Patent Application
20230038638
An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on June 14, 2021, is 92 kilobytes in size, and is titled CTBI_001_04US_SeqList_ST25.txt.
The complement system includes the classical, lectin and alternative pathways, and is tightly controlled by a number of regulators. Complement Factor I (CFI) is one such regulator, and acts to regulate the complement system by cleaving C4b and C3b proteins, thereby inactivating these proteins. Such cleavage results in inhibition of the classical, lectin and alternative pathways, respectively, thus ultimately preventing the assembly of the C3 and C5 convertase enzymes. CFI is encoded as a proenzyme and is then activated by proteolytic cleavage into a heterodimeric glycoprotein having a heavy chain and a light chain that are connected by a disulfide linkage. The light chain (also referred to as the B chain) comprises the serine protease domain (SPD) responsible for the cleavage of C3b and C4b, and contains a catalytic triad (His362, Asp411, and Ser507) within a region referred to as the active site. The heavy chain (also referred to as the A chain) comprises four domains: the FI membrane attack complex (FIMAC) domain, the scavenger receptor cysteine-rich domain SRCR (also called the CD5 domain) domain, the low density lipoprotein receptor 1 domain (LDLr1), and the low density lipoprotein receptor 2 domain (LDLr2). CFI is processed into its active form post-translationally by the addition of six Asn-linked glycans and proteolytic activation by furin, thereby excising a RRKR linker to generate the two chain mature protein.
With respect to its ability to cleave C3b or C4b, CFI is proteolytically active when it forms ternary complexes with its cofactors; Factor H (FH) or Complement Receptor 1 (CR1, also called CD35) and its physiological substrates, C3b and C4b. FH is an example of a soluble member of the group of proteins called regulators of complement activation (RCA). The formation of the complex made by CFI and FH and subsequent cleavage of C3b together act to regulate the alternative pathway of the complement system. Continuous regulation of C3b levels by CFI acts to maintain the balance between the classical and alternative pathways. For instance, removal of CFI has been shown to cause an immediate activation, resulting in over-activity, of the alternative pathway. CR1 is an example of a monomeric single-pass type I membrane glycoprotein that is a member of the group of proteins called regulators of complement activation (RCA). Formation of the complex made between CFI and CR1 and the subsequent cleavage of C3b and C4b act to regulate the alternative or the classical and lectin pathways, respectively.
Dysregulated CFI, mutated and dysfunctional CFI, or CFI deficiency have been implicated in diseases involving the complement system, and needed are methods for modulating or inhibiting particular points of regulation within the complement system. Provided here are compositions and methods to address the dysfunction and/or dysregulation in the complement system.
In one aspect, the disclosure provides a complement factor I (CFI) variant comprising at least one modification with respect 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 characteristic as compared to the wild type CFI. In some embodiments, the improved characteristic is selected from an increase in half-life or bioavailability, or increase or decrease in any one or more of activity, substrate specificity, potency, substrate affinity, cofactor affinity and catalytic capability In some embodiments, the improved characteristic is an increase in activity. In some embodiments, the improved characteristic is a change in substrate specificity.
In some embodiments, the increase in activity comprises an increase in the 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 the cleavage of C3b, and does not comprise an increase in the cleavage of C4b. In some embodiments, the increase in the cleavage of C3b is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold as compared to the wild type CFI or a fusion construct comprising wild type CFI.
In some embodiments, the increase in activity comprises an increase in the cleavage of C4b as compared to the wild type CFI or a fusion construct comprising wild type CFI, and does not comprise an increase in the cleavage of C3b. In some embodiments, the increase in the cleavage of C4b is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold as compared to the wild type CFI or a fusion construct comprising wild type CFI.
In some embodiments, the increase in the cleavage of C3b and C4b each is increased by at least or about 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 as compared to the wild type CFI or a fusion construct comprising wild type CFI.
In some embodiments, the increase in activity comprises an increase in the generation of iC3b. In some embodiments, an increase in activity comprises an increase in the generation of C3dg and/or C3c from iC3b.
In some embodiments, the increase in activity comprises a reduction in the levels of C3b Δ-chain In some embodiments, the increase in activity comprises an increase in the proteolysis of a peptide substrate. In some embodiments, an increase in activity comprises a reduction in the levels or function of membrane attack complex (MAC). In some embodiments, the increase in activity results in a reduction of an amplification of the complement system. In some embodiments, the improved characteristic is a decrease in activity for C3b and/or C4b.
In some embodiments, the improved characteristic is an increase in specificity for a substrate. In some embodiments, the increase in specificity comprises an increase in the specificity for 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 the specificity for 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 the specificity for 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 the specificity for C3b, as compared to wild type CFI or a fusion construct comprising wild type CFI. In some embodiments, the increase in the specificity for C3b is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold as compared to the wild type CFI or a fusion construct comprising wild type CFI.
In some embodiments, the increase in specificity comprises an increase in the specificity for C4b, as compared to wild type CFI or a fusion construct comprising wild type CFI. In some embodiments, the increase in the specificity for C4b is increased by at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 100-fold, at least or about 150-fold, at least or about 500-fold, or at least or about 1000-fold as compared to the wild type CFI or a fusion construct comprising wild type CFI.
In some embodiments, the modification with respect to a wild type CFI comprises any one or more of: 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, an insertion of one or more amino acid residues, an insertion of one or more CFI domains, and a swapping of one or more CFI domains. In some embodiments, the CFI variant comprises 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 CFI selected from: the serine protease domain (SPD), the Factor I membrane attack complex (FIMAC) domain, the SRCR domain, the low density lipoprotein receptor 1 (LDLr1) domain, and the low density lipoprotein receptor 2 (LDLr2) domain.
In some embodiments, the CFI variant comprises at least one modification corresponding to a wild type human CFI. In some embodiments, the CFI variant comprises at least one modification corresponding to a 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 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 of a corresponding region from a non-human species CFI. In some embodiments, the non-human species is mouse. In some embodiments, the CFI variant is a chimera, and wherein the modification comprises the substitution of one or more amino acid residues of the 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.
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 positions corresponding to positions K14, Y20, D26, F29, R35, E38, M220, K221, S250, L304, P305, K306, L307, and S308 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CFI variant comprises a substitution in a 200 loop of the CFI (SEQ ID NO: 13) for a 200 loop of trypsin having amino acid residues NG, wherein the 200 loop occurs between positions corresponding to position 514 and position 520 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 of the substitutions selected from K14A, Y20A, Y20F, D26A, F29A, R35A, E38A, M220A, K221Q, S250A, S250L, L304G, P305G, K306G, L307G, and S308G, 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 comprises one or more of the combination of substitutions M220A; K221Q, and L304G; P305G; K306G; L307G; S308G, 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 comprises one or more modifications at a 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 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. In some embodiments, the CFI variant comprises one or more modifications at a C-terminal region is a deletion 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 amino acid residues (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 are substituted for a linker. In some embodiments, the CFI variant comprises one or more of the substitutions selected from T377G, W381A, P384A, P384G, Y403F, A405S, G406R, G406A, Y408L, Q409D, Q409H, D425A, D425K, D425R, G556A, G556S, R557A, R557K, P558G, P558L, P558S, F559L, I560V, and Y563H, and/or a deletion of P384, 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 comprises one or more modifications at one or more N-linked glycosylation sites of the CFI. In some embodiments, the CFI variant comprises one or more modifications is a removal of an 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 positions corresponding to positions N52, N85, N159, N446, N476, and N518 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 of the substitutions selected from N52Q, N85Q, N159Q, N446Q, N476Q, and N518Q, 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 comprises one or more of combination of substitutions selected from N52Q; N85Q; N159Q, N446Q; N476Q; N518Q, 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 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 variant comprises one or more modifications at any one or more of the autolysis loop, the 99 loop, the S1 pocket entrance, or the activation loop of SPD, or any one or more of the domains presented in
In some embodiments, the CFI variant comprises a substitution of an autolysis loop of the CFI (REKDNERVFS, SEQ ID NO: 9) for an autolysis loop of trypsin (NTASSGADYPDE, SEQ ID NO: 10), wherein the autolysis loop occurs between positions corresponding to position 456 and position 465 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 an autolysis loop of the CFI (REKDNERVFS, SEQ ID NO: 9) for an autolysis loop of a mouse CFI (RGKDNQKVYS, SEQ ID NO: 11), wherein the autolysis loop occurs between positions corresponding to position 456 and position 465 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 of the substitutions selected from K14A, K312A, R314A, I322T, I322Y, I322V, V323I, V323G, V323A, K326A, R327A, R327P, R327N, A328C, K340G, D341A, G344R, G344K, G344Y, I345G, T346R, T346K, T346H, A361G, L364G, Y372G, W381K, W381G, P384A, P384G, V390G, N402E, N404G, G406D, G406E, G406F, G406H, G406I, G406K, G406L, G406M, G406N, G406P, G406Q, G406S, G406T, G406V, G406W, G406Y, Y408L, Y408F, Y408G, Y408P, Y408D, Y408A, Y408N, Y408T, Y408K, Y408R, Y408H, Y408I, Y408E, Y408M, Y408Q, Y408S, Y408W, Y408Y, Y408V, Q409G, E416A, K418G, N422K, D425A, D425K, D425R, D425G, R456A, R456N, E457G, E457A, E457D, E457F, E457H, E457I, E457K, E457L, E457M, E457N, E457P, E457Q, E457R, E457S, E457T, E457W, E457Y, E457V, K458A, E461Q, E461K, E461R, E461H, E461G, E461A, E461D, E461F, E461I, E461L, E461M, E461N, E461P, E461S, E461T, E461W, E461Y, E461V, R462K, R462A, R462D, F464Y, 5465G, Q467K, Q467R, W468C, G469L, T495F, Y496L, D497E, 5499G, I500K, A502S, K504Q, K504E, K504R, K504A, K504G, K504L, K504P, K504H, K504D, K504F, K504I, K504M, K504N, K504S, K504T, K504V, K504W, K504Y, D506A, D506V, D506E, D506G, S507A, E530D, E530G, E530F, E530Y, N531G, N531A, E530D, E530G, E530F, E530Y, E530R, E530K, N531D, N531E, N531F, N531H, N531I, N531K, N531L, N531M, N531P, N531Q, N531R, N531S, N531T, N531V, N531W, N531Y, G533A, K534Q, P535A, P535K, E536N, E536A, F537K and F537R, 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 comprises one or more of the combination substitutions selected from K326A; 327A, N531G; P535A, E457G; E461Q; R462K; F464Y, Y408L; N531G; E457G, Y408L; N531G; E457G; E461Q, Y408L; N531G; E457G; E461Q-R462K; F464Y, Y408L; N531G; P535A, K14A; D425R, E530D; N531G; G533A; K534Q; P535K; E536N, A5025; K504Q; F537K, T495F; Y496L; D497E; S499G; I500K, G533A; K534Q; P535K; E536N; F537K, T495F; Y496L; D497E; S499G; I500K; G533A; K534Q; P535K; E536N; F537K, Q467K; F537K, E530G; N531G, E530D; F537K, E457G; E461Q, E457G; E461G, Y408L; N531G; E457G; E461Q, N531G; E457G; E461Q, I322V; V323I, I322V; V323I; R327P, A328C; W468C, A328C; W468C; K326Y; R327N, Y408L; N531G; E461Q, Y408L; N531G; E457G; E461Q; R462K, Y408L; N531G; E457G; E461Q; F464Y, Y408L; N531G; E457G; R462K; F464Y, Y408L; N531G; E461Q; R462K; F464Y, Y408L; E457G; E461Q; R462K; F464Y, E457G; N531G; E461Q; R462K; F464Y, Y408L; E457G; E461Q; R462K, N531G; E457G; E461Q; F464Y, E416A; D425R, Y408L; 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, G406D; N531G, G406D; P535A, G406D; Y408L; N531G, G406D; Y408L; P535A, G406D; N531G; P535A, G406D; Y408L; N531G; P535A, K340G; I345G, L364G; Y372G, W381G; V390G, W381G; P384A; V390G, W381G; P384G; V390G, N404G; Q409G, K418G; D425G, T346R; K504E; E530R, T346K; K504D; E530K, G344R; Y408L; N531G, G344K; Y408L; N531G, T346R; Y408L; N531G, T346K; Y408L; N531G, K504D; Y408L; N531G, K504E; Y408L; N531G, Y408L; E530R; N531G, Y408L; E530K; N531G, T346R; Y408L; K504E; E530R; N531G, T346K; Y408L; K504D; E530K; N531G, Y408L; S507A; N531G, Y408L; N531G; E457G; E461Q; R462K; F464Y; S507A, E457G; S507A, and N531G; P535A; S507A, 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 comprises one or more modifications at an active site of the CFI. In some embodiments, the CFI variant comprises any modification 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 a 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 arrangement from N-terminus to C-terminus as (A chain)-(optional linker)-(B chain). In some embodiments, the CFI variant comprises an A chain and a B chain, wherein the CFI variant comprises a structural arrangement from N-terminus to C-terminus as (B chain)-(optional linker)-(A chain). In some embodiments, the CFI variant comprises modifications at one or more of C309 and C435, 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 comprises substitutions C309S; C435S, 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 B chain and the A chain are further linked by a disulfide bond. 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 a disulfide bond. 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 variant comprises one or more modifications presented in Table 7. In some embodiments, the CFI variant is more easily activated as compared to the wild type CFI. In some embodiments, the CFI variant comprises a modification at any one or more positions corresponding to positions I317, R318, R319, K320, and R321 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 of the substitutions selected from I317D, R318D, R319D, K320D, and R321K, 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 comprises substitutions I317D, R318D, R319D, K320D, and R321K, 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 comprises two or more modifications described herein. In some embodiments, the CFI variant comprises any one or more of the modifications presented in Table 9. In some embodiments, the CFI variant comprises one or more of the combination of substitutions selected from Y408; N531G, E38A; D425R, Y20F; D425R, S250A; D425R, Y408F; N531G, Y408L; N531G; E457G; E461Q; R462K; F464Y, K14A; Y20F, K14A; E38A, K14A; S250A, K14A; D425A, Y20F; E38A, Y20F; S250A, Y20F; D425A, E38A; S250A, E38A; D425A, S250A; D425A, K14A; N531G; P535A, Y20F; N531G; P535A, E38A; N531G; P535A, S250A; N531G; P535A, D425A; N531G; P535A, Y20F; Y408L; N531G; E457G; E461Q; R462K; F464Y, E38A; Y408L; N531G; E457G; E461Q; R462K; F464Y, S250A; Y408L; N531G; E457G; E461Q; R462K; F464Y, D425R; Y408L; N531G; E457G; E461Q; R462K; F464Y, Y20F; E38A; S250A; D425A, Y20F; E38A; S250A; D425A; Y408L; N531G; E457G; E461Q; R462K; F464Y, Y20F; 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, K504E; Y408L; N531G, E457G; N531G; D425K, Y408F; N531G, Y408L; E457G; N531G; D425K, Y408L; E457G; P535G; D425K, Y408L; E457G; N531G; K534Q, Y408L; N531G, R462K; F464Y, and Y408L; P535G; D425K, 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 comprises each one of the SPD, the FIMAC domain, the SRCR domain, the LDLr1 domain, and the LDLr2 domain, and any other domains presented in
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 a 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 variant thereof. In some embodiments, the CFI variant is activated by furin or variant thereof in vitro. In some embodiments, the CFI variant is activated by furin or variant thereof during recombinant production in a host cell. In some embodiments, the activation by furin or variant thereof during production in a host cell is by overexpression of furin or a variant thereof. In some embodiments, the CFI variant is activated by furin or variant thereof after production and secretion by a host cell, optionally in the media.
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; the fusion construct may comprise further 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 peptide repeats.
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 a modified albumin or albumin derivative. In some embodiments, the half-life extender is a wild type albumin. In some embodiments, the half-life extender is a 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 arrangement 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 arrangement 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 with at least 80% sequence identity thereto.
In some embodiments, the second component is at least one domain, or part of a domain of Factor H. In some embodiments, the at least one Factor H domain comprises any one or more of complement control protein (CCP) domains 1-20 of Factor H. In some embodiments, 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. In some embodiments, the at least one Factor H domain comprises each of the CCP domains 1-20 of Factor H. In some embodiments, the at least one Factor H domain comprises CCP1, CCP 2, CCP3, and CCP4. In some embodiments, the at least one Factor H domain comprises CCP2, CCP3, and CCP4. In some embodiments, the at least one Factor H domain comprises CCP2 and CCP3. In some embodiments, the amino acid sequence of the at least one domain of Factor H is, or is derived from, the sequence set forth in SEQ ID NO: 8. In some embodiments, the at least one Factor H domain comprises CCP domains 1-4 and 19-20 of Factor H.
In some embodiments, the second component is at least one domain, or part of a domain of Complement Receptor 1 (CR1). In some embodiments, the 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 part of a domain of a Complement Receptor I (CRI) and at least one domain, or part 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 selected from albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, and Fc. In some embodiments, the half-life extender is a repetitive peptide sequence. In some embodiments, the CFI variant comprises at least one modification with respect to a 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 of 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 a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In another aspect, the disclosure provides a fusion construct comprising a first component and a second component, wherein the first component comprises a wild type CFI or 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 an 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 a human serum albumin comprising an amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, the fusion construct comprises an amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence comprising at least 80% identity thereto. In some embodiments, the fusion construct consists of an amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7.
In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement from N-terminus to C-terminus (SEQ ID NO: 7)-(optional linker)-(SEQ ID NO: 5). In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement 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 sequence of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein the fusion construct comprises a structural arrangement 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 sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement from N-terminus to C-terminus (SEQ ID NO: 5)-(optional linker)-(SEQ ID NO: 7). In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement 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 sequence of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein the fusion construct comprises a structural arrangement 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 characteristic as compared to a free wild type CFI (not part of a fusion construct), or as compared to a fusion construct comprising a wild type CFI. In some embodiments, the improved characteristic is selected from an increase in half-life or bioavailability, or an increase or decrease in any one or more of activity, substrate specificity, potency, substrate affinity, cofactor affinity and catalytic capability. In some embodiments, the improved characteristic is an increase in activity. In some embodiments, the increase in activity comprises an increase in the cleavage of C3b and/or C4b. In some embodiments, the improved characteristic is an increase in substrate specificity.
In some embodiments, the increase in activity comprises an increase in the cleavage of C3b as compared to a wild type CFI not part of a fusion construct, or compared to a fusion construct comprising a wild type CFI. In some embodiments, the increase in activity comprises an increase in the cleavage of C3b and does not comprise an increase in the cleavage of C4b, as compared to a wild type CFI not part of a fusion construct, or compared to a fusion construct comprising a wild type CFI. In some embodiments, the increase in the cleavage of C3b is increased 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 as compared to a wild type CFI not part of a fusion construct, or compared to a fusion construct comprising a wild type CFI.
In some embodiments, the increase in activity comprises an increase in the cleavage of C4b as compared to a wild type CFI not part of a fusion construct, or compared to a fusion construct comprising a wild type CFI., In some embodiments, the increase in activity comprises an increase in the cleavage of C4b as compared to a wild type CFI not part of a fusion construct, or compared to a fusion construct comprising a wild type CFI and does not comprise an increase in the cleavage of C3b. In some embodiments, the increase in the cleavage of C4b is increased 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 as compared to a wild type CFI not part of a fusion construct, or compared to a fusion construct comprising a wild type CFI.
In some embodiments, the increase in activity comprises an increase in generation of iC3b. In some embodiments, the increase in activity comprises an increase in the generation of C3dg from iC3b. In some embodiments, the increase in activity comprises a reduction in the levels of C3b α-chain In some embodiments, the increase in activity comprises an increase in the hydrolysis of a peptide substrate or proteolysis of a macromolecular protein substrate.
In some embodiments, the improved characteristic is a decrease in activity with respect to C4b or C3b substrates.
In some embodiments, the fusion construct has at least one improved characteristic as compared to a free wild type CFI, without the presence of Factor H and/or without the presence of CR1. In some embodiments, the fusion construct has at least one improved characteristic as compared to a free wild type CFI, and wherein the at least one improved characteristic is further improved by the presence of exogenous Factor H and/or exogenous CR1.
In one aspect, the disclosure provides a pharmaceutical composition comprising any one of the CFI variants described herein, or any one of the fusion constructs described herein, and optionally a pharmaceutically acceptable excipient.
In another aspect, the disclosure provides a method of modulating the complement system, comprising contacting a sample in vitro or contacting a tissue in vivo with any one of the CFI variants described herein, or any one of the fusion constructs described herein. In some embodiments, the method is in vitro. In some embodiments, the method is in vivo.
In some embodiments, the method results in the increase in the cleavage of C3b, C4b, generation of iC3b, generation of C3dg, and/or C4c. In some embodiments, the method results in a decrease in hemolysis. In some embodiments, the method results in the reduction or level of MAC. In some embodiments, the method results in the reduction of the amplification of the complement system. In some embodiments, the method results in the increase in the hydrolysis of a peptide substrate, or an increase in the proteolysis of a macromolecular protein substrate.
In another aspect, the disclosure provides a method of treating a non-ocular condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the CFI variants described herein, or any one of the fusion constructs described herein, or any one of the pharmaceutical compositions described herein. Such treatment as contemplated herein includes both administration of a CFI variant of the disclosure or fusion construct of the disclosure, as well as administration of one or more nucleic acids encoding for a CFI variant of the disclosure or a fusion construct of the disclosure. Accordingly, provided herein are pharmaceutical compositions comprising the CFI variants of the disclosure, CFI fusion constructs of the disclosure, as well as pharmaceutical compositions comprising one or more nucleic acids encoding for CFI variants of the disclosure and encoding for fusion constructs of the disclosure.
In some embodiments, the non-ocular condition is characterized by a deficiency of CFI. In some embodiments, the non-ocular condition is characterized by dysregulation 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 renal injury, acute respiratory distress syndrome, bacterial meningitis, brain hemorrhage, burns, coronavirus infection, Epstein-Barr virus infection, hematopoietic stem cell transplantation, ischemia reperfusion injury, Lyme disease, myocardial infarction, organ transplantation, periodontitis, pneumonia, pre-eclampsia, 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, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic kidney failure, chronic obstructive pulmonary disease, Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), Granulomatosis with Polyangiitis (GPA), Guillain-Barré Syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy, lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy (MMN), multiple sclerosis (MS), non-insulin dependent diabetes, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, post-transplant lymphoproliferative disease, protein losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), ulcerative colitis, Amyotrophic lateral sclerosis (ALS), warm autoimmune hemolytic anemia (wAIHA), cold agglutinin disease (CAD), and Immune-Complex Membranoproliferative Glomerulonephritis (IC-MPGN), Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA), Huntington's disease and chronic inflammatory demyelinating polyneuropathy (CIDP).
In some embodiments, the non-ocular condition is non-oncological.
In some embodiments, the non-ocular condition is oncological. In some embodiments, the non-ocular condition is characterized by solid tumors, or by liquid tumors. In some embodiments, the non-ocular condition is characterized by solid tumors, and is selected from the group consisting of: colorectal tumors, hormone-refractory prostate cancer, melanoma, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic pancreas cancer, metastatic stomach cancer, nasopharyngeal carcinoma, non-small cell lung cancer, pancreas tumors, squamous cell carcinoma, and stomach tumors. In some embodiments, the non-ocular condition is characterized by liquid tumors, and is selected from the group consisting of: acute myelogenous leukemia, B-cell lymphoma, and Hodgkin's disease.
In some embodiments, the CFI variant, the fusion construct, or the pharmaceutical composition is administered to the subject subcutaneously, or intravenously. In some embodiments, the administration is a subcutaneous administration. In some embodiments, the subcutaneous administration is a daily, twice a week, or weekly, or every other week.
In another aspect, the disclosure provides a method of treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the CFI variants described herein, or any one of the fusion constructs described herein, or any one of the pharmaceutical compositions described herein. Such treatment as contemplated herein includes both administration of a CFI variant of the disclosure or fusion construct of the disclosure, as well as administration of one or more nucleic acids encoding for a CFI variant of the disclosure or a fusion construct of the disclosure. Accordingly, provided herein are pharmaceutical compositions comprising the CFI variants of the disclosure, CFI fusion constructs of the disclosure, as well as pharmaceutical compositions comprising one or more nucleic acids encoding for CFI variants of the disclosure and encoding for fusion constructs of the disclosure.
In some embodiments, the ocular condition is characterized by a deficiency of CFI. In some embodiments, the ocular condition is characterized by dysregulation of the complement system. In some embodiments, the ocular condition is selected from the group consisting of: diabetic macular edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, Stargardt Disease, uveitis, and vitreoretinopathy. In some embodiments, the ocular condition is non-oncological.
In another aspect, the disclosure provides a cell comprising one or more nucleic acids encoding a wild type CFI or variant thereof, and comprising one or more a nucleic acids encoding furin.
In another aspect, the disclosure provides a method of generating a wild type CFI or a variant thereof, in an activated state, the method comprising producing the CFI or a variant thereof recombinantly in a cell comprising one or more nucleic acids encoding the CFI or variant thereof, and comprising one or more nucleic acids encoding furin.
The disclosure provides compositions and methods useful for modulating the signaling and amplification of the complement system. By providing complement factor I (CFI) variants and CFI-containing fusion constructs that are more or less active on one or more physiological substrates of CFI, and/or more stable than plasma-derived CFI, a modulation of the complement system is observed. Such modulation includes an increased amount of C3b cleavage and/or C4b cleavage, thus reducing complement activation, and reducing the amplification of the complement pathways. For example, some CFI variants can alter levels of regulators 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, on the alternative pathway of the complement system, or on both pathways. The disclosure also provides methods of making and using these variants and constructs, for example in treating a disease or condition associated with complement dysregulation, e.g. treating an overactive complement response.
A. Complement Factor I Variants
Provided herein are Complement Factor I variants (CFI), such variants comprising one or more modifications with respect to a wild type CFI, referred to herein as “CFI variants.” As used herein, a “modification” to a wild type CFI includes: a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion (i.e. addition) of one or more amino acid residues, an insertion (i.e. addition) of one or more domains, an inversion of or one or more domains, and a substitution of one or more domains.
The CFI variants of the disclosure do not directly act on C3, for example, the variants of the disclosure do not directly cleave C3, do not directly inhibit C3, do not directly inhibit the activation of C3, and do not directly reduce the activation of C3.
As used herein, a wild type CFI refers to any naturally occurring full-length CFI that is not a disease-causing CFI, with or without a signal sequence, and which may be of any species.
In some embodiments, a wild type CFI is plasma-derived. In some embodiments, a wild type CFI is a human wild type CFI. In some embodiments, a wild type, human CFI having a signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 1 (as shown in Table 1 below). In some embodiments, a wild type CFI is a human CFI. In some embodiments, a wild type, human CFI does not include a signal sequence. In some embodiments, a wild type CFI without a signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 5 (as shown in Table 1 below).
A wild type CFI comprises a heavy chain and a light chain, which are also referred to as the A-chain and B-chain, respectively.
A CFI variant of the disclosure includes one or more of a deletion of one or more amino acid residues of a wild type CFI, a deletion of one or more CFI domains of a wild type CFI, a substitution of one or more amino acid residues of a wild type CFI, an insertion of one or more amino acid residues to a wild type CFI, an inversion of one or more CFI domains of a wild type CFI, and an insertion of one or more domains to a wild type CFI.
The CFI variants of the disclosure may be generated by the introduction of one or more modifications to a CFI base molecule, wherein the domains of the CFI base molecule correspond to those domains found in a wild type CFI, e.g. as put forth in
In some embodiments, the CFI variants provided herein modulate the activity of the complement system and have at least one improved characteristic as compared to a 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 capability, cofactor affinity (e.g. affinity for Factor H and/or CR1), substrate specificity and substrate affinity (e.g. affinity for C3b and/or C4b). In some embodiments, the improved characteristic is increased half-life. In some embodiments, the improved characteristic is an increase in activity, discussed further in detail, in subsequent sections below. In other embodiments, the improved characteristic is a change in substrate specificity for C3b and/or C4b, allowing for tunability of the CFI variant.
Provided in Table 1 are exemplary base molecules that may be used for the generation of any of the CFI variants. The base molecules of Table 1 were used to generate the CFI variants disclosed herein, having any one or more of the modifications discussed further herein. The base molecules provided herein may be useful for modulation of the complement system without further modification, or may be useful for modulation of the complement system with further modification. For example, any one of the base molecules provided in Table 1 may be further modified to include one or more modifications, such as 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 molecules of Table 1 may be further part of a fusion construct, further described below.
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In some embodiments, a base molecule itself may be a CFI variant, for example in some embodiments, a CFI variant comprising only the serine protease domain (CFI-SPD) itself is a CFI variant. In some embodiments, the CFI variants are derived from any base molecule of Table 1, and comprise modifications to loops corresponding to the loops of an unmodified CFI. In some embodiments, the CFI variants are derived from any base molecule of Table 1, and comprise substitution mutations. In some embodiments, the CFI variants are derived from any base molecule of Table 1, and comprise a deletion of one or more domains of CFI. In some embodiments, the CFI variants are derived from any base molecule of Table 1, and comprise an inversion of the A-chain and the B-chain of the 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 a wild type CFI of any species. For example, the amino acid sequence of the at least one CFI domain may comprise the 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 with respect 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, substitution mutations of one or more amino acid residues, an addition of one or more amino acid residues, the deletion of one or more domains of CFI, the substitution of one or more domains of CFI, or the 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: a serine protease domain (SPD), a Factor I membrane attack complex (FIMAC) domain, a scavenger receptor cysteine-rich domain (SRCR), a low density lipoprotein receptor 1 (LDLr1), and low density lipoprotein receptor 2 (LDLr2) domains. In some embodiments, the any one or more CFI domains are that of a human CFI. In some embodiments, the any one or more CFI domains comprise an amino acid sequence derived from the sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise all domains of a wild type CFI, i.e., each one of the SPD, the FIMAC domain, the SRCR domain, the LDLr1 domain, and the LDLr2 domain, and comprises a modification in any one or more of these domains with respect to the wild type CFI.
In some embodiments, the CFI variants do not comprise all of the domains corresponding to that of the wild type CFI. In some embodiments, the CFI variants comprise the SPD. In some embodiments, the CFI variants comprise only the SPD, 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 the SPD of a human CFI. In some embodiments, the CFI-SPD comprises no further modifications with respect to that of a wild type CFI SPD. In some embodiments, the CFI-SPD comprises one or more modifications with respect to that of a wild type CFI SPD. In some embodiments, the CFI-SPD comprises at least one modification with respect 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 of amino acid residues with respect to a CFI having the amino acid sequence set forth in SEQ ID NO: 5. For example, a CFI variant that includes substitutions at positions N531 and P535 will have substitutions at positions N531 and P535 in the amino acid sequence set forth in SEQ ID NO: 5.
Provided herein are CFI variants comprising or consisting of at least one modification with respect to a wild type CFI, wherein the CFI variant is capable of increasing complement system inhibition, and wherein the CFI variant has at least one improved characteristic as compared to the wild type CFI. Examples of improved characteristic include, but are not limited to, an increase in half-life, an increase in bioavailability or an increase or decrease in any one or more of activity, substrate specificity, potency, substrate affinity, cofactor affinity and catalytic capability. In exemplary embodiments, an improved characteristic is increased half-life. In other exemplary embodiments, an improved characteristic is increased, or altered substrate specificity.
Without limitation, the disclosure contemplates the exemplary CFI variants described in Table 13. The variants of Table 13 include modified CFIs, as well as CFI fusion constructs, described herein. For avoidance of doubt, unless otherwise indicated, where a residue number is indicated, it refers to SEQ ID NO: 5 (wild type human CFI), or a sequence corresponding thereto. For avoidance of doubt, by way of example a variant whose description is K14A indicates that the disclosure provides a CFI variant comprising a K14A substitution, e.g. a CFI variant comprising a K14A substitution in SEQ ID NO: 5 (or a sequence corresponding thereto); the disclosure also provides for a CFI variant consisting of a K14A substitution, e.g. a CFI variant, wherein SEQ ID NO: 5 has a K14A substitution.
In some embodiments, a CFI variant of the disclosure 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.
CFI variants of the disclosure can 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, e.g. substitutions, deletions, insertions and fusions. Modification, e.g. substitutions, for a given variant may be represented in one of many ways recognized by the skilled artisan. For example, a hCFI variant having 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 instances, a CFI variant having substitutions at T377G and N422K may be referred to as “hCFI; T377G; N422K” or CFI variant (T377G; N422K).” As described herein, variants with other modifications, such as deletions, or combinations of modifications, such as deletions, fusions and substitutions, can conform to similar styles of nomenclature. Tables disclosing variants (e.g. Tables 13, 7.1, and 7.2) include the following symbols and abbreviations and associated meanings: HSA=human serum albumin; CFI=complement factor I; Δ=Deletion of the amino acid range noted; →=Deletion of noted sequence and replaced with noted amino acids; Cr1=CR1 fusion; Fh=FH fusion; G(#) denotes a linker of sequence GGSSGG (SEQ ID NO: 6) repeated the indicated number of instances.
The activity and specificity of the CFI variants provided herein can be tuned for particular applications and therapeutic indications. For example, activity and specificity can be tuned by selection of C3b degraders, or C4b degraders, or degraders of both C3b and C4b. As used herein, protease activity for a substrate refers to the ability of a CFI variant of the disclosure to cleave its substrates, C4b and C3b. This can be expressed as an increase in C4b degrader activity, protease activity towards C4b, C3b degrader activity, protease activity towards C3b and the like.
As used herein a C3b degrader is a CFI variant that is capable of cleaving C3b; likewise, a C4b degrader is a CFI variant that is capable of cleaving C4b. The use of C3b degrader does not imply that it does not degrade C4b. A CFI variant can be both a C3b degrader, and a C4b degrader, and can show specificity for one over the other.
The CFI variants provided herein have modified characteristics that include increases or decreases in protease activity for a substrate as well increases or decreases in substrate specificity.
As used herein, specificity for a substrate, also referred to as substrate specificity, refers to the specificity for one over the other that a CFI variant demonstrates. If a substrate specificity of a CFI variant is about 1, then the specificity for both C4b and C3b are equal. If the specificity of a CFI variant is 2-fold higher for C4b, then it is deemed to demonstrate increased specificity of cleavage for C4b, as compared to C3b. Specificity for C4b in the examples provided herein, is expressed as a ratio of the percent maximum cleavage of C4b divided by the ratio of percent maximum cleavage of C3b. Likewise, specificity for C3b in the examples provided herein, is expressed as a ratio of the percent maximum cleavage of C3b divided by the ratio of percent maximum cleavage of C4b. An increase in protease activity for one substrate by a greater fold increase as compared to another substrate is an example of an increase in specificity for that substrate.
In some embodiments amino acids modifications (e.g. substitutions) either increase activity, confer specificity or both. In some embodiments, an increase in C4b degrader activity comprises an increase in the cleavage of C4b, (and the generation of a cleavage product such as C4c) and an increase in the specificity towards C4b comprises an increase in the cleavage of C4b and a decrease in the cleavage of C3b (and the generation of a cleavage product such as iC3b), as compared to wild type CFI or a fusion construct comprising wild type CFI.
In some embodiments the combination of two or more modifications (e.g. substitutions) confers unexpected increases in activity that are synergistic or additive.
In some embodiments the combination of one or more modifications confers unexpected increases or decreases in activity that are synergistic when C4b is the substrate and additive or less than additive when C3b is the substrate.
In some embodiments the combination of one or more modifications confers unexpected increases or decreases in activity that are synergistic when C3b is the substrate and additive or less than additive when C4b is the substrate.
Accordingly, a modified characteristic can be achieved by selection of one or more modifications that confer increased C3b degrader activity and decrease C4b degrader activity (increase in C3b substrate specificity) or, alternatively, confer increased C4b degrader activity and decrease C3b degrader activity (increase in C4b substrate specificity) or, alternatively, provide increased activity as degraders of both C3b and C4b (no change in specificity, but increase in activity for both substrates).
Accordingly, a modified characteristic can be achieved by selection of one or more modifications that confer increased C3b degrader activity and no change in C4b degrader activity (increase in C3b substrate specificity) or, alternatively, confer increased C4b degrader activity and no change in C3b degrader activity (increase in C4b substrate specificity).
Accordingly, a modified characteristic can be achieved by selection of one or more modifications that confer a decrease in C3b degrader activity and no change in C4b degrader activity (increase in C4b substrate specificity) or, alternatively, confer a decrease in C4b degrader activity and no change in C3b degrader activity (increase in C3b substrate specificity).
Modifications providing increased activity and specificity are typically concentrated in, but not bound by limitation, to structural regions critical in for CFI function. Exemplary structural regions where modifications (e.g. substitutions) lead to at least one improved characteristic are the C-terminal extension, the A:B interface, the surface representing an interface with cofactors and modifications (e.g. substitutions) within the active site of the SPD including surface loops that provide an interface with the C3b and C4b substrates and the CR1 and FH cofactors (
Without being bound to theory or mechanism, provided herein are CFI variants having one or more combinations of any of the amino acid modifications detailed below, wherein the CFI variants have at least one improved characteristic. CFI variants with combined modifications (e.g. substitutions) comprise two or more modifications in one or more regions of CFI selected from, but not limited to the structural regions of the C-terminal extension, the A:B interface, the interface with cofactors and the active site, including surface loops that provide an interface with cofactors and the C3b or C4b substrates.
In some embodiments CFI variants comprising two or more substitutions exhibit changes in activity, substrate specificity, or both. In some embodiments, an increase in activity comprises an increase in the cleavage of C4b, and/or the generation of C4c and specificity comprises a limited increase or a decrease in the cleavage of C3b, and/or the generation of iC3b as compared to wild type CFI (or compared to a fusion construct comprising wild type CFI, e.g. SEQ ID NO: 21). In some embodiments the combination of two or more substitutions confers unexpected increases in activity that are synergistic when C4b is the substrate and additive or less than additive when C3b is the substrate.
In some embodiments amino acids substitutions either increase activity, confer specificity or both and may switch between C3b selectivity and C4b selectivity. In some embodiments, an increase in activity comprises an increase in the cleavage of C4b, and/or the generation of C4c and selectivity comprises a decrease in the cleavage of C3b, and/or the generation of iC3b as compared to wild type CFI. In some embodiments, an increase in activity comprises an increase in the cleavage of C3b, and/or the generation of iC3b and specificity comprises a decrease in the cleavage of C4b, and/or the generation of C4c as compared to wild-type CFI. In some embodiments the nature of the amino acid substitution defines whether the CFI variant displays characteristics of specificity for C3b or specificity for C4b.
Exemplary variants of the disclosure are tested for differences in activity, and for differences in specificity. Exemplary data are provided in at least Table 7.2.
In some embodiments, the CFI variant exhibits increased activity, wherein the increase in activity comprises an increase in the C3b degrader activity by a CFI variant of the disclosure (with a concomitant increase in a C3b cleavage product). In some embodiments, a CFI variant of the disclosure exhibits increase C3b degrader 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21. In some embodiments, this increase in C3b degrader activity is accompanied by an increase also in C4b degrader activity. In some embodiments, this increase in C3b degrader activity is not accompanied by an increase also in C4b degrader activity, and there may even be a decrease in C4b degrader activity.
In some embodiments, the CFI variant exhibits increased activity, wherein the increase in activity comprises an increase in the C4b degrader activity by a CFI variant of the disclosure (with a concomitant increase in a C4b cleavage product). In some embodiments, a CFI variant of the disclosure exhibits increase C4b degrader 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21. In some embodiments, this increase in C4b degrader activity is accompanied by an increase also in C3b degrader activity. In some embodiments, this increase in C4b degrader activity is not accompanied by an increase also in C3b degrader activity, and there may even be a decrease in C3b degrader activity.
In some embodiments, the CFI variant exhibits increased activity, wherein the increase in activity comprises an increase in both C3b and C4b degrader activity. In some embodiments, a CFI variant of the disclosure exhibits both increased C3b and increased C4b degrader 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21. The increase in degrader activity of one substrate may be the same, but need not be.
In some embodiments, the CFI variant exhibits increased specificity for a substrate, wherein the increase in specificity is for C3b (over C4b). In some embodiments, a CFI variant of the disclosure exhibits increased specificity for C3b 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21.
In some embodiments, the CFI variant exhibits increased specificity for a substrate, wherein the increase in specificity is for C4b (over C3b). In some embodiments, a CFI variant of the disclosure exhibits increased specificity for C4b 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21 which has an about equal specificity for both C3b and C4b.
In some embodiments, the CFI variant exhibits decreased specificity for a substrate, wherein the decrease in specificity is for C3b (over C4b). In some embodiments, a CFI variant of the disclosure exhibits decreased specificity for C3b 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21 which has an about equal specificity for both C3b and C4b.
In some embodiments, the CFI variant exhibits decreased specificity for a substrate, wherein the decrease in specificity is for C4b (over C3b). In some embodiments, a CFI variant of the disclosure exhibits decreased specificity for C4b 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 750-fold at least or about 800-fold, at least or about 850-fold, at least or about 900-fold, at least or about 950-fold at least or even at least about 1000-fold, as compared to a wild type CFI, or a fusion construct comprising a wild type CFI, e.g. SEQ ID NO: 21 which has an about equal specificity for both C3b and C4b.
In some embodiments, exemplary amino acid residues where one or more substitutions may confer improved or unexpected characteristics compared 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 disclosure displaying one or more improved characteristics include CFI variants comprising two or more combinations of T377G, N422K, E457G, E461Q, or N531G as compared to wild type CFI (or 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 a single amino acid substitution of T377G, E457G or E461Q show at least a 2-fold increase in protease activity towards both C4b and C3b as compared to wild type CFI (or 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 a single substitution such as N531G show at least a 5-fold increased protease activity towards C4b and at least a 3-fold increased activity towards C3b, as compared to wild type CFI (or 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 a single substitution such as N422K show little or no change in protease activity towards C4b but show at least a 2-fold increased protease activity towards C3b as compared to wild type CFI (or 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, as compared to 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), exemplary CFI variants comprising or consisting of or consisting of two substitutions such as E457G and N531G show at least a 27-fold increase in activity towards C4b and an at least 4-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of two substitutions such as T377G and N531G show at least a 16-fold increase in activity towards C4b and an at least 4-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of two substitutions such as T377G and E457G show at least a 15-fold increase in activity towards C4b and an at least 4-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of two substitutions such as T377G and E457G show at least a 15-fold increase in activity towards C4b and an at least 4-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of two substitutions such as T377G and N422K or N422K and E457G show at least an 8-fold increase in activity towards C4b and an at least a 5-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of three substitutions such as T377G and E457G and N531G show at least a 100-fold increase in activity towards C4b and an at least 6-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of three substitutions such as T377G and E461Q and N531G show at least a 60-fold increase in activity towards C4b and an at least 5-fold increase in activity towards C3b.
In some embodiments, as compared to 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), exemplary CFI variants comprising or consisting of three substitutions such as T377G and N422K and N531G show at least a 45-fold increase in activity towards C4b and an at least 8-fold increase in activity towards C3b.
In some embodiments, exemplary CFI variants of the disclosure displaying one or more improved characteristics are CFI variants comprising or consisting of N531G, P535A and R557A as compared to wild type CFI (or 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 a single amino acid substitution of R557A show at least a wild-type activity towards C4b and a 20-fold reduction in C3b activity as compared to wild type CFI (or 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 a single substitution such as N531G show at least a 5-fold increased activity towards C4b and at least a 3-fold increased activity towards C3b, as compared to wild type CFI (or 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, as compared to 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), CFI variants comprising or consisting of two substitutions such as N531G and P535A show at least a 5-fold increased activity towards C4b and a 3-fold increase in C3b activity, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), variants comprising or consisting of three substitutions such as N531G and P535A and R557A show at least an 18-fold increase in activity towards C4b and 2.5-fold reduction in C3b activity, 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 disclosure displaying one or more improved characteristics are CFI variants comprising or consisting of D425R, E457G and E530Y, as compared to wild type CFI (or 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 a single amino acid substitution of E457G or E530Y show at least a wild-type activity towards C3b and C4b. In some embodiments, as compared to 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), variants comprising or consisting of three substitutions such as D425R and E457G and E530Y show at least an 8-fold increase in activity towards C3b and near wild type activity towards C4b, 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 disclosure displaying one or more improved characteristics are CFI variants comprising or consisting of R557A, R557M, R557P, and R557G, as compared to wild type CFI (or 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, as compared to 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), an exemplary CFI variant such as one consisting or comprising a R557A substitution shows at least a wild-type activity towards C4b and a 20-fold reduction in C3b activity, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), a variants comprising or consisting of or consisting of R557M and R557P show at least a 3-fold increase in activity towards C4b and 5-fold to 10-fold reductions in C3b activity, respectively, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), a variant such as R557G shows at least a 2-fold activity towards C4b and a 20-fold reduction in C3b activity, 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 disclosure displaying one or more improved characteristics are CFI variants comprising or consisting of E457T, E457Q, E457G or E457A, as compared to wild type CFI (or 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, as compared to 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), an exemplary CFI variant comprising or consisting of a substitution such as E457T shows at least 2.6-fold increased activity towards C3b and a 5-fold reduction in C4b activity, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), an exemplary CFI variant comprising or consisting of a substitutions E457Q or E457G show at least a wild-type activity towards both C3b and C4b, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), an exemplary CFI variant comprising or consisting of a substitution such as E457A shows at least 2.7-fold increased activity towards C4b and a 1.6-fold increase in C3b activity, 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 disclosure displaying one or more improved characteristics are CFI variants comprising or consisting of E530F, E530Y, or E530R substitutions, as compared to wild type CFI (or 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, as compared to 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), an exemplary CFI variant comprising or consisting of a substitution such as E530Y shows at least a 1.6-fold increase in activity towards C3b and near wild type activity on C4b activity, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), an exemplary CFI variant comprising or consisting of a substitution such as E530F shows at least a 1.6-fold increase in activity towards C3b and 3-fold reduction in C4b activity, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, as compared to 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), an exemplary CFI variant comprising or consisting of a substitution such as E530R shows at least 1.8-fold increased activity towards C3b and a 5-fold reduction of C4b activity, 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 disclosure displaying one or more improved characteristics are CFI variants comprising or consisting of E457G, E461Q, N531G or R557A substitutions, as compared to wild type CFI (or 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.
Exemplary CFI variants comprising or consisting of a single amino acid substitution of E457G or E461Q show at least a 2-fold increased activity towards C4b and C3b as compared to 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). Exemplary CFI variants comprising or consisting of a single substitution such as N531G show at least a 5-fold increased activity towards C4b and at least a 3-fold increased activity towards C3b. Exemplary CFI variants comprising or consisting of a single amino acid substitution of R557A show at least a wild-type activity towards C4b and a 20-fold reduction in C3b activity. In some embodiments, as compared to 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), variants comprising or consisting of two substitutions such as E457G and N531G show at least a 27-fold increase in activity towards C4b and an at least 4-fold increase in activity towards C3b. In some embodiments, as compared to 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), variants comprising or consisting of two substitutions such as E457G and E461Q show at least a 5-fold increase in activity towards C4b and C3b. In some embodiments, as compared to 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), variants comprising or consisting of two substitutions such as E461Q and N531G show at least a 12-fold increase in activity towards C4b and an at least 5-fold increase in activity towards C3b. In some embodiments, as compared to 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), variants comprising or consisting of two substitutions such as E457G and E461Q and N531G and R557A show at least a 12-fold increase in activity towards C4b and an at least 1.5-fold increase in activity towards C3b. These differences are as compared to wild type CFI (or compared to a fusion construct comprising wild type CFI, e.g. SEQ ID NO: 21).
In some embodiments, the CFI variant having has increased activity, wherein the increased activity comprises increased cleavage of C3b and/or specificity for C3b over C4b. In some embodiments, the CFI variant having increased cleavage of C3b and/or specificity for C3b over C4b comprises one or more substitutions in an 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 in 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; C3b interface region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the active site; C3b interface region is selected from one or more of T346, E401, and N531. In some embodiments, the amino acid position is a position within the autolysis loop; cofactor interface in a CFI having an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the autolysis loop; cofactor interface is selected from one or more of E457 and E461. These differences are as compared to wild type CFI (or 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, the CFI variant having has increased activity, wherein in the increased activity comprises increased cleavage of C4b and/or specificity for C4b over C3b. In some embodiments, the CFI variant having an increase in the cleavage of C4b comprises one or more substitutions in an 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 a 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 in 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; C4b interface region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the C-terminal extension; 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 position within the C4b interface region is E401. In some embodiments, the amino acid position is a position within the active site; C4b interface region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the active site; C4b interface region is G406. In some embodiments, the amino acid position is a position within the autolysis loop; 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 autolysis loop; 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; 51 entrance frame region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the active site; S1 entrance frame region is N531. In some embodiments, the amino acid position is a position within the S1 entrance frame region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the S1 entrance frame region is K534. These differences are as compared to wild type CFI (or 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, the improved characteristic is an increase in activity, wherein the increase in activity comprises an increase in the cleavage of C3b and/or C4b. In some embodiments, the CFI variants provided herein are C3b degraders, referring to the ability of the CFI variants to increase C3b cleavage. In some embodiments, the CFI variants provided herein are C4b degraders, referring to the ability of the CFI variants to increase C4b cleavage. In some embodiments, the CFI variants provided herein are C3b and C4b degraders, referring to the ability of the CFI variants to increase cleavage of both C3b and C4b.
In some embodiments, the CFI variant having has increased activity, wherein the increased activity comprises increased cleavage of C3b and C4b. In some embodiments, the CFI variant having an increase in the cleavage of C3b and C4b comprises one or more substitutions in 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 position within the substrate interface region is E401. In some embodiments, the amino acid position is a position within the active site; substrate interface region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the active site; 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 in 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 autolysis loop; 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 autolysis loop; 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; S1 entrance frame region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, position within the active site; S1 entrance frame region is N531. These differences are as compared to wild type CFI (or 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, the CFI variant having has increased activity, wherein the increase in activity comprises an increase in the cleavage of C3b by a CFI variant of the disclosure and does not comprise or minimally comprises an increase in the cleavage of C4b. In some embodiments, the CFI variant having an increase in the cleavage of C3b and does not comprise or minimally comprises an increase in the cleavage of C4b comprises one or more substitutions at positions selected from T346, E392, N422, E416, and E401 in amino acid positions set forth in SEQ ID NO: 5. In some embodiments, the amino acid position is a position within the active site; C3b interface region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the active site; 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 in 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 as compared to wild type CFI (or 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, the CFI variant has increased activity, wherein the increase in activity comprises an increase in the cleavage of C4b by a CFI variant of the disclosure and does not comprise or minimally comprises an increase in the cleavage of C3b. In some embodiments, the CFI variant having an increase in the cleavage of C4b and does not comprise or minimally comprises an increase in the cleavage of C3b comprises one or more substitutions in 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 a 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 one or more positions selected from 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 entrance frame region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the S1 entrance frame region is K534. In some embodiments, the amino acid position is a position within the c-terminal extension; C4b interface region in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the position within the C-terminal extension; C4b interface region is Y553. In some embodiments, the amino acid position is a position within the C-terminal extension region in 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 as compared to wild type CFI (or 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, the CFI variants of the disclosure that are specific C3b degraders are useful for the treatment of diseases.
In some embodiments, the CFI variants of the disclosure that are specific C4b degraders are useful for the treatment of diseases.
In some embodiments, the CFI variants of the disclosure that are both C4b and C3b degraders, and show an improved characteristic as compared to wild type CFI (e.g. increased activity for both C4b and C3b) are useful for the treatment of diseases.
For example, the diseases that may be treated by use of the C4b degraders include, but are not limited to a non-ocular condition. 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)-associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic kidney failure, chronic obstructive pulmonary disease (COPD), Cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), Granulomatosis with Polyangiitis (GPA), Guillain-Barré 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, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), warm Autoimmune hemolytic anemia (wAIHA), Immune-Complex Membranoproliferative Glomerulonephritis (IC-MPGN), and ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA) and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington disease and ischemia reperfusion injuries.
In some embodiments, the CFI variants provided here are degraders of both C3b and C4b and are useful for the treatment of diseases.
In some embodiments, an increase in activity comprises an increase in the generation of C3dg and/or C3c from iC3b. Exemplary CFI variants of the disclosure displaying this improved characteristic are a CFI variant that comprises the substitutions N531G+P535A, D425A, or D425R, 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 increase in activity comprises a reduction in the levels of C3b Δ-chain. An exemplary variant of the disclosure displaying this improved characteristic is a CFI variant that comprises the N531G+P535A substitutions, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. Other variants that exhibit similar improved characteristics are provided in Table 13, and discussed in the examples.
In some embodiments, an increase in activity comprises 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, e.g. such peptide substrates are useful in an assay format. Exemplary CFI variants of the disclosure displaying this improved characteristic are a CFI variant that comprises the modifications L307G, E457G, E461Q, E457G+E461Q+R462K+F464Y, N531G, N531A, P535A, N531G+P535A, Y408L, Y408L+N531G, Y408F+N531G, Y408L+N531G+E457G+E461Q+R462K+F464Y, A(Kl-P305)+N531G, Δ(K1-P305)+N531G+P535A, or the autolysis loop swap of 456-REKDNERVFS (SEQ ID NO: 9)-465 -->NTASSGADYPDE (SEQ ID NO: 10), wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. Other variants that exhibit similar improved characteristics are provided in Table 13, and discussed in the examples.
In some embodiments, an increase in activity comprises a reduction in the levels or function of membrane attack complex (MAC). In some embodiments, a reduction or even inhibition of hemolysis is correlated with the reducing in the levels of MAC, and accordingly, in some embodiments, an increase in activity comprises a decrease (partial or complete) in the observed hemolysis.
In some embodiments, an increase in activity comprises a reduction in the amplification of the complement system for the production of C3b. An exemplary variant of the disclosure displaying this improved characteristic is a CFI variant that comprises the N531G+P535A substitutions, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5. Other variants that exhibit similar improved characteristics are provided in Table 13, and discussed in the examples.
In some embodiments, the CFI variants are sialylated. In some embodiments, the CFI variants are further sialylated as compared to a wild type CFI. In some embodiments the CFI variants are sialylated by in vitro methods post-translationally.
In some embodiments, the CFI variants are activated variants (i.e. in an active two chain form). In some embodiments, the CFI variants are activated by furin (the term furin is inclusive of furin variants). In some embodiments, the CFI variants are activated by furin during production in a host cell. In some embodiments, the activation by furin during production in a host cell is achieved by overexpression of furin, e.g. by stable or transient transfection. In some embodiments, the CFI variant is activated by furin after production and secretion by a host cell, i.e. post-translationally.
References to modifications, such as substitutions, in the following sections are modifications with respect to the amino acid sequence of human CFI as set forth in SEQ ID NO: 5. However, it should be understood that modifications to corresponding amino acid residues of any non-human species may also be made.
Provided herein are CFI variants comprising one or more modifications at the interface of the heavy and light chains, also referred to as the A:B chain interface, and variants that cause a disruption to the A:B chain interface.
Without being bound to theory or mechanism, the serine protease domain (SPD) of CFI is thought to be kept in a zymogen-like state, via numerous 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.
Accordingly in some embodiments, provided herein are A:B chain interface CFI variants. Specifically, 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 at the A:B chain interface or cause a disruption to the A:B chain interface. The base molecule for the CFI variants presented in Table 2 may be wild type human CFI. It is noted that not all of the A:B chain interface CFI variants of the disclosure are provided in Table 2, and additional variants may be provided in at least the Examples and/or Table 13.
In some embodiments, the CFI variants comprise or consist of any one or more of the modifications presented in Table 2. In some embodiments, the CFI variants comprise a modification at any one or more positions corresponding to positions K14, Y20, D26, F29, R35, E38, M220, K221, S250, L304, P305, K306, L307, and S308 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise or consist of a substitution in a 200 loop of the CFI (MDANNVT, SEQ ID NO: 13) for a 200 loop of trypsin having amino acid residues NG, wherein the 200 loop occurs between positions corresponding to position 514 and position 520 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise or consist of any one or more of the substitutions selected from K14A, Y20A, Y20F, D26A, F29A, R35A, E38A, M220A, K221Q, S250A, S250L, L304G, P305G, K306G, L307G, and S308G, 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 variants comprise or consist of any one or more of the combination of substitutions M220A and K221Q, and L304G+P305G+K306G+L307G+S308G, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In the complex formed between CFI and C3b, the C-terminal extension region is positioned in a cavity between the A and B chain of the bound and slightly twisted CFI molecule. This suggests that the C-terminal extension of CFI could be an important regulatory region for the activation of CFI upon binding to C3b.
Accordingly, 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 at the C-terminal region or extension of CFI. The base molecule for the CFI variants presented in Table 3 may be a wild type human CFI. It is noted that not all of the C-terminal region CFI variants of the disclosure are provided in Table 3, and additional variants may be provided in at least the Examples and/or Table 13.
In some embodiments, the CFI variants comprise any one or more of the modifications presented in Table 3.
In some embodiments, the CFI variants comprise or consist of 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.
In some embodiments, the CFI variants comprise or consist of a deletion 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, amino acid residues, the CFI variants comprise or consist of a substitution in a 110 loop of the CFI (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 are substituted for a linker, e.g. GG.
In some embodiments, the CFI variants comprise or consist of any one or more of the substitutions selected from T377G, W381A, P384A, P384G, Y403F, A405S, G406R, G406A, Y408L, Q409D, Q409H, D425A, D425K, D425R, G556A, G556S, R557A, R557K, P558G, P558L, P558S, F559L, I560V, and Y563H, and/or a deletion of P384, 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 variants comprise or consist of any one or more of the combination modifications selected from P558S+F559L+I560V+Y563H, A405S+G406R+Y408L+Q409D, A405S+G406A+Y408L+Q409D, G406A+Y408L, and W381A+ΔP384, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Provided herein are CFI variants comprising at least one CFI domain, wherein the at least one CFI domain comprises one or more modifications at N-linked glycosylation sites of CFI.
In some embodiments, the modification at the N-linked glycosylation site is a removal of one or more N-linked glycosylation sites of a CFI.
Accordingly, provided herein are N-linked glycosylation site CFI variants. Specifically, 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 at the N-linked glycosylation site of CFI. The base molecule for the CFI variants presented in Table 4 may be wild type human CFI. It is noted that not all of the N-linked glycosylation site variants of the disclosure are provided in Table 4, and additional variants may be provided in at least the Examples and/or Table 13.
Without being bound by any theory or mechanism, exemplary CFI variants comprising an N-linked glycosylation site modification may include the following variants.
In some embodiments, the CFI variants comprise any one or more of the modifications presented in Table 4.
In some embodiments, the CFI variants comprise or consist of a modification at any one or more positions corresponding to positions N52, N85, N159, N446, N476, and N518 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise or consist of any one or more of the substitutions selected from N52Q, N85Q, N159Q, N446Q, N476Q, and N518Q, 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 variants comprise or consist of any one or more of combination of substitutions selected from N52Q+N85Q+N159Q, N446Q+N476Q+N518Q, and N52Q+N85Q+N159Q+N446Q+N476Q+N518Q, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Provided herein are CFI variants comprise or consist of at least one CFI domain, wherein the at least one CFI domain is the serine protease domain (SPD) of CFI, and wherein the CFI variant comprises one or more modifications at the SPD.
In the crystal structure of free CFI, cleavage of the activation loop did not result in the insertion of the newly formed N-terminal (Ile322), which is the next step in the classical activation of serine proteases. Instead, the crystal structure suggests that the C-terminal region of the cleaved activation loop remains in a tightly bent loop structure on the surface of CFI, in the same area that the uncleaved activation loop would have remained. This prevents the insertion into the activation pocket, and thus, maturation of the active site (referred to as classical serine protease activation via induced conformational rearrangements). Upon proteolytic activation of the SPD of CFI the new N-terminus of the activation loop is generally released and inserted into the activation pocket such that the cleaved activation loop forces a full activation of CFI in solution. Thus, mutations in the C-terminal region of the activation loop should not affect cleavage by furin, as the region is beyond the 3′ positions relative to the scissile bond.
Accordingly, provided herein are SPD CFI variants. In some embodiments, the CFI variants comprising one or more modifications within regions of the SPD of CFI (
It is noted that not all of the SPD CFI variants of the disclosure are provided in Table 5, and additional variants may be provided in at least the Examples and/or Table 13.
In some embodiments, the CFI variants comprise any one or more modifications presented in Table 5.
In some embodiments, the CFI variants comprise an autolysis loop substitution. The autolysis loop of serine proteases is part of the activation domain and are involved in substrate specificity. Trypsin has a longer autolysis loop than CFI, and several key residues are unique between the autolysis loops of trypsin and CFI. Differences may also occur between the autolysis loops from different species, such as between mouse and human. The mouse CFI autolysis loop may include a large number of differences as compared to the CFI autolysis loop of human CFI. Exemplary CFI variants may include a CFI variant wherein the autolysis loop of human CFI is swapped with that of human trypsin or swapped with that of mouse CFI. Such autolysis loop variants may help to identify critical residues that are involved in C3b and/or C4b cleavage activity. Accordingly, in some embodiments, provided herein are CFI variants, 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 of a corresponding region from a non-human species CFI. In some embodiments, the non-human species CFI is mouse CFI. Provided also herein are CFI variants wherein the CFI variant is a chimera, and wherein the modification comprises the substitution of one or more amino acid residues of the 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.
An exemplary autolysis loop CFI variant includes a trypsin autolysis loop substitution, comprising a substitution of an autolysis loop of the CFI (REKDNERVFS, SEQ ID NO: 9) for an autolysis loop of trypsin (NTASSGADYPDE, SEQ ID NO: 10), wherein the autolysis loop occurs between positions corresponding to position 456 and position 465 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Another exemplary autolysis loop CFI variant includes a mouse CFI autolysis loop swap, wherein 456REKDNERVFS465 (SEQ ID NO: 9) swapped to RGKDNQKVYS (SEQ ID NO: 11), wherein the autolysis loop occurs between positions corresponding to position 456 and position 465 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise or consist of one or more modifications at any one or more of the activation loop (residues 322-326 of SEQ ID NO: 5), 37-Loop (residues 342-344 of SEQ ID NO: 5), 60-Loop (residues 366-372 of SEQ ID NO: 5), 70-Loop (residues 377-389 of SEQ ID NO: 5), 99-Loop (residues 403-410 of SEQ ID NO: 5), 110-Loop (residues 418-426 of SEQ ID NO: 5), 150-Autolysis Loop (residues 455-463 of SEQ ID NO: 5), 180-Loop Oxyanion Stabilizing (residues 494-509 of SEQ ID NO: 5), and/or 22-Loop S1 Entrance Frame (residues 529-536 of SEQ ID NO: 5) of SPD.
In some embodiments, the CFI variants comprise or consist of a modification at any one or more positions corresponding to 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, E530, N531, G533, K534, P535, E536, and F537 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise or consist of any one or more of the substitutions selected from K14A, K312A, R314A, I322T, I322Y, I322V, V323I, V323G, V323A, K326A, R327A, R327P, R327N, A328C, K340G, D341A, G344R, G344K, G344Y, I345G, T346R, T346K, T346H, A361G, L364G, Y372G, W381K, W381G, P384A, P384G, V390G, N402E, N404G, G406D, G406E, G406F, G406H, G406I, G406K, G406L, G406M, G406N, G406P, G406Q, G406S, G406T, G406V, G406W, G406Y, Y408L, Y408F, Y408G, Y408P, Y408D, Y408A, Y408N, Y408T, Y408K, Y408R, Y408H, Y408I, Y408E, Y408M, Y408Q, Y408S, Y408W, Y408Y, Y408V, Q409G, E416A, K418G, N422K, D425A, D425K, D425R, D425G, R456A, R456N, E457G, E457A, E457D, E457F, E457H, E457I, E457K, E457L, E457M, E457N, E457P, E457Q, E457R, E457S, E457T, E457W, E457Y, E457V, K458A, E461Q, E461K, E461R, E461H, E461G, E461A, E461D, E461F, E461I, E461L, E461M, E461N, E461P, E461S, E461T, E461W, E461Y, E461V, R462K, R462A, R462D, F464Y, 5465G, Q467K, Q467R, W468C, G469L, T495F, Y496L, D497E, S499G, I500K, A502S, K504Q, K504E, K504R, K504A, K504G, K504L, K504P, K504H, K504D, K504F, K504I, K504M, K504N, K504S, K504T, K504V, K504W, K504Y, D506A, D506V, D506E, D506G, S507A, E530D, E530G, E530F, E530Y, N531G, N531A, E530D, E530G, E530F, E530Y, E530R, E530K, N531D, N531E, N531F, N531H, N531I, N531K, N531L, N531M, N531P, N531Q, N531R, N531S, N531T, N531V, N531W, N531Y, G533A, K534Q, P535A, P535K, E536N, E536A, F537K and F537R, 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 variants comprise or consist of any one or more of the combination substitutions selected from K326A+327A, N531G+P535A, E457G+E461Q+R462K+F464Y, Y408L +N531G+E457G, Y408L+N531G+E457G+E461Q, Y408L+N531G+E457G+E461Q-R462K+F464Y, Y408L+N531G+P535A, K14A+D425R, E530D+N531G+G533A+K534Q+P535K+E536N, A502S+K504Q+F537K, T495F+Y496L+D497E+S499G+I500K, G533A+K534Q+P535K+E536N+F537K, T495F+Y496L+D497E+S499G+I500K+G533A+K534Q+P535K+E536N+F537K, Q467K+F537K, E530G+N531G, E530D+F537K, E457G+E461Q, E457G+E461G, Y408L+N531G+E457G+E461Q, N531G+E457G+E461Q, I322V+V323I, I322V+V3231+R327P, A328C+W468C, A328C+W468C+K326Y+R327N, Y408L+N531G+E461Q, Y408L+N531G+E457G+E461Q+R462K, Y408L+N531G+E457G+E461Q+F464Y, Y408L+N531G+E457G+R462K+F464Y, Y408L+N531G+E461Q+R462K+F464Y, Y408L+E457G+E461Q+R462K+F464Y, E457G+N531G+E461Q+R462K+F464Y, Y408L+E457G+E461Q+R462K, N531G+E457G+E461Q+F464Y, E416A+D425R, Y408L+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, G406D+N531G, G406D+P535A, G406D+Y408L+N531G, G406D+Y408L+P535A, G406D+N531G+P535A, G406D+Y408L+N531G+P535A, K340G+I345G, L364G+Y372G, W381G+V390G, W381G+P384A+V390G, W381G+P384G+V390G, N404G+Q409G, K418G+D425G, T346R+K504E+E530R, T346K+K504D+E530K, G344R+Y408L+N531G, G344K+Y408L+N531G, T346R+Y408L+N531G, T346K+Y408L+N531G, K504D+Y408L+N531G, K504E+Y408L+N531G, Y408L+E530R+N531G, Y408L+E530K+N531G, T346R+Y408L+K504E+E530R+N531G, T346K+Y408L+K504D+E530K+N531G, Y408L+S507A+N531G, Y408L+N531G+E457G+E461Q+R462K+F464Y+S507A, E457G+S507A, and N531G+P535A +S507A, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Provided herein are CFI variants comprising or consisting 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 at the active site of CFI. In some embodiments, the active site CFI variants may improve the catalytic potential of CFI. In some embodiments, the CFI active site variants may improve the catalytic potential of CFI by improving the active site (catalytic machinery) without affecting C3b or C4b binding or binding specificity, which is dominated by exosite and A-chain interactions.
Accordingly, provided herein are active site CFI variants. Specifically, provided herein are exemplary CFI variants comprising a modification 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 at the active site of CFI. The base molecule for the CFI variants presented in Table 6 may be wild type human CFI.
In some embodiments, the CFI variants comprise or consist of modifications presented in Table 6.
In some embodiments, the CFI variants comprise or consist 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 variants comprise or consist of a substitution S507A, wherein the position corresponds to position S507 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Provided herein are CFI variants, wherein the CFI comprises an A chain and a B chain, and comprise an inversion of the A chain and the B chain. In some embodiments, the CFI variants without a chain inversion (the individual chains optionally comprising one or more modifications) comprise a structural arrangement from N-terminus to C-terminus, or C-terminus to N-terminus, as (A chain)-(optional linker)-(B chain). In some embodiments, the CFI variants comprise an inversion of the A chain and the B chain (the individual chains optionally comprising one or more modifications), such that the structural arrangement from N-terminus to C-terminus, or C-terminus to N-terminus, is (B chain)-(optional linker)-(A chain). The optional linkers may be of any suitable length, e.g. of at least one amino acid. A 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 linkers known in the art. An exemplary linker is GGSSGGn (SEQ ID NO: 6), wherein n is any number from about 1 to about 20. Exemplary linkers of 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.
Accordingly, provided herein are CFI variants, wherein the CFI comprises an A chain and a B chain, and wherein the structural arrangement from N-terminus to C-terminus, or C-terminus to N-terminus, is (B chain)-(optional 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 an inversion of the A and B chains of CFI.
Without being bound by theory or mechanism, exemplary CFI variants comprising an inversion of the A and B chains may comprise the amino acid sequences set forth in SEQ ID NOs: 17, 18, 19, or 20. The chains may be held together by optional linkers. The linkers between the A chain and the B chain of the inversion variants may be of any suitable length of at least one amino acid. A 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 linkers known in the art. It should be understood that, while the exemplary inversion variants shown in Table 7 include glycine polymer linkers, any suitable flexible linkers may be used for a CFI variant having an A-B chain inversion.
In some embodiments, the CFI variants comprise a substitution at C309 and/or C435, 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 variants comprise substitutions C309S and C435S, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Additional CFI Variants—Useful for Modulation and/or Evaluation of the Complement System
In some embodiments, there are CFI variants provided that, while useful for modulation of the complement system, may also be useful for evaluation of activity of the complement system, e.g. can be considered tool proteins, in addition to having therapeutic value.
For example, these other CFI variants may allow for various tests using the CFI fusion constructs. An exemplary such CFI variant may be non-activatable to serve as a control. Another exemplary such CFI variant may provide an easier activation of a fusion construct.
In some embodiments, such additional CFI variants provided herein comprise a modification to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the envisioned CFI variants provided herein are derived from a wild type mouse CFI. In some embodiments, the envisioned CFI variants provided herein are derived from a wild type human CFI. In some embodiments, the envisioned CFI variants provided herein are further derived from a CFI-SPD.
In exemplary embodiments, the CFI variants comprises any one or more of the exemplary modifications presented in Table 8. Such CFI variants may be useful for providing a control for or further study of any CFI variants provided herein. Such CFI variants may also provide therapeutic utility.
Exemplary CFI variants may include a non-activatable CFI variant, which may serve as a control.
In some embodiments, the CFI variants comprise any one or more of the modifications presented in Table 8.
In some embodiments, the CFI variants comprise or consist of a modification at any one or more positions corresponding to positions I317, R318, R319, K320, and R321 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the CFI variants comprise or consist of any one or more of the substitutions selected from I317D, R318D, R319D, K320D, and R321K, 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 variants are more easily activated as compared to the wild type CFI. In some embodiments, the CFI variants are more easily activated as compared to the wild type CFI, and comprise or consist of substitutions I317D, R318D, R319D, K320D, and R321K, 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 variants are not activatable, and comprise or consist of least one modification with respect to a wild type CFI. In some embodiments, the CFI variants are not activatable, and comprise a modification at a position corresponding to position R321 of a CFI having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CFI variants comprise 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.
Provided herein are CFI variants comprising or consisting of two or more modifications with respect to a wild type CFI. The modifications occur in the same or different domains of CFI. In some embodiments, the modifications include two or more substitutions. In some embodiments, the modifications include a substitution and a deletion. In some embodiments, the modifications include a substitution and an addition. In some embodiments, the modifications include a deletion and an addition. In some embodiments, the modifications include a substitution, a deletion, and an addition. As used herein, such variants collectively may be referred to as CFI combination variants.
Accordingly, provided herein are CFI combination variants. Specifically, 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 a wild type human CFI or a CFI-SPD. It should be understood that any of the CFI variants provided herein may comprise any combination of any of the modifications provided herein, such as, for example, any of the modifications presented in Tables 2-8 and in Table 13.
Without being bound by any theory or mechanism, exemplary combination CFI variants may include the following variants.
In some embodiments, the CFI variants comprise or consist of any one or more of the modifications presented in Table 9.
In some embodiments, the CFI variants comprise or consist of any one or more of the combination substitutions selected from Y408+N531G, E38A+D425R, Y20F+D425R, S250A+D425R, Y408F+N531G, Y408L+N531G+E457G+E461Q+R462K+F464Y, K14A+Y20F, K14A+E38A, K14A+S250A, K14A+D425A, Y20F+E38A, Y20F+S250A, Y20F+D425A, E38A+S250A, E38A+D425A, S250A+D425A, K14A+N531G+P535A, Y20F+N531G+P535A, E38A+N531G+P535A, S250A+N531G+P535A, D425A+N531G+P535A, Y20F+Y408L+N531G+E457G+E461Q+R462K+F464Y, E38A+Y408L+N531G+E457G+E461Q+R462K+F464Y, S250A+Y408L+N531G+E457G+E461Q+R462K+F464Y, D425R+Y408L+N531G+E457G+E461Q+R462K+F464Y, Y20F+E38A+S250A+D425A, Y20F+E38A+S250A+D425A+Y408L+N531G+E457G+E461Q+R462K+F464Y, Y20F+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, K504E+Y408L+N531G, E457G+N531G+D425K, Y408F+N531G, Y408L+E457G+N531G+D425K, Y408L+E457G+P535G+D425K, Y408L+E457G+N531G+K534Q, Y408L+N531G, R462K+F464Y, and Y408L+P535G+D425K, wherein the positions correspond to positions in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
Substitutions with Minimal Impact on Activity and Specificity
Certain CFI variants exhibit little or no differences compared to wild type CFI protease activity or substrate specificity. In some cases, the substitutions even decreased activity as compared to wild type CFI. Some substitutions that individually exhibited little or no difference in protease activity or substrate specificity as single site substitutions are provided in Table 15. However, it should be understood that the listing of the substitutions here do not indicate that one or more of these substitutions used in combination with another substitution my exhibit a different effect on CFI substrate specificity and CFI protease activity.
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 component and second component are fused (e.g. contiguous or separated by an optional linker). These fusion constructs are referred to herein as “CFI fusion constructs” or simply as “fusion constructs.” In some embodiments, the fusion construct comprises additional components, e.g. a third component, a fourth component, etc.
In some embodiments, the first component comprises a wild type CFI of any species, either a full length or domain thereof. In some embodiments, the first component comprises a CFI variant of the disclosure, described in detail in the preceding section. It is noted that the second component may increase the activity or alter the specificity of the CFI portion (first component) or its half-life. The second component may also allow for CFI portion (first component) to act within the complement system without the presence of an exogenous cofactor (e.g. a cofactor such as Factor H (FH) or CR1). As used herein, an exogenous cofactor for CFI is one that is not fused to CFI. It should be understood that a fusion construct may act within the complement system without the presence of FH and/or CR1, but the activity of the fusion construct may also be further increased with the presence of FH, and/or CR1, either as a part of the fusion construct or provided exogenously.
Provided herein are fusion constructs comprising a first component comprising any one of the CFI variants provided herein. It should be understood that the CFI variant may be any one of the CFI variants presented in Tables 2-9 or Table 13,or may comprise any combination of the modifications that are 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 disclosure may be held together by optional linkers. They may be of any suitable length of at least one amino acid. A 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 linkers 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 linkers are protease-sensitive cleavable linkers. Exemplary linkers linking 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.
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 CFI has a relatively short half-life, it may be advantageous in some embodiments to increase the half-life of CFI. As used herein, “CFI” is used to connotate either the wild type CFI, or variants thereof. By using a second component that is a half-life extender, the activity of CFI may increase, or it may improve another characteristic of the CFI as compared to a wild type CFI. For example, a wild type CFI or a CFI variant may have their half-life extended by fusing the CFI to a half-life extender.
Exemplary half-life extenders include, but are not limited to albumin, such as human serum albumin, PEG, a non-biodegradable polymer, a biodegradable polymer, 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 not a protein, and is a half-life extender, such as PEG. In some embodiments, the half-life extender is comprising peptide repeats.
In some embodiments, the second component is a half-life extender, and is albumin. It is noted that as used herein, albumin refers to any albumin such as any serum albumin, or an albumin variant, or albumin derivative. As an example, a variant of albumin includes 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 at least one modification corresponding to the amino acid sequence of an albumin of any non-human species. In exemplary embodiments, the albumin is human serum albumin (HSA) and is provided in SEQ ID NO: 7.
Exemplary fusion constructs comprising wild type CFI and HSA are referred to herein, as “CFI-HSA” and are discussed in further detail below.
In some embodiments, a fusion construct of the disclosure comprises albumin and a CFI variant of the disclosure.
In some embodiments, a wild type CFI or a CFI variant of the disclosure is the first component of a fusion construct, and wherein this CFI portion comprises an A chain and a B chain In some embodiments, the fusion construct comprises a structural arrangement from N-terminus to C-terminus (A chain)-(optional linker)-(B chain)-(optional linker)-(Second Component). In some embodiments, the fusion construct comprises an inversion of the A and B chains in its CFI component, such that the structural arrangement from N-terminus to C-terminus, is (B chain)-(optional linker)-(A chain)-(optional linker)-(Second Component).
In some embodiments, a wild type CFI or a CFI variant of the disclosure is the first component of a fusion construct, and wherein this CFI portion comprises an A chain and a B chain In some embodiments, the fusion construct comprises a structural arrangement from N-terminus to C-terminus, as (Second Component)-(optional linker)-(A chain)-(optional linker)-(B chain). In some embodiments, the fusion construct comprises an inversion of the A and B chains in its CFI component, such that the structural arrangement from N-terminus to C-terminus is (Second Component)-(optional linker)-(B chain)-(optional linker)-(A chain).
In some embodiments, provided herein are fusion constructs comprising at least a first component, wherein the first component is any of the wild type CFI or CFI variants provided herein (CFI portion), and a second component, wherein the first component and second component are fused, and wherein the second component is fused to the N-terminal end of the CFI portion. In some embodiments, the second component is fused to the C-terminal end of the CFI portion. In some embodiments, the second component is fused to the C-terminal end of the CFI portion, and a third component is further fused to the N-terminal end of the CFI portion. In some embodiments, the second component is fused to the N-terminal end of the CFI portion, and a third component is further fused to the C-terminal end of the CFI portion.
Accordingly, 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 structural arrangement from N-terminus to C-terminus, or C-terminus to N-terminus, is (Second Component)-(optional linker)-(B chain)-(optional linker)-(A chain). Such chain inversions are presented in Table 7 above. Table 7 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 an inversion of the A and B chains of CFI.
In some embodiments, the CFI variants comprise a substitution at C309 and/or C435, 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 variants comprise substitutions C309S and C435S, 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 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 CR1. Fusion constructs comprising at least one CFI domain and Factor H are discuss in further detail below. In some embodiments, the second component comprises at least one domain of Factor H and at least one domain of CR1. Fusion constructs comprising at least one CFI domain, at least one Factor H domain, and at least one CR1 domain are discussed in further detail below.
Provided herein are fusion constructs comprising a first component and a second component. In some embodiments, the first component comprises a wild type CFI, whereas in some embodiments the first component comprises a CFI variant of the disclosure. 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 embodiments, 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 the amino acid sequences of the components that may be used to generate CFI fusion constructs provided herein.
Turning 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. Wild type CFI used for fusion with a second component may comprise the amino acid sequence of SEQ ID NO: 5, which does not include the leader sequence present in SEQ ID NO: 1. A mouse Ig kappa chain V-III region MOPC 63 leader sequence (SEQ ID NO: 2) may instead be used for the 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.
MKLLHVFLLFLCFHLRFCKVTYTSQEDLVEKKCLAKKYTHLSC
C. Complement Factor I and Albumin Fusion Constructs
In some embodiments, provided herein are fusion constructs comprising a first component that is a wild type CFI, and second component that is albumin, e.g. serum albumin, e.g. human serum albumin.
In some embodiments, the albumin is human serum albumin (HSA), and the CFI is a wild type CFI, and such fusion constructs are referred to herein as “CFI-HSA.”
In some embodiments, a CFI-HSA may have an extended half-life with respect to a CFI not part of a fusion construct. An exemplary CFI-HSA construct can be generated by linking an albumin with wild type CFI by a flexible linker. In some embodiments, the CFI-HSA comprises the amino acid sequence set forth in SEQ ID NO: 21, or comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
In some embodiments, the fusion construct comprises a structural arrangement from N-terminus to C-terminus as (Albumin)-(optional linker)-(WT CFI A chain)-(optional linker)-(WT CFI B chain).
In some embodiments, the fusion construct comprises a structural arrangement from N-terminus to C-terminus as (WT CFI A chain)-(optional linker)-(WT CFI B chain)-(optional linker)-(Albumin).
In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement from N-terminus to C-terminus (SEQ ID NO: 7)-(optional linker)-(SEQ ID NO: 5). In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement 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 sequence of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein the fusion construct comprises a structural arrangement 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 sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement from N-terminus to C-terminus (SEQ ID NO: 5)-(optional linker)-(SEQ ID NO: 7). In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 wherein the fusion construct comprises a structural arrangement 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 sequence of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein the fusion construct comprises a structural arrangement 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 an amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence comprising at least 80% identity thereto. In some embodiments, the fusion construct consists of an amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the fusion construct comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7. In some embodiments it is noted that albumin fusion (e.g. N-terminal albumin fusion) to a wild type CFI provides solubility and facilitates activation of CFI-HSA. When activation of CFI to the mature two-chain protein with furin is carried out post translationally and activation is compared between CFI-HSA and a wild type CFI without an albumin (WT-CFI), it is observed that furin activates the CFI-HSA significantly better, and almost completely. It is observed that the CFI-HSA protein remains as a monomer with no evidence of aggregates. There is a significant and unexpected benefit of the amino terminal HSA fusion for maintaining solubility, monodispersity and efficient furin activation of a CFI-HSA construct. There is a significant improvement of bioavailability through, for example, improved half-life.
Accordingly provided herein are methods of increasing the activation of a CFI, comprising fusing a HSA to a wild type CFI, wherein the fusion is a N-terminal fusion prior to activation with furin; and activating with furin. In some embodiments the activation with furin is carried out in a cell during recombinant production of CFI variant, or CFI fusion construct of the dislcosure. In some embodiments the activation with furin is carried out in vitro.
In some embodiments, provided herein are fusion constructs comprising a first component that is a CFI variant of the disclosure, and second component that is albumin, e.g. serum albumin, e.g. human serum albumin.
In some embodiments, provided herein are fusion constructs 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 CFI selected from: the SPD, the FIMAC domain, the SRCR domain, the LDLr1, and the LDLr2 domains. In some embodiments, the 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, the any one or more domains of CFI correspond to the domains of a wild type CFI. In some embodiments, the at least one CFI domain comprises each one of the SPD, the FIMAC domain, the SRCR domain, and the LDLr1 and LDLr2 domains. In some embodiments, the at least one CFI domain of the CFI-HSA construct comprises only the SPD.
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), like CFI, is a protein involved in the complement pathway. FH is cofactor of CFI that forms a complex with CFI and C3b to catalyze C3b cleavage by CFI. As noted above, full-length FH comprises 20 domains.
In some embodiments, the second component of the fusion constructs of the disclosure is at least one Factor H domain, or part of a domain of FH. In some embodiments, the at least one FH domain comprises CCP domains 1-20 of FH. In some embodiments, the at least one domain of FH correspond to that of a wild type FH comprising the amino acid sequence set forth in SEQ ID NO: 4.
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. The CCP domains 1-4 and 19-20 are referred to as “mini Factor H” (mini FH).
Based on the structure of the complex formed by C3b-CFI and mini FH, several domains relevant for the function of FH were identified. The following types of exemplary FH-CFI fusion constructs were generated as base molecules in order to drive FH-independent CFI cleavage activity:
(a) FH domains 1-8 fused with CFI (Factor H-CPPs1-8+CFI)
(b) FH domains 1-4, 19-20, and 5-8 fused with CFI (Factor H-CPPs1-4+19-20+5-8+CFI)
(c) FH domains 1-8 fused with only the LDLr2 CFI domain (Factor H-CPPs1-8+LDLR2-CFI)
(d) FH domains 1-4, 19-20, and 5-8 fused with only the LDLr2 CFI domain (Factor H-CPPs1-4+19-20+5-8+LDLr2-CFI)
(e) FH domains 1-4 fused with human serum albumin (HSA) and the serine protease domain (SPD) of CFI (CFI-HSA(SPD)-factor H-CCP1-4)
(f) FH domains 2-4 fused with human serum albumin (HSA) and the serine protease domain (SPD) of CFI (CFI-HSA(SPD)-factor H-CCP2-4)
(g) FH domains 2-3 fused with human serum albumin (HSA) and the serine protease domain (SPD) of CFI (CFI-HSA(SPD)-factor H-CCP2-3).
Table 11a lists exemplary Factor H-containing fusion construct base molecules.
In some embodiments, a 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 CCP 1-4 of FH. In some embodiments, the CFI variant comprises a modification at any one or more positions corresponding to positions Y408, N531, E457, E461, R462, and F464 in a CFI having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, a 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 CCP 1-4 of FH. In some embodiments, the CFI variant comprises a modification at any one or more positions corresponding to positions Y408, E457, E461, R462, F464, S507, N531, P535 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 substitutions selected from Y408L, E457G, E461Q, R462K, F464Y, S507A, N531G, and P535A, 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 comprises any one or more of combination of substitutions selected from Y408L+N531G, Y408L+N531G+E457G, Y408L+N531G+E457G+E461Q+R462K +F464Y, Y408L+S507A+N531G, Y408L+N531G+E457G+E461Q+R462K+F464Y+S507A, E457G+S507A, and N531G+P535A+S507A, wherein the positions correspond to positions in a 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 (CR1). CR1 is also referred to as CD35. CR1, like CFI, is a protein involved in the complement pathway. CR1 is a cofactor of CFI. Accordingly, in some embodiments, a fusion construct comprising specific domains of CR1 fused to at least one CFI domain may allow for C3b and/or C4b cleavage independent of exogenous cofactor. An exogenous CR1 cofactor may be defined as any CR1 or portion thereof that is not fused to any CFI domain, and may be a wild type CR1, or may be CCP domains 1-3 or 15-17 of CR1. A wild type CR1 as used herein refers to any naturally occurring CR1 which is not a disease-causing CR1. In some embodiments, the CR1 is a human CR1.
In some embodiments, the second component of the fusion constructs of the disclosure is at least one CR1 domain, or part of a domain of CR1. In some embodiments, the at least one CR1 domain comprises CCP domains 15-17 of CR1. In some embodiments, the at least one CR1 domain comprises CCP domains 1-3 of CR1. In some embodiments, the fusion constructs of the disclosure comprising at least one CR1 domain also include fusion with albumin. In some embodiments, the fusion constructs of the disclosure comprising at least one CR1 domain also include fusion with albumin, and/or at least one domain of Factor H. In some embodiments, the at least one CR1 domain comprises CR1 CCP domain 15. In some embodiments, the at least one CR1 domain comprises CR1 CCP domain 16. In some embodiments, the at least one CR1 domain comprises CR1 CCP domain 17. In some embodiments, the at least one CR1 domain comprises CR1 CCP domains 15-16. In some embodiments, the at least one CR1 domain comprises CR1 CCP domains 16-17. In some embodiments, an exemplary fusion construct comprises a CFI having the modification N531G fused with CCP domains 15-17 of CR1. In some embodiments, the exemplary fusion construct comprises a CFI having the modification N531G fused with CCP domains 15-17 of CR1, and is further fused with albumin.
Table 11b lists exemplary CR1-containing fusion constructs and the corresponding sequence of an exemplary fusion construct comprising a wild type CFI and CR1 CCP domains 15-17.
F. Combination 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 include at least one CFI domain. As noted above, some exemplary fusion constructs comprising a first component comprising CFI, a second component, and a third component may include a fusion construct comprising albumin, at least one CFI domain, and at least one domain of Factor H (FH).
Other exemplary fusion constructs provided herein comprise 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 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, a wild type CFI or CFI variant, and at least one FH domain, and at least one CRI domain. The fusion constructs comprising at least one FH domain and at least one CR1 domain can comprise an orientation including an FH domain fused to a CR1 domain, alternating FH and CR1 domains, one or more sequential FH domains fused to one or more sequential CR1 domains, one or more sequential CR1 domains fused to one more FH domains, or combinations thereof. In some embodiments, the fusion construct comprises a wild type CFI or CFI variant, hCR1; CCP15; CCP16; CCP17, and hFH; CCP1; CCP2; CCP3; CCP4. In some embodiments, the fusion construct comprises a wild type CFI or 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 a wild type CFI or 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 a wild type CFI or CFI variant and hCR1; CCP15; CCP16; CCP17; hFH; CCP4. It is understood that any of the fusion constructs may further comprise one or more linkers as described herein. In some embodiments, the fusion construct comprises a wild type CFI or CFI variant, at least one FH domain, at least one CRI domain, and a linker region. It is understood that any of the fusion constructs may further comprise a human serum albumin. In some embodiments, the fusion construct comprises a human serum albumin, a wild type CFI or CFI variant, at least one FH domain, and at least one CRI domain.
In some embodiments, provided herein are fusion constructs comprising a first component comprising at least one CFI domain, a second component, and a third component, wherein the second component is at least one domain of FH, and the third component is 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).
II. Generation of CFI Variants and CFI Fusion Constructs
Provided herein are methods and compositions for generating CFI variants and CFI fusion constructs. Accordingly provided are nucleic acids and vectors encoding any of the CFI variants or fusion constructs of the disclosure. Also provided are cells comprising one or more nucleic acids encoding a CFI or variant thereof, and fusion constructs of the disclosure.
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 can include transcription regulatory elements, such as enhancers or promoters, operably linked to the nucleic acid sequence encoding the CFI variant or fusion construct of the disclosure.
Cell lines can be developed to express production of the CFI and the variants and fusion constructs described herein. Cell lines for producing CFI, CFI can be accomplished using any host cell capable of expressing the CFI variants, and CFI fusions constructs described herein. Host cells can be mammalian cells, insect cells, fungal cells, plant cells, and/or bacterial cells. For expression of the CFI variants and fusion constructs, the host cell line can be transiently or stably transfected or transduced with expression vectors encoding the CFI, CFI variants, and CFI fusions. Vectors can be, for example, plasmids or viral vectors. In some embodiments, the host cell line is a mammalian cell line. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.
CFI variants and fusion constructs described herein can be recombinantly expressed in mammalian cell lines known in the art for producing biologic products, e.g. Chinese hamster ovary (CHO) cells. Mammalian cells can be transfected or transduced with an expression vector encoding the CFI variants and fusion constructs described herein using any method known in the art.
Provided herein are methods of generating a CFI or a variant thereof in an activated state; the method comprising producing the CFI in a cell comprising one or more nucleic acid encoding the CFI or variant thereof, and an expression cassette for furin.
Provided herein are methods for production and purification of CFI variants and fusion constructs described herein. CFI variants and fusion constructs described herein may be purified from conditioned media by standard methods known in the art. In some embodiments CFI variants and fusion constructs may be purified by chromatography on affinity matrices. In some embodiments the affinity matrix is CaptureSelect™ human albumin affinity matrix. In some embodiments CFI variants and fusion constructs may be purified by chromatography on cation and/or anion exchange matrices and optionally size exclusion chromatography. CFI variants and fusion constructs may optimally be buffer exchanged into 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.
The CFI variants and fusion constructs of the disclosure may be used for modulating the complement system.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of modulating the classical and lectin complement pathway.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of modulating the alternate complement pathway.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of decreasing the amplification of the complement system.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the cleavage of C3b.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the cleavage of C4b.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the generation of C4c.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the generation of iC3b.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the generation of C3dg from iC3b.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the generation of C3c from iC3b.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of reducing the level of C3b α-chain.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the hydrolysis of a peptide substrate.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the proteolysis of a macromolecular protein substrate.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of reducing in the level or function of membrane attack complex (MAC).
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable reducing observed hemolysis.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the cleavage of C3b in the absence of cofactor, e.g. in a cofactor independent manner.
As discussed herein, in some embodiments, a CFI variant or CFI fusion construct of the disclosure is capable of increasing the cleavage of C4b in the absence of cofactor, e.g. in a cofactor independent manner.
The CFI variants and fusion constructs of the disclosure may be used for therapeutics in a subject. As used herein, a subject includes any mammalian subject 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 Modulation of the Complement System
Provided herein is 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 or fusion constructs provided herein. In some embodiments, the sample is plasma.
B. CFI Variants and Fusion Constructs for Treatment of Non-Ocular Conditions
In some embodiments, the CFI variants or fusion constructs provided herein are useful for treating a non-ocular condition in a subject. In some embodiments, provided herein is a method of treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the CFI variants or fusion constructs provided herein, or the pharmaceutical composition provided herein below.
In some embodiments, the non-ocular condition is characterized by a deficiency of CFI. In some embodiments, the non-ocular condition is characterized by dysregulation 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 renal injury, acute respiratory distress syndrome, bacterial meningitis, brain hemorrhage, burns, coronavirus infection, Epstein-Barr virus infection, hematopoietic stem cell transplantation, ischemia reperfusion injury, Lyme disease, myocardial infarction, organ transplantation, periodontitis, pneumonia, pre-eclampsia, schistosomiasis, sepsis, 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, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic kidney failure, chronic obstructive pulmonary disease (COPD), Cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), Granulomatosis with Polyangiitis (GPA), Guillain-Barré 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, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), and ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA) and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington disease and ischemia reperfusion injuries.
In some embodiments, the CFI variants or fusion constructs provided herein have an improved characteristic as compared to a wild type CFI. In some embodiments, the improved characteristic is an increase in activity, wherein the increase in activity comprises an increase in the cleavage of C3b and/or C4b. The potency and specificity of the CFI variant provided herein can be tuned for particular therapeutic indications. In some embodiments, the CFI variants or fusion constructs provided herein are C3b degraders. In some embodiments, the C3b degraders are useful for the treatment of diseases. In some embodiments, the CFI variants provided herein are C4b degraders and are useful for the treatment of diseases. For example, the diseases that may be treated by use of the C4b degraders include, but are not limited to a non-ocular condition. 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)-associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic kidney failure, chronic obstructive pulmonary disease (COPD), Cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMG), Granulomatosis with Polyangiitis (GPA), Guillain-Barré Syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy, lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy (MMN), multiple sclerosis (MS), non-insulin dependent diabetes, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), warm Autoimmune hemolytic anemia (wAIHA), Immune-Complex Membranoproliferative Glomerulonephritis (IC-MPGN), and ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA) and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington disease and ischemia reperfusion injuries.
In some embodiments, the non-ocular condition is non-oncological.
In some embodiments, the non-ocular condition is oncological. In some embodiments, the non-ocular condition is oncological, and is characterized by solid tumors, or by liquid tumors. In some embodiments, the non-ocular condition is characterized by solid tumors, and is selected from the group consisting of: colorectal tumors, hormone-refractory prostate cancer, melanoma, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic pancreas cancer, metastatic stomach cancer, nasopharyngeal carcinoma, non-small cell lung cancer, pancreas tumors, squamous cell carcinoma, and stomach tumors. In some embodiments, the non-ocular condition is characterized by liquid tumors, 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 Treatment of Ocular Conditions
In some embodiments, the CFI variants or fusion constructs provided herein are useful for treating an ocular condition in a subject. In some embodiments, provided herein is a method of treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the CFI variants or fusion constructs provided herein, or the pharmaceutical composition provided herein below.
In some embodiments, the ocular condition is characterized by a deficiency of CFI. In some embodiments, the ocular condition is characterized by dysregulation of the complement system.
In some embodiments, the ocular condition is characterized by the presence of a dysfunctional CFI gene. In some embodiments, the ocular condition is characterized by dysregulation of the complement system and low CFI levels.
In some embodiments, the ocular condition selected from the group consisting of: diabetic macular edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, Stargardt Disease, uveitis, and vitreoretinopathy.
In some embodiments, wherein the ocular condition is non-oncological.
D. Combination Therapies
The administration of any one of the therapeutic CFI variants or fusion constructs provided herein may be a monotherapy, or may be in combination with any other known drugs or treatments. The other known drugs or treatments may be for conditions associated with dysregulation of the complement system, or may be associated with a CFI deficiency. In some embodiments, the conditions may be ocular. In some embodiments, the conditions may be non-ocular. In some embodiments, the therapeutic CFI variants or fusion constructs provided herein are co-administered with one or more C5 inhibitors. In some embodiments, the C5 inhibitor is eculizumab. In some embodiments, the C5 inhibitor is cemdisiran.
E. Administration
The CFI variants and fusion constructs described herein may be delivered as polypeptide-based therapies, or nucleic-acid based therapies.
Such treatment as contemplated herein includes both administration of a CFI variant of the disclosure or fusion construct of the disclosure, as well as administration of one or more nucleic acids encoding for a CFI variant of the disclosure or a fusion construct of the disclosure. Accordingly, provided herein are pharmaceutical compositions comprising the CFI variants of the disclosure, CFI fusion constructs of the disclosure, as well as pharmaceutical compositions comprising one or more nucleic acids encoding for CFI variants of the disclosure and encoding for fusion constructs of the disclosure.
Accordingly provided herein are nucleic acids encoding the CFI variants and fusions constructs of the disclosure and are delivered as a part of a nucleic acid-based gene therapy to a subject in need. In some embodiments, the nucleic acid encoding for a CFI variant or fusion construct of the disclosure is delivered as a part of a viral vector based gene therapy (e.g. lentiviral-based therapy, adenoviral-based therapy, adeno-associated viral-based therapy, and the like). In some embodiments, the nucleic acid encoding for a CFI variant or fusion construct of the disclosure is delivered as a naked nucleic acid. In some embodiments, the nucleic acid encoding for a CFI variant or fusion construct of the disclosure is delivered inside a liposome. In some embodiments, the nucleic acid encoding for a CFI variant or fusion construct of the disclosure is delivered as a part of a nanoparticle. In some embodiments, the nucleic acid encoding for a CFI variant or fusion construct of the disclosure is delivered as a part of a virus-like particle.
In some embodiments, the CFI variants and fusion constructs described herein may be delivered as polypeptide-based therapeutics.
The in vivo administration of the therapeutic CFI variants or fusion constructs described herein (protein or nucleic acid based therapeutics) may be carried out intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraventricularly, intranasally, transmucosally, through implantation, or through inhalation. Administration of the therapeutic fusion constructs may be performed with any suitable excipients, carriers, or other agents to provide suitable or improved tolerance, transfer, delivery, and the like.
In exemplary embodiments, administration of the therapeutic CFI variants or fusion constructs described herein is a subcutaneous administration. In some embodiments, the subcutaneous administration is a daily, every other day, twice weekly, or weekly administration.
In some embodiments, administration of the therapeutic CFI variants or fusion constructs described herein is an intravenous administration.
As generally contemplated herein, the CFI variants or fusion constructs described herein are delivered in an activated two chain form. However, in some instances, inactive CFI variants or fusion constructs can be delivered in an inactive single chain form. In some embodiments, what is delivered comprises both single chain inactive and two chain active forms.
F. Dosages
In some embodiments, any of the therapeutic CFI variants or fusion constructs described herein may be administered to a subject in need thereof in a dosage of about 0.1 mg/kg to about 10 mg/kg. In some embodiments, the dosage is about 1 mg/kg. In some embodiments, administration of the therapeutic CFI variants or fusion constructs described herein is a subcutaneous administration, at a dosage of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, administration of the therapeutic CFI variants or fusion constructs described herein is an intravenous administration, at a dosage of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, administration of the therapeutic CFI variants or fusion constructs described herein is daily administration, every other day administration, weekly administration, or twice weekly administration.
In some embodiments, the target level of the therapeutic fusion constructs in plasma may be 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, 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 μg/ml, about 13.5 μg/ml, about 14 μg/ml, about 14.5 μg/ml, 15 μg/ml, about 15.5 μg/ml, about 16 μg/ml, about 16.5 μg/ml, about 17 μg/ml, about 17.5 μg/ml, about 18 μg/ml, about 18.5 μg/ml, about 19 μg/ml, about 19.5 μg/ml, about 20 μg/ml, about 20.5 μg/ml, about 21 μg/ml, about 21.5 μg/ml, about 22 μg/ml, about 22.5 μg/ml, about 23 μg/ml, about 23.5 μg/ml, about 24 μg/ml, about 24.5 μg/ml, 25 μg/ml, about 25.5 μg/ml, about 26 μg/ml, about 26.5 μg/ml, about 27 μg/ml, about 27.5 μg/ml, about 28 μg/ml, about 28.5 μg/ml, about 29 μg/ml, about 29.5 μg/ml, about 30 μg/ml. Exemplary fusion constructs that may be administered to a subject in need thereof to achieve a target level of about 20 μg/ml may include CFI-HSA, comprising a CFI corresponding to a wild type CFI.
G. Formulations
Pharmaceutical compositions containing a CFI variant or fusion constructs of the disclosure can be formulated in any conventional manner by mixing a selected amount of the polypeptide with one or more physiologically acceptable carriers or excipients, for use in the treatments provided herein. Selection of the carrier or excipient is within the skill of the administering profession and can depend upon a number of parameters. These include, for example, the mode of administration and disorder treated. The pharmaceutical compositions provided herein can be formulated for single dosage (direct) administration or for dilution or other modification. The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment. Typically, the compositions are formulated for single dosage administration, but not necessarily.
H. Pharmaceutical Compositions
The disclosure also provides pharmaceutical compositions comprising any one of the CFI variants or fusion constructs disclosed herein, and optionally a pharmaceutical acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical compositions may be formulated to be compatible with their intended routes of administration. In some embodiments, the pharmaceutical compositions of the disclosure 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 disclosure also provides a kit or article of manufacture comprising any one of the CFI variants or fusion constructs disclosed herein, or any pharmaceutical composition disclosed herein. In some embodiments, the kits may further include instructional materials for carrying out any of the methods disclosed herein. In some embodiments, the kits may further include sterile containers or vials for holding the fusion constructs and/or pharmaceutical compositions disclosed herein. In some embodiments, the kits may further include sterile delivery devices for administering the fusion constructs and/or pharmaceutical compositions disclosed herein. In some embodiments, an article of manufacture comprises any pharmaceutical composition of the disclosure.
For Example 1, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
A wild type CFI-HSA protein was expressed in Chinese hamster ovary (CHO) cells, purified with anti-albumin affinity purification, activated with furin, and purified by sizing columns. The activated CFI-HSA protein was subjected to in vitro sialylation to increase the total sialylation of CFI-HSA. Finally, the sialylated protein was purified using anti-albumin affinity purification and polished by size-exclusion column chromatography.
The CFI-HSA gene (SEQ ID NO: 21) was synthesized (ThermoFisher Scientific, Geneart, Regensburg, Germany), with the human serum albumin at the amino terminus of the CFI protein. The protein was made with the signal sequence of SEQ ID NO: 2, which was removed during expression. The amino terminal albumin tag was connected to the CFI gene through a linker (SEQ ID NO: 6). The gene of CFI-HSA was inserted into an expression vector (Lake Pharma, Hayward, Calif.) utilizing standard molecular biology techniques. The resulting plasmid DNA was transformed into E. coli. The transfected E. coli were grown in 200 ml of LB media for expression of plasmid DNA and harvested utilizing standard techniques. The plasmid DNA was run on an agarose gel for quality assessment and sequence confirmed before proceeding to transfection.
1.0 liter of suspension TunaCHO™ cells were seeded in a shake flask and were expanded using serum-free chemically defined medium. On the day of transfection, the expanded cells were seeded into a new flask with fresh medium. The plasmid DNA was transiently transfected into the CHO cells using Lipofectamine 2000 (ThermoFisher Scientific). The cells were maintained as a batch-fed culture until the end of the production run. The protein was expressed for 14 days at 37° C. at 125 RMP with 8% CO2 concentration. Cells were centrifuged and supernatant was collected for purification of secreted CFI-HSA at the end of 14 days expression.
The supernatant with expressed CFI-HSA protein was passed through a 10 ml gravity flow column of CaptureSelect™ human albumin affinity matrix (ThermoFisher Scientific). Column-bound protein was washed with 10 column volume of 20 mM sodium phosphate buffer. Bound CFI-HSA protein was eluted in two steps: first, with 3 column volume of 20 mM Tris-HCl, pH 7.0 buffer with and 2 M MgCl2, and second, with 3 column volume of 20 mM citric acid, pH 3.0. Elution from both steps 1 and 2 was collected in 5 ml fractions. Each fraction of the step 2 elution was neutralized with 10% of neutralization buffer (1.5 M tris-HCL pH 7.4). All fractions were analyzed by reducing and non-reducing SD S-PAGE electrophoresis and bands were visualized by SimplyBlue™ SafeStain (ThermoFisher Scientific). CFI-HSA runs as a 130 kDa band on a non-reducing gel and as 102 kDa and 28 kDa bands on a reducing gel. Fractions with maximum CFI-HSA concentration and purity were pooled for further processing.
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 referred to as the furin recognition sequence), resulting in a heavy and light chain connected by a disulfide bond. The furin-processed, mature, two-chain protein is the activated form of the CFI protein.
Cleavage of CFI-HSA for producing the protein in its activated form was performed by incubation of 4 μg of recombinant furin per mg of purified CFI-HSA in Tris-NaCl (tris buffered saline), 2.5 mM CaCl2 and 0.5% CHAPS at 30° C. for 18 hours. The CFI-HSA protein concentration was maintained at 1.4 mg/ml. This results in more than 90% activation of the protein. The activated protein was separated from inactivated CFI-HSA, and other proteins by size-exclusion chromatography. Size exclusion chromatography (SEC) was performed using a HiLoad 16/600 Superdex 200 column (GE Healthcare Life Sciences) and phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) as the mobile phase. Collected fractions were 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 subject to in vitro sialylation. Briefly, the sialylation was carried out in a two-step enzymatic reaction. First, a galactosylation reaction of CFI-HSA was performed in a 200 μl volume utilizing a 1:200 molar ratio of galactosyltransferase (GalT1) enzyme and CFI-HSA in 10 mM UDP-Galactose, 5 mM MnCl2, and 100 mM MES, pH 6.5 buffer. Galactosylated CFI-HSA was purified from the reaction mixture by CaptureSelect™ Human Albumin affinity chromatography, as described earlier. Next, the sialylation reaction was performed in a 250 μl volume utilizing a 1:50 molar ratio of enzyme alpha 2,6-sialyl transferase and purified CFI-HSA in 80 μM Alkaline phospahatase, 6.1 mM CMP-NANA, 10 mM ZnCl2 and 200 mM MES buffer, pH 6.5 at 37° C. for 1 hour. The sialylated CFI-HSA protein was purified from the reaction mixture by CaptureSelect™ Human Albumin affinity chromatography. The extent and characteristics of the sialic acid chain on CFI-HSA was determined by utilizing an Agilent/Prozyme Analytical service, GS-SAP method for total sialic acid quantitation (Agilent GS48), and mass spectrophotometric (MS) analysis (Lake Pharma analytical service), described in further detail below.
Briefly, total sialic acid quantitation was performed by mixing 20 μl of each sample with 10 μl of release reagent in a 96 well plate. The reaction mixture was incubated for 2 hours at 80° C. The samples were cooled to room temperature and 10 μl of labeling reagent was added to each sample for a further incubation of 3 hours at 50° C. The samples were again cooled down to room temperature and 160 μl of de-ionized (dI) water was added to bring the total volume to 200 μl. 10 μl of sample was injected in the Agilent UHPLC Poroshell C18 column to run at a flow rate of 0.4 ml/minute at 30° C. in 4% methanol, 8% acetonitrile in water (Line A1) and 100% ACN (Line B1). The peaks were recorded at 373/448 nm wavelength. A standard curve of total peak area versus picomoles (pmol) of sialic acid was generated by running 1-2000 pmol of NANA (N-acetylneuraminic acid, Neu5Ac) supplied with the kit on the same column Total sialic acid of each sample was quantitated by comparing the peak area of samples against the standard curve. The sialylation obtained is summarized in Table 1.1 below.
The mass spectrometric analysis 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. The digested samples were analyzed by Waters ACQUITY UPLC coupled to a Xevo G2-XS-QTOF mass spectrometer using a protein BEH C18 column. The performed analysis is summarized in Table 1.2 below.
Purified CFI-HSA protein was subjected to size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 column (GE Healthcare Life Sciences) and phosphate buffer saline as the mobile phase. Collected fractions were analyzed by CE-SDS (LabChip GXII, Perkin Elmer). Fractions containing the target protein were pooled, and the concentration was brought to 5 mg/ml, and the samples were flash frozen for storage at −80° C.
The DNA of CFI-HSA variants was generated either by synthesis or by site-directed mutagenesis utilizing standard techniques. The proteins were expressed in 250 ml of suspension in TunaCHO™ cells, as described herein with reference to wild type CFI-HSA protein, with the exception that the expression was done for 7 days instead of 14 days. After 7 days, the cells were centrifuged, and conditioned media was passed through a gravity flow column of CaptureSelect™ human albumin affinity matrix (ThermoFisher Scientific). Column-bound protein was washed with 10 column volume of 20 mM sodium phosphate buffer. Bound CFI-HSA protein was eluted with 3 column volume of 20 mM Tris-HCl, pH 7.0 buffer with and 2 M MgCl2 in 5 ml fractions. CFI-HSA or its variants were buffer exchanged (either by dialysis or a spin concentrator) into 30 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2, pH 7.4. Recombinant human furin, at a molar ratio of 1:25 (furin:CFI-HSA), was added to CFI-HSA and the reaction mixture was incubated at 30° C. for 16 hours. Two micrograms of the activation mixture was run on a 9% SDS-PAGE gel to assess the activation efficiency. Generally, more than 80% activation was achieved.
Activation was compared between CFI-HSA and wild-type CFI without an albumin, or other fusion tag (WT-CFI). A gene construct for WT-CFI was expressed essentially as described above for CFI-HSA. The recombinant WT-CFI protein showed moderate purity by reduced SDS-PAGE, however, significant High Molecular Weight Species (HMWS) and aggregates under reduced and non-reduced conditions were observed (
For Example 2, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
The proteolytic activity of wild type CFI-HSA and CFI variant-HSA fusions (referred collectively herein as “CFI-HSA proteins”) was tested by following the cleavage of chromogenic substrates by use of a chromophore. The S-2288 (Chromogenix) peptide substrate was selected for this assay as it is sensitive to a broad spectrum of serine proteases. The peptidolytic activity of the CFI-HSA proteins were measured by the rate of generation of p-nitroaniline (pNA) upon substrate cleavage, which occurred spectrophotometrically at 405 nm.
The CFI-HSA proteins were diluted to an initial concentration of 400 nM in 100 μl d of HBS/BSA (30 mM HEPES, 140 mM NaCl, 0.2% BSA, pH 7.4) in a non-coated 96-well microplate (Nunc). A working stock of 4 mM S-2288 was made in HBS/BSA in a separate tube. The microplate and diluted chromogenic substrate were pre-warmed to 37° C. for 5 minutes. The assay was initiated by the addition of 100 μl of pre-warmed S-2288 to the wells of the microplate containing the CFI-HSA proteins. This resulted in a final concentration of 200 nM of the CFI-HSA proteins, and 2 mM of S-2288 substrate in a 200 μl reaction volume. The rate of substrate cleavage was recorded every 30 seconds for 3 hours at 37° C. at 405 nm, using a microplate reader (Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific). Peptide hydrolysis activity of wild type CFI-HSA was normalized as 100% in order to calculate the percentage of peptidolysis activity of the CFI-HSA variants. The results are summarized in Table 2.1 below.
For Example 3, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
The C3b cleavage assay is a functional assay used to determine the ability of wild type CFI-HSA and CFI-HSA variants (referred collectively herein as “CFI-HSA proteins”) to cleave its natural substrate, C3b. Briefly, the CFI-HSA proteins were incubated with C3b and a truncated Factor H (mini FH) at 37° C. for analysis of C3b cleavage. Mini FH has been previously shown to be functionally active and support the CFI-mediated C3b cleavage (J Immunol. 2013 Jul. 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, the master reaction mixture was set up at room temperature containing the final concentrations of 500 nM of mini FH and 5 nM of the CFI-HSA proteins in HBS buffer (30 mM HEPES, 140 mM NaCl pH 7.4). The master reaction mixtures were transferred to 37° C. and allowed to equilibrate for 5 minutes. The cleavage reaction was initiated by the addition of C3b to a final concentration of 0.5 μM. 20 μl samples from the master mixtures were withdrawn for each time point measured, and quenched by the addition of 5× SDS reducing sample buffer. Samples were run on a 9% SDS-PAGE gel and C3b cleavage was visualized by Coomassie staining The amount of C3b cleavage that occurred was quantitated by densitometry. The C3b cleavage activity of wild type CFI-HSA was normalized as 100% in order to calculate the percentage of C3b cleavage activity of the CFI-HSA variants. The results of the C3b cleavage assay are summarized in Table 3.1 below.
To compare the rate of C3b cleavage by each CFI-HSA variant to that of the wild type CFI-HSA, a time course for C3b cleavage by the CFI-HSA proteins was performed in parallel. Disappearance of the C3(alpha)' band was observed as an indication of C3b cleavage. C3b includes two chains, (alpha)' and beta. When the disappearance of the C3(alpha)' band at a molecular weight of 114 kDa was observed, both SDS-PAGE and densitometry of the relevant stained band, corrected for the average background staining (lane intensity outside the band), were performed.
The apparent rate for loss of band intensity was estimated by fitting a simple exponential decay formula to the band intensity data as a function of time, thereby extracting an apparent rate constant (k) of C3b cleavage. The relative rate of C3b cleavage by the CFI-HSA variants was calculated by dividing with the corresponding WT rate: k(variant)/k(WT control). This procedure was performed on 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.
For Example 4, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
A C3dg assay was used to determine the cleavage of C3b caused 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 a time-resolved immuno-fluorometric assay (TRIFMA). Briefly, the complement pathway in human serum was activated by using 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, utilizing a C3dg antibody on a microtiter plate. Bound C3dg was detected by a combination of a biotinylated C3dg antibody and Europium-labelled streptavidin, and measured by time-resolved fluorometry.
MaxiSorb microtiter plates (Nunc) were coated with 100 μl monoclonal IgM rat anti-human C3dg antibody at 2 μg/ml in 15 mM Na2CO3, 35 mM NaHCO3, pH 9.6 coating buffer by overnight incubation at room temperature. The remaining protein binding sites were blocked by incubation with HSA at 1 mg/ml in TBS. Unbound HSA was washed with TBS-Tween.
Test samples were diluted in a 1 to 6 dilution of human serum to desired concentrations in a 100 μl volume with dilution buffer (0.14 M NaCl, 10 mM Tris, 14 mM sodium azide, with 0.05% (v/v) Tween 20 (TBS/Tween), 1 mg/ml HSA and 0.1 mg/ml of heat aggregated IgG. Four-fold, six point dilutions were made for each CFI-HSA variant to cover the variants concentration range from 3132 nM to 3 nM. The reaction mixture was incubated at 37° C. for 90 minutes and quenched by 10 mM EDTA. To capture the generated C3dg, 100 μl of each reaction mixture were added to the antibody-coated microtiter wells and incubated overnight at 4° C. To detect the bound C3dg, 100 μl of biotinylated rabbit anti-C3dg antibody (DAKO) was added at 0.5 μg/ml to the wells and incubated for 2 hours at room temperature. After washing with the 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 enhancement buffer (Ampliqon) was added to each well. Plates were read using a DELFIA-reader Victor5+(Perkin Elmer) by time-resolved fluorometry. The results are summarized in Table 4.1 below.
These results showed that exemplary CFI-HSA variants had a higher percentage of C3b cleavage activity than wild type CFI-HSA, or plasma-derived CFI.
For Example 5, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
A hemolysis assay is used for the measurement of hemolytic function of a compound that uses the complement pathway. Complement Factor I (CFI) mediates C3b cleavage with its cofactor Factor H (FH) within the alternate alternative pathway of the complement pathwaysystem. To test the hemolytic function of wild type CFI-HSA and CFI-HSA variants (referred collectively herein as “CFI-HSA proteins”) in the alternate alternative pathway, C3-deficient human serum spiked with human C3 was incubated with CFI-HSA and rabbit Alsevers solution, and total hemolysis was measured spectrophotometrically. The hemolysis assay was performed on wild type CFI-HSA and plasma-derived CFI (CFI-PD) with or without FH in order to understand the effect of the cofactor FH on total hemolysis.
Briefly, 12 ml of rabbit red blood cells (RBC) was washed twice with GVB buffer (Gelatin Veronal buffer: Sigma, with 8 mM EGTA and 10 mM MgCl2) and resuspended in 12 ml of ice cold GVB buffer. C3-deficient human serum was spiked with 1 μM of human C3, based on previous observations that 1 μM of C3 supports maximum hemolysis in this system. Three-fold eight-point serial dilutions of CFI-HSA in GVB buffer was done to achieve concentrations ranging from 260 μg/ml to 0.11 μg/ml in the reaction mixture. First, in a 96 well plate, 50 μl reaction mixture for each concentration point was prepared by adding 62.8% human serum, different concentrations of CFI-HSA with or without 200 μg/mL FH. The hemolysis reaction was started by adding 50 μl of rabbit RBC and incubated in a microtiter plate at 37° C. for 30 minutes. All assays were done in triplicates and all dilutions were done in GVB buffer. For a maximum hemolysis control, de-ionized water was added to the RBC, and 0.154 M NaCl was added to the RBC for a no hemolysis control. After incubation, the plate was centrifuged at 2000 rpm for 5 minutes and 90 μl of supernatant was transferred to another 96 well plate. The percent hemolysis was quantitated by measuring optical density (OD) of lysed RBC at 412 nm.
The absorbances at 412 nm were converted to a percentage of hemolysis, utilizing maximum hemolysis from the control as 100% and the buffer control 0%. The results of the hemolysis assay are summarized in Table 5.1 below.
These data showed that, at higher concentrations, both CFI-HSA and CFI-PD are active in the hemolysis assay. The inhibitory activity of CFI-HSA on the alternative pathway was similar to that of CFI-PD in the hemolysis assay. The hemolysis assay also showed that the inhibitory effect of CFI, both CFI-HSA and CFI-PD, on the alternative pathway increased significantly with cofactor FH.
The capacity to inhibit classical pathway hemolysis by CFI variants was measured. Sheep red blood cells were activated by anti-SRBC antibodies (Amboceptor, Testline, UK). The SRBCs were suspended in gelatin veronal buffer (GVB). In the assay plates, a dilution series of the CFI variants were added followed by the activated SRBC and Factor B and I depleted serum at ˜1% (v/v). The activated SRBC were incubated with test articles for 30 mins. The cells were pelleted and the supernatant transferred to a separate plate for absorbance readings at 412 nM. Percentage lysis was calculated as follows: 100* (Absorbance test sample)/(Absorbance no CFI (0% inhibition)). Data was plotted and analyzed using four parameter non-linear regression (GraphPad Software, USA). IC50 values were calculated for data from individual plates and averages were performed on logIC50 values and transformed to concentration (nM) as summarized in Table 5.3.
The capacity to inhibit alternative pathway hemolysis by CFI cariants was measured. Sheep red blood cells were activated by anti-SRBC antibodies (Amboceptor, Testline, UK). The SRBCs were suspended in 8% (v/v) of normal human serum depleted of Factors B and H to which was added eculizumab to deposit C3b. The activated SRBC with deposited C3b were incubated with full-length Factor H (Complement Technologies, USA) and the test articles. After a 10 min incubation Factors B and D (Complement Technologies, USA) were added and incubated for a further 10 min. Finally, guinea pig serum (Sigma-Aldrich, UK) was added and incubated for 20 min. The cells were pelleted and the supernatant transferred to a separate plate for absorbance readings at 412 nM. Percentage lysis was calculated as follows: 100* (Absorbance test sample)/(Absorbance no CFI (0% inhibition)). Data was plotted and analyzed using four parameter non-linear regression (GraphPad Software, USA). IC50 values were calculated for data from individual plates and averages were performed on logIC50 values and transformed to concentration (nM) as summarized in Table 5.3.
For Example 6, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
A Complement Factor I (CFI) fusion construct and a free wild type CFI were tested for concentration levels in plasma after a single subcutaneous dose in African green monkeys. The fusion construct comprised a human serum albumin (HSA) and a wild type CFI (CFI-HSA).
For Example 7, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
First, for each CFI-HSA variant, the master reaction mixture was set up at room temperature containing the final concentrations of 500 nM of mini FH and 500 nM of C3b in HBS buffer (30 mM HEPES, 140 mM NaCl pH 7.4). The master reaction mixtures were transferred to 37° C. and allowed to equilibrate for 5 minutes. The cleavage reaction was initiated by the addition of CFI-HSA protein to a final concentration of 5 nM. A sample volume corresponding to 0.6 ug of C3b was withdrawn from the master mixtures for each time point measured and quenched by the addition of 5× SDS reducing sample buffer. Samples were run on a 9 or 10% SDS-PAGE gel and C3b cleavage was visualized by Coomassie staining The amount of C3b cleavage that occurred was quantitated by densitometry. The C3b cleavage activity of wild type CFI-HSA was normalized as 100% in order to calculate the percentage of C3b cleavage activity of the CFI-HSA variants.
Another example of the cleavage reactions was performed as follows. C3b cleavage reactions were performed using 1 nM CFI (variant or wild type), 500 nM cofactor mini FH, and 500 nM soluble human C3b incubated for 10 minutes at 37° C. in HEPES buffered saline (HBS). The reaction was quenched by the addition of 1 M NaCl in HBS. The reactions were further diluted to a final concentration of 5 nM C3b in buffer (HBS, 0.5M NaCl, 0.05% Tween 20) before proceeding with an iC3b ELISA. The C3b cleavage activity was determined from the amount of iC3b generated in the cleavage reaction. The amount of iC3b formed was assayed using the MicroVue iC3b A006 ELISA kit (Quidel). The ELISA assay consists of a microplate coated with an iC3b specific monoclonal antibody for capture of formed iC3b during the reactions and detection of bound iC3b using an HRP-conjugated anti-iC3b antibody and a chromogenic substrate. The absorbance recorded is a relative measure of the iC3b product generated in the cleavage reactions. The fold difference of C3b cleavage activity of CFI variants relative to a reference molecule, CFI-HSA wild type, was calculated by dividing the background-corrected absorbance from CFI-HSA variants by the background-corrected absorbance for CFI-HSA wild type. Table 7.1 summarizes these results, presenting the fold difference of the median value for each CFI variant relative to the median value of the reference molecule. The fold differences were also calculated from SDS-PAGE gels. Samples from a C3b cleavage time course were run on a 9 or 10% SDS-PAGE gel and C3b cleavage was visualized by Coomassie staining The amount of C3b cleavage that occurred was quantitated by densitometry and the data plotted and an apparent rate constant (k) for loss of band intensity determined by fit to an exponential decay. The fold difference of C3b cleavage activity of CFI variants relative to a reference molecule, the CFI-HSA wild type, was calculated by dividing the k-value from the CFI-HSA variants by the k-value for CFI-HSA wild type.
C3b cleavage by CFI variants was further characterized by determining the EC50 for the C3b cleavage. Briefly, C3b cleavage reactions were performed using 25 nM mini FH, 75 nM soluble human C3b and a dilution series of the CFI variants. Reaction mixtures at each of the concentrations of the CFI variants were incubated for 5 min at 37° C. in HBS. The reaction was quenched by the addition of 1 M NaCl in HBS. The reactions were further diluted to a final concentration of 5 nM C3b in buffer (HBS, 0.5M NaCl, 0,05% Tween 20) before proceeding with an iC3b ELISA. The amount of iC3b generated in the reaction was determined using the MicroVue iC3b A006 ELISA kit (Quidel). The ELISA assay consists of a microplate coated with an iC3b specific monoclonal antibody for capture of formed iC3b during the reactions and detection of bound iC3b using an HRP-conjugated anti-iC3b antibody and a chromogenic substrate. The absorbance recorded is a relative measure of the iC3b product generated in the cleavage reactions. The EC50 values were calculated using a four-parameter non-linear regression fit without constraints in GraphPad Prism. Table 7.2 below summarizes the results of the iC3b ELISA titration analyses. EC50 values above 500 nM were set to be 500 nM. The cleavage reactions were also performed in the absence of mini FH where noted and analyzed in the same fashion as those containing mini FH.
CFI regulates the classical complement pathway by proteolytic inactivation of the C4b protein. CR1, a C3b/C4b receptor, and C4 binding protein (C4BP) act as cofactors for the CFI-catalyzed cleavage reaction of C4b. The C4b cleavage assay is a functional assay to determine the ability of CFI and variants thereof for C4b cleavage activity in the presence of either the CR1 or C4BP cofactors. Complement factor protein C2, which binds specifically to C4b and not to the CFI-cleaved product iC4b, was used for C4b capturing. The CFI-catalyzed cleavage of C4b was measured by measuring the decrease in the concentration of C4b bound to C2 protein, immobilized on an ELISA plate. The captured C4b protein was detected by Anti-C4c polyclonal rabbit Ab (DAKO, #A0065) in an ELISA assay. C4b cleavage activity by CFI-HSA was normalized as 100% to calculate the percentage of C4b cleavage activity of CFI variants.
For each CFI-HSA variant, the master reaction mixture was set up at room temperature containing the final concentrations of 250 nM cofactor (CR1 domains 1-3) and 250 nM human C4b in HBS buffer (30 mM HEPES, 140 mM NaCl pH 7.4). The master reaction mixtures were transferred to 37° C. and allowed to equilibrate for 5 minutes. The cleavage reaction was initiated by the addition of CFI-HSA protein to a final concentration of 250 nM. A sample volume corresponding to 0.6 ug of C3b was withdrawn from the master mixtures for each time point measured and quenched by the addition of 5× SDS reducing sample buffer followed by incubation at 95° C. for 5 minutes. Samples were run on a 9 or 10% SDS-PAGE gel and C4b cleavage was visualized by Coomassie staining The amount of C4b cleavage that occurred was quantitated by densitometry. The C4b cleavage activity of wild type CFI-HSA was normalized as 100% in order to calculate the percentage of C4b cleavage activity of the CFI-HSA variants.
Another example of the C4b cleavage activity assay was performed as follows, to determine the C4b cleavage activity of CFI variants relative to a reference molecule, CFI-HSA wild type. The cleavage reaction was performed with 250 nM of the CFI variants in the presence of 250 nM of cofactor (CR1 domains 1-3) and 250 nM human C4b, which was incubated for 30 minutes at 37° C. The reaction mixture was diluted 20-fold before addition to a blocked ELISA plate coated with a mouse monoclonal anti-C4c antibody. The absorbance recorded from the ELISA plate is a relative measure of the C4c product generated in the cleavage reactions and therefore a measure of C4b cleavage activity. The fold difference of C4b cleavage activity of CFI variants relative to a reference molecule, CFI-HSA wild type, was calculated by dividing the background-corrected absorbance from CFI-HSA variants by the background-corrected absorbance for CFI-HSA wild type. Table 7.1 below summarizes the fold differences of the C4b cleavage activity assay of CFI variants relative to the CFI-HSA reference molecule, as measured by C4c ELISA screen with CR1.
The EC50 of the C4b cleavage by CFI variants was measured. The assay was performed using 250 nM cofactor (CR1 domains 1-3), 250 nM human C4b and a dilution series of the CFI variants. The reaction mixtures were incubated for 30 minutes at 37° C. and then the reaction mixture was diluted 20-fold before beginning the ELISA. The amount of generated C4c was measured by ELISA using a mouse monoclonal antibody specific towards C4c. The absorbance recorded from the ELISA plate is a relative measure of the C4c product generated in the cleavage reactions and therefore a measure of C4b cleavage activity. The EC50 values were calculated using a four-parameter non-linear regression fit without constraints in GraphPad Prism. EC50 values above 1000 nM were set to be 1000 nM The cleavage reactions were also performed in the absence of CR1 where noted and analyzed in the same fashion as those containing CR1. Tables 7.2 summarizes the results of the C4c ELISA titration with the CR1 cofactor.
C4b cleavage reactions were carried out as described above in the absence of cofactor for a panel of CFI variants (Table 7.3). The results show that CFI variants with a C-terminal fusion protein that include a human CR1 domain maintained their ability to cleave C4b in the absence of cofactor in the reaction mixture. In contrast, the CFI variants lacking a CR1 C-terminal fusion did not maintain their ability to cleave C4b. These results suggest that CFI variants with a C-terminal CR1 fusion can be CR1 cofactor independent.
C3b cleavage reactions were carried out as described above in the absence of cofactor for a panel of CFI variants (Table 7.4). The results show that CFI variants with a C-terminal fusion protein that include a human CR1 domain maintained their ability to cleave C3b in the absence of cofactor in the reaction mixture. In contrast, the CFI variants lacking a CR1 C-terminal fusion did not maintain their ability to cleave C3b. These results suggest that CFI variants with a C-terminal CR1 fusion can be CR1 cofactor independent.
The specificity for C4b cleavage versus C3b cleavage and C3b cleavage versus C4b cleavage was calculated in two different ways. For the single point assays listed in Table 7.1, the baseline-subtracted median values used to calculate the fold difference values were used. Values below 0.01 were adjusted to 0.01. Each single median value for C4b and C3b was converted to a percent maximum using the following formula: 100%*(variant value/max value among all variants). Specificity for C4b was calculated as the ratio of the percent maximum C4b divided by percent maximum C3b. Specificity for C3b was calculated as the ratio of the percent maximum C3b divided by percent maximum C4b.
For the assays where EC50 values were determined, the specificity was calculated by normalizing to CFI-HSA. For C4b cleavage the max value was set at 1000 nM and all values above that were set to 1000 nM. For C3b cleavage the max value was set at 500 nM and all values above that were set to 500 nM. Specificity for C4b was calculated as follows: (C4b EC50 CFI-HSA/C4b EC50 variant)/(C3b EC50 CFI-HSA/C3b EC50 variant). Specificity for C3b was calculated as follows: (C3b EC50 CFI-HSA/C3b EC50 variant)/(C4b EC50 CFI-HSA/C4b EC50 variant). Results are reported in Table 7.2.
For Example 8, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
For Example 9, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
Various hemolytic assays were performed to assess the activity of CFI-HSA, CFI variants as compared to plasma-derived CFI. Hemolysis mediated through the classical pathway (CP) and alternative pathway (AP) was assessed. Briefly, the summary of the assays performed and the focus of the assays are presented in Table 9.1 below.
Generally, the above results demonstrate that, along with the advantages of the half-life extension and higher production of the recombinantly-produced CFI-HSA, the CFI-HSA performs as well as plasma-derived CFI. An illustrative application for the CFI-HSA can therefore be use in an enzyme replacement therapy for endogenous CFI in complement-related disorders.
Further, the CFI variant E461G tested relative to CFI-HSA.
For Example 10, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
For Example 11, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
The ocular pharmacokinetics of the N-terminal albumin fusion of wild-type CFI-HSA were examined after intravitreal dosing to six African Green Monkeys (AGMs). The 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). The left eye (OS) of all six animals was injected with an equivalent volume of 100 μL of sterile PBS for injection as a vehicle control. Non-terminal, vitreous humor samples (100 μL) were taken on days 1, 7, 14, 21 and 28 post dosing. Vitreous humor CFI-HSA drug concentrations were determined using a quantitative electrochemiluminescence (ECL) antigen assay optimized for measuring CFI-HSA in vitreous humor of AGMs. The assay employs coating of anti-CFI antibody (clone OX21, LS Bio, Seattle Wash.) at 2 μg/ml on the Meso Scale Discovery (MSD, Rockville, Mass.) assay plate to capture the CFI-HSA levels. Detection of the captured CFI-HSA is performed with a goat polyclonal anti-HSA antibody (Abcam, Cambridge, Mass.) at 0.5 μg/ml conjugated with SULFO-TAG which emits light [electrochemiluminescence (ECL)] on application of an electric potential. The ECL relative light units (RLU) is measured on a MESO® SECTOR S 600 Reader and the unknown CFI-HSA concentrations in vitreous humor are interpolated from a standard curve ranging from 0.05 μg/ml to 40 μg/ml Factor I-HSA. Data are provided in Table 11.1.
Non-compartmental analysis yielded apparent ocular terminal half-lives of 3.6 and 4.1 days for the 250 and 500 μg dose levels, respectively.
Complement Component 3a (C3a) levels in vitreous humor were determined by ELISA using the Quidel kit for C3a ELISA (
For Example 12, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
The pharmacokinetics of the N-terminal albumin fusion of wild-type CFI (CFI-HSA) was examined after intravenous and subcutaneous administration to CD-1 mice. Employing a sparse sampling design with up to two samples per mouse and three mice sampled at each timepoint, CD-1 mice were divided into four groups and treated with a single dose of either the plasma purified wild type CFI, or the recombinant wild-type CFI-HSA.
To compare the circulating half-life in plasma and bioavailability of the plasma-derived CFI and CFI-HSA, animals were dosed with either plasma-derived CFI or CFI-HSA both intravenously and subcutaneously. Plasma-derived CFI was administered at 1.3 mg/kg intravenously (group 1) and 6.5 mg/kg subcutaneously (group 2). CFI-HSA was administered at 3 mg/kg intravenously (group 3) and subcutaneously (group 4). An additional 3 animals received a single dose of an equivalent volume of PBS delivered subcutaneously as a vehicle control (group 5; not shown). Blood (˜30-50 μL) was collected in EDTA at various time points from 5 minutes to 144 hours post dosing and plasma separated by centrifugation.
CFI-HSA and plasma CFI concentrations were determined with a quantitative electrochemiluminescence (ECL) antigen assay for CFI-HSA and plasma CFI in CD-1 mouse EDTA plasma. For the CFI assay, the mouse monoclonal anti-Factor I antibody (MAB12907, Abnova, Taipei City, Taiwan) is coated at 2 μg/ml on the Meso Scale Discovery (MSD, Rockville, Mass.) assay plate to capture the plasma CFI. Detection of the captured CFI is performed with a mouse monoclonal anti-CFI antibody (clone 3R/8, CABT-47940MH, Creative diagnostic, Shirley N.Y.) at 0.5 μg/ml conjugated with SULFO-TAG which emits light [electrochemiluminescence (ECL)] on application of an electric potential. For the CFI-HSA assay, the mouse monoclonal anti-CFI antibody (clone 3R/8, CABT-47940MH) is coated at 1 μg/ml on the Meso Scale Discovery (MSD, Rockville, Mass.) assay plate to capture the CFI-HSA. Detection of the captured CFI-HSA is performed with a rabbit polyclonal anti-HSA antibody (ab24207, Abcam, Cambridge, Mass.) at 2 μg/ml conjugated with SULFO-TAG which emits light [electrochemiluminescence (ECL)] on application of an electric potential. The ECL relative light units (RLU) is measured on a MESO® SECTOR S 600 Reader and unknown plasma CFI and CFI-HSA concentrations are interpolated from the standard curves. Pharmacokinetic parameters were derived from the analysis of plasma CFI and CFI-HSA concentrations and provided in Table 12.1.
The circulation half-life following intravenous infusion of CFI-HSA was longer (˜22 hours) than the non-fusion version plasma CFI protein (˜13 hours), indicating that fusion of HSA to the CFI protein increases the half-life of compared to unfused CFI. Importantly, the bioavailability of CFI (53.6%) was similar to the CFI-HSA (46.7%), indicating that fusion of HSA to the CFI protein did not adversely affect bioavailability of CFI after subcutaneous dosing (Table 12.1). Fusing HSA to CFI protein increases half-life by ˜2-fold compared to the non-fusion version CFI protein after intravenous (
For Example 13, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
A rat model of peripheral nerve injury was developed to study complement involvement in Wallerian degeneration due to mechanical damage of the myelinated sciatic nerve. Male CD Sprague Dawley rats (Charles River Laboratories) weighing between 300 and 350 g at enrollment were anesthetized with a mixture of 2 to 2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen, and placed on a heating pad to maintain body temperature. Both legs underwent a sterile surgery to expose the sciatic nerve. One leg underwent a sciatic nerve injury (SNI) by clamping the sciatic nerve three times for 10 seconds using Dumont #7 forceps. The contralateral leg received no clamp injury and served as an internal control for each subject.
Immediately following induction of SNI, animals received an intravenous injection of CFI-HSA (Y408L; N531G variant) 4mg/kg (n=10), CFI-HSA (Y408L; N531G variant) 1.25 mg/kg (n=10), or control article (1× PBS; n=10) at a dose volume of 4 mL/kg. A subcutaneous injection of slow-release buprenorphine (0.01 mg/kg) was also administered for pain management. 4 or 24 hours after SNI, 5 animals from each treatment group were sacrificed by exsanguination.
At sacrifice, a 1cm (0.5 cm proximal and distal to the site of injury) piece of nerve was collected from the injured (ipsilateral) and sham legs, snap frozen, and stored at −80 ° C. until processed for mass spectrometry analysis (Phenoswitch Bioscience, Canada). K2-EDTA plasma samples were collected prior to SNI (baseline) as well as 1, 4, and 24 hours (where applicable) after SNI for evaluation of complement component fragments by mass spectrometry (MS). Cytokine and chemokine levels (Rat 27 plex Multiplex Immunoassay analyzed with a BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX, performed by Eve Technologies, Calgary, Canada) were assessed in K2-EDTA plasma collected at baseline (vehicle only), 4, and 24 hours (where applicable) after SNI. At sacrifice, whole blood and serum were collected for clinical pathology evaluation [complete blood counts (CBC) and serum chemistry; Biovet Inc., Canada].
Samples were denatured and precipitated, with a wash and buffer exchange before N-terminal labeling via reductive amine dimethylation. Samples were then digested with trypsin (or a mix of trypsin and chymotrypsin) before analysis via LC-MS/MS using SWATH. SWATH data was integrated on an ion library produced for each species and sample type. Top 10 peptides per protein contained in the ion library were integrated, and a peptide centric analysis was carried out for specific quantification of C3, C5, C4 and CFB N-terminal labeled peptides.
Cleavage products resulting from CFI-HSA (Y408L; N531G variant) catalytic activity on C3b were monitored in the nerve tissue (membrane-bound fragments) (
Overall, mass spectrometry results confirmed that mechanical nerve injury can trigger complement response at the site of nerve tissue. In addition, CFI-HSA (Y408L; N531G variant) showed greater cleavage activity on surface bound C3b than circulating C3b when compared to vehicle, suggesting CFI-HSA (Y408L; N531G variant) may perform better on surface-bound C3 where CR1 and C4bp cofactor are present.
For Example 14, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
The efficacy of a panel of CFI variants on complement activation in a sciatic nerve (SN) injury (SNI) rat model was determined Immediately following induction of SNI, animals received an IV injection with a CFI variant (n=6 for each variant) from a panel of CFI variants (Table 14.1), or a control article (1X 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 (Rat 27 plex Multiplex Immunoassay analyzed with a BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX, performed by Eve Technologies, Calgary, Canada) were assessed in K2-EDTA plasma collected at 4 and 24 hours after SNI. At sacrifice, serum was collected for serum chemistry [Biovet Inc., Canada].
The activity of CFI variants was monitored by detecting CFI cleavage products (C3dg and C3f) using mass spectrometry. N-terminal labelled C3f (S[2Me]EETK[2Me]QNEGF) (SEQ ID NO: 28) is the product of CFI cleavage of C3b and N-terminal labelled C3dg (E[2Me]DVPAADLSDQVPDTDSETR) (SEQ ID NO: 24) is the product of CFI cleavage of iC3b. Total activated C3f was determined as the percent of C3f peptides with N-terminal labeling (S[2Me]EETK[2Me]QNEGF) (SEQ ID NO: 28) multiplied by the total peptide signal size of C3f (SEETKQNEGF) (SEQ ID NO: 28).
A 2.5-fold increase in nerve C3dg levels 24-hours after injury were observed in vehicle-treated animals and no effect of CFI variant treatment was detected (
To compare the in vitro activity of CFI variants with the wild-type CFI, the iC3b EC50 (Table 14.2, column A) and the IC50 in classical pathway hemolysis (Table 14.2, column B) of wild-type CFI was divided by the iC3b EC50 and IC50 of each variant. To compare the in vivo increase in plasma C3f with in vitro data, total N-terminal labelled C3f at each time-point was divided by the baseline N-terminal labelled circulating C3f signal in plasma for each animal to provide an estimate of CFI-mediated cleavage of fluid-phase and surface-bound C3b (Table 14.2, column C and D). Plasma-derived CFI was selected as the closest approximation to activity of endogenous rodent CFI. The results of these data transformations are summarized in the table below.
The resulting fold-changes from both the CP hemolysis assay and the iC3b ELISA assay measures yielded similar rankings to circulating C3f levels at 4 hours. By 24 hours after injury the relative improvements in C3 cleavage activity between variants was less discernable. In this setting, Variant 2 and Variant 3 markedly outperformed the other CFI variants 4 hours after SNI while Variant 4 outperformed all variants at 24 hours. Overall, these data suggest that the addition of D425R and E416A substitutions into Variant 3 do not dramatically improve in vivo C3b cleavage. However, the addition of E457G and D425R into Variant 4 would result in a faster in vivo cleavage activity that may not be sustained over time. Further work is needed to confirm the accuracy of CP Hemolysis to predict circulating CFI activity in rodents, but the trend suggests this assay may provide a close estimate to acute circulating cleavage activity in rats.
Untreated, rats undergoing surgery and nerve pinch demonstrated strong increases in circulating macrophage inflammatory protein-1 alpha (MIP-1α) compared to historical baseline (
CFI variant effect on limiting complement activation in a model of cecal ligation and puncture (CLP)-induced sepsis in rats was assessed. For Example 15, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
A rat model of non-aseptic sepsis was used to study complement involvement following a cecal ligation and puncture (CLP) surgery. This surgery provides three facets of complement activation and inflammation (mechanical damage, bacterial exposure, and ischemic injury) that make it particularly relevant as a screening tool for other indications. Male CD Sprague Dawley rats (Charles River Laboratories) weighing between 300 and 350 g at enrollment were anesthetized with a mixture of 2 to 2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen, and placed on a heating pad to maintain body temperature. Sepsis was induced by a CLP surgical procedure. A midline incision was made in the abdominal wall, the cecum exteriorized, and ligated with a nylon suture (4-0) proximal to the ileo-cecal valve, then perforated using a 16-gauge needle passed through the distal portion of the cecum resulting in a small amount of cecum contents entering the abdominal cavity. The abdominal wall and skin were then sutured.
Immediately following the CLP procedure, animals received an intravenous injection of Variant 1 [CFI-HSA (E457G; N531G variant)] 4.25 mg/kg (n=6), Variant 2 [CFI-HSA (E457G; N531G variant with C-term CCP15; CCP16; CCP17 fusion)] 5 mg/kg (n=6), Variant 3 [CFI-HSA (E457G; E461Q; N531G; Δ(558-PFISQYNV (SEQ ID NO: 14)-565) variant)] 4.25 mg/kg (n=6), or control article (1× PBS; n=6) at a dose volume of 5 mL/kg. No sham arm was performed. 16 hours after CLP surgery all animals were sacrificed by exsanguination. K2-EDTA plasma samples were collected the day prior to enrolment (baseline), 3, and 16 hours after CLP for evaluation of complement component fragments by mass spectrometry (MS) and cytokine/chemokine levels (Rat 27 plex Multiplex Immunoassay analyzed with a BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX, performed by Eve Technologies, Calgary, Canada). Whole blood and serum were collected for clinical pathology evaluation [complete blood counts (CBC) and serum chemistry; Biovet Inc., Canada] at baseline and 16 hours.
The thrombocytopenia was observed for vehicle-treated animals 16 hours post-injury. A trend towards protection against thrombocytopenia was observed in Variant 1 and Variant 2 treated animals (
The sepsis inflammatory cytokine tumor necrosis factor alpha (TNFα) is released rapidly following the CLP surgery (
Evaluation of the therapeutic effects of CFI variants in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. For Example 16, reference to CFI-HSA refers to human serum albumin fused to the N-terminal end of wild type CFI (SEQ ID NO: 21).
Purpose: 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)] to limit complement mediated acute pulmonary inflammation in a mouse model of ARDS induced by a single administration of lipopolysaccharide (LPS).
A mouse model of aseptic ARDS was used to study complement involvement following an intratracheal instillation (IT) of LPS. Male C57BL/6 mice (Charles River Laboratories) weighing 20 to 25 g at enrolment were anesthetized under isoflurane and intratracheally instilled with 50 μg LPS (1 mg/mL LPS isolated from E. coli 0111:B4 in 0.9% saline solution, Sigma).
Three hours following the CLP procedure, animals received an intravenous injection of 5 mg/kg Variant 1 (n=8), 5 mg/kg Variant 2 (n=8) or control article (1× PBS; n=10) at a dosing volume of 5 mL/kg. To evaluate the potential impacts of repeat daily dosing, 27 hours post-LPS IT, Variant 2 treated animals received a second 5 mg/kg dose. A sham arm was subjected to a 50 μL intratracheal instillation of 0.9% saline solution (n=5) without any IV treatment. Variant 1 treated animals were sacrificed 24 hours post-LPS IT while Variant 2 treated animals were sacrificed 48 hours post-LPS IT.
K2-EDTA plasma, lung tissue, and bronchoalveolar lavage fluid (BALF) samples were collected at sacrifice for evaluation of complement component fragments by mass spectrometry (MS). BALF was harvested in three 300 μL perfusions of the right lung with cold PBS 1× containing Protease Inhibitor 1× (SigmaFAST®). Cytokine and chemokine levels (Mouse 31 plex Multiplex Immunoassay analyzed with a BioPlex 200 Cytokine Array, Assay Kit Millipore MILLIPLEX, performed by Eve Technologies, Calgary, Canada) were assessed in K2-EDTA plasma, BALF, and lung tissue (homogenized in PBS 1×+0.1% Triton X-100 with protease cocktail inhibitors) collected at sacrifice. At sacrifice, whole blood and serum were collected for clinical pathology evaluation [complete blood counts (CBC) and serum chemistry; Biovet Inc., Canada]. A cell count differential was performed on BALF samples to assess leukocyte recruitment to the lung.
LPS is a known alternative complement pathway inducing agent. We assessed CFI activity on circulating C3b cleavage products using mass spectrometry. Percent activated C3f was determined as the percent of the C3f peptide with N-terminal labeling (S[2Me]EETK[2Me]QNEGF) (SEQ ID NO: 28) multiplied by the total peptide signal size of C3f (SEETKQNEGF) (SEQ ID NO: 28). In the BALF, increased cleavage release of C3f was observed at 24 hours in all LPS-treated animals and sustained up to 48 hours (
This application claims priority to U.S. Provisional Application No. 63/038,874 filed on Jun. 14, 2020, U.S. Provisional Application No. 63/122,437 filed on Dec. 7, 2020, U.S. Provisional Application No. 63/124,698 filed on Dec. 11, 2020, and U.S. Provisional Application No. 63/179,160 filed on Apr. 23, 2021, the contents of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 63179160 | Apr 2021 | US | |
| 63124698 | Dec 2020 | US | |
| 63122437 | Dec 2020 | US | |
| 63038874 | Jun 2020 | US |
