activity against both substrates is increased).

[0044] In some embodiments, the altered characteristics can be achieved by selection of one or more modifications that confer increased C3b degrading agent activity and leave C4b degrading agent activity unchanged (increased C3b substrate specificity) or that confer increased C4b degrading agent activity and leave C3b degrading agent activity unchanged (increased C4b substrate specificity).

[0045] In some embodiments, altered characteristics can be achieved by selection of one or more modifications that confer increased C3b degrader activity and leave C4b degrader activity unchanged (increased C4b substrate specificity) or that confer decreased C4b degrader activity and leave C3b degrader activity unchanged (increased C3b substrate specificity).

[0046] Modifications that provide increased activity and specificity are typically, but not limited to, focused in structural regions important for CFI function. Exemplary structural regions with modifications (e.g., substitutions) that can result in at least one improved characteristic are the C-terminal extension, the A:B interface, the surface that represents the interface with the cofactor and modifications (e.g., substitutions) within the active site of the SPD, including surface loops that provide interfaces with the C3b and C4b substrates, and the CR1 and FH cofactors (see FIG. 1).

[0047] Without being bound by theory or mechanism, provided herein are CFI variants having a combination of one or more of any of the amino acid modifications detailed below, which have at least one improved characteristic. CFI variants having combined modifications (e.g., substitutions) include two or more modifications in one or more regions of CFI selected from, but not limited to, the structural region of the C-terminal extension, the A:B interface, and the interface with cofactors and the active site (including surface loops that provide interfaces with cofactors and C3b or C4b substrates).

[0048] In some embodiments, CFI variants containing two or more substitutions exhibit altered activity, substrate specificity, or both. In some embodiments, increased activity comprises increased cleavage of C4b and / or generation of C4c, and specificity comprises a limited increase or decrease in cleavage of C3b and / or generation of iC3b, compared to wild-type CFI (or compared to a fusion construct containing wild-type CFI, e.g., SEQ ID NO: 21). In some embodiments, the combination of two or more substitutions confers an unexpected increase in activity that is synergistic when C4b is a substrate and additive or less than additive when C3b is a substrate.

[0049] In some embodiments, the amino acid substitutions increase activity, confer specificity, or both, and may switch between C3b and C4b selectivity. In some embodiments, the increased activity comprises increased cleavage of C4b and / or increased generation of C4c, and the selectivity comprises decreased cleavage of C3b and / or generation of iC3b, as compared to wild-type CFI. In some embodiments, the increased activity comprises increased cleavage of C3b and / or increased generation of iC3b, and the specificity comprises decreased cleavage of C4b and / or generation of C4c, as compared to wild-type CFI. In some embodiments, the nature of the amino acid substitutions defines whether the CFI variant exhibits the characteristic of specificity for C3b or specificity for C4b.

[0050] Exemplary variants of the disclosure are tested for differences in activity and for differences in specificity. Exemplary data are provided in Table 7.2.

[0051] In some embodiments, the CFI variants exhibit increased activity, where increased activity includes increased C3b degrader activity (with a concomitant increase in C3b cleavage products) by the CFI variants of the disclosure. In some embodiments, the CFI variants of the disclosure exhibit increased activity that is at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 25-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 75-fold, at least or about The C3b degrader activity of the antibody or antibody fragments may be increased by at least or about 100-fold, at least or about 150-fold, at least or about 200-fold, at least or about 250-fold, at least or about 300-fold, at least or about 350-fold, at least or about 400-fold, at least or about 450-fold, at least or about 500-fold, at least or about 550-fold, at least or about 600-fold, at least or about 650-fold, at least or about 700-fold, at least or about 750-fold, at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold, or even at least or about 1000-fold. In some embodiments, this increase in C3b degrader activity is also accompanied by an increase in C4b degrader activity. In some embodiments, this increase in C3b degrader activity is not accompanied by an increase in C4b degrader activity, and may even be a decrease in C4b degrader activity.

[0052] In some embodiments, the CFI variants exhibit increased activity, where increased activity includes increased C4b degrader activity (with a concomitant increase in C4b cleavage products) by the CFI variants of the disclosure. In some embodiments, the CFI variants of the disclosure exhibit increased activity that is at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 25-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 75-fold, at least or about The C4b degrader activity of the antibody or antibody fragments may be increased by at least or about 100-fold, at least or about 150-fold, at least or about 200-fold, at least or about 250-fold, at least or about 300-fold, at least or about 350-fold, at least or about 400-fold, at least or about 450-fold, at least or about 500-fold, at least or about 550-fold, at least or about 600-fold, at least or about 650-fold, at least or about 700-fold, at least or about 750-fold, at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold, or even at least or about 1000-fold. In some embodiments, this increase in C4b degrader activity is also accompanied by an increase in C3b degrader activity. In some embodiments, this increase in C4b degrader activity is not accompanied by an increase in C3b degrader activity, and may even be a decrease in C3b degrader activity.

[0053] In some embodiments, the CFI variants exhibit increased activity, including increased activity of both C3b and C4b degrader activity. In some embodiments, the CFI variants of the disclosure exhibit increased activity, including increased activity of both C3b and C4b degrader activity, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 25-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 75 ... 0 fold, at least or about 150 fold, at least or about 200 fold, at least or about 250 fold, at least or about 300 fold, at least or about 350 fold, at least or about 400 fold, at least or about 450 fold, at least or about 500 fold, at least or about 550 fold, at least or about 600 fold, at least or about 650 fold, at least or about 700 fold, at least or about 750 fold, at least or about 800 fold, at least or about 850 fold, at least or about 900 fold, at least or about 950 fold, and even at least or about 1000 fold increased C3b degrader activity and increased C4b degrader activity. The increase in degrader activity for one substrate may be, but need not be, the same.

[0054] In some embodiments, the CFI variants exhibit increased specificity for a substrate, the increased specificity being for C3b (over C4b). In some embodiments, the CFI variants of the disclosure exhibit increased specificity that is at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 25-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 75-fold, at least or about In some embodiments, the antibody exhibits an increase in specificity for C3b of at least or about 100-fold, at least or about 150-fold, at least or about 200-fold, at least or about 250-fold, at least or about 300-fold, at least or about 350-fold, at least or about 400-fold, at least or about 450-fold, at least or about 500-fold, at least or about 550-fold, at least or about 600-fold, at least or about 650-fold, at least or about 700-fold, at least or about 750-fold, at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold, and even at least or about 1000-fold.

[0055] In some embodiments, the CFI variants exhibit increased specificity for a substrate, the increased specificity being for C4b (over C3b). In some embodiments, the CFI variants of the disclosure exhibit increased specificity that is at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 25-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4 ...-fold, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about The antibody exhibits an increase in specificity for C4b of at least or about 75 fold, at least or about 100 fold, at least or about 150 fold, at least or about 200 fold, at least or about 250 fold, at least or about 300 fold, at least or about 350 fold, at least or about 400 fold, at least or about 450 fold, at least or about 500 fold, at least or about 550 fold, at least or about 600 fold, at least or about 650 fold, at least or about 700 fold, at least or about 750 fold, at least or about 800 fold, at least or about 850 fold, at least or about 900 fold, at least or about 950 fold, and even at least or about 1000 fold.

[0056] In some embodiments, the CFI variants exhibit reduced specificity for a substrate, the reduced specificity being for C3b (over C4b). In some embodiments, the CFI variants of the disclosure ...), at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 25-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about These antibodies exhibit a decrease in specificity for C3b of at least or about 75 fold, at least or about 100 fold, at least or about 150 fold, at least or about 200 fold, at least or about 250 fold, at least or about 300 fold, at least or about 350 fold, at least or about 400 fold, at least or about 450 fold, at least or about 500 fold, at least or about 550 fold, at least or about 600 fold, at least or about 650 fold, at least or about 700 fold, at least or about 750 fold, at least or about 800 fold, at least or about 850 fold, at least or about 900 fold, at least or about 950 fold, and even at least or about 1000 fold.

[0057] In some embodiments, the CFI variants exhibit reduced specificity for a substrate, the reduced specificity being for C4b (over C3b). In some embodiments, the CFI variants of the disclosure exhibit reduced specificity for a substrate, the reduced specificity being for C4b (over C3b). In some embodiments, the CFI variants of the disclosure exhibit reduced specificity for a substrate, the reduced specificity being for C4b (over C3b). In some embodiments, the CFI variants of the disclosure exhibit reduced specificity for a substrate, the reduced specificity being for C4b (over C3b). In some embodiments, the CFI variants of the disclosure exhibit reduced specificity for a substrate, the reduced specificity being for C4b (over C3b). In some embodiments, the CFI variants of the disclosure exhibit reduced specificity for a substrate, the reduced specificity being for C4b (over C3b). These antibodies exhibit a decrease in specificity for C4b of at least or about 75 fold, at least or about 100 fold, at least or about 150 fold, at least or about 200 fold, at least or about 250 fold, at least or about 300 fold, at least or about 350 fold, at least or about 400 fold, at least or about 450 fold, at least or about 500 fold, at least or about 550 fold, at least or about 600 fold, at least or about 650 fold, at least or about 700 fold, at least or about 750 fold, at least or about 800 fold, at least or about 850 fold, at least or about 900 fold, at least or about 950 fold, and even at least or about 1000 fold.

[0058] In some embodiments, the CFI variants have increased activity, the increased activity comprising increased cleavage of C4b over C3b and / or increased specificity for C4b. In some embodiments, the CFI variants with increased cleavage of C4b comprise one or more substitutions at an amino acid position set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the A:B interface region in a CFI having an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the cofactor interface region of a CFI having an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C-terminal extension region of a CFI having an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C-terminal extension;C4b interface region of a CFI having an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C4b interface region of a CFI having an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the active site;C4b interface region of a CFI having an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the autolysis loop;cofactor interface region region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the active site;S1 entrance frame region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the S1 entrance frame region of CFI having the amino acid sequence set forth in SEQ ID NO:5. These differences are as compared to wild-type CFI (or as compared to a fusion construct comprising wild-type CFI, e.g., SEQ ID NO:21), and the positions correspond to positions of CFI having the amino acid sequence set forth in SEQ ID NO:5.

[0059] In some embodiments, the improved characteristic is increased activity, where increased activity comprises increased cleavage of C3b and / or C4b. In some embodiments, the CFI variants provided herein are C3b degraders and refer to the ability of the CFI variants to increase C3b cleavage. In some embodiments, the CFI variants provided herein are C4b degraders and refer to the ability of the CFI variants to increase C4b cleavage. In some embodiments, the CFI variants provided herein are C3b and C4b degraders and refer to the ability of the CFI variants to increase cleavage of both C3b and C4b.

[0060] In some embodiments, the CFI mutant has increased activity, the increased activity comprises increased cleavage of C3b and C4b. In some embodiments, the CFI mutant with increased cleavage of C3b and C4b comprises one or more substitutions at the amino acid position set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the substrate interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the position within the substrate interface region is E401. In some embodiments, the amino acid position is a position within the active site; substrate interface region region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the cofactor interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C-terminal extension region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the autolytic loop; cofactor interface region region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the active site; S1 entrance frame region of CFI having the amino acid sequence set forth in SEQ ID NO:5. These differences are compared to wild-type CFI (or compared to a fusion construct containing wild-type CFI, e.g., SEQ ID NO:21), and the positions correspond to the positions of a CFI having the amino acid sequence shown in SEQ ID NO:5.

[0061] In some embodiments, the CFI variants have increased activity, where increased activity includes increased cleavage of C3b by the CFI variants of the present disclosure, and does not include or minimally includes increased cleavage of C4b. In some embodiments, the amino acid position is a position within the active site:C3b interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the cofactor interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C-terminal extension region of CFI having the amino acid sequence set forth in SEQ ID NO:5. These differences are as compared to wild-type CFI (or as compared to a fusion construct comprising a wild-type CFI, e.g., SEQ ID NO:21), and the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID NO:5.

[0062] In some embodiments, the CFI variants have increased activity, where the increased activity includes increased cleavage of C4b by the CFI variants of the present disclosure, and does not include or minimally includes increased cleavage of C3b. In some embodiments, the CFI variants having increased cleavage of C4b and does not include or minimally includes increased cleavage of C3b include one or more substitutions at an amino acid position set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the A:B interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the cofactor interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the S1 entrance frame region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C-terminal extension;C4b interface region of CFI having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the amino acid position is a position within the C-terminal extension region of CFI having the amino acid sequence set forth in SEQ ID NO:5. These differences are compared to wild-type CFI (or compared to a fusion construct containing wild-type CFI, e.g., SEQ ID NO:21), and the positions correspond to the positions of a CFI having the amino acid sequence shown in SEQ ID NO:5.

[0063] In some embodiments, CFI variants of the disclosure that are specific C3b degrading agents are useful in the treatment of disease.

[0064] In some embodiments, CFI variants of the disclosure that are specific C4b degrading agents are useful in the treatment of disease.

[0065] In some embodiments, CFI variants of the disclosure that are both C4b and C3b degraders and exhibit improved characteristics compared to wild-type CFI (e.g., increased activity against both C4b and C3b) are useful for treating diseases.

[0066] For example, diseases that may be treated by the use of C4b degrading agents include, but are not limited to, non-ocular conditions, hi some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), immune-mediated hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease (COPD), cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), granulomatosis with polyangiitis (GPA), Guillain-Barre syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy (IgAN), lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy. and systemic chronic indications selected from the group consisting of myeloma, myeloma syndrome (MMN), multiple sclerosis (MS), non-insulin-dependent diabetes mellitus, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein-losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), ulcerative colitis, warm autoimmune hemolytic anemia (wAIHA), immune complex membranoproliferative glomerulonephritis (IC-MPGN), and ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA), and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington's disease, and ischemia-reperfusion injury.

[0067] In some embodiments, the CFI variants provided herein are degraders of both C3b and C4b and are useful in the treatment of disease.

[0068] In some embodiments, the increased activity comprises increased production of C3dg and / or C3c from iC3b. Exemplary variants that exhibit such improved characteristics are provided in Table 3 and discussed in the Examples.

[0069] In some embodiments, the increased activity comprises a decreased level of the C3bα chain. Exemplary variants that exhibit such improved characteristics are provided in Table 3 and discussed in the Examples.

[0070] In some embodiments, the increased activity comprises hydrolysis of a peptide substrate or proteolysis of a polymeric protein substrate. In some embodiments, the polymeric protein substrate is C3b. In some embodiments, the polymeric protein substrate is C4b. In some embodiments, the peptide substrate is a chromogenic substrate, e.g., such peptide substrates are useful in assay formats. Exemplary variants that exhibit such improved characteristics are provided in Table 3 and discussed in the Examples.

[0071] In some embodiments, increased activity includes a decrease in the level or function of the membrane attack complex (MAC). In some embodiments, a decrease or even inhibition of hemolysis correlates with a decrease in the level of MAC, and thus in some embodiments, increased activity includes a decrease (partial or complete) in observed hemolysis.

[0072] In some embodiments, the increased activity comprises a decreased amplification of the complement system for the production of C3b. Exemplary variants that exhibit such improved characteristics are provided in Table 3 and discussed in the Examples.

[0073] In some embodiments, the CFI variants are sialylated. In some embodiments, the CFI variants are further sialylated compared to wild-type CFI. In some embodiments, the CFI variants are post-translationally sialylated by in vitro methods.

[0074] In some embodiments, the CFI mutant is an activated mutant (i.e., active two-chain form). In some embodiments, the CFI mutant is activated by furin (the term furin includes furin mutants). In some embodiments, the CFI mutant is activated by furin during production in a host cell. In some embodiments, activation by furin during production in a host cell is achieved by overexpression of furin, e.g., by stable or transient transfection. In some embodiments, the CFI mutant is activated by furin following production and secretion by the host cell, i.e., post-translationally.

[0075] References to modifications such as substitutions in the following paragraphs are to the amino acid sequence of human CFI as shown in SEQ ID NO: 5. However, it should be understood that modifications to the corresponding amino acid residues of any non-human species may also be made.

[0076] A:B-strand interface CFI mutants Provided herein are CFI mutants that contain one or more modifications in the heavy and light chain interface, also referred to as the A:B chain interface, that cause a disruption in the A:B chain interface.

[0077] Without wishing to be bound by theory or mechanism, it is believed that the serine protease domain (SPD) of CFI is held in a zymogen-like state through multiple interactions with its own A-strand. Naturally occurring CFI can cleave peptide or protein substrates at a relatively slow rate, but the rate of cleavage by CFI is increased by disrupting the A:B-strand interface.

[0078] C-terminal region mutant In the complex formed between CFI and C3b, the C-terminal extension is located in a cavity between the A and B chains of the bound, slightly twisted CFI molecule, suggesting that the C-terminal extension of CFI may be an important regulatory region for the activation of CFI upon binding to C3b.

[0079] N-linked glycosylation site mutants Provided herein are CFI variants comprising at least one CFI domain, wherein the at least one CFI domain comprises one or more modifications at N-linked glycosylation sites of CFI.

[0080] In some embodiments, the modification in an N-linked glycosylation site is the removal of one or more N-linked glycosylation sites of the CFI.

[0081] Serine protease domain mutants Provided herein are CFI variants that comprise or consist of at least one CFI domain, the at least one CFI domain being the serine protease domain (SPD) of CFI, and the CFI variants comprise one or more modifications in the SPD.

[0082] In the crystal structure of free CFI, cleavage of the activation loop did not result in the insertion of the newly formed N-terminus (Ile322), which is the next step in classical activation of serine proteases. Instead, the crystal structure suggests that the C-terminal region of the cleaved activation loop remains in a strongly curved loop structure on the surface of CFI, in the same region where the uncleaved activation loop would have remained. This prevents insertion into the activation pocket and thus prevents active site maturation (called classical serine protease activation by induced conformational rearrangement). When the SPD of CFI is activated upon proteolysis, the new N-terminus of the activation loop is generally released and the cleaved activation loop is inserted into the activation pocket to fully activate CFI in solution. Therefore, mutations in the C-terminal region of the activation loop should not affect cleavage by furin, since this region is 3' beyond the scissile bond.

[0083] Thus, SPD CFI mutants are provided herein. In some embodiments, CFI mutants comprising one or more modifications within the SPD region of CFI (FIG. 1) may comprise one or more modifications in any one or more of the activation loop (residues 322-326 of SEQ ID NO:5), the 37-loop (residues 342-344 of SEQ ID NO:5), the 60-loop (residues 366-372 of SEQ ID NO:5), the 70-loop (residues 377-389 of SEQ ID NO:5), the 99-loop (residues 403-410 of SEQ ID NO:5), the 110-loop (residues 418-426 of SEQ ID NO:5), the 150-autolysis loop (residues 455-463 of SEQ ID NO:5), the 180-loop oxyanion stabilization (residues 494-509 of SEQ ID NO:5), and / or the 220-loop S1 entrance frame (residues 529-536 of SEQ ID NO:5). Specifically, provided herein are CFI variants that include an SPD of CFI, the CFI variants including any one or more of the modifications set forth in Table 5. Table 5 presents exemplary CFI variants that include one or more modifications to the amino acid sequence set forth in SEQ ID NO:5, where the one or more modifications are in the SPD. The base molecules of the CFI variants set forth in Table 5 can be wild-type, human CFI, or CFI-SPD, where the SPD corresponds to that of wild-type CFI (also called Δ(K1-P305) or A-chain deleted), or CFI fused to another complement regulator, such as Factor H (FH-CFI) or CR1 (CR1-CFI), which are discussed in more detail herein when referring to fusion constructs.

[0084] In some embodiments, the CFI variants include autolysis loop substitutions. The autolysis loop of a serine protease is part of the activation domain and is responsible for substrate specificity. Trypsin has a longer autolysis loop than CFI, and some key residues are unique between the autolysis loop of trypsin and the autolysis loop of CFI. Differences may also occur between autolysis loops from different species, such as between mouse and human. The mouse CFI autolysis loop may include multiple differences compared to the CFI autolysis loop of human CFI. Exemplary CFI variants may include CFI variants in which the autolysis loop of human CFI is exchanged with the autolysis loop of human trypsin or exchanged with the autolysis loop of mouse CFI. Such autolysis loop variants may help identify key residues involved in C3b and / or C4b cleavage activity. Thus, in some embodiments, CFI variants are provided herein that are chimeras that include one or more domains from human CFI, where the human CFI further includes substitutions of one or more amino acid residues for amino acid residues in the corresponding regions from a non-human species CFI. In some embodiments, the non-human species CFI is mouse CFI. Also provided herein are CFI variants that are chimeric and in which the modification comprises the replacement of one or more amino acid residues of CFI with amino acid residues from the corresponding region of a non-CFI serine protease. In some embodiments, the non-CFI serine protease is trypsin.

[0085] An exemplary autolytic loop CFI mutant comprises a trypsin autolytic loop substitution, which comprises a substitution of the autolytic loop of CFI (REKDNERVFS, SEQ ID NO:9) for the autolytic loop of trypsin (NTASSGADYPDE, SEQ ID NO:10), wherein the autolytic loop occurs between positions corresponding to positions 456 and 465 in CFI having the amino acid sequence set forth in SEQ ID NO:5.

[0086] Another exemplary autolysis loop CFI mutant is 456 REKDNERVFS 465(SEQ ID NO: 9) is swapped for RGKDNQKVYS (SEQ ID NO: 11), and the autolysis loop occurs between positions corresponding to positions 456 and 465 of CFI having the amino acid sequence shown in SEQ ID NO:5.

[0087] Active site mutants Provided herein are CFI mutants that comprise or consist of one or more modifications in the active site of CFI. In some embodiments, provided herein are CFI mutants that comprise at least one CFI domain, where the at least one CFI domain comprises a modification to the amino acid sequence set forth in SEQ ID NO:5, where the modification is in the active site of CFI. In some embodiments, the active site CFI mutants may improve the catalytic ability of CFI. In some embodiments, the CFI active site mutants may improve the catalytic ability of CFI by improving the active site (catalytic mechanism) without affecting C3b or C4b binding or binding specificity, which is governed by exosite and A-chain interactions.

[0088] AB chain inversion CFI mutant Provided herein are CFI variants, where the CFI comprises an A chain and a B chain, and where the A chain and the B chain are inverted. In some embodiments, the CFI variants without chain inversion (each chain optionally includes one or more modifications) comprise an N-terminal to C-terminal or C-terminal to N-terminal structural arrangement of (A chain)-(optional linker)-(B chain). In some embodiments, the CFI variants comprise an A chain and a B chain inversion (each chain optionally includes one or more modifications) in an N-terminal to C-terminal or C-terminal to N-terminal structural arrangement of (B chain)-(optional linker)-(A chain). The optional linker may be of any suitable length, for example at least one amino acid in length. The linker may be a flexible linker, or may be a peptide of about 1 to about 20 amino acid residues in length, and the amino acid residue may include a glycine residue. The linker may also optionally include a serine residue. Exemplary flexible linkers can include, but are not limited to, glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, or any other suitable flexible linkers known in the art. An exemplary linker is (GGSS) n (SEQ ID NO:28), (GGSSGG) n (SEQ ID NO: 29), and (GGSS) n GG (SEQ ID NO: 30), and n is any number from about 1 to about 2. Exemplary linkers can be 1 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 3 to 15, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 15, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 15, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 15, 6 to 10, 6 to 9, 6 to 8, or 6 to 7 amino acids in length.

[0089] Without being bound by theory or mechanism, exemplary CFI mutants comprising an inversion of A and B chains may comprise the amino acid sequence shown in SEQ ID NO: 17, 18, 19, or 20. The chains may be held together by an optional linker. The linker between the A and B chains of the inversion mutant may be at least one amino acid of any suitable length. The linker may be a flexible linker and may be a peptide of about 1 to about 10, 3 to 11 to about 20, or 1 to about 40 amino acid residues in length, and the amino acid residue may comprise a glycine residue. The linker may also optionally comprise a serine residue. Exemplary flexible linkers may include, but are not limited to, glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, or any other suitable flexible linkers known in the art.

[0090] Additional CFI variants - useful for regulating and / or evaluating the complement system In some embodiments, there are CFI variants that are useful for regulating the complement system, but may also be useful for assessing the activity of the complement system, e.g., they can be considered tool proteins in addition to having therapeutic value.

[0091] For example, these other CFI variants may allow for a variety of tests using CFI fusion constructs. Exemplary such CFI variants may be inactivatable to serve as controls. Another exemplary such CFI variant may provide for easier activation of the fusion construct.

[0092] In some embodiments, such additional CFI mutants provided herein comprise modifications to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the contemplated CFI mutants provided herein are derived from wild-type mouse CFI. In some embodiments, the contemplated CFI mutants provided herein are derived from wild-type human CFI. In some embodiments, the contemplated CFI mutants provided herein are further derived from CFI-SPD.

[0093] Exemplary CFI mutants can include non-activatable CFI mutants, which may provide particular utility, for example, as decoys or controls.

[0094] CFI combination mutants Provided herein are CFI mutants that contain or consist of two or more modifications relative to wild-type CFI. The modifications occur in the same or different domains of CFI. In some embodiments, the modifications include two or more substitutions. In some embodiments, the modifications include substitutions and deletions. In some embodiments, the modifications include substitutions and additions. In some embodiments, the modifications include deletions and additions. In some embodiments, the modifications include substitutions, deletions and additions. As used herein, such mutants may be collectively referred to as CFI combination mutants.

[0095] B. Fusion constructs containing complement factor I Provided herein are fusion constructs that include at least a first component (CFI portion) that includes at least one domain of complement factor I, and at least a second component, where the first component is fused to the second and subsequent components (e.g., contiguous or separated by an optional linker). These fusion constructs are referred to herein as "CFI fusion constructs" or simply "fusion constructs." In some embodiments, the fusion constructs include additional components, e.g., a third component, a fourth component, a fifth component, etc. The exemplary construct in FIG. 7 shows a fusion construct having two N-terminal components, two C-terminal components, and a core component, i.e., at least five components.

[0096] FIG. 7 shows a schematic diagram of an exemplary CFI fusion construct, in which a "core" domain may comprise any one or more of the CFI variants of the present disclosure, flanked at either or both the N-terminus and C-terminus by one or more domains (one or more components). The depicted domains may optionally be linked by linkers. For example, the photograph in FIG. 7 shows two N-terminal and two C-terminal components, a five-component fusion construct. The variants provided in Table 3 follow this format. Exemplary first component CFI core domains are shown in Table 2 and Table 3 below.

[0097] [Table 3-1]

[0098] [Table 3-2]

[0099] [Table 3-3]

[0100] [Table 3-4]

[0101] [Table 3-5]

[0102] [Table 3-6]

[0103] [Table 3-7]

[0104] In some embodiments, the first component comprises a wild-type CFI of any species, either full length or a domain thereof (the "core" domain). In some embodiments, the first component comprises a CFI variant of the present disclosure detailed in the previous section, e.g., Table 3, and also provided in Table 2. It should be noted that the second and subsequent components may increase the activity of the CFI portion (first component) or may alter the specificity or half-life thereof. The second and subsequent components may also enable the CFI portion (first component) to act within the complement system in the absence of an exogenous cofactor (e.g., a cofactor such as factor H (FH) or CR1). As used herein, an exogenous cofactor of CFI is one that is not fused to CFI. It should be understood that the fusion construct may act within the complement system in the absence of FH and / or CR1, and that the activity of the fusion construct may also be further increased by the presence of FH and / or CR1, either as part of the fusion construct or provided exogenously.

[0105] Provided herein are fusion constructs that include a first component that includes any one of the CFI variants provided herein. It should be understood that the CFI variant can be any one of the CFI variants shown in Tables 2 and 3.

[0106] In some embodiments, the second and subsequent components of the fusion construct are proteins. In some embodiments, the second and / or subsequent components are not proteins.

[0107] The components of the fusion construct of the present disclosure may be held together by an optional linker, as shown in FIG. 7. They may be of any suitable length of at least one amino acid. The linker may be a flexible linker and may be a peptide of about 1 to about 20 amino acid residues in length, and the amino acid residue may include a glycine residue. The linker may also optionally include a serine residue. Exemplary flexible linkers may include, but are not limited to, glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, or any other suitable flexible linkers known in the art. An exemplary linker is GGSSGG n (SEQ ID NO: 51), where n is any number from about 1 to about 20. Other exemplary linkers include (GGSS) n (GGSS) n GG is included, which indicates n repeated times GGSS followed by the linker GG (SEQ ID NO: 27). In some embodiments, the linker is a protease-sensitive cleavable linker. Exemplary linkers linking fusion constructs can be 1-50, 5-50, 10-50, 15-50, 20-50, 25-50, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-15, 6-10, 6-9, 6-8, or 6-7 amino acids in length.

[0108] CFI+half-life extender fusion construct In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant (first component) and a second component, the second component being a half-life extender. Since naturally occurring CFI has a relatively short half-life, in some embodiments, it may be advantageous to increase the half-life of CFI. As used herein, "CFI" is used to connote either wild-type CFI or a mutant thereof. By using a second component that is a half-life extender, the activity of CFI may be increased or another characteristic of CFI may be improved compared to wild-type CFI. For example, wild-type CFI or a CFI mutant may have their half-life extended by fusing CFI to a half-life extender.

[0109] Exemplary half-life extenders include, but are not limited to, albumin, such as human serum albumin, PEG, non-biodegradable polymers, biodegradable polymers, and Fc. In some embodiments, the second component is a protein and a half-life extender, such as albumin or Fc. In some embodiments, the second component is not a protein and is a half-life extender, such as PEG. In some embodiments, the half-life extender is a peptide repeat.

[0110] In some embodiments, the second component is a half-life extender and is albumin. Note that as used herein, albumin refers to any albumin, such as any serum albumin, or an albumin variant, or an albumin derivative. As an example, an albumin variant includes any albumin that contains at least one modification corresponding to the amino acid sequence shown in SEQ ID NO: 7 (wild-type human serum albumin (HSA)), or at least one modification corresponding to the amino acid sequence of any non-human species of albumin. In an exemplary embodiment, the albumin is human serum albumin (HSA), and is provided in SEQ ID NO: 7.

[0111] An exemplary fusion construct comprising wild-type CFI and HSA is referred to herein as "CFI-HSA" and is discussed in further detail below.

[0112] In some embodiments, a fusion construct of the present disclosure comprises albumin and a CFI variant of the present disclosure. Exemplary constructs are provided in Table 3.

[0113] Structural arrangement of fusion constructs An exemplary structural arrangement is shown in FIG.

[0114] In some embodiments, a wild-type CFI or a CFI variant of the disclosure is the first component of a fusion construct, and the CFI portion comprises chain A and chain B. In some embodiments, the fusion construct comprises a structural arrangement from N-terminus to C-terminus as (chain A)-(optional linker)-(chain B)-(optional linker)-(second component). In some embodiments, the fusion construct comprises an inversion of chain A and chain B in its CFI component, such that the structural arrangement from N-terminus to C-terminus is (chain B)-(optional linker)-(chain A)-(optional linker)-(second component).

[0115] In some embodiments, a wild-type CFI or a CFI variant of the disclosure is the first component of a fusion construct, and the CFI portion comprises chain A and chain B. In some embodiments, the fusion construct comprises a structural arrangement from N-terminus to C-terminus as (second component)-(optional linker)-(chain A)-(optional linker)-(chain B). In some embodiments, the fusion construct comprises an inversion of chain A and chain B in its CFI component, such that the structural arrangement from N-terminus to C-terminus is (second component)-(optional linker)-(chain B)-(optional linker)-(chain A).

[0116] In some embodiments, provided herein are fusion constructs that include at least a first component (CFI moiety) that is either a wild-type CFI or a CFI mutant provided herein, and a second component, where the first and second components are fused, and the second component is fused to the N-terminus of the CFI moiety. In some embodiments, the second component is fused to the C-terminus of the CFI moiety. In some embodiments, the second component is fused to the C-terminus of the CFI moiety, and a third component is further fused to the N-terminus of the CFI moiety. In some embodiments, the second component is fused to the N-terminus of the CFI moiety, and a third component is further fused to the C-terminus of the CFI moiety.

[0117] 2A-2D show a model of a fusion construct comprising albumin and a CFI variant, where the CFI variant comprises an AB chain inversion. FIG. 2A-2B shows a first version of a model of a fusion construct comprising human serum albumin (HSA) and the A and B chains of CFI, and FIG. 2C-2D shows a second version thereof, where the A and B chains comprise an inversion. The first version of the AB chain inversion CFI variant comprises an interdomain disulfide bond. The second version does not comprise an interdomain disulfide bond. Both versions of the inversion variant may be constructed in a head-to-tail fashion as (HSA)-(optional linker)-(B chain)-(optional linker)-(A chain).

[0118] Thus, provided herein are CFI variants, which are a first component of a fusion construct comprising a first component and a second component, and which are fused to a second component, and which comprise an A chain and a B chain, and which have a structural arrangement from the N-terminus to the C-terminus or the C-terminus to the N-terminus of: (second component)-(optional linker)-(B chain)-(optional linker)-(A chain).

[0119] In some embodiments, the second component is at least one domain of factor H. Fusion constructs comprising at least one CFI domain and factor H are discussed in more detail below. In some embodiments, the second component is at least one domain of CR1. Fusion constructs comprising at least one CFI domain and factor H are discussed in more detail below. In some embodiments, the second component comprises at least one domain of factor H and at least one domain of CR1. Fusion constructs comprising at least one CFI domain, at least one factor H domain, and at least one CR1 domain are discussed in more detail below.

[0120] Components of the fusion construct Provided herein are fusion constructs comprising a first component and an additional component. In some embodiments, the first component comprises wild-type CFI or a CFI variant of the present disclosure. In some embodiments, the component comprises a half-life extender. In some embodiments, the second component comprises at least one domain of factor H (FH), at least one domain of CR1, or a mixture of FH and CR1 domains. In some embodiments, the fusion construct further comprises an additional component. In some embodiments, the first, second, and third (or more) components are any one or more of the components presented in Table 4. Table 4 shows various exemplary components and amino acid sequences of the components that may be used to generate the CFI fusion constructs provided herein.

[0121] With reference to Table 4, SEQ ID NO:1 is the amino acid sequence of wild-type plasma-derived human CFI, designated "CFI-PD," and has a leader sequence. The wild-type CFI used in fusion with a second component may comprise the amino acid sequence of SEQ ID NO:5, which does not include the leader sequence present in SEQ ID NO:1. Alternatively, the mouse Ig kappa chain V-III region MOPC 63 leader sequence (SEQ ID NO:2) may be used in recombinant production of any of the CFI fusion constructs provided herein. In some embodiments, provided herein are CFI fusion constructs comprising at least one CFI domain, wherein the at least one CFI domain comprises the amino acid sequence set forth in SEQ ID NO:5.

[0122] [Table 4-1]

[0123] [Table 4-2]

[0124] C. Complement Factor I and Albumin Fusion Constructs CFI mutant + albumin fusion constructs In some embodiments, provided herein is a fusion construct comprising a first component which is a CFI variant of the present disclosure (see, e.g., Table 2) and a second component which is an albumin, e.g., serum albumin, e.g., human serum albumin.

[0125] In some embodiments, provided herein is a fusion construct comprising at least one CFI domain and a second component, the second component being HSA, and the at least one CFI domain comprises any one or more domains of CFI selected from SPD, FIMAC domain, SRCR domain, LDLr1, and LDLr2 domain. In some embodiments, any one or more domains of CFI comprise an amino acid sequence set forth in SEQ ID NO:5 or comprise an amino acid sequence derived from SEQ ID NO:5. In some embodiments, any one or more domains of CFI correspond to domains of wild-type CFI. In some embodiments, at least one CFI domain comprises one each of SPD, FIMAC domain, SRCR domain, and LDLr1 and LDLr2 domain. In some embodiments, at least one CFI domain of the CFI-HSA construct comprises only SPD.

[0126] FIG. 3A shows a model of an exemplary fusion construct comprising HSA fused to CFI, where CFI comprises a CFI variant comprising an SPD, a FIMAC domain, a SRCR domain, and one each of an LDLr1 and an LDLr2 domain. Thus, both A and B chains are included in CFI in this model. The FIMAC domain, the SRCR domain, and the LDLr1 and LDLr2 domains are both A chains or heavy chains, while the SPD is B chains or light chains. In some embodiments, the amino acid residues of any one or more domains of the fusion construct may correspond to those of wild-type CFI. In some embodiments, the amino acid residues of any one or more domains of the fusion construct may comprise one or more modifications relative to those of wild-type CFI.

[0127] FIG. 3B shows a model of an exemplary fusion construct including HSA fused to a CFI portion, where the CFI includes only a serine protease domain (SPD). The exemplary fusion construct shown in FIG. 3B may be referred to as "HSA-SPD" and includes an activation loop at amino acid residues 322-326, an autolysis loop at amino acid residues 455-463, and an S1 entrance frame at amino acid residues 529-536. In some embodiments, any one or more amino acid residues in the activation loop, autolysis loop, and S1 entrance frame of the fusion construct may correspond to amino acid residues of a wild-type SPD of CFI. In some embodiments, any one or more amino acid residues in the activation loop, autolysis loop, and S1 entrance frame of the fusion construct may include one or more modifications relative to the wild-type SPD of CFI.

[0128] D. Complement Factor I and Factor H Fusion Constructs In some embodiments, fusion constructs are provided herein that include wild-type CFI (or a variant thereof) fused to at least one domain of factor H. Factor H (FH), like CFI, is a protein involved in the complement pathway. FH is a cofactor of CFI that forms a complex with CFI and C3b to catalyze the cleavage of C3b by CFI. As described above, full-length FH includes 20 domains. FIG. 4A shows a schematic diagram of FH showing its 20 domains, each of which is a complement control protein (CCP) domain, each of which is connected in a head-to-tail configuration by a short linker. The CCP domains are numbered 1-20 starting from the N-terminus. CCPs 1-4 form a complex with C3b, and CCPs 19-20 form a complex with C3d. Without being bound by any theory or mechanism, it is believed that FH is important for efficient cleavage of C3b by CFI. Thus, in some embodiments, fusion constructs comprising specific domains of FH fused to at least one CFI domain may allow C3b cleavage independent of exogenous FH. Exogenous FH may be defined as any FH that is not fused to any CFI domain, and may be wild-type FH. Wild-type FH as used herein refers to any naturally occurring FH that is not disease-causing FH. In some embodiments, FH is human FH. In some embodiments, wild-type FH comprises the amino acid sequence shown in SEQ ID NO:4.

[0129] In some embodiments, the additional component of the fusion construct of the present disclosure is at least one factor H domain, or a portion of a domain of FH. In some embodiments, the at least one FH domain comprises CCP domains 1-20 of FH. In some embodiments, the at least one domain of FH corresponds to a domain of wild-type FH comprising the amino acid sequence set forth in SEQ ID NO:4.

[0130] In some embodiments, provided herein is a fusion construct comprising at least one CFI domain and an additional component, the additional component being at least one factor H domain, the at least one factor H domain comprising complement control protein (CCP) domains 1-4 and 19-20 of factor H. CCP domains 1-4 and 19-20 are referred to as "mini-factor H" (mini-FH). FIG. 4B shows a schematic diagram of mini-FH showing CCP domains 1-4 connected by a Gly connector to domains 19-20 comprising a His tag. In some embodiments, the mini-FH is human mini-FH. In some embodiments, the amino acid sequence of mini-FH comprises the amino acid sequence set forth in SEQ ID NO:8.

[0131] Based on the structure of the complex formed by C3b-CFI and mini-FH, several domains relevant to the function of FH are available. To drive FH-independent CFI cleavage activity, the following types of exemplary FH-CFI fusion constructs were generated as base molecules: (a) FH domains 1–8 fused to CFI (Factor H-CCP1–8+CFI). (b) FH domains 1–4 fused to CFI (Factor H-CCP1–4+CFI). (c) FH domains 19–20 fused to CFI (Factor H-CCP19–20+CFI). (d) FH domains 5–8 fused to CFI (Factor H-CCP1–4+19–20+5–8+CFI). (e) FH domains 1-4, 19-20, and / or 5-8 fused to CFI (f) FH domains 1–4 and 19–20 fused to CFI (Factor H-CCP1–4+19–20+CFI). (g) FH domains 1–4, 19–20, and 5–8 fused to CFI (Factor H-CCP1–4+19–20+5–8+CFI). (h) FH domains 1–8 fused to the LDLr2 CFI domain only (Factor H-CCP1–8+LDLR2-CFI). (i) FH domains 1–4, 19–20, and 5–8 fused to the LDLr2 CFI domain only (Factor H-CCP1–4 + 19–20 + 5–8 + LDLr2-CFI). (j) FH domains 1-4 fused to human serum albumin (HSA) and the serine protease domain (SPD) of CFI (CFI-HSA(SPD)-Factor H-CCP1-4) (k) FH domains 2-4 fused to human serum albumin (HSA) and the serine protease domain (SPD) of CFI (CFI-HSA(SPD)-Factor H-CCP2-4). (l) FH domains 2–3 fused to human serum albumin (HSA) and the serine protease domain (SPD) of CFI (CFI-HSA(SPD)-Factor H-CCP2–3).

[0132] The fusion can further be fused to albumin, such as HAS.

[0133] FIG. 5 shows a model of an exemplary fusion construct comprising FH and CFI, and comprising CCP domains 1-8 of FH fused to CFI, where the FH portion of the fusion construct is a truncated mini-FH, and the CFI comprises wild-type CFI. The wild-type CFI comprises one each of the SPD, FIMAC domain, SRCR domain, LDLr1 and LDLr2 domain. The exemplary fusion construct shown in FIG. 5 is also referred to herein as Factor H-CPP1-8+CFI. Both A and B chains are included in CFI. The FIMAC domain, SRCR domain, and LDLr1 and LDLr2 domains are both A chain or heavy chain, while the SPD is B chain or light chain. FH comprises a linker comprising domains 1-4 and domains 5-8. In some embodiments, any one or more amino acid residues of the domains of FH and / or CFI of the fusion construct may correspond to amino acid residues of wild-type FH or wild-type CFI, respectively. In some embodiments, any one or more amino acid residues of the FH and / or CFI domains of the fusion construct may contain one or more modifications relative to the wild-type FH or wild-type CFI domain, respectively.

[0134] Table 5A lists exemplary Factor H-containing fusion constructs.

[0135] [Table 5]

[0136] In some embodiments, the CFI variant is a first component of a fusion construct comprising a first component and a second component, the CFI variant is fused to a second component, the second component being at least one factor H domain, and the FH domain comprising CCP1-4 of FH.

[0137] In some embodiments, the CFI variant is a first component of a fusion construct comprising a first component and a second component, the CFI variant is fused to a second component, the second component being at least one factor H domain, and the FH domain comprising CCP1-4 of FH.

[0138] CR1 fusion constructs are discussed in the following section. It should be noted that FH and CR1 chimeric constructs can also be generated and act as one component of the fusion construct of the present disclosure. For example, referring to construct number 90 in Table 3, such components can be: HHHHHHSG (SEQ ID NO: 38); hCR1; CCP15; CCP16; CCP17; hFH; CCP4; GGGGGGGGGGGG (SEQ ID NO: 37); hFH; CCP19; CCP20.

[0139] E. Complement Factor I and Complement Receptor 1 Fusion Constructs In some embodiments, provided herein is a fusion construct comprising wild-type CFI (or a variant thereof) fused to at least one domain of complement receptor 1 (CR1). CR1 is also referred to as CD35. CR1, like CFI, is a protein involved in the complement pathway. CR1 is a cofactor of CFI. Thus, in some embodiments, a fusion construct comprising a particular domain of CR1 fused to at least one CFI domain may enable C3b and / or C4b cleavage independent of exogenous cofactors. An exogenous CR1 cofactor may be defined as any CR1 or part thereof that is not fused to any CFI domain, and may be wild-type CR1, or may be CCP domains 1-3 or 15-17 of CR1. Wild-type CR1 as used herein refers to any naturally occurring CR1 that is not a disease-causing CR1. In some embodiments, the CR1 is human CR1.

[0140] In some embodiments, at least a second component of the fusion construct of the present disclosure is at least one CR1 domain, or a portion of a domain of CR1. In some embodiments, at least one CR1 domain comprises CCP domains 8-10 of CR1. In some embodiments, at least one CR1 domain comprises CCP domains 15-17 of CR1. In some embodiments, at least one CR1 domain comprises CCP domains 1-3 of CR1. In some embodiments, a fusion construct of the present disclosure comprising at least one CR1 domain also comprises a fusion with albumin. In some aspects, a fusion construct of the present disclosure comprising at least one CR1 domain also comprises a fusion with albumin and / or at least one domain of factor H. In some embodiments, at least one CR1 domain comprises CR1 CCP domain 15. In some embodiments, at least one CR1 domain comprises CR1 CCP domain 16. In some embodiments, at least one CR1 domain comprises CR1 CCP domain 17. In some embodiments, at least one CR1 domain comprises CR1 CCP domains 15-16. In some embodiments, at least one CR1 domain comprises CR1 CCP domains 16-17.

[0141] Table 5B lists exemplary CR1-containing fusion constructs and the corresponding sequences of exemplary fusion constructs containing wild-type CFI and CR1 CCP domains 15-17.

[0142] [Table 6-1]

[0143] [Table 6-2]

[0144] F. Combinatorial Fusion Constructs In some embodiments, provided herein are fusion constructs comprising at least one domain of Complement Factor I (CFI), a second component, a third component, a fourth component, and a fifth component, as provided in Figure 7 and Table 3. These exemplary fusion constructs may include combinations of components fused to each other, each comprising at least one "core" CFI domain. As noted above, some exemplary fusion constructs comprising a first component, a second component, and a third component comprising CFI may include fusion constructs comprising albumin, at least one CFI domain, and at least one domain of Factor H (FH).

[0145] FIG. 5 shows a schematic diagram of three exemplary fusion constructs including HSA, at least one CFI domain, and various domains of factor H. Each of the exemplary fusion constructs shown may include a CFI-HSA portion including a leader sequence, HSA, wild-type CFI as previously described herein, and various domains of FH (referred to as "CCP portion" in FIG. 5). As described above, the CFI-HSA portion may be constructed similarly with a GGSSGG (SEQ ID NO: 6) linker fusing the HSA and SPD of CFI to each other. An exemplary fusion, referred to herein as the CFI-HSA-FH_CCP1-4 fusion construct, includes wild-type CFI and the CCP domains of FH 1-4. CFI-HSA-FH_CCP1-4 also includes a GGSSGG (SEQ ID NO: 6) linker, which is a combination of a GGSSGG-linker (SEQ ID NO: 6) and a Gly-only linker that connects the CCP4 and CCP19 domains together in the mini-factor H. Other exemplary fusion constructs shown include CCP domains 2-4 of FH and CCP domains 2-3 of FH. The lengths of the linkers used in the exemplary fusion constructs are shown, with conservative minimum lengths shown in brackets. It should be understood that any other suitable flexible linkers may also be used.

[0146] Other exemplary fusion constructs provided herein include wild-type CFI or CFI mutant, at least one FH domain, and at least one CRI domain. In some embodiments, the fusion construct includes wild-type CFI or CFI mutant, at least one FH domain, and at least one CRI domain. In some embodiments, the fusion construct includes human serum albumin, wild-type CFI or CFI mutant, at least one FH domain, and at least one CRI domain. The fusion construct including at least one FH domain and at least one CR1 domain can include an orientation including an FH domain fused to a CR1 domain, alternating FH and CR1 domains, one or more consecutive FH domains fused to one or more consecutive CR1 domains, one or more consecutive CR1 domains fused to one or more FH domains, or a combination thereof. In some embodiments, the fusion construct includes wild-type CFI or CFI mutant, hCR1; CCP15; CCP16; CCP17, and hFH; CCP1; CCP2; CCP3; CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant, and hCR1;CCP15;hFH;CCP2;CCP3;CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant, and hFH;CCP1;hCR1;CCP16;hFH;CCP3;CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant, and hCR1;CCP15;CCP16;hFH;CCP3;CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant, and hFH;CCP1;hCR1;CCP16;CCP17;hFH;CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant, and hCR1;CCP15;CCP16;CCP17;hFH;CCP4. It is understood that any of the fusion constructs may further comprise one or more linkers as described herein. In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant, at least one FH domain, at least one CRI domain, and a linker region.It is understood that any of the fusion constructs may further comprise human serum albumin. In some embodiments, the fusion construct comprises human serum albumin, wild-type CFI or a CFI variant, at least one FH domain, and at least one CRI domain.

[0147] In some embodiments, provided herein is a fusion construct comprising a first component comprising at least one CFI domain, a second component, and a third component, wherein the second component is at least one domain of FH, and the third component is an optional half-life extender. In some embodiments, the third component is a protein (e.g., serum albumin or Fc). In some embodiments, the third component is not a protein (e.g., PEG).

[0148] II. Generation of CFI mutants and CFI fusion constructs Methods and compositions for generating CFI variants and CFI fusion constructs are provided herein. Accordingly, nucleic acids and vectors encoding any of the disclosed CFI variants or fusion constructs are provided. Cells comprising one or more nucleic acids encoding CFI or variants thereof, and the disclosed fusion constructs are also provided.

[0149] Provided herein are nucleic acids encoding the CFI variants and fusion constructs described herein.

[0150] Provided herein are expression vectors encoding the CFI variants and fusion constructs described herein. The expression vectors can include a transcriptional regulatory element, such as an enhancer or promoter, operably linked to a nucleic acid sequence encoding a CFI variant or fusion construct of the present disclosure.

[0151] Cell lines can be developed to express the production of CFI and mutant and fusion constructs described herein. Cell lines for producing CFI, CFI can be accomplished using any host cell capable of expressing the CFI mutants and CFI fusion constructs described herein. The host cells can be mammalian, insect, fungal, plant, and / or bacterial cells. For expression of CFI mutants and fusion constructs, the host cell lines can be transiently or stably transfected or transduced with expression vectors encoding CFI, CFI mutants, and CFI fusions. The vectors can be, for example, plasmids or viral vectors. In some embodiments, the host cells are mammalian cell lines. In some embodiments, the host cells are Chinese Hamster Ovary (CHO) cells.

[0152] The CFI variants and fusion constructs described herein can be recombinantly expressed in mammalian cell lines known in the art for producing biological products, such as Chinese Hamster Ovary (CHO) cells. Mammalian cells can be transfected or transduced with expression vectors encoding the CFI variants and fusion constructs described herein using any method known in the art.

[0153] Provided herein is a method for producing an activated CFI or a mutant thereof, comprising producing CFI in a cell comprising one or more nucleic acids encoding CFI or a mutant thereof and an expression cassette for furin.

[0154] Methods are provided herein for the production and purification of the CFI variants and fusion constructs described herein. The CFI variants and fusion constructs described herein may be purified from conditioned medium by standard methods known in the art. In some embodiments, the CFI variants and fusion constructs may be purified by chromatography on an affinity matrix. In some embodiments, the affinity matrix is ​​CaptureSelect™ human albumin affinity matrix. In some embodiments, the CFI variants and fusion constructs may be purified by chromatography on a cation exchange matrix and / or anion exchange matrix, and optionally size exclusion chromatography. The CFI variants and fusion constructs may be optimally buffer exchanged into any suitable buffer known in the art. Purity may be assessed by any method known in the art, including gel electrophoresis, orthogonal HPLC methods, staining and spectrophotometric techniques.

[0155] III. Use of CFI Variants and CFI Fusion Constructs The CFI variants and fusion constructs of the present disclosure may be used to regulate the complement system.

[0156] As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the present disclosure can regulate both the classical and lectin complement pathways.

[0157] As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the present disclosure are capable of regulating the alternative complement pathway.

[0158] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the present disclosure can reduce amplification of the complement system.

[0159] As discussed herein, in some embodiments, a CFI mutant or CFI fusion construct of the present disclosure is capable of increasing cleavage of C3b.

[0160] As discussed herein, in some embodiments, a CFI mutant or CFI fusion construct of the present disclosure is capable of increasing cleavage of C4b.

[0161] As discussed herein, in some embodiments, a CFI mutant or CFI fusion construct of the present disclosure can increase the production of C4c.

[0162] As discussed herein, in some embodiments, a CFI mutant or CFI fusion construct of the present disclosure can increase production of iC3b.

[0163] As discussed herein, in some embodiments, a CFI mutant or CFI fusion construct of the present disclosure can increase the production of C3dg from iC3b.

[0164] As discussed herein, in some embodiments, the CFI mutants or CFI fusion constructs of the present disclosure can increase the generation of C3c from iC3b.

[0165] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the present disclosure can reduce levels of the C3b alpha chain.

[0166] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the present disclosure is capable of increasing hydrolysis of a peptide substrate.

[0167] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the present disclosure can increase proteolysis of a macromolecular protein substrate.

[0168] As discussed herein, in some embodiments, a CFI mutant or CFI fusion construct of the disclosure can reduce the levels or function of the membrane attack complex (MAC).

[0169] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the present disclosure can reduce observed hemolysis.

[0170] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the present disclosure can increase cleavage of C3b in the absence of a cofactor, e.g., in a cofactor-independent manner.

[0171] As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure can increase cleavage of C4b in the absence of a cofactor, e.g., in a cofactor-independent manner.

[0172] The CFI variants and fusion constructs of the present disclosure may be used to treat a subject. As used herein, a subject includes any mammalian subject, including primates, rodents, livestock, zoo animals, and pets. In some embodiments, the mammalian subject is a human subject. In some embodiments, the mammalian subject is a non-human primate.

[0173] A. CFI Mutants and Fusion Constructs for Modulation of the Complement System Provided herein are methods of modulating the complement system, comprising contacting a sample in vitro or a tissue in vivo with any one of the CFI variants or fusion constructs provided herein. In some embodiments, the sample is plasma.

[0174] B. Treatment of Non-ocular Symptoms In some embodiments, the CFI variants or fusion constructs provided herein are useful for treating a non-ocular condition in a subject. In some embodiments, provided herein is a method of treating an ocular condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the CFI variants or fusion constructs provided herein, or a pharmaceutical composition provided herein below.

[0175] In some embodiments, the non-ocular condition is characterized by a deficiency in CFI. In some embodiments, the non-ocular condition is characterized by dysregulation of the complement system.

[0176] In some embodiments, the non-ocular condition is an acute systemic indication, hi some embodiments, the non-ocular condition is an acute systemic indication selected from the group consisting of acute glomerulonephritis, acute kidney injury, acute respiratory distress syndrome, bacterial meningitis, cerebral hemorrhage, burns, coronavirus infection, Epstein-Barr virus infection, hematopoietic stem cell transplantation, ischemia-reperfusion injury, Lyme disease, myocardial infarction, organ transplant, periodontitis, pneumonia, pre-eclampsia, schistosomiasis, sepsis, stroke, thromboembolism, and traumatic brain injury.

[0177] In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is a chronic systemic indication. In some embodiments, the non-ocular condition is a chronic systemic indication. In some embodiments, the non-ocular condition is a chronic systemic indication. In some embodiments, the non-ocular condition is a chronic systemic indication. In some embodiments, the non-ocular condition is a chronic systemic indication. In some embodiments, the non-ocular condition is a chronic systemic indication. and systemic chronic indications selected from the group consisting of chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington's disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein-losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), ulcerative colitis, and ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA), and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington's disease, and ischemia-reperfusion injury.

[0178] In some embodiments, the CFI variants or fusion constructs provided herein have improved characteristics compared to wild-type CFI. In some embodiments, the improved characteristics are increased activity, which includes increased cleavage of C3b and / or C4b. The potency and specificity of the CFI variants provided herein can be tailored for a particular therapeutic indication. In some embodiments, the CFI variants or fusion constructs provided herein are C3b degrading agents. In some embodiments, the C3b degrading agents are useful for treating a disease. In some embodiments, the CFI variants provided herein are C4b degrading agents and are useful for treating a disease. For example, diseases that may be treated by the use of C4b degrading agents include, but are not limited to, non-ocular conditions. In some embodiments, the non-ocular condition is a systemic chronic indication.In some embodiments, the non-ocular condition is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), immune-mediated hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease (COPD), cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), granulomatosis with polyangiitis (GPA), Guillain-Barre syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy, lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy ( and systemic chronic indications selected from the group consisting of ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA), and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington's disease, and ischemia-reperfusion injury.

[0179] In some embodiments, the non-ocular condition is non-neoplastic.

[0180] In some embodiments, the non-ocular condition is neoplastic. In some embodiments, the non-ocular condition is neoplastic and is characterized by a solid tumor or a liquid tumor. In some embodiments, the non-ocular condition is characterized by a solid tumor and is selected from the group consisting of colorectal tumor, hormone refractory prostate cancer, melanoma, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic pancreatic cancer, metastatic gastric cancer, nasopharyngeal carcinoma, non-small cell lung cancer, pancreatic tumor, squamous cell carcinoma, and gastric tumor. In some embodiments, the non-ocular condition is characterized by a liquid tumor and is selected from the group consisting of acute myeloid leukemia, B-cell lymphoma, and Hodgkin's disease.

[0181] C. CFI Mutants and Fusion Constructs for Treatment of Ocular Conditions In some embodiments, the CFI variants or fusion constructs provided herein are useful for treating an ocular condition in a subject. In some embodiments, provided herein are methods of treating an ocular condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the CFI variants or fusion constructs provided herein, or a pharmaceutical composition provided herein below.

[0182] In some embodiments, the ocular condition is characterized by a deficiency in CFI. In some embodiments, the ocular condition is characterized by dysregulation of the complement system.

[0183] In some embodiments, the ocular condition is characterized by the presence of a dysfunctional CFI gene, hi some embodiments, the ocular condition is characterized by dysregulation of the complement system and low CFI levels.

[0184] In some embodiments, the ocular condition is selected from the group consisting of diabetic macular edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open-angle glaucoma, polypoidal choroidal vasculopathy, Stargardt's disease, uveitis, and vitreoretinopathy.

[0185] In some embodiments, the ocular condition is non-neoplastic.

[0186] D. Combination Therapy Administration of any one of the therapeutic CFI variants or fusion constructs provided herein may be a monotherapy or may be combined with any other known drug or treatment. The other known drug or treatment may be for a condition associated with dysregulation of the complement system or may be associated with CFI deficiency. In some embodiments, the condition may be ocular. In some embodiments, the condition may be non-ocular. In some embodiments, the therapeutic CFI variants or fusion constructs provided herein are co-administered with one or more C5 inhibitors. In some embodiments, the C5 inhibitor is eculizumab. In some embodiments, the C5 inhibitor is semiziran.

[0187] E. Administration The CFI variants and fusion constructs described herein may be delivered as polypeptide-based therapies or nucleic acid-based therapies.

[0188] Such treatments contemplated herein include both administration of a CFI variant of this disclosure or a fusion construct of this disclosure, as well as administration of one or more nucleic acids encoding a CFI variant of this disclosure or a fusion construct of this disclosure. Accordingly, provided herein are pharmaceutical compositions comprising a CFI variant of this disclosure, a CFI fusion construct of this disclosure, as well as pharmaceutical compositions comprising one or more nucleic acids encoding a CFI variant of this disclosure and encoding a fusion construct of this disclosure.

[0189] Thus, nucleic acids encoding the CFI variants and fusion constructs of the present disclosure are provided herein and delivered to a subject in need thereof as part of a nucleic acid-based gene therapy. In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the present disclosure are delivered as part of a viral vector-based gene therapy (e.g., lentivirus-based therapy, adenovirus-based therapy, adeno-associated virus-based therapy, etc.). In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the present disclosure are delivered as naked nucleic acids. In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the present disclosure are delivered inside a liposome. In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the present disclosure are delivered as part of a nanoparticle. In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the present disclosure are delivered as part of a virus-like particle.

[0190] In some embodiments, the CFI variants and fusion constructs described herein may be delivered as a polypeptide-based therapy.

[0191] In vivo administration of the therapeutic CFI variants or fusion constructs (protein or nucleic acid based therapeutics) described herein may be performed intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraarterially, intraventricularly, intranasally, transmucosally, implanted, or by inhalation. Administration of the therapeutic fusion constructs may be performed using any suitable excipient, carrier, or other agent to provide suitable or improved tolerance, translocation, delivery, and the like.

[0192] In exemplary embodiments, administration of a therapeutic CFI mutant or fusion construct described herein is subcutaneous administration. In some embodiments, subcutaneous administration is daily, every other day, twice weekly, or once weekly.

[0193] In some embodiments, administration of a therapeutic CFI mutant or fusion construct described herein is intravenous administration.

[0194] As generally contemplated herein, the CFI mutants or fusion constructs described herein are delivered in an activated, two-chain form. In some cases, however, an inactive CFI mutant or fusion construct can be delivered in an inactive, single-chain form. In some embodiments, what is delivered includes both the single-chain inactive form and the two-chain active form.

[0195] F. Dosage In some embodiments, any of the therapeutic CFI variants or fusion constructs described herein may be administered to a subject in need thereof at a dosage of about 0.05 mg / kg to about 10 mg / kg. In some embodiments, the dosage is about 1 mg / kg. In some embodiments, administration of a therapeutic CFI variant or fusion construct described herein is subcutaneous at a dosage of about 0.05 mg / kg, 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, administration of a therapeutic CFI variant or fusion construct described herein is intravenously administered at a dose of about 0.05 mg / kg, 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, administration of a therapeutic CFI variant or fusion construct described herein is daily, every other day, weekly, or twice weekly.

[0196] In some embodiments, the target level of a therapeutic fusion construct in plasma is about 0.05 mg / kg, 0.1 μg / mL, about 0.5 μg / mL, about 1 μg / mL, about 1.5 μg / mL, about 2 μg / mL, about 2.5 μg / mL, about 3 μg / mL, about 3.5 μg / mL, about 4 μg / mL, about 4.5 μg / mL, 5 μg / mL, about 5.5 μg / mL, about 6 μg / mL L, approximately 6.5μg / mL, approximately 7μg / mL, approximately 7.5μg / mL, approximately 8μg / mL, approximately 8.5μg / mL, approximately 9μg / mL, approximately 9.5μg / mL, approximately 10μg / mL, approximately 10.5 μg / mL, approximately 11 μg / mL, approximately 11.5 μg / mL, approximately 12 μg / mL, approximately 12.5 μg / mL, approximately 13 μg / mL, approximately 13.5 μg / mL, approximately 14 μg / mL, approximately 14.5 μg / mL, 15μg / mL, approximately 15.5μg / mL, approximately 16μg / mL, approximately 16.5μg / mL, approximately 17μg / mL, approximately 17.5μg / mL, approximately 18μg / mL, approximately 18.5μ g / mL, approximately 19μg / mL, approximately 19.5μg / mL, approximately 20μg / mL, approximately 20.5μg / mL, approximately 21μg / mL, approximately 21.5μg / mL, approximately 22μg / mL, approximately 22.5μ The target level may be about 10 μg / mL, about 25 μg / mL, about 50 μg / mL, about 100 μg / mL, about 150 μg / mL, about 200 μg / mL, about 250 μg / mL, or even about 300 μg / mL. An exemplary fusion construct that may be administered to a subject in need thereof to achieve a target level of about 20 μg / mL may include CFI-HSA, which contains a CFI corresponding to wild-type CFI.

[0197] G. Preparations Pharmaceutical compositions containing the disclosed CFI variants or fusion constructs can be formulated in any conventional manner by mixing a selected amount of the polypeptide with one or more physiologically acceptable carriers or excipients for use in the treatments provided herein. The choice of carrier or excipient is within the skill of the administration specialist and can depend on several parameters. These include, for example, the mode of administration and the disorder being treated. The pharmaceutical compositions provided herein can be formulated for single dose (direct) administration or for dilution or other modification. The concentration of the compound in the formulation is effective for delivery of an amount effective for the intended treatment upon administration. Typically, but not necessarily, the composition is formulated for single administration.

[0198] H. Pharmaceutical Compositions The present disclosure also provides a pharmaceutical composition comprising any one of the CFI mutants or fusion constructs disclosed herein, and optionally a pharma- ceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical compositions may be formulated to be compatible with their intended route of administration. In some embodiments, the pharmaceutical compositions of the present disclosure are suitable for administration to human subjects or other non-human primates. In an exemplary embodiment, the pharmaceutical composition is formulated for subcutaneous administration.

[0199] I. Kits and Articles of Manufacture for Therapeutic CFI Mutants and Fusion Constructs The present disclosure also provides kits or articles of manufacture comprising any one of the CFI mutants or fusion constructs disclosed herein, or any pharmaceutical composition disclosed herein. In some embodiments, the kit may further comprise instructional materials for practicing any of the methods disclosed herein. In some embodiments, the kit may further comprise a sterile container or vial for holding the fusion construct and / or pharmaceutical composition disclosed herein. In some embodiments, the kit may further comprise a sterile delivery device for administering the fusion construct and / or pharmaceutical composition disclosed herein. In some embodiments, the article of manufacture comprises any pharmaceutical composition of the present disclosure. EXAMPLES

[0200] Example 1: Expression, purification, activation, and in vitro sialylation of CFI mutants and fusion proteins overview In Example 1, reference to CFI-HSA refers to human serum albumin fused to the N-terminus of human wild-type CFI (SEQ ID NO:21).

[0201] Wild-type CFI-HSA protein was expressed in Chinese Hamster Ovary (CHO) cells, purified by anti-albumin affinity purification, activated with furin, and purified by sizing column. The activated CFI-HSA protein was subjected to in vitro sialylation to increase the total sialylation of CFI-HSA. Finally, the sialylated protein was purified using anti-albumin affinity purification and finalized by size-exclusion column chromatography.

[0202] Expression The CFI-HSA gene (SEQ ID NO: 21) with human serum albumin at the amino terminus of the CFI protein was synthesized (ThermoFisher Scientific, Geneart, Regensburg, Germany). The protein was made with a signal sequence of SEQ ID NO: 2 that was removed during expression. The amino-terminal albumin tag was connected to the CFI gene via a linker (SEQ ID NO: 6). The gene for CFI-HSA was inserted into an expression vector (Lake Pharma, Hayward, CA) using standard molecular biology techniques. The resulting plasmid DNA was transformed into E. coli. The transfected E. coli was grown in 200 mL LB medium for expression of the plasmid DNA and harvested using standard techniques. The plasmid DNA was run on an agarose gel for quality assessment and sequence confirmation before proceeding to transfection.

[0203] 1.0 liter of suspension TunaCHO™ cells were seeded into shake flasks and grown using serum-free synthetic medium. On the day of transfection, the grown cells were seeded into new flasks containing fresh medium. Plasmid DNA was transiently transfected into CHO cells using Lipofectamine 2000 (ThermoFisher Scientific). Cells were maintained as batch-fed cultures until the end of the production run. Protein was expressed for 14 days at 37°C, 125 RMP with 8% CO2 concentration. Cells were centrifuged and the supernatant was collected for purification of secreted CFI-HSA after 14 days of expression.

[0204] purification The supernatant containing the expressed CFI-HSA protein was passed through a 10 mL gravity-flow column of CaptureSelect™ human albumin affinity matrix (ThermoFisher Scientific). The protein bound to the column was washed with 10 column volumes of 20 mM sodium phosphate buffer. The bound CFI-HSA protein was eluted in two steps: first, with 3 column volumes of 20 mM Tris-HCl, pH 7.0 buffer containing 2 M MgCl2, and second, with 3 column volumes of 20 mM citric acid, pH 3.0. The eluates from both steps 1 and 2 were collected in 5 mL fractions. Each fraction of the step 2 elution was neutralized with 10% neutralization buffer (1.5 M tris-HCL pH 7.4). All fractions were analyzed by reducing and non-reducing SDS-PAGE electrophoresis, and bands were visualized by SimplyBlue™ SafeStain (ThermoFisher Scientific). CFI-HSA migrates as a 130 kDa band on non-reducing gels and as 102 kDa and 28 kDa bands on reducing gels. Fractions with the greatest CFI-HSA concentration and purity were pooled for further processing.

[0205] Furin activation CFI-HSA is expressed as an inactive single-chain precursor protein and is activated by another serine protease, furin. Furin is an endoprotease that cleaves CFI at its conserved RRKR sequence (also called the furin recognition sequence), resulting in disulfide-linked heavy and light chains. The furin-processed mature two-chain protein is the activated form of the CFI protein.

[0206] Cleavage of CFI-HSA to produce the protein in its activated form was performed by incubating 4 μg of recombinant furin per mg of purified CFI-HSA in Tris-NaCl, 2.5 mM CaCl2, and 0.5% CHAPS for 18 h at 30°C. The CFI-HSA protein concentration was maintained at 1.4 mg / mL. This results in more than 90% activation of the protein. The activated protein was separated from the inactivated CFI-HSA and other proteins by size-exclusion chromatography. Size-exclusion chromatography (SEC) was performed using a HiLoad 16 / 600 Superdex 200 column (GE Healthcare Life Sciences) and phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) as the mobile phase. The collected fractions were analyzed by CE-SDS (LabChip GXII, Perkin Elmer). Fractions containing the target protein were pooled and analyzed by SE-UPLC.

[0207] In vitro sialylation The activated CFI-HSA protein was subjected to in vitro sialylation. Briefly, sialylation was performed in a two-step enzymatic reaction. First, the galactosylation reaction of CFI-HSA was carried out in a volume of 200 μL utilizing a 1:200 molar ratio of galactosyltransferase (GalT1) enzyme and CFI-HSA in 10 mM UDP-galactose, 5 mM MnCl2, and 100 mM MES, pH 6.5 buffer. Galactosylated CFI-HSA was purified from the reaction mixture by CaptureSelect™ human albumin affinity chromatography as previously described. The sialylation reaction was then carried out in a 250 μL volume at 37° C. for 1 h using a 1:50 molar ratio of the enzyme α2,6-sialyltransferase and purified CFI-HSA in 80 μM alkaline phosphatase, 6.1 mM CMP-NANA, 10 mM ZnCl2, and 200 mM MES buffer, pH 6.5. Sialylated CFI-HSA protein was purified from the reaction mixture by CaptureSelect™ human albumin affinity chromatography. The extent and characteristics of sialic acid chains on CFI-HSA were determined by utilizing Agilent / Prozyme analytical services, the GS-SAP method for total sialic acid quantification (Agilent GS48), and mass spectrophotometric (MS) analysis (Lake Pharma analytical services), as described in more detail below.

[0208] Briefly, total sialic acid quantification was performed by mixing 20 μL of each sample with 10 μL of releasing reagent in a 96-well plate. The reaction mixture was incubated at 80° C. for 2 h. The samples were cooled to room temperature and 10 μL of labeling reagent was added to each sample for further incubation at 50° C. for 3 h. The samples were cooled again to room temperature and 160 μL of deionized (dI) water was added to a total volume of 200 μL. 10 μL of sample was injected onto an Agilent UHPLC Poroshell C18 column and run at 30° C. in 4% methanol, 8% acetonitrile in water (line A1) and 100% ACN (line B1) at a flow rate of 0.4 mL / min. Peaks were recorded at 373 / 448 nm wavelength. A standard curve of total peak area versus picomoles (pmol) of sialic acid was generated by running 1-2000 pmol of NANA (N-acetylneuraminic acid, Neu5Ac) supplied with the kit on the same column. Total sialic acid in each sample was quantified by comparing the peak area of ​​the sample to the standard curve.

[0209] Mass spectrometry was performed by a standard trypsin Q-TOF mass spectrometer. Briefly, all samples were treated with DTT and iodoacetamide, reduced, alkylated, and then digested with trypsin. The digested samples were analyzed by a Waters ACQUITY UPLC coupled to a Xevo G2-XS-QTOF mass spectrometer using a protein BEH C18 column.

[0210] Finishing The purified CFI-HSA protein was subjected to size-exclusion chromatography (SEC) using a HiLoad 16 / 600 Superdex 200 column (GE Healthcare Life Sciences) and phosphate-buffered saline as the mobile phase. The collected fractions were analyzed by CE-SDS (LabChip GXII, Perkin Elmer). The fractions containing the target protein were pooled, the concentration was brought to 5 mg / mL, and the samples were flash-frozen for storage at -80°C.

[0211] Expression and purification of CFI-HSA mutants DNA for the CFI-HSA variants was generated either synthetically or by site-directed mutagenesis using standard techniques. Proteins were expressed in 250 mL suspensions of TunaCHO™ cells as described herein for wild-type CFI-HSA protein, except that expression was carried out for 7 days instead of 14 days. After 7 days, cells were centrifuged and conditioned medium was passed over a gravity-flow column of CaptureSelect™ human albumin affinity matrix (ThermoFisher Scientific). Proteins bound to the column were washed with 10 column volumes of 20 mM sodium phosphate buffer. Bound CFI-HSA protein was eluted in 5 mL fractions with 3 column volumes of 20 mM Tris-HCl, pH 7.0 buffer containing 2 M MgCl2. CFI-HSA or its variants were buffer exchanged (either by dialysis or spin concentrator) into 30 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2, pH 7.4. Recombinant human furin was added to CFI-HSA at a molar ratio of 1:25 (furin:CFI-HSA) and the reaction mixture was incubated for 16 hours at 30° C. Two micrograms of the activation mixture was run on a 9% SDS-PAGE gel to assess activation efficiency. In general, >80% activation was achieved.

[0212] The N-terminal albumin fusion provides solubility and promotes activation of CFI-HSA.

[0213] The activity of the CFI mutants of the present disclosure and fusion proteins containing such mutants can be compared to wild-type CFI or to each other. To do so, a genetic construct for WT-CFI was expressed essentially as described above for CFI-HSA. The purity of the recombinant protein can be assessed by SDS-PAGE under reducing and non-reducing conditions, either in the presence or absence of furin. It has previously been shown in PCT application PCT / US2021 / 037278 that there is a significant and unexpected benefit of an N-terminal HSA tag (in SEQ ID NO: 21) to maintain solubility, monodispersity and efficient furin activation. The same can be assessed for the CFI mutants and fusion proteins of the present disclosure.

[0214] Example 2: Characterization by peptide lytic activity assay In Example 2, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0215] The proteolytic activity of wild-type CFI-HSA and CFI mutant-HSA fusions (collectively referred to herein as "CFI-HSA proteins") can be tested by following the cleavage of a chromogenic substrate through the use of a chromophore. The S-2288 (Chromogenix) peptide substrate is chosen for this assay because it is sensitive to a wide range of serine proteases. The peptidolytic activity of the CFI-HSA protein is measured by the rate of production of p-nitroaniline (pNA) upon substrate cleavage, which occurs spectrophotometrically at 405 nm.

[0216] CFI-HSA protein is diluted to an initial concentration of 400 nM in 100 μL of HBS / BSA (30 mM HEPES, 140 mM NaCl, 0.2% BSA, pH 7.4) in a non-coated 96-well microplate (Nunc). A working stock of 4 mM S-2288 is made in HBS / BSA in a separate tube. The microplate and diluted chromogenic substrate are pre-warmed to 37° C. for 5 minutes. The assay is initiated by adding 100 μL of pre-warmed S-2288 to the wells of the microplate containing CFI-HSA protein. This results in a final concentration of 200 nM CFI-HSA protein and 2 mM S-2288 substrate in a 200 μL reaction volume. The rate of substrate cleavage is recorded every 30 seconds for 3 hours at 37° C. at 405 nm using a microplate reader (Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific). To calculate the percentage of peptidolytic activity of the CFI-HSA mutants, the peptidolytic activity of wild-type CFI-HSA is normalized as 100%.

[0217] Example 3: Characterization by C3b cleavage assay In Example 3, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0218] The C3b cleavage assay is a functional assay that can be used to determine the ability of wild-type CFI, wild-type CFI-HSA and mutant CFI, and CFI-HSA mutants (collectively referred to herein as "CFI-HSA proteins") to cleave their natural substrate C3b. An exemplary protocol is provided herein. For the analysis of C3b cleavage, CFI-HSA protein is incubated with C3b and truncated factor H (mini-FH) at 37°C. Mini-FH has previously been shown to be functionally active and support CFI-mediated C3b cleavage (J Immunol. 2013 Jul 15; 191(2): 912-21). Cleavage of C3b into smaller fragments is then monitored over time by SDS-PAGE.

[0219] First, for each CFI-HSA variant, a master reaction mixture is prepared at room temperature containing a final concentration of 500 nM mini-FH and 5 nM CFI-HSA protein in HBS buffer (30 mM HEPES, 140 mM NaCl pH 7.4). The master reaction mixture is transferred to 37 °C and equilibrated for 5 min. The cleavage reaction is initiated by adding C3b to a final concentration of 0.5 μM. 20 μL samples are withdrawn from the master mixture for each measured time point and quenched by the addition of 5 × SDS reducing sample buffer. Samples are run on a 9% SDS-PAGE gel and C3b cleavage is visualized by Coomassie staining. The amount of C3b cleavage that has occurred is quantified by densitometry. To calculate the percentage of C3b cleavage activity of the CFI-HSA variants, the C3b cleavage activity of wild-type CFI-HSA is normalized as 100%.

[0220] A time course of C3b cleavage by CFI-HSA protein can be performed in parallel to compare the rate of C3b cleavage by each CFI-HSA mutant with that of wild-type CFI-HSA. If disappearance of the C3(alpha)' band with molecular weight of 114 kDa is observed (on SDS-PAGE), it is indicative of C3b cleavage. This is because C3b contains two chains, (alpha)' and beta. Densitometry of the relevant stained bands, corrected for the average background staining (lane intensity outside the bands), can be performed to quantitatively assess.

[0221] The apparent rate of loss of band intensity can be estimated by fitting a simple exponential decay equation to the band intensity data as a function of time, thereby extracting the apparent rate constant (k) for C3b cleavage. The relative rate of C3b cleavage by the CFI-HSA mutant is calculated by dividing by the corresponding WT rate: k(mutant) / k(WT control). This procedure is performed in multiple independent SDS-PAGE experiments, and the average of k(mutant) / k(WT control) is calculated, along with the associated standard deviation.

[0222] The percentage of C3bα chain remaining after incubation over time is measured to assess the activity of the tested CFI mutants compared to wild-type CFI. Since even small differences in C3b cleavage can cause diseases such as atypical hemolytic uremic syndrome (aHUS), mutants that show differences may be useful to increase the activity of the complement system to combat C3-induced diseases.

[0223] Example 4: Quantitative analysis of CFI-HSA C3b cleavage activity by measuring C3dg formation by time-resolved immunofluorescence assay (TRIFMA) In Example 4, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0224] The C3dg assay can be used to determine the cleavage of C3b caused by complement factor I (CFI), or its mutants or fusion proteins. The formation of C3dg can be used as a quantitative analysis of CFI-HSA C3b cleavage activity, and in one possible assay, it is measured by time-resolved immunofluorescence assay (TRIFMA). An exemplary protocol is provided herein.

[0225] The complement pathway in human serum is activated by using heat-aggregated IgG. The effect of plasma-derived CFI or CFI-HSA proteins, including CFI-HSA mutants, on C3b cleavage is measured by capturing C3dg on a microtiter plate using a C3dg antibody. Bound C3dg is detected by a combination of biotinylated C3dg antibody and europium-labeled streptavidin and measured by time-resolved fluorometry.

[0226] MaxiSorb microtiter plates (Nunc) are coated with 100 μL monoclonal IgM rat anti-human C3dg antibody at 2 μg / mL in 15 mM Na2CO3, 35 mM NaHCO3, pH 9.6 coating buffer by overnight incubation at room temperature. Remaining protein binding sites are blocked by incubation with 1 mg / mL HSA in TBS. Unbound HSA is washed off with TBS-Tween.

[0227] Dilute test samples to the desired concentrations in 1-6 dilutions of human serum in a 100 µL volume containing dilution buffer (0.14 M NaCl, 10 mM Tris, 14 mM sodium azide, 0.05% (v / v) Tween 20 (TBS / Tween), 1 mg / mL HSA and 0.1 mg / ml heat-aggregated IgG. Four-fold, six-point dilutions are performed for each CFI-HSA variant, covering a variant concentration range of 3132 nM to 3 nM. Incubate the reaction mixture at 37 °C for 90 min and add 10 mM Quench with EDTA. To capture the generated C3dg, 100 μL of each reaction mixture is added to the antibody-coated microtiter wells and incubated overnight at 4 °C. To detect the bound C3dg, 100 μL of biotinylated rabbit anti-C3dg antibody (DAKO) is added to the wells at 0.5 μg / mL and incubated for 2 h at room temperature. After washing with Eu3+-streptavidin combination (Perkin Elmer), 25 μM EDTA is added to the wells and incubated for 1 h at room temperature (1 / 1000). After washing, 200 μL of enhancement buffer (Ampliqon) is added to each well. The plates are read by time-resolved fluorometry using a DELFIA-reader Victor5+ (Perkin Elmer).

[0228] Example 5: Characterization by hemolysis assay Hemolysis assay can be used for the measurement of hemolytic function of compounds using the complement pathway. Complement factor I (CFI) mediates C3b cleavage by its cofactor factor H (FH) in the alternative pathway of the complement pathway system. To test hemolytic function in the alternative pathway, C3-deficient human serum spiked with human C3 is incubated with the mutant / fusion protein of the present disclosure and rabbit Alsevers solution, and total hemolysis is measured spectrophotometrically. The results of the hemolysis assay can be performed with or without cofactor FH to understand the effect of FH on total hemolysis. An exemplary protocol is provided herein. Briefly, 12 mL of rabbit red blood cells (RBCs) are washed twice with GVB buffer (gelatin veronal buffer: Sigma, containing 8 mM EGTA and 10 mM MgCl2) and resuspended in 12 mL of ice-cold GVB buffer. Based on previous observations that 1 μM C3 supports maximal hemolysis in this system, C3-deficient human serum is spiked with 1 μM human C3. A 3-fold 8-point serial dilution of the mutant / fusion protein in GVB buffer is performed to achieve concentrations ranging from 260 μg / mL to 0.11 μg / mL in the reaction mixture. First, in a 96-well plate, 50 μL of reaction mixture for each concentration point is prepared by adding 62.8% human serum, different concentrations of mutant / fusion protein with or without 200 μg / mL FH. The hemolysis reaction is initiated by adding 50 μL of rabbit RBCs and incubated for 30 min at 37 °C in a microtiter plate. All assays are performed in triplicate and all dilutions are performed in GVB buffer. For maximum hemolysis control, deionized water is added to the RBCs and for no hemolysis control, 0.154 M NaCl is added to the RBCs. After incubation, the plate is centrifuged at 2000 rpm for 5 min and 90 μL of the supernatant is transferred to another 96-well plate. The hemolysis rate is quantified by measuring the optical density (OD) at 412 nm of the lysed RBCs.

[0229] The absorbance at 412 nm is converted to percentage hemolysis, with maximum hemolysis from the controls set as 100% and the buffer control set as 0%.

[0230] 9A-9B show dose-response curves generated from hemolytic assays for CFI with and without its cofactor Factor H, respectively. Dose-response curves were generated by nonlinear regression analysis and curve fitting to a four-parameter sigmoidal curve in Prism software. Data are presented as 50% alternative pathway activity (AP) of wild-type CFI-HSA with FH, and plasma-derived CFI with FH. 50 ) was shown.

[0231] Figures 9C-D show dose-response curves for the percentage of hemolysis inhibition measured by plasma-derived CFI and wild-type CFI-HSA in the classical and alternative pathways, respectively, demonstrating that plasma-derived CFI and wild-type CFI-HSA function similarly in human serum.

[0232] These data showed that at higher concentrations, both CFI-HSA and CFI-PD were active in the hemolytic assay. The inhibitory activity of CFI-HSA against the alternative pathway was similar to that of CFI-PD in the hemolytic assay. The hemolytic assay also showed that the inhibitory effect of CFIs, both CFI-HSA and CFI-PD, against the alternative pathway was significantly increased with the cofactor FH.

[0233] The ability to inhibit classical pathway hemolysis by mutants / fusion proteins can be measured. An exemplary protocol is provided herein. Sheep red blood cells are activated with anti-SRBC antibodies (Amboceptor, Testline, UK). SRBCs are suspended in gelatin veronal buffer (GVB). In the assay plate, a dilution series of the CFI mutant is added, followed by activated SRBCs and factor B and I depleted serum at approximately 1% (v / v). The activated SRBCs are incubated with the test substance for 30 minutes. The cells are pelleted and the supernatant is transferred to a separate plate for absorbance reading at 412 nM. Percent lysis is calculated as follows: 100 *(Absorbance of test sample) / (Absorbance without CFI (0% inhibition)). Data are plotted and analyzed using a four-parameter nonlinear regression (GraphPad Software, USA). IC 50 Values ​​were calculated for data from individual plates and the average was calculated as logIC 50 values ​​and convert to concentrations (nM).

[0234] The ability to inhibit alternative pathway hemolysis by mutants / fusion proteins can also be measured. An exemplary protocol is provided herein. Sheep red blood cells are activated by anti-SRBC antibodies (Amboceptor, Testline, UK). SRBCs are suspended in 8% (v / v) factor B and factor H depleted normal human serum to which eculizumab is added to deposit C3b. Activated SRBCs with deposited C3b are incubated with full length factor H (Complement Technologies, USA) and the test substance. After 10 minutes of incubation, factors B and D (Complement Technologies, USA) are added and incubated for another 10 minutes. Finally, guinea pig serum (Sigma-Aldrich, UK) is added and incubated for 20 minutes. Cells are pelleted and the supernatant transferred to another plate for absorbance reading at 412 nM. Percent lysis is calculated as follows: 100 * (Absorbance of test sample) / (Absorbance without CFI (0% inhibition)). Data are plotted and analyzed using a 4-parameter non-linear regression (GraphPad Software, USA). IC50 values ​​are calculated for data from individual plates and averages are performed on the logIC50 values ​​and converted to concentration (nM).

[0235] Example 6: Pharmacokinetic modeling to determine dosing in humans based on non-human primate data The variants / fusion proteins can be tested for concentration levels in plasma after a single subcutaneous administration in a model system, e.g., African green monkeys. The fusion constructs included human serum albumin (HSA) and wild-type CFI (CFI-HSA). FIG. 8A is a graph showing the measured concentrations of the CFI-HSA fusion construct compared to free CFI after subcutaneous administration to monkeys at a dose of 1 mg / kg. The CFI-HSA fusion construct was shown to be able to achieve a target level of about 20 μg / mL. Measurable concentrations of the CFI-HSA fusion construct were sustained for up to 14 days, with a target concentration of about 20 μg / mL measured for about 7 days. These data support weekly subcutaneous administration of the CFI-HSA fusion construct for therapeutic use. These data support that weekly subcutaneous administration can be used for therapeutic purposes in humans for CFI-HSA, and comparable modeling can be performed for the variants and variant fusion constructs of the present disclosure. FIG. 8B shows the graph shown in FIG. 8A, with individual data points shown along the curve for added clarity.

[0236] Example 7: Characterization by C3b and C4b cleavage assays C3b cleavage reaction In Example 7, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0237] One exemplary protocol for the cleavage reaction using SDS-PAGE is as follows: First, a master reaction mixture containing 500 nM final concentration of mini-FH and 500 nM C3b in HBS buffer (30 mM HEPES, 140 mM NaCl pH 7.4) for each CFI-HSA variant was prepared at room temperature. The master reaction mixture was transferred to 37° C. and equilibrated for 5 min. The cleavage reaction was initiated by adding CFI-HSA protein to a final concentration of 5 nM. A sample volume corresponding to 0.6 μg of C3b was removed from the master mixture for each measured time point and quenched by the addition of 5×SDS reducing sample buffer. Samples were run on a 9 or 10% SDS-PAGE gel and C3b cleavage was visualized by Coomassie staining. The amount of C3b cleavage that occurred was quantified by densitometry. To calculate the percentage of C3b cleavage activity of the CFI-HSA mutants, the C3b cleavage activity of wild-type CFI-HSA was normalized as 100%.

[0238] Another exemplary protocol for cleavage reaction, as measured by ELISA, was performed as follows: C3b cleavage reaction was performed with 1 nM CFI (mutant or wild type), 500 nM cofactor mini-FH, and 500 nM soluble human C3b, incubated for 10 min at 37° C. in HEPES-buffered saline (HBS). The reaction was quenched by adding 1 M NaCl in HBS. Before proceeding with iC3b ELISA, the reaction was further diluted in buffer (HBS, 0.5 M NaCl, 0.05% Tween 20) to a final concentration of 5 nM C3b. C3b cleavage activity was determined from the amount of iC3b generated in the cleavage reaction. The amount of iC3b formed was assayed using MicroVue iC3b A006 ELISA kit (Quidel). The ELISA assay consists of a microplate coated with an iC3b-specific monoclonal antibody to capture iC3b formed during the reaction, and detection of bound iC3b using an HRP-conjugated anti-iC3b antibody and a chromogenic substrate. The recorded absorbance is a relative measure of the iC3b product generated in the cleavage reaction. The fold difference in C3b cleavage activity of the CFI variants relative to the reference molecule, CFI-HSA wild type, was calculated by dividing the background-corrected absorbance from the CFI-HSA variants by the background-corrected absorbance for CFI-HSA wild type. Table 7.3 summarizes these results and shows the median fold difference of each CFI variant relative to the median of the reference molecule. The fold difference was also calculated from SDS-PAGE gels. Samples from the C3b cleavage time course were run on 9 or 10% SDS-PAGE gels, and C3b cleavage was visualized by Coomassie staining. The amount of C3b cleavage that occurred was quantified by densitometry, the data were plotted, and the apparent rate constant (k) for loss of band intensity was determined by fitting to an exponential decay. The fold difference in C3b cleavage activity of the CFI mutant relative to the reference molecule, CFI-HSA wild type, was calculated by dividing the k value from the CFI-HSA mutant by the k value for CFI-HSA wild type.

[0239] C3b cleavage by the CFI mutants was further characterized by determining the EC50 for C3b cleavage. Briefly, C3b cleavage reactions were performed using 25 nM miniFH, 75 nM soluble human C3b, and a dilution series of the CFI mutants. The reaction mixtures of each concentration of CFI mutants were incubated in HBS at 37° C. for 5 min. The reaction was quenched by the addition of 1 M NaCl in HBS. Before proceeding with the iC3b ELISA, the reactions were further diluted in buffer (HBS, 0.5 M NaCl, 0.05% Tween 20) to a final concentration of 5 nM C3b. The amount of iC3b produced in the reaction was determined using a MicroVue iC3b A006 ELISA kit (Quidel). The ELISA assay consisted of a microplate coated with an iC3b-specific monoclonal antibody to capture the iC3b formed in the reaction, and detection of bound iC3b using an HRP-conjugated anti-iC3b antibody and a chromogenic substrate. The absorbance recorded is a relative measure of the iC3b product generated in the cleavage reaction. EC50 values ​​were calculated using an unconstrained four-parameter nonlinear regression fit in GraphPad Prism. Table 7.4 below summarizes the results of the iC3b ELISA titration analysis. EC50 values ​​above 500 nM were set to 500 nM. Cleavage reactions were also performed in the absence of mini-FH and displayed and analyzed in the same manner as those containing mini-FH.

[0240] C4b cleavage reaction CFI regulates the classical complement pathway by proteolytic inactivation of C4b protein. CR1, C3b / C4b receptor, and C4 binding protein (C4BP) act as cofactors for the CFI-catalyzed cleavage reaction of C4b. The C4b cleavage assay is a functional assay to determine the ability of CFI and its mutants for C4b cleavage activity in the presence of either CR1 or C4BP cofactor. Complement factor protein C2, which specifically binds to C4b but not to the CFI cleavage product iC4b, was used for C4b capture. CFI-catalyzed cleavage of C4b was measured by measuring the decrease in the concentration of C4b bound to C2 protein immobilized on an ELISA plate. The captured C4b protein was detected by anti-C4c polyclonal rabbit Ab (DAKO, #A0065) in an ELISA assay. The C4b cleavage activity by CFI-HSA was normalized as 100% to calculate the percentage of C4b cleavage activity of the CFI mutants.

[0241] For each CFI-HSA variant, a master reaction mixture was prepared at room temperature containing a final concentration of 250 nM cofactors (CR1 domains 1-3) and 250 nM human C4b in HBS buffer (30 mM HEPES, 140 mM NaCl pH 7.4). The master reaction mixture was transferred to 37 °C and equilibrated for 5 min. The cleavage reaction was initiated by adding CFI-HSA protein to a final concentration of 250 nM. A sample volume corresponding to 0.6 μg of C3b was removed from the master mixture for each measured time point and quenched by the addition of 5 × SDS reducing sample buffer followed by incubation at 95 °C for 5 min. Samples were run on 9 or 10% SDS-PAGE gels and C4b cleavage was visualized by Coomassie staining. The amount of C4b cleavage that occurred was quantified by densitometry. To calculate the percentage of C4b cleavage activity of the CFI-HSA mutants, the C4b cleavage activity of wild-type CFI-HSA was normalized as 100%.

[0242] Another example of a C4b cleavage activity assay was performed as follows to determine the C4b cleavage activity of the CFI mutants relative to the reference molecule, CFI-HSA wild type. The cleavage reaction was performed with 250 nM of the CFI mutants in the presence of 250 nM of cofactor (CR1 domains 1-3) and 250 nM of human C4b, which was incubated at 37°C for 30 minutes. The reaction mixture was diluted 20-fold and then added to a blocked ELISA plate coated with a mouse monoclonal anti-C4c antibody. The absorbance recorded from the ELISA plate is a relative measure of the C4c product generated in the cleavage reaction and thus a measure of the C4b cleavage activity. The fold difference in the C4b cleavage activity of the CFI mutants relative to the reference molecule, CFI-HSA wild type, was calculated by dividing the background-corrected absorbance from the CFI-HSA mutant by the background-corrected absorbance for CFI-HSA wild type. Table 7.3 below summarizes the fold difference in C4b cleavage activity assay of CFI variants compared to the CFI-HSA reference molecule as measured by C4c ELISA screening with CR1.

[0243] EC of C4b cleavage by CFI mutants 50 was measured. The assay was performed using 250 nM cofactor (CR1 domains 1-3), 250 nM human C4b and a dilution series of the CFI variants. The reaction mixture was incubated at 37°C for 30 minutes, then the reaction mixture was diluted 20-fold before starting the ELISA. The amount of C4c generated was measured by ELISA using a mouse monoclonal antibody specific for C4c. The absorbance recorded from the ELISA plate is a relative measure of the C4c product generated in the cleavage reaction and therefore a measure of the C4b cleavage activity. EC50 values ​​were calculated using an unconstrained four-parameter nonlinear regression fit in GraphPad Prism. EC50 values ​​above 1000 nM were set to 1000 nM. Cleavage reactions were also performed in the absence of CR1 and recorded and analyzed in the same manner as those containing CR1. Table 7.4 summarizes the results of the C4c ELISA titration with CR1 cofactor.

[0244] Activity in the absence of cofactors C4b cleavage reactions were performed as described above in the absence of cofactors for a panel of CFI mutants (Table 7.1). The results show that CFI mutants with C-terminal fusion proteins containing the human CR1 domain maintained their ability to cleave C4b in the absence of cofactors in the reaction mixture. In contrast, CFI mutants lacking the CR1 C-terminal fusion did not maintain their ability to cleave C4b. These results suggest that CFI mutants with C-terminal CR1 fusions may be CR1 cofactor independent.

[0245] [Table 7]

[0246] C3b cleavage reactions were performed as described above in the absence of cofactors for a panel of CFI mutants (Table 7.2). The results show that CFI mutants with C-terminal fusion proteins containing the human CR1 domain maintained their ability to cleave C3b in the absence of cofactors in the reaction mixture.

[0247] [Table 8]

[0248] Single-point screening of CFI mutants for C4b and C3b cleavage The fold difference in C3b cleavage activity of the CFI mutants relative to the reference molecule, CFI-HSA wild type, was calculated. The background corrected absorbance from the CFI-HSA mutants was divided by the background corrected absorbance for CFI-HSA wild type. The results are shown in Table 7.3.

[0249] [Table 9]

[0250] Specificity for C4b cleavage versus C3b cleavage and C3b cleavage versus C4b cleavage was determined. Specificity was calculated by normalizing EC50 values ​​to CFI-HSA. For C4b cleavage, the maximum was set to 1000 nM and all values ​​above that were set to 1000 nM. For C3b cleavage, the maximum was set to 500 nM and all values ​​above that were set to 500 nM. The results are shown in Table 7.4.

[0251] [Table 10]

[0252] Example 8: Preparation and selection of CFI mutants against C3b, C4b, or both C3b and C4b In Example 8, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0253] For assays where EC50 values ​​were determined, the specificity of C4b cleavage vs. C3b cleavage and C3b cleavage vs. C4b cleavage was calculated by normalizing to CFI-HSA as shown in Table 7.4. The specificity is shown in Table 8.1. The specificity for C4b was calculated as follows: (C4b EC50 CFI-HSA / C4b EC50 mutant) / (C3b EC50 CFI-HSA / C3b EC50 mutant). The specificity for C3b was calculated as follows: (C3b EC50 CFI-HSA / C3b EC50 mutant) / (C4b EC50 CFI-HSA / C4b EC50 mutant). The results are shown in Table 8.1.

[0254] [Table 11]

[0255] Example 10: Intravitreal injection In Example 10, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0256] Pharmacokinetics of CFI-HSA after intravitreal injection The ocular pharmacokinetics of an N-terminal albumin fusion of wild-type CFI-HSA was investigated after intravitreal administration to six African green monkeys (AGM). Six animals were divided into two groups treated at two dose levels: one group received a single intravitreal injection of 500 μg CFI-HSA (right eye, OD, N=3) and the other group received a single intravitreal injection of 250 μg CFI-HSA (right eye, OD, N=3). The left eye (OS) of all six animals was injected with an equal volume of 100 μL of sterile PBS for injection as a vehicle control. Non-terminal vitreous humor samples (100 μL) were collected on days 1, 7, 14, 21, and 28 after administration. Vitreous fluid CFI-HSA drug concentrations were determined using a quantitative electrochemiluminescence (ECL) antigen assay optimized to measure CFI-HSA in the vitreous fluid of AGMs. The assay captures CFI-HSA levels using coating of 2 μg / mL of anti-CFI antibody (clone OX21, LS Bio, Seattle, WA) onto Meso Scale Discovery (MSD, Rockville, MA) assay plates. Detection of captured CFI-HSA is performed using 0.5 μg / mL of goat polyclonal anti-HSA antibody (Abcam, Cambridge, MA) conjugated with SULFO-TAG, which emits light upon application of an electrical potential [electrochemiluminescence (ECL)]. ECL relative light units (RLU) are measured with a MESO™ SECTOR S 600 reader, and unknown CFI-HSA concentrations in the vitreous fluid are interpolated from a standard curve ranging from 0.05 μg / mL to 40 μg / mL of factor I-HSA.

[0257] Non-compartmental analysis yielded apparent ocular terminal half-lives of 3.6 and 4.1 days for the 250 μg and 500 μg dose levels, respectively.

[0258] Complement component 3a (C3a) levels in the vitreous humor were determined by ELISA using the Quidel kit for C3a ELISA (Figure 10). CFI-HSA fusion protein dose-dependently reduced ocular C3a levels up to 7 days after intraocular injection. Increasing C3b degradation by CFI-HSA reduces complex formation between C3b and Bb, leading to reduced C3 cleavage into C3a and C3b via the amplification loop of the alternative pathway. Other mutants and fusion constructs can be tested in this same model for their activity.

[0259] Example 11: CFI-HSA in mouse plasma and plasma CFI pharmacokinetics In Example 11, references to CFI-HSA refer to human serum albumin fused to the N-terminus of wild-type CFI (SEQ ID NO:21).

[0260] The pharmacokinetics of an N-terminal albumin fusion of wild-type CFI (CFI-HSA) is examined following intravenous and subcutaneous administration to CD-1 mice. Using a sparse sampling design with a maximum of two samples per mouse and three mice sampled at each time point, CD-1 mice are divided into four groups and treated with a single dose of either plasma-purified wild-type CFI or recombinant wild-type CFI-HSA.

[0261] To compare the plasma circulating half-life and bioavailability of plasma-derived CFI and CFI-HSA, animals are administered either plasma-derived CFI or CFI-HSA both intravenously and subcutaneously. Plasma-derived CFI is administered intravenously at 1.3 mg / kg (group 1) and subcutaneously at 6.5 mg / kg (group 2). CFI-HSA is administered intravenously (group 3) and subcutaneously (group 4) at 3 mg / kg. An additional three animals receive a single dose of an equal volume of PBS delivered subcutaneously as a vehicle control (group 5; not shown). Blood (approximately 30-50 μL) is collected into EDTA at various time points from 5 min to 144 h post-dose, and plasma is separated by centrifugation.

[0262] CFI-HSA and plasma CFI concentrations are determined using a quantitative electrochemiluminescence (ECL) antigen assay for CFI-HSA and plasma CFI in CD-1 mouse EDTA plasma. For the CFI assay, mouse monoclonal anti-factor I antibody (MAB12907, Abnova, Taipei City) is coated at 2 μg / mL on a Meso Scale Discovery (MSD, Rockville, Massachusetts) assay plate to capture plasma CFI. Detection of captured CFI is performed using 0.5 μg / mL mouse monoclonal anti-CFI antibody (clone 3R / 8, CABT-47940MH, Creative diagnostics, Shirley, NY) conjugated with SULFO-TAG, which emits light when a potential is applied [electrochemiluminescence (ECL)]. For the CFI-HSA assay, mouse monoclonal anti-CFI antibody (clone 3R / 8, CABT-47940MH) is coated onto Meso Scale Discovery (MSD, Rockville, Sachusetts) assay plates at 1 μg / mL to capture CFI-HSA. Detection of captured CFI-HSA is performed using 2 μg / mL rabbit polyclonal anti-HSA antibody (ab24207, Abcam, Cambridge, Massachusetts) conjugated with SULFO-TAG, which emits light upon application of a potential [electrochemiluminescence (ECL)]. ECL relative light units (RLU) are measured on a MESO™ SECTOR S 600 reader, and unknown plasma CFI and CFI-HSA concentrations are interpolated from the standard curve.

[0263] The circulating half-life of CFI-HSA after intravenous injection is longer (about 22 hours) than that of the unfused plasma CFI protein (about 13 hours), indicating that the fusion of HSA to the CFI protein increases the half-life compared to the unfused CFI. Importantly, the bioavailability of CFI (53.6%) is similar to that of CFI-HSA (46.7%), indicating that the fusion of HSA to the CFI protein did not adversely affect the bioavailability of CFI after subcutaneous administration. The fusion of HSA to the CFI protein increases the half-life by about 2-fold compared to the unfused CFI protein after intravenous administration (Figure 11) or subcutaneous administration (Figure 12). Similar exposure is achieved with a 2-fold lower dose of the fusion protein CFI-HSA compared to CFI after subcutaneous injection (Figure 12).

[0264] The circulation of other mutants and fusion proteins can be tested in a similar manner.

[0265] Example 12: Biological activity of CFI-HSA following intravenous administration in a rodent model of complement activation A rat model of peripheral nerve injury was developed to study the involvement of complement in Wallerian degeneration due to mechanical injury of the myelinated sciatic nerve. Male CD Sprague Dawley rats (Charles River Laboratories), weighing 300–350 g at the time of enrollment, were anesthetized with a mixture of 2–2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen and placed on a heating pad to maintain body temperature. Aseptic surgery was performed on both legs to expose the sciatic nerve. Sciatic nerve injury (SNI) was performed on one leg by clamping the sciatic nerve three times for 10 seconds using Dumont #7 forceps. The contralateral leg received no clamp injury and served as an internal control for each subject.

[0266] Immediately after induction of SNI, animals can receive an intravenous injection of the variant or fusion protein of the present disclosure. A subcutaneous injection of sustained release buprenorphine (0.01 mg / kg) is also administered for pain management. Four or 24 hours after SNI, five animals from each treatment group are sacrificed by exsanguination.

[0267] At the time of sacrifice, 1 cm tissue strips (0.5 cm proximal and distal to the injury site) are taken from the injured (ipsilateral) and sham legs, snap frozen, and stored at -80°C until processed for mass spectrometry (Phenoswitch Bioscience, Canada). K2-EDTA plasma samples are collected before SNI (baseline) and 1, 4, and 24 hours (if applicable) after SNI for evaluation of complement component fragments by mass spectrometry (MS). Cytokine and chemokine levels (rat 27-plex multiplex immunoassay analyzed with BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX performed by Eve Technologies, Calgary, Canada) are evaluated in K2-EDTA plasma collected at baseline (vehicle only), 4 hours, and 24 hours (if applicable) after SNI. At the time of sacrifice, whole blood and serum are collected for clinical pathology evaluation [complete blood count (CBC) and serum chemistry; Biovet Inc, Canada].

[0268] Mass spectrometry of in vivo samples Samples are denatured and precipitated, washed and buffer exchanged prior to N-terminal labeling by reductive amine dimethylation. Samples are then digested with trypsin (or a mixture of trypsin and chymotrypsin) and then analyzed by LC-MS / MS using SWATH. SWATH data are integrated into ion libraries generated for each species and sample type. The top 10 peptides per protein contained in the ion libraries are integrated and peptide-centric analysis is performed for specific quantification of C3, C5, C4 and CFB N-terminally labeled peptides.

[0269] Cleavage products resulting from catalytic activity on C3b are monitored by mass spectrometry in neural tissues (membrane-bound fragments) (Figure 21A) and circulation (soluble fragments in plasma) (Figure 21B). CFI-HSA (Y408L;N531G mutant) cleavage activity results in two major cleavage fragments detected by mass spectrometry: C3dg and C3f. Cleavage of surface-bound C3b by CFI results in surface-bound C3dg fragments and soluble C3f fragments. Both fragments are soluble when formed from soluble C3b. Thus, detection of C3dg in plasma may result from cleavage of soluble C3b, while detection of C3dg in tissues may result from cleavage of membrane-bound C3b. N-terminally labeled C3dg (E[2Me]DVPAADLSDQVPDTDSETR) (SEQ ID NO: 24) is the product of CFI cleavage of iC3b. The activity of the CFI mutants is determined as the percentage of C3dg peptide with an N-terminal label (termed "activated C3dg") multiplied by the total signal size of C3dg (EDVPAADLSDQVPDTDSETR) (SEQ ID NO: 48).

[0270] Example 13: In vivo activity in a rat model of peripheral nerve injury The efficacy of a panel of CFI variants against complement activation in a sciatic nerve (SN) injury (SNI) rat model can be determined. Immediately after induction of SNI, animals are intravenously injected with variants or fusion proteins or control (1x PBS) at a dose volume of 5mL / kg. 24 hours after SNI, all animals are sacrificed by exsanguination. Cytokine and chemokine levels (rat 27-plex multiplex immunoassay analyzed with BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX performed by Eve Technologies, Calgary, Canada) are assessed in K2-EDTA plasma collected 4 and 24 hours after SNI. At the time of sacrifice, serum is collected for serum chemistry [Biovet Inc, Canada].

[0271] The activity of the mutants and fusion proteins is monitored by detecting the CFI cleavage products (C3dg and C3f) using mass spectrometry. N-terminally labeled C3f (S[2Me]EETK[2Me]QNEGF) (SEQ ID NO: 48) is the product of CFI cleavage of C3b, and N-terminally labeled C3dg (E[2Me]DVPAADLSDQVPDTDSETR) (SEQ ID NO: 24) is the product of CFI cleavage of iC3b. Total activated C3f is determined as the percentage of C3f peptides with N-terminal label (S[2Me]EETK[2Me]QNEGF) (SEQ ID NO: 48) multiplied by the total peptide signal size of C3f (SEETKQNEGF) (SEQ ID NO: 49).

[0272] Example 14: In vivo activity of CFI mutants in the cecal ligation and puncture model The effects of CFI variants and fusion proteins on limiting complement activation in a model of cecal ligation and puncture (CLP) induced sepsis in rats can be assessed.

[0273] A rat model of non-sterile sepsis can be used to study complement involvement after cecal ligation and puncture (CLP) surgery. This surgery provides three facets of complement activation and inflammation (mechanical injury, bacterial exposure, and ischemic injury), which are particularly relevant as screening tools for other indications. Male CD Sprague Dawley rats (Charles River Laboratories), weighing 300-350 g at the time of enrollment, are anesthetized with a mixture of 2-2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen and placed on a heating pad to maintain body temperature. Sepsis is induced by a CLP surgical procedure. A midline incision is made in the abdominal wall, and the cecum is exposed and ligated with a nylon suture (4-0) proximal to the ileocecal valve, then perforated using a 16-gauge needle passed through the distal portion of the cecum to allow a small amount of cecal contents to enter the peritoneal cavity. The abdominal wall and skin are then sutured.

[0274] Immediately after the CLP procedure, animals receive an intravenous injection of the selected mutant or fusion protein or control substance (1x PBS) at a dose volume of 5mL / kg. 16 hours after the CLP procedure, all animals are sacrificed by exsanguination. K2-EDTA plasma samples are collected the day before enrollment (baseline), 3 hours and 16 hours after CLP for evaluation of complement component fragments by mass spectrometry (MS) and cytokine / chemokine levels (rat 27-plex multiplex immunoassay analyzed with BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX performed by Eve Technologies, Calgary, Canada). Whole blood and serum are collected at baseline and 16 hours for clinical pathology evaluation [complete blood count (CBC) and serum chemistry; Biovet Inc, Canada].

[0275] Example 15: In vivo activity of CFI mutants in acute respiratory distress syndrome Evaluation of the therapeutic effects of CFI mutants and fusion proteins in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model can be studied.

[0276] Objective: The aim of this study was to evaluate the efficacy of mutant and fusion proteins to limit complement-mediated acute pulmonary inflammation in a mouse model of ARDS induced by a single dose of lipopolysaccharide (LPS).

[0277] To study complement involvement following intratracheal instillation (IT) of LPS, a mouse model of germ-free ARDS can be used. Male C57BL / 6 mice (Charles River Laboratories) weighing 20-25 g at the time of enrollment are anesthetized under isoflurane and instilled intratracheally with 50 µg of LPS (1 mg / mL LPS isolated from E. coli 0111:B4 in 0.9% saline solution, Sigma).

[0278] Three hours after the CLP procedure, animals receive an intravenous injection of the 5 mutant or fusion protein or control substance (1x PBS) at a dose volume of 5 mL / kg. To evaluate the potential effects of repeated daily administration, animals can receive a second 5 mg / kg dose 27 hours after LPS IT. The sham arm is subjected to intratracheal instillation of 50 μL of 0.9% saline (n=5) without IV treatment.

[0279] K2-EDTA plasma, lung tissue, and bronchoalveolar lavage fluid (BALF) samples are collected at sacrifice for evaluation of complement component fragments by mass spectrometry (MS). BALF is collected in three 300 μL perfusions of the right lung with cold PBS 1× containing protease inhibitor 1× (SigmaFAST™). Cytokine and chemokine levels (Mouse 31 plex Multiplex immunoassays analyzed with BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX performed by Eve Technologies (Calgary, Canada)) are evaluated in K2-EDTA plasma, BALF, and lung tissue (homogenized in PBS 1× + 0.1% Triton X-100 containing protease inhibitor mixture) collected at sacrifice. At the time of sacrifice, whole blood and serum are collected for clinical pathology evaluation [complete blood count (CBC) and serum chemistry; Biovet Inc, Canada]. Cell sorting is performed on BALF samples to assess leukocyte recruitment to the lungs.

[0280] LPS is a known inducer of the alternative complement pathway. CFI mutants or fusion activity against circulating C3b cleavage products can be assessed using mass spectrometry. The percentage of activated C3f is determined as the percentage of C3f peptides with N-terminal label (S[2Me]EETK[2Me]QNEGF) (SEQ ID NO: 48) multiplied by the total peptide signal size of C3f (SEETKQNEGF) (SEQ ID NO: 49).

Claims

1. A complement factor I (CFI) variant comprising at least one modification relative to wild-type complement factor I (CFI), capable of regulating the complement system and having at least one improved characteristic compared to wild-type CFI, the CFI variant being selected from those shown in Table 2 or Table 3.

2. 2. The CFI variant of claim 1, wherein the improved characteristic is selected from an increase in half-life or bioavailability, or an increase or decrease in any one or more of activity, substrate specificity, potency, substrate affinity, cofactor affinity and catalytic ability.

3. The CFI variant of claim 2 , wherein the improved characteristic is increased activity.

4. The CFI mutant of claim 3 , wherein the increased activity comprises increased cleavage of C3b and / or C4b compared to wild-type CFI.

5. 5. The CFI mutant of claim 4, wherein said increased activity comprises increased cleavage of C3b and does not comprise increased cleavage of C4b.

6. The CFI mutant of claim 4 or 5, wherein the increase in C3b cleavage is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold compared to wild-type CFI.

7. 5. The CFI mutant of claim 4, wherein said increased activity comprises increased cleavage of C4b but not increased cleavage of C3b compared to wild-type CFI.

8. The CFI mutant of claim 4 or 7, wherein the increase in C4b cleavage is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold compared to wild-type CFI.

9. The CFI mutant of claim 4, wherein the increase in cleavage of C3b and C4b is at least or about at least 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold, respectively, compared to wild-type CFI.

10. The CFI mutant of any one of claims 3 to 6 and 9, wherein the increased activity comprises increased production of iC3b.

11. The CFI mutant of any one of claims 3 to 6 and 9 to 10, wherein the increased activity comprises increased production of C3dg and / or C3c from iC3b.

12. The CFI variant according to any one of claims 3 to 6 and 10 to 11, wherein said increased activity comprises a reduced level of the C3bα chain.

13. The CFI variant according to any one of claims 3 to 12, wherein said increased activity comprises increased proteolysis of a peptide substrate.

14. The CFI mutant of any one of claims 3 to 13, wherein said increased activity comprises a decrease in the level or function of the membrane attack complex (MAC).

15. A CFI variant according to any one of claims 3 to 14, wherein said increased activity results in a decreased amplification of the complement system.

16. 3. The CFI variant of claim 2, wherein said improved characteristic is reduced activity against C3b and / or C4b.

17. The CFI variant of claim 2 , wherein the improved characteristic is increased specificity for a substrate.

18. 18. The CFI variant of claim 17, wherein the increased specificity comprises increased specificity for C3b or C4b compared to wild-type CFI.

19. 18. The CFI variant of claim 17, wherein the increased specificity comprises increased specificity for C3b and / or C4b compared to wild-type CFI.

20. 18. The CFI variant of claim 17, wherein the increased specificity comprises increased specificity for C3b compared to wild-type CFI.

21. 18. The CFI variant of claim 17, wherein the increased specificity comprises increased specificity for C4b compared to wild-type CFI.

22. 19. The CFI variant of claim 18, wherein the increase in specificity for C3b is at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold, compared to wild-type CFI.

23. 19. The CFI variant of claim 18, wherein the increase in specificity for C4b is at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold, compared to wild-type CFI.

24. The CFI mutant of any one of claims 1 to 23, wherein the modification to the wild-type CFI comprises any one or more of the following: deletion of one or more amino acid residues, deletion of one or more CFI domains, substitution of one or more amino acid residues, insertion of one or more amino acid residues, insertion of one or more CFI domains, and swapping of one or more CFI domains.

25. 25. The CFI variant of any one of claims 1 to 24, comprising any one or more domains of CFI selected from the serine protease domain (SPD), the factor I membrane attack complex (FIMAC) domain, the SRCR domain, the low density lipoprotein receptor 1 (LDLr1) domain, and the low density lipoprotein receptor 2 (LDLr2) domain.

26. A CFI variant according to any one of claims 1 to 25, comprising at least one modification corresponding to wild-type human CFI.

27. A CFI variant according to any one of claims 1 to 25, comprising at least one modification corresponding to wild-type non-human CFI.

28. A CFI variant according to any one of claims 1 to 25, comprising at least one modification corresponding to wild-type CFI having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

5.

29. A CFI variant according to any one of claims 1 to 28, which is a chimera comprising one or more domains derived from human CFI, said human CFI further comprising a substitution of one or more amino acid residues for amino acid residues in the corresponding region derived from a non-human species CFI.

30. 30. The CFI variant of claim 29, wherein the non-human species is mouse.

31. 31. The CFI variant of any one of claims 1 to 30, which is chimeric and wherein the modification comprises the replacement of one or more amino acid residues of the CFI with amino acid residues from the corresponding region of a non-CFI serine protease.

32. 32. The CFI variant of claim 31, wherein the non-CFI serine protease is trypsin.

33. 33. A CFI variant according to any one of claims 1 to 32, comprising an A chain and a B chain and comprising one or more modifications at the interface between the A chain and the B chain.

34. A CFI variant according to any one of claims 1 to 32, comprising one or more modifications in the C-terminal region of said CFI variant.

35. A CFI variant according to any one of claims 1 to 32, comprising one or more modifications at one or more N-linked glycosylation sites of said CFI.

36. 36. The CFI variant of claim 35, wherein the one or more modifications is removal of an N-linked glycosylation site.

37. 33. The CFI variant of any one of claims 1 to 32, comprising one or more modifications in the SPD domain of said CFI, optionally comprising one or more modifications in any one or more of the autolysis loop, the 99 loop, the S1 pocket entrance, or the activation loop of SPD, or any one or more of the domains shown in Figure 1.

38. 38. The CFI mutant of claim 37, comprising a substitution of the autolysis loop of CFI (REKDNERVFS, SEQ ID NO: 9) for the autolysis loop of trypsin (NTASSGADYPDE, SEQ ID NO: 10), wherein the autolysis loop occurs between positions corresponding to positions 456 and 465 in CFI having the amino acid sequence shown in SEQ ID NO:

5.

39. A CFI mutant according to any one of claims 1 to 32, comprising one or more modifications in the active site of said CFI.

40. 33. A CFI variant according to any one of claims 1 to 32, comprising an A chain and a B chain, with the structural arrangement from N-terminus to C-terminus as (A chain)-(optional linker)-(B chain).

41. 33. A CFI variant according to any one of claims 1 to 32, comprising an A chain and a B chain, with the structural arrangement from N-terminus to C-terminus as (B chain)-(optional linker)-(A chain).

42. 33. The CFI mutant of any one of claims 1 to 32, which is more readily activated compared to the wild-type CFI.

43. 33. A CFI variant according to any one of claims 1 to 32, comprising each one of the SPD, FIMAC, SRCR, LDLr1 and LDLr2 domains, and any other domains shown in Figure 1.

44. 33. The CFI variant of any one of claims 1 to 32, which does not contain all of the SPD, FIMAC domain, SRCR domain, LDLr1 domain and LDLr2 domain.

45. 45. The CFI variant of claim 44, comprising an SPD.

46. 46. ​​The CFI variant of claim 45, comprising the amino acid sequence shown in SEQ ID NO:

12.

47. 47. The CFI variant of any one of claims 1 to 46, which is sialylated.

48. 48. The CFI variant of any one of claims 1 to 47, which is active.

49. 49. The CFI mutant of claim 48, which is activated by furin or a mutant thereof.

50. 50. The CFI mutant of claim 49, which is activated in vitro by furin or a mutant thereof.

51. 50. The CFI mutant of claim 49, which is activated by furin or a mutant thereof during production in a host cell.

52. 52. The CFI mutant of claim 51, wherein said activation by furin or a mutant thereof during production in a host cell is due to overexpression of furin or a mutant thereof.

53. 50. The CFI mutant of claim 49, which is activated by furin or a mutant thereof after production and secretion by the host cell, optionally in the culture medium.

54. 54. The CFI variant of any one of claims 1 to 53, which is a first component of a fusion construct comprising a first component and at least a second component, and which is fused to said second component.

55. 55. The CFI variant of claim 54, wherein the second component is a protein.

56. 55. The CFI variant of claim 54, wherein the second component is not a protein.

57. 57. The CFI variant of any one of claims 54 to 56, wherein the second component is a half-life extender.

58. 58. The CFI variant of claim 57, wherein the half-life extender comprises peptide repeats.

59. 55. The CFI variant of claim 54, wherein the second component is a half-life extender selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc.

60. 60. The CFI variant of claim 59, wherein the half-life extender is a modified albumin or albumin derivative.

61. 60. The CFI variant of claim 59, wherein the half-life extender is wild-type albumin.

62. 60. The CFI variant of claim 59, wherein the half-life extender is human serum albumin or a variant thereof.

63. 63. The CFI variant of any one of claims 54 to 62, comprising an A chain and a B chain, said fusion construct comprising the structural arrangement N-terminal to C-terminal or C-terminal to N-terminal as (second component)-(optional linker)-(A chain)-(optional linker)-(B chain).

64. 63. The CFI variant of any one of claims 54 to 62, comprising an A chain and a B chain, said fusion construct comprising the structural arrangement N-terminal to C-terminal or C-terminal to N-terminal as (second component)-(optional linker)-(B chain)-(optional linker)-(A chain).

65. 55. The CFI variant of claim 54, wherein the second component is at least one domain or part of a domain of factor H.

66. 66. The CFI variant of claim 65, wherein said at least one factor H domain comprises any one or more of factor H complement control protein (CCP) domains 1-20.

67. 67. The CFI variant of claim 65 or 66, wherein the amino acid sequence of at least one factor H domain is or is derived from the sequence shown in SEQ ID NO:

4.

68. 68. The CFI variant of any one of claims 65 to 67, wherein said at least one factor H domain comprises each of CCP domains 1 to 20 of factor H.

69. 68. The CFI variant of any one of claims 65 to 67, wherein the at least one factor H domain comprises CCP1, CCP2, CCP3, and CCP4.

70. 68. The CFI variant of any one of claims 65 to 67, wherein the at least one factor H domain comprises CCP2, CCP3, and CCP4.

71. 68. The CFI variant of any one of claims 65 to 67, wherein said at least one factor H domain comprises CCP2 and CCP3.

72. 67. The CFI mutant of claim 65 or 66, wherein the amino acid sequence of at least one domain of factor H is or is derived from the sequence shown in SEQ ID NO:

8.

73. 73. The CFI variant of claim 72, wherein said at least one factor H domain comprises CCP domains 1-4 and 19-20 of factor H.

74. 55. The CFI variant of claim 54, wherein the second component is at least one domain or portion of a domain of Complement Receptor 1 (CR1).

75. 75. The CFI variant of claim 74, wherein at least one domain of CR1 is any one or more of CR1 CCP domains 15-17.

76. 55. The CFI variant of claim 54, wherein the second component comprises at least one domain or portion of a domain of complement receptor I (CRI) and at least one domain or portion of a domain of factor H.

77. 55. The CFI variant of claim 54, wherein the second component is a domain or portion of a domain of at least one of CR1 and FH.

78. 78. The CFI variant of any one of claims 54 to 77, wherein the fusion construct further comprises a third component.

79. 80. The CFI variant of claim 78, wherein the third component is a protein.

80. 80. The CFI variant of claim 78, wherein the third component is not a protein.

81. 76. The CFI variant of any one of claims 54 to 75, further comprising a third component, said third component optionally being a half-life extender selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc.

82. 82. The CFI variant of claim 81, wherein the half-life extender is a repeating peptide sequence.

83. A fusion construct comprising at least a first component, a second component, and a third component, wherein the first component comprises wild-type CFI or a mutant thereof (CFI mutant), the second component comprises FH or CR1, and the third component comprises FH or CR1.

84. 84. The fusion construct of claim 83, wherein the first component comprises a wild-type CFI comprising the amino acid sequence set forth in SEQ ID NO:

5.

85. 85. The fusion construct of claim 83 or 84, comprising albumin.

86. 86. The fusion construct of claim 85, wherein the albumin is human serum albumin.

87. The fusion construct of claim 86, wherein the human serum albumin comprises the amino acid sequence set forth in SEQ ID NO:

7.

88. 84. The fusion construct of claim 83, wherein the first component comprises a CFI mutant.

89. The fusion construct of claim 88, wherein the CFI mutant is a CFI mutant according to any one of claims 1 to 49.

90. A fusion construct according to any one of claims 83 to 89, having at least one improved characteristic compared to free wild-type CFI (not part of the fusion construct).

91. The fusion construct of any one of claims 83 to 90, wherein the improved characteristic is selected from an increase in half-life or bioavailability, or an increase or decrease in any one or more of activity, substrate specificity, potency, substrate affinity, cofactor affinity and catalytic ability.

92. 92. The fusion construct of claim 91, wherein the improved characteristic is increased activity.

93. The fusion construct of claim 92, wherein the increased activity comprises increased cleavage of C3b and / or C4b.

94. The fusion construct of claim 93, wherein said increased activity comprises increased cleavage of C3b and does not include increased cleavage of C4b.

95. The fusion construct of claim 93 or 94, wherein the increase in C3b cleavage is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold compared to wild-type CFI that is not part of the fusion construct or compared to a fusion construct comprising wild-type CFI.

96. The fusion construct of claim 93, wherein said increased activity comprises increased cleavage of C4b and does not comprise increased cleavage of C3b compared to wild-type CFI.

97. The fusion construct of claim 93 or 96, wherein the increase in C4b cleavage is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold compared to wild-type CFI that is not part of the fusion construct or compared to a fusion construct comprising wild-type CFI.

98. The fusion construct of any one of claims 92 to 95 and 97, wherein the increased activity comprises increased production of iC3b.

99. The fusion construct of any one of claims 92 to 95 and 97 to 98, wherein the increased activity comprises increased production of C3dg from iC3b.

100. The fusion construct of any one of claims 92 to 95 and 97 to 99, wherein the increased activity comprises a decreased level of C3bα chain.

101. The fusion construct of any one of claims 92 to 100, wherein the increased activity comprises increased hydrolysis of a peptide substrate or proteolysis of a macromolecular protein substrate.

102. 92. The fusion construct of claim 91, wherein the improved characteristic is reduced activity.

103. A fusion construct according to any one of claims 83 to 102, having at least one improved characteristic compared to free wild-type CFI in the absence of factor H and / or in the absence of CR1.

104. The fusion construct of any one of claims 83 to 102, having at least one improved characteristic compared to free wild-type CFI, said at least one improved characteristic being further improved by the presence of exogenous factor H and / or exogenous CR1.

105. A fusion construct comprising any of the fusion constructs in Table 3.

106. A pharmaceutical composition comprising a CFI variant according to any one of claims 1 to 82 or a fusion construct according to any one of claims 83 to 105, and optionally a pharma- ceutically acceptable excipient.

107. A method of modulating the complement system comprising contacting a sample in vitro or a tissue in vivo with a CFI variant according to any one of claims 1 to 82 or a fusion construct according to any one of claims 83 to 104.

108. 108. The method of claim 107, wherein the method is in vitro.

109. 108. The method of claim 107, wherein the method is in vivo.

110. 110. The method of any one of claims 107 to 109, which results in increased cleavage of C3b, C4b, production of iC3b, production of C3dg and / or C4c.

111. 110. The method of any one of claims 107 to 109, which results in reduced hemolysis.

112. 110. The method of any one of claims 107 to 109, which results in a reduction in the level of MAC.

113. The method according to any one of claims 107 to 109, which results in a reduced amplification of the complement system.

114. 110. A method according to any one of claims 107 to 109, which results in increased hydrolysis of a peptide substrate or increased proteolysis of a macromolecular protein substrate.

115. A method of treating a non-ocular condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a CFI variant according to any one of claims 1 to 82, or a fusion construct according to any one of claims 83 to 105, or a pharmaceutical composition according to claim 106.

116. 116. The method of claim 115, wherein the non-ocular condition is characterized by a deficiency in CFI.

117. 117. The method of claim 115 or 116, wherein the non-ocular condition is characterized by dysregulation of the complement system.

118. The method of any one of claims 115 to 117, wherein the non-ocular symptom is a systemic acute symptom.

119. The method of claim 118, wherein the non-ocular condition is a systemic acute indication selected from the group consisting of acute glomerulonephritis, acute kidney injury, acute respiratory distress syndrome, bacterial meningitis, cerebral hemorrhage, burns, coronavirus infection, Epstein-Barr virus infection, hematopoietic stem cell transplantation, ischemia-reperfusion injury, Lyme disease, myocardial infarction, organ transplantation, periodontitis, pneumonia, preeclampsia, schistosomiasis, sepsis, stroke, thromboembolism, ischemia-reperfusion injury, and traumatic brain injury.

120. The method of any one of claims 115 to 117, wherein the non-ocular condition is a systemic chronic indication.

121. The non-ocular conditions include Alzheimer's disease, antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uraemic syndrome (aHUS), immune-mediated hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease, Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), granulomatosis with polyangiitis (GPA), Guillain-Barre syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy, lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy (MMN), multiple sclerosis (MS), non-insulin dependent diabetes mellitus, degenerative diseases, myasthenia gravis ...

121. The method of claim 120, wherein the chronic systemic indication is selected from the group consisting of chronic arthropathy, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, post-transplant lymphoproliferative disease, protein-losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), ulcerative colitis, amyotrophic lateral sclerosis (ALS), warm autoimmune hemolytic anemia (wAIHA), cold agglutinin disease (CAD), and immune complex membranoproliferative glomerulonephritis (IC-MPGN), Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA), Huntington's disease, and chronic inflammatory demyelinating polyneuropathy (CIDP).

122. 118. The method of any one of claims 115 to 117, wherein the non-ocular condition is non-neoplastic.

123. 118. The method of any one of claims 115 to 117, wherein the non-ocular condition is neoplastic.

124. 123. The method of claim 122, wherein the non-ocular condition is characterized by a solid tumor or by a liquid tumor.

125. 125. The method of claim 124, wherein the non-ocular condition is characterized by a solid tumor and is selected from the group consisting of colorectal tumor, hormone refractory prostate cancer, melanoma, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic pancreatic cancer, metastatic gastric cancer, nasopharyngeal carcinoma, non-small cell lung cancer, pancreatic tumor, squamous cell carcinoma, and gastric tumor.

126. 125. The method of claim 124, wherein the non-ocular condition is characterized by a liquid tumor and is selected from the group consisting of acute myeloid leukemia, B-cell lymphoma, and Hodgkin's disease.

127. The method of any one of claims 107 to 126, wherein the CFI variant, the fusion construct or the pharmaceutical composition is administered subcutaneously or intravenously to the subject.

128. 128. The method of claim 127, wherein the administration is subcutaneous administration.

129. 129. The method of claim 128, wherein the subcutaneous administration is daily, or weekly, or biweekly.

130. A method of treating an ocular condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a CFI variant according to any one of claims 1 to 82, a fusion construct according to any one of claims 83 to 105, or a pharmaceutical composition according to claim 106.

131. 131. The method of claim 130, wherein the ocular condition is characterized by a deficiency in CFI.

132. 132. The method of claim 130 or 131, wherein the ocular condition is characterized by dysregulation of the complement system.

133. 133. The method of any one of claims 130 to 132, wherein the ocular condition is selected from the group consisting of diabetic macular edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, Stargardt's disease, uveitis, and vitreous retinopathy.

134. 134. The method of any one of claims 130 to 133, wherein the ocular condition is non-neoplastic.

135. A cell comprising one or more nucleic acids encoding wild-type CFI or a mutant thereof, and comprising one or more nucleic acids encoding furin.

136. A method for producing wild-type CFI or a mutant thereof in an activated state, comprising recombinantly producing CFI or a mutant thereof in a cell comprising one or more nucleic acids encoding CFI or a mutant thereof and comprising one or more nucleic acids encoding furin.

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