NZ747180B2 - Conjugated c1 esterase inhibitor and uses thereof - Google Patents

Abstract

The present invention provides, among other things, a conjugated Cl-INH for improved treatment of complement-mediated disorders, including hereditary angioedema (HAE). In some embodiments, a conjugated Cl-INH provided by the present invention is a PEGylated Cl-INH. In some embodiments, a conjugated Cl-INH provided by the present invention is a polysialic acid (PSA) conjugated Cl-INH.

Description

CONJUGATED C1 ESTERASE TOR AND USES THEREOF RELATED APPLICATIONS This application claims priority to, and the benefit of, US. provisional application number 62/318,003 filed on April 4, 2016, the content of which is hereby orated by reference in its entirety.

BACKGROUND ibitor (Cl-INH), also known as C1 esterase inhibitor, is the largest member of the serpin protein superfamily. It is a heavily glycosylated serine proteinase inhibitor having the main function of inhibiting the spontaneous activation of the complement system. Cl—INH regulates the complement cascade system, plays a key role in the regulation of the contact (kallikrein-kinin) amplification cascade, and participates in the regulation of the ation and fibrinolytic systems. Kamaukhova, E., C] -Esterase Inhibitor: Biological Activities and eutic Applications. J Hematol Thromb Dis, 1: 113 (2013).

Dysfunction and/or deficiency of C1-INH in subjects has been correlated with a y of autoimmune disease due to the e of Cl-INH to inhibit the activation of the complement system. An example of such a disease is hereditary angioedema (HAE), a rare, but potentially life-threatening disorder characterized by unpredictable and recurrent attacks of in?ammation. Symptoms of HAE attacks include swelling of the face, mouth and/or airway that occur spontaneously or are triggered by mild trauma. Such swelling can also occur in any part of the body. In some cases, HAE is associated with low plasma levels of Cl—inhibitor, while in other cases the n circulates in normal or elevated amounts but it is dysfunctional. In addition to the episodes of in?ammation, it also can cause more serious or life threatening indications, such as autoimmune diseases or lupus erythematosus.

CINRYZE®, a human plasma derived C1 esterase tor, has been approved for prophylactic use and treatment of acute attacks of HAE. Berinert® (also a plasma-derived human Cl-INH, CSL Behring) is indicated for treatment of acute HAE attack. Ruconest® (conestat alfa, Pharming N.V.) is a recombinant Cl-INH expressed in engineered rabbits is indicated for IV administration for ent of acute HAE attack. Ruconest® has the same amino acid sequence as human plasma derived Cl-INH, but it is made in transgenic rabbits.

Ruconest has an extremely short half-life of about 2.4-2.7 hours. See Ruconest® FDA Label and Prescribing Information.

There s a need for improved Cl esterase inhibitors for the treatment and prophylaxis of various C1 esterase mediated indications.

SUMMARY The present ion provides, among other things, improved long—acting Cl esterase inhibitor that can be used to effectively treat various complement-mediated disorders including HAE.

In particular, the present invention provides C1 esterase inhibitor conjugates (also referred to as "conjugated Cl esterase inhibitors") that exhibit comparable or even longer ife than plasma derived C1-INH. The present invention is, in part, based on the surprising discovery that PEGylated and polysialylated Cl-INH can have extended serum half-life of, e.g., at least 4 days. It is contemplated that long serum half-life of a conjugated Cl-INH leads to superior in viva efficacy and permits a preferable dosing regimen and route of administration. For example, the conjugated Cl-INH described herein may be stered subcutaneously or intravenously with reduced frequency compared to currently approved Cl-INH therapeutics, while still achieving desired efficacy (e.g., prophylaxis). The conjugated Cl tor proteins described herein may be produced using plasma derived or recombinantly produced Cl-INH. Therefore, conjugated Cl-INH described herein can be manufactured in a cost-effective manner and not dependent on blood supply. Because they can be recombinantly produced in cultured cells, they offer more consistency in production and final t than those products purified from human blood, human blood components (e. g. plasma), or animal milk. Thus, the present invention provides conjugated Cl esterase inhibitors that are safer, more ive for treatment of HAE and other complement—mediated In one aspect, the present invention provides a conjugated Cl-INH sing a Cl-INH n and at least one PEG moiety covalently linked to the Cl-INH protein. In some embodiments, the Cl-INH protein comprises at least one glycan e and the at least one PEG moiety is covalently linked to the at least one glycan residue. In some ments, the at least one PEG moiety is covalently linked to the Cl-INH protein via an oxime linkage.

In some embodiments, the at least one PEG moiety forms a covalent oxime link to a glycan residue or an amine group of Cl-INH. In some embodiments, the at least one PEG moiety forms a nt oxime link to a glycan residue. In some embodiments, the at least one PEG moiety forms a covalent oxime link to an amine group of .

In some embodiments, the glycan residue is a sialic acid residue or a galactose e of C1-INH. In some embodiments, the glycan residue is a sialic acid residue.

In some embodiments, the Cl-INH protein suitable for the present invention is recombinantly produced or plasma derived.

In some embodiments, the Cl-INH protein includes a C1-INH domain that has an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID N021, SEQ ID N022, SEQ ID NO:37, or SEQ ID NO:38.

In some embodiments, the Cl-INH protein is a fusion protein. In some embodiments, the fusion protein includes an EC domain directly or indirectly fused to a C1- INH domain. In some embodiments, the Fc domain is derived from IgGl. In some embodiments, the Fc domain comprises amino acid substitutions corresponding to L234A and L235A according to EU numbering. In some embodiments, the Fc domain ses one or more amino acid substitutions at ons corresponding to Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and/or Asn434 of IgG1 according to EU numbering.

In some embodiments, the fusion protein includes an albumin domain ly or indirectly fused to a Cl-INH domain.

In some embodiments, the t invention provides a Cl-INH protein that has a glycosylation profile comprising no more than about 50% (e.g., no more than 45%, 40%, 35 %, 30%, 25%, 20%, 15 %, 10%, or 5%) neutral glycan species.

In some embodiments, the present invention provides a Cl-INH protein that has a glycosylation profile comprising between about 5% and about 25% neutral glycan In some embodiments, the present invention es a Cl-INH protein that comprises, on average, at least about 30% (e.g., at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) charged glycans per molecule.

In some embodiments, the C1-INH protein contains less than about 20% (e.g., less than 15%, 10%, or 5%) of one or more of mannose, a—galactose, NGNA, or oligomannose-type glycosylation.

In some embodiments, the C1-INH protein has a glycosylation profile comprising one or more of the ing: between about 5% and about 30% neutral glycan species; between about 10% and about 30% mono—sialylated glycan s; between about % and about 50% lylated glycan s; between about 15% and about 35% tri- sialylated glycan species; and/or between about 5% and about 15% tetra-sialylated glycan species.

In some embodiments, the C1-INH protein has a glycosylation profile comprising: no more than 30% neutral glycan species; between about 20% and about 30% mono-sialylated glycan species; between about 30% and about 40% di-sialylated glycan s; between about 10% and about 20% tri-sialylated glycan species; and, between about % and about 10% tetra-sialylated glycan s.

In some ments, the C1-INH protein comprises, on average, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 sialylated glycan residues per molecule.

In some embodiments, the C1-inhibitor polypeptide comprises, on average, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mole sialic acid per mole of protein.

In some embodiments, a Cl-INH protein with a glycosylation profile described herein is a fusion protein. In certain embodiments, a C1-INH protein with a glycosylation profile described herein is an unconj ugated protein.

In some embodiments, a PEG ated to a C1-INH protein has a molecular weight between about 1 KDa and 50 KDa, n about 1 KDa and 40 KDa, between about KDa and 40 KDa, between about 1 KDa and 30 KDa, between about 1 KDa and 25 KDa, between about 1 KDa and 20 KDa, between about 1 KDa and 15 KDa, between about 1 KDa and 10 KDa, or between about 1 KDa and 5 KDa. In some embodiments, a PEG conjugated to a C1-INH protein has a molecular weight of or greater than about 1 KDa, 2 KDa, 3 KDa, 4 KDa, 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa, 45 KDa, or 50 KDa. In some embodiments, a PEG conjugated to a C1-INH protein has linear or ed ures. In some embodiments, the branched PEG moiety can have 2, 3, 4, or 5 arm branches.

In some ments, the conjugated C1-INH has a PEG/Cl-INH ration between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between 1 to about 10, or between about 1 to about 5.

In some embodiments, the conjugated C1-INH has a ife comparable to or greater than a plasma-derived human C1-INH protein. In some embodiments, the half-life of the conjugated Cl-INH is in the range of 100%-500% of the half-life of the plasma—derived Cl—INH protein. In some embodiments, the conjugated C1—INH protein has a half-life of at least about 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 hours.

In some embodiments, the conjugated C1-INH has a half-life of at least about 3, 4, 5, 6,7, 8,9, 10, 11, 12, 13, or 14 days.

In some embodiments, the conjugated C1-INH has a specific activity in the range of 50%-150% of the specific activity of plasma-derived human C1-INH protein.

In another aspect, the present invention provides a method of producing a conjugated C1 esterase inhibitor (Cl-INH), sing steps of providing a C1-INH protein sing at least one glycan residue and/or at least one amine group, and providing a PEG moiety under conditions that permit the PEG moiety to react with the at least one glycan residue and/or the at least one amine group to form a linkage, thereby ing the conjugated C1-INH.

In some ments, the PEG moiety comprises PEG-CHg-O-NHZ. In some specific embodiments, the at least one glycan residue is a sialic acid residue. In further embodiments, the at least one glycan e is a galactose residue.

In some embodiments, the method described herein further includes a step of oxidizing the at least one glycan residue prior to reacting with the PEG moiety. In some embodiments, the oxidizing step ses use of periodate oxidation. In some embodiments, the periodate oxidation is carried out with a molar ratio of periodate to C1-INH at between about 20:1 to about 50: 1. In some embodiments, the molar ratio of periodate to PEG is between about 2.5 to about 40. In some embodiments, the molar ratio of PEG to Cl- INH is between 25:1 and 100:1.

In further embodiments, the present method further comprises a step of purifying the ated Cl-INH. In some embodiments, the purifying step includes one or more of anion exchange, tangential ?ow filtration, diafiltration, and dialysis.

In a further aspect, the present invention provides a pharmaceutical composition comprising a conjugated Cl esterase inhibitor (Cl-INH), and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition comprising a conjugated Cl-INH is liquid. In other embodiments, the pharmaceutical composition comprising a conjugated C1-INH is lized.

In yet another , the present invention provides a kit comprising a pharmaceutical composition comprising conjugated Cl-INH (e.g., in a liquid and lized form). In some embodiments, the kit contains a syringe. In some embodiments, the syringe is preloaded with the pharmaceutical composition comprising ated Cl-INH.

In some embodiments, n the pharmaceutical composition is lyophilized, the kit further comprises a reconstitution buffer.

In still another aspect, the present invention provides a method of treating a complement-mediated disorder comprising administering to a t in need of treatment the pharmaceutical composition of conjugated Cl esterase inhibitor (Cl-INH).

In a d aspect, the present invention es a use of a composition sing a ated Cl-esterase inhibitor (Cl-INH) in the manufacture of a medicament for treating a complement-mediated disorder.

In some embodiments, the complement-mediated disorder is selected from hereditary angioedema, antibody mediated rejection, neuromyelitis optica spectrum disorders, traumatic brain injury, spinal cord injury, ic brain injury, burn injury, toxic epidermal necrolysis, multiple sclerosis, amyotrophic l sclerosis (ALS), Parkinson’s disease, , chronic in?ammatory demyelinating polyneuropathy (CIDP), enia gravis, and/or multifocal motor neuropathy.

In some embodiments, the present invention es a composition comprising a conjugated C1 esterase inhibitor (Cl-INH) comprising: a Cl-INH protein comprising at least one glycan residue; at least one polysialic acid (PSA) moiety. In some embodiments, the at least one polysialic acid (PSA) moiety is covalently linked to the at least one glycan residue.

In another aspect, the present invention provides a composition comprising a conjugated Cl esterase inhibitor (Cl-INH) comprising a Cl-INH protein sing at least one glycan residue; and at least one polysialic acid (PSA) moiety. In some embodiments, the at least one polysialic acid (PSA) moiety is ntly linked to the Cl-INH protein via an oxime linkage or a hydrazone linkage. In some embodiments, the polysialic acid (PSA) moiety is covalently linkted to the Cl-INH protein via an oxime linkage. In some embodiments, the polysialic acid (PSA) moiety is covalently linked to the Cl-INH protein via an oxime linkage. In some embodiments, the oxime e is n the PSA moiety and a glycan residue or an amine group of .

In some embodiments, the glycan residue is a sialic acid residue.

In some embodiments, the Cl-INH protein is recombinantly ed or plasma derived.

In some embodiments, the Cl-INH protein comprises a Cl-INH domain having an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:37, or SEQ ID NO:38.

In some embodiments, the Cl-INH n is a fusion n. In some embodiments, the fusion protein may comprise an Fc domain directly or indirectly fused to a Cl-INH domain. In some embodiments, the Fc domain may be derived from IgGl. In some embodiments, the Fc domain may comprise amino acid substitutions corresponding to L234A and L235A according to EU numbering. In some ments, the fusion protein may comprise an albumin domain directly or indirectly fused to a Cl-INH domain.

In some embodiments, the Cl-INH protein has a ylation profile comprising no more than about 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15 %, 10%, or 5% neutral glycan species, prior to PEGylation.

In some embodiments, the Cl-INH protein has a glycosylation profile comprising between about 5% and about 25% neutral glycan species, prior to PEGylation.

In some embodiments, the Cl-INH n comprises, on average, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% charged glycans per molecule.

In some embodiments, the Cl-INH protein contains less than about 20%, 15%, %, or 5% of one or more of mannose, (x—galactose, NGNA, or oligomannose-type glycosylation, prior to conjugation with PSA.

In some embodiments, prior to conjugation with PSA, the Cl—INH protein has a glycosylation profile comprising one or more of the following: between about 5% and about % neutral glycan species; n about 10% and about 30% mono-sialylated glycan species; between about 30% and about 50% di-sialylated glycan species; between about 15% and about 35% tri—sialylated glycan species; or n about 5% and about 15% tetra- sialylated glycan s.

In some embodiments, prior to conjugation with PSA, the Cl-INH protein has a glycosylation e comprising: no more than 30% neutral glycan species; between about % and about 30% mono-sialylated glycan species; between about 30% and about 40% di— sialylated glycan species; between about 10% and about 20% tri-sialylated glycan species; and n about 5% and about 10% tetra-sialylated glycan species.

In some embodiments, the Cl—INH protein comprises, on e, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 sialylated glycan residues per molecule.

In some embodiments, the C1-INH protein comprises, on e, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, , 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mole sialic acid per mole of protein In some embodiments, the PSA has a molecular weight between about 1 KDa and 50 KDa, between about 1 KDa and 40 KDa, between about 5 KDa and 40 KDa, between about 1 KDa and 30 KDa, between about 1 KDa and 25 KDa, between about 1 KDa and 20 KDa, between about 1 KDa and 15 KDa, between about 1 KDa and 10 KDa, or between about 1 KDa and 5 KDa.

In some embodiments, the PSA has a molecular weight of about 1 KDa, 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa, 45 KDa, or 50 KDa.

In some embodiments, the conjugated C1-INH has a PSA/Cl-INH ratio of between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5.

In some embodiments, the conjugated C1-INH has a half-life comparable or greater that than a plasma derived human C1-INH.

In some embodiments, the conjugated C1-INH has a half-life in the range of 100%-500% of the half-life of the plasma derived Cl-INH.

In some ments, the conjugated C1-INH has a half-life of at least about 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, or 170 hours.

In some embodiments, the ated C1-INH has a half-life of at least about 3, 4, 5, 6,7, 8,9, 10, 11, 12, 13, or 14 days.

In some embodiments, the conjugated C1-INH has a specific activity in the range of 50%-150% of the specific activity of plasma d human C-INH.

In a further aspect, the present invention provides a method of producing a ated C1 esterase inhibitor (Cl-INH). In some embodiments, the method comprises steps of: providing a C1-INH protein comprising at least one glycan residue and/or at least one amine group; and ing a polysialic acid (PSA) moiety under conditions that permit the PSA moiety to react with the at least one glycan residue and/or the at least one amine group to form a linkage, thereby producing the conjugated . In some embodiments, the at least one glycan residue is a sialic acid residue.

In some embodiments, the method further comprises a step of oxidizing the at least one glycan residue prior to reacting with the PSA moiety. In some embodiments, the oxidizing step ses periodate ion. In some embodiments, the periodate oxidation may be carried out with a molar ratio of periodate to C1-INH at between about 20:1 to about 50: 1. In some embodiments, the molar ratio of periodate to PSA may be between about 2.5 to about 40.

In some embodiments, the molar ratio of PSA to C1—INH is n about :1 and 100:1.

In some embodiments, the method further comprises a step of purifying the conjugated C1-INH.

In some embodiments, the purifying step comprises one or more of anion exchange, tangential ?ow filtration tration, and dialysis.

In yet r aspect, the present ion provides conjugated Cl esterase tor (Cl-INH) produced by a method of an above aspect or embodiment.

In still another aspect, the present invention es a pharmaceutical composition comprising a conjugated Cl esterase inhibitor (Cl-INH) of an above aspect or embodiment and a pharmaceutically acceptable carrier. In some ments, the composition of the pharmaceutical composition is liquid. In some ments, the composition of the pharmaceutical composition is lyophilized.

In one aspect, the present invention provides a kit sing a pharmaceutical composition of an above aspect or embodiment and a syringe. In some embodiments, the syringe is preloaded with the pharmaceutical composition. In some embodiments, the pharmaceutical composition is lyophilized and the kit further comprises a reconstitution buffer.

In another aspect, the present invention provides a method of treating a complement-mediated disorder comprising administering to a subject in need of treatment a pharmaceutical composition of an above aspect or embodiment. In some ments, the ment-mediated disorder is selected from hereditary angioedema, antibody mediated rejection, neuromyelitis optica spectrum disorders, traumatic brain , spinal cord injury, ischemic brain , burn injury, toxic mal necrolysis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, stroke, chronic in?ammatory demyelinating polyneuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

In a further aspect, the present invention provides a use of a composition comprising a conjugated Cl-esterase inhibitor of an above aspect or ment, in the manufacture of a medicament for treating a complement mediated disorder. In some ments, the complement-mediated er is selected from hereditary angioedema, antibody mediated rejection, neuromyelitis optica spectrum disorders, traumatic brain injury, spinal cord injury, ischemic brain injury, burn injury, toxic epidermal necrolysis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, stroke, chronic in?ammatory demyelinating polyneuropathy (CIDP), myasthenia gravis, and/or multifocal motor neuropathy.

Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not tion. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings are for illustration purposes only, not for limitation.

Figure 1 is a schematic representation of Cl-INH. From right to left the three domains are the signal e, the inus, also referred to as N—terminal domain, and the serpin . N-linked glycans are shown as long al lines with diamond heads and ed glycans are shown as short vertical lines.

Figure 2 depicts the mature Cl-INH amino acid sequence and potential sites for PEGylation.

Figure 3 depicts a schematic of a chemical equation depicting an exemplary amine mediated PEGylation.

Figure 4A is a schematic of a al equation depicting an exemplary glycan mediated aminoxy PEGylation. Figure 4B is a schematic of a chemical equation depicting an exemplary sialic acid mediated (SAM) aminoxy PEGylation.

Figure 5 depicts a schematic of a chemical equation depicting an ary galactose mediated (GAM) PEGylation.

Figure 6, panels A and B, depicts the s of a inary rat study of Cl- INH PEGylated (either 5 KDa or 40 KDa) via amino groups compared with sialic acid. rhCl-INH and Cinryze are provided as a comparator. Panel C depicts an SDS-PAGE gel of Cl-INH PEGylated with either 5 KDa or 40 KDa PEG.

Figure 7 depicts a schematic of exemplary PEGylation process A.

Figure 8 depicts a schematic of exemplary PEGylation process B. s 9A—E depict schematics summarizing l exemplary PEGylation protocols suitable for PEGylating Cl-INH.

Figure 10A depicts the Cl—INH-PEG IC50 of 5KSAM KHR5 octyl load samples. Figure 10B depicts the Cl-INH-PEG IC50 before and after removal of free PEG by Figure 11 depicts the chromatography results of an exemplary 40 KDa PEGylated C1-INH purification from free PEG and other contaminants.

Figure 12 depicts the chromatography results of an exemplary 20 KDa ted C1-INH purification from free PEG and other contaminants.

Figure 13 depicts the chromatography s of an exemplary 5 KDa PEGylated Cl-INH purification from free PEG and other contaminants.

Figure 14 depicts the results of a Non-Human Primate (NHP) PK Study of IV stered PEGylated rhC1 INH V. rhC1 INH.

Figure 15 depicts the results of a NHP PK study in which varied Cl-INH-PEG loads were administered to the NHP.

Figure 16 depicts the results of an IV v. SC NHP study of PEGylated rhC1— Figure 17 depicts the results of a rat PK titer analysis on Cl-INH-PEG samples with varied 5KPEG loading.

Figure 18, panels A-E, depicts a series of gels and graphs that depict the purity of C1-INH-PEG. Panels A and B are barium-iodine stained SDS-PAGE gels used to detect free PEG in C1-INH-PEG samples. Panels C and D are RP—HPLC graphs that were used to detect free PEG 1K and 2K in -PEG samples. Panel E depicts two SDS-PAGE gels loaded with C1-INH samples.

Figure 19, panels A-C, depicts a series of graphs and gels that depict purity, IC50, and PK data of Cl-INH-PEG samples ated with SAM process. Panel A is an IC50 graph of various Cl—INH samples. Panel B is an SDS-PAGE gel that depicts C1-INH sample purity and ated Cl-INH sample IC50 values. Panel C is a graph that depicts PK values from a rat study in which the rats ed intravenous C1-INH-PEG and non- PEGylated C1-INH.

Figure 20, panels A, B, and C, s a series of graphs that depict C1-INH IC50 values.

Figure 21 depicts a schematic for an exemplary amine coupling PEGylation process for Cl-INH.

Figure 22, panels A-D, depicts a series of gels and graphs that depict the purity of C1-INH-PEG. Panel A depicts a barium iodine stained SDS-PAGE gel used to detect free PEG in Cl-INH-PEG s. Panel B depicts an RP-HPLC graph for the ion of free PEG 1K and 2K. Panels C and D depict purification chromatograms for free NHS—PEG20K (Panel C) and NHS-PEG40K (Panel D).

Figure 23, panels A-C, depicts a series of graphs and gels that depict purity, IC50, and PK data of Cl-INH samples. Panel A is an IC50 graph of various Cl-INH samples.

Panel B is a graph that depicts PK values from a rat study in which the rats received intravenous Cl-INH-PEG and non—PEGylated Cl-INH. Panel C is an SDS-PAGE gel that depicts Cl-INH sample purity and associated Cl-INH sample IC50 values.

Figure 24, panels A and B, depicts a gel and a graph that depicts the purity of Cl-INH-PSA produced with the sialic acid mediated (SAM) process. Panel A is an SDS gel, and Panel B is an IC50 graph of Cl-INH—PSA.

Figure 25, panels A, B, and C, depicts a series of graphs that show PK values from a rat study in which the rats ed intravenous Cl-INH-PEG, Cl-INH-PSA, e- PEG, Cl-INH, or Cinryze.

DEFINITIONS In order for the present invention to be more readily understood, n terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

Animal: As used herein, the term "animal" refers to any member of the animal m. In some ments, "animal" refers to , at any stage of development. In some embodiments, "animal" refers to non—human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e. g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

Approximately or about: As used in this application, the terms "about" and ximately" are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal ?uctuations appreciated by one of ordinary skill in the relevant art. As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either ion (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Bioavailabilizy: As used , the term "bioavailability" lly refers to the percentage of the administered dose that reaches the blood stream of a subject.

Biologically active: As used herein, the phrase "biologically active" refers to a characteristic of any agent that has activity in a biological system, and particularly in an sm. For ce, an agent that, when stered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a peptide is biologically active, a portion of that e that shares at least one biological activity of the peptide is typically referred to as a "biologically active" portion.

Carrier or diluent: As used herein, the terms "carrier" or "diluent" refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) carrier or diluting substance useful for the preparation of a pharmaceutical formulation. Exemplary diluents e sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. ate-buffered saline), sterile saline on, Ringer's solution or dextrose solution.

CI-inhibitor or C1 esterase inhibitor or C1 -INH: As used herein, the term "Cl-inhibitor" or "C1 esterase inhibitor" or "Cl-INH" can all be used interchangeably and refer to any wild-type, , naturally occurring, recombinant produced, and/or ed Cl-INH proteins (e.g., Cl-INH proteins with one or more amino acid mutations, ons, truncations, insertions, and/or fusion proteins) that retain substantial C1-INH biological activity unless otherwise specified. A "Cl-inhibitor" or "C1 esterase inhibitor" or "Cl-INH" may be a fusion protein. In some embodiments, a Cl-INH fusion protein comprises a C1- INH polypeptide or domain and an Fc domain. In some embodiments, a Cl-INH fusion protein comprises a Cl-INH polypeptide or domain and an albumin domain. In some embodiments, the fusion protein r comprises a linker. A Cl-INH protein may be recombinantly expressed in recombinant cells. In certain embodiments, the Cl-INH is sed in mammalian cells, preferably CHO cells, or human cells, preferably HT1080 or HEK cells.

Conjugate: As used herein, the term "conjugate" may refer to a moiety covalently ed to a protein ly or indirectly. Typically, where a protein is attached to a conjugate, it may be referred to as a conjugated protein or protein conjugate. In some embodiments, a conjugate described herein is hylene glycol (PEG). Where a protein is attached to a PEG moiety, it may be referred to as a PEGylated protein.

Functional equivalent or derivative: As used herein, the term "functional equivalent" or ional derivative" denotes, in the context of a functional derivative of an amino acid ce, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. A functional derivative or equivalent may be a l derivative or is prepared synthetically. Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The substituting amino acid desirably has chemico-physical properties which are r to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.

Fusion protein: As used herein, the term "fusion protein" or "chimeric protein" refers to a protein created through the joining of two or more originally separate proteins, or portions thereof. In some embodiments, a linker or spacer will be present between each protein.

Half-Life: As used herein, the term "half-life" is the time required for a quantity such as n tration or activity to fall to half of its value as ed at the beginning of a time period.

Hereditary angioedema or HAE: As used herein, the term "hereditary dema" or "HAE" refers to a blood disorder terized by unpredictable and recurrent attacks of in?ammation. HAE is typically associated with Cl-INH deficiency, which may be the result of low levels of Cl-INH or Cl-INH with impaired or decreased ty. Symptoms include, but are not limited to, swelling that can occur in any part of the body, such as the face, extremities, genitals, gastrointestinal tract and upper airways.

Improve, increase, or reduce: As used herein, the terms "improve,73 ‘s'increase" or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the ent described herein, or a measurement in a control subject (or multiple control subject) in the e of the treatment described herein. A "control subject" is a subject af?icted with the same form of disease as the t being treated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term "in vitro" refers to events that occur in an cial environment, e.g., in a test tube or reaction vessel, in cell culture, etc, rather than within a multi-cellular organism.

In Vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism, such as a human and a man animal. In the context of cell- based s, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

Linker: As used herein, the term "linker" refers to, in a fusion protein, an amino acid sequence other than that appearing at a particular position in the natural protein and is generally designed to be ?exible or to interpose a structure, such as an d—helix, between two protein moieties. A linker is also referred to as a . A linker or a spacer typically does not have biological function on its own.

Polypeptide: The term "polypeptide" as used herein refers to a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ry skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified. As used herein, the terms "polypeptide" and "peptide" are used inter-changeably.

Prevent: As used herein, the term "prevent" or "prevention", when used in connection with the occurrence of a disease, disorder, and/or ion, refers to ng the risk of ping the disease, disorder and/or condition. See the definition of "risk." Protein: The term "protein" as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary al association with other polypeptides in order to form the discrete functioning unit, the terms "polypeptide" and "protein" may be used interchangeably. If the discrete functional unit is sed of more than one polypeptide that physically associate with one another, the term "protein" refers to the multiple polypeptides that are ally coupled and function together as the discrete unit.

Risk: As will be understood from context, a "risk" of a disease, disorder, and/or condition comprises a likelihood that a particular individual will develop a disease, disorder, and/or ion (e. g., muscular dystrophy). In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a nce sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event (e.g., muscular dystrophy). In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular dual. In some embodiments, relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

Subject: As used herein, the term "subject" refers to a human or any non- human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human ting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be af?icted with or is susceptible to a disease or disorder but may or may not display symptoms of the e or disorder.

Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that ical and chemical phenomena rarely, if ever, go to tion and/or proceed to teness or achieve or avoid an absolute result. The term antially" is therefore used herein to capture the potential lack of completeness nt in many biological and chemical phenomena.

Substantial gy: The phrase "substantial homology" is used herein to refer to a comparison n amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally ered to be "substantially gous" if they contain homologous residues in corresponding positions. gous residues may be identical es. Alternatively, homologous residues may be non—identical residues will appropriately similar structural and/or functional characteristics.

For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non-polar" side chains Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution.

As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in cial computer programs such as BLASTN for nucleotide ces and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol, 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein se search programs", Nucleic Acids Res. 25:3389—3402, 1997; Baxevanis, et al., Bioinformatics : A Practical Guide to the Analysis ofGenes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying gous sequences, the ms mentioned above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are gous over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical es in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, ing those available in commercial computer programs such as BLASTN for tide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol, 215(3): 0, 1990; Altschul, et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389—3402, 1997; Baxevanis et al., ormatics .‘ A Practical Guide to the Analysis ofGenes and Proteins, Wiley, 1998; and Misener, et al., (eds), Bioinformatics Methods and ols (Methods in lar Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of ty. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some ments, the relevant stretch is a complete ce. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more Su?eringfrom: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, er, and/or condition.

Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, condition, or event (for example, DMD) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a c polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or ty of a protein associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, condition, and/or event (5) having undergone, planning to undergo, or requiring a transplant.

In some embodiments, an individual who is susceptible to a disease, er, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Therapeutically e?ective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or tible to a disease, disorder, and/or ion, to treat, se, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or ts only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

ED DESCRIPTION OF CERTAIN EMBODIMENTS The present invention es, among other things, a conjugated Cl—INH for improved treatment of complement-mediated disorders, including hereditary angioedema (HAE). In ular, a conjugated Cl-INH provided by the present invention is a PEGylated C l -INH.

It is contemplated that a conjugated C1-INH (e.g., a PEGylated C1-INH, or a polysialic acid (PSA) conjugated Cl-INH) has extended half-life compared to unconj ugated (e. g., un—PEGylated) but otherwise identical . According to the present invention, any Cl-INH proteins may be conjugated (e.g., ted, or PSA ated) including, but not limited to, plasma-derived or recombinantly sed Cl-INH proteins. In some embodiments, a Cl-INH protein that may be ated (e.g., PEGylated, or PSA conjugated) is a fusion protein. As described below, the result of conjugation (e.g., PEGylation, or PSA conjugated) according to the present invention extends in vivo half-life while retaining ctedly good bioavailability and/or bioactivity of the Cl-INH protein.

Therefore, conjugated (e.g., PEGylated, or PSA conjugated) Cl-INH provided herein permits improved treatment of HAE and other complement-mediated diseases, disorders or conditions by, e.g., reducing dosing frequency and increasing prophylactic efficacy. s aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated ise. The disclosures of all of the art cited herein are incorporated by reference in their entirety.

Cl-INH Proteins The present invention may be used to conjugate any C1-INH proteins. Human Cl-INH is an important anti-in?ammatory plasma protein with a wide range of inhibitory and non-inhibitory biological activities. By sequence homology, ure of its inal domain, and mechanism of protease inhibition, it belongs to the serpin superfamily, the largest class of plasma protease inhibitors, which also includes antithrombin, teinase inhibitor, plasminogen activator inhibitor, and many other structurally similar proteins that te diverse physiological systems. Cl-INH is an inhibitor of proteases in the complement system, the contact system of kinin tion, and the intrinsic ation pathway. Cai, S. & Davis, A. E., Complement Regulatory Protein Cl Inhibitor Binds to Selectins and Interferes with Endothelial-Leukocyte Adhesion, J Immunol, 171:4786-4791 . Specifically, Cl-INH has been shown to inhibit Clr and Cls of the complement system. Cl-INH is also a major regulator of coagulation factors XI and XII, as well as kallikrein and other serine proteases of the coagulation and fibrinolytic systems including tissue type plasminogen activator and plasmin.

Low plasma content of Cl-INH or its dysfunction result in the activation of both complement and contact plasma cascades, and may affect other systems as well. A decrease in Cl-INH plasma content to levels lower than 55 ug/mL (~25% of normal) has been shown to induce spontaneous activation of C1.

A schematic ing the structure of Cl-INH is provided in Figure 1. The signal peptide, N-terminal domain, and serpin domain are shown. Cl-INH is The 22 amino acid signal e is required for ion and cleaved from the rest of the Cl-INH protein.

Cl-INH has two domains: a C-terminal domain having 365 amino acids, which is a typical serpin domain, and an inal domain having 113 amino acids. The protein is stabilized by two disulfide bridges which t the domains. These disulfide bridges are formed by Cys101 of the N-terminal domain which forms a disulfide bond with Cys406 of the C- terminal (serpin) domain and Cys108 of the N-terminal domain which forms a disulfide bond with Cys183 of C—terminal . The serpin domain is responsible for the protease ty of C1-INH. Pl-Pl’ denotes the Arg444-Thr445 scissile bond.

More than 26% of the weight of the glycosylated protein is carbohydrate. The glycans are unevenly distributed over human C1—INH. The N-terminus is heavily glycosylated, having three N-linked (shown as long vertical lines with diamond heads) and at least seven O-linked (shown as short vertical lines) carbohydrate groups. Three N—attached glycans are ed to asparagine residues Asn2l6, Asn23l, and Asn330 in the serpin domain (shown as long vertical lines with diamond heads). Although the functional role of the exceptionally long and heavily glycosylated N-terminal domain is still r, it may be essential for the protein’s conformational stability, recognition, affinity to xins and ins, and nce. The intrinsic heterogeneity of the carbohydrate moiety greatly contributes to the heterogeneity of the whole Cl-INH, one of the reasons why production of a recombinant Cl-INH ing the properties of plasma-derived Cl—INH is ult.

As used herein, Cl-INH proteins suitable for conjugation and use according to the present invention comprise a Cl-INH polypeptide or domain with wild—type or modified amino acid sequences (e.g., Cl-INH proteins with amino acid mutations, deletions, truncations, and/or insertions) that retain substantial Cl-INH biological activity. Typically, a Cl-INH protein is produced using recombinant technology, but may also be plasma—derived.

In some embodiments, a Cl-INH polypeptide or domain le for the present invention includes an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical or homologous to the wild-type human C1-INH protein (amino acids 1-478) (amino acids 1-97 are underlined): NPNATSSSSQ 2DPESL§ QDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITAN TTDEPTTQ 2PTTEPTT§ 2PTI§ 2PT§ 2PTT§ QLPTDSFTQ 2PTTGSFCPGPVTLCSDLESHSTEAV LGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSY PKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDA NLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHF KNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDME QALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCG LTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQ HKFPVFMGRVYDPRA (SEQ ID NO:1).

In some embodiments, a Cl-INH polypeptide or domain suitable for the present ion es an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical or homologous to the mature wild-type human Cl-INH protein (amino acids 98-478): GSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASL LTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTF VNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIY LSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLS HNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQD MLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVA RTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA (SEQ ID NO:2).

In some embodiments, a C1-INH ptide or domain suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical or homologous to a human Cl-INH protein (amino acids 1-478) having an E165Q mutation (mutated amino acid bolded and underlined): SSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITAN TTDEPITQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSECPGPVTLCSDLESHSTEAV LGDALVDESLKLYHAESAMKKVETNMAESPFSIASLLTQVLLGAGENTKTNLESILS YPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSD ANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPF HFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLED MEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNL CGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWD QQHKFPVFMGRVYDPRA (SEQ ID NO:37).

In some ments, a Cl-INH polypeptide or domain suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical or homologous to a mature human Cl-INH protein (amino acids 98-478) having an E165Q mutation (mutated amino acid bolded and underlined): GSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASL LTQVLLGAGENTKTNLESILSYPKDETCVHQALKGETTKGVTSVSQIEHSPDLAIRDT FVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAI YLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQL SHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQ DMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISV ARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA (SEQ ID NO:38).

Homologues or analogues of human Cl-INH proteins can be prepared according to methods for altering polypeptide sequence known to one of ry skill in the art such as are found in nces that compile such methods. As will be appreciated by those of ordinary skill in the art, two sequences are lly considered to be antially homologous" if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. atively, homologous residues may be non—identical residues will appropriately similar structural and/or functional characteristics. For example, as is well known by those of ordinary skill in the art, n amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non- polar" side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution. In some embodiments, conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In some embodiments, a "conservative amino acid tution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.

As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Moi. Biol, 215(3): 403-410, 1990; Altschul, et al., s in Enzymology; Altschul, et al., d BLAST and PSI-BLAST: a new generation of n database search programs", Nucleic Acids Res. 25:3389—3402, 1997; Baxevanis, et al., Bioinformatics : A Practical Guide to the is ofGenes and Proteins, Wiley, 1998; and Misener, et al., (eds), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In on to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology.

In some embodiments, a Cl-INH polypeptide or domain suitable for the present ion may be a truncated C1-INH protein. For example, a C1-INH polypeptide or domain suitable for the present invention includes a portion or a fragment of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:37 or SEQ ID NO:38. .

Cl-INH Fusion Proteins In some embodiments, Cl—INH proteins that can be conjugated according to the present invention include Cl-INH fusion proteins. A Cl-INH fusion protein may include a Cl-INH domain (also ed to as a Cl—INH polypeptide) and another domain or moiety that typically can facilitate a therapeutic effect of Cl-INH by, for example, enhancing or increasing half-life, stability, potency, and/or delivery of Cl-INH protein, or reducing or eliminating immunogenicity, clearance, or ty. Such suitable domains or moieties for a Cl-INH fusion protein include but are not limited to Fc s and albumin domains. A suitable fusion domain or moiety (e.g., a Fc or albumin domain) may be directly or indirectly linked, fused or attached to the N-terminus, inus or internally to a Cl-INH protein.

The following sections describe exemplary Cl-INH fusion proteins that may be conjugated.

In some embodiments, a suitable Cl-INH fusion protein contains an Fc domain or a portion thereof that binds to the FcRn receptor. As a non—limiting e, a le Fc domain may be derived from an immunoglobulin subclass such as IgG. In some embodiments, a suitable Fc domain is derived from IgGl, IgG2, IgG3, or IgG4. In some embodiments, a suitable Fc domain is d from IgM, IgA, IgD, or IgE. Particularly suitable Fc domains include those derived from human or zed antibodies. In some embodiments, a suitable Fc domain is a modified Fc portion, such as a modified human Fc portion.

Cl—inhibitor Fc fusion proteins may exist as dimers, as shown in In some embodiments, an Fc domain suitable for the present invention may include an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) cal to the wild-type human IgG1 Fc domain: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID N023).

In some embodiments, a suitable Fc domain may include one or more mutations that reduce or eliminate ment activation and/or antibody-dependent cell- mediated cytotoxicity (ADCC) activity (also referred to as "effector on"). For example, suitable Fc domains may include mutations corresponding to L234A and L235A (LALA) of IgG1, according to EU numbering. An exemplary human IgG1 Fc domain having a LALA mutation (mutated residues underlined) is shown below: DKTHTCPPCPAPE?GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:4).

In some embodiments, an EC domain suitable for the present invention includes an amino acid ce at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:4 while maintaining mutations corresponding to L234A and L235A (LALA) of IgG1, according to EU numbering.

It is contemplated that improved binding between Fc domain and the FcRn receptor results in prolonged serum half-life. Thus, in some embodiments, a suitable Fc domain ses one or more amino acid mutations that lead to improved binding to FcRn. s mutations within the Fc domain that effect improved binding to FcRn are known in the art and can be adapted to practice the present invention. In some embodiments, a le Fc domain comprises one or more ons at one or more positions corresponding to Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433, and/or Asn 434 of human IgG1, according to EU numbering.

For example, a suitable Fc domain may contain mutations of H433K (His433Lys) and/or N434F (Asn434Phe). As a non-limiting example, a suitable Fc domain may n mutations H433K (His433Lys) and N434F (Asn434Phe). Additional amino acid substitutions that can be included in a Fc domain include those described in, e.g., US. Patent Nos. 6,277,375; 8,012,476; and 8,163,881, which are incorporated herein by reference.

In some embodiments, an EC domain suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human IgG1 Fc domain while maintaining one or more ons corresponding to Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433, and/or Asn 434 of human IgG1, according to EU numbering lined below): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALQHYTQKSLSLSPGK (SEQ ID NO:5).

In some embodiments, an EC domain le for the present invention es an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human IgG1 Fc domain while maintaining mutations ponding to L234A and L235A (LALA) of IgG1, and one or more mutations corresponding to Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433, and/or Asn 434 of human IgG1, ing to EU numbering (mutated residues underlined): DKTHTCPPCPAPE?GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALQHYTQKSLSLSPG K (SEQ ID NO:6).

In some embodiments, an EC domain derived from IgG4 is used for the present invention. Without wishing to be bound by any theory, IgG4 is reported to have lower complement activation than WT IgGl. Thus, in some embodiments, a wild-type human IgG4 Fc domain is used in the present invention. In some embodiments, an EC domain suitable for the present invention is derived from human IgG4 with a mutation corresponding to an $228P substitution in the core hinge region sequence according to the EU index. This substitution has also been referred to as S241P according to Kabat et al (1987 Sequences of ns of immunological interest. United States Department of Health and Human Services, Washington DC.). Without wishing to be bound by any , it is contemplated that this substitution has the effect of making the sequence of the core of the hinge region the same as that of a Wild-type IgGl or IgG2 isotype antibody and results in the production of the homogenous form of the IgG4 antibody and hence abrogates the dissociation and reassociation of the heavy chains which often leads to the tion of heterodimeric IgG4 antibodies. In addition, IgG4 derived Fc s may be used for stability at high concentrations.

Thus, in some embodiments, an F0 domain suitable for the present invention includes an amino acid ce at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the wild-type human IgG4 Fc domain: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO:9).

In some embodiments, an EC domain suitable for the present invention es an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the human IgG4 Fc domain while maintaining a on corresponding to an SZ41P substitution according to EU numbering (mutated residue underlined): ESKYGPPCPECPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS MKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 10).

In some embodiments, an Fc domain described herein may include a signal peptide. An exemplary signal e suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to METPAQLLFLLLLWLPDTTG.

For example, a suitable Fc domain may have an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human IgG1 Fc domain with a signal peptide, and having mutations that enhance the binding to the FcRn receptor (signal peptide and mutated residues underlined): METPAQLLFLLLLWLPDTTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALQHYTQKSLSLSPGK (SEQ ID NO:7).

In some embodiments, an EC domain suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human IgG1 Fc domain with a signal peptide, and having both LALA and mutations that enhance the binding to the FcRn receptor (mutated residues underlined): METPAEQLLFLLLLWLPDTTGDKTHTCPPCPAPE?GGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEAL?HYTQKSLSLSPGK (SEQ ID NO:8).

Exemplary CI-INH—Fc Fusion Proteins In particular embodiments, a suitable Cl-INH fusion protein includes a C1- INH polypeptide or domain and an Fc domain. In some embodiments, a suitable Cl-INH fusion protein includes a linker that associates the Cl-INH ptide or domain with the Fc domain. In certain embodiments, as shown in Fc moieties may be directly fused to the N-terminal region of the full length (1-478 aa) as well as mature (98-478) Cl-inhibitor.

As non-limiting examples, suitable Cl-INH Fc fusion proteins may have an amino acid ce shown below: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREES QYNSTYRVVSVLTVLHQ QDWLNGKEYKCKVSNKALPAPIE KGQ QPREPQ QVYTLPPSRDELTKNS QVSLTCLVKGFYPSDIAVEWESNGQ QPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQ 2g QGNVFSCSVMHEALHNHYTQ 2K8LSLSPG ENPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITA NTTDEPFTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEA VLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILS CVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSD ANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPF HFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLED PSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNL CGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWD QQHKFPVFMGRVYDPRA (SEQ ID NO:11) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQ QYNSTYRVVSVLTVLHQ KEYKCKVSNKALPAPIE KTISKAKGE QPREPE QVYTLPPSRDELTKNQ QVSLTCLVKGFYPSDIAVEWESNGE QPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQ 2g 2GNVFSCSVMHEALHNHYT§ 2K5LSLSPG EGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIAS LLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRD TFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNA IYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQ LSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTS QDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAIS VARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA (SEQ ID NO: 12) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQ 2YNSTYRVVSVLTVLH§ 2DWLNGKEYKCKVSNKALPAPI EKTISKAKGQ QPREPE QVYTLPPSRDELTKNE QVSLTCLVKGFYPSDIAVEWESNGQ QPEN PVLDSDGSFFLYSKLTVDKSRWSQQGNVFSCSVMHEALHNHYTQQKSLSLSP G_KNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKIT ANTTDEPTTQF’I‘TEPTTQPTIQPTQPTTQLPTDSPTQF’I‘TGSFCPGPVTLCSDLESHSTE AVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESI LSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNS DANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEP FHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLED MEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNL CGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWD QQHKFPVFMGRVYDPRA (SEQ ID NO:13) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREESQYNSTYRVVSVLTVLHEQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQ QPREPE 2VYTLPPSRDELTKNQ QVSLTCLVKGFYPSDIAVEWESNGQ QPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 2g CSVMHEALHNHYTQ QKSLSLSP GPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSI ASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIR DTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLN AIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQL QLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTT SQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAI SVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA (SEQ ID NO:14) ESKYGPPCPSCPAPEELGGPSVELEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQEN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS KAKG PREP VYTLPPS EEMTKN VSLTCLVKGFYPSDIAVEWESNG PEN NYKTTPPVLDSDGSEELYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSL gNPNATSSSSQDPESLQDRGEGKVATTVISKMLEVEPILEVSSLPTTNSTTNSATKIT ANTTDEPTTQPITEPTTQPTIQPTQPTTQLPTDSPTQPITGSECPGPVTLCSDLESHSTE AVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESI FTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNS DANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEP FHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLED MEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNL CGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWD QQHKFPVFMGRVYDPRA (SEQ ID NO:32) ESKYGPPCPSCPAPEELGGPSVELEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQEN WYVDGVEVHNAKTKPREEQPNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKG PREP VYTLPPS EEMTKN VSLTCLVKGFYPSDIAVEWESNG PEN NYKTTPPVLDSDGSEELYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSL gGSFCPGPVTLCSDLESHSTEAVLGDALVDPSLKLYHAESAMKKVETNMAESPESI VLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIR DTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLN AIYLSAKWKTTEDPKKTRMEPEHEKNSVIKVPMMNSKKYPVAHEIDQTLKAKVGQL QLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTT SQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAI SVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA (SEQ ID N0233) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSg QEDPEVg QFN WYVDGVEVHNAKTKPREEQ QFNSTYRVVSVLTVLHQ QDWLNGKEYKCKVSNKGLPSS MKGEQPREPEQVYTLPPSSQEEMTKNQ2VSLTCLVKGFYPSDIAVEWESNGQQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQ QEGNVFSCSVMHEALHNHYTQ QKSLSLSL G_KNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKIT ANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTE AVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESI LSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNS DANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEP FHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLED MEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNL CGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWD QQHKFPVFMGRVYDPRA (SEQ ID NO:15) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSg QEDPEVE QFN WYVDGVEVHNAKTKPREEE RVVSVLTVLHQ KEYKCKVSNKGLPSS IEKTISKAKGQ QPREPQ PSS 2EEMTKN§ 2VSLTCLVKGFYPSDIAVEWESNGQ QPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQ QEGNVFSCSVMHEALHNHYTQ QKSLSLSL ?GSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSI ASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIR DTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLN AIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQL QLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTT SQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAI SVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA (SEQ ID N0216).

In some embodiments, a suitable Cl-INH Fc fusion protein has an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO:ll, SEQ ID N0212, SEQ ID NO:l3, SEQ ID NO:l4, SEQ ID N0215, SEQ ID NO:l6, SEQ ID NO:32, or SEQ ID NO:33.

It is contemplated that a -Fc fusion protein may be provided in various urations ing homodimeric or monomeric configurations. For example, a suitable homodimeric configuration may be designed to have the C—terminal end of fusion partner (e.g., a Cl-INH polypeptide plus linker) attached to the inal end of both Fc polypeptide strands. A suitable monomeric configuration may be designed to have the C- terminal end of fusion partner (e.g, a Cl—INH polypeptide plus linker) fused to one PC dimer. ric, also referred to herein as monovalent, forms may be used for certain applications and routes of administration, e.g., subcutaneous administration. A monomeric configuration may decrease steric hindrance, increase half-life, and/or may increase bioavailability.

Without wishing to be bound by any theory, it is contemplated that monovalent forms may be particularly useful for Cl-INH-Fc fusion constructs because Cl- INH is a suicide inhibitor. Since it is a suicide inhibitor, the binding of one Cl-INH "arm" of a dimer Fc fusion will result in increased rate of clearance of the bound Cl-INH fusion protein, even in the event that a second arm remain unbound.

An advantage of the Fc fusion proteins, both monomeric and dimeric, is that Fc expression was found to occur at higher levels than expression of Cl-INH alone. Activity assays comparing the dimeric Cl—INH—Fc constructs with Cl-INH t the Fc fusion have been shown to have similar Clq binding activity. The inclusion of a linker was also tested and found not to affect the ability of Cl-INH-Fc fusion n to bind its target.

Methods of making monomeric antibody fusion proteins include those described in, e. g., PCT Publication Nos. WO2011/063348; WO2012/020096; WO20132’138643; WO2014087299; Dumont, J. et a1., Monomeric Fc Fusions: Impact on Pharmacokinetic and Biological Activity of Protein Therapeutics, Biodrugs, 20(3): 151-160 (2006); Ishino, T. et a1, Protein ure and Folding: Half-life Extension of Biotherapeutics Modality by osylation for the Engineering a ric Fc Domain, J. Biol. Chem., 288:16529-16537 (2013), the disclosures of which are incorporated herein by reference.

Monovalent Cl-inhibitor can be made by using a d containing the Fc- C1 co transfected with a plasmid expressing Fc alone. In addition, it could be made by using a dual er plasmid with one promoter generating Fc-Cl and the other promoter generating Fc alone. Monovalent Fc could also be made using bispecific technology where ic amino acids in the hinge region of the Fc are mutated to impart stability of the Fc region (e.g. Knob and hole technology or other stabilizing mutations which drive formation of the monovalent C1).

Albumin Domains In some embodiments, a le Cl-INH fusion protein contains an albumin domain. Albumin is a soluble, monomeric protein which comprises about lf of the blood serum protein. Albumin functions ily as a carrier protein for steroids, fatty acids, and thyroid es and plays a role in stabilizing extracellular ?uid volume.

Albumin has a globular unglycosylated serum protein of molecular weight 66,500. Albumin is synthesized in the liver as preproalbumin which has an N—terminal peptide that is removed before the t protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin.

Albumin is made up of three homologous s (I-III), and each of these is comprised of two subdomains (A and B). The principal regions of ligand binding to human serum albumin are located in cavities in subdomains IIA and IIIA, which are formed mostly of hydrophobic and positively charged residues and exhibit similar chemistry. Human serum albumin has 585 amino acids and a lar mass of 66,500 Da. The amino acids include cysteines, all but one of which are involved in the ion of 17 stabilizing disulfide bonds.

Typically, Albumin has a prolonged serum half-life of 19 days. FcRn controls the long serum half—life of albumin. FcRn is a dual binding receptor that, in addition to albumin, binds IgG, and ts both proteins from intracellular degradation. The C- terminal domain of the albumin molecule has been shown to be important for binding to FcRn. In particular, domain IIIB is shown to be important for binding to FcRn. In some embodiments, lack of domain IIIB or mutations of 464His, 510His, and 535His abolishes FcRn binding.

Typically, Albumin fusion proteins of the invention are monomeric. In some embodiments, this feature may be an age over the dimeric Fc fusion embodiments for the s described above with regard to monomeric Fc fusion embodiments.

In some embodiments, an albumin polypeptide suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the wild-type human serum albumin: MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQ CPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARR HPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCA SLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDR ADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYKTTLEKCCAAADPHECY AKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPI‘LVEVS GSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVN ALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATK EQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASRAALGL (SEQ ID NO: 17).

In some embodiments, an albumin polypeptide suitable for the present invention includes an amino acid sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the D3 domain of wild-type human serum albumin: METPAQLLFLLLLWLPDTTGVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ VSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRV TKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASRAALGL (SEQ ID NO:20).

Linker 0r Spacer A Cl-INH polypeptide or domain may be directly or indirectly linked to an EC domain or an albumin domain. In some embodiments, a suitable Cl-INH fusion n contains a linker or spacer that joins a Cl-INH polypeptide or domain and an EC or albumin domain. An amino acid linker or spacer is lly ed to be ?exible or to interpose a structure, such as an alpha—helix, between the two protein moieties. A linker or spacer can be relatively short, or can be longer. lly, a linker or spacer contains for example 3-100 (e.g., 5-100, 10-100, 20-100 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, -50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20) amino acids in length. In some embodiments, a linker or spacer is equal to or longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. Typically, a longer linker may decrease steric hindrance. In some embodiments, a linker will comprise a mixture of glycine and serine residues. In some embodiments, the linker may additionally se ine, proline, and/or alanine residues. Thus, in some embodiments, the linker comprises between 10-100, 10-90, 10-80, 10-70, 10-60, 10—50, 10-40, 10-30, 10-20, 10-15 amino acids. In some embodiments, the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids. In some embodiments, the linker is not a linker ting of ALEVLFQGP (SEQ ID NO: 37).

As non-limiting examples, s or spacers suitable for the present invention include but are not limited to GGG linker and GGGGSGGGGS ((GGGGS)2 linker SEQ ID NO:27). In some embodiments, the linker comprises the sequence GGG and/or the sequence of SEQ ID NO:27.

Other suitable s include GGAAAAAGGGGGGAP (GAG linker, SEQ ID NO:34); AAAAAGGGGGMGGGGGAAAAAGGGGGG? (GAG2 linker, SEQ ID NO:35); and MGGGGGAAAAAGGGGGMGGGGGAAAAAGGGGGMGGGGGAAAAAGGG GG? (GAG3 linker, SEQ ID NO:36).

Suitable linkers or spacers also include those having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the above exemplary s, e.g., GGG linker, GGGGSGGGGS ((GGGGS)2 linker SEQ ID NO:27), GAG linker (SEQ ID NO:34), GAG2 linker (SEQ ID NO:35), or GAG3 linker (SEQ ID NO:36). Additional linkers suitable for use with some embodiments may be found in US2012/0232021, filed on March 2, 2012, the disclosure of Which is hereby incorporated by reference in its entirety.

Typically, a linker is included that associates the Cl-INH polypeptide or domain with the Fc or albumin domain Without ntially affecting or reducing the ability of the Cl-INH polypeptide or domain to bind to any of its e ligands (e.g., Cls, etc.).

Glycosylation/Glycan Mapping (Pro?le) of C1-INH Proteins According to the present invention, a Cl-INH protein may be conjugated via a glycan residue and/or an amine group. In ular, a Cl—INH protein may be conjugated at a glycan residue such as, for example, a sialic acid residue or a galactose residue. Thus, a Cl-INH protein suitable for conjugation according to the t invention may be characterized with distinct glycan maps, in particular, sialic acid content. In some embodiments, a Cl-INH protein has a glycosylation profile similar to that of plasma-derived . In some embodiments, a Cl-INH protein has a glycosylation profile that is distinct from that of plasma-derived Cl-INH.

Without wishing to be bound by any theory, it is thought that glycan map ing glycan linkage along with the shape and xity of the branch structure may impact in vivo clearance, bioavailability, and/or efficacy.

Typically, a glycan map may be determined by tic digestion and subsequent chromatographic is. Various enzymes may be used for tic digestion including, but not limited to, suitable glycosylases, peptidases (e. g., Endopeptidases, Exopeptidases), proteases, and phosphatases. In some embodiments, a suitable enzyme is alkaline phosphatase. In some embodiments, a le enzyme is neuraminidase. Glycans may be detected by chromatographic analysis. For example, glycans may be detected by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection PAD) or size exclusion High Performance Liquid Chromatography (HPLC). The quantity of glycan represented by each peak on a glycan map may be calculated using a standard curve of glycan according to methods known in the art and disclosed herein.

In some embodiments, Cl-INH proteins may be characterized with a glycan map. The ve amount of glycan corresponding to each peak group may be determined based on the peak group area relative to the corresponding peak group area in a predetermined reference standard. Various reference standards for glycan mapping are known in the art and can be used to ce the t invention. In some embodiments, Cl-INH proteins may be characterized with a glycan map comprising five or fewer peak groups ed from the peak groups indicative of neutral, mono-sialylated, di-sialylated, tri— sialylated or tetra-sialylated Cl-INH protein.

In some embodiments, C1-INH proteins have a glycosylation profile comprising at least one of the following: neutral glycan species, ialylated species, di- sialylated species, alylated species and/or tetra-sialylated species. In some embodiments, Cl-INH proteins have a glycosylation profile comprising l glycan species, mono-sialylated species, di-sialylated species, tri—sialylated species and ialylated species. In some ments, Cl-INH proteins have a glycosylation profile comprising no more than about 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15 %, 10%, or 5% neutral glycan species. In some ments, Cl-INH proteins have a glycosylation profile comprising between about 5% and about 30% neutral glycan species. In some embodiments, Cl-INH proteins have a glycosylation profile comprising between about 5% and about 25% neutral glycan s. In some embodiments, Cl—INH ns have a glycosylation profile comprising between about 10% and about 20% neutral glycan species. In some embodiments, Cl-INH proteins comprises, on average, at least about 80% charged glycans per molecule (e.g., greater than about 85%, 90%, 95% or 99% s per molecule). In some embodiments, Cl-INH proteins have a glycosylation profile comprising between about % and about 30% mono-sialylated s. In some embodiments, C1-INH proteins have a glycosylation profile comprising between about 30% and about 50% di-sialylated species. In some embodiments, C1—INH proteins have a glycosylation profile comprising between about % and about 35% tri-sialylated species. In some embodiments, Cl-INH proteins have a ylation profile comprising between about 5% and about 15% tetra-sialylated species.

In some embodiments, Cl—INH proteins have a glycosylation profile comprising no more than 30% neutral glycan species, n about 20% and about 30% mono—sialylated glycan species, between about 30% and about 40% di-sialylated glycan species, between about 10% and about 20% tri—sialylated glycan species, and between about 5% and about 10% tetra- sialylated glycan species.

In some embodiments, C1-INH proteins have a sialylation e similar to that of plasma-derived C1-INH. In some embodiments, C1-INH proteins have a sialylation profile distinct than that of plasma—derived C1-INH. In some embodiments, C1-INH proteins have a ation profile that renders a half-life similar to or longer than that of plasma- derived Cl-INH. In some embodiments, Cl-INH proteins comprise, on average, at least about 10, 11, 12, 13, or 14 sialylated glycan residues per molecule. In some embodiments, Cl-INH proteins comprise, on average, at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, , 26, 27, 28, or 29 sialylated glycan residues per le. In some embodiments, C1-INH proteins comprise, on average, at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 sialylated glycan residues per molecule.

In some embodiments, C1—INH proteins contain less than about 20%, 15%, %, or 5% of one or more of mannose, (x—galactose, N-glycolylneuraminic acid (NGNA), or oligomannose-type ylation. In some embodiments, C1-INH proteins contain no more than about 20%, 15%, 10%, or 5% of one or more of e, (x—galactose, N- glycolylneuraminic acid (NGNA), or oligomannose-type glycosylation.

In some embodiments, Cl-INH ns have a glycosylation profile that is not immunogenic. In some embodiments, C1-INH proteins have a ylation profile that does not increase serum clearance rate when compared with plasma—derived human Cl-INH.

In some embodiments, Cl-INH proteins have a glycosylation profile that decreases serum clearance rate when ed with plasma-derived human Cl-INH. In some embodiments, C1-INH ns have a glycosylation profile that decreases serum clearance rate when compared with conestat alfa.

Various methods of manipulating the glycosylation profile of ns are known in the art. These s as well as others yet to be discovered are plated by the instant invention. Methods of manipulating the glycosylation profile of Cl-INH proteins and polypeptides of the invention include in vitro, in situ, and in vivo methods. In some embodiments the glycosylation profile of expressed proteins or polypeptides is altered through post-expression chemical cation of the expressed protein or polypeptide. In some embodiments the cell culture conditions are manipulated to achieve sion of proteins having a desired glycosylation profile. These cell culture conditions include l of the production and culture process including length of culture, additives to culture , and/or co—expression of genes to enhance glycosylation. Selection of host cells and specific clones of transfected host cells may also be used to enhance glycosylation. Some methods of ing glycosylation e purification processes to enrich for ns or polypeptides having the desired glycosylation profile.

In some embodiments, cells engineered to express Cl-INH proteins can also be engineered to modify glycosylation, in particular, increase ation of the expressed C1— INH. For example, cells may be engineered to express a logous enzyme in the glycosylation pathway (wild-type or mutated) to e desired glycosylation, e.g., to increase sialylation. In some embodiments, cells may also be engineered to overexpress an endogenous enzyme to achieve desired glycosylation, e.g., to increase sialylation. In some embodiments, cells are engineered to reduce or prevent expression of endogenous enzymes that reduce, inhibit, or degrade sialylation (e.g., with an antisense construct).

The various glycosylation patterns/glycan maps and in particular, sialylation profiles or levels, described herein may be applicable to a Cl-INH domain or polypeptide alone or in a fusion protein context (e.g., a Cl-INH—Fc or Cl-INH-albumin fusion protein).

Cl-INH proteins with glycosylation ns/glycan maps and in particular, sialylation es or levels, described herein may be ated or unconjugated. It is plated that a desired glycosylation pattem/glycan map including a desired sialylation profile or level may extend in vivo half-life of Cl—INH protein. In particular, a desired glycosylation pattern/glycan map including a desired sialylation profile or level, in combination with Fc or albumin fusion, may achieve desired in vivo half-life of Cl—INH protein described in this application even without conjugation. Conjugation (e.g., PEGylation) however further extends in vivo ife of C1-INH ns including those with desired glycosylation pattern or sialylation level.

PEGylation ing to the present invention, a chemical or biological moiety can be conjugated, directly or indirectly, to a Cl-INH protein described herein. In particular, such a moiety is a hylene glycol (PEG) moiety including, but not limited to, mono- or poly- (e. g., 2-4) PEG moieties. As used herein, a process of conjugating a PEG moiety, directly or indirectly, to a protein is referred to as PEGylation. PEGylation can result in increased half— life of Cl—INH, as described herein.

PEGylation can be carried out by any suitable reaction known in the art.

Methods for preparing a PEGylated protein can lly include (a) reacting a polypeptide with polyethylene glycol (such as a reactive ester or de derivative of PEG) under conditions whereby the ptide s ed to one or more PEG groups; and (b) obtaining the reaction product(s). In general, the conditions for the ons can be determined case by case based on known parameters and the desired result.

There are a number of PEG attachment methods available to those skilled in the art and bed in, for example, EP 0 401 384; Malik et al., Exp. Hematol., 20:1028- 1035 (1992); EP 0 154 316; EP 0 401 384; WO 21; and WO 95/34326. For example, the step of PEGylating a eutic molecule described herein can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule.

Target sites Activated PEGs PEG-NHS, PEG-Aldehyde, PEG-p- N . 1 . _ erm1na amino groupt Nitrophenyloxycarbonyl -NH of Lysine PEG-NIIS, PEG-Aldehyde, PEG-p- 2 Nitrophenyloxycarbonyl carboxylic group PEG-NH2 Thiol/cysteine PEG-Maleimide, PEG-Iodoacetamide Glycan/aldehyde (sralic ac1d PEG-Aminoxy, PEG-Hydrazide and terminal galatose) In some embodiments, a PEG moiety for conjugation is an activated PEG. For example, a suitable PEG moiety may include an y functional group. In some embodiments, a suitable PEG moiety may include a hydrazide functional group. In some embodiments, a suitable PEG moiety may include a maleimide or iodoacetamide functional group. In some embodiments, a suitable PEG moiety may include an N-hydroxysuccinimide (NHS) ester. Thus, a PEG moiety may be ated to a Cl-INH protein via an oxime linkage, an amide linkage, a hydrazone linkage, a thioether linkage or other type of linkages.

In some embodiments, a PEG moiety may have linear or branched structures.

For example, a PEG moiety may include 2, 3, 4, or 5 arm branches. A suitable PEG-NHS moiety may include linear PEG—NHS 1K, linear PEG-NHS 2K, linear PEG—NHS 5K, branched PEG-NHS 5K, branched PEG-NHS 20K, or branched PEG-NHS 40K. As a further example, a PEG—aminoxy moiety may include linear or branched PEG-aminoxy 2K, PEG- aminoxy 5K, PEG—aminoxy 5K, PEG-aminoxy 10K, PEG-aminoxy 20K, or PEG-aminoxy In some embodiments, the PEG is conjugated to Cl—INH via one or more amino acid residues of the Cl-INH n. See Figure 3.

In some embodiments, the PEG is conjugated to Cl—INH via one or more galactose es of the Cl-INH protein. In some embodiments, one or more galactose residues of the Cl-INH protein are oxidized before the PEG is conjugated to the galactose In some embodiments, the PEG is conjugated to Cl—INH via one or more sialic acid residues of the Cl-INH protein. In some embodiments one or more of the sialic acid residues of the Cl-INH protein are ed before the PEG is conjugated to the sialic acid residues.

In some embodiments, the PEG is conjugated to oxidized sialic acid via an oxime linkage. In some embodiments, the PEG is conjugated to oxidized sialic acid via a hydrazone linkage.

A Cl-INH n may be PEGylated at various levels ing to the present invention. For example, the molar ratio of PEG to Cl—INH may range between about :1 and 100:1; between about 10:1 and 100:1; between about 15:1 and 100:1; between about 2021 and 100:1; between about 25:1 and 10021; n about 30:1 and 100:1; between about 40:1 and 100:1; between about 50:1 and 100:1; between about 10:1 and 90:1; n about 1021 and 80:1; between about 10:1 and 70:1; between about 10:1 and 60:1; between about :1 and 50:1; between about 10:1 and 40:1; between about 15:1 and 35:1; or between about 2021 and 30: 1. In some embodiments, the molar ratio of PEG to Cl-INH may be at least about 1:1, at least about 5:1, at least about 10:1; at least about 15:1; at least about 20:1; at least about 25: 1; at least about 30:1; at least about 35: 1; at least about 40: 1; at least about 45:1; or at least about 50:1.

In some embodiments, the molar ratio of PEG to sialic acid is at least about 1:1, at least about 1:5, at least about 1:10, at least about 1:15, at least about 1:20, at least about 1:25, at least about 1:30, at least about 1:35, at least about 1:40 at least about 1:45, at least about 1:50. In some embodiments, the molar ratio of PEG to sialic acid is between about 1:1 and about 1:50, between about 1:1 and about 1:45, between about 1:1 and about 1:40, between about 1:1 and about 1:35, between about 1:1 and about 1:30, n about 1:1 and about 1:25, between about 1:1 and about 1:20, between about 1:1 and about 1:15, between about 1:1 and about 1:10, or between about 1:1 and about 1:5.

Polysialic Acid Conjugation Polysialic acid (PSA), also referred to as colominic acid (CA), is a naturally occurring ccharide. It is a lymer of N—acetylneuraminic acid with 0t(2—>8) ketosidic linkage and contains Vicinal diol groups at its ducing end. It is negatively charged and a natural constituent of the human body.

PSAs consist of polymers (generally homopolymers) of ylneuraminic acid. The secondary amino group normally bears an acetyl group, but it may instead bear a glycolyl group. Possible substituents on the hydroxyl groups include acetyl, lactyl, ethyl, sulfate, and phosphate groups.

PSAs and modified PSAs (mPSAs) generally comprise linear polymers consisting essentially of ylneuraminic acid moieties linked by 2,8- or 2,9- glycosidic linkages or combinations of these (e.g. ating 2,8- and 2,9- linkages). In some embodiments, the glycosidic linkages of PSAs and mPSAs, are oc-2,8. Such PSAs and mPSAs are derived from colominic acids. Typical PSAs and mPSAs comprise at least 2, preferably at least 5, more preferably at least 10 and most preferably at least 20 N-acetylneuraminic acid es. Thus, they may comprise from 2 to 300 N—acetylneuraminic acid moieties, preferably from 5 to 200 N—acetylneuraminic acid moieties, or most preferably from 10 to 100 N-acetylneuraminic acid moieties. PSAs and CAs preferably are essentially free of sugar moieties other than N-acetylneuraminic acid. In some embodiments, PSAs comprise at least 90%, at least 95% and or at least 98% N-acetylneuraminic acid moieties.

Where PSAs comprise es other than N-acetylneuraminic acid (as, for example in mPSAs) these are preferably located at one or both of the ends of the polymer chain. Such "other" moieties may, for example, be moieties derived from terminal N- neuraminic acid moieties by oxidation or reduction.

For example, terminal N-acetylneuraminic acid unit is converted to an aldehyde group by reaction with sodium periodate. Additionally, terminal N—acetylneuraminic acid unit is subjected to reduction to reductively open the ring at the reducing terminal N-acetylneuraminic acid unit, whereby a vicinal diol group is formed, followed by oxidation to convert the vicinal diol group to an aldehyde group.

Different PSA derivatives can be prepared from oxidized PSA containing a single de group at the ducing end. The preparation of aminooxy PSA is bed, for example, in /166622, the contents of which are hereby incorporated by reference. PSA-NH2 containing a al amino group can be prepared by reductive amination with NH4Cl and PSA-SH containing a terminal sulfhydryl group by reaction of PSA-NH2 with othiolane (Traut's reagent), both procedures are described in US 7,645,860 B2. PSA hydrazine can be prepared by reaction of oxidized PSA with hydrazine according to US 7,875,708 B2. PSA hydrazide can be prepared by reaction of oxidized PSA with adipic acid azide ( nic acids (a sub-class of PSAs) are lymers of N- acetylneuraminic acid (NANA) with 0t (2—>8) ketosidic linkage, and are produced, inter alia, by particular strains of ichia coli possessing K1 antigen. Colominic acids have many physiological functions. They are important as a raw material for drugs and cosmetics.

As used herein, "sialic acid moieties" includes sialic acid monomers or polymers ("polysaccharides") which are soluble in an aqueous solution or suspension and have little or no negative impact, such as side effects, to mammals upon administration of the PSA-blood coagulation protein conjugate in a pharmaceutically ive amount. The polymers are characterized, in one aspect, as having 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid units. In certain aspects, different sialic acid units are combined in a chain.

In some embodiments, the sialic acid portion of the polysaccharide compound is highly hydrophilic, and in another embodiment the entire compound is highly hydrophilic.

Hydrophilicity is conferred primarily by the pendant carboxyl groups of the sialic acid units, as well as the hydroxyl groups. The ride unit may contain other functional groups, such as, amine, hydroxyl or sulphate groups, or combinations thereof. These groups may be present on naturally-occurring ride compounds, or uced into derivative polysaccharide compounds.

The naturally occurring polymer PSA is ble as a polydisperse ation showing a broad size distribution (e.g. Sigma C-5762) and high polydispersity (PD). Because the polysaccharides are usually ed in bacteria carrying the inherent risk of copurifying endotoxins, the purification of long sialic acid polymer chains may raise the probability of increased endotoxin content. Short PSA molecules with 1-4 sialic acid units can also be synthetically prepared (Kang SH et al., Chem Commun. 2000;227—8; Ress DK and Linhardt RJ, Current Organic Synthesis. 2004;1131-46), thus minimizing the risk of high endotoxin levels. However PSA preparations with a narrow size bution and low polydispersity, which are also endotoxin-free, can now be manufactured. Polysaccharide compounds of particular use for the present disclosure are, in one , those produced by bacteria. Some of these naturally-occurring polysaccharides are known as glycolipids. In some embodiments, the polysaccharide compounds are substantially free of terminal galactose units.

In some embodiments, the PSA is conjugated to Cl—INH via one or more sialic acid residues of the Cl-INH protein. In some embodiments one or more of the sialic acid residues of the C1-INH protein are oxidized before the PSA is conjugated to the sialic acid residues.

In some embodiments, the PSA is conjugated to oxidized sialic acid via an oxime linkage. In some embodiments, the PSA is conjugated to oxidized sialic acid via a hydrazone linkage.

A Cl-INH protein may be conjugated with PSA at various levels according to the present invention. For example, the molar ratio of PSA to Cl-INH may range between about 5:1 and 100:1; between about 10:1 and 100:1; between about 15:1 and 100:1; n about 20:1 and 100:1; between about 25:1 and 100:1; between about 30:1 and 100:1; between about 40:1 and 100:1; n about 50:1 and 100:1; between about 10:1 and 90:1; between about 10:1 and 80:1; between about 10:1 and 70:1; between about 10:1 and 60:1; between about 10:1 and 50:1; between about 10:1 and 40:1; between about 15:1 and 35:1; or between about 20:1 and 30:1. In some embodiments, the molar ratio of PSA to Cl-INH may be at least about 1:1, at least about 5:1, at least about 10:1; at least about 15:1; at least about 20:1; at least about 25:1; at least about 30:1; at least about 35:1; at least about 40:1; at least about 45:1; or at least about 50:1.

In some embodiments, the molar ratio of PSA to sialic acid is at least about 1:1, at least about 125, at least about 1:10, at least about 1:15, at least about 1:20, at least about 1:25, at least about 1:30, at least about 1:35, at least about 1:40 at least about 1:45, at least about 1:50. In some embodiments, the molar ratio of PSA to sialic acid is between about 1:1 and about 1:50, between about 1:1 and about 1:45, between about 1:1 and about 1:40, n about 1:1 and about 1:35, between about 1:1 and about 1:30, between about 1:1 and about 1:25, between about 1:1 and about 1:20, between about 1:1 and about 1:15, between about 1:1 and about 1:10, or between about 1:1 and about 1:5.

Extended half-life According to the present invention, conjugation (e.g., PEGylation or PSA conjugated) extends in Vivo half-life of . Typically, conjugated (e.g., PEGylated or PSA conjugated) C1-INH has a half-life longer than the unconjugated (e.g., un-PEGylated or non-PSA conjugated) C1-INH. In some embodiments, conjugated (e.g., PEGylated or PSA ated) C1-INH has a half-life comparable to or greater than a plasma-derived human C1—INH protein. In some embodiments, the half-life of the conjugated (e.g., PEGylated or PSA conjugated) C1-INH is in the range of about 80%-500%, 0%, 100%-500%, 110%-500%, 120%-500%, 80%-400%, 90%-300%, 100%-300%, 50%, 100%-200%, or 100%-150% of the half-life of the plasma-derived C1-INH protein.

In some embodiments, the conjugated (e.g., PEGylated or PSA conjugated) C1-INH protein has a half—life of at least about 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 hours. In some embodiments, conjugated (e.g., PEGylated or PSA ated) Cl—INH has an in vivo half—life of or greater than about 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, 10 days, 11 days, 12 days, 13 days, or 14, days. In some embodiments, a ated (e.g., PEGylated or PSA conjugated) C1- INH protein has an in viva half-life ranging between about 0.5 and 14 days, 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, n 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days and 4 days, between 2 day and 3 days, between 2.5 days and 10 days, between 2.5 days and 9 days, between 2.5 days and 8 days, between 2.5 days and 7 days, between 2.5 days and 6 days, between 2.5 days and 5 days, between 2.5 days and 4 days, between 3 days and 10 days, n 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, between 3 days and 6 days, between 3 days and 5 days, between 3 days and 4 days, between 3.5 days and 10 days, between 3.5 days and 9 days, between 3.5 days and 8 days, between 3.5 days and 7 days, between 3.5 days and 6 days, between 3.5 days and 5 days, between 3.5 days and 4 days, between 4 days and 10 days, between 4 days and 9 days, between 4 days and 8 days, between 4 days and 7 days, between 4 days and 6 days, between 4 days and 5 days, between 4.5 days and 10 days, between 4.5 days and 9 days, between 4.5 days and 8 days, between 4.5 days and 7 days, between 4.5 days and 6 days, n 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between .5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, n 7 days and 10 days, between 8 days and 10 days, between 9 days and 10 days, between 10 days and 11 days, between 11 days and 12 days, between 12 days and 13 days, between 13 days and 14 days.

Pharmaceutical compositions The present invention further provides a ceutical composition containing a conjugated Cl-INH described herein and a physiologically able carrier.

The carrier and conjugated Cl-INH protein are typically e and formulated to suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are not d to water, salt solutions (e.g., NaCl), , buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, ium te, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc, as well as combinations thereof. The ceutical preparations can, if desired, be mixed with auxiliary agents (e. g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for cing osmotic pressure, buffers, coloring, ?avoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

A le pharmaceutical composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. A ition can be a liquid solution, suspension, emulsion, , pill, capsule, sustained release formulation, or powder. A composition can also be formulated as a suppository, with traditional binders and rs such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, ium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.

A pharmaceutical composition or medicament can be formulated in accordance with the routine ures as a pharmaceutical composition adapted for administration to human beings. For e, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed er in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette ting the ty of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

A conjugated Cl-INH described herein can be ated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino l, histidine, procaine, etc.

A preferred formulation comprises 50 mM NaPO4 (pH 7.2), 50 mM Sorbitol, and 150 mM Glycine. The formulation may be liquid, or may be lyophilized and reconstituted prior to stration.

Routes ofAdministration A conjugated Cl-INH described herein (or a ition or ment containing a conjugated Cl-INH described herein) is administered by any appropriate route.

In some embodiments, a conjugated Cl-INH or a pharmaceutical composition containing the same is administered systemically. Systemic administration may be intravenous, intradermal, ranial, hecal, inhalation, transdermal (topical), cular, intramuscular, subcutaneous, intramuscular, oral, and/or transmucosal administration. In some embodiments, a conjugated Cl-INH or a pharmaceutical composition containing the same is administered subcutaneously. As used herein, the term "subcutaneous ", is defined as a layer of loose, irregular tive tissue immediately beneath the skin. For e, the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, the thigh region, abdominal region, gluteal region, or scapular region. In some embodiments, a conjugated Cl-INH or a pharmaceutical composition containing the same is administered intravenously. In some embodiments, a conjugated Cl— INH or a pharmaceutical composition containing the same is administered . In some embodiments, a conjugated Cl-INH or a pharmaceutical composition containing the same is administered intracranially. In some embodiments, a conjugated Cl—INH or a pharmaceutical composition containing the same is administered intrathecally. More than one route can be used concurrently, if desired.

In some embodiments, a conjugated Cl-INH or a ceutical composition containing the same is administered to the subject by aneous (i.e., beneath the skin) administration. For such purposes, the formulation may be injected using a syringe.

However, other devices for administration of the formulation are available such as injection devices (e.g., the Inject-easeTM and Genj ectTM s); injector pens (such as the GenPenTM); needleless s (e.g., MediJectorTM and BioJectorTM); and subcutaneous patch delivery systems. Thus, the present invention further es a kit containing a pharmaceutical composition comprising conjugated Cl-INH (e.g., in a liquid and lyophilized form) and an injection device such as a syringe. In some embodiments, the syringe is preloaded with the pharmaceutical composition comprising conjugated Cl-INH. Wherein the ceutical composition is lyophilized, the kit may further include a reconstitution buffer.

The present invention contemplates single as well as le administrations of a therapeutically effective amount of a conjugated C1-INH or a pharmaceutical composition containing the same described herein. A conjugated Cl-INH or a ceutical composition containing the same can be administered at regular intervals, depending on the nature, severity and extent of the subject’s condition (e.g., hereditary angioedema). In some embodiments, a therapeutically effective amount of a conjugated C1- INH or a pharmaceutical composition containing the same may be administered periodically at regular intervals (e.g., once every year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (once every month), biweekly (once every two weeks), weekly, daily or continuously).

In some embodiments, administration results only in a localized effect in an individual, while in other embodiments, administration results in effects throughout multiple portions of an individual, for example, systemic effects. Typically, stration results in delivery of a conjugated Cl-INH to one or more target tissues. In some ments, the conjugated Cl-INH is delivered to one or more target tissues including, but not d to, heart, brain, skin, blood, spinal cord, ed muscle (e. g., skeletal muscle), smooth muscle, kidney, liver, lung, and/or spleen. In some ments, the conjugated Cl-INH is red to the heart. In some embodiments, the conjugated Cl-INH is delivered to the central nervous system, particularly the brain and/or spinal cord. In some embodiments, the conjugated Cl-INH is delivered to triceps, tibialis anterior, soleus, cnemius, biceps, trapezius, deltoids, quadriceps, and/or diaphragm.

Dosage Forms and Dosing Regimen In some embodiments, a composition is administered in a therapeutically effective amount and/or according to a dosing regimen that is correlated with a ular desired outcome (e. g., with prophylaxis of a complement-mediated chronic e, such as HAE).

Particular doses or amounts to be administered in ance with the present invention may vary, for example, depending on the nature and/or extent of the desired outcome, on particulars of route and/or timing of administration, and/or on one or more characteristics (e.g., weight, age, personal history, c characteristic, lifestyle parameter, severity of cardiac defect and/or level of risk of cardiac , etc., or combinations f).

Such doses or amounts can be determined by those of ordinary skill. In some embodiments, an appropriate dose or amount is determined in accordance with rd clinical techniques.

Alternatively or additionally, in some embodiments, an appropriate dose or amount is determined through use of one or more in vitro or in viva assays to help identify desirable or optimal dosage ranges or amounts to be administered.

In s embodiments, a conjugated Cl-INH is administered at a eutically effective amount. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., prophylaxis, ng, modulating, , preventing and/or ameliorating the underlying disease or condition). Generally, the amount of a eutic agent (e.g., a conjugated Cl-INH) stered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate s depending on these and other related factors. In addition, both objective and tive assays may optionally be employed to identify optimal dosage ranges. In some particular embodiments, appropriate doses or amounts to be administered may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In some embodiments, a composition is provided as a ceutical formulation. In some embodiments, a pharmaceutical formulation is or comprises a unit dose amount for administration in accordance with a closing regimen correlated with achievement of the reduced incidence or risk of an HAE attack.

In some embodiments, a formulation comprising a conjugated Cl-INH described herein administered as a single dose. In some embodiments, a ation comprising a conjugated Cl-INH described herein is administered at regular intervals.

Administration at an val," as used herein, indicates that the therapeutically ive amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In some embodiments, a formulation comprising a conjugated Cl-INH described herein is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice , thrice weekly, daily, twice daily, or every six hours. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual.

A therapeutically effective amount is commonly stered in a dosing regimen that may comprise multiple unit doses. For any particular eutic protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical . Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific Cl-INH employed; the duration of the treatment; and like factors as is well known in the medical arts.

As used herein, the term thly" means stration once per two months (126., once every two months); the term "monthly" means stration once per month; the term "triweekly" means administration once per three weeks (i.e., once every three weeks); the term "biweekly" means administration once per two weeks (i.€., once every two weeks); the term "weekly" means administration once per week; and the term "daily" means administration once per day.

In some embodiments, a ation sing a conjugated Cl-INH described herein is administered at regular intervals indefinitely. In some embodiments, a formulation comprising a conjugated Cl-INH described herein is administered at regular intervals for a defined period.

It is to be r understood that for any particular subject, ic dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the enzyme ement therapy and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the d invention.

Combination Therapy In some embodiments, a conjugated Cl-INH is administered in combination with one or more known therapeutic agents (e.g., corticosteroids) currently used for treatment of a complement-mediated e. In some embodiments, the known therapeutic agent(s) is/are administered according to its rd or approved dosing regimen and/or schedule. In some embodiments, the known therapeutic agent(s) is/are administered according to a n that is d as compared with its standard or approved dosing regimen and/or schedule. In some embodiments, such an altered regimen differs from the standard or approved dosing regimen in that one or more unit doses is altered (e.g., reduced or increased) in amount, and/or in that dosing is altered in frequency (e.g., in that one or more intervals n unit doses is expanded, resulting in lower frequency, or is reduced, resulting in higher frequency).

Complement-mediated Disorders Conjugated Cl-INH and pharmaceutical composition containing the same may be used to treat HAE and various other ment—mediated disorders.

In some embodiments, the ated ns provided by the invention are suitable for acute attacks associated with complement-mediated disorders, e.g., NMOSD AMR, and HAE events. These attacks may be long or short. In some embodiments, the disease or disorder is chronic. In some embodiments the compositions and methods of the ion are used prophylactically. Exemplary complement-mediated disease that may be treated using the compositions and s disclosed herein include, but are not limited to, hereditary angioedema, antibody mediated rejection, neuromyelitis optica spectrum disorders, traumatic brain injury, spinal cord , ischemic brain injury, burn injury, toxic epidermal necrolysis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, stroke, chronic inflammatory demyelinating uropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

EXAMPLES Other features, s, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the es, while indicating embodiments of the t invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples.

Example 1. PEGylation 0f Cl-INH This example illustrates exemplary methods suitable for PEGylation of Cl- INH proteins. Three different PEGylation strategies were explored. Exemplary PEGylation scheme are shown in Figures 3-5. These were conjugation of PEG to sialic acid residues (sialic acid mediated [SAM] chemistry), conjugation of PEG to galactose acid residues (galactose ed [GAM] chemistry), and amine mediated conjugation of PEG.

Aminoxy-PEGs were utilized in order to form a more stable oxime linkage.

PEGylation was performed utilizing ques developed based on methods described in Park et al., Carbohydrate-Mediated Polyethylene Glycol ation of TSH Improves Its Pharmacological Properties. Endocrinology, March 2013, 154(3):1373—1383. d sialic acid residues on a glycosylated protein typically result in increased half-life compared to a protein with fewer or no sialic acid residues while terminal ose residues on carbohydrate chains are known to cause receptor mediated clearance and decrease the serum half-life of proteins. Accordingly, l efforts focused on GAM PEG conjugation in order to block receptor ed clearance of C1 INH. While all three approaches appeared to be promising, amine and SAM PEGylation were singly found to yield the greatest degree of Cl-INH PEGylation with minimal and acceptable loss in y. GAM PEGylation was less efficient and more heterogeneous in comparison.

Initial in vivo PK study was conducted to evaluate ted Cl-INH.

Specifically, SAM 5 KDa and 40 KDa PEGylated Cl-INH was compared with amino PEGylated Cl-INH in a rat PK study. See Figure 6, panels A-C. PEGylated Cl-INH was quantified using an antigen assaying using a Cl-INH to prepare the standard curve. The samples were also analyzed by Western blot to check for potential degradation. Doses of 1 mg/kg IV and 3 mg/kg were in the range of Cinryze® in humans (2—3 mg/kg). These studies demonstrated that the PEGylated proteins had a 3-4 fold increase in half-life, likely due to a decrease in clearance.

Further pharmacokinetic studies were performed with Cl-INH-PEG using 1 mg/kg intravenous administration to male SD rats. These data are presented in Table 1 below.

C1JNH Ff.Gv??? ParsA-gggg‘gggxgx; Sisal»: Amid Mediates: {SAM} u"~.........=..."""~..:...........................,......................................................................r.............‘..........................

V v . . . ‘i‘srmér a: f};- 3 "*E‘Et‘} is Eéxzear, W fr‘EtZ} is Emma 35:9; Table l. Pharmacokinetic parameters of Cl-INH-PEG intravenously stered to male Sprague Dawley (SD) rats.

In addition, in NHP studies, subcutaneous bioavailability was observed to be about 30-40%, which was an unexpected improvement over an unconjugated recombinant Cl-INH n.

Therefore, PEGylated Cl-INH appears to have increased half-life and sufficient bioavailability suitable for eutic use.

Example 2. Exemplary PEGylation Protocols Process A Purified Cl—INH was dialyzed into 100 mM sodium e at pH 5.6.

Periodate oxidation was carried out for 30 minutes at 40C. The reaction was quenched with glycerol for 15 minutes at 4°C. The oxidized Cl-INH was dialyzed into acetate buffer. The material was then PEGylated overnight at 40C, followed by a glycine . Free PEG was removed by anion exchange. An exemplary tic of Process A is provided in Figure 7.

Cl-INH-PEG 40 KDa prepared by Process A, was further ed using the following method.

About 1 mg of 40 KDa PEG amine conjugated to Cl—INH was diluted 20 fold with sample dilution buffer (5 mM NaPO4 at pH 7.00). The resulting on exhibited a conductivity of 0.716 mS/cm. The sample was loaded onto a 10 mL GigaCap Q (650) column XK16. A ?ow rate was 150 cm/h for the entire process. The column was washed extensively with sample dilution buffer and the protein was eluted with a 10 column volume gradient to 500 mM NaCl. 2 mL fractions were collected and the samples analyzed by SDS- PAGE. The chromatography results are depicted in Figure 11. Peak ons were then pooled and dialized into formulation buffer (50 mM Phosphate (pH=7.l), 150 mM Glycine, 50 mM Sorbitol), concentrated to 21.0 mg/ml, and quantitated by 280 nm absorbance (Nano— drop).

A similar purification was performed on a Cl-INH—PEG 20 KDa preparation, depicted in Figure 12 and a Cl-INH-PEG 5 KDa preparation, depicted in Figure 13.

Quantitation of all of the samples was performed on a rop instrument using the extinction coefficient and molecular weight derived from the protein’s amino acid sequence. The results are shown below in Table 2: -Conc. Total n Total Protein at Start-0 (m_ ml) (ml) (m_) (m_) Recover C1-INH- 40 C1-INH- 20 kDa PEG C1-INH - 5 kDa 2.2 0.15 Table 2: Quantitation of Cl-INH PEGylation process samples.

Process B Purified Cl-INH was exchanged into 100 mM sodium acetate at pH 5.6 via TFF buffer exchange. Periodate oxidation was d out for 30 minutes at room temperature. Periodate was provided at 40x molar excess. Up to 4 mg/mL Cl-INH was present in the reaction. The reaction was quenched with glycerol for 15 s at room temperature. The material was then PEGylated overnight at room temperature. PEG was provided at 100x molar . Up to 2 mg/mL Cl-INH was present in the reaction. Free PEG was d by TFF buffer exchange. An ary schematic of Process B is provided in Figure 8.

Other exemplary PEGylation protocols suitable for PEGylating C1—INH are summarized in Figures 9A-E.

SAM Process—PEG SK In this process, about 200 mL of octyl load material (~ 0.9 mg/ml Cl—INH in Tris/ammonium sulphate solution) was buffer exchanged into 100 mM sodium acetate, pH5.6 using Pellicon XL, Biomax, 30kDa (PES) TFF cassette with 10x diavolume ge. 40 uM C1-INH (3.7 mg/ml) was treated with 1.6 mM sodium periodate (40x) for 30 minutes at room temperature with gentle stirring (50 ml reaction, in 100 mM sodium acetate, pH 5.6).

The reaction was quenched with 1.5 % glycerol for 15 s at room temp. 21.6 uM Cl-INH (2mg/ml) was d with 2.16 mM 5kDa—PEG (100x) gently stirring overnight at room temp (92.5 ml reaction, in 100mM sodium acetate, pH 5.6).

The on was then quenched with 2.16 mM glycine (100x) for 1 hour at room temperature.

TFF diafiltration removal of free PEG was done using a Pellicon XL, Biomax 100kDa MWCO (PES) TFF cassette with 10x diavolume exchange into 50 mM sodium phosphate, 150 mM glycine, and 50 mM sorbitol, at pH 7.1. The product was then filter sterilized using a .22 uM, PES, Millipore steri?ip filter. The IC50 of the PEGylated samples are shown in Figure 10, panels A and B, and in Figure 20, panels A—B.

The yield after each process step is presented below in Table 3: concentration Ste D (m ml) Octyl load .93 TFF into acetate 4.2 Oxidation 3.6 PEG lation 2 TFF to stora_e buffer 8.25 82.5 m_, sterile filtration Table 3: Octyl load PEGylation step .

SAM Process—PEG linear 2K, 5K, branched 5K, 10K, 20K, 40K The SAM process was also used to e Cl-INH—PEG with the following kinds of PEG: linear 2K, linear 5K, branched 5K, branched 10K, branched 20K, and branched 40K.

Cl-INH PEGylated with PEG 2K, 5K and 10 K were purified with Amicon centrifugal filter (cut-off 30K). Cl—INH ted with PEG-aminoxy 20K or 40K was purified by AKTA system for free PEG removal. Characterization of the Cl-INH is shown in Figure 18, panels A-E. Cl—INH-PEG produced by the SAM process was assayed for purity and potency, and PK was evaluated in rat models. These data are presented in Figure 19, panels A-C. onal characterization and IC50 values of the PEGylated samples are shown in Figure 24, panels A and B.

The SAM PEGylation conditions for C1-INH—PEG generation is shown in Table 4 below.

NaIO4 equivalent Branched Branched Branched ——_—— Table 4. SAM PEGylation conditions for -PEG tion.

PEGylation via Amine Coupling Process C1-INH-PEG was also prepared with an amine coupling process. A schematic representation of an exemplary PEGylation via amino coupling s is ed in Figure Cl—INH PEGylated with PEGlK, linear 5K and branched 5K were purified by Amicon centrifugal filter (cut-off 30K). A barium—iodine stain was used to detect free PEG for PEGSK moieties, and RP—HPLC was utilized to detect free PEGlK and 2K. C1-INH PEGylated with NHS-PEG20K and 40K were purified by the AKTA pure chromatography system. Characterization of the PEGylated Cl-INH is shown in Figure 22, panels A—D.

-PEG ed by the amine coupling process was assayed for purity, potency, and PK was evaluated in a rat model. These data are presented in Figure 23, panels - The PEGylation conditions for Cl-INH-PEG generation via the amine coupling process is shown in Table 5 below.

Protein conc. PEG Temp. Time PEGMW (mm "M ———-—- ———-—- Branched 5K——-_- Branched * * Branched Table 5. PEGylation conditions for Cl-INH-PEG generation via the amine coupling process.* PEGylation with 100 x PEG20K had a low conversion ratio and was reprocessed with r 40X PEG20K.

Example 3: Non-Human Primate PK Study of IV Administered PEGylated C1-INH Non-human primates (NHP) (cynomolgus monkeys) were divided into two groups and intravenously dosed with recombinant human Cl-INH (rhCl-INH) at 30 mg/kg or PEGylated rhCl-INH at 5 mg/kg. Exemplary results of the study are summarized in Figure 14 and Table 6.

In NHP, PEGylated rhCl-INH displayed 6-fold lower clearance and 3-fold longer terminal ife compared to NH. A similar trend was also observed in rat studies, which showed a 4—fold decrease in clearance and a 4-fold increase in half-life.

Dose CL VZ T1/2 Table 6: NHP PK Study of PEGylated rhCl INH v. rhC1 INH results aone of the three monkeys in the study showed increased elimination rate after 408 hr and was not included in PK ation In?uence ofPEG Load 0n PK 0fNHP Administered IV C1-INH Further PK studies were conducted with NHP. NHP received IV administered -PEG at 5x, 10x, 20x and 40x loads. Exemplary results are shown in Figure 15.

Example 4: NHP IV v. SC PK of PEGylated Cl-INH NHP were divided into two groups and intravenously dosed with PEGylated Cl-INH at 5 mg/kg or subcutaneously (SC) dosed with PEGylated C1-INH at 10 mg/kg. The s of the study are summarized in Figure 16 and Table 7. Functional activity (SA = 4.8 U/mg) of the PEGylated C1-INH was ined over the time course of the study.

Significantly and unexpectedly, in NHP, PEGylated C1-INH exhibited a bioavailability of 85%, with half-life comparable to that of IV administration. The preclinical data collected thus far supports potential for once weekly or even less frequent dosing.

Dose CmaX TmaX AUCinf F (mg/kg) (Hg/mL) (hr) (Hg/mL-hl‘) (%) SC 10 3 94 72 25599 85 Table 7: IV V. SC dosing of PEGylated rhCl-INH in NHP F = 58% for hrCl-inh in NHP following SC dosing Example 5: Oxidation/titration to test minimal PEG to maximized PK profile The DT-1215 titer assay used was an ELISA based method which captures PEG-rCl—INH protein from serum samples with an anti-PEG antibody. The protein was then ed with a labeled anti—Cl-INH protein. PEG—rCl-INH was used to prepare the standard curve. Figures 17 depicts the results of a DT-1215 titer analysis and sample specific activity.

Tables 8 and 9 provide further data. ive specific group lot sample potentcy ty (vs parent) (U/mg) [TJUOUJCD KHR3 2.5x 1.52 92.11 6.54 KHR3 5X 1.61 86.96 6.17 KHR3 10X 1.61 86.96 6.17 KHR3 20X 1.77 79.10 5.62 KHR2 40X 2.05 68.29 4.85 C36R14- parent 1.4 100 7.1 Table 8: The change in ic activity observed at different levels of periodate treatment.

As seen in Table 9 (below) the change in the periodate level ed in a different ratio of PEG to C1 INH. % rCl PEG/rCl Sample Activity mol/mol C1-INH Table 9: The half-life achieved at different levels of PEG compared to the unconjugated C1 e 6: Physical Characterization of PEGylated Cl-INH Purity of PEGylated preparation was analyzed using SEC and SEC-MALs.

CD spectra of 0.1 mg/ml PEG-Cl-INH proteins were measured at 25 0C. CD data were processed by AVIV and CDNN softwares. No significant change is observed when proteins are PEGylated based on the CD a and secondary structure analysis. ing to the C-terminal crystal structure of Cl—INH (ZOAY), 27 % l and 30% beta-sheet.

ASKAmine B40K -D40K SAM ESK SAM C1-INH PEG-Cl- AMINE - PEG-Cl- PEG-C1- INH PEG-Cl-INH INH INH Helix 29.60% 32.20% Antlparalle____ 9.10% Parallel 8.90% Beta-Turn 17.10% Rndm.Coil 35.50% 33.80% Total Sum 101.60% % 100.50% 103.60% 103.60% 101.00% Table 10: Data demonstrates that PEGylation Does Not Alter Cl-INH Secondary Structure.

The melting temperature (Tm) of PEGylated C1-INH was measured by nanoDSF. PEGylation was found not to dramatically change C1-INH thermal stability. The Tm of 40 KDa amino-PEGylated C-INH was measured to be 20C higher than the other conjugates tested. The data are presented in Table 11.

. . In?ection Point #1 Sample ID Sample Description Sample Lot# for Ratio (Unfolding) A1 5K Amine PEG Cl-INH ‘ CS19875 57.7OC B 1 40K Amine PEG C l-INH 6 59.5OC 1 _ 570°C 1 40K SAM PEG Cl-INH CSl9878 57.40C E 5K SAM PEG C1-INH 5K-SAM-C1-INH-KH-R1 565°C C1-INH1 C1 INH SHIRE DT615 57.30C Table 11: Tm analysis of PEGylated Cl—INH. r magnetic resonance (NMR) was used to characterize the PEGylation level. PEGylation on amine was low, about 3 PEG moeities per Cl-INH. Sialic acid can be heavily PEGylated to reach saturation for the 5K PEG reactant. 40K PEGylated on Sialic acid reaches ~ 9 PEGs per le. PEGylated level was quantified at different periodate concentration. The data is presented in Table 12.

Sample name PEG/Cl-INH Ratio -INH MW"< Comments A 3.2 101 5K Amine PEG B 3.2 213 40K Amine PEG C 28.3 226.5 5K SAM PEG D 9.3 457 40K SAM PEG R1 25.3 211.5 5K SAM PEG R2 21.2 191 5K SAM PEG R3A 2.5 97.5 2.5X Periodate R3B 5 110 5X Periodate R3C 11.5 142.5 10X Periodate R3D 19.5 182.5 20X Periodate R4 21.2 191 TFE s Table 12: NMR characterization of PEGylated Cl—INH preparations.

Example 7: Characterization of C1-INH-PSA Cl-INH was conjugated with polysialic acid (PSA) via the Sialic acid ed (SAM) process. Characterization of the Cl-INH-PSA produced by the SAM process was assayed for purity and potency. These data are presented in Figure 24, panels A and B. The data te that while free PSA does not interfere with the potency assay itself, Cl-INH potency was reduced by ~4-7 fold under the PSA2ClINH conditions tested here.

PK studies were med in rat using C1-INH-PSA, Cl-INH-PEG, and Cinryze®-PEG. The data are presented in Figure 25, panels A-C.

EQUIVALENTS AND SCOPE Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific ments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims (81)

1. A composition comprising a conjugated C1 esterase inhibitor (C1-INH) comprising: a C1-INH protein comprising a polyethylene glycol (PEG) moiety; and wherein the polyethylene glycol (PEG) moiety is covalently linked to the C1-INH protein via an oxime linkage, wherein the oxime linkage is between the PEG moiety and a glycan residue of C1-INH, wherein the glycan e is a sialic acid residue, and wherein the conjugated C1-INH has an extended in vivo ife compared to unconjugated C1-INH.

2. The composition of claim 1, wherein the C1-INH protein is recombinantly produced or plasma derived.

3. The composition of claim 1 or claim 2, wherein the C1-INH protein comprises a C1- INH domain having an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% cal to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:37, or SEQ ID NO:38.

4. The composition of any one of claims 1-3, wherein the C1-INH protein is a fusion protein.

5. The ition of claim 3, wherein the fusion n comprises an Fc domain directly or indirectly fused to a C1-INH domain.

6. The composition of claim 5, n the Fc domain is derived from IgG1.

7. The composition of claim 5 or claim 6, wherein the Fc domain comprises amino acid substitutions ponding to L234A and L235A according to EU numbering.

8. The composition of claim 4, wherein the fusion protein comprises an albumin domain directly or indirectly fused to a C1-INH domain.

9. The composition of any one of claims 1-8, wherein the C1-INH protein has a glycosylation e sing no more than about 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15 %, 10%, or 5% neutral glycan species, prior to PEGylation.

10. The composition of any one of claims 1-9, wherein the C1-INH protein has a glycosylation profile comprising between about 5% and about 25% neutral glycan species, prior to PEGylation.

11. The composition of any one of claims 1-10, wherein the C1-INH protein comprises, on average, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% charged glycans per molecule.

12. The composition of any one of claims 1-11, wherein the C1-INH protein contains less than about 20%, 15%, 10%, or 5% of one or more of mannose, a–galactose, NGNA, or oligomannose-type glycosylation, prior to PEGylation.

13. The composition of any one of claims 1-12, wherein, prior to PEGylation, the C1-INH protein has a glycosylation profile comprising one or more of the following: between about 5% and about 30% neutral glycan species; between about 10% and about 30% mono-sialylated glycan s; between about 30% and about 50% di-sialylated glycan species; between about 15% and about 35% tri-sialylated glycan species; or between about 5% and about 15% tetra-sialylated glycan species.

14. The composition of any one of claims 1-13, n, prior to PEGylation, the C1-INH protein has a glycosylation profile comprising: no more than 30% l glycan species; between about 20% and about 30% mono-sialylated glycan species; between about 30% and about 40% di-sialylated glycan species; between about 10% and about 20% alylated glycan species; and between about 5% and about 10% tetra-sialylated glycan s.

15. The composition of any one of claims 1-14, n the C1-INH protein comprises, on average, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 sialylated glycan residues per molecule.

16. The composition of any one of claims 1-15, wherein the C1-INH protein comprises, on average, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mole sialic acid per mole of protein

17. The composition of any one of claims 1-16, n the PEG has a molecular weight n about 1 KDa and 50 KDa, n about 1 KDa and 40 KDa, between about 5 KDa and 40 KDa, between about 1 KDa and 30 KDa, between about 1 KDa and 25 KDa, between about 1 KDa and 20 KDa, between about 1 KDa and 15 KDa, between about 1 KDa and 10 KDa, or n about 1 KDa and 5 KDa.

18. The composition of any one of claims 1-17, wherein the PEG has a molecular weight of about 1 KDa, 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa, 45 KDa, or 50 KDa.

19. The composition of any one of claims 1-18, wherein the conjugated C1-INH has a PEG/C1-INH ratio of between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5.

20. The composition of any one of claims 1-19, wherein the conjugated C1-INH has a half-life comparable or r that than a plasma derived human C1-INH.

21. The composition of any one of claims 1-20, wherein the conjugated C1-INH has a half-life in the range of 100%-500% of the half-life of the plasma derived C1-INH.

22. The composition of any one of claims 1-21, wherein the conjugated C1-INH has a half-life of at least about 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 hours.

23. The composition of any one of claims 1-22, wherein the conjugated C1-INH has a half-life of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

24. The composition of any one of claims 1-23, wherein the conjugated C1-INH has a specific activity in the range of 50%-150% of the specific activity of plasma d human C-INH.

25. A method of producing a conjugated C1 esterase inhibitor (C1-INH), said method comprising steps of: ing a C1-INH protein comprising a glycan residue; and ing a PEG moiety under conditions that permit the PEG moiety reacts with the glycan e to form an oxime linkage, thereby producing the conjugated C1-INH, wherein the glycan residue is a sialic acid residue, and wherein the conjugated C1-INH has an extended in vivo half-life compared to ugated C1-INH.

26. The method of claim 25, wherein the PEG moiety comprises PEG-CH2-O-NH2.

27. The method of claim 25 or claim 26, wherein the method further comprises a step of oxidizing the at least one glycan residue prior to reacting with the PEG moiety.

28. The method of claim 27, wherein the oxidizing step comprises periodate oxidation.

29. The method of claim 28, wherein the periodate oxidation is carried out with a molar ratio of periodate to C1-INH at between about 20:1 to about 50:1.

30. The method of claim 29, wherein the molar ratio of periodate to PEG is between about 2.5 to about 40.

31. The method of any one of claims 25-30, wherein the molar ratio of PEG to C1-INH is between about 25:1 and 100:1.

32. The method of any one of claims 25-31, wherein the method r comprises a step of purifying the conjugated C1-INH.

33. The method of claim 32, wherein the purifying step comprises one or more of anion exchange, tangential flow filtration diafiltration, and dialysis.

34. A ceutical composition comprising a conjugated C1 esterase inhibitor (C1- INH) of any one of claims 1-24, and a pharmaceutically acceptable carrier.

35. The pharmaceutical composition of claim 34, wherein the composition is liquid.

36. The pharmaceutical composition of claim 34, wherein the composition is lyophilized.

37. A kit comprising a pharmaceutical composition of any of claims 34-36, and a syringe.

38. The kit of claim 37, n the syringe is preloaded with the ceutical composition.

39. The kit of claim 37, wherein the pharmaceutical composition is lyophilized and the kit further comprises a reconstitution buffer.

40. Use of a ition comprising a conjugated C1-esterase inhibitor of any one of claims 1-24, in the manufacture of a medicament for treating a complement mediated disorder.

41. The use of claim 40, wherein the complement-mediated er is selected from hereditary angioedema, antibody mediated rejection, yelitis optica spectrum disorders, tic brain injury, spinal cord injury, ischemic brain injury, burn injury, toxic epidermal necrolysis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, stroke, chronic inflammatory demyelinating uropathy , myasthenia gravis, and/or ocal motor neuropathy.

42. A composition comprising a conjugated C1 esterase inhibitor (C1-INH) comprising: a C1-INH protein comprising at least one glycan residue; and a polysialic acid (PSA) moiety, wherein the polysialic acid (PSA) moiety is ntly linked to the C1-INH protein via an oxime linkage, wherein the oxime linkage is between the PSA moiety and the glycan residue of C1-INH, wherein the glycan residue is a sialic acid residue, and wherein the conjugated C1-INH has an extended in vivo half-life compared to unconjugated C1-INH.

43. The composition of claim 42, wherein the C1-INH protein is recombinantly produced or plasma derived.

44. The composition of claim 42 or claim 43, wherein the C1-INH protein ses a C1-INH domain having an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:37, or SEQ ID NO:38.

45. The composition of any one of claims 42-44, wherein the C1-INH protein is a fusion protein.

46. The ition of claim 45, wherein the fusion protein comprises an Fc domain directly or indirectly fused to a C1-INH domain.

47. The composition of claim 46, wherein the Fc domain is derived from IgG1.

48. The composition of claim 46 or claim 47, wherein the Fc domain comprises amino acid substitutions corresponding to L234A and L235A according to EU numbering.

49. The composition of claim 45, wherein the fusion protein comprises an albumin domain directly or ctly fused to a C1-INH .

50. The composition of any one of claims 42-49, wherein the C1-INH protein has a glycosylation profile comprising no more than about 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15 %, 10%, or 5% neutral glycan species, prior to PEGylation.

51. The composition of any one of claims 42-50, wherein the C1-INH protein has a glycosylation profile comprising between about 5% and about 25% neutral glycan species, prior to tion.

52. The ition of any one of claims 42-51, wherein the C1-INH protein comprises, on average, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% charged glycans per molecule.

53. The composition of any one of claims 42-52, wherein the C1-INH protein contains less than about 20%, 15%, 10%, or 5% of one or more of mannose, a–galactose, NGNA, or oligomannose-type glycosylation, prior to conjugation with PSA.

54. The composition of any one of claims 42-53, wherein, prior to conjugation with PSA, the C1-INH protein has a glycosylation profile sing one or more of the following: between about 5% and about 30% neutral glycan species; between about 10% and about 30% mono-sialylated glycan s; between about 30% and about 50% di-sialylated glycan s; between about 15% and about 35% tri-sialylated glycan species; or between about 5% and about 15% tetra-sialylated glycan species.

55. The composition of any one of claims 42-54, wherein, prior to conjugation with PSA, the C1-INH n has a glycosylation profile comprising: no more than 30% neutral glycan species; between about 20% and about 30% mono-sialylated glycan species; between about 30% and about 40% di-sialylated glycan species; between about 10% and about 20% tri-sialylated glycan species; and between about 5% and about 10% tetra-sialylated glycan species.

56. The ition of any one of claims 42-55, wherein the C1-INH n comprises, on average, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 sialylated glycan residues per molecule.

57. The composition of any one of claims 42-55, wherein the C1-INH protein comprises, on average, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mole sialic acid per mole of protein

58. The composition of any one of claims 42-57, wherein the PSA has a molecular weight between about 1 KDa and 50 KDa, n about 1 KDa and 40 KDa, between about 5 KDa and 40 KDa, between about 1 KDa and 30 KDa, between about 1 KDa and 25 KDa, between about 1 KDa and 20 KDa, between about 1 KDa and 15 KDa, between about 1 KDa and 10 KDa, or between about 1 KDa and 5 KDa.

59. The composition of any one of claims 42-58, wherein the PSA has a molecular weight of about 1 KDa, 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa, 45 KDa, or 50 KDa.

60. The composition of any one of claims 42-59, wherein the conjugated C1-INH has a PSA/C1-INH ratio of between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5.

61. The composition of any one of claims 42-60, wherein the conjugated C1-INH has a half-life comparable or greater that than a plasma derived human C1-INH.

62. The ition of any one of claims 42-60, wherein the conjugated C1-INH has a half-life in the range of 100%-500% of the half-life of the plasma derived .

63. The composition of any one claims 42-62, wherein the conjugated C1-INH has a halflife of at least about 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 hours.

64. The ition of any one of claims 42-62, wherein the conjugated C1-INH has a half-life of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

65. The composition of any one of claims 42-62, wherein the conjugated C1-INH has a specific activity in the range of 50%-150% of the specific activity of plasma derived human C-INH.

66. A method of producing a conjugated C1 esterase inhibitor (C1-INH), said method comprising steps of: providing a C1-INH n comprising a glycan residue; and ing a polysialic acid (PSA) moiety under conditions that permit the PSA moiety to react with the glycan residue to form an oxime linkage, thereby producing the conjugated C1-INH, wherein the glycan residue is a sialic acid residue, and n the conjugated C1- INH has an extended in vivo half-life compared to unconjugated C1-INH.

67. The method of claim 66, wherein the method further comprises a step of ing the at least one glycan residue prior to ng with the PSA moiety.

68. The method of claim 67, n the oxidizing step comprises periodate oxidation.

69. The method of claim 68, wherein the periodate oxidation is carried out with a molar ratio of periodate to C1-INH at between about 20:1 to about 50:1.

70. The method of claim 69, wherein the molar ratio of periodate to PSA is between about 2.5 to about 40.

71. The method of any one of claims 66-70, wherein the molar ratio of PSA to C1-INH is between about 25:1 and 100:1.

72. The method of any one of claims 66-71, wherein the method further comprises a step of ing the conjugated C1-INH.

73. The method of claim 72, wherein the purifying step comprises one or more of anion exchange, tangential flow filtration diafiltration, and dialysis.

74. A pharmaceutical composition comprising a conjugated C1 esterase inhibitor (C1- INH) of any one of claims 42-65, and a pharmaceutically acceptable carrier.

75. The ceutical composition of claim 74, wherein the composition is liquid.

76. The pharmaceutical composition of claim 74, wherein the composition is lyophilized.

77. A kit comprising a pharmaceutical composition of any of claims 74-76, and a syringe.

78. The kit of claim 77, wherein the syringe is preloaded with the ceutical composition.

79. The kit of claim 77, wherein the ceutical composition is lyophilized and the kit further comprises a reconstitution .

80. Use of a composition comprising a conjugated erase inhibitor of any one of claims 42-65, in the manufacture of a medicament for treating a complement mediated disorder.

81. The use of claim 80, wherein the complement-mediated disorder is selected from hereditary angioedema, antibody mediated rejection, neuromyelitis optica spectrum disorders, traumatic brain injury, spinal cord injury, ischemic brain injury, burn injury, toxic epidermal necrolysis, le sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, stroke, chronic inflammatory demyelinating polyneuropathy (CIDP), enia gravis, and/or multifocal motor neuropathy. hr et a1. JBC 200.7 289; 21700