CN116635528A - Complement factor I related compositions and methods - Google Patents

Abstract

The present application provides Complement Factor I (CFI) variants that exhibit at least one improved feature relative to wild-type CFI. The CFI variants of the application may exhibit adjustable specificity and activity. Also included are CFI-containing fusion constructs comprising at least one domain of CFI, e.g., wild-type full-length CFI fused to human serum albumin. Methods of making and using such CFI variants and fusion constructs are also included. The CFI variants and fusion constructs provided herein may be useful in the treatment of diseases or disorders associated with deregulation of the complement system or insufficient CFI.

Description

Complement factor I related compositions and methods

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application Ser. No. 63/038,874, U.S. provisional application Ser. No. 63/122,437, U.S. provisional application Ser. No. 63/124,698, and U.S. provisional application Ser. No. 63/179,160, filed on month 4 and 23 of 2021, filed on month 6 and month 12 of 2020, filed on day 7 of 2020, and incorporated herein by reference in their entirety.

Reference to sequence Listing

An electronic version of the sequence listing is filed herewith, the contents of which are incorporated by reference in their entirety. Electronic version was created at 2021, 6-month 14, 92 kilobytes in size and titled ctbi_001_04wo_seqlist_st25.txt.

Background

The complement system includes the classical pathway, the lectin pathway, and the alternative pathway, and is tightly controlled by a variety of modulators. Complement Factor I (CFI) is one such modulator and is used to modulate the complement system by cleaving C4b and C3b proteins, thereby inactivating these proteins. Such cleavage causes inhibition of the classical pathway, lectin pathway and alternative pathway, respectively, thereby ultimately hampering the assembly of C3 convertases and C5 convertases. CFI is encoded as a pre-enzyme and then activated by proteolytic cleavage into a heterodimeric glycoprotein having heavy and light chains linked by disulfide bonds. The light chain (also called the B chain) comprises Serine Protease Domains (SPDs) responsible for cleaving C3B and C4B, and contains catalytic triplets (His 362, asp411 and Ser 507) within the region called the active site. The heavy chain (also called the a chain) comprises four domains: FI-attack membrane complex (FIMAC) domain, scavenger receptor cysteine-rich domain SRCR (also known as CD5 domain) domain, low density lipoprotein receptor 1 domain (LDLr 1) and low density lipoprotein receptor 2 domain (LDLr 2). CFI is post-translationally processed into its active form by the addition of six Asn-linked glycans and proteolytic activation by furin (furin), thereby cleaving the RRKR linker to produce a double-stranded mature protein.

In terms of its ability to cleave C3b or C4b, when CFI forms a ternary complex with its cofactor; CFI is proteolytically active when Factor H (FH) or complement receptor 1 (CR 1, also known as CD 35) and its physiological substrates C3b and C4 b. FH is an example of a soluble member of the proteome called complement activation modulator (RCA). The complex formation by CFI and FH and subsequent cleavage of C3b together serve to modulate the alternative pathway of the complement system. Continuous regulation of C3b content by CFI acts to maintain a balance between classical and alternative pathways. For example, removal of CFI has been shown to cause immediate activation of the alternative pathway, causing overactivity. CR1 is an example of a monomeric single pass type I membrane glycoprotein that is a member of the proteome known as complement activation modulator (RCA). The formation of complexes formed between CFI and CR1 and subsequent cleavage of C3b and C4b are used to modulate the alternative or classical pathways and lectin pathway, respectively.

Dysregulated CFI, mutated and dysfunctional CFI or CFI deficiency has been associated with diseases involving the complement system, and what is needed is a method for modulating or inhibiting specific regulatory points within the complement system. Provided herein are compositions and methods for addressing dysfunctions and/or disorders in the complement system.

Disclosure of Invention

In one aspect, the invention provides a Complement Factor I (CFI) variant comprising at least one modification relative to wild-type CFI, wherein the CFI variant is capable of modulating the complement system, and wherein the CFI variant has at least one improved feature compared to wild-type CFI. In some embodiments, the improved feature 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 capacity. In some embodiments, the improvement is characterized by an increase in activity. In some embodiments, the improvement is characterized by a change in substrate specificity.

In some embodiments, the increased activity comprises increased cleavage of C3b and/or C4b as compared to wild-type CFI or a fusion construct comprising wild-type CFI. In some embodiments, the increase in activity comprises an increase in cleavage of C3b, and no increase in cleavage of C4 b. In some embodiments, the increase in cleavage of 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 greater than the wild-type CFI or fusion construct comprising the wild-type CFI.

In some embodiments, the increase in activity comprises an increase in cleavage of C4b and no increase in cleavage of C3b as compared to wild-type CFI or a fusion construct comprising wild-type CFI. In some embodiments, the increase in cleavage of 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 greater than the wild-type CFI or fusion construct comprising the wild-type CFI.

In some embodiments, the increase in cleavage of C3b and 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, respectively, as compared to the wild-type CFI or fusion construct comprising the wild-type CFI.

In some embodiments, the increase in activity comprises an increase in production of iC3 b. In some embodiments, the increase in activity comprises an increase in C3dg and/or C3C from iC3 b.

In some embodiments, the increased activity comprises a decreased content of C3b alpha chains. In some embodiments, the increased activity comprises increased proteolysis of the peptide substrate. In some embodiments, the increased activity comprises a decrease in the content or function of a tapping complex (MAC). In some embodiments, the increased activity results in decreased amplification of the complement system. In some embodiments, the improvement is characterized by a decrease in the activity of C3b and/or C4 b.

In some embodiments, the improvement is characterized by an increased substrate specificity. In some embodiments, the increase in specificity comprises an increase in specificity of C3b or C4b as compared to wild-type CFI or a fusion construct comprising wild-type CFI. In some embodiments, the increase in specificity comprises an increase in specificity of C3b and/or C4b as compared to wild-type CFI or a fusion construct comprising wild-type CFI. In some embodiments, the increase in specificity comprises an increase in specificity of C3b as compared to wild-type CFI or a fusion construct comprising wild-type CFI.

In some embodiments, the increase in specificity comprises an increase in specificity of C3b as compared to wild-type CFI or a fusion construct comprising wild-type CFI. In some embodiments, the increase in specificity of 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 greater than the wild-type CFI or fusion construct comprising the wild-type CFI.

In some embodiments, the increase in specificity comprises an increase in specificity of C4b as compared to wild-type CFI or a fusion construct comprising wild-type CFI. In some embodiments, the increase in specificity of 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 greater than the wild-type CFI or fusion construct comprising the wild-type CFI.

In some embodiments, the modification relative to the wild-type CFI comprises any one or more of: 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 CFI domains, and exchange of one or more CFI domains. In some embodiments, the CFI variants comprise any one or more of the modifications presented in tables 2-9 and 13.

In some embodiments, the CFI variant comprises any one or more domains of a CFI selected from the group consisting of: serine Protease Domain (SPD), factor I tapping complex (FIMAC) domain, SRCR domain, low density lipoprotein receptor 1 (LDLr 1) domain, and low density lipoprotein receptor 2 (LDLr 2) domain.

In some embodiments, the CFI variant comprises at least one modification corresponding to wild-type human CFI. In some embodiments, the CFI variant comprises at least one modification corresponding to wild-type non-human CFI. In some embodiments, the CFI variant comprises at least one modification corresponding to a wild-type CFI having the amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO. 5.

In some embodiments, the CFI variant is a chimeric comprising one or more domains from a human CFI, and wherein the human CFI further comprises a substitution of one or more amino acid residues for amino acid residues from a corresponding region of a CFI of a non-human species. In some embodiments, the non-human species is a mouse. In some embodiments, the CFI variant is a chimeric, and wherein the modification comprises replacing one or more amino acid residues of CFI with an amino acid residue from a corresponding region of a non-CFI serine protease. In some embodiments, the non-CFI serine protease is trypsin.

In some embodiments, the CFI variant comprises an a-chain and a B-chain, wherein the CFI variant comprises one or more modifications at the interface of the a-chain and the B-chain.

In some embodiments, the CFI variant comprises one or more of the modifications presented in table 2. In some embodiments, the CFI variant comprises a modification at any one or more of positions K14, Y20, D26, F29, R35, E38, M220, K221, S250, L304, P305, K306, L307, and S308 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises a substitution of the trypsin loop 200 having the amino acid residue NG in loop 200 of CFI (SEQ ID NO: 13), wherein the loop 200 is located between positions corresponding to positions 514 and 520 in CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CFI variant comprises one or more of the substitutions selected from K14A, Y20A, Y20F, D3526A, F29A, R A, E38A, M220A, K221Q, S250A, S L, L304G, P305G, K306G, L G and S308G, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises the substitution M220A; K221Q and L304G; P305G; K306G; L307G; one or more of the combinations of S308G, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

In some embodiments, the CFI variant comprises one or more modifications at the C-terminal region of the CFI variant. In some embodiments, the CFI variant comprises one or more of the modifications presented in table 3. In some embodiments, the CFI variant comprises a modification at any one or more of positions T377, W381, P384, Y403, a405, G406, Y408, Q409, D425, G556, R557, P558, P559, I560, and Y563 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises one or more modifications at the C-terminal region that are deletions of amino acid residues (PFISQYNV, SEQ ID NO: 14) between positions corresponding to positions 558 to 565 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CFI variant comprises a substitution of the linker with an amino acid residue (DGNK, SEQ ID NO: 15) between positions corresponding to positions 420 to 424 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CFI variant comprises one or more substitutions selected from T377G, W381A, P384A, P384G, Y403F, A405S, G406R, G406A, Y L, Q409D, Q409H, D425A, D425K, D425R, G556A, G556S, R557A, R K, P558G, P558L, P558S, F559L, I560V and Y563H, and/or a deletion of P384, wherein the position corresponds to a position in the CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variant comprises one or more modifications at one or more N-linked glycosylation sites of CFI. In some embodiments, the CFI variant comprises one or more modifications that are removal of the N-linked glycosylation site. In some embodiments, the CFI variant comprises one or more of the modifications presented in table 4. In some embodiments, the CFI variant comprises a modification at any one or more of positions N52, N85, N159, N446, N476, and N518 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises one or more substitutions selected from N52Q, N85Q, N159Q, N446Q, N476Q and N518Q, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises a polypeptide selected from N52Q; N85Q; N159Q, N446Q; N476Q; one or more of the combinations of substitutions of N518Q, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

In some embodiments, the CFI variant comprises one or more modifications in the SPD domain of the CFI. In some embodiments, the CFI variant comprises one or more of the modifications presented in table 5. In some embodiments, the CFI variants comprise one or more modifications at any one or more of the autolytic loop, loop No. 99, S1 pocket entry, or activation loop of the SPD or any one or more of the domains presented in fig. 1. In some embodiments, the CFI variant comprises modifications at any one or more of positions K14, K312, R314, I322, V323, K326, R327, a328, K340, D341, G344, I345, T346, a361, L364, Y372, W381, P384, V390, N402, N404, G406, Y408, Q409, E416, K418, N422, D425, E457, K458, R456, E461, R462, F464, S465, Q467, W468, G469, T495, Y496, D497, S499, I500, a502, K504, D506, S507, E530, N531, G533, K534, P535, E536, and F537 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises a substitution of the trypsin's autolytic loop (NTASSGADYPDE, SEQ ID NO: 10) by the CFI's autolytic loop (REKDNERVFS, SEQ ID NO: 9), wherein the autolytic loop is located between positions corresponding to positions 456 and 465 in the CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CFI variant comprises a substitution of the autolytic loop of CFI (REKDNERVFS, SEQ ID NO: 9) for the autolytic loop of mouse CFI (RGKDNQKVYS, SEQ ID NO: 11), wherein the autolytic loop is located between positions corresponding to position 456 and position 465 in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variant comprises one or more substitutions selected from the group consisting of: k14,312,314,322,322,323,323,323,327,327,340,341,344,344,346,346,346,372,381,381,384,384,384,384,406,406,406,406,406,406,406,406,406,408,408,418,425, 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 495 497.5.90.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.506.506.506.530.530.531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 or 533.535 536.536K and F537R, wherein said position corresponds to a sequence having the amino acid sequence of SEQ ID NO:5, and the position in CFI of the amino acid sequence set forth in SEQ ID NO.

In some embodiments, the CFI variant comprises one or more of the combination substitutions selected from the group consisting of: K326A; R327A, N531G; P535A, E457G; E461Q; R462K; F464Y, Y L; N531G; E457G, Y L; N531G; E457G; E461Q, Y L; N531G; E457G; E461Q-R462K; F464Y, Y L; N531G; P535A, K a; d425R, E D; N531G; G533A; K534Q; P535K; E536N, A S502; K504Q; F537K, T F; Y496L; D497E; S499G; I500K, G533A; K534Q; P535K; E536N; F537K, T F; Y496L; D497E; S499G; I500K; G533A; K534Q; P535K; E536N; F537K, Q467K; F537K, E G530; N531G, E D; F537K, E457G; E461Q, E457G; E461G, Y L; N531G; E457G; E461Q, N531G; E457G; E461Q, I V322; V323I, I V322; V323I; R327P, A328C; W468C, A328C; W468C; K326Y; R327N, Y L; N531G; E461Q, Y L; N531G; E457G; E461Q; R462K, Y L; N531G; E457G; E461Q; F464Y, Y L; N531G; E457G; R462K; F464Y, Y L; N531G; E461Q; R462K; F464Y, Y L; E457G; E461Q; R462K; F464Y, E457G; N531G; E461Q; R462K; F464Y, Y L; E457G; E461Q; R462K, N531G; E457G; E461Q; F464Y, E416A; d425R, Y L; N531G; E457G; E461Q; R462K; F464Y; S507A, E457G; E461G, K312A; R314A, G469L; R456N; E457T; k458A, G469L; R456N; k458A, G469L; R456N; K458A; e461G, G469L; R456N; K458A; E461G; F537K, G406D; Y408L, G D; N531G, G D; P535A, G406D; Y408L; N531G, G D; Y408L; P535A, G406D; N531G; P535A, G406D; Y408L; N531G; P535A, K G; I345G, L364G; Y372G, W381G; V390G, W381G; P384A; V390G, W381G; P384G; V390G, N G; Q409G, K G; d425G, T346R; K504E; E530R, T K; K504D; E530K, G344R; Y408L; N531G, G344K; Y408L; N531G, T346R; Y408L; N531G, T K; Y408L; N531G, K D; Y408L; N531G, K E; Y408L; N531G, Y L; E530R; N531G, Y L; E530K; N531G, T346R; Y408L; K504E; E530R; N531G, T K; Y408L; K504D; E530K; N531G, Y L; S507A; N531G, Y L; N531G; E457G; E461Q; R462K; F464Y; S507A, E457G; S507A and N531G; P535A; S507A, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

In some embodiments, the CFI variant comprises one or more modifications at the active site of CFI. In some embodiments, the CFI variant comprises any of the modifications presented in table 6. In some embodiments, the CFI variant comprises a modification at a position corresponding to position S507 in a CFI having the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises substitution S507A, wherein the position corresponds to position S507 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises an a-chain and a B-chain, wherein the CFI variant comprises a structural configuration in the form of (a-chain) - (optionally a linker) - (B-chain) from N-terminus to C-terminus. In some embodiments, the CFI variant comprises an a-chain and a B-chain, wherein the CFI variant comprises a structural configuration in the form of (B-chain) - (optionally linker) - (a-chain) from N-terminus to C-terminus. In some embodiments, the CFI variant comprises a modification at one or more of C309 and C435, wherein the position corresponds to a position in the CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises the substitution C309S; C435S, wherein said position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the B chain and the a chain are further linked by disulfide bonds. In some embodiments, the CFI variant comprises the amino acid sequence set forth in SEQ ID NO. 17 or SEQ ID NO. 18. In some embodiments, the B chain and the a chain are not further linked by disulfide bonds. In some embodiments, the CFI variant comprises the amino acid sequence set forth in SEQ ID NO. 19 or SEQ ID NO. 20.

In some embodiments, the CFI variants comprise one or more modifications presented in table 7. In some embodiments, the CFI variant is more susceptible to activation than the wild-type CFI. In some embodiments, the CFI variant comprises modifications at any one or more of positions I317, R318, R319, K320 and R321 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises one or more of the substitutions selected from I317D, R318D, R319D, K D and R321K, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises the substitutions I317D, R318D, R319D, K D and R321K, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises two or more modifications described herein. In some embodiments, the CFI variant comprises one or more of the modifications presented in table 9. In some embodiments, the CFI variant comprises one or more of the following combinations of substitutions selected from: y408; N531G, E a; d425R, Y F; d425R, S a; d425R, Y F; N531G, Y L; N531G; E457G; E461Q; R462K; F464Y, K a; Y20F, K a; E38A, K a; S250A, K a; d425A, Y F; E38A, Y F; S250A, Y F; d425A, E a; S250A, E a; d425A, S a; d425A, K a; N531G; P535A, Y F; N531G; P535A, E a; N531G; P535A, S a; N531G; P535A, D a; N531G; P535A, Y F; Y408L; N531G; E457G; E461Q; R462K; F464Y, E a; Y408L; N531G; E457G; E461Q; R462K; F464Y, S a; Y408L; N531G; E457G; E461Q; R462K; F464Y, D425R; Y408L; N531G; E457G; E461Q; R462K; F464Y, Y F; E38A; S250A; d425A, Y F; E38A; S250A; d425A; Y408L; N531G; E457G; E461Q; R462K; F464Y, Y F; E38A; S250A; d425A; Y408L; N531G; E457G; e461Q, I317D; R318D; R319D; K320D; R321K; E457G; E461Q-R462K; F464Y, I317D; R318D; R319D; K320D; R321K; E457G; E461Q-R462K; F464Y, I317D; R318D; R319D; K320D; R321K; Y408L; N531G; E457G; E461Q; R462K; F464Y, K504D; Y408L; N531G, K E; Y408L; N531G, E457G; N531G; d425K, Y F; N531G, Y L; E457G; N531G; d425K, Y L; E457G; P535G; d425K, Y L; E457G; N531G; K534Q, Y L; N531G, R462K; F464Y and Y408L; P535G; d425K, wherein said position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some implementations, the CFI variants include each of the SPD, FIMAC domain, SRCR domain, LDLr1 domain, and LDLr2 domain, and any other domain presented in fig. 1. In some embodiments, the CFI variant does not include all of the SPD, FIMAC domain, SRCR domain, LDLr1 domain, and LDLr2 domain. In some embodiments, the CFI variant comprises an SPD. In some embodiments, the CFI variant comprises the amino acid sequence set forth in SEQ ID NO. 12.

In some embodiments, the CFI variant comprises or consists of any one or more of the modifications presented in table 13, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant is sialylated. In some embodiments, the CFI variant is further sialylated as compared to wild-type CFI.

In some embodiments, the CFI variant is in an activated form. In some embodiments, the CFI variant is activated by furin or a variant thereof. In some embodiments, the CFI variant is in vitro activated by furin or a variant thereof. In some embodiments, the CFI variant is activated by furin or a variant thereof during recombinant production in the host cell. In some embodiments, the activation by furin or a variant thereof during production of the host cell is by overexpression of furin or a variant thereof. In some embodiments, the CFI variant is activated by furin or a variant thereof after production and secretion by a host cell, optionally in a medium.

In some embodiments, the CFI variant is a first component of a fusion construct comprising the first component and a second component, and the CFI variant is fused to the second component; the fusion construct may comprise other components. In some embodiments, the second component is a protein. In some embodiments, the second component is not a protein. In some embodiments, the second component is a half-life extender. In some embodiments, the half-life extender comprises a peptide repeat.

In some embodiments, the second component is a half-life extender selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc. In some embodiments, the half-life extender is modified albumin or an albumin derivative. In some embodiments, the half-life extender is wild-type albumin. In some embodiments, the half-life extender is human serum albumin or a variant thereof.

In some embodiments, the CFI variant comprises an a-chain and a B-chain, and wherein the fusion construct comprises a structural configuration from N-terminus to C-terminus or C-terminus to N-terminus as (second component) - (optional linker) - (a-chain) - (optional linker) - (B-chain). In some embodiments, the CFI variant comprises an a-chain and a B-chain, and wherein the fusion construct comprises a structural configuration from N-terminus to C-terminus or C-terminus to N-terminus as (second component) - (optional linker) - (B-chain) - (optional linker) - (a-chain). In some embodiments, the fusion construct comprises or consists of the amino acid sequence set forth in SEQ ID NO. 21 or a sequence having at least 80% sequence identity thereto.

In some embodiments, the second component is at least one domain or portion of a domain of factor H. In some embodiments, at least one factor H domain comprises any one or more of the Complement Control Protein (CCP) domains 1-20 of factor H. In some embodiments, the amino acid sequence of at least one factor H domain is or is derived from the sequence set forth in SEQ ID NO. 4. In some implementations, at least one factor H domain includes each of CCP domains 1-20 of factor H. In some implementations, at least one factor H domain includes CCP1, CCP2, CCP3, and CCP4. In some implementations, at least one factor H domain includes CCP2, CCP3, and CCP4. In some implementations, at least one factor H domain includes CCP2 and CCP3. In some embodiments, the amino acid sequence of at least one factor H domain is or is derived from the sequence set forth in SEQ ID NO. 8. In some implementations, at least one factor H domain includes CCP domains 1-4 and 19-20 of factor H.

In some embodiments, the second component is at least one domain or portion of a domain of complement receptor 1 (CR 1). In some embodiments, at least one domain of CR1 is any one or more of CR1 CCP domains 15-17. In some embodiments, 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.

In some embodiments, the fusion construct further comprises a third component. In some embodiments, the third component is a protein. In some embodiments, the third component is not a protein.

In some embodiments, the CFI variant comprises a third component, wherein the third component is a half-life extender, optionally selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc. In some embodiments, the half-life extender is a repeat peptide sequence. In some embodiments, the CFI variant comprises at least one modification relative to wild-type CFI, wherein the CFI variant is not activatable. In some embodiments, the CFI variant comprises a modification at a position corresponding to position R321 in a CFI having the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises a substitution R321A, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

In another aspect, the invention provides a fusion construct comprising a first component and a second component, wherein the first component comprises wild-type CFI or a variant thereof (CFI variant), and wherein the second component comprises a half-life extender. In some embodiments, the first component comprises a wild-type CFI comprising the amino acid sequence set forth in SEQ ID NO. 5.

In some embodiments, the second component is albumin. In some embodiments, the second component is human serum albumin. In some embodiments, the second component comprises human serum albumin comprising the amino acid sequence set forth in SEQ ID NO. 7.

In some embodiments, the fusion construct comprises the amino acid sequence set forth in SEQ ID NO. 21 or an amino acid sequence having at least 80% identity thereto. In some embodiments, the fusion construct consists of the amino acid sequence set forth in SEQ ID NO. 21. In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7.

In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configuration from N-terminus to C-terminus (SEQ ID NO. 7) - (optionally linker) - (SEQ ID NO. 5). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configurations from N-terminus to C-terminus (SEQ ID NO. 7) - (linker) - (SEQ ID NO. 5). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, wherein the fusion construct comprises the structural configurations from N-terminus to C-terminus (SEQ ID NO. 7) - (SEQ ID NO. 6) - (SEQ ID NO. 5). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configuration from N-terminus to C-terminus (SEQ ID NO. 5) - (optionally linker) - (SEQ ID NO. 7). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configurations from N-terminus to C-terminus (SEQ ID NO. 5) - (linker) - (SEQ ID NO. 7). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, wherein the fusion construct comprises a structural configuration from N-terminus to C-terminus (SEQ ID NO. 5) - (SEQ ID NO. 6) - (SEQ ID NO. 7).

In some embodiments, the first component comprises a CFI variant. In some embodiments, the CFI variant is any CFI variant described herein. In some embodiments, the CFI variant comprises or consists of any one or more of the modifications presented in table 13, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the fusion construct has at least one improved feature compared to the free wild-type CFI (rather than a portion of the fusion construct) or compared to the fusion construct comprising the wild-type CFI. In some embodiments, the improved feature 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. In some embodiments, the improvement is characterized by an increase in activity. In some embodiments, the increase in activity comprises an increase in C3b and/or C4b cleavage. In some embodiments, the improvement is characterized by an increase in substrate specificity.

In some embodiments, the increase in activity comprises an increase in cleavage of C3b compared to the wild-type CFI, but not a portion of the fusion construct, or compared to the fusion construct comprising the wild-type CFI. In some embodiments, the increase in activity comprises an increase in cleavage of C3b and does not comprise an increase in cleavage of C4b compared to the wild-type CFI, but not a portion of the fusion construct, or compared to the fusion construct comprising the wild-type CFI. In some embodiments, the increase in cleavage of 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 greater than the wild-type CFI, but not a portion of the fusion construct, or as compared to a fusion construct comprising the wild-type CFI.

In some embodiments, the increase in activity comprises an increase in cleavage of C4b compared to the wild-type CFI, but not a portion of the fusion construct, or compared to the fusion construct comprising the wild-type CFI. In some embodiments, the increase in activity comprises an increase in cleavage of C4b and no increase in cleavage of C3b compared to the wild-type CFI, but not a portion of the fusion construct, or compared to the fusion construct comprising the wild-type CFI. In some embodiments, the increase in cleavage of 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 greater than the wild-type CFI, but not a portion of the fusion construct, or as compared to a fusion construct comprising the wild-type CFI.

In some embodiments, the increase in activity comprises an increase in production of iC3 b. In some embodiments, the increase in activity comprises an increase in C3dg generated from iC3 b. In some embodiments, the increased activity comprises a decreased content of C3b alpha chains. In some embodiments, the increased activity comprises increased hydrolysis of a peptide substrate or increased proteolysis of a macromolecular protein substrate.

In some embodiments, the improvement is characterized by reduced activity relative to a C4b or C3b substrate.

In some embodiments, the fusion construct has at least one improved feature compared to the free wild-type CFI in the absence of factor H and/or in the absence of CR 1. In some embodiments, the fusion construct has at least one improved feature compared to the free wild-type CFI, and wherein the at least one improved feature is further improved by the presence of exogenous factor H and/or exogenous CR 1.

In one aspect, the invention provides a pharmaceutical composition comprising any of the CFI variants described herein, or any of the fusion constructs described herein, and optionally a pharmaceutically acceptable excipient.

In another aspect, the invention provides a method of modulating the complement system, the method comprising contacting a sample in vitro or contacting a tissue in vivo with any of the CFI variants described herein or any of the fusion constructs described herein. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

In some embodiments, the method results in increased cleavage of C3b, C4b, increased production of iC3b, increased production of C3dg and/or C4C. In some embodiments, the method results in reduced hemolysis. In some embodiments, the method results in a decrease in the content of MAC. In some embodiments, the method results in reduced amplification of the complement system. In some embodiments, the method results in increased hydrolysis of the peptide substrate or increased proteolysis of the macromolecular protein substrate.

In another aspect, the invention provides a method of treating a non-ocular disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any of the CFI variants described herein, or any of the fusion constructs described herein, or any of the pharmaceutical compositions described herein. Such treatment as contemplated herein includes administration of both the CFI variants of the invention or the fusion constructs of the invention, as well as administration of one or more nucleic acids encoding the CFI variants of the invention or the fusion constructs of the invention. Accordingly, provided herein are pharmaceutical compositions comprising the CFI variants of the invention, the CFI fusion constructs of the invention, and pharmaceutical compositions comprising one or more nucleic acids encoding the CFI variants of the invention and encoding the fusion constructs of the invention.

In some embodiments, the non-ocular condition is characterized by insufficient CFI. In some embodiments, the non-ocular condition is characterized by a disorder of the complement system.

In some embodiments, the non-ocular condition is a systemic acute indication. In some embodiments, 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, burn injury, coronavirus infection, epstein-Barr virus (Epstein-Barr virus) infection, hematopoietic stem cell transplantation, ischemia reperfusion injury, lyme disease (Lyme disease), myocardial infarction, organ transplantation, periodontitis, pneumonia, preeclampsia, schistosomiasis, sepsis, stroke, thromboembolism, ischemia-reperfusion injury, and traumatic brain injury.

In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is a systemic chronic indication selected from the group consisting of: alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, anti-phospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous Pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease, crohn's disease, diabetic neuropathy, systemic myasthenia gravis (gMG), granulomatous Polyangiitis (GPA), green-Barre syndrome (GBS), hereditary Angioedema (HAE), suppurative sweat gland (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, osteoarthritis, pancreatitis, parkinson's disease, paroxysmal nocturnal hemuria, post-transplant lymphoproliferative disorder, protein-lost bowel disease, psoriasis, gangrene-type abscess, rheumatoid arthritis, schizophrenia (SZ), systemic Lupus Erythematosus (SLE), immune Thrombocytopenia (ITP), ulcerative colitis, amyotrophic Lateral Sclerosis (ALS), warm autoimmune hemolytic anemia (wAIHA), condensed Colletotrichosis (CAD), and immune complex membranoproliferative glomerulonephritis (IC-MPGN), lambert-eaton muscle weakness syndrome (lamert-Eaton myasthenic syndrome, LEMS), CHAPLE syndrome (CD 55 deficiency), thrombotic Microangiopathy (TMA), huntington's disease, and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP).

In some embodiments, the non-ocular condition is non-oncogenic.

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

In some embodiments, the CFI variant, fusion construct or pharmaceutical composition is administered to the subject subcutaneously or intravenously. In some embodiments, the administration is subcutaneous administration. In some embodiments, the subcutaneous administration is once daily, twice a week, or once weekly, or once every other week.

In another aspect, the invention provides a method of treating an ocular disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any of the CFI variants described herein, or any of the fusion constructs described herein, or any of the pharmaceutical compositions described herein. Such treatment as contemplated herein includes administration of both the CFI variants of the invention or the fusion constructs of the invention, as well as administration of one or more nucleic acids encoding the CFI variants of the invention or the fusion constructs of the invention. Accordingly, provided herein are pharmaceutical compositions comprising the CFI variants of the invention, the CFI fusion constructs of the invention, and pharmaceutical compositions comprising one or more nucleic acids encoding the CFI variants of the invention and encoding the fusion constructs of the invention.

In some embodiments, the ocular disorder is characterized by insufficient CFI. In some embodiments, the ocular disorder is characterized by a disorder of the complement system. In some embodiments, the ocular disorder is selected from the group consisting of: diabetic Macular Edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, stargardt disease (Stargardt Disease), uveitis, and vitreoretinopathy. In some embodiments, the ocular condition is non-oncogenic.

In another aspect, the invention provides a cell comprising one or more nucleic acids encoding wild-type CFI or a variant thereof and comprising one or more nucleic acids encoding furin.

In another aspect, the invention provides a method of producing wild-type CFI or a variant thereof in an activated state, the method comprising recombinantly producing CFI or a variant thereof in a cell comprising one or more nucleic acids encoding CFI or a variant thereof and comprising one or more nucleic acids encoding furin.

Drawings

FIG. 1 is a schematic representation of the domains of wild-type Complement Factor I (CFI) showing the heavy and light chains. The heavy chain (a chain) includes FIMAC, SRCR, LDLr, LDLr2 domains and linkers. The light chain (B chain) includes a serine protease domain. The light chain region includes an activating ring, 37-ring, 60-ring, 70-ring, 99-ring, 110-ring, 150-autolytic ring, 190-ring, oxyanion stabilizing and/or 220-ring S1 inlet framework.

Figures 2A-2D depict an exemplary model of a fusion construct of the invention between albumin (e.g., serum albumin, e.g., human Serum Albumin (HSA)) and CFI comprising a CFI variant, wherein the CFI variant comprises an a-B strand inversion.

Fig. 3 depicts a model of an exemplary CFI albumin (e.g., serum albumin, such as Human Serum Albumin (HSA)) fusion construct comprising serum albumin fused to a CFI, wherein the CFI comprises a wild-type CFI.

FIG. 4 depicts a model of an exemplary CFI-HSA fusion construct comprising HSA fused to the serine protein domain of CFI.

Fig. 5A depicts a schematic diagram showing the Factor H (FH) for its 20 domains.

FIG. 5B depicts a schematic diagram of a cofactor H, which shows domains 1-4 linked to domains 19-20 of FH.

FIG. 6 depicts a model of an exemplary fusion construct comprising factor H and part of the CFI, domains 1-8 comprising FH fused to the CFI, wherein the CFI comprises a wild-type CFI.

Fig. 7 depicts a schematic representation of three exemplary fusion constructs of the invention, each comprising HSA, at least one CFI domain, and various domains of factor H, the portions being connected by optional/exemplary linkers.

Figures 8A-8B are graphs depicting the relative percentages of human and mouse C3B cleavage when comparing various CFI variant fusion constructs (HSA and FH) to CFI wild-type fusion constructs.

Fig. 9 is a graph depicting the activity of a fusion construct of the invention comprising a CFI variant (n531 g+p535A) compared to the activity of wild-type CFI.

FIG. 10 is a graph depicting the half maximal Effective Concentration (EC) of a fusion construct of the invention comprising a variant of CFI (N531 G+P535A) compared to a fusion construct comprising wild-type CFI ₅₀ ) Is a graph of (2).

FIGS. 11A-11B depict dose response curves generated for hemolysis assays of plasma-derived CFI (CFI-PD) and CFI-HSA wild-type with or without its cofactor, factor H, respectively.

FIGS. 11C-11D depict dose response curves of percent hemolysis inhibition measured in classical and alternative pathways by plasma-derived CFI (CFI-PD) and CFI-HSA wild-type, respectively.

FIGS. 12A-12B are graphs depicting measured concentrations of wild-type CFI-HSA fusion construct compared to free plasma purified CFI after a single subcutaneous administration to a monkey at a dose of 1 mg/kg.

Figure 13 depicts a scatter plot showing fold changes in activity for C4b, fold changes in activity for C3b, and engineering specificity, showing that various CFI variants are tunable and selected for C3b, C4b, or both. A: specificity for C4 b; b: specificity for both; and C: specificity for C3 b.

Fig. 14A depicts a dose response curve showing the effect of the presence of CR1 on C4b degradation. CR1 in the presence of the CFI variants of the invention as exogenous cofactor supply, or with the CFI variants fusion.

Fig. 14B depicts a bitmap showing fold changes in activity for C4B, fold changes in activity for C3B, and engineering specificity of CFI variants, as shown in previous fig. 13, with dots representing CFI-CR1 fusions of fig. 14A, indicated by arrows.

Fig. 14C depicts a dose response curve showing classical pathway activity of CFI variants of the invention in the presence and absence of exogenous CR1 cofactor. Both exogenous CR1 and fusion CR1 enhance classical pathway activity.

Figure 14D depicts a dose response showing classical pathway activity of a CFI variant of the invention fused to CR1 in the presence and absence of an exogenous CR1 cofactor.

Figures 14E-14F depict scatter plots of fold change in activity for C4b and C3b for various CFI variants provided herein, indicating further adjustability of the CFI variants tested.

FIGS. 15A-15B depict graphs depicting C3B degradation and C4B degradation of CFI and plasma-derived CFI, respectively.

FIGS. 15C-15D depict graphs of hemolysis assays for CFI-HSA and plasma derived CFI. Where AP represents the alternate path focus analysis and CP+AP represents the alternate and classical path focus analysis.

FIGS. 15E-15F depict the results of a hemolysis assay using the E461G variant, CFI-HSA and plasma derived CFI. Where CP represents the classical path focus analysis and cp+ap represents the alternative and classical path focus analysis.

Fig. 16A depicts a graph predicting a Pharmacokinetic (PK) profile of human exposure after multiple subcutaneous administrations of CFI-HSA.

Fig. 16B-16C depict predicted concentrations of CFI-HSA over time for single administration (fig. 16B) or multiple administration (fig. 16C) compared to the predicted pharmacokinetic profile of fig. 16A.

FIG. 17 is an image of a stained SDS-PAGE gel showing the effect of fusion of HSA with the N-terminus of CFI on solubility, aggregation and activation compared to wild type CFI without the fusion tag.

Fig. 18 depicts a curve showing the amount of C3a detected in samples from vitreous humor after intravitreal Injection (IVT) CFI-HSA at doses of 250 μg or 500 μg in an african green monkey primate model.

FIG. 19 depicts a plot of the amount of factor I detected in plasma at indicated time points following intravenous injection of 3mg/kg CFI-HSA (factor I-HSA) or 1.3mg/kg CFI (factor I) in CD1 mice.

FIG. 20 depicts a plot of the levels of factor I detected in plasma at indicated time points after subcutaneous injection of 3mg/kg CFI-HSA (factor I-HSA) or 6.5mg/kg CFI (factor I) in CD1 mice.

Figure 21 depicts graphs and figures showing CFI cleavage product (C3 dg) content of C3B (membrane-bound fragment) in CFI-HSA (hCFI; Y408L; N531G variant) produced by mass spectrometry on nerve tissue (figure 21A) and circulation (soluble fragment in plasma) in a rat model of sciatic nerve injury (figure 21B).

Fig. 22 depicts a graph showing the content of CFI cleavage products (C3 dg) detected in nerve tissue 24 hours after administration of CFI variants in a rat model of injury to sciatic nerve.

Figure 23 depicts a graph showing CFI cleavage product content detected by mass spectrometry in plasma of animals treated with CFI variants following sciatic nerve injury. The CFI cleavage products detected included C3dg (FIG. 23A) and C3f content (FIG. 23B).

Fig. 24 depicts a graph showing circulating macrophage inflammatory protein 1 alpha (MIP-1 alpha) after sciatic nerve injury following intravenous injection with CFI variants in a sciatic nerve injury rat model.

Fig. 25 depicts a graph showing changes in tumor necrosis factor alpha (tnfalpha) content at 16 hours (fig. 25A) and 3 hours (fig. 25B) after Cecal Ligation and Puncture (CLP) surgery and intravenous administration of CFI variants in a rat CLP model.

Fig. 26 depicts graphs showing% C3f activation in lung (fig. 26A), bronchoalveolar lavage (BALF) (fig. 26B) and plasma (fig. 26C) from animal collection 24 hours and 48 hours after intratracheal Instillation (IT) LPS and intravenous administration of CFI variants in a mouse model of Acute Respiratory Distress Syndrome (ARDS).

Detailed Description

The present invention provides compositions and methods suitable for modulating signaling and amplification of the complement system. Modulation of the complement system is observed by providing Complement Factor I (CFI) variants and fusion constructs containing CFI that have more or less activity on one or more physiological substrates of CFI and/or are more stable than plasma derived CFI. 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 amount of modulator within 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 pathways of the complement system, or both pathways. The invention also provides methods of making and using these variants and constructs, e.g., to treat diseases or conditions associated with complement dysregulation, e.g., to treat overactivated complement response.

I. Complement factor I proteins suitable for modulating the complement system

A. Complement factor I variants

Provided herein are complement factor I variants (CFIs), such variants comprising one or more modifications relative to wild-type CFI, referred to herein as "CFI variants. As used herein, "modification" for 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 of one or more amino acid residues (i.e., an addition), an insertion of one or more domains (i.e., an addition), an inversion of one or more domains, and a substitution of one or more domains.

The CFI variants of the invention do not directly act on C3, e.g., the variants of the invention do not directly cleave C3, do not directly inhibit activation of C3 and do not directly reduce activation of C3.

As used herein, wild-type CFI refers to any naturally occurring full length CFI that is not a pathogenic CFI, with or without a signal sequence, and can be of any species.

In some embodiments, the wild-type CFI is plasma-derived. In some embodiments, the wild-type CFI is a human wild-type CFI. In some embodiments, the wild-type human CFI having a signal sequence comprises the amino acid sequence set forth in SEQ ID NO. 1 (as set forth in Table 1 below). In some embodiments, the wild-type CFI is human CFI. In some embodiments, the wild-type human CFI does not include 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 (as set forth in Table 1 below).

Wild-type CFI comprises heavy and light chains, also known as a and B chains, respectively. Fig. 1 depicts a schematic diagram showing CFIs of two chains. The heavy chain (a chain) has four domains: FI-attack membrane complex (FIMAC) domain (residues 36 to 90 of SEQ ID NO: 5), SRCR domain further consists of a plurality of scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein 1 domain (LDLr 1) and low density lipoprotein 2 domain (LDLr 2). The light chain (B chain) consists of Serine Protease Domain (SPD). The interface between these chains is called the A-B chain interface.

The CFI variants of the invention include one or more of the following: deletion of one or more amino acid residues of wild-type CFI, deletion of one or more CFI domains of wild-type CFI, substitution of one or more amino acid residues of wild-type CFI, insertion of one or more amino acid residues into wild-type CFI, inversion of one or more CFI domains of wild-type CFI, and insertion of one or more domains into wild-type CFI.

The CFI variants of the invention may be produced by introducing one or more modifications to a CFI base molecule, wherein the domains of the CFI base molecule correspond to those found in wild-type CFI, e.g. as set forth in fig. 1. The CFI base molecule may be a wild-type CFI of any species, or the CFI base molecule may comprise only a portion of a wild-type CFI, having only some domains of a wild-type CFI of any species (e.g., already CFI variants). 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 a wild-type non-human primate CFI. In some embodiments, the CFI base molecule comprises only some domains of wild-type human CFI.

In some embodiments, the CFI variants provided herein modulate the activity of the complement system and have at least one improved feature 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 capacity, cofactor affinity (e.g., affinity for factor H and/or CR 1), substrate specificity, and substrate affinity (e.g., affinity for C3b and/or C4 b). In some embodiments, the improvement is characterized by an extended half-life. In some embodiments, the improvement is characterized by an increase in activity as discussed in further detail in the following section. In other embodiments, the improvement is characterized by a change in the substrate specificity of C3b and/or C4b, thereby allowing for the adjustability of the CFI variant.

Provided in table 1 are exemplary base molecules that can be used to generate any of the CFI variants. The base molecules of table 1 are used to produce CFI variants disclosed herein with any one or more of the modifications further discussed herein. The base molecules provided herein may be suitable for modulating the complement system without further modification, or may be suitable for modulating the complement system with further modification. For example, any of the base molecules provided in table 1 may be further modified to include one or more modifications, such as a deletion of one or more amino acid residues, a deletion of one or more CFI domains, a substitution of one or more amino acid residues, or an addition of one or more amino acid residues or CFI domains. The base molecule of table 1 may be another part of a fusion construct, described further below.

Table 1: base molecules for producing CFI variants

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In some embodiments, the base molecule itself may be a CFI variant, e.g., in some embodiments, a CFI variant comprising only the serine protease domain (CFI-SPD) itself is a CFI variant. In some embodiments, the CFI variant is derived from any of the base molecules of table 1 and comprises modifications of the loop corresponding to the loop of the unmodified CFI. In some embodiments, the CFI variant is derived from any of the base molecules of table 1 and comprises a substitution mutation. In some embodiments, the CFI variant is derived from any of the base molecules of table 1 and comprises a deletion of one or more domains of CFI. In some embodiments, the CFI variant is derived from any of the base molecules of table 1 and comprises an inversion of the a and B chains of CFI. Examples of such inversions are provided in Table 9, and include, but are not limited to, SEQ ID NOs 17, 18, 19 and 20.

In some embodiments, provided herein are CFI variants comprising at least one CFI domain, wherein the at least one CFI domain corresponds to those of wild-type CFI of any species. For example, the amino acid sequence of at least one CFI domain may comprise an amino acid sequence derived from a 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 include 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.

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 the group consisting of: serine Protease Domain (SPD), factor I tapping complex (FIMAC) domain, scavenger receptor cysteine-rich domain (SRCR), low density lipoprotein receptor 1 (LDLr 1) and low density lipoprotein receptor 2 (LDLr 2) domain. In some embodiments, any one or more of the CFI domains is a CFI domain of a human CFI. In some embodiments, any one or more of the CFI domains comprises an amino acid sequence derived from the sequence set forth in SEQ ID NO. 5.

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

In some embodiments, the CFI variant does not comprise all domains corresponding to wild-type CFI. In some embodiments, the CFI variant comprises an SPD. In some embodiments, the CFI variant comprises only SPDs, wherein the a chain of the CFI has been deleted, referred to herein as "CFI-SPD". In some embodiments, the CFI-SPD comprises the amino acid sequence set forth in SEQ ID NO:12 (as shown in Table 1), which is an SPD of human CFI. In some embodiments, the CFI-SPD does not comprise further modifications relative to a wild-type CFI SPD. In some embodiments, the CFI-SPD comprises one or more modifications relative to a wild-type CFI SPD. In some embodiments, the CFI-SPD comprises at least one modification relative to the amino acid sequence set forth in SEQ ID NO. 12.

Exemplary variants of CFI are described in further detail below. Exemplary CFI variants comprise one or more substitutions relative to the amino acid residues of CFI having the amino acid sequence set forth in SEQ ID NO. 5. For example, a CFI variant comprising substitutions at positions N531 and P535 would have substitutions at positions N531 and P535 in the amino acid sequence set forth in SEQ ID NO. 5.

Exemplary CFI variants

Provided herein are CFI variants comprising or consisting of at least one modification relative to wild-type CFI, wherein the CFI variant is capable of increasing complement system inhibition, and wherein the CFI variant has at least one improved feature compared to wild-type CFI. Examples of improved characteristics include, but are not limited to, any one or more of increased half-life, increased bioavailability or activity, substrate specificity, potency, substrate affinity, cofactor affinity, and catalytic ability. In an exemplary embodiment, the improvement is characterized by an increased half-life. In other exemplary embodiments, the improvement is characterized by increased or altered substrate specificity.

Without limitation, the present invention encompasses the exemplary CFI variants described in table 13. The variants of table 13 include modified CFIs, as well as the CFI fusion constructs described herein. For the avoidance of doubt, unless indicated otherwise, where the number of residues is indicated it refers to SEQ ID NO 5 (wild type human CFI) or a sequence corresponding thereto. For the avoidance of doubt, for example, variants described as being indicative of K14A, the invention provides CFI variants comprising a K14A substitution, for example CFI variants comprising a K14A substitution in SEQ ID No. 5 (or a sequence corresponding thereto); the invention also provides CFI variants, e.g., CFI variants, consisting of a K14A substitution, wherein SEQ ID NO. 5 has a K14A substitution.

In some embodiments, the CFI variants of the invention comprise or consist of: any one or more of the modifications presented in table 13, wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID No. 5.

The CFI variants of the invention 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. Modification (e.g., substitution) of a given variant may be indicated in one of many ways recognized by those of skill in the art. For example, hCFI variants with substitutions at T377G and N422K may be referred to as having substitutions: "T377G and N422K", "T377G-N422K", "T377G+N422K", "T377G/N422K", or "T377G; N422K "and are used interchangeably herein. In some cases, CFI variants with substitutions at T377G and N422K may be referred to as "hCFI; T377G; N422K "or CFI variants (T377G; N422K)". As described herein, variants with other modifications (such as deletions) or combinations of modifications (such as deletions, fusions, and substitutions) may conform to similar patterns of nomenclature. Table disclosure variants (e.g., tables 13, 7.1 and 7.2) include the following symbols and abbreviations and related meanings: HSA = human serum albumin; CFI = complement factor I; delta = deletion of the amino acid range; -deleting the sequence and replacing with the amino acid; cr1=cr1 fusion; fh=fh fusion; g (#) represents a linker repeating the sequence GGSSGG (SEQ ID NO: 6) of the indicated number of examples.

TABLE 13 exemplary CFI variants

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The activity and specificity of the CFI variants provided herein can be modulated for specific applications and therapeutic indications. For example, activity and specificity may be modulated by selecting a C3b degrading agent or a C4b degrading agent or a degrading agent of both C3b and C4b. As used herein, protease activity against a substrate refers to the ability of the CFI variants of the invention to cleave their substrates, C4b and C3 b. This can be expressed as an increase in C4b degradant activity, protease activity against C4b, C3b degradant activity, protease activity against C3b, and the like.

As used herein, a C3b degrading agent is a CFI variant capable of cleaving C3 b; likewise, a C4b degrading agent is a CFI variant capable of cleaving C4b. The use of a C3b degrading agent does not suggest that it does not degrade C4b. CFI variants may be both C3b and C4b degrading agents, and may exhibit specificity for one relative to the other.

The CFI variants provided herein have modified characteristics that include increased or decreased protease activity of the substrate and increased or decreased substrate specificity.

As used herein, specificity for a substrate (also referred to as substrate specificity) refers to the specificity that CFI variants demonstrate for one compared to the other. If the substrate specificity of the CFI variant is about 1, the specificity is equal for both C4b and C3 b. If the specificity of the CFI variant is 2-fold higher for C4b, it is then considered to exhibit increased cleavage specificity for C4b compared to C3 b. Specific expression of C4b in the examples provided herein is the ratio of the percent maximum cleavage of C4b divided by the percent maximum cleavage of C3 b. Likewise, in the examples provided herein, the specific expression of C3b is the ratio of the maximum percent cleavage of C3b divided by the maximum percent cleavage of C4b. An example of a substrate with a doubled protease activity compared to another substrate is one with an increased specificity for that substrate.

In some embodiments, amino acid modifications (e.g., substitutions) increase activity, confer specificity, or both. In some embodiments, the increased activity of the C4b degrading agent comprises an increased cleavage of C4b (and production of cleavage products such as C4C) and the increased specificity for C4b comprises an increased cleavage of C4b and a decreased cleavage of C3b (and production of cleavage products such as iC3 b) as compared to wild-type CFI or a fusion construct comprising wild-type CFI.

In some embodiments, a combination of two or more modifications (e.g., substitutions) imparts a surprising increase in synergistic or additive activity.

In some embodiments, the combination of one or more modifications imparts an unexpected increase or decrease in activity that is synergistic when C4b is the substrate and additive or less additive when C3b is the substrate.

In some embodiments, the combination of one or more modifications imparts an unexpected increase or decrease in activity that is synergistic when C3b is the substrate and additive or less additive when C4b is the substrate.

Thus, the modified feature may be achieved by selecting one or more modifications that confer increased C3b degradant activity and decreased C4b degradant activity (increased C3b substrate specificity), or alternatively, confer increased C4b degradant activity and decreased C3b degradant activity (increased C4b substrate specificity), or alternatively, provide increased activity (unchanged specificity, but increased activity of both substrates) as degradants of both C3b and C4 b.

Thus, the modified feature may be achieved by selecting one or more modifications that confer increased C3b degradant activity without an alteration in C4b degradant activity (increased C3b substrate specificity), or alternatively, confer increased C4b degradant activity without an alteration in C3b degradant activity (increased C4b substrate specificity).

Thus, the modified feature may be achieved by selecting one or more modifications that confer reduced activity of the C3b degradant and no change in activity of the C4b degradant (increased C4b substrate specificity), or alternatively confer reduced activity of the C4b degradant and no change in activity of the C3b degradant (increased C3b substrate specificity).

Modifications that provide increased activity and specificity are typically focused on, but are not limited to, structural regions critical to CFI function. Exemplary structural regions in which modification (e.g., substitution) results in at least one improved feature are the C-terminal extension, the a: B interface, a surface representing the cofactor within the active site with SPD and the interface of modification (e.g., substitution), including the surface loop providing the interface with C3B and C4B substrates and CR1 and FH cofactors (fig. 1).

Without being bound by theory or mechanism, provided herein are CFI variants having one or more combinations of any of the amino acid modifications described in detail below, wherein the CFI variants have at least one improved feature. Variants of CFI with combinatorial modifications (e.g., substitutions) include two or more modifications in one or more regions of CFI selected from, but not limited to, C-terminal extension, a: B interface, interface with cofactor and active site, including surface loops providing an interface with cofactor and C3B or C4B substrate.

In some embodiments, CFI variants comprising two or more substitutions exhibit a change in activity, substrate specificity, or both. In some embodiments, the increased activity comprises an increased cleavage of C4b, and/or the production of C4C and specifically the cleavage of C3b, and/or the limited increase or decrease in the production of iC3b, as compared to the wild-type CFI (or as compared to a fusion construct comprising the wild-type CFI (e.g., SEQ ID NO: 21)). In some embodiments, a combination of two or more modifications imparts an unexpected increase in activity that is synergistic when C4b is the substrate and additive or less additive when C3b is the substrate.

In some embodiments, amino acid substitutions increase activity, confer specificity, or both, and may switch between C3b selectivity and C4b selectivity. In some embodiments, the increased activity comprises increased cleavage of C4b and/or increased production of C4C, and optionally comprises decreased cleavage of C3b and/or decreased production of iC3b, as compared to wild-type CFI. In some embodiments, the increased activity comprises increased cleavage of C3b and/or increased production of iC3b, and the specificity comprises decreased cleavage of C4b and/or decreased production of C4C, as compared to wild-type CFI. In some embodiments, the nature of the amino acid substitution defines whether the CFI variant exhibits a specificity for C3b or a characteristic of specificity for C4 b.

Exemplary variants of the invention were tested for differences in activity and differences in specificity. Exemplary data are provided in at least table 7.2.

In some embodiments, the CFI variants exhibit increased activity, wherein the increase in activity comprises increasing C3b degradant activity (with an increase in C3b cleavage product) by the CFI variants of the invention. In some embodiments, the CFI variants of the invention exhibit an increase in C3b degrading agent activity 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 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 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 800-fold, at least or about 900-fold, or at least or about 1000-fold, or at least or about 900-fold, or at least or about 900-fold, as compared to wild-type CFI or fusion construct comprising wild-type CFI (e.g.g.g.g. SEQ ID NO: 21). In some embodiments, this increase in C3b degradant activity is also accompanied by an increase in C4b degradant activity. In some embodiments, this increase in C3b degradant activity is also accompanied by an increase in C4b degradant activity, and the C4b degradant activity may even decrease.

In some embodiments, the CFI variants exhibit increased activity, wherein the increase in activity comprises increasing C4b degradant activity (with an increase in C4b cleavage product) by the CFI variants of the invention. In some embodiments, the CFI variants of the invention exhibit an increase in C4b degrading agent activity 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 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 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 800-fold, at least or about 900-fold, or at least or about 1000-fold, or at least or about 900-fold, or at least or about 900-fold, as compared to wild-type CFI or fusion construct comprising wild-type CFI (e.g.g.g.g. SEQ ID NO: 21). In some embodiments, this increase in C4b degradant activity is also accompanied by an increase in C3b degradant activity. In some embodiments, this increase in C4b degradant activity is also accompanied by an increase in C3b degradant activity, and the C3b degradant activity may even decrease.

In some embodiments, the CFI variant exhibits increased activity, wherein the increased activity comprises increased activity of C3b and C4b degradants. In some embodiments, the CFI variants of the invention exhibit an increase in C3b and increased C4b degradant activity 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 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 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 9-fold, at least or about 30-fold, at least or about 700-fold, at least or at least about 900-fold, at least or about 900-fold, or even at least about 800-fold, or at least or about 900-fold, or about 1000-fold, or at least or about 900-fold, as compared to wild-type CFI or fusion constructs comprising wild-type CFI (e.g.g.g.seq ID NO: 21). The increase in the degradant activity of the substrate may be the same, but need not be.

In some embodiments, the CFI variant exhibits increased specificity for the substrate, wherein the increase in specificity is for C3b (over C4 b). In some embodiments, the CFI variants of the invention exhibit 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 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 40-fold, at least or about 800-fold, at least or about 900-fold, or at least or about 1000-fold, or at least, or about fold, as compared to a wild-type CFI or fusion construct comprising wild-type CFI (e.g.g.g.g. SEQ ID: 21).

In some embodiments, the CFI variant exhibits increased specificity for the substrate, wherein the increase in specificity is for C4b (over C3 b). In some embodiments, the CFI variants of the invention exhibit 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 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 700-fold, at least or about 900-fold, at least or at least about 900-fold, or at least or about 900-fold, or at least compared to wild-type CFI or fusion constructs comprising wild-type CFI (e.g.g., SEQ ID NO: 21) having a specificity that is about equal to both C3b and C4 b.

In some embodiments, the CFI variant exhibits reduced specificity for the substrate, wherein the reduction in specificity is for C3b (over C4 b). In some embodiments, the CFI variants of the invention exhibit 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 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 700-fold, at least or about 900-fold, at least or at least about 900-fold, at least or about 900-fold, or at least about 900-fold, or at least or about 100-fold, compared to wild-type CFI or fusion constructs comprising wild-type CFI (e.g.g.g., SEQ ID: 21) that are both.

In some embodiments, the CFI variant exhibits reduced specificity for the substrate, wherein the reduction in specificity is for C4b (over C3 b). In some embodiments, the CFI variants of the invention exhibit 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 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 700-fold, at least or about 900-fold, at least or at least about 900-fold, or at least or about 900-fold, or at least compared to wild-type CFI or fusion constructs comprising wild-type CFI (e.g.g., SEQ ID NO: 21) having a specificity that is about equal to both C3b and C4 b.

In some embodiments, exemplary amino acid residues in which one or more substitutions may confer improved or unexpected characteristics, as compared to, include, but are not limited to, L307, T377, G406, Y408, E416, N422, D425, E457, E461, K504, E530, P535, R557, P558, and combinations thereof, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5 (or a sequence corresponding thereto).

In some embodiments, exemplary CFI variants of the invention exhibiting one or more improved characteristics compared to wild-type CFI (or compared to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21) comprise: a CFI variant comprising two or more combinations of T377G, N422K, E457G, E461Q or N531G, wherein said positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, an exemplary CFI variant comprising or consisting of a single amino acid substitution of T377G, E457G or E461Q, as compared to a wild-type CFI (or as compared to a fusion construct comprising a wild-type CFI, e.g., SEQ ID No. 21), exhibits at least a 2-fold increase in protease activity for both C4b and C3b, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, exemplary CFI variants comprising or consisting of a single substitution such as N531G exhibit at least a 5-fold increase in protease activity against C4b and at least a 3-fold increase in activity against C3b compared to wild-type CFI (or compared to fusion constructs comprising wild-type CFI, e.g., SEQ ID No. 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, exemplary CFI variants comprising or consisting of a single substitution such as N422K exhibit little or NO change in protease activity against C4b, but exhibit at least a 2-fold increase in protease activity against C3b, as compared to wild-type CFI (or as compared to a fusion construct comprising wild-type CFI, e.g., SEQ ID No. 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, exemplary CFI variants comprising or consisting of two substitutions (such as E457G and N531G) exhibit at least 27-fold increase in activity against C4b and at least 4-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of two substitutions (such as T377G and N531G) exhibit at least 16-fold increase in activity against C4b and at least 4-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of two substitutions (such as T377G and E457G) exhibit at least 15-fold increase in activity against C4b and at least 4-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of two substitutions (such as T377G and E457G) exhibit at least 15-fold increase in activity against C4b and at least 4-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of two substitutions (such as T377G and N422K or N422K and E457G) exhibit at least 8-fold increase in activity against C4b and at least 5-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of three substitutions (such as T377G and E457G and N531G) exhibit at least 100-fold increase in activity against C4b and at least 6-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of three substitutions (such as T377G and E461Q and N531G) exhibit at least a 60-fold increase in activity against C4b and at least a 5-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants comprising or consisting of three substitutions (such as T377G and N422K and N531G) exhibit at least 45-fold increase in activity against C4b and at least 8-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21).

In some embodiments, exemplary CFI variants of the invention that exhibit one or more improved characteristics compared to wild-type CFI (or compared to fusion constructs comprising wild-type CFI, e.g., SEQ ID No. 21) are CFI variants comprising or consisting of N531G, P535A and R557A, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, an exemplary CFI variant comprising or consisting of a single amino acid substitution of R557A exhibits at least wild-type activity against C4b and a 20-fold decrease in C3b activity compared to wild-type CFI (or compared to a fusion construct comprising wild-type CFI, e.g., SEQ ID No. 21), wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, exemplary CFI variants comprising or consisting of a single substitution such as N531G exhibit at least a 5-fold increase in activity against C4b and at least a 3-fold increase in activity against C3b compared to wild-type CFI (or compared to fusion constructs comprising wild-type CFI, e.g., SEQ ID No. 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, a variant of CFI comprising or consisting of two substitutions (such as N531G and P535A) exhibits at least a 5-fold increase in activity against C4b and a 3-fold increase in C3b activity compared to a wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, variants comprising or consisting of three substitutions (such as N531G and P535A and R557A) exhibit at least 18-fold increase in activity against C4b and 2.5-fold decrease in C3b activity compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants of the invention that exhibit one or more improved characteristics compared to wild-type CFI (or compared to fusion constructs comprising wild-type CFI, e.g., SEQ ID No. 21) are CFI variants comprising or consisting of D425R, E457G and E530Y, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, exemplary CFI variants comprising or consisting of a single amino acid substitution of E457G or E530Y exhibit at least wild-type activity against C3b and C4 b. In some embodiments, variants comprising or consisting of three substitutions (such as D425R and E457G and E530Y) exhibit at least an 8-fold increase in activity towards C3b and a near wild-type activity towards C4b, as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants of the invention that exhibit one or more improved characteristics compared to wild-type CFI (or compared to a fusion construct comprising wild-type CFI, e.g., SEQ ID No. 21) are CFI variants comprising or consisting of R557A, R557M, R557P and R557G, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, an exemplary CFI variant, such as one consisting of or comprising an R557A substitution, exhibits at least wild-type activity against C4b and a 20-fold decrease in C3b activity as compared to a wild-type CFI (e.g., a wild-type CFI of SEQ ID NO:5, a fusion construct comprising SEQ ID NO:5, or a fusion construct of SEQ ID NO: 21), wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, variants comprising or consisting of R557M and R557P, respectively, exhibit at least a 3-fold increase in activity against C4b and a 5-fold to 10-fold decrease in C3b activity, as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising or fusion construct of SEQ ID NO:5 or SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, variants such as R557G exhibit at least 2-fold activity against C4b and 20-fold decrease in C3b activity compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants of the invention that exhibit one or more improved characteristics compared to wild-type CFI (or compared to a fusion construct comprising wild-type CFI, e.g., SEQ ID NO: 21) are CFI variants comprising or consisting of E457T, E457Q, E457G or E457A, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants comprising or consisting of substitutions such as E457T exhibit at least a 2.6-fold increase in activity against C3b and a 5-fold decrease in C4b activity, as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants comprising or consisting of substitutions E457Q or E457G exhibit at least wild-type activity against both C3b and C4b, as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants comprising or consisting of substitutions such as E457A exhibit at least a 2.7-fold increase in activity against C4b and a 1.6-fold increase in C3b activity compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, an exemplary CFI variant of the invention that exhibits one or more improved characteristics compared to a wild-type CFI (or compared to a fusion construct comprising a wild-type CFI, e.g., SEQ ID No. 21) is a CFI variant comprising or consisting of E530F, E Y or E530R, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, an exemplary CFI variant comprising or consisting of a substitution such as E530Y exhibits at least a 1.6-fold increase in activity against C3b and a C4b activity that is close to wild-type activity, as compared to a wild-type CFI (e.g., a wild-type CFI of SEQ ID NO:5, a fusion construct comprising SEQ ID NO:5, or a fusion construct of SEQ ID NO: 21), wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, exemplary CFI variants comprising or consisting of substitutions such as E530F exhibit at least a 1.6-fold increase in activity against C3b and a 3-fold decrease in C4b activity as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, an exemplary CFI variant comprising or consisting of a substitution such as E530R exhibits at least a 1.8-fold increase in activity against C3b and a 5-fold decrease in C4b activity as compared to a wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising or fusion construct of SEQ ID NO:5 or fusion construct of SEQ ID NO: 21), wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, an exemplary CFI variant of the invention that exhibits one or more improved characteristics compared to a wild-type CFI (or compared to a fusion construct comprising a wild-type CFI, e.g., SEQ ID No. 21) is a CFI variant comprising or consisting of a E457G, E Q, N G or R557A substitution, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

Exemplary CFI variants comprising or consisting of a single amino acid substitution of E457G or E461Q exhibit at least a 2-fold increase in activity against C4b and C3b compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21). Exemplary CFI variants comprising or consisting of a single substitution such as N531G exhibit at least a 5-fold increase in activity against C4b and at least a 3-fold increase in activity against C3 b. An exemplary CFI variant comprising or consisting of a single amino acid substitution of R557A exhibits at least wild-type activity against C4b, and a 20-fold reduction in C3b activity. In some embodiments, variants comprising or consisting of two substitutions (such as E457G and N531G) exhibit at least a 27-fold increase in activity against C4b and at least a 4-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21). In some embodiments, variants comprising or consisting of two substitutions (such as E457G and E461Q) exhibit at least 5-fold increased activity against C4b and C3b compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21). In some embodiments, variants comprising or consisting of two substitutions (such as E461Q and N531G) exhibit at least a 12-fold increase in activity against C4b and at least a 5-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21). In some embodiments, variants comprising or consisting of two substitutions (such as E457G and E461Q and N531G and R557A) exhibit at least a 12-fold increase in activity against C4b and at least a 1.5-fold increase in activity against C3b as compared to wild-type CFI (e.g., wild-type CFI of SEQ ID NO:5, fusion construct comprising SEQ ID NO:5, or fusion construct of SEQ ID NO: 21). These differences are compared to wild-type CFI (or to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21).

In some embodiments, the CFI variant has increased activity, wherein the increased activity comprises increased cleavage of C3b and/or increased specificity of C3b relative to C4 b. In some embodiments, the CFI variant with increased cleavage of C3b and/or increased specificity of C3b relative to C4b comprises one or more substitutions in the amino acid positions set forth in SEQ ID NO. 5. In some embodiments, the amino acid position is selected from one or more of E392, E416, D420, N422, D425, P558, T346, E401, G406, E457, E461, and N531 in a 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 in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the cofactor interface region is selected from one or more of E392, D420, and N422. In some embodiments, the amino acid position is a position within the C-terminal extension region of a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the c-terminal extension region is selected from one or more of E416, D425, and P558. In some embodiments, the amino acid position is a position within the active site; the C3b interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the active site; the C3b interface region is selected from one or more of T346, E401, and N531. In some embodiments, the amino acid position is a position in the autolytic loop; cofactor interface in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the ring is self-dissolving; the cofactor interface is selected from one or more of E457 and E461. These differences are compared to wild-type CFI (or to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variant has increased activity, wherein the increased activity comprises increased cleavage of C4b and/or increased specificity of C4b relative to C3 b. In some embodiments, the cleaved CFI variants with increased C4b comprise one or more substitutions in the amino acid positions set forth in SEQ ID NO. 5. In some embodiments, the amino acid position is selected from one or more of L307, T377, D420, D425, Y553, R557, P558, E401, G406, E457, E461, E487, N531, and K534 in a 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 A:B interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the A:B interface region is selected from one or more of L307 and E487. In some embodiments, the amino acid position is a position within the cofactor interface region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the cofactor interface region is selected from one or more of T377 and D420. In some embodiments, the amino acid position is a position within the C-terminal extension region of a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the C-terminal extension region is selected from one or more of D425, R557, and P558. In some embodiments, the amino acid position is a position within the c-terminal extension; the C4b interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the C-terminal extension; the C4b interface region is Y553. In some embodiments, the amino acid position is a position within the C4b interface region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the C4b interface region is E401. In some embodiments, the amino acid position is a position within the active site; the C4b interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the active site; the C4b interface region is G406. In some embodiments, the amino acid position is a position within the self-dissolving loop; cofactor interface regions in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the ring is self-dissolving; the cofactor interface region is selected from one or more of E457 and E461. In some embodiments, the amino acid position is a position within the active site; the S1 entry frame in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the active site; the S1 portal frame area is N531. In some embodiments, the amino acid position is a position within the S1 entry framework region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the S1 portal frame region is K534. These differences are compared to wild-type CFI (or to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the improvement is characterized by an increase in activity, wherein the increase in activity comprises an increase in cleavage of C3b and/or C4 b. In some embodiments, a CFI variant provided herein is a C3b degradation product, referring to the ability of the CFI variant to increase C3b cleavage. In some embodiments, the CFI variants provided herein are C4b degradants, referring to the ability of the CFI variant to increase C4b cleavage. In some embodiments, the CFI variants provided herein are C3b and C4b degrading agents, referring to the ability of the CFI variants to increase cleavage of both C3b and C4 b.

In some embodiments, the CFI variant has increased activity, wherein the increased activity comprises increased cleavage of C3b and C4 b. In some embodiments, the CFI variants with increased cleavage of C3b and C4b comprise one or more substitutions in the amino acid positions set forth in SEQ ID NO. 5. In some embodiments, the amino acid position is selected from one or more of E392, E420, E401, G406, D420, D425, P558, E457, D459, N460, E461, and N531 in a 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 substrate interface region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the substrate interface region is E401. In some embodiments, the amino acid position is a position within the active site; a substrate interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the active site; the substrate interface region is G406. In some embodiments, the amino acid position is a position within the cofactor interface region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the cofactor interface region is D420. In some embodiments, the amino acid position is a position within the C-terminal extension region of a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the c-terminal extension region is selected from one or more of D425 and P558. In some embodiments, the amino acid position is a position within the self-dissolving loop; cofactor interface regions in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the ring is self-dissolving; the cofactor interface region is selected from one or more of E457, D459, N460, and E461. In some embodiments, the amino acid position is a position within the active site; the S1 entry frame in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the active site; the S1 portal frame area is N531. These differences are compared to wild-type CFI (or to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variants have increased activity, wherein the increase in activity comprises increasing cleavage of C3b by the CFI variants of the invention and no or minimal increase in cleavage of C4 b. In some embodiments, the cleaved CFI variant with increased cleavage of C3b and NO or minimal increased cleavage of C4b comprises one or more substitutions at positions selected from T346, E392, N422, E416 and E401 in the amino acid positions set forth in SEQ ID NO. 5. In some embodiments, the amino acid position is a position within the active site; the C3b interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the active site; the C3b interface region is T346. In some embodiments, the amino acid position is a position within the cofactor interface region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the cofactor interface region is selected from one or more of E392 and N422. In some embodiments, the amino acid position is a position within the c-terminal extension region of a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the C-terminal extension region is E416. These differences are compared to wild-type CFI (or to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variants have increased activity, wherein the increase in activity comprises increasing cleavage of C4b by the CFI variants of the invention and no or minimal increase in cleavage of C3 b. In some embodiments, the cleaved CFI variant with increased cleavage of C4b and without or minimally comprising increased cleavage of C3b comprises one or more substitutions in the amino acid positions set forth in SEQ ID NO. 5. In some embodiments, the amino acid position is selected from L307, T377, E460, E487, K534, Y553, and R557. In some embodiments, the amino acid position is a position within the A:B interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the A:B interface region is selected from one or more of L307 and E487. In some embodiments, the amino acid position is a position within the cofactor interface region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the cofactor interface region is T377. In some embodiments, the amino acid position is a position within the S1 entry framework region in a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the location within the S1 portal frame region is K534. In some embodiments, the amino acid position is a position within the c-terminal extension; the C4b interface region in CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the C-terminal extension; the C4b interface region is Y553. In some embodiments, the amino acid position is a position within the C-terminal extension region of a CFI having the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the position within the C-terminal extension region is R557. These differences are compared to wild-type CFI (or to fusion constructs comprising wild-type CFI, e.g., SEQ ID NO: 21), wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variants of the invention as specific C3b degrading agents are useful for treating diseases.

In some embodiments, the CFI variants of the invention as specific C4b degrading agents are useful for treating diseases.

In some embodiments, CFI variants of the invention that are C4b and C3b degrading agents and that exhibit improved characteristics compared to wild-type CFI (e.g., increased activity for both C4b and C3 b) are useful in treating diseases.

For example, diseases treatable by the use of C4b degrading agents include, but are not limited to, non-ocular disorders. In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is a systemic chronic indication selected from the group consisting of: alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), anti-neutrophil cytoplasmic antibody (ANCA) related vasculitis, anti-phospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous Pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic Obstructive Pulmonary Disease (COPD), condensed Collectinopathy (CAD), crohn's disease, diabetic neuropathy, systemic myasthenia gravis (gMG), granulomatosis with polyangiitis (GPA), green-Barlich 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 (MMN), multiple Sclerosis (MS), non-insulin dependent diabetes mellitus, osteoarthritis, pancreatitis, parkinson's disease, paroxysmal Nocturnal Hemoglobinuria (PNH), post-transplantation lymphoproliferative disorder, protein-lost bowel disease, psoriasis, gangrene abscess, rheumatoid arthritis, schizophrenia (SZ), systemic Lupus Erythematosus (SLE), immune Thrombocytopenia (ITP), warm autoimmune hemolytic anemia (wAIHA), immune complex membranous proliferative glomerulonephritis (IC-MPGN), ulcerative colitis, lambert-eaton muscle weakness syndrome (LEMS), CHAPLE syndrome (CD 55 deficiency), thrombotic Microangiopathy (TMA) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), huntington's disease and ischemia reperfusion injury.

In some embodiments, the CFI variants provided herein are degradants of both C3b and C4b and are useful in the treatment of diseases.

In some embodiments, the increase in activity comprises an increase in C3dg and/or C3C from iC 3b. Exemplary CFI variants of the invention exhibiting this improved feature are CFI variants comprising the substitution N531g+p535A, D a or D425R, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the increased activity comprises a decreased content of C3b alpha chains. An exemplary variant of the invention exhibiting this improved feature is a CFI variant comprising a N531g+p535A substitution, wherein said position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5. Other variations exhibiting similar improved features are provided in table 13 and discussed in the embodiments.

In some embodiments, the activity increases hydrolysis of a peptide substrate or proteolysis of a macromolecular protein substrate. In some embodiments, the macromolecular protein substrate is C3b. In some embodiments, the macromolecular protein substrate is C4b. In some embodiments, the peptide substrate is a chromogenic substrate, for example, such peptide substrate is suitable for use in an assay format. Exemplary CFI variants of the invention exhibiting this improved feature are autolytic loop exchanged CFI variants comprising the modification L307G, E457G, E461Q, E457g+e461q+r462k+f464Y, N531G, N531A, P535A, N531g+p535A, Y408L, Y l+n531G, Y408f+n531G, Y l+n531g+e457g+e461q+r462k+f464Y, Δ (K1-P305) +n531G, Δ (K1-P305) +n531g+p535A, or 456-REKDNERVFS-465- > NTASSGADYPDE, wherein said position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. Other variations exhibiting similar improved features are provided in table 13 and discussed in the embodiments.

In some embodiments, the increased activity comprises a decrease in the content or function of a tapping complex (MAC). In some embodiments, a reduction or even inhibition of hemolysis is associated with a reduction in MAC content, and thus, in some embodiments, an increase in activity comprises a reduction (partial or complete) in the observed hemolysis.

In some embodiments, the increased activity comprises a decrease in amplification of the complement system for the production of C3b. An exemplary variant of the invention exhibiting this improved feature is a CFI variant comprising a N531g+p535A substitution, wherein said position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5. Other variations exhibiting similar improved features are provided in table 13 and discussed in the embodiments.

In some embodiments, the CFI variant is sialylated. In some embodiments, the CFI variant is further sialylated as compared to wild-type CFI. In some embodiments, the CFI variant is sialylated post-translationally by an in vitro method.

In some embodiments, the CFI variant is an activated variant (i.e., in an active double-stranded form). In some embodiments, the CFI variant is activated by furin (the term furin includes furin variants). In some embodiments, the CFI variant is activated by furin during production of the host cell. In some embodiments, activation by furin during production in the host cell is achieved by overexpression of furin, e.g., by stable or transient transfection. In some embodiments, the CFI variant is activated by furin after production and secretion by the host cell, i.e., post-translationally.

Reference to a modification (such as a substitution) in the following sections is a modification relative to the amino acid sequence of human CFI as set forth in SEQ ID NO: 5. However, it will be appreciated that modifications may also be made to the corresponding amino acid residues of any non-human species.

A: B chain interface CFI variants

Provided herein are CFI variants comprising one or more modifications at the interface of the heavy and light chains (also known as a: B chain interface) and variants that disrupt the a: B chain interface.

Without being bound by theory or mechanism, it is believed that the Serine Protease Domain (SPD) of CFI remains in a proenzyme-like state via a number of interactions with its own a chain. Although naturally occurring CFI can cleave peptide or protein substrates at a relatively slow rate, the rate of cleavage by CFI is increased by disrupting the a: B chain interface.

Thus, in some embodiments, provided herein are variants of a: B chain interface CFI. In particular, provided herein are exemplary CFI variants comprising any one or more of the modifications presented in table 2. Table 2 presents CFI variants comprising one or more modifications to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are located at or disrupt the A:B chain interface. The base molecule for the CFI variants presented in table 2 may be wild-type human CFI. It should be noted that not all a: B chain interface CFI variants of the invention are provided in table 2, and additional variants may be provided in at least examples and/or table 13.

Table 2: illustrative A-B chain interface CFI variants

In some embodiments, the CFI variant comprises or consists of any one or more of the modifications presented in table 2. In some embodiments, the CFI variant comprises a modification at any one or more of positions K14, Y20, D26, F29, R35, E38, M220, K221, S250, L304, P305, K306, L307, and S308 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of a substitution in the 200 th loop of CFI (MDANNNVT, SEQ ID NO: 13) having the 200 th loop of trypsin of amino acid residue NG, wherein the 200 th loop is located between positions corresponding to positions 514 and 520 in the CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variant comprises or consists of any one or more of the substitutions selected from K14A, Y20A, Y20F, D3526A, F29A, R A, E38A, M220A, K221Q, S250A, S L, L G, P305G, K306G, L307G and S308G, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of any one or more of the substitutions M220A in combination with K221Q and l304g+p305g+k305g+l307 g+s308G, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

C-terminal region variants

In the complex formed between CFI and C3B, the C-terminal extension region is disposed in the cavity between the a and B chains of the bound and slightly twisted CFI molecule. This suggests that C-terminal extension of CFI may be an important regulatory region for activating CFI when bound to C3 b.

Thus, provided herein are C-terminal region CFI variants. Table 3 presents exemplary CFI variants comprising or consisting of one or more modifications to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are located at the C-terminal region or extension of the CFI. The base molecule for the CFI variants presented in table 3 may be wild-type human CFI. It should be noted that not all C-terminal region CFI variants of the invention are provided in table 3, and additional variants may be provided in at least examples and/or table 13.

Table 3: exemplary C-terminal CFI variants

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In some embodiments, the CFI variant comprises any one or more of the modifications presented in table 3.

In some embodiments, the CFI variant comprises or consists of a modification at any one or more of positions T377, W381, P384, Y403, a405, G406, Y408, Q409, D425, G556, R557, P558, P559, I560, and Y563 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of a deletion of amino acid residues (PFISQYNV, SEQ ID NO: 14) between positions corresponding to positions 558 through 565 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the amino acid residue, CFI variant, comprises or consists of a substitution in loop 110 of CFI (DGNK, SEQ ID NO: 15) between positions 420 to 424 in a CFI corresponding to the amino acid sequence set forth in SEQ ID NO:5, the substitution being substituted with a linker (e.g., GG).

In some embodiments, the CFI variant comprises or consists of any one or more of substitutions selected from T377G, W381A, P384A, P384G, Y403F, A405S, G406R, G406A, Y L, Q409D, Q409H, D425A, D425K, D425R, G556A, G556S, R557A, R K, P558G, P558L, P558S, F559L, I560V and Y563H, and/or a deletion of P384, wherein the position corresponds to a position in the CFI having the amino acid sequence set forth in SEQ ID NO 5.

In some embodiments, the CFI variant comprises or consists of any one or more of the combination modifications selected from P558s+f559l+i560v+y563H, A405s+g406r+y408l+q409D, A s+g406a+y408l+q409D, G406a+y408L, and W381a+Δp384, wherein the position corresponds to a position in the CFI having the amino acid sequence set forth in SEQ ID No. 5.

N-linked glycosylation site variants

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

In some embodiments, the modification at the N-linked glycosylation site is removal of one or more N-linked glycosylation sites of the CFI.

Thus, provided herein are N-linked glycosylation site CFI variants. In particular, provided herein are exemplary CFI variants comprising or consisting of any one or more of the modifications presented in table 4. Table 4 presents exemplary CFI variants comprising one or more modifications to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are located at N-linked glycosylation sites of the CFI. The base molecule for the CFI variants presented in table 4 may be wild-type human CFI. It should be noted that not all N-linked glycosylation site variants of the invention are provided in table 4, and additional variants may be provided in at least examples and/or table 13.

Table 4: exemplary N-linked glycosylation site CFI variants

Without being bound by any theory or mechanism, exemplary CFI variants comprising modification of the N-linked glycosylation site may include the following variants.

In some embodiments, the CFI variant comprises any one or more of the modifications presented in table 4.

In some embodiments, the CFI variant comprises or consists of a modification at any one or more of positions N52, N85, N159, N446, N476 and N518 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of any one or more of the substitutions selected from N52Q, N85Q, N159Q, N446Q, N476Q and N518Q, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of any one or more of a combination of substitutions selected from n52q+n85q+n159Q, N446q+n476q+n518Q and n52q+n85q+n159q+n446q+n476q+n518Q, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO 5.

Serine protease domain variants

Provided herein are CFI variants comprising or consisting of at least one CFI domain, wherein the at least one CFI domain is a Serine Protease Domain (SPD) of CFI, and wherein the CFI variants comprise one or more modifications at the SPD.

In the crystal structure of free CFI, cleavage of the activation loop does not lead to insertion of the newly formed N-terminus (Ile 322), which is the next step in classical activation of serine proteases. Instead, the crystal structure indicates that the C-terminal region of the cleaved activation ring remains in a tightly curved ring structure on the surface of the CFI, as the same region that the uncleaved activation ring would remain. This prevents insertion into the activation pocket and thus prevents active site maturation (referred to as classical serine protease activation via induced conformational rearrangement). After hydrolytic activation of the SPD protein of CFI, the new N-terminus of the activation loop is typically released and inserted into an activation bag, such that the cleaved activation loop promotes complete activation of CFI in solution. Thus, a mutation in the C-terminal region of the activation loop should not affect cleavage by furin, as this region exceeds the 3' position relative to the scissile bond.

Thus, SPD CFI variants are provided herein. In some embodiments, a CFI variant comprising one or more modifications within a region of an SPD comprising a CFI (fig. 1) may comprise one or more modifications located at any one or more of: an activation loop (residues 322-326 of SEQ ID NO: 5), a 37-loop (residues 342-344 of SEQ ID NO: 5), a 60-loop (residues 366-372 of SEQ ID NO: 5), a 70-loop (residues 377-389 of SEQ ID NO: 5), a 99-loop (residues 403-410 of SEQ ID NO: 5), a 110-loop (residues 418-426 of SEQ ID NO: 5), a 150-autolysis loop (residues 455-463 of SEQ ID NO: 5), a 180-loop oxyanion stabilization (residues 494-509 of SEQ ID NO: 5), and/or a 220-loop S1 entrance frame (residues 529-536 of SEQ ID NO: 5). In particular, provided herein are CFI variants comprising SPDs of CFI, and wherein the CFI variants comprise any one or more of the modifications presented in table 5. Table 5 presents exemplary CFI variants comprising one or more modifications to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are SPDs. The base molecule for the CFI variant presented in table 5 may be wild-type, human CFI, or CFI-SPD, wherein the SPD corresponds to the CFI of the wild-type CFI (also known as delta (K1-P305) or a chain deleted), or a fused CFI using another complement regulator, such as factor H (FH-CFI) or CR1 (CR 1-CFI) (discussed in further detail herein when referring to fusion constructs).

It should be noted that not all SPD CFI variants of the present invention are provided in table 5, and additional variants may be provided in at least embodiments and/or table 13.

Table 5: exemplary serine protease domain CFI variants

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In some embodiments, the CFI variant comprises any one or more of the modifications presented in table 5.

In some embodiments, the CFI variant comprises an autolytic loop substitution. The autolytic loop of serine proteases is part of the activation domain and is involved in substrate specificity. Trypsin has a longer autolytic loop than CFI and there are several unique key residues between the autolytic loop of trypsin and CFI. Differences from different species, such as between mice and humans, can also occur between autolytic loops. The mouse CFI autolytic loop may include a number of differences compared to the CFI autolytic loop of the human CFI. Exemplary CFI variants may include CFI variants wherein the autolytic loop of human CFI exchanges with the autolytic loop of human trypsin or with the autolytic loop of mouse CFI. Such autolytic loop variants may help identify key residues involved in C3b and/or C4b cleavage activity. Thus, in some embodiments, provided herein are CFI variants, wherein the CFI variants are chimeras comprising one or more domains from a human CFI, and wherein the human CFI further comprises a substitution of one or more amino acid residues from an amino acid residue of a corresponding region of a non-human species CFI. In some embodiments, the non-human species CFI is a mouse CFI. Also provided herein are CFI variants, wherein the CFI variants are chimeras, and wherein the modification comprises substitution of one or more amino acid residues of CFI with amino acid residues from a corresponding region of a non-CFI serine protease. In some embodiments, the non-CFI serine protease is trypsin.

Exemplary autolytic loop CFI variants include trypsin autolytic loop substitutions comprising an autolytic loop of CFI (REKDNERVFS, SEQ ID NO: 9) substituted trypsin (NTASSGADYPDE, SEQ ID NO: 10), wherein the autolytic loop is located between positions corresponding to positions 456 and 465 in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

Another exemplary autolytic loop CFI variant includes a mouse CFI autolytic loop switch, wherein ⁴⁵⁶ REKDNERVFS ⁴⁶⁵ (SEQ ID NO: 9) to RGKDNQKVYS (SEQ ID NO: 11), wherein the autolytic loop is located between positions corresponding to position 456 and position 465 in the CFI having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the CFI variant comprises one or more modifications at any one or more of the following: an activation loop (residues 322-326 of SEQ ID NO: 5), a 37-loop (residues 342-344 of SEQ ID NO: 5), a 60-loop (residues 366-372 of SEQ ID NO: 5), a 70-loop (residues 377-389 of SEQ ID NO: 5), a 99-loop (residues 403-410 of SEQ ID NO: 5), a 110-loop (residues 418-426 of SEQ ID NO: 5), a 150-autolysis loop (residues 455-463 of SEQ ID NO: 5), a 180-loop oxyanion stabilization (residues 494-509 of SEQ ID NO: 5), and/or a 22-loop S1 entrance frame (residues 529-536 of SEQ ID NO: 5).

In some embodiments, the CFI variant comprises or consists of a modification at any one or more of positions K14, K312, R314, I322, V323, K326, R327, a328, K340, D341, G344, I345, T346, a361, L364, Y372, W381, P384, V390, N402, N404, G406, Y408, Q409, E416, K418, N422, D425, E457, K458, R456, E461, R462, F464, S465, Q467, W468, G469, T495, Y496, D497, S499, I500, a502, K504, D506, S507, E530, N531, G533, K534, P535, E536, and F537 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of any one or more of the substitutions selected from the group consisting of: k14,312,314,322,322,323,323,323,327,327,340,341,344,344,346,346,346,372,381,381,384,384,384,384,406,406,406,406,406,406,406,406,406,408,408,418,425, 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 495 497.5.90.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.506.506.506.530.530.531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 or 533.535 536.536K and F537R, wherein said position corresponds to a sequence having the amino acid sequence of SEQ ID NO:5, and the position in CFI of the amino acid sequence set forth in SEQ ID NO.

In some embodiments, the CFI variant comprises or consists of any one or more of the combination substitutions selected from: K326A+R327 G+P535 G+E461Q+R462K+F464 408L+N531 G+E457408 L+N531G+E457G+E461Q-R462K+F464 408L+N531G+P535 14 A+D5D+N531 G+G533A+K534Q+P535K+E536 502S+K504Q+F537 495F+Y496L+D497E+S499G+I500 A+K4Q+P535 K+E536 N+F5375F+Y496 L+D497E+S499 G+I500K+G534 A+K534Q+P535K+E536 N+F537+F5375G+N531+Q5530. D+F537 457G+E461 408L+N531G+E457G+E461 V+V323 V+V323 I+R326C+W468 328C+W468C+K326Y+R327 408L+N531G+E461 408L+N531G+E457G+E461Q+R462 408L+N531G+E457G+E461Q+F464 408L+N531G+E457G+R462K+F464 408L+N531G+E461Q+R462K+F464 408L+E457G+E461Q+R462K+F464 G+N531G+E461Q+R462 K+R462 K+F464 408L+E457G+E461Q+R462 531G+E457G+E461Q+F464 416A+D425 L+N531G+E457G+E461Q+R462K+F464Y+S507 457G+E461 312A+R314 469L+R456N+E457T+K458 469L+R456N+K458 469L+R456N+K458A+E461 469L+R456N+K458a+e461g+f537 406d+y408 d+n531 d+p535 d+y315406 d+y1200l+n531 d+y408l+p535 d+n531g+p535 d+y408l+n531g+p535 g+340 g+i345 g+y 370g+v393381 g+p384 a+v39393g+p384G. G+V390 404G+Q409 418G+D425 Rb4K+E530 K+K4D+E530 R+Y454K+Y454L+N531 344K+Y408L+N531 346 R+Y45408 L+N531 K+Y4512L+N531 504 D+Y4512E+Y4512L+N531 L+E12E+N1205R+N531+Y454K+K4R+N12L+N531+N12L+S5A+N531 G+E11111G+E1JE1JK+R1XY1K+F1Y+S5R5XY111TK+S5G+S507 G+S507A and N531G+P535A+S507A, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

Active site variants

Provided herein are CFI variants comprising or consisting of one or more modifications at the active site of CFI. In some embodiments, provided herein are CFI variants comprising at least one CFI domain, wherein the at least one CFI domain comprises a modification to the amino acid sequence set forth in SEQ ID No. 5, wherein the modification is located at the active site of the CFI. In some embodiments, active site CFI variants may improve the catalytic potential of CFI. In some embodiments, CFI active site variants can improve the catalytic potential of CFI by improving the active site (catalytic mechanism) without affecting the binding or binding specificity of C3b or C4b, which is governed by the exosite and a-chain interactions.

Thus, provided herein are active site CFI variants. In particular, provided herein are exemplary CFI variants comprising the modifications presented in table 6. Table 6 presents CFI variants comprising one or more modifications to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are located at the active site of the CFI. The base molecule for the CFI variants presented in table 6 may be wild-type human CFI.

Table 6: exemplary active site CFI variants

Modification from WT hCFI	Description of variants, modification purposes
		S507A	Active site (S195A)

In some embodiments, the CFI variant comprises or consists of the modifications presented in table 6.

In some embodiments, the CFI variant comprises or consists of a modification at a position corresponding to position S507 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of substitution S507A, wherein the position corresponds to position S507 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

Inverted CFI variants of the a-B chain

Provided herein are CFI variants, wherein CFI comprises an a-strand and a B-strand, and comprises an inversion of the a-strand and the B-strand. In some embodiments, the CFI variant without strand inversion (the individual strand optionally comprising one or more modifications) comprises a structural configuration from N-terminus to C-terminus or C-terminus to N-terminus as (a-strand) - (optionally linker) - (B-strand). In some embodiments, the CFI variants comprise inversions of the a-and B-strands (the individual strands optionally comprising one or more modifications) such that the N-terminal to C-terminal or C-terminal to N-terminal structure is configured as (B-strand) - (optionally linker) - (a-strand). The optional linker may have any suitable length, for example at least one amino acid in length. The linker may be a flexible linker and may be a peptide of about 1 to about 20 amino acid residues in length, wherein the amino acid residues may comprise glycine residues. The linker may also optionally comprise serine residues. 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 linker known in the art. An exemplary linker is GGSSGG ⁿ Wherein n is any number from about 1 to about 20. Exemplary linkers 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.

Thus, provided herein are CFI variants, wherein the CFI comprises an a-chain and a B-chain, and wherein the structure from N-terminus to C-terminus or C-terminus to N-terminus is configured as (B-chain) - (optionally linker) - (a-chain). Such fusion constructs are presented in table 7. Table 7 presents exemplary CFI variants comprising or consisting of one or more modifications relative to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are inversions of the A and B strands of CFI.

Table 7: exemplary CFI strand inversion variants

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Without being bound by theory or mechanism, an inverted exemplary CFI variant comprising an a-chain and a B-chain may comprise the amino acid sequence set forth in SEQ ID NOs 17, 18, 19 or 20. The chains may be joined together by optional linkers. The linker between the a and B chains of the inverted variant may have 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 10, 3-11 to about 20, or 1 to about 40 acid residues in length, wherein the amino acid residues may comprise glycine residues. The linker may also optionally comprise serine residues. 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 linker known in the art. It should be appreciated that although the exemplary inversion variants shown in table 7 include glycine polymer linkers, any suitable flexible linker may be used for CFI variants with a-B chain inversion.

In some embodiments, the CFI variant comprises a substitution at C309 and/or C435, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variants comprise substitutions C309S and C435S, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

Additional CFI variants-suitable for modulation and/or assessment of the complement system

In some embodiments, CFI variants are present, provided that when applicable to modulate the complement system, but may also be applicable to assess the activity of the complement system, e.g., in addition to having therapeutic value, they may be considered tool proteins.

For example, these other CFI variants may allow for various tests using CFI fusion constructs. Exemplary such CFI variants may not be activated to serve as controls. Another exemplary such CFI variant may provide for easier activation of the fusion construct.

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

In exemplary embodiments, the CFI variants comprise any one or more of the exemplary modifications presented in table 8. Such CFI variants may be suitable for providing controls or further studies for any of the CFI variants provided herein. Such CFI variants may also provide therapeutic utility.

Table 8: other exemplary CFI variants

Exemplary CFI variants may include non-activatable CFI variants, which may serve as controls.

In some embodiments, the CFI variants comprise any one or more of the modifications presented in table 8.

In some embodiments, the CFI variant comprises or consists of a modification at any one or more of positions I317, R318, R319, K320 and R321 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises or consists of any one or more of the substitutions selected from the group consisting of I317D, R318D, R319D, K D and R321K, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant is more susceptible to activation than the wild-type CFI. In some embodiments, the CFI variant is more susceptible to activation than a wild-type CFI and comprises or consists of the substitutions I317D, R318D, R319D, K D and R321K, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant is not activatable and comprises or consists of at least one modification relative to wild-type CFI. In some embodiments, the CFI variant is not activatable and comprises a modification at a position corresponding to position R321 in a CFI having the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the CFI variant comprises a substitution R321A, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

CFI combinatorial variants

Provided herein are CFI variants comprising or consisting of two or more modifications relative to wild-type CFI. Modifications occur in the same or different domains of the CFI. In some embodiments, the modification comprises two or more substitutions. In some embodiments, the modifications include substitutions and deletions. In some embodiments, modifications include substitutions and additions. In some embodiments, the modifications include deletions and additions. In some embodiments, modifications include substitutions, deletions, and additions. As used herein, these variants collectively may be referred to as CFI combination variants.

Thus, provided herein are CFI combination variants. In particular, provided herein are exemplary CFI variants comprising any one or more of the modifications presented in table 9. Table 9 presents CFI variants comprising two or more modifications to the amino acid sequence set forth in SEQ ID NO. 5. The base molecule for the CFI variants presented in table 9 may be wild-type human CFI or CFI-SPD. It is to be understood that any of the CFI variants provided herein may include any combination of any of the modifications provided herein, such as any of the modifications presented in tables 2-8 and 13.

Table 9: exemplary combinatorial CFI variants

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Without being bound by any theory or mechanism, exemplary combinatorial CFI variants may include the following variants.

In some embodiments, the CFI variant comprises or consists of any one or more of the modifications presented in table 9.

In some embodiments, the CFI variant comprises or consists of any one or more of the combination substitutions selected from the group consisting of: y408+n531 38a+d425 20f+d425 250a+d425 f+n531 408l+n531g+e457g+e461q+r462k+f464 14a+y20 14 a+e3814 a+s25014 a+d425 20 f+e330f+s20f+s250 f+s250 f+d425 20f+d425 a+d425 250a+d425 14a+n531g+p535 f+n531g+p535 a+n531g+p535 a+p535 a+n531g+p535 a+n5g+n5f+y408 l+n531 g+e12q+r12k+f464 38 a+y12l+n12g+e457 g+e461q+r462k+f464 250 a+y8k+n531 g+e462 g+e462 k+f464 r+m464 r+n5k+n5k+m12g+m12g+mK+m8k+m8k+m5k+g5k+m5g+m5g+m5g+m5g+n5g+n5g+n5g+n5k+n5g+n5g+n5g5g+ n5g5g+ n5g+ g+ k5g+ k5g+ g+ k5g+ k5g+ g+ g+ k5g+ g+ k+ k5k+ k5k+ k+ k+ k5+ k+ k+ 5+ 5+ k+ k+ 5+ k+ 5+ e5+ e5+ E5+ E5+ E5E5E5E5ER5ER5E5EEERE5EEEEEEEEEEEE+ EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE 250A+D425A+Y408L+N531G+E457G+E461Q+R462K+F464 20F+E38A+S250A+D425A+Y408L+N531G+E457G+E461 317 D+R315D+R315D+K405K+E457 G+E461Q-R462K+F464 317 D+R313D+K315D+R321 K+E457G+E461Q-R462K+F464 D+R313D+R313D+R405D+R404K+Y408L+N531 G+E405K+E404E+Y404E+Y404L+N531+Y111D+R405D+R405D+R405D+R405D+R405D+R3D+R405D+R405K+R3D+R405K+R11D+R5K+R5K+R8R5K+R5K+R5K+R5K+R5K+R5K+R5K+R4R5K+R5K+R5K R5K+R5K R5K+R5K R5K R5K R5and R5K R5K R5Kand R5K R5Kand R5Kand RKand RKand RKKand RKand RKand RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR wherein said position corresponds to SEQ ID NO:5, and the position in CFI of the amino acid sequence set forth in SEQ ID NO.

Substitution with minimal impact on activity and specificity

Some CFI variants exhibit little or no difference in protease activity or substrate specificity compared to wild-type CFI. In some cases, substitution even reduced activity compared to wild-type CFI. Some substitutions that exhibit little or no difference in protease activity or substrate specificity alone are provided in table 15 as single site substitutions. However, it is to be understood that the list of substitutions herein does not indicate that one or more of these substitutions used in combination with another substitution may exhibit different effects on CFI substrate specificity and CFI protease activity.

TABLE 15 substitution with little or no difference in activity or specificity

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B. Fusion constructs comprising complement factor I

Provided herein are fusion constructs comprising at least a first component (CFI portion) comprising at least one domain of complement factor I; and a second component, wherein the first and second components are fused (e.g., consecutively or separately via an optional linker). These fusion constructs are referred to herein as "CFI fusion constructs" or simply "fusion constructs". In some embodiments, the fusion construct comprises additional components, such as a third component, a fourth component, and the like.

In some embodiments, the first component comprises wild-type CFI of any species, full length, or a domain thereof. In some embodiments, the first component comprises a CFI variant of the invention, described in detail in the preceding section. It should be noted that the second component may increase activity or alter the specificity of the CFI moiety (first component) or its half-life. The second component may also allow the CFI moiety (first component) to function within the complement system in the absence of exogenous cofactors (e.g., cofactors such as Factor H (FH) or CR 1). As used herein, an exogenous cofactor for CFI is a cofactor that is not fused to CFI. It will be appreciated that the fusion construct may function within the complement system in the absence of FH and/or CR1, but that the activity of the fusion construct may also be further increased or exogenously provided as part of the fusion construct in the presence of FH and/or CR 1.

Provided herein are fusion constructs comprising a first component comprising any of the CFI variants provided herein. It is to be understood that the CFI variants may be any of the CFI variants presented in tables 2-9 or table 13, or may comprise any combination of the modifications presented in tables 2-9 or table 13.

In some embodiments, the second component of the fusion construct is a protein. In some embodiments, the second component is not a protein.

The components of the fusion constructs of the invention may be joined together by an optional linker. It may have 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, wherein the amino acid residues may comprise glycine residues. The linker may also optionally comprise serine residues. 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 linker known in the art. An exemplary linker is GGSSGGn (SEQ ID NO: 6), wherein n is any number from about 1 to about 20. In some embodiments, the linker is a protease-sensitive cleavable linker. Exemplary linkers connecting the 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.

CFI+ half-life extender fusion constructs

In some embodiments, the fusion construct comprises a wild-type CFI or CFI variant (first component) and a second component, and wherein the second component is a half-life extender. Because naturally occurring CFIs have a relatively short half-life, it may be advantageous in some embodiments to increase the half-life of CFIs. As used herein, "CFI" is used to encompass wild-type CFI or variants thereof. By using a second component that is a half-life extender, the activity of CFI can be increased, or CFI can improve another feature of CFI compared to wild-type CFI. For example, a wild-type CFI or CFI variant may have its half-life by fusing CFI with a half-life extender.

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

In some embodiments, the second component is a half-life extender and is albumin. It should be noted that albumin, as used herein, refers to any albumin, such as any serum albumin, or albumin variant, or albumin derivative. For example, variants of albumin include any albumin comprising at least one modification corresponding to the amino acid sequence set forth in SEQ ID NO. 7 (wild type Human Serum Albumin (HSA)) or comprising at least one modification corresponding to the amino acid sequence of albumin of any non-human species. In an exemplary embodiment, albumin is Human Serum Albumin (HSA) and is provided in SEQ ID NO. 7.

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

In some embodiments, the fusion constructs of the invention comprise albumin and the CFI variants of the invention.

Structural configuration of fusion constructs

In some embodiments, the wild-type CFI or CFI variant of the invention is the first component of a fusion construct, and wherein this CFI portion comprises an a-strand and a B-strand. In some embodiments, the fusion construct comprises a structural configuration of (a chain) - (optionally linker) - (B chain) - (optionally linker) - (second component) from N-terminus to C-terminus. In some embodiments, the fusion construct comprises inversion of the a and B chains in their CFI components such that the structure from the N-terminus to the C-terminus is configured as (B chain) - (optional linker) - (a chain) - (optional linker) - (second component).

In some embodiments, the wild-type CFI or CFI variant of the invention is the first component of a fusion construct, and wherein this CFI portion comprises an a-strand and a B-strand. In some embodiments, the fusion construct comprises a structural configuration in the form of (second component) - (optional linker) - (a-strand) - (optional linker) - (B-strand) from N-terminus to C-terminus. In some embodiments, the fusion construct comprises inversion of the a and B chains in their CFI components such that the structure from the N-terminus to the C-terminus is configured as (second component) - (optional linker) - (B chain) - (optional linker) - (a chain).

In some embodiments, provided herein are fusion constructs comprising at least a first component and a second component, wherein the first component is any one of the wild-type CFI or CFI variants provided herein (CFI portion), wherein the first component and the second component are fused, and wherein the second component is fused to the N-terminus of the CFI portion. In some embodiments, the second component is fused to the C-terminus of the CFI moiety. In some embodiments, the C-terminus of the CFI portion of the second component is fused, and the third component is further fused to the N-terminus of the CFI portion. In some embodiments, the second component is fused to the N-terminus of the CFI moiety, and the third component is further fused to the C-terminus of the CFI moiety.

Figures 2A-2D depict models of fusion constructs comprising albumin and CFI variants, wherein the CFI variants comprise a-B strand inversion. Fig. 2A-2B depict a first version and fig. 2C-2D depict a second version of a model of a fusion construct comprising a and B chains of Human Serum Albumin (HSA) and CFI, wherein the a and B chains comprise inversions. The first version of the A-B strand inverted CFI variant comprises an inter-domain disulfide bond. The second version does not contain inter-domain disulfide bonds. Two versions of the inverted variant can be constructed in a head-to-tail fashion: (HSA) - (optionally linker) - (B chain) - (optionally linker) - (a chain).

Thus, provided herein are CFI variants, wherein the CFI variant is a first component of a fusion construct comprising a first component and a second component, and the CFI variant is fused to the second component, and wherein the CFI comprises an a-chain and a B-chain, and wherein the structure from N-terminus to C-terminus or C-terminus to N-terminus is configured as (second component) - (optional linker) - (B-chain) - (optional linker) - (a-chain). Such a chain inversion is presented in table 7 above. Table 7 presents exemplary CFI variants comprising one or more modifications to the amino acid sequence set forth in SEQ ID NO. 5, wherein the one or more modifications are inversions of the A and B chains of CFI.

FIGS. 2A-2B depict models of exemplary CFI variants including albumin fusions, and inverted variants including modifications V311-V565-G (13) -K1-G310. In some embodiments, such fusion constructs comprising albumin and comprising chain inverted CFI comprise the amino acid sequence set forth in SEQ ID NO 17 or 18.

FIGS. 2C-2D depict models of exemplary CFI variants with albumin fusion, and inverted variants V311-V565-G (10) -K1-G310+C309S+C435S. In some embodiments, such fusion constructs comprising albumin and comprising chain inverted CFI comprise the amino acid sequence set forth in SEQ ID NO 19 or 20.

In some embodiments, the CFI variant comprises a substitution at C309 and/or C435, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variants comprise substitutions C309S and C435S, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5. 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 further detail below. In some embodiments, the second component is at least one domain of CR 1. Fusion constructs comprising at least one CFI domain and factor H are discussed in further detail below. In some implementations, the second component includes at least one domain of factor H and at least one domain of CR 1. Fusion constructs comprising at least one CFI domain, at least one factor H domain, and at least one CR1 domain are discussed in further detail below.

Components of fusion constructs

Provided herein are fusion constructs comprising a first component and a second component. In some embodiments, the first component comprises a wild-type CFI, while in some embodiments, the first component comprises a CFI variant of the invention. In some embodiments, the second 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 a third component. In some implementations, the first, second, and third (or more) components are any one or more of the components presented in table 10. Table 10 presents various exemplary components and amino acid sequences that can be used to generate the components of the CFI fusion constructs provided herein.

Referring to Table 10, SEQ ID NO:1 is the amino acid sequence of wild-type plasma derived human CFI, referred to as "CFI-PD", and has a leader sequence. The wild type CFI for fusion with the second component may comprise the amino acid sequence of SEQ ID NO. 5, excluding the leader sequence present in SEQ ID NO. 1. The mouse Ig kappa chain V-III region MOPC 63 leader sequence (SEQ ID NO: 2) may alternatively be used for 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.

Table 10: components of exemplary CFI fusion constructs

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C. Complement factor I and albumin fusion constructs

Wild-type CFI+albumin fusion constructs

In some embodiments, provided herein are fusion constructs comprising a first component (which is a wild-type CFI) and a second component (which is albumin, such as serum albumin, e.g., human serum albumin).

In some embodiments, the albumin is Human Serum Albumin (HSA), and CFI is wild-type CFI, and such fusion constructs are referred to herein as "CFI-HSA.

In some embodiments, CFI-HSA may have an extended half-life for CFI, but not a portion of the fusion construct. Exemplary CFI-HSA constructs can be produced by ligating albumin with wild-type CFI through a flexible linker. In some embodiments, CFI-HSA comprises the amino acid sequence set forth in SEQ ID NO. 21, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence.

Fig. 3 can depict a model of an exemplary CFI-HSA fusion construct comprising HSA fused to CFI, wherein CFI comprises wild-type CFI.

In some embodiments, the fusion construct comprises a structural configuration in the form of (albumin) - (optional linker) - (WT CFI a chain) - (optional linker) - (WT CFI B chain) from N-terminus to C-terminus.

In some embodiments, the fusion construct comprises a structural configuration in the form of (WT CFI a chain) - (optionally linker) - (WT CFI B chain) - (optionally linker) - (albumin) from N-terminus to C-terminus.

In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configuration from N-terminus to C-terminus (SEQ ID NO. 7) - (optionally linker) - (SEQ ID NO. 5). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configurations from N-terminus to C-terminus (SEQ ID NO. 7) - (linker) - (SEQ ID NO. 5). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, wherein the fusion construct comprises the structural configurations from N-terminus to C-terminus (SEQ ID NO. 7) - (SEQ ID NO. 6) - (SEQ ID NO. 5). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configuration from N-terminus to C-terminus (SEQ ID NO. 5) - (optionally linker) - (SEQ ID NO. 7). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7, wherein the fusion construct comprises the structural configurations from N-terminus to C-terminus (SEQ ID NO. 5) - (linker) - (SEQ ID NO. 7). In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, wherein the fusion construct comprises a structural configuration from N-terminus to C-terminus (SEQ ID NO. 5) - (SEQ ID NO. 6) - (SEQ ID NO. 7).

In some embodiments, the fusion construct comprises the amino acid sequence set forth in SEQ ID NO. 21 or an amino acid sequence having at least 80% identity thereto. In some embodiments, the fusion construct consists of the amino acid sequence set forth in SEQ ID NO. 21. In some embodiments, the fusion construct comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7. In some embodiments, it should be noted that albumin fusion (e.g., N-terminal albumin fusion) to wild-type CFI provides solubility and promotes CFI-HSA activation. When CFI activation to mature double-stranded protein was performed posttranslationally in the absence of albumin (WT-CFI) and compared between CFI-HSA and wild-type CFI without albumin (WT-CFI), furin was observed to significantly preferentially activate CFI-HSA and almost fully activate CFI-HSA. It was observed that the CFI-HSA protein remained as monomer and no sign of aggregation. Amino-terminal HSA fusion presents significant and unexpected benefits for maintaining solubility, monodispersity, and efficient furin activation of the CFI-HSA construct. For example, bioavailability is significantly improved via improved half-life.

Accordingly, provided herein are methods of increasing activation of CFI comprising fusing HSA to wild-type CFI, wherein the fusion is an N-terminal fusion prior to activation by furin; activated by furin. In some embodiments, activation by furin is performed in cells during recombinant production of the CFI variants or CFI fusion constructs of the invention. In some embodiments, activation by furin is performed in vitro.

CFI variant+albumin fusion constructs

In some embodiments, provided herein are fusion constructs comprising a first component (which is a CFI variant of the invention) and a second component (which is albumin, e.g., serum albumin, e.g., human serum albumin).

In some embodiments, provided herein is a fusion construct comprising at least one CFI domain and a second component, wherein the second component is HSA, and wherein the at least one CFI domain comprises any one or more domains of a CFI selected from the group consisting of: SPD, FIMAC domain, SRCR domain, LDLr1, and LDLr2 domain. In some embodiments, any one or more domains of CFI comprise the 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 the CFI correspond to domains of a wild-type CFI. In some implementations, the at least one CFI domain includes each of an SPD, FIMAC domain, SRCR domain, and LDLr1 and LDLr2 domains. In some embodiments, at least one CFI domain of the CFI-HSA construct comprises only SPD.

Fig. 3 may depict a model of an exemplary fusion construct comprising HSA fused to a CFI, wherein the CFI comprises a CFI variant comprising each of an SPD, FIMAC domain, SRCR domain, and LDLr1 and LDLr2 domains. Thus, in this model, both the a-chain and the B-chain are included in the CFI. Together, the FIMAC domain, SRCR domain, and LDLr1 and LDLr2 domains are the a-chain or heavy chain, while the SPD is the B-chain or light chain. In some embodiments, the amino acid residues of any one or more domains of the fusion construct may correspond to amino acid residues 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 the domain of the wild-type CFI.

Fig. 4 depicts a model of an exemplary fusion construct comprising HSA fused to a CFI portion, wherein the CFI comprises only Serine Protease Domain (SPD). The exemplary fusion construct depicted in FIG. 4 may be referred to as "HSA-SPD" and includes an activation loop at amino acid residues 322-326, an autolytic loop at amino acid residues 455-463, and an S1 entry frame at amino acid residues 529-536. In some embodiments, the amino acid residues of any one or more of the activation loop, the autolytic loop, and the S1 entry framework of the fusion construct may correspond to the amino acid residues of the wild-type SPD of the CFI. In some embodiments, the amino acid residues of any one or more of the activation loop, the autolytic loop, and the S1 entry framework of the fusion construct may comprise one or more modifications relative to the wild-type SPD of the CFI.

D. Complement factor I and factor H fusion constructs

In some embodiments, provided herein are fusion constructs comprising a wild-type CFI (or variant thereof) fused to at least one domain of factor H. Factor H (FH), such as CFI, is a protein involved in the complement pathway. FH is a cofactor for CFI, which forms a complex with CFI and C3b to catalyze C3b cleavage by CFI. As noted above, the full-length FH includes 20 domains. Fig. 5A depicts a schematic diagram of FH showing its 20 domains, each of which is a Complement Control Protein (CCP) domain, and each of which is connected by a short linker in a head-to-tail configuration. The CCP fields begin numbered 1-20 at the N-terminus. CCP 1-4 is complexed with C3b, and CCP 19-20 is complexed with C3 d. Without being bound by any theory or mechanism, FH is believed to be important for efficient C3b cleavage by CFI. Thus, in some embodiments, fusion constructs comprising a specific domain of FH fused to at least one CFI domain may allow C3b to lyse independently of exogenous FH. Exogenous FH may be defined as any FH that does not fuse with any CFI domain, and may be wild-type FH. As used herein, wild-type FH refers to any naturally occurring FH that is not the FH that causes the disease. In some embodiments, FH is human FH. In some embodiments, wild type FH contains the SEQ ID NO. 4 set forth in the amino acid sequence.

In some embodiments, the invention of the fusion construct second component is at least one factor H domain or FH domain part. In some embodiments, at least one FH domain includes CCP domains 1-20 of FH. In some embodiments, FH at least one domain corresponding to the amino acid sequence set forth in SEQ ID NO. 4 wild type FH domain.

In some embodiments, provided herein are fusion constructs comprising at least one CFI domain and a second component, wherein the second component is at least one factor H domain, and wherein the at least one factor H domain comprises Complement Control Protein (CCP) domains 1-4 and 19-20 of factor H. CCP domains 1-4 and 19-20 are referred to as "micro factor H" (micro FH). FIG. 5B depicts a schematic diagram of a miniature FH showing CCP domains 1-4 connected to domains 19-20 by Gly linkers, the domains including His tags. In some embodiments, miniature FH is human miniature FH. In some embodiments, miniature FH amino acid sequence containing SEQ ID NO. 8 set forth in the amino acid sequence.

Based on the structure of the complex formed by C3b-CFI and miniature FH, several domains associated with the function of FH were identified. An exemplary FH-CFI fusion construct of the following type was generated as a base molecule to drive FH-dependent CFI lytic activity:

(a) FH domain 1-8 fused with CFI (factor H-CPP 1-8+CFI)

(b) FH domains 1-4, 19-20 and 5-8 fused to CFI (factor H-CPP 1-4+19-20+5-8+CFI)

(c) FH domain 1-8 fused only to LDLr2 CFI domain (factor H-CPP 1-8+LDLR2-CFI)

(d) FH domains 1-4, 19-20 and 5-8 fused only to LDLr2 domain (factor H-CPP)

1-4+19-20+5-8+LDLr2-CFI)

(e) FH domains 1-4 fused to Serine Protease Domain (SPD) of Human Serum Albumin (HSA) and CFI

(CFI-HSA (SPD) -factor H-CCP 1-4)

(f) FH domains 2-4 fused to Serine Protease Domain (SPD) of Human Serum Albumin (HSA) and CFI

(CFI-HSA (SPD) -factor H-CCP 2-4)

(g) FH domain 2-3 fused to the Serine Protease Domain (SPD) of Human Serum Albumin (HSA) and CFI (CFI-HSA (SPD) -factor H-CCP 2-3).

Fig. 6 depicts a model of an exemplary fusion construct comprising FH and CFI, comprising CCP domains 1-8 of FH fused to CFI, wherein the FH portion of the fusion construct is truncated mini-FH, and wherein CFI comprises wild-type CFI. The wild-type CFI includes each of SPD, FIMAC domain, SRCR domain, LDLr1, and LDLr2 domains. The exemplary fusion construct shown in FIG. 6 is also referred to herein as factor H-CPPs1-8+CFI. Both the a-chain and the B-chain are included in the CFI. Together, the FIMAC domain, SRCR domain, and LDLr1 and LDLr2 domains are the a-chain or heavy chain, while the SPD is the B-chain or light chain. FH contains domains 1-4 and contains the domain 5-8 of the joint. In some embodiments, fusion constructs of FH and/or CFI any one or more domain of the amino acid residues corresponding to wild-type FH or wild-type CFI amino acid residues. In some embodiments, fusion constructs of FH and/or CFI any one or more domain of amino acid residues can include relative to the wild type FH or wild type CFI domain of one or more modification.

Table 11a lists exemplary fusion construct base molecules containing factor H.

Table 11a: construct base molecules containing factor H fusions

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In some embodiments, the CFI variant is a first component of a fusion construct comprising a first component and a second component, and the CFI variant is fused to the second component, wherein the second component is at least one factor H domain, wherein the FH domain comprises CCPs 1-4 of FH. In some embodiments, the CFI variant comprises modifications at any one or more of positions Y408, N531, E457, E461, R462, and F464 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant is a first component of a fusion construct comprising a first component and a second component, and the CFI variant is fused to the second component, wherein the second component is at least one factor H domain, wherein the FH domain comprises CCPs 1-4 of FH. In some embodiments, the CFI variant comprises a modification at any one or more of positions Y408, E457, E461, R462, F464, S507, N531, P535 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises any one or more of the substitutions selected from Y408L, E457G, E461Q, R462K, F464Y, S507A, N531G and P535A, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

In some embodiments, the CFI variant comprises any one or more of the combinations of substitutions selected from y408l+n531G, Y l+n531g+e457G, Y l+n531g+e457g+e461q+r462k+f464Y, Y408l+s507a+n531G, Y408l+n531g+e457g+e461q+r462k+f464y+s507A, E457g+s507A and n531g+p535a+s507A, wherein the position corresponds to a position in the CFI having the amino acid sequence set forth in SEQ ID NO 5.

E. Complement factor I and complement receptor 1 fusion constructs

In some embodiments, provided herein are fusion constructs comprising a wild-type CFI (or variant thereof) fused to at least one domain of complement receptor 1 (CR 1). CR1 is also known as CD35.CR1, such as CFI, is a protein involved in the complement pathway. CR1 is a cofactor for CFI. Thus, in some embodiments, fusion constructs comprising a specific domain of CR1 fused to at least one CFI domain may allow C3b and/or C4b to be independent of exogenous cofactors. Exogenous CR1 cofactor can be defined as any CR1 or a portion thereof that does not fuse with any CFI domain, and can be wild-type CR1, or can 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 pathogenic CR1. In some embodiments, CR1 is human CR1.

In some embodiments, the second component of the fusion construct of the invention is at least one CR1 domain or a portion of a CR1 domain. In some embodiments, at least one CR1 domain comprises CCP domains 15-17 of CR 1. In some embodiments, at least one CR1 domain comprises CCP domains 1-3 of CR 1. In some embodiments, the fusion constructs of the invention comprising at least one CR1 domain further comprise a fusion with albumin. In some embodiments, the fusion constructs of the invention comprising at least one CR1 domain further comprise a fusion with albumin, and/or at least one factor H domain. In some embodiments, at least one CR1 domain comprises a CR1 CCP domain 15. In some embodiments, at least one CR1 domain comprises a CR1 CCP domain 16. In some embodiments, at least one CR1 domain comprises a 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. In some embodiments, an exemplary fusion construct comprises a CFI with modified N531G fused to CCP domains 15-17 of CR 1. In some embodiments, an exemplary fusion construct comprises a CFI with modified N531G fused to CCP domains 15-17 of CR1, and further fused to albumin.

Table 11b lists the corresponding sequences of exemplary CR 1-containing fusion constructs and exemplary fusion constructs comprising wild-type CFI and CR1 CCP domains 15-17.

Table 11b: constructs containing complement factor 1 fusions

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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, and a third component. These exemplary fusion constructs may comprise a combination of components fused together and each comprise at least one CFI domain. As noted above, some exemplary fusion constructs comprising a first component, a second component, and a third component comprising CFI may comprise a fusion construct comprising albumin, at least one CFI domain, and at least one domain of Factor H (FH).

Fig. 7 depicts a schematic illustration of three exemplary fusion constructs comprising HSA, at least one CFI domain, and different domains of factor H. Each of the exemplary fusion constructs shown may include: a CFI-HSA portion comprising a leader sequence HSA; wild-type CFI as described previously herein; and the variation domain of FH (referred to as the "CCP-part" in FIG. 7). As noted above, the CFI-HSA portion may be constructed with GGSSGG (SEQ ID NO: 6) linkers fusing the HSA and SPD of the CFI together. An exemplary fusion, referred to herein as a CFI-HSA-FH_CCP1-4 fusion construct, includes the CCP domains of wild-type CFI and FH 1-4. CFI-HSA-FH_CCP1-4 also contains GGSSGG (SEQ ID NO: 6) linker, which is a combination of GGSSGG-linker (SEQ ID NO: 6) and Gly-only linker of CCP4 domain and CCP19 domain linked together in micro-factor H. Other exemplary fusion constructs shown include CCP domain 2-4 of FH and CCP domain 2-3 of FH. The linker lengths used in the exemplary fusion constructs are shown, with the conservative minimum lengths shown in parentheses. It should be appreciated that any other suitable flexible joint may be used.

Other exemplary fusion constructs provided herein include a wild-type CFI or CFI variant, at least one FH domain, and at least one CRI domain. In some embodiments, the fusion construct comprises a wild-type CFI or CFI variant, at least one FH domain, and at least one CRI domain. In some embodiments, the fusion construct comprises human serum albumin, wild type CFI or CFI variant and at least one FH domain and at least one CRI domain. A fusion construct comprising at least one FH domain and at least one CR1 domain may comprise an orientation comprising 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 comprises wild-type CFI or a CFI variant hCR1; CCP15; CCP16; CCP17 and hFH; CCP1; CCP2; CCP3; CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI variant and hCR1; CCP15; hFH; CCP2; CCP3; CCP4. In some embodiments, the fusion construct comprises a wild-type CFI or CFI variant and hFH; CCP1; hCR1; CCP16; hFH; CCP3; CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI variant and hCR1; CCP15; CCP16; hFH; CCP3; CCP4. In some embodiments, the fusion construct comprises a wild-type CFI or CFI variant and hFH; CCP1; hCR1; CCP16; CCP17; hFH; CCP4. In some embodiments, the fusion construct comprises wild-type CFI or a CFI variant and hCR1; CCP15; CCP16; CCP17; hFH; CCP4. It is to be understood that any fusion construct may further comprise one or more linkers as described herein. In some embodiments, the fusion construct comprises a wild-type CFI or CFI variant, at least one FH domain, at least one CRI domain, and a linker region. It will be appreciated that any fusion construct may further comprise human serum albumin. In some embodiments, the fusion construct comprises human serum albumin, wild type CFI or CFI variant, at least one FH domain, and at least one CRI domain.

In some embodiments, this paper provides a first component, a second component and a third component fusion construct, the first component comprising at least one CFI domain, wherein the second component is FH at least one domain, and the third component is any 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).

Generation of CFI variants and CFI fusion constructs

Provided herein are methods and compositions for generating CFI variants and CFI fusion constructs. Nucleic acids and vectors encoding either the CFI variants or fusion constructs of the invention are thus provided. Also provided are cells comprising one or more nucleic acids encoding CFI or variants thereof, and fusion constructs of the invention.

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

Provided herein are expression vectors encoding the CFI variants and fusion constructs described herein. Expression vectors may include transcriptional regulatory components, such as enhancers or promoters, operably linked to a nucleic acid sequence encoding a CFI variant or fusion construct of the invention.

Cell lines can be developed to express CFI and variants described herein and the generation of fusion constructs. The cell lines used to produce CFI, CFI may be implemented using any host cell capable of expressing the CFI variants and CFI fusion constructs described herein. The host cell may be a mammalian cell, an insect cell, a fungal cell, a plant cell, and/or a bacterial cell. Host cell lines can be transiently or stably transfected or transduced with expression vectors encoding CFI, CFI variants, and CFI fusions relative to expression of CFI variants and fusion constructs. The vector may be, for example, a plastid or viral vector. In some embodiments, the host cell strain is a mammalian cell strain. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell.

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

Provided herein are methods of producing CFI or variants thereof in an activated state; the method comprises producing CFI in a cell comprising one or more nucleic acids encoding CFI or variants thereof and an expression cassette for furin.

Provided herein are methods for producing and purifying CFI variants and fusion constructs described herein. The CFI variants and fusion constructs described herein can be purified from conditioned media by standard methods known in the art. In some embodiments, the CFI variants and fusion constructs can be purified by chromatography on an affinity matrix. In some embodiments, the affinity matrix is CaptureSelect ^TM Human albumin affinity matrix. In some embodiments, CFI variants and fusion constructs can be purified by chromatography on cation and/or anion exchange matrices and optionally size exclusion chromatography. The CFI variants and fusion constructs may be optimally buffer exchanged for any suitable buffer known in the art. Purity can be assessed by any method known in the art, including gel electrophoresis, orthogonal HPLC methods, staining, and spectrophotometric techniques.

Use of CFI variants and CFI fusion constructs

The CFI variants and fusion constructs of the invention are useful for modulating the complement system.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of modulating the classical and lectin complement pathways.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of modulating the alternative complement pathway.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of reducing amplification of the complement system.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing cleavage of C3 b.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing cleavage of C4 b.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing C4C production.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing the production of iC3 b.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing production of C3dg from iC3 b.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing production of C3C from iC3 b.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of reducing the content of C3b alpha chains.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing hydrolysis of a peptide substrate.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing proteolysis of a macromolecular protein substrate.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of reducing the content or function of an tapping complex (MAC).

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of reducing the observed hemolysis.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing cleavage of C3b in the absence of cofactors, e.g., in a cofactor independent manner.

As discussed herein, in some embodiments, the CFI variants or CFI fusion constructs of the invention are capable of increasing cleavage of C4b in the absence of cofactors, e.g., in a cofactor independent manner.

The CFI variants and fusion constructs of the invention are useful as therapeutic agents in individuals. As used herein, an individual includes any mammalian individual and includes primates, rodents, domestic animals, 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.

A. CFI variants and fusion constructs for modulating 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 of the CFI variants or fusion constructs provided herein. In some embodiments, the sample is plasma.

B. CFI variants and fusion constructs for the treatment of non-ocular disorders

In some embodiments, the CFI variants or fusion constructs provided herein are useful for treating a non-ocular disorder in an individual. In some embodiments, provided herein is a method of treating an ocular disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any one of the CFI variants or fusion constructs provided herein, or a pharmaceutical composition provided below.

In some embodiments, the non-ocular condition is characterized by insufficient CFI. In some embodiments, the non-ocular condition is characterized by a disorder of the complement system.

In some embodiments, the non-ocular condition is a systemic acute indication. In some embodiments, 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, burn injury, coronavirus infection, epstein barr virus infection, hematopoietic stem cell transplantation, ischemia reperfusion injury, lyme disease, myocardial infarction, organ transplantation, periodontitis, pneumonia, preeclampsia, schistosomiasis, septicemia, stroke, thromboembolism and traumatic brain injury.

In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is a systemic chronic indication selected from the group consisting of: alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, anti-phospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous Pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic Obstructive Pulmonary Disease (COPD), condenser-borne disease (CAD), crohn's disease, diabetic neuropathy, systemic myasthenia gravis (gMG), granulomatous associated polyangiitis (GPA), green-Barre syndrome (GBS), hereditary Angioedema (HAE), suppurative sweat gland (HS), igA nephropathy (IgAN), lupus Nephritis (LN), membranous glomerulonephritis (MN) Microscopic Polyangiitis (MPA), motor neuron disease, multifocal Motor Neuropathy (MMN), multiple Sclerosis (MS), non-insulin dependent diabetes mellitus, osteoarthritis, pancreatitis, parkinson's disease, paroxysmal Nocturnal Hemuria (PNH), post-transplant lymphoproliferative disorder, protein-lost bowel disease, psoriasis, pustule gangrene, rheumatoid arthritis, schizophrenia (SZ), systemic Lupus Erythematosus (SLE), immune Thrombocytopenia (ITP) and ulcerative colitis, lanbert-Eatone's muscle weakness syndrome (LEMS), CHAPLE's syndrome (CD 55 deficiency), thrombotic Microangiopathy (TMA) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), huntington's disease and ischemia reperfusion injury.

In some embodiments, the CFI variants or fusion constructs provided herein have improved characteristics compared to wild-type CFI. In some embodiments, the improvement is characterized by an increase in activity, wherein the increase in activity comprises an increase in cleavage of C3b and/or C4 b. The potency and specificity of the CFI variants provided herein may be adjusted 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 agent is useful for treating a disease. In some embodiments, the CFI variants provided herein are C4b degrading agents and are useful in the treatment of diseases. For example, diseases treatable by the use of C4b degrading agents include, but are not limited to, non-ocular disorders. In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is a systemic chronic indication selected from the group consisting of: alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), anti-neutrophil cytoplasmic antibody (ANCA) related vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous Pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic Obstructive Pulmonary Disease (COPD), condensed Collectinopathy (CAD), crohn's disease, diabetic neuropathy, systemic myasthenia gravis (gMG), granulomatosis with polyangiitis (GPA), green-Barlich syndrome (GBS), hereditary Angioedema (HAE), suppurative sweat gland (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, osteoarthritis, pancreatitis, parkinson's disease, paroxysmal Nocturnal Hemoglobinuria (PNH), post-transplantation lymphoproliferative disorder, protein-lost bowel disease, psoriasis, gangrene abscess, rheumatoid arthritis, schizophrenia (SZ), systemic Lupus Erythematosus (SLE), immune Thrombocytopenia (ITP), warm autoimmune hemolytic anemia (wAIHA), immune complex membranous proliferative glomerulonephritis (IC-MPGN), ulcerative colitis, lambert-eaton muscle weakness syndrome (LEMS), CHAPLE syndrome (CD 55 deficiency), thrombotic Microangiopathy (TMA) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), huntington's disease and ischemia reperfusion injury.

In some embodiments, the non-ocular condition is non-oncogenic.

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

C. CFI variants and fusion constructs for treating ocular disorders

In some embodiments, the CFI variants or fusion constructs provided herein are useful for treating an ocular disorder in an individual. In some embodiments, provided herein is a method of treating an ocular disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any one of the CFI variants or fusion constructs provided herein, or a pharmaceutical composition provided below.

In some embodiments, the ocular disorder is characterized by insufficient CFI. In some embodiments, the ocular disorder is characterized by a disorder of the complement system.

In some embodiments, the ocular disorder is characterized by the presence of a dysfunctional CFI gene. In some embodiments, the ocular disorder is characterized by a deregulation of the complement system and a low CFI content.

In some embodiments, the ocular disorder is selected from the group consisting of: diabetic Macular Edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, stark disease, uveitis, and vitreoretinopathy.

In some embodiments, wherein the ocular disorder is non-oncogenic.

D. Combination therapy

Administration of any of the therapeutic CFI variants or fusion constructs provided herein may be monotherapy, or may be combined with any other known drugs or treatments. Other known drugs or treatments may be used for conditions associated with deregulation of the complement system, or may be associated with insufficient CFI. In some embodiments, the disorder may be ocular. In some embodiments, the disorder 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 (eculizumab). In some embodiments, the C5 inhibitor is cemdeiiran.

E. Application of

The CFI variants and fusion constructs described herein may be delivered in the form of polypeptide-based therapies or nucleic acid-based therapies.

Such treatment as contemplated herein includes administration of both the CFI variants of the invention or the fusion constructs of the invention, as well as administration of one or more nucleic acids encoding the CFI variants of the invention or the fusion constructs of the invention. Accordingly, provided herein are pharmaceutical compositions comprising the CFI variants of the invention, the CFI fusion constructs of the invention, and pharmaceutical compositions comprising one or more nucleic acids encoding the CFI variants of the invention and encoding the fusion constructs of the invention.

Thus, provided herein are nucleic acids encoding the CFI variants and fusion constructs of the invention, and delivered to an individual in need thereof as part of nucleic acid-based gene therapy. In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the invention are delivered as part of viral vector-based gene therapy (e.g., lentiviral-based therapy, adenovirus-based therapy, adeno-associated virus-based therapy, and the like). In some embodiments, the nucleic acid encoding the CFI variants or fusion constructs of the invention is delivered in the form of a naked nucleic acid. In some embodiments, nucleic acids encoding the CFI variants or fusion constructs of the invention are delivered inside liposomes. In some embodiments, the nucleic acid encoding the CFI variants or fusion constructs of the invention is delivered as part of a nanoparticle. In some embodiments, the nucleic acid encoding the CFI variants or fusion constructs of the invention is delivered as part of a virus-like particle.

In some embodiments, the CFI variants and fusion constructs described herein may be delivered in the form of a polypeptide-based therapeutic.

In vivo administration of the therapeutic CFI variants or fusion constructs described herein (protein or nucleic acid based therapeutics) may be performed intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraventricular, intranasally, transmucosally, via implantation, or via inhalation. Administration of the therapeutic fusion construct may be performed using any suitable excipient, carrier, or other agent to provide suitable or improved tolerability, transfer, delivery, and the like.

In exemplary embodiments, the administration of the therapeutic CFI variants or fusion constructs described herein is subcutaneous administration. In some embodiments, the subcutaneous administration is daily, every other day, twice weekly, or once weekly.

In some embodiments, the administration of the therapeutic CFI variants or fusion constructs described herein is intravenous administration.

As generally contemplated herein, the CFI variants or fusion constructs described herein are delivered in an activated double stranded form. However, in some cases, inactive CFI variants or fusion constructs may be delivered in inactive single-stranded form. In some implementations, the delivered content includes both single-stranded inactive forms and double-stranded active forms.

F. Dosage of

In some embodiments, any of the therapeutic CFI variants or fusion constructs described herein may be administered to an individual in need thereof at a dose of about 0.1mg/kg to about 10 mg/kg. In some embodiments, the dose is about 1mg/kg. In some embodiments, the therapeutic CFI variants or fusion constructs described herein are administered subcutaneously at a dose of about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.5mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 8.5mg/kg, about 9mg/kg, about 9.5mg/kg, or about 10 mg/kg. In some embodiments, administering a therapeutic CFI variant or fusion construct described herein is intravenously administered at a dose of about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.5mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 8.5mg/kg, about 9mg/kg, about 9.5mg/kg, or about 10 mg/kg. In some embodiments, the administration of the therapeutic CFI variants or fusion constructs described herein is daily, every other day, once a week, or twice a week.

In some embodiments of the present invention, in some embodiments, the therapeutic fusion construct may be present in the plasma at a target level of about 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, about 5 μg/ml, about 5.5 μg/ml, about 6 μg/ml, about 6.5 μg/ml, about 7 μg/ml, about 7.5 μg/ml, about 8 μg/ml, about 8.5 μg/ml, about 9 μg/ml, about 9.5 μg/ml, about 10 μg/ml, about 10.5 μg/ml, about 11 μg/ml, about 11.5 μg/ml, about 12 μg/ml, about 12.5 μg/ml about 13. Mu.g/ml, about 13.5. Mu.g/ml, about 14. Mu.g/ml, about 14.5. Mu.g/ml, about 15. Mu.g/ml, about 15.5. Mu.g/ml, about 16. Mu.g/ml, about 16.5. Mu.g/ml, about 17. Mu.g/ml, about 17.5. Mu.g/ml, about 18. Mu.g/ml, about 18.5. Mu.g/ml, about 19. Mu.g/ml, about 19.5. Mu.g/ml, about 20. Mu.g/ml, about 20.5. Mu.g/ml, about 21. Mu.g/ml, about 21.5. Mu.g/ml, about 22. Mu.5. Mu.g/ml, about 22.5. Mu.g/ml, about 23. Mu.g/ml, about 23.5. Mu.g/ml, about 24.5. Mu.g/ml, about 25. Mu.g/ml, about 25.5. Mu.g/ml, about 26. Mu.g/ml, about 26.5. Mu.g/ml, about 27. Mu.g/ml, about 27.5. Mu.g/ml, about 28. Mu.g/ml, about 28.5. Mu.g/ml, about 29. Mu.g/ml, about 29.5. Mu.g/ml, about 30. Mu.g/ml. An exemplary fusion construct that can be administered to an individual in need thereof to achieve a target content of about 20 μg/ml can include CFI-HSA comprising a CFI corresponding to wild-type CFI.

G. Formulations

Pharmaceutical compositions containing the CFI variants or fusion constructs of the invention may be formulated in any conventional manner for use in the treatments provided herein by mixing a selected amount of the polypeptide with one or more physiologically acceptable carriers or excipients. The choice of carrier or excipient is within the skill of the application profession and can depend on a variety of parameters. These parameters include, for example, the mode of administration and the disorder being treated. The pharmaceutical compositions provided herein may be formulated for single dose (direct) administration or for dilution or other modification. The concentration of the compound in the formulation is effective to deliver an amount effective for the desired treatment when administered. Typically, the compositions are formulated for single dose administration, but are not required.

H. Pharmaceutical composition

The invention also provides pharmaceutical compositions comprising any of the CFI variants or fusion constructs disclosed herein, and optionally a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical composition may be formulated to be compatible with its intended route of administration. In some embodiments, the pharmaceutical compositions of the invention are suitable for administration to a human subject or other non-human primate. In exemplary embodiments, the pharmaceutical composition is formulated for subcutaneous administration.

I. Kits and articles of manufacture for therapeutic CFI variants and fusion constructs

The invention also provides kits or articles of manufacture comprising any of the CFI variants disclosed herein or the fusion constructs disclosed herein or any of the pharmaceutical compositions disclosed herein. In some embodiments, the kit may further comprise instructional materials for performing any of the methods disclosed herein. In some embodiments, the kit may further comprise a sterile container or vial for containing 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 constructs and/or pharmaceutical compositions disclosed herein. In some embodiments, the article of manufacture comprises any of the pharmaceutical compositions of the present invention.

Examples

Example 1: CFI-HSA expression, purification, activation and in vitro sialylation

SUMMARY

For example 1, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

The wild-type CFI-HSA protein was expressed in Chinese Hamster Ovary (CHO) cells, purified by anti-albumin affinity purification, furin activated and purified by a fractionation column. The activated CFI-HSA protein is subjected to in vitro sialylation to increase the total sialylation of CFI-HSA. Finally, sialylated proteins were purified using anti-albumin affinity purification and polished by size exclusion column chromatography.

Expression of

The CFI-HSA gene (SEQ ID NO: 21) (Simer's Feishi technology, geneart, ridge, germany (ThermoFisher Scientific, geneart, regensburg, germany)) was synthesized in which human serum albumin was located at the amino terminus of the CFI protein. A protein was prepared with the signal sequence of SEQ ID NO. 2, which was removed during expression. The amino-terminal albumin tag is linked to the CFI gene via a linker (SEQ ID NO: 6). The CFI-HSA gene was inserted into an expression vector (Lake Pharma, haiword, canada) using standard molecular biology techniques. The resulting plastid DNA was transformed into E.coli (E.coli). Transfected E.coli was grown in 200ml LB medium to express plastid DNA and collected using standard techniques. Plastid DNA was manipulated on agarose gel to facilitate quality assessment and sequence confirmation prior to transfection.

1.0 liter of suspension TunaCHO ^TM Cells were seeded in shake flasks and expanded using a chemically defined medium without serum. On the day of transfection, the expanded cells were inoculated into a new flask with fresh medium. Plasmid DNA was transiently transfected into CHO cells using lipofectamine 2000 (sameifer technology). The cells were maintained as fed-batch cultures until the end of the production run. Protein in the presence of 8% CO ₂ At a concentration of 125RMPAnd expressed for 14 days. The cells were centrifuged and the supernatant was collected for purification of the secreted CFI-HSA at the end of 14 days of expression.

Purification

Supernatant with CFI-HSA-expressing protein was passed through CaptureStelelect ^TM A 10ml gravity flow column of human albumin affinity matrix (sameimer technology). The column bound protein was washed with 10 column volumes of 20mM sodium phosphate buffer. The bound CFI-HSA protein is eluted in two steps:first, 3 column volumes of 20mM Tris-HCl, pH 7.0 buffer and 2M MgCl2 were used, and second, 3 column volumes of 20mM citric acid, pH 3.0 were used. The eluate from both steps 1 and 2 was collected in 5ml fractions. Each fraction of the eluate from step 2 was neutralized with 10% neutralization buffer (1.5M tris-HCl pH 7.4). All fractions were analyzed by reducing and non-reducing SDS-PAGE electrophoresis and purified by Simply blue ^TM SafeStain (Semer Fidelity technology) observes bands. CFI-HSA was run on a non-reducing gel with a 130kDa band and on a reducing gel with 102kDa and 28kDa bands. The fractions with maximum CFI-HSA concentration and purity were pooled for further processing.

Furin activation

CFI-HSA is expressed as an inactive single-chain precursor protein and is activated by furin another serine protease. Furin is an endoprotease that cleaves CFI at its conserved RRKR sequence (also known as the furin recognition sequence), producing heavy and light chains linked by disulfide bonds. The furin-treated, mature, double-stranded protein is an activated form of CFI protein.

CFI-HSA cleavage for production of proteins in activated form was performed by cleavage in Tris-NaCl (triple buffered saline), 2.5mM CaCl ₂ In 0.5% CHAPSIncubation of 4. Mu.g recombinant furin/mg purified CFI-HSA was performed for 18 hours. The CFI-HSA protein concentration was maintained at 1.4 mg/ml. This causes protein activation by more than 90%. The activated protein was separated from the unactivated CFI-HSA and other proteins by size exclusion chromatography. Using a HiLoad 16/600Superdex 200 column (GE healthcare Life sciences (GE Healthcare Life Science)) and phosphate buffered saline (PBS, 137mM NaCl,2.7mM KCl,10mM Na) ₂ HPO ₄ ，2mM KH ₂ PO ₄ pH 7.4) as mobile phase. The collected eluate was analyzed by CE-SDS (LabChip GXII, perkin Elmer). Fractions containing the target protein were pooled and analyzed by SE-UPLC.

In vitro sialylation

The activated CFI-HSA protein was subjected to in vitro sialylation. Briefly, sialylation proceeds in a two-step enzymatic reaction. First, 10mM UDP-galactose, 5mM MnCl was used ₂ And 100mM MES, pH 6.5 buffer in a 1:200 molar ratio of galactosyltransferase (GalT 1) enzyme to CFI-HSA, in a volume of 200. Mu.l. As previously described, by CaptureSelect ^TM Human albumin affinity chromatography galactosylated CFI-HSA was purified from the reaction mixture. Subsequently, 80. Mu.M alkaline phosphatase, 6.1mM CMP-NANA, 10mM ZnCl was used ₂ And 200mM MES buffer, 1:50 molar ratio of enzyme alpha 2, 6-sialyltransferase and purified CFI-HSA in pH 6.5 in 250. Mu.l volume, inThe sialylation reaction was carried out for 1 hour. By CaptureSelect ^TM Human albumin affinity chromatography the sialylated CFI-HSA protein was purified from the reaction mixture. The extent and character of sialic acid chains on CFI-HSA was determined by using Agilent/Prozyme analysis service, GS-SAP method for total sialic acid quantification (Agilent GS 48) and mass spectro-luminance (MS) analysis (Lake Pharma analysis service), described in further detail below.

Briefly, total sialic acid quantification was performed by mixing 20 μl of each sample with 10 μl of release reagent in a 96 well plate. The reaction mixture is reacted in the presence ofIncubate for 2 hours. Samples were cooled to room temperature and 10 μl of labeling reagent was added to each sample for use at +.>The culture was further carried out for 3 hours. The sample was again cooled to room temperature and 160 μl of deionized (dI) water was added to bring the total volume to 200 μl. 10 μl of sample was injected into Agilent UHPLC Poroshell C column at a flow rate of 0.4ml/min in 4% methanol, 8% acetonitrile in water (line A1) and 100% ACN (line B1 In->And (5) operating downwards. Peaks were recorded at 373/448nm wavelength. A standard curve of total peak area versus picomolar (pmol) of sialic acid was generated by supplying 1-2000pmol NANA (N-acetylneuraminic acid, neu5 Ac) with the kit on the same column. The total sialic acid of each sample was quantified by comparing the peak areas of the samples relative to a standard curve. The sialylation obtained is summarized in table 1.1 below.

Table 1.1: sialylation assay results

Proteins	Sialic acid (Neu 5 Ac) pmol/ug protein
		Recombinant CFI-HSA	35±0.7
Recombinant CFI-HSA in vitro sialylation	69±2.2
		Bovine fetuin control	222±2.6

Mass spectrometry was performed by a standard trypsin Q-TOF mass spectrometer. Briefly, all samples were treated, reduced and alkylated by DTT and iodoacetamide, followed by trypsin digestion. Digested samples were analyzed by Waters acid UPLC coupled to Xex G2-XS-QTOF mass spectrometer using a protein BEH C18 column. The analysis performed is summarized in table 1.2 below.

Table 1.2: peptide analysis results

Polishing (Polishing)

Purified CFI-HSA protein was Size Exclusion Chromatographed (SEC) using a HiLoad 16/600Superdex 200 column (GE healthcare life sciences) and phosphate buffered saline as mobile phase. The collected eluate was analyzed by CE-SDS (LabChip GXII, perkin Elmer). Fractions containing the target protein were pooled and brought to a concentration of 5mg/ml, and the samples were flash frozen for storage at-80 ℃.

Expression and purification of CFI-HSA variants

DNA for the CFI-HSA variants was generated synthetically or by site-directed mutagenesis using standard techniques. Protein expression in TunaCHO ^TM In 250ml suspension in cells, as described herein with reference to wild-type CFI-HSA protein, the difference is that expression is performed for 7 days instead of 14 days. After 7 days, the cells were centrifuged and the conditioned medium was passed through CaptureStlect ^TM Gravity flow column of human albumin affinity matrix (sameire feichi technology). The column bound protein was washed with 10 column volumes of 20mM sodium phosphate buffer. The bound CFI-HSA protein was buffered with 3 column volumes of 20mM Tris-HCl, pH 7.0 and 2M MgCl ₂ Is eluted in 5ml fractions. Buffer exchange of CFI-HSA or variants thereof (by dialysis or spin concentrator) to 30mM HEPES, 150mM NaCl, 2.5mM CaCl ₂ pH 7.4. Recombinant human furin was added to CFI-HSA at a molar ratio of 1:25 (furin: CFI-HSA) and inThe reaction mixture was incubated for 16 hours. Two micrograms of the activation mixture was run on a 9% sds-PAGE gel to evaluate activation efficiency. Generally, more than 80% activation is achieved.

N-terminal albumin fusion provides solubility and promotes CFI-HSA activation

Comparison of activation between CFI-HSA and wild-type CFI without albumin or other fusion tag (WT-CFI). WT-CFI gene construct The constructs were expressed substantially as described above for CFI-HSA. Recombinant WT-CFI proteins exhibited moderate purity by reduced SDS-PAGE, however, significant High Molecular Weight Species (HMWS) and aggregates under reducing and non-reducing conditions were observed (fig. 17). For CFI-HSA with N-terminal HSA tag added, tunaCHO was used as described above ^TM Transient expression of the cells followed by purification showed no HMWS or aggregates on reduced and non-reduced SDS-PAGE. As shown in fig. 17, activation of the purified recombinant CFI with furin produced further increases in aggregates and HMWS with almost complete polydispersity. In addition, substantially no activated CFI was observed by reducing SDS-PAGE. In contrast, CFI-HSA activated effectively by addition of furin was almost entirely under the same conditions, and the CFI-HSA protein remained monomeric under non-reducing conditions, with no sign of aggregates and HMWS (fig. 17). There is a significant and unexpected benefit to the N-terminal HSA tag to maintain solubility, monodispersity, and efficient furin activation when compared to CFI without any fusion tag.

Example 2: characterization of CFI-HSA variants by peptide lytic Activity analysis

For example 2, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

By testing the proteolytic activity of wild type CFI-HSA and CFI variant HSA fusion (collectively referred to herein as "CFI-HSA protein") by using chromophores after cleavage of the chromogenic substrate. The S-2288 (Chromogenix) peptide substrate was chosen for this analysis because it is sensitive to a broad range of serine proteases. The peptide-degrading activity of the CFI-HSA protein was measured by the rate of p-nitroaniline (pNA) production upon cleavage of the substrate, which was shown to occur at 405nm by spectrophotometry.

In an uncoated 96-well microplate (Nunc), the CFI-HSA protein was diluted to 400nM initial concentration in 100. Mu.l of HBS/BSA (30mM HEPES,140mM NaCl,0.2%BSA,pH 7.4). A working stock of 4mM S-2288 was prepared in HBS/BSA in separate tubes. Preheating the microplate and diluted chromogenic substrate to a temperatureAnd then maintained for 5 minutes. Analysis was initiated by adding 100. Mu.l of pre-warmed S-2288 to wells of a microplate containing CFI-HSA protein. Thus, a final concentration of 200nM CFI-HSA protein and a reaction volume of 200. Mu.l of 2mM S-2288 substrate were obtained. Using a microplate reader (Multiskan) ^TM GO micro-disc spectro-luminance meter, sameifeishier technology), in +.>Substrate cleavage rate was recorded every 30 seconds at 405nm for 3 hours. The percentage of the peptide lytic activity of the wild type CFI-HSA variant was calculated by normalizing the peptide lytic activity of the wild type CFI-HSA to 100%. The results are summarized in table 2.1 below.

Table 2.1: peptide decomposition analysis

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Example 3: CFI-HSA variant characterization for C3b cleavage analysis

For example 3, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

The C3b cleavage assay is a functional assay for determining the ability of wild-type CFI-HSA and CFI-HSA variants (collectively referred to herein as "CFI-HSA proteins") to cleave its native substrate C3 b. Briefly, CFI-HSA protein together with C3b and truncated factor H (miniature FH)The following incubation was performed to analyze C3b cleavage. Miniature FH has been shown to be functionally active and support CFI mediated C3b cleavage (J immunol.2013, 7 months 15; 191 (2): 912-21). The cleavage of C3b into smaller fragments was then monitored over time by SDS-PAGE.

First, for each CFI-HSA variant, it was set at room temperatureThe main reaction mixture containing miniature FH at a final concentration of 500nM and CFI-HSA protein at 5nM was set in HBS buffer (30mM HEPES,140mM NaCl,pH 7.4). Transferring the main reaction mixture toAnd allowed to equilibrate for 5 minutes. The cleavage reaction was started by adding C3b to a final concentration of 0.5 μm. 20 μl of samples from the master mix at each time point measured were extracted and quenched by addition of 5×SDS reduction sample buffer. Samples were run on 9% SDS-PAGE gels and C3b cleavage was visualized by Coomassie (Coomassie) staining. The amount of C3b cleavage that occurs was quantified by densitometry. The C3b cleavage activity of wild-type CFI-HSA was normalized to 100% to calculate the percent C3b cleavage activity of the CFI-HSA variant. The results of the C3b cleavage analysis are summarized in table 3.1 below.

Table 3.1: c3b cleavage assay

Figures 8A-8B are graphs depicting the relative percentages of human and mouse C3B cleavage when comparing various CFI variant fusion constructs to CFI wild-type fusion constructs. These results show that each variant tested has a higher percentage of C3b cleavage in both human and mouse compared to the fusion construct comprising wild-type CFI.

To compare the rate of C3b cleavage of each CFI-HSA variant with the rate of wild-type CFI-HSA variant, the time course of C3b cleavage by the CFI-HSA protein was performed simultaneously. The disappearance of the C3 (α)' band was observed as an indication of C3b cleavage. C3b includes both (α)' and β chains. When disappearance of the C3 (. Alpha.)' band of molecular weight 114kDa was observed, SDS-PAGE and densitometry of the relevant stained bands were performed correcting for average background staining (lane intensity outside the band).

The apparent rate of loss of band intensity is estimated by fitting a simple exponential decay to the band intensity data over time, thereby extracting the apparent rate constant (k) of C3b splitting. The relative rate of C3b cleavage by CFI-HSA variants was determined by using the corresponding WT rates: k (variant)/k (WT control). This step was performed for 3 independent SDS-PAGE experiments and the average of k (variant)/k (WT control) was calculated along with the accompanying standard deviation. These results are summarized in table 3.2 below.

Table 3.2: c3b cleavage assay

Fig. 9 is a graph depicting the activity of fusion constructs comprising CFI variants comprising substituted N531g+p535A fused to HSA compared to the activity of wild type CFI-HSA. The percentage of C3b alpha chains remaining after incubation over time was measured to assess the activity of the tested CFI variants compared to wild-type CFI. The tested CFI variants exhibited about 2-fold to about 3-fold increased activity compared to wild-type CFI. Since subtle differences in C3b cleavage can cause diseases such as atypical hemolytic uremic syndrome (aHUS), these results demonstrate that CFI-HSA variants can be useful for increasing the activity of the complement system against C3-induced diseases.

Example 4: quantitative analysis of CFI-HSA C3b cleavage Activity by time resolved immunofluorescence analysis (TRIFMA) by measuring C3dg formation

For example 4, reference to CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

C3dg analysis was used to determine C3b cleavage by Complement Factor I (CFI). The formation of C3dg was used as a quantitative analysis of CFI-HSA C3b cleavage activity and was measured by time resolved immunofluorescence analysis (TRIFMA). Briefly, the complement pathway in human serum is activated by the use of heat-aggregated IgG. The effect of plasma-derived CFI or CFI-HSA proteins (including CFI-HSA variants) on C3b cleavage was measured by capturing C3dg on a microtiter plate, using C3dg antibodies. Binding of C3dg was detected by a combination of biotin-labeled C3dg antibody and europium-labeled streptavidin, and measured by time-resolved fluorometry.

By incubation at room temperature overnight, 100. Mu.l of monoclonal IgM rat anti-human C3dg antibody was incubated at 2. Mu.g/ml in 15mM Na ₂ CO ₃ 、35mM NaHCO ₃ MaxiSorb microtiter plates (Nunc) were coated in pH 9.6 coating buffer. The remaining protein binding sites were blocked by incubation with HSA at 1mg/ml in TBS. Unbound HSA was washed with TBS-Tween.

Diluting the test samples with dilution buffer (0.14M NaCl, 10mM Tris, 14mM sodium azide, and 0.05% (v/v) Tween 20 (TBS/Tween), 1mg/ml HSA and 0.1mg/ml heat aggregated IgG in a volume of 100. Mu.l at 1 to 6 fold dilution of human serum to the desired concentration four-fold, six-point dilutions were performed for each CFI-HSA variant to cover variant concentrations in the range of 3132nM to 3nMIncubate for 90 minutes and quench with 10mM EDTA. To capture the generated C3dg, 100 μl of each reaction mixture was added to the antibody coated microtiter wells and at +.>The incubation was performed overnight. To detect bound C3dg, 100 μl of biotin-labeled rabbit anti-C3 Dg Antibody (DAKO) was added to the wells at 0.5 μg/ml and incubated for 2 hours at room temperature. After washing with eu3+ -streptavidin combination (perkin elmer), 25 μm EDTA was added to the wells and incubated for 1 hour at room temperature (1/1000). After washing, 200 μl of enhancement buffer (Ampliqon) was added to each well. Culture plates were read by time resolved fluorometry using DELFIA reader victori5+ (perkin elmer). The results are summarized in table 4.1 below.

Table 4.1: quantitative analysis of C3dg (TRIFMA)

FIG. 10 is a graph depicting half maximal Effective Concentration (EC) of fusion constructs comprising CFI variants compared to fusion constructs comprising wild-type CFI ₅₀ ) Is a graph of (2). The CFI variants tested were those comprising the substitution N531g+p535A fused to HSA. The TRIFMA analysis showed that the tested CFI variants exhibited about 18-fold improvement in activity over the wild type.

These results demonstrate that exemplary CFI-HSA variants have a higher percentage of C3b cleavage activity than wild-type CFI-HSA or plasma derived CFI.

Example 5: characterization of CFI-HSA variants by hemolysis analysis

For example 5, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Hemolysis assays are used to measure the hemolytic function of compounds using the complement pathway. Complement Factor I (CFI) uses its cofactor H (FH) to mediate C3b cleavage within the alternative pathway of the complement pathway. To test the hemolysis function of wild-type CFI-HSA and CFI-HSA variants (collectively referred to herein as "CFI-HSA proteins") in the alternative pathway, C3 deficient human serum plus human C3 was incubated with CFI-HSA and rabbit ark solution (rabbit Alsevers solution) and total hemolysis was measured spectrophotometrically. Hemolysis assays were performed on wild-type CFI-HSA and plasma-derived CFI (CFI-PD) in the presence or absence of FH to understand the effect of cofactor FH on total hemolysis.

Briefly, GVB buffer (gelatin Buddha buffer (Gelatin Veronal buffer)) was used to prepare sigma (sigma), 8mM EGTA and 10mM MgCl ₂ ) 12ml rabbit Red Blood Cells (RBC) were washed twice and resuspended in 12ml ice-cold GVB buffer. Based on previous observations, the addition of 1 μΜ human C3 to the C3 deficient human serum, 1 μmc3, supported the greatest hemolysis in this system. Three-fold eight-point serial dilutions of CFI-HSA were prepared in GVB buffer to achieve concentrations in the reaction mixture ranging from 260 μg/ml to 0.11 μg/ml. First, in a 96-well plate, by adding 62.8% human serum, different concentrations of CFI-HSA with or without 200g/mL FH preparation of each concentration point of 50 u l reaction mixture. Hemolysis was initiated by adding 50 μl rabbit RBC and was performed inIncubate in microtiter plates for 30 minutes. All assays were performed in triplicate and all dilutions were performed in GVB buffer. For the most hemolyzed control, deionized water was added to RBCs and 0.154M NaCl was added to RBCs for the non-hemolyzed control. After incubation, the culture dish was centrifuged at 2000rpm for 5 minutes and 90 μl of supernatant was transferred to another 96 well culture dish. Percent hemolysis was quantified by measuring the Optical Density (OD) of lysed RBCs at 412 nm.

The absorbance at 412nm was converted to percent hemolysis using the maximum hemolysis from the control as 100% and buffer control as 0%. The results of the hemolysis analysis are summarized in table 5.1 below.

Table 5.1: hemolysis analysis results

FIGS. 11A-11B depict dose response curves generated for CFI autolysis assays with and without cofactor factor H, respectively. Dose response curves were generated by nonlinear regression analysis and fitted to 4-parameter sigmoid curves in prism software. Table 5.2 below summarizes the results of the absorbance measurements in the assays, showing 50% of the alternative pathway activities of wild-type CFI-HSA and FH and plasma-derived CFI and FH (AP) ₅₀ )。

FIGS. 11C-11D depict dose response curves of percent hemolysis inhibition measured in classical and alternative pathways by plasma-derived CFI and CFI-HSA wild-type, respectively. These figures show that plasma-derived CFI and CFI-HSA wild-type proceed similarly in human serum.

Table 5.2: alternative pathway Activity

	AP 50 (nM)
		CFI-HSA+FH	990±82
CFI-PD+FH	723±84

These data show that at higher concentrations, CFI-HSA and CFI-PD are active in hemolysis assays. The inhibitory activity of CFI-HSA on the alternative pathway was similar to that of CFI-PD in the hemolysis assay. Hemolysis analysis also showed that the inhibition of the alternative pathway by CFI (both CFI-HSA and CFI-PD) increased significantly with cofactor FH.

The ability to inhibit classical pathway hemolysis by CFI variants was measured. Sheep erythrocytes are activated by anti-SRBC antibodies (mediator, testline, uk). SRBC was suspended in Gelatin Verona Buffer (GVB). In the assay tray, a dilution series of CFI variants was added followed by addition of SRBC activated at about 1% (v/v) and factor B and I depleted serum. The activated SRBC were incubated with the test object for 30 minutes. Cells were pelleted and the supernatant transferred to individual culture plates for aspiration brightness readings at 412 nM. Percent dissolution was calculated as follows: 100 ^* (suction brightness test sample)/(suction brightness no CFI (0% inhibition)). Data were plotted and analyzed using four parameter nonlinear regression (GraphPad software, usa). IC for calculating data from individual trays ₅₀ Value and log IC ₅₀ Values were averaged and converted to concentrations (nM) as summarized in table 5.3.

The ability to inhibit alternative pathway hemolysis by CFI variants was measured. Sheep erythrocytes are activated by anti-SRBC antibodies (mediator, testline, uk). SRBC are suspended in 8% (v/v) of normal human serum depleted of factors B and H, to which Exclusive is added to deposit C3B. Activated SRBC with deposited C3b with full-length H (complement technology, U.S.A.) The test objects are incubated together. After incubation for 10min, factors B and D (complement technology, usa) were added and incubated for an additional 10min. Finally, guinea pig serum (Sigma-Aldrich, uk) was added and incubated for 20 minutes. Cells were pelleted and the supernatant transferred to individual culture plates for aspiration brightness readings at 412 nM. Percent dissolution was calculated as follows: 100 ^* (suction brightness test sample)/(suction brightness no CFI (0% inhibition)). Data were plotted and analyzed using four parameter nonlinear regression (GraphPad software, usa). IC for calculating data from individual trays ₅₀ Value and log IC ₅₀ Values were averaged and converted to concentrations (nM) as summarized in table 5.3.

TABLE 5.3 IC for one set of variants in classical and alternative paths ₅₀ Value of

Example 6: determination of pharmacokinetic models for human administration based on non-human primate data

For example 6, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

After a single subcutaneous dose in african green monkeys, the plasma concentration levels of Complement Factor I (CFI) fusion construct and free wild-type CFI were tested. The fusion construct comprises Human Serum Albumin (HSA) and wild-type CFI (CFI-HSA). FIG. 12A is a graph depicting measured concentrations of CFI-HSA fusion constructs compared to free CFI after subcutaneous administration to monkeys at a dose of 1 mg/kg. The CFI-HSA fusion construct exhibited a target content of about 20. Mu.g/ml. The concentration of the measurable CFI-HSA fusion construct was maintained for up to 14 days, and the target concentrate of about 20 μg/ml was measured for about 7 days. These data support weekly subcutaneous administration of CFI-HSA fusion constructs for therapeutic use. Data using non-human primates was used to model human plasma concentrations and is shown in table 6.1 below. These data support that weekly subcutaneous administration can be used for therapeutic purposes in humans.

Fig. 12B depicts the chart shown in fig. 12A, with individual data points shown along the curve for additional clarity.

Table 6.1: model plasma concentrations of CFI-HSA fusion constructs in humans based on non-human primate data

Frequency of administration	Average value (μg/ml)	Peak value (μg/ml)	Trough (mu g/ml)
				Daily of	135	142	128
Every other day	68	75	61
				Twice a week	45	52	38
Once a week	19	27	13

Example 7: c3b and C4b cleavage analysis of CFI-HSA and CFI variant characteristics

C3b cleavage reaction

For example 7, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

First, for each CFI-HSA variant, a main reaction mixture containing a final concentration of 500nM of miniature FH and 500nM of C3b in HBS buffer (30mM HEPES,140mM NaCl,pH 7.4) was set at room temperature. Transferring the main reaction mixture toAnd allowed to equilibrate for 5 minutes. The cleavage reaction was started by adding CFI-HSA protein to a final concentration of 5 nM. Sample volumes corresponding to 0.6ug c3b were extracted from the master mix at each time point measured and quenched by addition of 5 x SDS reduction sample buffer. Samples were run on 9 or 10% SDS-PAGE gels and C3b cleavage was visualized by Coomassie staining. The amount of C3b cleavage that occurs was quantified by densitometry. The C3b cleavage activity of wild-type CFI-HSA was normalized to 100% to calculate the percent C3b cleavage activity of the CFI-HSA variant.

Another embodiment of the cleavage reaction is carried out as follows. In 1nM CFI (variant or wild type), 500nM cofactor miniature FH and 500nM soluble human C3b, inThe C3b cleavage reaction was performed by incubation in HEPES Buffered Saline (HBS) for 10 min. The reaction was quenched by addition of HBS containing 1M NaCl. The reaction was further diluted to a final concentration of 5nM C3b in buffer (HBS, 0.5M NaCl, 0.05% Tween 20) before performing the iC3b ELISA. The amount of C3b cleavage activity from the iC3b produced in the cleavage reaction. The amount of iC3b formed was analyzed using a MicroVue iC3b A006ELISA kit (Quidel). The ELISA assay consisted of a microplate coated with iC3 b-specific monoclonal antibodies for capturing the formed iC3b during the reaction, and bound iC3b was detected using HRP-conjugated anti-iC 3b antibodies and chromogenic substrate. The recorded absorbance was that produced in the cleavage reactionRelative measure of iC3b product. The fold difference in C3b cleavage activity of the CFI variant relative to the reference molecule CFI-HSA wild-type was calculated by dividing the background corrected absorbance of the CFI-HSA variant by the background corrected absorbance of the CFI-HSA wild-type. Table 7.1 summarizes these results, presenting a fold difference in median for 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 observed by Coomassie staining. The amount of C3b cleavage that occurs is quantified by densitometry and the plotted data and apparent rate constant (k) of the loss of band intensity determined by fitting exponential decay. The fold difference in C3b cleavage activity of the CFI variant relative to the reference molecule (CFI-HSA wild type) was calculated by dividing the k value from the CFI-HSA variant by the k value of the CFI-HSA wild type.

C3b cleavage by CFI variants was further characterized by determining EC50 for C3b cleavage. Briefly, C3b cleavage reactions were performed using 25nM miniature FH, 75nM soluble human C3b, and a series of diluted CFI variants. Allowing the reaction mixture of CFI variants at each concentration to react inThe incubation was performed in HBS for 5min. The reaction was quenched by addition of HBS containing 1M NaCl. The reaction was further diluted to a final concentration of 5nM C3b in buffer (HBS, 0.5M NaCl, 0.05% Tween 20) before performing the iC3b ELISA. The amount of iC3b produced in the reaction was determined using the MicroVue iC3b A006ELISA kit (Quidel). The ELISA assay consisted of a microplate coated with iC3 b-specific monoclonal antibodies for capturing the formed iC3b during the reaction, and bound iC3b was detected using HRP-conjugated anti-iC 3b antibodies and chromogenic substrate. The absorbance is recorded as a relative measure of the iC3b product produced in the cleavage reaction. EC50 values were calculated using a four-parameter nonlinear regression fit, without limitation in GraphPad Prism. Table 7.2 below summarizes the results of iC3b ELISA titration analysis. EC50 values above 500nM were set to 500nM. The cleavage reaction was also performed in the absence of micro-FH, with attention and analysis in the same way as that containing micro-FH.

C4b cleavage reaction

CFI modulates the classical complement pathway by proteolytic inactivation of C4b proteins. CR1, C3b/C4b receptor and C4 binding protein (C4 BP) act as cofactors for the CFI catalytic cleavage reaction of C4b. The C4b cleavage assay is a functional assay that determines the ability of CFI and its variants to be used for C4b cleavage activity in the presence of CR1 or C4BP cofactors. Complement factor protein C2, which specifically binds to C4b but not CFI cleavage product iC4b, was used to capture C4b. CFI catalytic cleavage of C4b was measured by measuring the decrease in concentration of C4b bound to C2 protein immobilized on ELISA culture plates. Captured C4b protein was detected by anti-C4C polyclonal rabbit Ab (DAKO, #a0065). The percent C4b cleavage activity of the CFI variant was calculated by normalizing the C4b cleavage activity of CFI-HSA to 100%.

For each CFI-HSA variant, a master reaction mixture was set at room temperature containing 250nM cofactor (CR 1 domains 1-3) and a final concentration of 250nM human C4b in HBS buffer (30mM HEPES,140mM NaCl pH 7.4). Transferring the main reaction mixture toAnd allowed to equilibrate for 5 minutes. The cleavage reaction was initiated by adding CFI-HSA protein to a final concentration of 250 nM. Sample volumes corresponding to 0.6ug C3b were extracted from the master mix at each time point measured and quenched by addition of 5 XSDS reduction sample buffer, followed by +. >Incubate for 5 minutes. Samples were run on 9 or 10% SDS-PAGE gels and C4b cleavage was visualized by Coomassie staining. The amount of C4b cleavage that occurs was quantified by densitometry. The C4b cleavage activity of wild-type CFI-HSA was normalized at 100% to calculate the percent C4b cleavage activity of the CFI-HSA variant.

Another example of a C4b cleavage activity assay is performed below to determine the C4b cleavage activity of the CFI variant relative to the reference molecule CFI-HSA wild-type. Cleavage reactions were performed with 250nM CFI variants in the presence of 250nM cofactor (CR 1 domains 1-3) and 250nM human C4bIncubate for 30 minutes. The reaction mixture was diluted 20-fold prior to addition to the blocked ELISA plates coated with mouse monoclonal anti-C4C antibody. The absorbance recorded from the ELISA plate is a relative measure of the C4C product produced in the cleavage reaction, and as such is a measure of the C4b cleavage activity. The fold difference in C4b cleavage activity of the CFI variant relative to the reference molecule CFI-HSA wild-type was calculated by dividing the background corrected absorbance of the CFI-HSA variant by the background corrected absorbance of the CFI-HSA wild-type. Table 7.1 below outlines the fold differences in C4b cleavage activity analysis of CFI variants relative to the CFI-HSA reference molecule, as measured by C4cELISA screening using CR 1.

Measurement of EC of CFI variant cleavage C4b ₅₀ . Analysis was performed using 250nM cofactor (CR 1 domains 1-3), 250nM human C4b, and a dilution series of CFI variants. The reaction mixture is reacted in the presence ofIncubate for 30 minutes and then dilute the reaction mixture 20-fold before starting ELISA. The amount of C4C produced 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 produced in the cleavage reaction, and as such is a measure of the C4b cleavage activity. EC50 values were calculated using a four-parameter nonlinear regression fit, without limitation in GraphPad Prism. EC50 values exceeding 1000nM were set to 1000nM. The cleavage reaction was also carried out in the absence of CR1, where indicated and analyzed in the same manner as those containing CR 1. Table 7.2 summarizes the results of the C4C ELISA titrated with CR1 cofactor.

CFI variant activity in the absence of cofactors

The C4b cleavage reaction was performed in the absence of cofactors for a set of CFI variants as described above (table 7.3). The results demonstrate that CFI variants with C-terminal fusion proteins comprising the human CR1 domain maintain their ability to cleave C4b in the absence of cofactors in the reaction mixture. In contrast, CFI variants lacking CR 1C-terminal fusion do not maintain their ability to cleave C4 b. These results indicate that CFI variants with C-terminal CR1 fusion can be independent of CR1 cofactor.

TABLE 7.3 cleavage of CFI variants of C4b in the absence of cofactors

The C3b cleavage reaction was performed in the absence of cofactors for a set of CFI variants as described above (table 7.4). The results demonstrate that CFI variants with C-terminal fusion proteins comprising the human CR1 domain maintain their ability to cleave C3b in the absence of cofactors in the reaction mixture. In contrast, CFI variants lacking CR 1C-terminal fusion do not maintain their ability to cleave C3 b. These results indicate that CFI variants with C-terminal CR1 fusion can be independent of CR1 cofactor.

TABLE 7.4 cleavage of CFI variants of C3b in the absence of cofactors

Single point screening of CFI variants for C4b and C3b cleavage

The specificity of C4b cleavage versus C3b cleavage and C3b cleavage versus C4b cleavage is calculated in two different ways. For the single point analysis listed in table 7.1, the fold difference was calculated using the median value subtracted from the baseline. Values below 0.01 are adjusted to 0.01. Each single median of C4b and C3b was converted to a percentage maximum using the following formula: 100% > (variant value/maximum among all variants). The specificity of C4b was calculated as the ratio of the maximum C4b percentage divided by the maximum C3b percentage. The specificity of C3b was calculated as the ratio of the maximum C3b percentage divided by the maximum C4b percentage.

Table 7.1: variant screening for C4b and C3b cleavage

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For the assays to determine EC50 values, specificity was calculated by normalization to CFI-HSA. For C4b cleavage, the maximum value was set at 1000nM and all values above were set at 1000nM. For C3b cleavage, the maximum was set at 500nM and all values above were set at 500nM. The C4b specificity was calculated as follows: (C4 b EC50CFI-HSA/C4b EC50 variant)/(C3 b EC50 CFI-HSA/C3b EC50 variant). The C3b specificity was calculated as follows: (C3 b EC50 CFI-HSA/C3b EC50 variant)/(C4 b EC50CFI-HSA/C4b EC50 variant). The results are reported in table 7.2.

TABLE 7.2 EC for variants in C4b and C3b cleavage assays ₅₀ Value of

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Example 8: scalability and selection of CFI variants for C3b, C4b or both C3b and C4b

For example 8, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Figure 13 depicts a scatter plot showing fold changes in activity for C4b, fold changes in activity for C3b, and engineering specificity, showing that various CFI variants are tunable and selected for C3b, C4b, or both. Each point in the bitmap represents a different CFI variant. Those that aggregate in region a are modulators of classical and lectin pathway specificity and have at least 10-fold specificity for C4b relative to C3 b. Those that aggregate in region B are central pathway modulators in region C and have increased activity for both C3B and C4B and at least 10-fold specificity for C3B relative to C4B, as compared to CFI as a wild-type alternative pathway-specific modulator. Those that aggregate in region C are alternative pathway-specific modulators compared to wild-type CFI, and have at least 10-fold specificity for C3b relative to C4b central pathway modulators, and increased activity for both C3b and C4 b.

Fig. 14A depicts a dose response curve showing the efficacy and specificity of C4b degradation and CFI variants characterized as C4b degrading agents. The C4b degrading agent is a CFI fusion of the CFI wild type and CCP domains 15-17 of CR1, which is linked by a flexible linker (GGSSGG) (SEQ ID NO: 6), and is also further fused to albumin. CFI-CR1 fusion was tested in the absence of exogenous CR1 cofactor, and wild-type CFI was tested in the presence and absence of exogenous CR1 cofactor.

Fig. 14B depicts a bitmap showing fold-changes in activity for C4B, fold-changes in activity for C3B, and engineering specificity for the CFI variants previously shown in fig. 13. The points representing the CFI-CR1 fusion of FIG. 14A are indicated by arrows. Together, FIGS. 14A-14B demonstrate the engineered C4B potency and specificity of CFI-CR1 fusion proteins.

Figures 14C-14D depict dose response curves showing the activity of CFI variants that rely on exogenous CR1 cofactors to enhance classical pathway activity compared to CFI variants that are active even in the absence of exogenous CR1 cofactors. These figures depict the concentration of test (M) and show classical pathway activity as measured by C4b degradation. The CFI variant of FIG. 14C is T495F+Y496L+D497E+S499G+I500K+G533A+K534Q+P535K+E536N+F537K, and the CFI variant of FIG. 14D is the CFI-CR1 fusion and CCP domain 15-17 of the CFI wild type of CR1, joined by a flexible linker (GGSSGG) (SEQ ID NO: 6). These figures demonstrate that C4b degradants can be engineered for enhanced potency and exogenous CR1 cofactor independence. These figures also show that CR1 can act similarly to exogenous CR1 cofactors when fused with CFI.

Figures 14E-14F depict scatter plots of fold change in activity for C4b and C3b for various CFI variants provided herein, indicating further adjustability of the CFI variants tested. Figure 14E depicts the results of screening assays performed on CFI variants, measured as fold changes relative to CFI-HSA, exhibiting fold changes in activity for C4b and fold changes in activity for C3 b. FIG. 14F depicts EC from 14E data ₅₀ Values.

Example 9: CFI-HSA activity compared to plasma derived CFI as measured by in vitro cleavage and hemolysis analysis of C3b and C4b

For example 9, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Various hemolysis assays were performed to assess the activity of CFI-HSA, CFI variants compared to plasma-derived CFI. Hemolysis mediated via Classical Pathway (CP) and Alternative Pathway (AP) was evaluated. Briefly, an overview of the analysis performed and the focus of the analysis are presented in table 9.1 below.

TABLE 9.1

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FIGS. 15A-15B depict graphs depicting C3B degradation and C4B degradation of CFI and plasma-derived CFI, respectively. CFI-HSA was capable of being expressed as 25.5nM (95% CI:21.9-29.6 nM) and 365nM (95% CI:297-448 nM) EC, respectively ₅₀ Values cleave both C3b and C4b in buffer. These data are summarized in table 9.2 below. The graphs shown in FIGS. 15A-15B are normalized and the data originates from non-NOT Analysis of standardized data. These results indicate that the cleavage activity by CFI-HSA is not significantly different from physiological (plasma-derived) CFI, suggesting that recombinant CFI-HSA as well as plasma-derived CFI may be expressed well and potentially act as a surrogate or supplement for CFI activity under physiological conditions.

TABLE 9.2

FIGS. 15C-15D depict graphs of hemolysis assays in AP+C3b and mixed AP+CP, respectively, by CFI-HSA and plasma-derived CFI. CFI-HSA was able to degrade human serum hemolysis assay C3b with 26.4nM (95% CI:15.5-44.7 nM) of IC ₅₀ Values completely inhibited complement-mediated lysis and were equivalent to physiological (plasma-derived) CFI. In addition, CFI-HSA was able to run at 426nM (95% CI:162-1120 nM) of IC in a CFI depleted human serum hemolysis assay ₅₀ Values completely inhibited complement-mediated lysis and were equivalent to physiological CFI. These data are summarized in table 9.3 below. These results again indicate that the activity of CFI-HSA is not significantly different from the physiological activity (plasma derived CFI).

TABLE 9.3

Generally, the above results demonstrate that CFI-HSA and plasma derived CFI are expressed well, along with the advantages of increased half-life and higher yield of recombinantly produced CFI-HSA. Illustrative uses of CFI-HSA are thus useful in enzyme replacement therapy of endogenous CFI in complement-associated disorders.

Furthermore, CFI variant E461G was tested against CFI-HSA. FIGS. 15E-15F depict the results of AP+CP analysis and CP analysis using the E461G variant, CFI-HSA, and plasma-derived CFI, respectively. These results indicate that E461G has engineered C3b potency and specificity.

Example 10: predicting human exposure pharmacokinetic profiles using multiple subcutaneous administrations of CFI-HSA

For example 10, reference to CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Fig. 16A depicts a graph predicting a Pharmacokinetic (PK) profile of human exposure after multiple subcutaneous administrations of CFI-HSA. Multiple administrations were once per week over a period of eight weeks. These results indicate that when a dose of 3mg/kg is used, starting at about 3-4 weeks, the blood CFI range is within the range of the normal population. Human iso-speed ratios scale to an index of 0.37 and a distribution volume of 0.88 based on PK model/clearance of rat and cynomolgus macaque populations.

FIGS. 16B-16C depict the concentration of CFI-HSA over time (FIG. 16B) compared to the predicted pharmacokinetic profile described above (FIG. 16C). These results demonstrate that the blood CFI range in the pharmacokinetic profile can be increased after multiple weekly administrations of CFI-HSA, while the CFI-HSA concentration follows the curve as shown in fig. 16B.

Example 11: half-life of CFI-HSA in non-human primate vitreous humor

For example 11, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Pharmacokinetics of CFI-HSA after intravitreal injection

The ocular pharmacokinetics of N-terminal albumin fusion of wild-type CFI-HSA was examined after intravitreal administration of six African Green Monkeys (AGM). Six animals were divided into two groups treated at 2 dose levels: one group received a single intravitreal injection of 500 μg of CFI-HSA (right eye, OD, n=3) and the other group received a single intravitreal injection of 250 μg of CFI-HSA (right eye, OD, n=3). All six animals were injected with an equal volume of 100 μl sterile PBS for left eye (OS) for vehicle control injection. Non-terminal vitreous humor samples (100 μl) were collected on days 1, 7, 14, 21, and 28 post-dosing. The vitreous CFI-HSA drug concentration was determined using quantitative Electrochemiluminescence (ECL) antigen analysis optimized for measuring CFI-HSA in AGM vitreous. This assay uses anti-CFI antibody (clone OX21, LS Bio, seattle, washington) coated at 2 μg/ml on Meso Scale Discovery (MSD, rocyvere (Rockville), MA) assay plates to capture CFI-HSA content. Use and application Light emission upon application of an electrical potential [ Electrochemiluminescence (ECL)]The detection of captured CFI-HSA was performed with goat polyclonal anti-HSA antibody (Ai Bokang (Abcam), cambridge (Cambridge), mass.) conjugated to SULFO-TAG at 0.5 μg/ml. At the position ofThe ECL Relative Light Units (RLU) were measured on a SECTOR S600 reader and the unknown CFI-HSA concentration in the vitreous was interpolated from a standard curve ranging from 0.05 μg/ml to 40 μg/ml factor I-HSA. The data are provided in table 11.1.

TABLE 11.1 CFI-HSA content at days 1, 7, 14, 21, and 28 after intravitreal administration

* BLQ measurement below analytical quantification limit

Non-compartmental analysis gave apparent ocular terminal half-lives of 3.6 and 4.1 days for 250 and 500 μg dose contents, respectively.

TABLE 11.2 evaluation of PK parameters for CFI-HSA in the vitreous humour of African green monkey

The complement component 3a (C3 a) content in the vitreous was determined by ELISA using the Quidel kit of C3a ELISA (fig. 18). At most 7 days after intraocular injection, the CFI-HSA fusion protein reduced ocular C3a content in a dose-dependent manner. The increase in C3b degradation by CFI-HSA reduces complex formation between C3b and Bb, such that the amplified loop via the alternative pathway reduces cleavage of C3 into C3a and C3b.

Example 12: CFI-HSA in mouse plasma and plasma CFI pharmacokinetics

For example 12, reference to CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

The pharmacokinetics of N-terminal albumin fusion of wild-type CFI (CFI-HSA) was examined after intravenous and subcutaneous administration of CD-1 mice. CD-1 mice were divided into four groups and treated with a single dose of plasma purified wild-type CFI or recombinant wild-type CFI-HSA using a sparse sampling design with at most two samples per mouse and three mice sampled at each time point.

To compare the circulating half-life in plasma and the bioavailability of plasma-derived CFI and CFI-HSA, animals were given plasma-derived CFI or CFI-HSA intravenously and subcutaneously. Plasma-derived CFI was administered intravenously (group 1) at 1.3mg/kg and subcutaneously (group 2) at 6.5 mg/kg. CFI-HSA was administered intravenously (group 3) and subcutaneously (group 4) at 3 mg/kg. An additional 3 animals received a single dose of equivalent volume of PBS delivered subcutaneously as vehicle control (group 5; not shown). Blood (about 30-50 μl) was collected in EDTA at various time points from 5 minutes to 144 hours post-dose and plasma was separated by centrifugation.

CFI-HSA and plasma CFI concentrations were determined in CD-1 mouse EDTA plasma using quantitative Electrochemiluminescence (ECL) antigen analysis of CFI-HSA and plasma CFI. For CFI analysis, mouse monoclonal anti-factor I antibodies (MAB 12907, innova (Abnova), taibei, taiwan) were coated at 2 μg/ml on Meso Scale Discovery (MSD, rocyville (Rockville), MA) analysis plates to capture plasma CFI. By and with the emission of light upon application of an electric potential [ Electrochemiluminescence (ECL) ]The detection of captured CFI was performed with 0.5. Mu.g/ml mouse monoclonal anti-HSA antibody conjugated to SULFO-TAG (asexual propagation 3R/8, CABT-47940MH, creative diagnosis (Creative diagnostic), new York Xueli City). For CFI-HSA analysis, mouse monoclonal anti-CFI antibody (clone 3R/8, CABT-47940 MH) was coated at 1 μg/ml on Meso Scale Discovery (MSD, rockville (Rockville), MA analysis plates to capture plasma CFI-HSA. By and with the emission of light upon application of an electric potential [ Electrochemiluminescence (ECL)]The detection of captured CFI-HSA was performed with rabbit polyclonal anti-HSA antibody conjugated to SULFO-TAG (ab 24207, ai Bokang, cambridge, mass.). At the position ofSECTOR S600 reader measures ECL Relative Light Units (RLU) and interpolates unknown plasma CFI and CFI-HSA concentrations from standard curves. Pharmacokinetic parameters were derived from analysis of plasma CFI and CFI-HSA concentrations and are provided in table 12.1.

Table 12.1. Pharmacokinetics of plasma CFI and CFI-HSA as assessed by measuring CFI antigen in plasma from CD-1 mice after single bolus IV and SC administration.

The longer circulation half-life (about 22 hours) after intravenous infusion of CFI-HSA compared to the non-fusion form of plasma CFI protein (about 13 hours), indicates that fusion of HSA with CFI protein increases half-life compared to unfused CFI. Importantly, the bioavailability of CFI (53.6%) was similar to CFI-HSA (46.7%), indicating that fusion of HSA with CFI protein did not adversely affect the bioavailability of CFI after subcutaneous administration (table 12.1). Fusion of HSA to CFI protein increased half-life by about 2-fold after intravenous (fig. 19) or subcutaneous administration (fig. 20) compared to the intravenous non-fusion form of CFI protein. Similar exposure was achieved with 2-fold lower doses of fusion protein CFI-HSA after subcutaneous injection (fig. 20) compared to CFI.

Example 13: CFI-HSA bioactivity following intravenous administration of complement-activated rodent models

For example 13, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

A rat model of peripheral nerve injury was developed to investigate complement involvement in Wo Leshi degeneration (Wallerian degeneration) due to mechanical injury of myelinated sciatic nerve. Male CD Shi Boge dori rats (Sprague Dawley rats) (charles river laboratories (Charles River Laboratories)) weighing between 300g and 350g were anesthetized with a mixture of 2 to 2.5% isoflurane USP (Abbot laboratories, montreal, canada) in oxygen at enrollment and placed on a heating pad to maintain body temperature. The legs were subjected to a sterile procedure to expose the sciatic nerve. One leg underwent Sciatic Nerve Injury (SNI) by clamping the sciatic nerve three times for 10 seconds using Dumont #7 forceps. The contralateral leg did not receive clamp injury and served as an internal control for each individual.

Immediately after SNI induction, animals received intravenous injection of CFI-HSA (Y408L; N531G variant) 4mg/kg (n=10), CFI-HSA (Y408L; N531G variant) 1.25mg/kg (n=10) or control (1 x pbs; n=10) at a dose volume of 4 mL/kg. Subcutaneous injections of slow-release buprenorphine (0.01 mg/kg) were also administered for pain management. 4 or 24 hours after SNI, 5 animals from each treatment group were sacrificed by exsanguination.

At the time of sacrifice, 1cm (0.5 cm near and 0.5cm far from the injury site) nerve segments were collected from the injury (ipsilateral) and leg prosthesis, rapidly frozen, and stored inDown to treatment for mass spectrometry (Phenoswitch Bioscience, canada). K2-EDTA plasma samples were collected at SNI (baseline) and 1, 4 and 24 hours after SNI (as applicable) to evaluate complement component fragments by Mass Spectrometry (MS). Cytokine and chemokine levels were assessed in K2-EDTA plasma collected at baseline (vehicle only), 4 and 24 hours (as applicable) after SNI (27 plex Multiplex immunoassay in rats, canada, using the BioPlex 200 cytokine array, assay kit millbo miplex, by Eve technique, calgary). At the time of sacrifice, whole blood and serum were collected for clinical pathology assessment [ whole blood count (CBC) and serum chemistry; biovet Inc., canada]。

Mass spectrometry of in vivo samples

Samples were denatured and precipitated with washing and buffer exchange followed by N-terminal labeling via reductive amine dimethyl ation. The sample was then digested with trypsin (or a mixture of trypsin and chymotrypsin) and subsequently analyzed via LC-MS/MS using SWATH. The SWATH data are integrated onto ion libraries generated for each species and sample type. The first 10 peptides per protein contained in the ion library were integrated and peptide center analysis was performed to specifically quantify C3, C5, C4 and CFB N-terminal tagged peptides.

The cleavage products resulting from the catalytic activity of CFI-HSA (Y408L; N531G variant) on C3B were monitored by mass spectrometry in the neural tissue (membrane-bound fragment) (FIG. 21A) and in the circulation (soluble fragment in plasma) (FIG. 21B). The cleavage activity of CFI-HSA (Y408L; N531G variant) was such that 2 major cleavage fragments were detected by mass spectrometry: c3dg and C3f. Cleavage of surface bound C3b by CFI will result in surface bound C3dg fragments and soluble C3f fragments. When formed from soluble C3b, both fragments are soluble. Thus, detection of C3dg in plasma may be due to cleavage of soluble C3b, whereas detection of C3dg in tissue may be caused by cleavage of membrane bound C3 b. The N-terminal tag C3dg (E2 Me) DVPAADLSDQVPDTDSETR) (SEQ ID NO: 24) is the CFI cleavage product of iC3 b. The activity of the CFI variant was determined as a percentage of the C3dg peptide, where the N-terminal tag (referred to as "activated C3 dg") multiplied by the total signal size of C3dg (EDVPAADLSDQVPDTDSETR) (SEQ ID NO: 24). The dose-dependent increase in activated C3dg fragment compared to plasma was significantly more pronounced in damaged nerve tissue, indicating that CFI-HSA (Y408L; N531G variant) may be more active in the surface-bound configuration than in the circulation. This effect was not detected at the early time point (4 hours), but was observed 24 hours after injury, indicating that tissue C3b formation was delayed, CFI-HSA (Y408L; N531G variant) exhibited slow lytic activity in vivo, or the observed effect was a result of CFI-HSA (Y408L; N531G variant) activity.

Overall, mass spectrometry results demonstrate that mechanical nerve damage can trigger complement responses at nerve tissue sites. In addition, CFI-HSA (Y408L; N531G variant) exhibited greater cleavage activity on surface-bound C3b than circulating C3b when compared to vehicle, indicating that CFI-HSA (Y408L; N531G variant) could be better expressed for surface-bound C3 in which CR1 and C4bp cofactors were present.

Example 14: in vivo Activity of CFI variants in a rat model of peripheral nerve injury

For example 14, mention of CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Efficacy of a set of CFI variants on complement activation in a Sciatic Nerve (SN) injury (SNI) rat model was determined. Immediately after SNI induction, animals received IV injection of CFI variants (n=6 for each variant) from a panel of CFI variants (table 14.1) or controls (1 x pbs; n=6) at a dose volume of 5 mL/kg. 24 hours after SNI, all animals were sacrificed by exsanguination. Cytokine and chemokine levels were assessed in K2-EDTA plasma collected 4 and 24 hours post SNI (27 plex Multiplex immunoassay in rats, canada, using the BioPlex 200 cytokine array, assay kit milbezels, by Eve technique). At the time of sacrifice, serum was collected for serum chemistry [ Biovet inc., canada ].

TABLE 14.1

The activity of CFI variants was monitored by detecting CFI cleavage products (C3 dg and C3 f) using mass spectrometry. CFI cleavage product with C3f (S2 Me) for C3b and C3dg (E2 Me) for DVPAADLSDQVPDTDSETR) for iC3 b. Total activated C3f was determined as the percentage of C3f peptide, where the N-terminal tag (S2 Me EETK 2Me QNEGF) multiplied by the total peptide signal size of C3f (SEETKQNEGF).

An increase in neural C3dg content of 2.5 fold 24 hours after injury was observed in vehicle-treated animals, and no effect of CFI variant treatment was detected (fig. 22). No significant increase in C3dg and C3f levels was detected in the plasma of vehicle treated animals (fig. 23A and 23B). However, a trend of increasing plasma C3f content was observed 4 hours after variant 2 and variant 3 treatments (fig. 23B), followed by an increase in plasma C3dg content at 24 hours (fig. 23A). Variant 4 showed delayed activity, where C3f and C3dg levels peaked in plasma at 24 hours. The higher content of plasma C3dg after 4 hours of variant 1 treatment indicated that cofactor fusion outperforms variant 4 at the previous time point for soluble C3b cleavage, but this effect did not persist for 24 hours (fig. 23A).

To compare the in vitro activity of CFI variants by wild-type CFI, iC3b EC of wild-type CFI was used ₅₀ (Table 14.2, column A), and IC for classical path hemolysis ₅₀ (Table 14.2, column B) divided by the iC3b EC for each variant ₅₀ IC (integrated circuit) ₅₀ . To compare in vivo and in vitro data for plasma C3f, total N-terminally labeled C3f at each time point was divided by baseline N-terminally labeled circulating C3f signal in plasma of each animal to provide estimates of CFI-mediated liquid phase and surface binding C3b cleavage (table 14.2, columns C and D). Plasma derived CFI was chosen as the closest approximation to the activity of endogenous rodent CFI. The results of these data transformations are summarized in the following table.

TABLE 14.2

The resulting fold change measured by CP hemolysis analysis and iC3b ELISA analysis produced a similar fractionation to circulating C3f content at 4 hours. The relative improvement in C3 cleavage activity between variants was less discernible 24 hours after injury. In this setting, variant 2 and variant 3 clearly outperformed the other CFI variant at 4 hours post SNI, while variant 4 outperformed all variants at 24 hours. Overall, these data indicate that adding D425R and E416A substitutions to variant 3 did not significantly increase C3b cleavage in vivo. However, the addition of E457G and D425R to variant 4 will result in faster in vivo cleavage activity, which may not persist over time. Further work was needed to confirm the accuracy of CP hemolysis to predict circulating CFI activity in rodents, but the trend suggests that this analysis may provide a close estimate of the massive circulating lytic activity in rats.

Untreated rats underwent surgery and nerve pinching indicated a strong increase in circulating macrophage inflammatory protein-1α (MIP-1α) compared to the historical baseline (fig. 24). MIP-1α has been shown to contribute to the pathogenesis of neuralgia in a model of similar sciatic nerve injury in mouse 1. The large increase decreased with administration of all CFI variants, but most significantly with cofactor fusion variant 1.

Example 15: in vivo Activity of CFI variants in cecal ligation and puncture models

The effect of Cecal Ligation and Puncture (CLP) -induced sepsis on CFI variants limiting complement activation in a model of CLP in rats was assessed. For example 15, reference to CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

Following Cecal Ligation and Puncture (CLP) surgery, complement participation was studied using a rat model of non-sterile sepsis. This procedure provides three aspects of complement activation and inflammation (mechanical destruction, bacterial exposure, and ischemic injury) that make it particularly relevant as a screening tool for other indications. Male CD Shi Boge dori rats (charles river laboratories) weighing between 300g and 350g were anesthetized with a 2 to 2.5% isoflurane USP (Abbot laboratories, montreal, canada) mixture in oxygen at the time of enrollment and placed on a heating pad to maintain body temperature. Sepsis is induced by CLP surgical procedures. A midline incision was made in the abdominal wall, the cecum was removed, and the cecum valve was accessed with a nylon suture (4-0) for engagement, followed by a 16-gauge needle penetration through the distal portion of the cecum, allowing a small amount of cecum content to enter the abdominal cavity. The abdominal wall and skin were then sutured.

Following the CLP procedure, animals received intravenous injection of variant 1[ cfi-HSA (E457G; N531G variant) ]4.25mg/kg (n=6), variant 2[ cfi-HSA (E457G; N531G variant fused to C-terminal CCP15; CCP16; CCP 17) ]5mg/kg (n=6), variant 3[ cfi-HSA (E457G; E461Q; N531G; delta (558-PFISQYNV-565 variant) ]4.25mg/kg (n=6), or control (1 x pbs; n=6) at a dosing volume of 5 mL/kg. No dummy treatment group was performed. All animals were sacrificed by exsanguination 16 hours after CLP surgery. One day prior to enrollment (baseline), K2-EDTA plasma samples were collected 3 and 16 hours after CLP to evaluate complement component fragments by Mass Spectrometry (MS) and cytokine/chemokine content (27 plex Multiplex immunoassay of rats by Eve technology, calgari, canada with a BioPlex 200 cytokine array, assay kit, millplex). At baseline and 16 hours, whole blood and serum were collected for clinical pathology assessment [ whole blood count (CBC) and serum chemistry; biovet Inc., canada ].

Thrombocytopenia was observed in vehicle-treated animals 16 hours after injury. Trends towards thrombocytopenia were observed in animals treated with variant 1 and variant 2 (fig. 25A). This protection was less pronounced in animals treated with variant 3, indicating that deletion of the C-terminal portion of CFI may not increase catalytic activity. This in vivo observation is consistent with the lower lytic activity of variant 3 observed in vitro via C4C and iC3b compared to variants 1 and 2. Variant 1 and variant 2 exhibited similar effects of thrombocytopenia, suggesting that addition of CR1 cofactor fusion to variant 1 may not increase in vivo activity.

The inflammatory cytokine tumor necrosis factor alpha (tnfα) was rapidly released after CLP surgery (fig. 25B). In untreated animals, tnfα increased from surgery by about 2.9-fold over 3 hours. In contrast, treatment with variant 2 significantly reduced these effects, with an average decrease in circulating tnfα content compared to baseline. Similar protective trends were observed in animals receiving variant 1. Rats treated with variant 3 still showed an increase in circulating tnfα (mean fold change of about 1.5), even to a lesser extent than untreated animals.

Example 16: in vivo Activity of CFI variants in acute respiratory distress syndrome

The therapeutic effect of CFI variants in a LPS-induced Acute Respiratory Distress Syndrome (ARDS) mouse model was evaluated. For example 16, reference to CFI-HSA refers to human serum albumin fused to the N-terminus of wild type CFI (SEQ ID NO: 21).

The purpose is as follows: the purpose of this study was to assess the efficacy of variant 1[ cfi-HSA (E457G; E461Q) ] and variant 2[ cfi-HSA (E457G; E461Q; N531G) ] in limiting complement-mediated acute lung inflammation in an ARDS mouse model induced by single administration of Lipopolysaccharide (LPS).

Following LPS intratracheal Instillation (IT), the sterile ARDS mouse model was used to study complement participation. Male C57BL/6 mice (Charles river laboratories) weighing between 20g and 25g were anesthetized under isoflurane at the time of enrollment and were instilled intratracheally with 50 μg LPS (1 mg/mL LPS isolated from E.coli 0111: B4 in 0.9% saline solution, sigma).

Three hours after CLP procedure, animals received intravenous injection of 5mg/kg of variant 1 (n=8), 5mg/kg of variant 2 (n=8) or control (1 x pbs; n=10) at a dosing volume of 5 mL/kg. To assess the potential effect of repeated daily dosing, animals treated with variant 2 received a second 5mg/kg dose 27 hours after LPS IT. The sham-treated group was subjected to 50 μl of an intratracheal instillation of 0.9% physiological saline solution (n=5) without any IV treatment. Animals treated with variant 1 were sacrificed 24 hours after LPS IT, while animals treated with variant 2 were sacrificed 48 hours after LPS IT.

Collection of K at the time of sacrifice ₂ EDTA plasma, lung tissue and bronchoalveolar lavage (BALF) samples to evaluate the complement component fragments by Mass Spectrometry (MS). Cold PBS 1X containing protease inhibitor 1X in three 300 μl right lung infusionsBALF was collected. K collected at the time of sacrifice ₂ Assessment of cytokine and chemokine content in EDTA plasma, BALF and lung tissue (homogenized with protease cocktail inhibitor in PBS 1X 0.1% triton X-100) (31 plex Multiplex immunoassay in mice, calgari, canada, using BioPlex 200 cytokine array, assay kit millbo MILLIPLEX, by Eve technique). At the time of sacrifice, whole blood and serum were collected for clinical pathology assessment [ whole blood count (CBC) and serum chemistry; biovet Inc., canada ]. Differential cell counts were performed on BALF samples to assess leukocyte recruitment to the lungs.

LPS is a known alternative complement pathway inducer. We used mass spectrometry to assess CFI activity on circulating C3b lysates. The percentage of activated C3f was determined as the percentage of C3f peptide, where the N-terminal tag (S2 Me EETK 2Me QNEGF) multiplied by the total peptide signal size of C3f (SEETKQNEGF). In BALF, an increase in the lytic release of C3f was observed in all LPS-treated animals for up to 48 hours at 24 hours (fig. 26B). Similar trends and schedules were observed in the lungs, independent of CFI variant treatment (fig. 26A). No lytic activity (C3 f release) of variants 1 and 2 was detected in bronchoalveolar fluid or lung tissue 24 hours and 48 hours after LPS administration. However, we cannot rule out the possibility that the C3f fragment has been detected at an earlier time point after LPS administration. In contrast, a significant increase in circulating C3f was detected in animals receiving both doses of variant 2 48 hours after LPS administration, indicating C3b cleavage in the cumulative enhanced circulation at the 5mg/kg dose.

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Val Asn Cys Ser Met Ala Gln Ile Gln Leu Cys Pro Pro Pro Pro Gln

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Ile Pro Asn Ser His Asn Met Thr Thr Thr Leu Asn Tyr Arg Asp Gly

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Glu Lys Val Ser Val Leu Cys Gln Glu Asn Tyr Leu Ile Gln Glu Gly

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Glu Glu Ile Thr Cys Lys Asp Gly Arg Trp Gln Ser Ile Pro Leu Cys

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Val Glu Lys Ile Pro Cys Ser Gln Pro Pro Gln Ile Glu His Gly Thr

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Ile Asn Ser Ser Arg Ser Ser Gln Glu Ser Tyr Ala His Gly Thr Lys

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Leu Ser Tyr Thr Cys Glu Gly Gly Phe Arg Ile Ser Glu Glu Asn Glu

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Thr Thr Cys Tyr Met Gly Lys Trp Ser Ser Pro Pro Gln Cys Glu Gly

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Leu Pro Cys Lys Ser Pro Pro Glu Ile Ser His Gly Val Val Ala His

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Met Ser Asp Ser Tyr Gln Tyr Gly Glu Glu Val Thr Tyr Lys Cys Phe

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Glu Gly Phe Gly Ile Asp Gly Pro Ala Ile Ala Lys Cys Leu Gly Glu

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Met Asp Gly Ala Ser Asn Val Thr Cys Ile Asn Ser Arg Trp Thr

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Gly Arg Pro Thr Cys Arg Asp Thr Ser Cys Val Asn Pro Pro Thr

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Val Gln Asn Ala Tyr Ile Val Ser Arg Gln Met Ser Lys Tyr Pro

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Ser Gly Glu Arg Val Arg Tyr Gln Cys Arg Ser Pro Tyr Glu Met

1070 1075 1080

Phe Gly Asp Glu Glu Val Met Cys Leu Asn Gly Asn Trp Thr Glu

1085 1090 1095

Pro Pro Gln Cys Lys Asp Ser Thr Gly Lys Cys Gly Pro Pro Pro

1100 1105 1110

Pro Ile Asp Asn Gly Asp Ile Thr Ser Phe Pro Leu Ser Val Tyr

1115 1120 1125

Ala Pro Ala Ser Ser Val Glu Tyr Gln Cys Gln Asn Leu Tyr Gln

1130 1135 1140

Leu Glu Gly Asn Lys Arg Ile Thr Cys Arg Asn Gly Gln Trp Ser

1145 1150 1155

Glu Pro Pro Lys Cys Leu His Pro Cys Val Ile Ser Arg Glu Ile

1160 1165 1170

Met Glu Asn Tyr Asn Ile Ala Leu Arg Trp Thr Ala Lys Gln Lys

1175 1180 1185

Leu Tyr Ser Arg Thr Gly Glu Ser Val Glu Phe Val Cys Lys Arg

1190 1195 1200

Gly Tyr Arg Leu Ser Ser Arg Ser His Thr Leu Arg Thr Thr Cys

1205 1210 1215

Trp Asp Gly Lys Leu Glu Tyr Pro Thr Cys Ala Lys Arg

1220 1225 1230

<210> 5

<211> 565

<212> PRT

<213> Chile person

<400> 5

Lys Val Thr Tyr Thr Ser Gln Glu Asp Leu Val Glu Lys Lys Cys Leu

1 5 10 15

Ala Lys Lys Tyr Thr His Leu Ser Cys Asp Lys Val Phe Cys Gln Pro

20 25 30

Trp Gln Arg Cys Ile Glu Gly Thr Cys Val Cys Lys Leu Pro Tyr Gln

35 40 45

Cys Pro Lys Asn Gly Thr Ala Val Cys Ala Thr Asn Arg Arg Ser Phe

50 55 60

Pro Thr Tyr Cys Gln Gln Lys Ser Leu Glu Cys Leu His Pro Gly Thr

65 70 75 80

Lys Phe Leu Asn Asn Gly Thr Cys Thr Ala Glu Gly Lys Phe Ser Val

85 90 95

Ser Leu Lys His Gly Asn Thr Asp Ser Glu Gly Ile Val Glu Val Lys

100 105 110

Leu Val Asp Gln Asp Lys Thr Met Phe Ile Cys Lys Ser Ser Trp Ser

115 120 125

Met Arg Glu Ala Asn Val Ala Cys Leu Asp Leu Gly Phe Gln Gln Gly

130 135 140

Ala Asp Thr Gln Arg Arg Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser

145 150 155 160

Thr Glu Cys Leu His Val His Cys Arg Gly Leu Glu Thr Ser Leu Ala

165 170 175

Glu Cys Thr Phe Thr Lys Arg Arg Thr Met Gly Tyr Gln Asp Phe Ala

180 185 190

Asp Val Val Cys Tyr Thr Gln Lys Ala Asp Ser Pro Met Asp Asp Phe

195 200 205

Phe Gln Cys Val Asn Gly Lys Tyr Ile Ser Gln Met Lys Ala Cys Asp

210 215 220

Gly Ile Asn Asp Cys Gly Asp Gln Ser Asp Glu Leu Cys Cys Lys Ala

225 230 235 240

Cys Gln Gly Lys Gly Phe His Cys Lys Ser Gly Val Cys Ile Pro Ser

245 250 255

Gln Tyr Gln Cys Asn Gly Glu Val Asp Cys Ile Thr Gly Glu Asp Glu

260 265 270

Val Gly Cys Ala Gly Phe Ala Ser Val Thr Gln Glu Glu Thr Glu Ile

275 280 285

Leu Thr Ala Asp Met Asp Ala Glu Arg Arg Arg Ile Lys Ser Leu Leu

290 295 300

Pro Lys Leu Ser Cys Gly Val Lys Asn Arg Met His Ile Arg Arg Lys

305 310 315 320

Arg Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln

325 330 335

Val Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile

340 345 350

Gly Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg Ala Ser Lys

355 360 365

Thr His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp Ile His Pro

370 375 380

Asp Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile Ile Phe His

385 390 395 400

Glu Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu

405 410 415

Met Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile

420 425 430

Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr

435 440 445

Cys Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn Glu Arg Val Phe

450 455 460

Ser Leu Gln Trp Gly Glu Val Lys Leu Ile Ser Asn Cys Ser Lys Phe

465 470 475 480

Tyr Gly Asn Arg Phe Tyr Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr

485 490 495

Asp Gly Ser Ile Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Val

500 505 510

Cys Met Asp Ala Asn Asn Val Thr Tyr Val Trp Gly Val Val Ser Trp

515 520 525

Gly Glu Asn Cys Gly Lys Pro Glu Phe Pro Gly Val Tyr Thr Lys Val

530 535 540

Ala Asn Tyr Phe Asp Trp Ile Ser Tyr His Val Gly Arg Pro Phe Ile

545 550 555 560

Ser Gln Tyr Asn Val

565

<210> 6

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 6

Gly Gly Ser Ser Gly Gly

1 5

<210> 7

<211> 585

<212> PRT

<213> Chile person

<400> 7

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu

580 585

<210> 8

<211> 415

<212> PRT

<213> Chile person

<400> 8

Met Arg Leu Leu Ala Lys Ile Ile Cys Leu Met Leu Trp Ala Ile Cys

1 5 10 15

Val Ala Glu Asp Cys Asn Glu Leu Pro Pro Arg Arg Asn Thr Glu Ile

20 25 30

Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr Pro Glu Gly Thr Gln Ala

35 40 45

Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser Leu Gly Asn Ile Ile Met

50 55 60

Val Cys Arg Lys Gly Glu Trp Val Ala Leu Asn Pro Leu Arg Lys Cys

65 70 75 80

Gln Lys Arg Pro Cys Gly His Pro Gly Asp Thr Pro Phe Gly Thr Phe

85 90 95

Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr Gly Val Lys Ala Val Tyr

100 105 110

Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly Glu Ile Asn Tyr Arg Glu

115 120 125

Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile Pro Ile Cys Glu Val Val

130 135 140

Lys Cys Leu Pro Val Thr Ala Pro Glu Asn Gly Lys Ile Val Ser Ser

145 150 155 160

Ala Met Glu Pro Asp Arg Glu Tyr His Phe Gly Gln Ala Val Arg Phe

165 170 175

Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly Asp Glu Glu Met His Cys

180 185 190

Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys Pro Lys Cys Val Glu Ile

195 200 205

Ser Cys Lys Ser Pro Asp Val Ile Asn Gly Ser Pro Ile Ser Gln Lys

210 215 220

Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln Tyr Lys Cys Asn Met Gly

225 230 235 240

Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val Cys Thr Glu Ser Gly Trp

245 250 255

Arg Pro Leu Pro Ser Cys Glu Glu Ala Gly Gly Gly Gly Gly Gly Gly

260 265 270

Gly Gly Gly Gly Gly Gly Lys Cys Gly Pro Pro Pro Pro Ile Asp Asn

275 280 285

Gly Asp Ile Thr Ser Phe Pro Leu Ser Val Tyr Ala Pro Ala Ser Ser

290 295 300

Val Glu Tyr Gln Cys Gln Asn Leu Tyr Gln Leu Glu Gly Asn Lys Arg

305 310 315 320

Ile Thr Cys Arg Asn Gly Gln Trp Ser Glu Pro Pro Lys Cys Leu His

325 330 335

Pro Cys Val Ile Ser Arg Glu Ile Met Glu Asn Tyr Asn Ile Ala Leu

340 345 350

Arg Trp Thr Ala Lys Gln Lys Leu Tyr Ser Arg Thr Gly Glu Ser Val

355 360 365

Glu Phe Val Cys Lys Arg Gly Tyr Arg Leu Ser Ser Arg Ser His Thr

370 375 380

Leu Arg Thr Thr Cys Trp Asp Gly Lys Leu Glu Tyr Pro Thr Cys Ala

385 390 395 400

Lys Arg Glu Asn Leu Tyr Phe Gln Gly His His His His His His

405 410 415

<210> 9

<211> 10

<212> PRT

<213> Chile person

<400> 9

Arg Glu Lys Asp Asn Glu Arg Val Phe Ser

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Chile person

<400> 10

Asn Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp Glu

1 5 10

<210> 11

<211> 10

<212> PRT

<213> Chile person

<400> 11

Arg Gly Lys Asp Asn Gln Lys Val Tyr Ser

1 5 10

<210> 12

<211> 260

<212> PRT

<213> Chile person

<400> 12

Lys Leu Ser Cys Gly Val Lys Asn Arg Met His Ile Arg Arg Lys Arg

1 5 10 15

Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val

20 25 30

Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly

35 40 45

Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr

50 55 60

His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp Ile His Pro Asp

65 70 75 80

Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu

85 90 95

Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met

100 105 110

Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile Pro

115 120 125

Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr Cys

130 135 140

Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn Glu Arg Val Phe Ser

145 150 155 160

Leu Gln Trp Gly Glu Val Lys Leu Ile Ser Asn Cys Ser Lys Phe Tyr

165 170 175

Gly Asn Arg Phe Tyr Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp

180 185 190

Gly Ser Ile Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys

195 200 205

Met Asp Ala Asn Asn Val Thr Tyr Val Trp Gly Val Val Ser Trp Gly

210 215 220

Glu Asn Cys Gly Lys Pro Glu Phe Pro Gly Val Tyr Thr Lys Val Ala

225 230 235 240

Asn Tyr Phe Asp Trp Ile Ser Tyr His Val Gly Arg Pro Phe Ile Ser

245 250 255

Gln Tyr Asn Val

260

<210> 13

<211> 7

<212> PRT

<213> Chile person

<400> 13

Met Asp Ala Asn Asn Val Thr

1 5

<210> 14

<211> 8

<212> PRT

<213> Chile person

<400> 14

Pro Phe Ile Ser Gln Tyr Asn Val

1 5

<210> 15

<211> 4

<212> PRT

<213> Chile person

<400> 15

Asp Gly Asn Lys

1

<210> 16

<211> 1135

<212> PRT

<213> Chile person

<400> 16

Lys Val Thr Tyr Thr Ser Gln Glu Asp Leu Val Glu Lys Lys Cys Leu

1 5 10 15

Ala Lys Lys Tyr Thr His Leu Ser Cys Asp Lys Val Phe Cys Gln Pro

20 25 30

Trp Gln Arg Cys Ile Glu Gly Thr Cys Val Cys Lys Leu Pro Tyr Gln

35 40 45

Cys Pro Lys Asn Gly Thr Ala Val Cys Ala Thr Asn Arg Arg Ser Phe

50 55 60

Pro Thr Tyr Cys Gln Gln Lys Ser Leu Glu Cys Leu His Pro Gly Thr

65 70 75 80

Lys Phe Leu Asn Asn Gly Thr Cys Thr Ala Glu Gly Lys Phe Ser Val

85 90 95

Ser Leu Lys His Gly Asn Thr Asp Ser Glu Gly Ile Val Glu Val Lys

100 105 110

Leu Val Asp Gln Asp Lys Thr Met Phe Ile Cys Lys Ser Ser Trp Ser

115 120 125

Met Arg Glu Ala Asn Val Ala Cys Leu Asp Leu Gly Phe Gln Gln Gly

130 135 140

Ala Asp Thr Gln Arg Arg Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser

145 150 155 160

Thr Glu Cys Leu His Val His Cys Arg Gly Leu Glu Thr Ser Leu Ala

165 170 175

Glu Cys Thr Phe Thr Lys Arg Arg Thr Met Gly Tyr Gln Asp Phe Ala

180 185 190

Asp Val Val Cys Tyr Thr Gln Lys Ala Asp Ser Pro Met Asp Asp Phe

195 200 205

Phe Gln Cys Val Asn Gly Lys Tyr Ile Ser Gln Met Lys Ala Cys Asp

210 215 220

Gly Ile Asn Asp Cys Gly Asp Gln Ser Asp Glu Leu Cys Cys Lys Ala

225 230 235 240

Cys Gln Gly Lys Gly Phe His Cys Lys Ser Gly Val Cys Ile Pro Ser

245 250 255

Gln Tyr Gln Cys Asn Gly Glu Val Asp Cys Ile Thr Gly Glu Asp Glu

260 265 270

Val Gly Cys Ala Gly Phe Ala Ser Val Thr Gln Glu Glu Thr Glu Ile

275 280 285

Leu Thr Ala Asp Met Asp Ala Glu Arg Arg Arg Ile Lys Ser Leu Leu

290 295 300

Pro Lys Leu Ser Cys Gly Val Lys Asn Arg Met His Ile Arg Arg Lys

305 310 315 320

Arg Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln

325 330 335

Val Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile

340 345 350

Gly Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg Ala Ser Lys

355 360 365

Thr His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp Ile His Pro

370 375 380

Asp Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile Ile Phe His

385 390 395 400

Glu Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu

405 410 415

Met Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile

420 425 430

Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr

435 440 445

Cys Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn Glu Arg Val Phe

450 455 460

Ser Leu Gln Trp Gly Glu Val Lys Leu Ile Ser Asn Cys Ser Lys Phe

465 470 475 480

Tyr Gly Asn Arg Phe Tyr Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr

485 490 495

Asp Gly Ser Ile Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Val

500 505 510

Cys Met Asp Ala Asn Asn Val Thr Tyr Val Trp Gly Val Val Ser Trp

515 520 525

Gly Glu Asn Cys Gly Lys Pro Glu Phe Pro Gly Val Tyr Thr Lys Val

530 535 540

Ala Asn Tyr Phe Asp Trp Ile Ser Tyr His Val Gly Arg Pro Phe Ile

545 550 555 560

Ser Gln Tyr Asn Val Gly Ser Ser Gly Gly Lys Val Thr Tyr Thr Ser

565 570 575

Gln Glu Asp Leu Val Glu Lys Lys Cys Leu Ala Lys Lys Tyr Thr His

580 585 590

Leu Ser Cys Asp Lys Val Phe Cys Gln Pro Trp Gln Arg Cys Ile Glu

595 600 605

Gly Thr Cys Val Cys Lys Leu Pro Tyr Gln Cys Pro Lys Asn Gly Thr

610 615 620

Ala Val Cys Ala Thr Asn Arg Arg Ser Phe Pro Thr Tyr Cys Gln Gln

625 630 635 640

Lys Ser Leu Glu Cys Leu His Pro Gly Thr Lys Phe Leu Asn Asn Gly

645 650 655

Thr Cys Thr Ala Glu Gly Lys Phe Ser Val Ser Leu Lys His Gly Asn

660 665 670

Thr Asp Ser Glu Gly Ile Val Glu Val Lys Leu Val Asp Gln Asp Lys

675 680 685

Thr Met Phe Ile Cys Lys Ser Ser Trp Ser Met Arg Glu Ala Asn Val

690 695 700

Ala Cys Leu Asp Leu Gly Phe Gln Gln Gly Ala Asp Thr Gln Arg Arg

705 710 715 720

Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu His Val

725 730 735

His Cys Arg Gly Leu Glu Thr Ser Leu Ala Glu Cys Thr Phe Thr Lys

740 745 750

Arg Arg Thr Met Gly Tyr Gln Asp Phe Ala Asp Val Val Cys Tyr Thr

755 760 765

Gln Lys Ala Asp Ser Pro Met Asp Asp Phe Phe Gln Cys Val Asn Gly

770 775 780

Lys Tyr Ile Ser Gln Met Lys Ala Cys Asp Gly Ile Asn Asp Cys Gly

785 790 795 800

Asp Gln Ser Asp Glu Leu Cys Cys Lys Ala Cys Gln Gly Lys Gly Phe

805 810 815

His Cys Lys Ser Gly Val Cys Ile Pro Ser Gln Tyr Gln Cys Asn Gly

820 825 830

Glu Val Asp Cys Ile Thr Gly Glu Asp Glu Val Gly Cys Ala Gly Phe

835 840 845

Ala Ser Val Thr Gln Glu Glu Thr Glu Ile Leu Thr Ala Asp Met Asp

850 855 860

Ala Glu Arg Arg Arg Ile Lys Ser Leu Leu Pro Lys Leu Ser Cys Gly

865 870 875 880

Val Lys Asn Arg Met His Ile Arg Arg Lys Arg Ile Val Gly Gly Lys

885 890 895

Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val Ala Ile Lys Asp Ala

900 905 910

Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly Gly Cys Trp Ile Leu

915 920 925

Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr His Arg Tyr Gln Ile

930 935 940

Trp Thr Thr Val Val Asp Trp Ile His Pro Asp Leu Lys Arg Ile Val

945 950 955 960

Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu Asn Tyr Asn Ala Gly

965 970 975

Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met Lys Lys Asp Gly Asn

980 985 990

Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile Pro Ala Cys Val Pro Trp

995 1000 1005

Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr Cys Ile Val Ser Gly

1010 1015 1020

Trp Gly Arg Glu Lys Asp Asn Glu Arg Val Phe Ser Leu Gln Trp

1025 1030 1035

Gly Glu Val Lys Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn

1040 1045 1050

Arg Phe Tyr Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp Gly

1055 1060 1065

Ser Ile Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys

1070 1075 1080

Met Asp Ala Asn Asn Val Thr Tyr Val Trp Gly Val Val Ser Trp

1085 1090 1095

Gly Glu Asn Cys Gly Lys Pro Glu Phe Pro Gly Val Tyr Thr Lys

1100 1105 1110

Val Ala Asn Tyr Phe Asp Trp Ile Ser Tyr His Val Gly Arg Pro

1115 1120 1125

Phe Ile Ser Gln Tyr Asn Val

1130 1135

<210> 17

<211> 575

<212> PRT

<213> Chile person

<400> 17

Val Lys Asn Arg Met His Ile Arg Arg Lys Arg Ile Val Gly Gly Lys

1 5 10 15

Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val Ala Ile Lys Asp Ala

20 25 30

Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly Gly Cys Trp Ile Leu

35 40 45

Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr His Arg Tyr Gln Ile

50 55 60

Trp Thr Thr Val Val Asp Trp Ile His Pro Asp Leu Lys Arg Ile Val

65 70 75 80

Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu Asn Tyr Asn Ala Gly

85 90 95

Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met Lys Lys Asp Gly Asn

100 105 110

Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile Pro Ala Cys Val Pro Trp

115 120 125

Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr Cys Ile Val Ser Gly Trp

130 135 140

Gly Arg Glu Lys Asp Asn Glu Arg Val Phe Ser Leu Gln Trp Gly Glu

145 150 155 160

Val Lys Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn Arg Phe Tyr

165 170 175

Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala

180 185 190

Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asp Ala Asn Asn

195 200 205

Val Thr Tyr Val Trp Gly Val Val Ser Trp Gly Glu Asn Cys Gly Lys

210 215 220

Pro Glu Phe Pro Gly Val Tyr Thr Lys Val Ala Asn Tyr Phe Asp Trp

225 230 235 240

Ile Ser Tyr His Val Gly Arg Pro Phe Ile Ser Gln Tyr Asn Val Gly

245 250 255

Gly Gly Gly Gly Gly Gly Gly Gly Gly Lys Val Thr Tyr Thr Ser Gln

260 265 270

Glu Asp Leu Val Glu Lys Lys Cys Leu Ala Lys Lys Tyr Thr His Leu

275 280 285

Ser Cys Asp Lys Val Phe Cys Gln Pro Trp Gln Arg Cys Ile Glu Gly

290 295 300

Thr Cys Val Cys Lys Leu Pro Tyr Gln Cys Pro Lys Asn Gly Thr Ala

305 310 315 320

Val Cys Ala Thr Asn Arg Arg Ser Phe Pro Thr Tyr Cys Gln Gln Lys

325 330 335

Ser Leu Glu Cys Leu His Pro Gly Thr Lys Phe Leu Asn Asn Gly Thr

340 345 350

Cys Thr Ala Glu Gly Lys Phe Ser Val Ser Leu Lys His Gly Asn Thr

355 360 365

Asp Ser Glu Gly Ile Val Glu Val Lys Leu Val Asp Gln Asp Lys Thr

370 375 380

Met Phe Ile Cys Lys Ser Ser Trp Ser Met Arg Glu Ala Asn Val Ala

385 390 395 400

Cys Leu Asp Leu Gly Phe Gln Gln Gly Ala Asp Thr Gln Arg Arg Phe

405 410 415

Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu His Val His

420 425 430

Cys Arg Gly Leu Glu Thr Ser Leu Ala Glu Cys Thr Phe Thr Lys Arg

435 440 445

Arg Thr Met Gly Tyr Gln Asp Phe Ala Asp Val Val Cys Tyr Thr Gln

450 455 460

Lys Ala Asp Ser Pro Met Asp Asp Phe Phe Gln Cys Val Asn Gly Lys

465 470 475 480

Tyr Ile Ser Gln Met Lys Ala Cys Asp Gly Ile Asn Asp Cys Gly Asp

485 490 495

Gln Ser Asp Glu Leu Cys Cys Lys Ala Cys Gln Gly Lys Gly Phe His

500 505 510

Cys Lys Ser Gly Val Cys Ile Pro Ser Gln Tyr Gln Cys Asn Gly Glu

515 520 525

Val Asp Cys Ile Thr Gly Glu Asp Glu Val Gly Cys Ala Gly Phe Ala

530 535 540

Ser Val Thr Gln Glu Glu Thr Glu Ile Leu Thr Ala Asp Met Asp Ala

545 550 555 560

Glu Arg Arg Arg Ile Lys Ser Leu Leu Pro Lys Leu Ser Cys Gly

565 570 575

<210> 18

<211> 578

<212> PRT

<213> Chile person

<400> 18

Val Lys Asn Arg Met His Ile Arg Arg Lys Arg Ile Val Gly Gly Lys

1 5 10 15

Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val Ala Ile Lys Asp Ala

20 25 30

Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly Gly Cys Trp Ile Leu

35 40 45

Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr His Arg Tyr Gln Ile

50 55 60

Trp Thr Thr Val Val Asp Trp Ile His Pro Asp Leu Lys Arg Ile Val

65 70 75 80

Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu Asn Tyr Asn Ala Gly

85 90 95

Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met Lys Lys Asp Gly Asn

100 105 110

Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile Pro Ala Cys Val Pro Trp

115 120 125

Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr Cys Ile Val Ser Gly Trp

130 135 140

Gly Arg Glu Lys Asp Asn Glu Arg Val Phe Ser Leu Gln Trp Gly Glu

145 150 155 160

Val Lys Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn Arg Phe Tyr

165 170 175

Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala

180 185 190

Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asp Ala Asn Asn

195 200 205

Val Thr Tyr Val Trp Gly Val Val Ser Trp Gly Glu Asn Cys Gly Lys

210 215 220

Pro Glu Phe Pro Gly Val Tyr Thr Lys Val Ala Asn Tyr Phe Asp Trp

225 230 235 240

Ile Ser Tyr His Val Gly Arg Pro Phe Ile Ser Gln Tyr Asn Val Gly

245 250 255

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Lys Val Thr Tyr

260 265 270

Thr Ser Gln Glu Asp Leu Val Glu Lys Lys Cys Leu Ala Lys Lys Tyr

275 280 285

Thr His Leu Ser Cys Asp Lys Val Phe Cys Gln Pro Trp Gln Arg Cys

290 295 300

Ile Glu Gly Thr Cys Val Cys Lys Leu Pro Tyr Gln Cys Pro Lys Asn

305 310 315 320

Gly Thr Ala Val Cys Ala Thr Asn Arg Arg Ser Phe Pro Thr Tyr Cys

325 330 335

Gln Gln Lys Ser Leu Glu Cys Leu His Pro Gly Thr Lys Phe Leu Asn

340 345 350

Asn Gly Thr Cys Thr Ala Glu Gly Lys Phe Ser Val Ser Leu Lys His

355 360 365

Gly Asn Thr Asp Ser Glu Gly Ile Val Glu Val Lys Leu Val Asp Gln

370 375 380

Asp Lys Thr Met Phe Ile Cys Lys Ser Ser Trp Ser Met Arg Glu Ala

385 390 395 400

Asn Val Ala Cys Leu Asp Leu Gly Phe Gln Gln Gly Ala Asp Thr Gln

405 410 415

Arg Arg Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu

420 425 430

His Val His Cys Arg Gly Leu Glu Thr Ser Leu Ala Glu Cys Thr Phe

435 440 445

Thr Lys Arg Arg Thr Met Gly Tyr Gln Asp Phe Ala Asp Val Val Cys

450 455 460

Tyr Thr Gln Lys Ala Asp Ser Pro Met Asp Asp Phe Phe Gln Cys Val

465 470 475 480

Asn Gly Lys Tyr Ile Ser Gln Met Lys Ala Cys Asp Gly Ile Asn Asp

485 490 495

Cys Gly Asp Gln Ser Asp Glu Leu Cys Cys Lys Ala Cys Gln Gly Lys

500 505 510

Gly Phe His Cys Lys Ser Gly Val Cys Ile Pro Ser Gln Tyr Gln Cys

515 520 525

Asn Gly Glu Val Asp Cys Ile Thr Gly Glu Asp Glu Val Gly Cys Ala

530 535 540

Gly Phe Ala Ser Val Thr Gln Glu Glu Thr Glu Ile Leu Thr Ala Asp

545 550 555 560

Met Asp Ala Glu Arg Arg Arg Ile Lys Ser Leu Leu Pro Lys Leu Ser

565 570 575

Cys Gly

<210> 19

<211> 575

<212> PRT

<213> Chile person

<400> 19

Val Lys Asn Arg Met His Ile Arg Arg Lys Arg Ile Val Gly Gly Lys

1 5 10 15

Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val Ala Ile Lys Asp Ala

20 25 30

Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly Gly Cys Trp Ile Leu

35 40 45

Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr His Arg Tyr Gln Ile

50 55 60

Trp Thr Thr Val Val Asp Trp Ile His Pro Asp Leu Lys Arg Ile Val

65 70 75 80

Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu Asn Tyr Asn Ala Gly

85 90 95

Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met Lys Lys Asp Gly Asn

100 105 110

Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile Pro Ala Ser Val Pro Trp

115 120 125

Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr Cys Ile Val Ser Gly Trp

130 135 140

Gly Arg Glu Lys Asp Asn Glu Arg Val Phe Ser Leu Gln Trp Gly Glu

145 150 155 160

Val Lys Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn Arg Phe Tyr

165 170 175

Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala

180 185 190

Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asp Ala Asn Asn

195 200 205

Val Thr Tyr Val Trp Gly Val Val Ser Trp Gly Glu Asn Cys Gly Lys

210 215 220

Pro Glu Phe Pro Gly Val Tyr Thr Lys Val Ala Asn Tyr Phe Asp Trp

225 230 235 240

Ile Ser Tyr His Val Gly Arg Pro Phe Ile Ser Gln Tyr Asn Val Gly

245 250 255

Gly Gly Gly Gly Gly Gly Gly Gly Gly Lys Val Thr Tyr Thr Ser Gln

260 265 270

Glu Asp Leu Val Glu Lys Lys Cys Leu Ala Lys Lys Tyr Thr His Leu

275 280 285

Ser Cys Asp Lys Val Phe Cys Gln Pro Trp Gln Arg Cys Ile Glu Gly

290 295 300

Thr Cys Val Cys Lys Leu Pro Tyr Gln Cys Pro Lys Asn Gly Thr Ala

305 310 315 320

Val Cys Ala Thr Asn Arg Arg Ser Phe Pro Thr Tyr Cys Gln Gln Lys

325 330 335

Ser Leu Glu Cys Leu His Pro Gly Thr Lys Phe Leu Asn Asn Gly Thr

340 345 350

Cys Thr Ala Glu Gly Lys Phe Ser Val Ser Leu Lys His Gly Asn Thr

355 360 365

Asp Ser Glu Gly Ile Val Glu Val Lys Leu Val Asp Gln Asp Lys Thr

370 375 380

Met Phe Ile Cys Lys Ser Ser Trp Ser Met Arg Glu Ala Asn Val Ala

385 390 395 400

Cys Leu Asp Leu Gly Phe Gln Gln Gly Ala Asp Thr Gln Arg Arg Phe

405 410 415

Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu His Val His

420 425 430

Cys Arg Gly Leu Glu Thr Ser Leu Ala Glu Cys Thr Phe Thr Lys Arg

435 440 445

Arg Thr Met Gly Tyr Gln Asp Phe Ala Asp Val Val Cys Tyr Thr Gln

450 455 460

Lys Ala Asp Ser Pro Met Asp Asp Phe Phe Gln Cys Val Asn Gly Lys

465 470 475 480

Tyr Ile Ser Gln Met Lys Ala Cys Asp Gly Ile Asn Asp Cys Gly Asp

485 490 495

Gln Ser Asp Glu Leu Cys Cys Lys Ala Cys Gln Gly Lys Gly Phe His

500 505 510

Cys Lys Ser Gly Val Cys Ile Pro Ser Gln Tyr Gln Cys Asn Gly Glu

515 520 525

Val Asp Cys Ile Thr Gly Glu Asp Glu Val Gly Cys Ala Gly Phe Ala

530 535 540

Ser Val Thr Gln Glu Glu Thr Glu Ile Leu Thr Ala Asp Met Asp Ala

545 550 555 560

Glu Arg Arg Arg Ile Lys Ser Leu Leu Pro Lys Leu Ser Ser Gly

565 570 575

<210> 20

<211> 578

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<213> Chile person

<400> 20

Val Lys Asn Arg Met His Ile Arg Arg Lys Arg Ile Val Gly Gly Lys

1 5 10 15

Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val Ala Ile Lys Asp Ala

20 25 30

Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly Gly Cys Trp Ile Leu

35 40 45

Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr His Arg Tyr Gln Ile

50 55 60

Trp Thr Thr Val Val Asp Trp Ile His Pro Asp Leu Lys Arg Ile Val

65 70 75 80

Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu Asn Tyr Asn Ala Gly

85 90 95

Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met Lys Lys Asp Gly Asn

100 105 110

Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile Pro Ala Ser Val Pro Trp

115 120 125

Ser Pro Tyr Leu Phe Gln Pro Asn Asp Thr Cys Ile Val Ser Gly Trp

130 135 140

Gly Arg Glu Lys Asp Asn Glu Arg Val Phe Ser Leu Gln Trp Gly Glu

145 150 155 160

Val Lys Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn Arg Phe Tyr

165 170 175

Glu Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala

180 185 190

Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asp Ala Asn Asn

195 200 205

Val Thr Tyr Val Trp Gly Val Val Ser Trp Gly Glu Asn Cys Gly Lys

210 215 220

Pro Glu Phe Pro Gly Val Tyr Thr Lys Val Ala Asn Tyr Phe Asp Trp

225 230 235 240

Ile Ser Tyr His Val Gly Arg Pro Phe Ile Ser Gln Tyr Asn Val Gly

245 250 255

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Lys Val Thr Tyr

260 265 270

Thr Ser Gln Glu Asp Leu Val Glu Lys Lys Cys Leu Ala Lys Lys Tyr

275 280 285

Thr His Leu Ser Cys Asp Lys Val Phe Cys Gln Pro Trp Gln Arg Cys

290 295 300

Ile Glu Gly Thr Cys Val Cys Lys Leu Pro Tyr Gln Cys Pro Lys Asn

305 310 315 320

Gly Thr Ala Val Cys Ala Thr Asn Arg Arg Ser Phe Pro Thr Tyr Cys

325 330 335

Gln Gln Lys Ser Leu Glu Cys Leu His Pro Gly Thr Lys Phe Leu Asn

340 345 350

Asn Gly Thr Cys Thr Ala Glu Gly Lys Phe Ser Val Ser Leu Lys His

355 360 365

Gly Asn Thr Asp Ser Glu Gly Ile Val Glu Val Lys Leu Val Asp Gln

370 375 380

Asp Lys Thr Met Phe Ile Cys Lys Ser Ser Trp Ser Met Arg Glu Ala

385 390 395 400

Asn Val Ala Cys Leu Asp Leu Gly Phe Gln Gln Gly Ala Asp Thr Gln

405 410 415

Arg Arg Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu

420 425 430

His Val His Cys Arg Gly Leu Glu Thr Ser Leu Ala Glu Cys Thr Phe

435 440 445

Thr Lys Arg Arg Thr Met Gly Tyr Gln Asp Phe Ala Asp Val Val Cys

450 455 460

Tyr Thr Gln Lys Ala Asp Ser Pro Met Asp Asp Phe Phe Gln Cys Val

465 470 475 480

Asn Gly Lys Tyr Ile Ser Gln Met Lys Ala Cys Asp Gly Ile Asn Asp

485 490 495

Cys Gly Asp Gln Ser Asp Glu Leu Cys Cys Lys Ala Cys Gln Gly Lys

500 505 510

Gly Phe His Cys Lys Ser Gly Val Cys Ile Pro Ser Gln Tyr Gln Cys

515 520 525

Asn Gly Glu Val Asp Cys Ile Thr Gly Glu Asp Glu Val Gly Cys Ala

530 535 540

Gly Phe Ala Ser Val Thr Gln Glu Glu Thr Glu Ile Leu Thr Ala Asp

545 550 555 560

Met Asp Ala Glu Arg Arg Arg Ile Lys Ser Leu Leu Pro Lys Leu Ser

565 570 575

Ser Gly

<210> 21

<211> 1156

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<213> Chile person

<400> 21

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser Ser Gly Gly Lys

580 585 590

Val Thr Tyr Thr Ser Gln Glu Asp Leu Val Glu Lys Lys Cys Leu Ala

595 600 605

Lys Lys Tyr Thr His Leu Ser Cys Asp Lys Val Phe Cys Gln Pro Trp

610 615 620

Gln Arg Cys Ile Glu Gly Thr Cys Val Cys Lys Leu Pro Tyr Gln Cys

625 630 635 640

Pro Lys Asn Gly Thr Ala Val Cys Ala Thr Asn Arg Arg Ser Phe Pro

645 650 655

Thr Tyr Cys Gln Gln Lys Ser Leu Glu Cys Leu His Pro Gly Thr Lys

660 665 670

Phe Leu Asn Asn Gly Thr Cys Thr Ala Glu Gly Lys Phe Ser Val Ser

675 680 685

Leu Lys His Gly Asn Thr Asp Ser Glu Gly Ile Val Glu Val Lys Leu

690 695 700

Val Asp Gln Asp Lys Thr Met Phe Ile Cys Lys Ser Ser Trp Ser Met

705 710 715 720

Arg Glu Ala Asn Val Ala Cys Leu Asp Leu Gly Phe Gln Gln Gly Ala

725 730 735

Asp Thr Gln Arg Arg Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr

740 745 750

Glu Cys Leu His Val His Cys Arg Gly Leu Glu Thr Ser Leu Ala Glu

755 760 765

Cys Thr Phe Thr Lys Arg Arg Thr Met Gly Tyr Gln Asp Phe Ala Asp

770 775 780

Val Val Cys Tyr Thr Gln Lys Ala Asp Ser Pro Met Asp Asp Phe Phe

785 790 795 800

Gln Cys Val Asn Gly Lys Tyr Ile Ser Gln Met Lys Ala Cys Asp Gly

805 810 815

Ile Asn Asp Cys Gly Asp Gln Ser Asp Glu Leu Cys Cys Lys Ala Cys

820 825 830

Gln Gly Lys Gly Phe His Cys Lys Ser Gly Val Cys Ile Pro Ser Gln

835 840 845

Tyr Gln Cys Asn Gly Glu Val Asp Cys Ile Thr Gly Glu Asp Glu Val

850 855 860

Gly Cys Ala Gly Phe Ala Ser Val Thr Gln Glu Glu Thr Glu Ile Leu

865 870 875 880

Thr Ala Asp Met Asp Ala Glu Arg Arg Arg Ile Lys Ser Leu Leu Pro

885 890 895

Lys Leu Ser Cys Gly Val Lys Asn Arg Met His Ile Arg Arg Lys Arg

900 905 910

Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val

915 920 925

Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly

930 935 940

Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg Ala Ser Lys Thr

945 950 955 960

His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp Ile His Pro Asp

965 970 975

Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile Ile Phe His Glu

980 985 990

Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile Ala Leu Ile Glu Met

995 1000 1005

Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu Pro Arg Ser Ile

1010 1015 1020

Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro Asn Asp

1025 1030 1035

Thr Cys Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn Glu Arg

1040 1045 1050

Val Phe Ser Leu Gln Trp Gly Glu Val Lys Leu Ile Ser Asn Cys

1055 1060 1065

Ser Lys Phe Tyr Gly Asn Arg Phe Tyr Glu Lys Glu Met Glu Cys

1070 1075 1080

Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala Cys Lys Gly Asp Ser

1085 1090 1095

Gly Gly Pro Leu Val Cys Met Asp Ala Asn Asn Val Thr Tyr Val

1100 1105 1110

Trp Gly Val Val Ser Trp Gly Glu Asn Cys Gly Lys Pro Glu Phe

1115 1120 1125

Pro Gly Val Tyr Thr Lys Val Ala Asn Tyr Phe Asp Trp Ile Ser

1130 1135 1140

Tyr His Val Gly Arg Pro Phe Ile Ser Gln Tyr Asn Val

1145 1150 1155

<210> 22

<211> 1376

<212> PRT

<213> Chile person

<400> 22

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys

20 25 30

Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala

35 40 45

Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn

50 55 60

Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu

65 70 75 80

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr

85 90 95

Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala

100 105 110

Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp

115 120 125

Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys

130 135 140

Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr

145 150 155 160

Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe

165 170 175

Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala

180 185 190

Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu

195 200 205

Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln

210 215 220

Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser

225 230 235 240

Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr

245 250 255

Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu

260 265 270

Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln

275 280 285

Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu

290 295 300

Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala

305 310 315 320

Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys

325 330 335

Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr

340 345 350

Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg

355 360 365

Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala

370 375 380

Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu

385 390 395 400

Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu

405 410 415

Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr

420 425 430

Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg

435 440 445

Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys

450 455 460

Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu

465 470 475 480

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys

485 490 495

Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu

500 505 510

Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr

515 520 525

Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys

530 535 540

Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

545 550 555 560

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu

565 570 575

Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly

580 585 590

Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser

595 600 605

Ser Gly Gly Lys Val Thr Tyr Thr Ser Gln Glu Asp Leu Val Glu Lys

610 615 620

Lys Cys Leu Ala Lys Lys Tyr Thr His Leu Ser Cys Asp Lys Val Phe

625 630 635 640

Cys Gln Pro Trp Gln Arg Cys Ile Glu Gly Thr Cys Val Cys Lys Leu

645 650 655

Pro Tyr Gln Cys Pro Lys Asn Gly Thr Ala Val Cys Ala Thr Asn Arg

660 665 670

Arg Ser Phe Pro Thr Tyr Cys Gln Gln Lys Ser Leu Glu Cys Leu His

675 680 685

Pro Gly Thr Lys Phe Leu Asn Asn Gly Thr Cys Thr Ala Glu Gly Lys

690 695 700

Phe Ser Val Ser Leu Lys His Gly Asn Thr Asp Ser Glu Gly Ile Val

705 710 715 720

Glu Val Lys Leu Val Asp Gln Asp Lys Thr Met Phe Ile Cys Lys Ser

725 730 735

Ser Trp Ser Met Arg Glu Ala Asn Val Ala Cys Leu Asp Leu Gly Phe

740 745 750

Gln Gln Gly Ala Asp Thr Gln Arg Arg Phe Lys Leu Ser Asp Leu Ser

755 760 765

Ile Asn Ser Thr Glu Cys Leu His Val His Cys Arg Gly Leu Glu Thr

770 775 780

Ser Leu Ala Glu Cys Thr Phe Thr Lys Arg Arg Thr Met Gly Tyr Gln

785 790 795 800

Asp Phe Ala Asp Val Val Cys Tyr Thr Gln Lys Ala Asp Ser Pro Met

805 810 815

Asp Asp Phe Phe Gln Cys Val Asn Gly Lys Tyr Ile Ser Gln Met Lys

820 825 830

Ala Cys Asp Gly Ile Asn Asp Cys Gly Asp Gln Ser Asp Glu Leu Cys

835 840 845

Cys Lys Ala Cys Gln Gly Lys Gly Phe His Cys Lys Ser Gly Val Cys

850 855 860

Ile Pro Ser Gln Tyr Gln Cys Asn Gly Glu Val Asp Cys Ile Thr Gly

865 870 875 880

Glu Asp Glu Val Gly Cys Ala Gly Phe Ala Ser Val Thr Gln Glu Glu

885 890 895

Thr Glu Ile Leu Thr Ala Asp Met Asp Ala Glu Arg Arg Arg Ile Lys

900 905 910

Ser Leu Leu Pro Lys Leu Ser Cys Gly Val Lys Asn Arg Met His Ile

915 920 925

Arg Arg Lys Arg Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu

930 935 940

Pro Trp Gln Val Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly

945 950 955 960

Ile Tyr Ile Gly Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg

965 970 975

Ala Ser Lys Thr His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp

980 985 990

Ile His Pro Asp Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile

995 1000 1005

Ile Phe His Glu Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile

1010 1015 1020

Ala Leu Ile Glu Met Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu

1025 1030 1035

Leu Pro Arg Ser Ile Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu

1040 1045 1050

Phe Gln Pro Asn Asp Thr Cys Ile Val Ser Gly Trp Gly Arg Glu

1055 1060 1065

Lys Asp Asn Glu Arg Val Phe Ser Leu Gln Trp Gly Glu Val Lys

1070 1075 1080

Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn Arg Phe Tyr Glu

1085 1090 1095

Lys Glu Met Glu Cys Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala

1100 1105 1110

Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asp Ala Asn

1115 1120 1125

Asn Val Thr Tyr Val Trp Gly Val Val Ser Trp Gly Glu Asn Cys

1130 1135 1140

Gly Lys Pro Glu Phe Pro Gly Val Tyr Thr Lys Val Ala Asn Tyr

1145 1150 1155

Phe Asp Trp Ile Ser Tyr His Val Gly Arg Pro Phe Ile Ser Gln

1160 1165 1170

Tyr Asn Val Gly Gly Ser Ser Gly Gly Gly His Cys Gln Ala Pro

1175 1180 1185

Asp His Phe Leu Phe Ala Lys Leu Lys Thr Gln Thr Asn Ala Ser

1190 1195 1200

Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr Glu Cys Arg Pro Glu

1205 1210 1215

Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu Asp Asn Leu Val

1220 1225 1230

Trp Ser Ser Pro Lys Asp Val Cys Lys Arg Lys Ser Cys Lys Thr

1235 1240 1245

Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Thr Asp Ile

1250 1255 1260

Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr Gly His Arg

1265 1270 1275

Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly Asn Ala

1280 1285 1290

Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro Cys

1295 1300 1305

Gly Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn

1310 1315 1320

Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn

1325 1330 1335

Pro Gly Ser Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro

1340 1345 1350

Ser Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser

1355 1360 1365

Gly Pro Ala Pro Gln Cys Ile Ile

1370 1375

<210> 23

<211> 603

<212> PRT

<213> Chile person

<400> 23

Met Lys Leu Ala His Leu Ser Leu Phe Leu Leu Ala Leu His Leu Ser

1 5 10 15

Ser Ser Arg Ser Pro Ser Ala Ser Asp Leu Pro Gln Glu Glu Leu Val

20 25 30

Asp Gln Lys Cys Leu Leu Gln Lys Tyr Thr His Arg Ser Cys Asn Lys

35 40 45

Val Phe Cys Gln Pro Trp Gln Arg Cys Ile Glu Gly Thr Cys Ile Cys

50 55 60

Lys Leu Pro Tyr Gln Cys Pro Arg Ala Gly Thr Pro Val Cys Ala Met

65 70 75 80

Asn Gly Arg Ser Tyr Pro Thr Tyr Cys His Gln Lys Ser Phe Glu Cys

85 90 95

Leu His Pro Glu Ile Lys Phe Ser His Asn Gly Thr Cys Ala Ala Glu

100 105 110

Gly Lys Phe Asn Val Ser Leu Ile Tyr Gly Arg Thr Lys Thr Glu Gly

115 120 125

Leu Val Gln Val Lys Leu Val Asp Gln Asp Glu Arg Met Phe Ile Cys

130 135 140

Lys Asn Ser Trp Ser Met Ala Glu Ala Asn Val Ala Cys Val Asp Leu

145 150 155 160

Gly Phe Pro Leu Gly Val Arg Asp Ile Gln Gly Ser Phe Asn Ile Ser

165 170 175

Gly Asn Leu His Ile Asn Asp Thr Glu Cys Leu His Val His Cys Arg

180 185 190

Gly Val Glu Thr Ser Leu Ala Glu Cys Ala Phe Thr Lys Arg Arg Thr

195 200 205

Glu Leu Ser Asn Gly Leu Ala Gly Val Val Cys Tyr Lys Gln Asp Ala

210 215 220

Asp Phe Pro Thr Ser Leu Ser Phe Gln Cys Val Asn Gly Lys His Ile

225 230 235 240

Pro Gln Glu Lys Ala Cys Asn Gly Val Asn Asp Cys Gly Asp Gln Ser

245 250 255

Asp Glu Leu Cys Cys Lys Gly Cys Arg Gly Asn Ala Ser Leu Cys Lys

260 265 270

Ser Gly Val Cys Ile Pro Asp Gln Tyr Lys Cys Asn Gly Glu Val Asp

275 280 285

Cys Ile Thr Gly Glu Asp Glu Ser Arg Cys Glu Glu Asp Arg Gln Gln

290 295 300

Asn Ile Pro Lys Gly Leu Ala Arg Ser Ala Gln Gly Glu Ala Glu Ile

305 310 315 320

Glu Thr Glu Glu Thr Glu Met Leu Thr Pro Gly Met Asp Asn Glu Arg

325 330 335

Lys Arg Ile Lys Ser Leu Leu Pro Lys Leu Ser Cys Gly Val Lys Arg

340 345 350

Asn Thr His Thr Arg Arg Lys Arg Val Ile Gly Gly Lys Pro Ala Asn

355 360 365

Val Gly Asp Tyr Pro Trp Gln Val Ala Ile Lys Asp Gly Gln Arg Ile

370 375 380

Thr Cys Gly Gly Ile Tyr Ile Gly Gly Cys Trp Ile Leu Thr Ala Ala

385 390 395 400

His Cys Val Arg Pro Ser Arg Ala His Ser Tyr Gln Val Trp Thr Ala

405 410 415

Leu Leu Asp Trp Leu Lys Pro Asn Ser Gln Leu Gly Ile Gln Thr Val

420 425 430

Lys Arg Val Ile Val His Glu Lys Tyr Asn Gly Ala Thr Phe Gln Asn

435 440 445

Asp Ile Ala Leu Ile Glu Met Lys Met His Thr Gly Lys Lys Glu Cys

450 455 460

Glu Leu Pro Asn Ser Val Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu

465 470 475 480

Phe Gln Pro Asn Asp Arg Cys Ile Ile Ser Gly Trp Gly Arg Gly Lys

485 490 495

Asp Asn Gln Lys Val Tyr Ser Leu Arg Trp Gly Glu Val Asp Leu Ile

500 505 510

Gly Asn Cys Ser Gln Phe Tyr Pro Asp Arg Tyr Tyr Glu Lys Glu Met

515 520 525

Gln Cys Ala Gly Thr Arg Asp Gly Ser Ile Asp Ala Cys Lys Gly Asp

530 535 540

Ser Gly Gly Pro Leu Val Cys Glu Asp Ile Asn Asn Val Thr Tyr Val

545 550 555 560

Trp Gly Ile Val Ser Trp Gly Glu Asn Cys Gly Lys Pro Glu Phe Pro

565 570 575

Gly Val Tyr Thr Arg Val Ala Asn Tyr Phe Asp Trp Ile Ser Tyr His

580 585 590

Val Gly Arg Ser Leu Val Ser Gln His Asn Val

595 600

<210> 24

<211> 20

<212> PRT

<213> Chile person

<400> 24

Glu Asp Val Pro Ala Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Asp

1 5 10 15

Ser Glu Thr Arg

20

<210> 25

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 25

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10

<210> 26

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 26

Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly

1 5 10

<210> 27

<211> 9

<212> PRT

<213> Chile person

<400> 27

Arg Pro Phe Ile Ser Gln Tyr Asn Val

1 5

<210> 28

<211> 10

<212> PRT

<213> Chile person

<400> 28

Ser Glu Glu Thr Lys Gln Asn Glu Gly Phe

1 5 10

Claims (186)

1. A Complement Factor I (CFI) variant comprising at least one modification relative to a wild-type CFI, wherein the CFI variant is capable of modulating the complement system, and wherein the CFI variant has at least one improved feature compared to a wild-type CFI.

2. The CFI variant of claim 1 wherein the improved feature 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 capacity.

3. The CFI variant of claim 2 wherein the improvement is characterized by an increase in activity.

4. A CFI variant according to claim 3 wherein the increased activity comprises increased cleavage of C3b and/or C4b compared to wild-type CFI.

5. The CFI variant of claim 4 wherein the increase in activity comprises an increase in cleavage of C3b and does not comprise an increase in cleavage of C4 b.

6. The CFI variant of any of claims 4 to 5, wherein the increase in cleavage of 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 greater than the wild-type CFI.

7. The CFI variant of claim 4 wherein the increase in activity comprises an increase in cleavage of C4b and no increase in cleavage of C3b compared to the wild-type CFI.

8. The CFI variant of any of claims 4 and 7, wherein the increase in cleavage of 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 greater than the wild-type CFI.

9. The CFI variant of claim 4 wherein the increase in cleavage of C3b and C4b is each 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 increase as compared to wild-type CFI.

10. A CFI variant according to any one of claims 3 to 6 and 9 wherein the increased activity comprises increased production of iC3 b.

11. A CFI variant according to any one of claims 3 to 6 and 10 to 10, wherein the increase in activity comprises an increase in C3dg and/or C3C generated from iC3 b.

12. A CFI variant according to any one of claims 3 to 6 and 10 to 11 wherein the increased activity comprises a reduced content of C3b alpha chains.

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

14. A CFI variant according to any one of claims 3 to 13 wherein the increased activity comprises a decreased content or function of a tapping complex (MAC).

15. The CFI variant of any of claims 3 to 14, wherein the increase in activity results in a decrease in amplification of the complement system.

16. The CFI variant of claim 2 wherein the improvement is characterized by a decrease in activity of C3b and/or C4 b.

17. The CFI variant of claim 2 wherein the improvement is characterized by an increase in substrate specificity.

18. The CFI variant of claim 17 wherein the increase in specificity comprises an increase in specificity of C3b or C4b as compared to wild-type CFI.

19. The CFI variant of claim 17 wherein the increase in specificity comprises an increase in specificity of C3b and/or C4b as compared to wild-type CFI.

20. The CFI variant of claim 17 wherein the increase in specificity comprises an increase in specificity of C3b as compared to wild-type CFI.

21. The CFI variant of claim 17 wherein the increase in specificity comprises an increase in specificity of C4b as compared to wild-type CFI.

22. The CFI variant of claim 18, wherein the increase in specificity of 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 greater than the wild-type CFI.

23. The CFI variant of claim 18, wherein the increase in specificity of 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 greater than the wild-type CFI.

24. A CFI variant according to any one of claims 1 to 23 wherein the modification to wild-type CFI comprises any one or more of: 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 CFI domains, and exchange of one or more CFI domains.

25. The CFI variant of any one of claims 1-24, wherein the CFI variant comprises any one or more of the modifications presented in tables 2-9 and table 13.

26. A CFI variant according to any one of claims 1 to 24, wherein the CFI variant comprises any one or more domains of a CFI selected from the group consisting of: serine Protease Domain (SPD), factor I tapping complex (FIMAC) domain, SRCR domain, low density lipoprotein receptor 1 (LDLr 1) domain, and low density lipoprotein receptor 2 (LDLr 2) domain.

27. The CFI variant of any one of claims 1 to 26, wherein the CFI variant comprises at least one modification corresponding to wild-type human CFI.

28. The CFI variant of any one of claims 1 to 26, wherein the CFI variant comprises at least one modification corresponding to wild-type non-human CFI.

29. A CFI variant according to any one of claims 1 to 26, wherein the CFI variant comprises at least one modification corresponding to a wild-type CFI having the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 5.

30. The CFI variant of any one of claims 1 to 29, wherein the CFI variant is a chimera comprising one or more domains from a human CFI, and wherein the human CFI further comprises a substitution of one or more amino acid residues for amino acid residues from a corresponding region of a non-human species CFI.

31. The CFI variant of claim 30 wherein the non-human species is a mouse.

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

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

34. The CFI variant of any one of claims 1-33, comprising an a-strand and a B-strand, wherein the CFI variant comprises one or more modifications at the interface of the a-strand and the B-strand.

35. The CFI variant of claim 34 comprising any one or more of the modifications presented in table 2.

36. A CFI variant according to any one of claims 34 to 35 comprising a modification at any one or more of positions K14, Y20, D26, F29, R35, E38, M220, K221, S250, L304, P305, K306, L307 and S308 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

37. A CFI variant according to any one of claims 34 to 36 comprising a substitution of the loop 200 of trypsin having amino acid residues NG in the loop 200 of CFI (SEQ ID NO: 13), wherein the loop 200 is located between positions corresponding to positions 514 and 520 in CFI having the amino acid sequence set forth in SEQ ID NO: 5.

38. A CFI variant according to any one of claims 34 to 37 comprising any one or more of the substitutions selected from K14A, Y20A, Y F, D3526A, F29A, R35A, E3538A, M220A, K221Q, S250A, S L, L304G, P305G, K G, L307G and S308G, wherein said position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

39. The CFI variant according to any one of claims 35 to 38 comprising the substitution M220A; K221Q and L304G; P305G; K306G; L307G; any one or more of the combinations of S308G, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ id No. 5.

40. A CFI variant according to any one of claims 1 to 33 comprising one or more modifications at the C-terminal region of the CFI variant.

41. A CFI variant according to claim 40 comprising any one or more of the modifications presented in Table 3.

42. The CFI variant of any of claims 40 to 41, comprising a modification at any one or more positions corresponding to positions T377, W381, P384, Y403, a405, G406, Y408, Q409, D425, G556, R557, P558, P559, I560 and Y563 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

43. The CFI variant of any of claims 40-42, wherein the one or more modifications at the C-terminal region are deletions of amino acid residues (PFISQYNV, SEQ ID NO: 14) between positions 558-565 in a CFI corresponding to the amino acid sequence set forth in SEQ ID NO: 5.

44. A CFI variant according to any one of claims wherein the linker is substituted with an amino acid residue (DGNK, SEQ ID NO: 15) between positions 420 to 424 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

45. A CFI variant according to any one of claims 40 to 44 comprising any one or more of substitutions selected from T377G, W381A, P384A, P384G, Y403F, A405S, G406R, G406 42408L, Q409D, Q409H, D425A, D425K, D425R, G556S, R557A, R557 558G, P558L, P558S, F559L, I560V and Y563H and/or a deletion of P384, wherein said position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID NO 5.

46. A CFI variant according to any one of claims 1 to 33 comprising one or more modifications at one or more N-linked glycosylation sites of the CFI.

47. A variant CFI according to claim 46, wherein said one or more modifications is the removal of an N-linked glycosylation site.

48. The CFI variant of claim 46 comprising any one or more of the modifications presented in table 4.

49. A CFI variant according to any one of claims 46 to 48 comprising a modification at any one or more of positions N52, N85, N159, N446, N476 and N518 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

50. A CFI variant according to any one of claims 46 to 49 comprising any one or more of the substitutions selected from N52Q, N85Q, N159Q, N446Q, N476Q and N518Q, wherein said position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

51. A CFI variant according to any one of claims 46 to 50 comprising a polypeptide selected from N52Q; N85Q; N159Q, N446Q; N476Q; any one or more of the combinations of substitutions of N518Q, wherein the positions correspond to positions in CFI having the amino acid sequence set forth in SEQ id No. 5.

52. A CFI variant according to any one of claims 1 to 33 comprising one or more modifications in the SPD domain of the CFI.

53. The CFI variant of claim 52 comprising any one or more of the modifications presented in table 5.

54. The CFI variant of any of claims 52-53, comprising one or more modifications at any one or more of the autolytic loop, loop No. 99, S1 pocket entry or activation loop of the SPD or any one or more of the domains presented in fig. 1.

55. The CFI variant of any of claims 52-54, comprising a modification at any one or more of positions K14, K312, R314, I322, V323, K326, R327, a328, K340, D341, G344, I345, T346, a361, L364, Y372, W381, P384, V390, N402, N404, G406, Y408, Q409, E416, K418, N422, D425, E457, K458, R456, E461, R462, F464, S465, Q467, W468, G469, T495, Y496, D497, S499, I500, a502, K504, D506, S507, E530, N531, G533, K534, P535, E536, and F537 in a CFI corresponding CFI having the amino acid sequence set forth in SEQ ID NO 5.

56. The CFI variant of any of claims 52 to 55, comprising a substitution of the trypsin's autolytic loop (NTASSGADYPDE, SEQ ID NO: 10) by the CFI autolytic loop (REKDNERVFS, SEQ ID NO: 9), wherein the autolytic loop is located between positions corresponding to positions 456 and 465 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

57. The CFI variant of any of claims 52-55, comprising a substitution of the autolytic loop of the CFI (REKDNERVFS, SEQ ID NO: 9) for the autolytic loop of a mouse CFI (RGKDNQKVYS, SEQ ID NO: 11), wherein the autolytic loop is located between positions corresponding to position 456 and position 465 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.

58. A CFI variant according to any one of claims 52 to 57 comprising any one or more of the substitutions selected from: k14,312,314,322,322,323,323,323,327,327,340,341,344,344,346,346,346,372,381,381,384,384,384,384,406,406,406,406,406,406,406,406,406,408,408,418,425, 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 461 495 497.5.90.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.504.506.506.506.530.530.531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 531 or 533.535 536.536K and F537R, wherein said position corresponds to a sequence having the amino acid sequence of SEQ ID NO:5, and the position in CFI of the amino acid sequence set forth in SEQ ID NO.

59. A CFI variant according to any one of claims 52 to 58 comprising any one or more of the combination substitutions selected from: K326A; R327A, N531G; P535A, E457G; E461Q; R462K; F464Y, Y L; N531G; E457G, Y L; N531G; E457G; E461Q, Y L; N531G; E457G; E461Q-R462K; F464Y, Y L; N531G; P535A, K a; d425R, E D; N531G; G533A; K534Q; P535K; E536N, A S502; K504Q; F537K, T F; Y496L; D497E; S499G; I500K, G533A; K534Q; P535K; E536N; F537K, T F; Y496L; D497E; S499G; I500K; G533A; K534Q; P535K; E536N; F537K, Q467K; F537K, E G530; N531G, E D; F537K, E457G; E461Q, E457G; E461G, Y L; N531G; E457G; E461Q, N531G; E457G; E461Q, I V322; V323I, I V322; V323I; R327P, A328C; W468C, A328C; W468C; K326Y; R327N, Y L; N531G; E461Q, Y L; N531G; E457G; E461Q; R462K, Y L; N531G; E457G; E461Q; F464Y, Y L; N531G; E457G; R462K; F464Y, Y L; N531G; E461Q; R462K; F464Y, Y L; E457G; E461Q; R462K; F464Y, E457G; N531G; E461Q; R462K; F464Y, Y L; E457G; E461Q; R462K, N531G; E457G; E461Q; F464Y, E416A; d425R, Y L; N531G; E457G; E461Q; R462K; F464Y; S507A, E457G; E461G, K312A; R314A, G469L; R456N; E457T; k458A, G469L; R456N; k458A, G469L; R456N; K458A; e461G, G469L; R456N; K458A; E461G; F537K, G406D; Y408L, G D; N531G, G D; P535A, G406D; Y408L; N531G, G D; Y408L; P535A, G406D; N531G; P535A, G406D; Y408L; N531G; P535A, K G; I345G, L364G; Y372G, W381G; V390G, W381G; P384A; V390G, W381G; P384G; V390G, N G; Q409G, K G; d425G, T346R; K504E; E530R, T K; K504D; E530K, G344R; Y408L; N531G, G344K; Y408L; N531G, T346R; Y408L; N531G, T K; Y408L; N531G, K D; Y408L; N531G, K E; Y408L; N531G, Y L; E530R; N531G, Y L; E530K; N531G, T346R; Y408L; K504E; E530R; N531G, T K; Y408L; K504D; E530K; N531G, Y L; S507A; N531G, Y L; N531G; E457G; E461Q; R462K; F464Y; S507A, E457G; S507A and N531G; P535A; S507A, wherein the position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

60. A CFI variant according to any one of claims 1 to 33 comprising one or more modifications at the active site of the CFI.

61. The CFI variant of claim 60 comprising any modification presented in table 6.

62. The CFI variant of any of claims 60 to 61, comprising a modification at a position corresponding to position S507 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

63. The CFI variant of any of claims 60 to 62, comprising substitution S507A, wherein the position corresponds to position S507 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

64. The CFI variant of any of claims 1-33 comprising an a-chain and a B-chain, wherein the CFI variant comprises a structural configuration in the form of (a-chain) - (optionally linker) - (B-chain) from N-terminus to C-terminus.

65. The CFI variant of any of claims 1 to 33 comprising an a-chain and a B-chain, wherein the CFI variant comprises a structural configuration in the form of (B-chain) - (optionally linker) - (a-chain) from N-terminus to C-terminus.

66. The CFI variant of any of claims 64-65, comprising a modification at one or more of C309 and C435, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

67. A CFI variant according to any one of claims 64 to 66 comprising the substitution C309S; C435S, wherein said position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID NO. 5.

68. A CFI variant according to any one of claims 64 to 67 wherein the B chain and the a chain are further linked by a disulfide bond.

69. A variant CFI according to claim 68 comprising the amino acid sequence set forth in SEQ ID NO. 17 or SEQ ID NO. 18.

70. The CFI variant of any of claims 64-67, wherein the B strand and the a strand are not further linked by a disulfide bond.

71. The CFI variant according to claim 70, comprising the amino acid sequence set forth in SEQ ID NO. 19 or SEQ ID NO. 20.

72. A CFI variant according to any one of claims 64 to 67 comprising any one or more of the modifications presented in table 7.

73. A CFI variant according to any one of claims 1 to 33, wherein the CFI variant is more susceptible to activation than the wild-type CFI.

74. The CFI variant of claim 73, comprising a modification at any one or more of positions I317, R318, R319, K320 and R321 in a CFI corresponding to the amino acid sequence set forth in SEQ ID No. 5.

75. A CFI variant according to any one of claims 73 to 74, comprising any one or more of the substitutions selected from I317D, R318D, R319D, K320D and R321K, wherein said positions correspond to those in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

76. The CFI variant of claim 75, comprising the substitutions I317D, R318D, R319D, K320D and R321K, wherein said positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

77. A CFI variant according to any one of claims 1 to 76 comprising any two or more modifications according to claims 34 to 76.

78. A CFI variant according to any one of claims 1 to 77 comprising any one or more of the modifications presented in table 9.

79. A CFI variant according to claim 77 comprising any one or more of the following combinations of substitutions selected from: y408; N531G, E a; d425R, Y F; d425R, S a; d425R, Y F; N531G, Y L; N531G; E457G; E461Q; R462K; F464Y, K a; Y20F, K a; E38A, K a; S250A, K a; d425A, Y F; E38A, Y F; S250A, Y F; d425A, E a; S250A, E a; d425A, S a; d425A, K a; N531G; P535A, Y F; N531G; P535A, E a; N531G; P535A, S a; N531G; P535A, D a; N531G; P535A, Y F; Y408L; N531G; E457G; E461Q; R462K; F464Y, E a; Y408L; N531G; E457G; E461Q; R462K; F464Y, S a; Y408L; N531G; E457G; E461Q; R462K; F464Y, D425R; Y408L; N531G; E457G; E461Q; R462K; F464Y, Y F; E38A; S250A; d425A, Y F; E38A; S250A; d425A; Y408L; N531G; E457G; E461Q; R462K; F464Y, Y F; E38A; S250A; d425A; Y408L; N531G; E457G; e461Q, I317D; R318D; R319D; K320D; R321K; E457G; E461Q-R462K; F464Y, I317D; R318D; R319D; K320D; R321K; E457G; E461Q-R462K; F464Y, I317D; R318D; R319D; K320D; R321K; Y408L; N531G; E457G; E461Q; R462K; F464Y, K504D; Y408L; N531G, K E; Y408L; N531G, E457G; N531G; d425K, Y F; N531G, Y L; E457G; N531G; d425K, Y L; E457G; P535G; d425K, Y L; E457G; N531G; K534Q, Y L; N531G, R462K; F464Y and Y408L; P535G; d425K, wherein said position corresponds to a position in CFI having the amino acid sequence set forth in SEQ ID No. 5.

80. The CFI variant according to any of claims 1-33, wherein the CFI variant comprises each of an SPD, FIMAC domain, SRCR domain, LDLr1 domain, and LDLr2 domain, and any other domain presented in fig. 1.

81. The CFI variant according to any of claims 1-33, wherein the CFI variant does not comprise all of an SPD, FIMAC domain, SRCR domain, LDLr1 domain, and LDLr2 domain.

82. The CFI variant of claim 81, wherein the CFI variant comprises an SPD.

83. The CFI variant of claim 82, comprising the amino acid sequence set forth in SEQ ID No. 12.

84. The CFI variant of claim 1 comprising or consisting of any one or more of the modifications presented in table 13.

85. A CFI variant according to any one of claims 1 to 84, wherein the CFI variant is sialylated.

86. A CFI variant according to any one of claims 1 to 85, wherein the CFI variant is further sialylated compared to wild type CFI.

87. A CFI variant according to any one of claims 1 to 86, wherein the CFI variant is active.

88. The CFI variant of claim 87, wherein the CFI variant is activated by furin (furin) or a variant thereof.

89. The CFI variant of claim 88, wherein the CFI variant is activated in vitro by furin or a variant thereof.

90. The CFI variant of claim 88, wherein the CFI variant is activated by furin or variant thereof during production in a host cell.

91. The CFI variant of claim 90, wherein activation by furin or variant thereof during production in a host cell is by overexpression of furin or variant thereof.

92. The CFI variant of claim 88, wherein the CFI variant is activated by furin or a variant thereof after production and secretion by a host cell, optionally in culture medium.

93. A CFI variant according to any one of claims 1 to 92, wherein the CFI variant is a first component of a fusion construct comprising a first component and a second component, and the CFI variant is fused to the second component.

94. A CFI variant according to claim 93 wherein the second component is a protein.

95. A CFI variant according to claim 93 wherein the second component is not a protein.

96. A CFI variant according to any one of claims 93 to 95, wherein the second component is a half-life extender.

97. A CFI variant according to claim 96 wherein the half-life extender comprises a peptide repeat.

98. The CFI variant of claim 93 wherein the second component is a half-life extender selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc.

99. A CFI variant according to claim 98, wherein the half-life extender is a modified albumin or albumin derivative.

100. A CFI variant according to claim 98, wherein the half-life extender is wild-type albumin.

101. The CFI variant of claim 98, wherein the half-life extender is human serum albumin or a variant thereof.

102. The CFI variant of any of claims 93-101, wherein the CFI variant comprises an a-chain and a B-chain, and wherein the fusion construct comprises a structural configuration in the form of (second component) - (optional linker) - (a-chain) - (optional linker) - (B-chain) from N-terminus to C-terminus or C-terminus to N-terminus.

103. The CFI variant of any of claims 93-101, wherein the CFI variant comprises an a-chain and a B-chain, and wherein the fusion construct comprises a structural configuration in the form of (second component) - (optional linker) - (B-chain) - (optional linker) - (a-chain) from N-terminus to C-terminus or C-terminus to N-terminus.

104. A CFI variant according to claim 101 comprising the amino acid sequence set forth in SEQ ID No. 21.

105. A CFI variant according to claim 93 wherein the second component is at least one domain or part of a domain of factor H.

106. The CFI variant of claim 105, wherein at least one factor H domain comprises any one or more of the Complement Control Protein (CCP) domains 1-20 of factor H.

107. A CFI variant according to any one of claims 105 to 106, wherein the amino acid sequence of the at least one factor H domain is or is derived from the sequence set forth in SEQ ID No. 4.

108. The CFI variant of any of claims 105-107, wherein the at least one factor H domain comprises each of CCP domains 1-20 of factor H.

109. The CFI variant of any of claims 105-107, wherein the at least one factor H domain comprises CCP1, CCP2, CCP3, and CCP4.

110. The CFI variant of any of claims 105-107, wherein the at least one factor H domain comprises CCP2, CCP3, and CCP4.

111. The CFI variant of any of claims 105-107, wherein the at least one factor H domain comprises CCP2 and CCP3.

112. A CFI variant according to any one of claims 105 to 106, wherein the amino acid sequence of at least one domain of factor H is or is derived from the sequence set forth in SEQ ID No. 8.

113. The CFI variant of claim 112, wherein the at least one factor H domain comprises CCP domains 1-4 and 19-20 of factor H.

114. A CFI variant according to claim 93 wherein the second component is at least one domain or part of a domain of complement receptor 1 (CR 1).

115. A CFI variant according to claim 114, wherein at least one domain of CR1 is any one or more of CR1 CCP domains 15-17.

116. The CFI variant of claim 93, 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.

117. A CFI variant according to any one of claims 93 to 115, wherein the fusion construct further comprises a third component.

118. A CFI variant according to claim 117, wherein the third component is a protein.

119. A CFI variant according to claim 117, wherein the third component is not a protein.

120. The CFI variant of any of claims 93-115, further comprising a third component, wherein the third component is a half-life extender, optionally selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc.

121. A CFI variant according to claim 120 wherein the half-life extender is a repeat peptide sequence.

122. A CFI variant comprising at least one modification relative to wild-type CFI, wherein the CFI variant is non-activatable.

123. A CFI variant according to claim 122, comprising a modification at a position corresponding to position R321 in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

124. The CFI of any of claims 122-123, comprising a substitution R321A, wherein the position corresponds to a position in a CFI having the amino acid sequence set forth in SEQ ID No. 5.

125. A fusion construct comprising a first component and a second component, wherein the first component comprises wild-type CFI or a variant thereof (CFI variant), and wherein the second component comprises a half-life extender.

126. The fusion construct according to claim 125, wherein the first component comprises a wild-type CFI comprising the amino acid sequence set forth in SEQ ID No. 5.

127. The fusion construct according to claim 126, wherein the second component is albumin.

128. The fusion construct of claim 127, wherein the second component is human serum albumin.

129. The fusion construct according to claim 128, wherein the second component comprises human serum albumin comprising the amino acid sequence set forth in SEQ ID No. 7.

130. The fusion construct according to claim 125, comprising the amino acid sequence set forth in SEQ ID No. 21 or an amino acid sequence having at least 80% identity thereto.

131. The fusion construct according to claim 125, consisting of the amino acid sequence set forth in SEQ ID No. 21.

132. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 7.

133. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 7, wherein the fusion construct comprises a structural configuration in the form of (SEQ ID No. 7) - (optionally linker) - (SEQ ID No. 5) from the N-terminus to the C-terminus.

134. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 7, wherein the fusion construct comprises a structural configuration in the form of (SEQ ID No. 7) - (linker) - (SEQ ID No. 5) from N-terminus to C-terminus.

135. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, wherein the fusion construct comprises a structural configuration in the form of (SEQ ID No. 7) - (SEQ ID No. 6) - (SEQ ID No. 5) from the N-terminus to the C-terminus.

136. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 7, wherein the fusion construct comprises a structural configuration in the form of (SEQ ID No. 5) - (optionally a linker) - (SEQ ID No. 7) from the N-terminus to the C-terminus.

137. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 7, wherein the fusion construct comprises a structural configuration in the form of (SEQ ID No. 5) - (linker) - (SEQ ID No. 7) from N-terminus to C-terminus.

138. The fusion construct according to claim 125, comprising the amino acid sequences of SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, wherein the fusion construct comprises a structural configuration in the form of (SEQ ID No. 5) - (SEQ ID No. 6) - (SEQ ID No. 7) from the N-terminus to the C-terminus.

139. The fusion construct according to claim 125, wherein the first component comprises a CFI variant.

140. The fusion construct according to claim 139, wherein the CFI variant is any of the CFI variants according to claims 1 to 88.

141. The fusion construct according to any one of claims 125-140, wherein the fusion construct has at least one improved feature compared to free wild-type CFI (not part of the fusion construct).

142. The fusion construct according to any one of claims 125-141, wherein the improved feature 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 capacity.

143. The fusion construct according to claim 142, wherein the improvement is characterized by an increase in activity.

144. The fusion construct according to claim 143, wherein the increase in activity comprises an increase in cleavage of C3b and/or C4 b.

145. The fusion construct of claim 144, wherein the increase in activity comprises an increase in cleavage of C3b and does not comprise an increase in cleavage of C4 b.

146. The fusion construct according to any one of claims 144 to 145, wherein the increase in cleavage of 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 greater than a wild-type CFI that is not part of the fusion construct, or as compared to a fusion construct comprising a wild-type CFI.

147. The fusion construct of claim 144, wherein the increase in activity comprises an increase in cleavage of C4b and no increase in cleavage of C3b compared to the wild-type CFI.

148. The fusion construct according to any one of claims 144 and 147, wherein the increase in cleavage of 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 greater than a wild-type CFI that is not part of the fusion construct, or as compared to a fusion construct comprising a wild-type CFI.

149. The fusion construct according to any one of claims 143 to 146 and 148, wherein the increase in activity comprises an increase in production of iC3 b.

150. The fusion construct according to any one of claims 143-146 and 148-149, wherein the increase in activity comprises an increase in C3dg generated from iC3 b.

151. The fusion construct according to any one of claims 143 to 146 and 148 to 150, wherein the increase in activity comprises a decrease in content of C3b alpha chains.

152. The fusion construct according to any one of claims 143 to 151, wherein the increase in activity comprises an increase in hydrolysis of a peptide substrate or an increase in proteolysis of a macromolecular protein substrate.

153. The fusion construct according to claim 142, wherein the improvement is characterized by a decrease in activity.

154. The fusion construct according to any one of claims 125-153, wherein the fusion construct has at least one improved feature in the absence of factor H and/or in the absence of CR1 compared to free wild-type CFI.

155. The fusion construct according to any one of claims 1-153, wherein the fusion construct has at least one improved feature compared to free wild-type CFI, and wherein the at least one improved feature is further improved by the presence of exogenous factor H and/or exogenous CR 1.

156. A pharmaceutical composition comprising any one of the CFI variants of claims 1-124, or any one of the fusion constructs of claims 125-155, and optionally a pharmaceutically acceptable excipient.

157. A method of modulating the complement system comprising contacting a sample in vitro or a tissue in vivo with any one of the CFI variants of claims 1-124 or any one of the fusion constructs of claims 125-155.

158. The method of claim 157, wherein the method is performed in vitro.

159. The method of claim 157, wherein the method is performed in vivo.

160. The method of any one of claims 157 to 159, wherein the method results in increased cleavage of C3b, C4b, increased production of iC3b, increased production of C3dg and/or C4C.

161. The method of any one of claims 157 to 159, wherein the method causes reduced hemolysis.

162. The method of any one of claims 157 to 159, wherein the method results in a reduction in the content of MAC.

163. The method of any one of claims 157-159, wherein the method results in reduced amplification of the complement system.

164. The method of any one of claims 157 to 159, wherein the method results in increased hydrolysis of a peptide substrate or increased proteolysis of a macromolecular protein substrate.

165. A method of treating a non-ocular disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any one of the CFI variants of claims 1-120, or any one of the fusion constructs of claims 125-155, or the pharmaceutical composition of claim 156.

166. The method of claim 165, wherein the non-ocular disorder is characterized by insufficient CFI.

167. The method of any one of claims 165-166, wherein the non-ocular disorder is characterized by a disorder of the complement system.

168. The method according to any one of claims 165-167, wherein the non-ocular disorder is a systemic acute indication.

169. The method of claim 168, wherein the non-ocular disorder is a systemic acute indication selected from the group consisting of: acute glomerulonephritis, acute kidney injury, acute respiratory distress syndrome, bacterial meningitis, cerebral hemorrhage, burn injury, coronavirus infection, epstein-Barr virus (Epstein-Barr virus) infection, hematopoietic stem cell transplantation, ischemia reperfusion injury, lyme disease (Lyme disease), myocardial infarction, organ transplantation, periodontitis, pneumonia, preeclampsia, schistosomiasis, sepsis, stroke, thromboembolism, ischemia-reperfusion injury, and traumatic brain injury.

170. The method according to any one of claims 165-167, wherein the non-ocular disorder is a systemic chronic indication.

171. The method of claim 170, wherein the non-ocular disorder is a systemic chronic indication selected from the group consisting of: alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, anti-phospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous Pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease, crohn's disease, diabetic neuropathy, systemic myasthenia gravis (gMG), granulomatous Polyangiitis (GPA), green-Barre syndrome (GBS), hereditary Angioedema (HAE), suppurative sweat gland (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, osteoarthritis, pancreatitis, parkinson's disease, paroxysmal nocturnal hemuria, post-transplant lymphoproliferative disorder, protein-lost bowel disease, psoriasis, gangrene abscess, rheumatoid arthritis, schizophrenia (SZ), systemic Lupus Erythematosus (SLE), immune Thrombocytopenia (ITP), ulcerative colitis, amyotrophic Lateral Sclerosis (ALS), warm autoimmune hemolytic anemia (wAIHA), condensed Colletotrichosis (CAD), immune complex membranoproliferative glomerulonephritis (IC-MPGN), lambert-eaton muscle weakness syndrome (LEMS), CHAPLE syndrome (CD 55 deficiency), thrombotic Microangiopathy (TMA), huntington's disease, and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP).

172. The method according to any one of claims 165-167, wherein the non-ocular disorder is non-oncogenic.

173. The method according to any one of claims 165-167, wherein the non-ocular disorder is oncogenic.

174. The method of claim 172, wherein the non-ocular disorder is characterized by a solid tumor or a liquid tumor.

175. The method of claim 174, wherein the non-ocular disorder is characterized by a solid tumor and is selected from the group consisting of: colorectal cancer, hormone refractory prostate cancer, melanoma, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic pancreatic cancer, metastatic gastric cancer, nasopharyngeal cancer, non-small cell lung cancer, pancreatic tumor, squamous cell carcinoma, and gastric tumor.

176. The method of claim 174, wherein the non-ocular disorder is characterized by a liquid tumor and is selected from the group consisting of: acute myelogenous leukemia, B-cell lymphoma, and hodgkin's disease.

177. The method of any one of claims 180 to 176, wherein the CFI variant, the fusion construct, or the pharmaceutical composition is administered subcutaneously or intravenously to the subject.

178. The method of claim 177, wherein the administration is subcutaneous administration.

179. The method of claim 178, wherein the subcutaneous administration is once daily or once weekly or once every other week.

180. A method of treating an ocular disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any one of the CFI variants of claims 1-120, or any one of the fusion constructs of claims 125-155, or the pharmaceutical composition of claim 156.

181. The method of claim 180, wherein the ocular disorder is characterized by insufficient CFI.

182. The method of any one of claims 180 to 181, wherein the ocular disorder is characterized by a disorder of the complement system.

183. The method according to any one of claims 180 to 182, wherein the ocular disorder is selected from the group consisting of: diabetic Macular Edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, stargardt disease (Stargardt Disease), uveitis, and vitreoretinopathy.

184. The method according to any one of claims 180 to 183, wherein the ocular disorder is non-oncogenic.

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

186. A method of producing a wild-type CFI or variant thereof in an activated state, the method comprising recombinantly producing the CFI or variant thereof in a cell comprising one or more nucleic acids encoding the CFI or variant thereof and comprising one or more nucleic acids encoding furin.