HK1261372A1 - Conjugated c1 esterase inhibitor and uses thereof - Google Patents

Description

Conjugated C1 esterase inhibitors and uses thereof

RELATED APPLICATIONS

This application claims priority and benefit from U.S. provisional application No. 62/318,003 filed on 4.4.2016, the entire contents of which are hereby incorporated by reference.

Background

C1 inhibitors (C1-INH), also known as C1 esterase inhibitors, are the largest members of the serine protease inhibitor protein superfamily. It is a heavily glycosylated serine protease inhibitor with a primary function of inhibiting the spontaneous activation of the complement system. C1-INH regulates the complement cascade, plays a key role in regulating the contact (kallikrein-kinin) amplification cascade, and is involved in the regulation of the coagulation and fibrinolytic systems. Karnaukhova, E., C1-Esterase Inhibitor: Biological Activities and Therapeutic applications.J. Hematol Thromb Dis,1:113 (2013).

Since C1-INH is unable to inhibit activation of the complement system, dysfunction and/or deficiency of C1-INH in a subject has been associated with various autoimmune diseases. An example of such a disease is Hereditary Angioedema (HAE), a rare but potentially life-threatening condition characterized by unpredictable and recurrent episodes of inflammation. Symptoms of HAE attacks include swelling of the face, mouth and/or airways, either naturally occurring or caused by mild trauma. This swelling can also occur in any part of the body. In some cases, HAE is associated with low plasma levels of C1 inhibitors, while in other cases the protein circulates in normal or elevated amounts, but it is dysfunctional. In addition to inflammatory episodes, it can also lead to more serious or life-threatening indications, such as autoimmune diseases or lupus erythematosus.

Is a human plasma derived inhibitor of the C1 esterase, which has been approved for prophylactic use and treatment of acute episodes of HAE.(also plasma-derived human C1-INH, CSL Behring) is indicated for the treatment of acute HAE episodes.(constellation alfa, Pharming n.v.) is recombinant C1-INH expressed in engineered rabbits suitable for IV administration to treat acute HAE episodes.Has the same amino acid sequence as human plasma-derived C1-INH, but is prepared in transgenic rabbits. Rucest has a very short half-life of about 2.4 to 2.7 hours. See alsoFDA labels and prescription information.

There remains a need for improved inhibitors of the C1 esterase for the treatment and prevention of various C1 esterase-mediated indications.

Disclosure of Invention

The present invention provides, inter alia, improved long-acting C1 esterase inhibitors that are useful for the effective treatment of various complement-mediated disorders, including HAE.

In particular, the present invention provides C1 esterase inhibitor conjugates (also referred to as "conjugated C1 esterase inhibitors") that exhibit half-lives comparable to or even longer than plasma-derived C1-INH. The present invention is based in part on the following surprising findings: pegylated and polysialylated C1-INH may have an extended (e.g., at least 4 days) serum half-life. It is contemplated that the long serum half-life of conjugated C1-INH results in excellent in vivo efficacy and allows for preferred dosing regimens and routes of administration. For example, the conjugated C1-INH described herein can be administered subcutaneously or intravenously at a reduced frequency compared to currently approved C1-INH therapeutics while still achieving the desired efficacy (e.g., prevention). The conjugated C1 inhibitor proteins described herein can be prepared using plasma-derived or recombinantly produced C1-INH. Thus, the conjugated C1-INH described herein can be manufactured in a cost-effective manner and is independent of blood supply. Because they can be produced recombinantly in cultured cells, they provide better consistency in production and final products than those purified from human blood, human blood components (e.g., plasma), or animal milk. Accordingly, the present invention provides safer, more effective conjugated C1 esterase inhibitors for the treatment of HAE and other complement-mediated disorders.

In one aspect, the present invention provides a conjugated C1-INH comprising a C1-INH protein and at least one PEG moiety covalently attached to the C1-INH protein. In some embodiments, the C1-INH protein comprises at least one glycan residue, and the at least one PEG moiety is covalently linked to the at least one glycan residue. In some embodiments, the at least one PEG moiety is covalently linked to the C1-INH protein through an oxime bond.

In some embodiments, the at least one PEG moiety forms a covalent oxime bond with a glycan residue or amine group of C1-INH. In some embodiments, the at least one PEG moiety forms a covalent oxime bond with the glycan residue. In some embodiments, the at least one PEG moiety forms a covalent oxime bond with the amine group of C1-INH.

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

In some embodiments, the C1-INH protein suitable for the present invention is recombinantly produced or plasma derived.

In some embodiments, the C1-INH protein includes a C1-INH domain having an amino acid sequence that is 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. 1, SEQ ID No. 2, SEQ ID No. 37, or SEQ ID No. 38.

In some embodiments, the C1-INH protein is a fusion protein. In some embodiments, the fusion protein comprises an Fc domain fused directly or indirectly to a C1-INH domain. In some embodiments, the Fc domain is derived from IgG 1. 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 comprises one or more amino acid substitutions at positions corresponding to Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and/or Asn434 according to EU numbering of IgG 1.

In some embodiments, the fusion protein comprises an albumin domain fused directly or indirectly to a C1-INH domain.

In some embodiments, the present invention provides a C1-INH protein having a glycosylation profile comprising no more than about 50% (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of neutral glycan species.

In some embodiments, the present invention provides a C1-INH protein having a glycosylation profile comprising about 5% to about 25% of neutral glycan species.

In some embodiments, the present invention provides a C1-INH protein comprising, 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, α -galactose, NGNA, or oligomannose-type glycosylation.

In some embodiments, the C1-INH protein has a glycosylation profile comprising one or more of: from about 5% to about 30% neutral glycan species, from about 10% to about 30% mono-sialylated glycan species, from about 30% to about 50% di-sialylated glycan species, from about 15% to about 35% tri-sialylated glycan species, or from about 5% to 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, from about 20% to about 30% mono-sialylated glycan species, from about 30% to about 40% di-sialylated glycan species, from about 10% to about 20% tri-sialylated glycan species, and from about 5% to about 10% tetra-sialylated glycan species.

In some embodiments, 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 moles of sialic acid per mole of protein.

In some embodiments, the C1-INH protein having a glycosylation profile described herein is a fusion protein. In certain embodiments, the C1-INH proteins having a glycosylation profile described herein are unconjugated proteins.

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

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

In some embodiments, the conjugated C1-INH has a half-life comparable to or greater than that of plasma-derived human C1-INH protein. In some embodiments, the half-life of the conjugated C1-INH is in the range of 100% -500% of the half-life of the plasma-derived C1-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 half-life of conjugated C1-INH is at least about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14 days.

In some embodiments, the specific activity of the conjugated C1-INH is 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 process for the preparation of a conjugated C1 esterase inhibitor (C1-INH), comprising the steps of: providing a C1-INH protein comprising at least one glycan residue and/or at least one amine group, and providing a PEG moiety to form a bond under conditions that allow the PEG moiety to react with the at least one glycan residue and/or the at least one amine group, thereby producing a conjugated C1-INH.

In some embodiments, the PEG moiety comprises PEG-CH₂-O-NH₂. In some embodiments, the at least one glycan residue is a sialic acid residue. In further embodiments, the at least one glycan residue is a galactose residue.

In some embodiments, the methods described herein further comprise the step of oxidizing the at least one glycan residue prior to reacting with the PEG moiety. In some embodiments, the oxidizing step comprises oxidizing with periodate. In some embodiments, periodate oxidation is performed at a periodate to C1-INH molar ratio of about 20:1 to about 50: 1. In some embodiments, the molar ratio of periodate to PEG is from about 2.5 to about 40. In some embodiments, the molar ratio of PEG to C1-INH is 25:1 to 100: 1.

In further embodiments, the method further comprises the step of purifying the conjugated C1-INH. In some embodiments, the purification step comprises one or more of anion exchange, tangential flow filtration, diafiltration, and dialysis.

In a further aspect, the present invention provides a pharmaceutical composition comprising a conjugated C1 esterase inhibitor (C1-INH) and a pharmaceutically acceptable carrier.

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

In another aspect, the invention provides kits comprising pharmaceutical compositions (e.g., in liquid and lyophilized forms) containing conjugated C1-INH. In some embodiments, the kit contains a syringe. In some embodiments, the syringe is pre-loaded with a pharmaceutical composition comprising conjugated C1-INH.

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

In another aspect, the invention provides a method of treating a complement-mediated disorder comprising administering to a subject in need of treatment a pharmaceutical composition of a conjugated C1 esterase inhibitor (C1-INH).

In a related aspect, the invention provides the use of a composition comprising a conjugated C1-esterase inhibitor (C1-INH) in the manufacture of a medicament for the treatment of a complement-mediated disorder.

In some embodiments, the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 inflammatory demyelinating multiple neuropathy (CIDP), myasthenia gravis, and/or multifocal motor neuropathy.

In some embodiments, the present invention provides compositions comprising a conjugated C1 esterase inhibitor (C1-INH), the conjugated C1 esterase inhibitor comprising: a C1-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 attached to the at least one glycan residue.

In another aspect, the invention provides a composition comprising a conjugated C1 esterase inhibitor (C1-INH), the conjugated C1 esterase inhibitor comprising: a C1-INH protein comprising 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 covalently attached to the C1-INH protein via an oxime or hydrazone bond. In some embodiments, the polysialic acid (PSA) moiety is covalently linked to the C1-INH protein via an oxime bond. In some embodiments, the polysialic acid (PSA) moiety is covalently linked to the C1-INH protein via an oxime bond. In some embodiments, the oxime bond is between the PSA moiety and a glycan residue or amine group of C1-INH.

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

In some embodiments, the C1-INH protein is recombinantly produced or plasma derived.

In some embodiments, the C1-INH protein comprises a C1-INH domain having an amino acid sequence at least 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 C1-INH protein is a fusion protein. In some embodiments, the fusion protein may comprise an Fc domain fused directly or indirectly to a C1-INH domain. In some embodiments, the Fc domain may be derived from IgG 1. In some embodiments, the Fc domain may comprise amino acid substitutions corresponding to L234A and L235A according to EU numbering. In some embodiments, the fusion protein may comprise an albumin domain fused directly or indirectly to a C1-INH domain.

In some embodiments, prior to pegylation, the C1-INH protein has a glycosylation profile comprising no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of neutral glycan species.

In some embodiments, prior to pegylation, the C1-INH protein has a glycosylation profile comprising about 5% to about 25% of neutral glycan species.

In some embodiments, 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.

In some embodiments, the C1-INH protein comprises less than about 20%, 15%, 10%, or 5% of one or more of mannose, α -galactose, NGNA, or oligomannose-type glycosylation prior to conjugation to PSA.

In some embodiments, prior to conjugation to PSA, the C1-INH protein has a glycosylation profile comprising one or more of: from about 5% to about 30% neutral glycan species, from about 10% to about 30% mono-sialylated glycan species, from about 30% to about 50% di-sialylated glycan species, from about 15% to about 35% tri-sialylated glycan species, or from about 5% to about 15% tetra-sialylated glycan species.

In some embodiments, prior to conjugation to PSA, the C1-INH protein has a glycosylation profile comprising: no more than 30% neutral glycan species, from about 20% to about 30% mono-sialylated glycan species, from about 30% to about 40% di-sialylated glycan species, from about 10% to about 20% tri-sialylated glycan species, and from about 5% to about 10% tetra-sialylated glycan species.

In some embodiments, 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-INH protein comprises an average of 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 moles of sialic acid per mole of protein

In some embodiments, the molecular weight of the PSA is about 1KDa to 50KDa, about 1KDa to 40KDa, about 5KDa to 40KDa, about 1KDa to 30KDa, about 1KDa to 25KDa, about 1KDa to 20KDa, about 1KDa to 15KDa, about 1KDa to 10KDa, or about 1KDa to 5 KDa.

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

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

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

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

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

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

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

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

In some embodiments, the molar ratio of PSA to C1-INH is about 25:1 to 100: 1.

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

In some embodiments, the purification step comprises one or more of anion exchange, tangential flow filtration diafiltration, and dialysis.

In another aspect, the present invention provides a conjugated C1 esterase inhibitor (C1-INH) prepared by the method of the above aspects or embodiments.

In another aspect, the present invention provides a pharmaceutical composition comprising a conjugated C1 esterase inhibitor (C1-INH) of the above aspects or embodiments and a pharmaceutically acceptable carrier. In some embodiments, the components of the pharmaceutical composition are liquids. In some embodiments, the components of the pharmaceutical composition are lyophilized.

In one aspect, the invention provides a kit comprising a pharmaceutical composition of the above aspects or embodiments 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 invention provides a method of treating a complement-mediated disorder comprising administering to a subject in need of treatment a pharmaceutical composition of the above aspects or embodiments. In some embodiments, the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 inflammatory demyelinating multiple neuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

In a further aspect, the invention provides the use of a composition comprising a conjugated C1-esterase inhibitor of the above aspects or embodiments in the manufacture of a medicament for the treatment of a complement-mediated disorder. In some embodiments, the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 inflammatory demyelinating multiple neuropathy (CIDP), myasthenia gravis, and/or multifocal motor neuropathy.

Other features, objects, and advantages of the invention will be apparent from 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 limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

Drawings

The drawings are only for purposes of illustration and are not intended to be limiting.

FIG. 1 is a schematic representation of C1-INH. From left to right, the three domains are the signal peptide, the N-terminus (also referred to as the N-terminal domain) and the serpin domain. N-linked glycans are shown as long vertical lines with diamond-shaped heads, and O-linked glycans are shown as short vertical lines.

FIG. 2 depicts the mature C1-INH amino acid sequence and potential sites for PEGylation.

Fig. 3 depicts a schematic depicting the chemical equation for an exemplary amine-mediated pegylation.

Fig. 4A is a schematic depicting the chemical equation for an exemplary glycan-mediated aminoxy pegylation. Fig. 4B is a schematic depicting the chemical equation for an exemplary Sialic Acid Mediated (SAM) aminoxy pegylation.

Fig. 5 depicts a schematic diagram depicting the chemical equation of an exemplary galactose-mediated (GAM) pegylation.

FIG. 6 (panels A and B) depicts the results of a preliminary rat study of C1-INH PEGylation (5kDa or 40kDa) by amino groups compared to sialic acid. rhC1-INH and Cinryze are provided as comparisons. Panel C depicts SDS-PAGE gels with C1-INH PEGylation with 5kDa or 40kDa PEG.

Fig. 7 depicts a schematic of an exemplary pegylation process a.

Fig. 8 depicts a schematic of an exemplary pegylation process B.

FIGS. 9A-E depict schematic diagrams summarizing several exemplary PEGylation schemes suitable for PEGylation of C1-INH.

FIG. 10A depicts the C1-INH-PEG IC50 of the 5KSAM KHR5 octyl loading sample. Fig. 10B depicts C1-INH-PEG IC50 before and after removal of free PEG by TFF.

Fig. 11 depicts the chromatographic results of an exemplary 40KDa pegylated C1-INH purified from free PEG and other contaminants.

Fig. 12 depicts the chromatographic results of an exemplary 20KDa pegylated C1-INH purified from free PEG and other contaminants.

Fig. 13 depicts the chromatographic results of an exemplary 5KDa pegylated C1-INH purified from free PEG and other contaminants.

Fig. 14 depicts the results of a non-human primate (NHP) PK study with IV administration of pegylated rhC 1INH and rhC1 INH.

Fig. 15 depicts the results of NHP PK studies, where different C1-INH-PEG loadings were administered to NHPs.

Fig. 16 depicts NHP study results of IV and SC administration of pegylated rhC 1-INH.

FIG. 17 depicts the results of rat PK titration analysis of C1-INH-PEG samples with various 5KPEG loadings.

FIG. 18(A-E panels) depicts a series of gels and graphs depicting the purity of C1-INH-PEG. Panel A and B are SDS-PAGE gels stained with barium-iodide (barium-iodide) for detection of free PEG in C1-INH-PEG samples. Panel C and D are RP-HPLC plots for detection of free PEG1K and PEG2K in C1-INH-PEG samples. E-Panel depicts two SDS-PAGE gels with C1-INH loaded samples.

Fig. 19 (panels a-C) depicts a series of graphs and gels depicting purity, IC50 and PK data for C1-INH-PEG samples conjugated by the SAM process. Panel A is a graph of IC50 for various C1-INH samples. Panel B is an SDS-PAGE gel depicting the purity of the C1-INH sample and the associated IC50 values for the C1-INH sample. Panel C is a graph depicting PK values from a rat study in which rats received intravenous C1-INH-PEG and unpegylated C1-INH.

FIG. 20(A, B and C plot) depicts a series of graphs depicting C1-INH IC50 values.

FIG. 21 depicts a schematic of an exemplary amine-coupled PEGylation process for C1-INH.

FIG. 22(A-D panels) depicts a series of gels and graphs depicting the purity of C1-INH-PEG. Panel A is a barium iodide stained SDS-PAGE gel for detection of free PEG in C1-INH-PEG samples. Panel B depicts the RP-HPLC profile for detection of free PEG1K and PEG 2K. Panel C and D depict purification chromatograms of free NHS-PEG20K (panel C) and NHS-PEG40K (panel D).

FIG. 23(A-C panels) depicts a series of graphs and gels depicting purity, IC50 and PK data for C1-INH samples. Panel A is a graph of IC50 for various C1-INH samples. Panel B is a graph depicting PK values from a rat study in which rats received intravenous C1-INH-PEG and unpegylated C1-INH. Panel C is an SDS-PAGE gel depicting the purity of the C1-INH sample and the associated IC50 values for the C1-INH sample.

FIG. 24 (panels A and B) depicts gels and graphs depicting the purity of C1-INH-PSA prepared by a Sialic Acid Mediated (SAM) process. Panel A is an SDS gel and panel B is an IC50 plot of C1-INH-PSA.

FIG. 25(A, B and C panels) depicts a series of graphs showing PK values from rat studies in which rats received intravenous C1-INH-PEG, C1-INH-PSA, Cinryze-PEG, C1-INH, or Cinryze.

Definition of

In order that the invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

Animals: as used herein, the term "animal" refers to any member of the kingdom animalia. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal 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, a cow, a primate, and/or a pig). In some embodiments, the animal includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, an insect, and/or a worm. In some embodiments, the animal can be a transgenic animal, a genetically engineered animal, and/or a clone.

About or about: as used in this patent application, the terms "about" and "approximately" are used as equivalents. Any numbers used in this patent application, and with or without and about/approximately, are intended to encompass any normal fluctuations as would be understood by one of ordinary skill in the relevant art. As used herein, the term "about" or "approximately" when applied to one or more stated values refers to a value similar to the stated reference value. In certain embodiments, the term "about" or "approximately" refers to a series 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 of any direction (greater than or less than) of the stated value, unless otherwise stated or otherwise apparent from the context (unless the number exceeds 100% of the possible values).

Bioavailability: as used herein, the term "bioavailability" generally refers to the percentage of an administered dose that reaches the bloodstream of a subject.

The biological activity is as follows: as used herein, the phrase "biologically active" refers to the characteristic of any agent that is active in a biological system, and in particular, in an organism. For example, an agent that has a biological effect on an organism is considered to be biologically active when administered to that organism. In particular embodiments, where a peptide is biologically active, a portion of the peptide that shares at least one biological activity of the peptide is generally referred to as a "biologically active" portion.

Carrier or diluent: as used herein, the term "carrier" or "diluent" refers to a pharmaceutically acceptable carrier or diluent material used to prepare a pharmaceutical formulation (e.g., safe and non-toxic for human administration). Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, ringer's solution, or dextrose solution.

A C1 inhibitor or a C1 esterase inhibitor or C1-INH: as used herein, unless otherwise indicated, the terms "C1-inhibitor" or "C1 esterase inhibitor" or "C1-INH" are all used interchangeably and refer to any wild-type, native, naturally occurring, recombinantly produced and/or modified C1-INH protein (e.g., C1-INH protein and/or fusion protein having one or more amino acid mutations, deletions, truncations, insertions) that retains the essential biological activity of C1-INH. The "C1-inhibitor" or "C1 esterase inhibitor" or "C1-INH" may be a fusion protein. In some embodiments, the C1-INH fusion protein comprises a C1-INH polypeptide or domain and an Fc domain. In some embodiments, the C1-INH fusion protein comprises a C1-INH polypeptide or domain and an albumin domain. In some embodiments, the fusion protein further comprises a linker. The C1-INH protein can be recombinantly expressed in recombinant cells. In certain embodiments, C1-INH is expressed 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 that is covalently attached, directly or indirectly, to a protein. Generally, when a protein is attached to a conjugate, it may be referred to as a conjugated protein or protein conjugate. In some embodiments, the conjugates described herein are polyethylene glycol (PEG). When a protein is attached to a PEG moiety, it may be referred to as a pegylated protein.

Functional equivalents or derivatives: as used herein, in the context of a functional derivative of an amino acid sequence, the term "functional equivalent" or "functional derivative" indicates a molecule that retains substantially similar biological activity (function or structure) as the original sequence. The functional derivatives or equivalents may be natural derivatives or synthetically prepared. 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 substituted amino acid desirably has similar chemical-physical properties as the substituted amino acid. Desirable similar chemical-physical properties include similarity in charge, loftiness, hydrophobicity, hydrophilicity, and the like.

Fusion protein: as used herein, the term "fusion protein" or "chimeric protein" refers to a protein produced by linking two or more originally isolated proteins or portions thereof. In some embodiments, there will be a linker or spacer between each protein.

Half-life: as used herein, the term "half-life" is the time required for an amount, e.g., protein concentration or activity, to fall to half its value as measured at the beginning of a time period.

Hereditary angioedema or HAE: as used herein, the term "hereditary angioedema" or "HAE" refers to a blood disorder characterized by unpredictable and recurrent episodes of inflammation. HAE is usually associated with C1-INH deficiency, which may be the result of low levels of C1-INH or C1-INH with impaired or reduced activity. Symptoms include, but are not limited to, swelling that can occur in any part of the body, such as the face, limbs, genitalia, gastrointestinal tract, and upper respiratory tract.

Improvement, increase or decrease: as used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, indicate a value relative to a baseline measurement, e.g., a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject having the same form of disease as the subject being treated, which is about the same age as the subject being treated.

In vitro: as used herein, the term "in vitro" refers to an event that occurs in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

In vivo: as used herein, the term "in vivo" refers to events occurring within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

Linker as used herein, the term "linker" refers to an amino acid sequence in a fusion protein other than that occurring at a particular position in the native protein, and is generally designed to be flexible or to interpose a structure, such as an α -helix, between two protein moieties.

Polypeptide: as used herein, the term "polypeptide" refers to a continuous 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 ordinary skill in the art will appreciate that the term is not limited to long chains, and may refer to the smallest chain comprising two amino acids linked together via a peptide bond. The polypeptide may be processed and/or modified as known to those skilled in the art. As used herein, the terms "polypeptide" and "peptide" are used interchangeably.

Prevention: as used herein, the term "preventing," when used in conjunction with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of development of the disease, disorder, and/or condition. See definition of "risk".

Protein: as used herein, the term "protein" refers to one or more polypeptides that serve as discrete units. The terms "polypeptide" and "protein" are used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides in order to form a discrete functional unit. If a discrete functional unit consists of more than one polypeptide physically bound to each other, the term "protein" refers to multiple polypeptides with discrete units physically coupled and acting together.

Risk: as will be understood from the context, "risk" of a disease, disorder, and/or condition includes the likelihood that a particular individual will develop a disease, disorder, and/or condition (e.g., muscular dystrophy). In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is 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, the risk is expressed as a risk relative to a risk associated with a reference sample or a reference sample set. In some embodiments, the reference sample or reference sample set has a known risk of a disease, disorder, condition, and/or event (e.g., muscular dystrophy). In some embodiments, the reference sample or set of reference samples is from an individual comparable to the specific individual. In some embodiments, the 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, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person provided to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

Essentially: as used herein, the term "substantially" refers to a qualitative condition that exhibits an overall or near overall degree or range of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena are rarely, if ever, completed and/or proceed to completion or to achieve or avoid absolute results. The term "substantially" is thus used herein to capture the integrity of the potential lack inherent to many biological and chemical phenomena.

Basic homology: the phrase "substantial homology" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues, with suitably similar structural and/or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and/or have "polar" or "nonpolar" side chains. Substitution of one amino acid for another of the same type can often be considered a "homologous" substitution.

As is well known in the art, amino acid or nucleic acid sequences can 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 procedures are described in the following documents: altschul et al, Basic local alignment search tool, J.mol.biol.,215(3): 403-; altschul et al, Methods in Enzymology; altschul et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic acids SRes.25:3389-3402, 1997; baxevanis et al, Bioinformatics A Practical Guide to the analysis of Genes and Proteins, Wiley, 1998; and Misener et al, (ed.), bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs generally provide an indication of the degree of homology. In some embodiments, two sequences are indicated as 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 homologous over the relevant residue stretch. In some embodiments, the relevant segment is the full sequence. In some embodiments, the relevant segment 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 one of ordinary skill in the art, two sequences are generally considered "substantially identical" if they contain identical residues at the corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can 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 procedures are described in the following documents: altschul et al, Basic local alignment search tool, J.mol.biol.,215(3): 403-; altschul et al, Methods in Enzymology; altschul et al, Nucleic acids SRes.25:3389-3402, 1997; baxevanis et al, Bioinformatics A Practical Guide to the analysis of Genes and Proteins, Wiley, 1998; and Misener et al, (ed.), bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above procedures generally provide an indication of the degree of identity. In some embodiments, two sequences are indicated as 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 the relevant residue segment. In some embodiments, the relevant segment is the full sequence. In some embodiments, the relevant segment 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.

Has the following symptoms: an individual suffering from a "disease, disorder, and/or condition" has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and/or condition.

Susceptible to: an individual "susceptible to" a disease, disorder, and/or condition has not yet 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 predisposed to a disease, disorder, condition, or event (e.g., DMD) may be characterized by one or more of the following: (1) mutations in genes associated with the development of diseases, disorders and/or conditions; (2) genetic polymorphisms associated with the development of a disease, disorder and/or condition; (3) increased and/or decreased expression and/or activity of a protein associated with a disease, disorder, and/or condition; (4) habits and/or lifestyles associated with the development of a disease, disorder, condition and/or event, (5) have undergone transplantation, are scheduled to undergo transplantation or require transplantation. In some embodiments, an individual who is predisposed to a disease, disorder, 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.

A therapeutically effective amount of: as used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and/or delay the onset of symptoms of a disease, disorder, and/or condition when administered to a subject suffering from or susceptible to the disease, disorder, and/or condition. It will be appreciated by one of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

Treatment: as used herein, the term "treating" refers to partially or completely alleviating, ameliorating, reducing, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of: one or more symptoms or characteristics of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of disease and/or exhibits only early signs of disease, for reducing the risk of developing pathology associated with the disease.

Detailed Description

The invention provides, inter alia, conjugated C1-INH for use in improving the treatment of complement-mediated disorders, including Hereditary Angioedema (HAE). In particular, the conjugated C1-INH provided by the present invention is PEGylated C1-INH.

It is contemplated that conjugated C1-INH (e.g., pegylated C1-INH or polysialic acid (PSA) conjugated C1-INH) has an extended half-life compared to unconjugated (e.g., ungegylated) but otherwise identical C1-INH. According to the present invention, any C1-INH protein may be conjugated (e.g., pegylated or PSA conjugated), including but not limited to plasma derived or recombinantly expressed C1-INH protein. In some embodiments, the C1-INH protein that can be conjugated (e.g., pegylated or PSA conjugated) is a fusion protein. As described below, the results of conjugation (e.g., pegylation or PSA conjugation) according to the present invention extend half-life in vivo while unexpectedly retaining good bioavailability and/or bioactivity of the C1-INH protein. Thus, the conjugated (e.g., pegylated or PSA conjugated) C1-INH provided herein allows for improved treatment of HAE and other complement-mediated diseases, disorders or conditions by, for example, reducing dosing frequency and increasing prophylactic efficacy.

The following sections describe various aspects of the invention in detail. The use of segments is not meant to limit the invention. Each segment may be suitable for use in any aspect of the invention. In this patent application, the use of "or" means "and/or" unless stated otherwise. The disclosures of all of the techniques cited herein are incorporated by reference in their entirety.

C1-INH protein

Human C1-INH, which belongs to the serine protease Inhibitor superfamily, the largest class of plasma protease inhibitors, which also includes antithrombin, α 1-protease inhibitors, plasminogen activator inhibitors, and many other structurally similar proteins that regulate different physiological systems C1-INH is the Complement system, the contact system for kinin production, and the protease Inhibitor in the intrinsic coagulation pathway Cai, S.Daprovis, A.E., comparative Regulation Protein C1 Inhibitor binding to receptors and intermediates with their intrinsic coagulation pathways.

Low plasma levels of C1-INH or its dysfunction lead to activation of both complement and the exposure plasma cascade, and may also affect other systems. The reduction of the plasma levels of C1-INH to levels below 55 μ g/mL (-25% of normal) has been shown to induce spontaneous activation of C1.

A schematic depicting the structure of C1-INH is provided in fig. 1. The signal peptide, N-terminal domain and serpin domain are shown. C1-INH is a 22 amino acid signal peptide required for secretion and cleaved from the remainder of the C1-INH protein. C1-INH has two domains: a C-terminal domain of 365 amino acids, which is a typical serpin domain, and an N-terminal domain of 113 amino acids. The protein is stabilized by two disulfide bridges connecting 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 the C-terminal domain. The serpin domain is responsible for the protease activity of C1-INH. P1-P1' indicates Arg444-Thr445 as a scissile bond.

More than 26% by weight of the glycosylated protein is carbohydrate. Glycans are unevenly distributed on 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 attached to asparagine residues Asn216, Asn231, and Asn330 (shown as long vertical lines with diamond-shaped heads) in the serpin domain. Although the functional role of the particularly long and heavily glycosylated N-terminal domain is still unclear, it may be important for the conformational stability of the protein, recognition, affinity for endotoxins and selectins, and clearance. The inherent heterogeneity of the carbohydrate fraction contributes greatly to the heterogeneity of the entire C1-INH, which is one of the difficult reasons for generating recombinant C1-INH that mimics the properties of plasma-derived C1-INH.

As used herein, C1-INH proteins suitable for conjugation and use according to the invention comprise C1-INH polypeptides or domains having wild-type and modified amino acid sequences (e.g., C1-INH proteins with amino acid mutations, deletions, truncations, and/or insertions) that retain the essential biological activity of C1-INH. Typically, the C1-INH protein is produced using recombinant techniques, but may also be of plasma origin.

In some embodiments, a C1-INH polypeptide or domain suitable for the present invention includes an amino acid sequence that is 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 wild-type human C1-INH protein (amino acids 1-478) (amino acids 1-97 are underlined):

NPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQP TTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:1)。

in some embodiments, a C1-INH polypeptide or domain suitable for the present invention includes an amino acid sequence that is 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, wild-type human C1-INH protein (amino acids 98-478):

GSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:2)。

in some embodiments, a C1-INH polypeptide or domain suitable for the present invention includes an amino acid sequence that is 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 C1-INH protein (amino acids 1-478) having the E165Q mutation (the mutated amino acid is bold and underlined):

NPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:37)。

in some embodiments, a C1-INH polypeptide or domain suitable for the present invention includes an amino acid sequence that is 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 an mature human C1-INH protein (amino acids 98-478) having the E165Q mutation (the mutated amino acid is bold and underlined):

GSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:38)。

homologues or analogues of human C1-INH protein may be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, for example as found in references compiling such methods. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues, with suitably similar structural and/or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and/or have "polar" or "nonpolar" side chains. Substitution of one amino acid for another of the same type can generally be considered a "homologous" substitution. In some embodiments, conservative substitutions of amino acids include substitutions in amino acids within the following groups: (a) m, I, L, V, respectively; (b) f, Y, W, respectively; (c) k, R, H, respectively; (d) a, G, respectively; (e) s, T, respectively; (f) q, N, respectively; and (g) E, D. In some embodiments, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.

As is well known in the art, amino acid or nucleic acid sequences can 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 procedures are described in the following documents: altschul et al, Basic local alignment search tool, J.mol.biol.,215(3): 403-; altschul et al, Methods in Enzymology; altschul et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic acids SRes.25:3389-3402, 1997; baxevanis et al, Bioinformatics A Practical Guide to the analysis of Genes and Proteins, Wiley, 1998; and Misener et al, (ed.), bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs generally provide an indication of the degree of homology.

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

C1-INH fusion protein

In some embodiments, C1-INH proteins that can be conjugated according to the invention include C1-INH fusion proteins. The C1-INH fusion protein may include a C1-INH domain (also referred to as a C1-INH polypeptide) and another domain or portion that may generally facilitate the therapeutic effect of C1-INH by, for example, enhancing or increasing the half-life, stability, efficacy and/or delivery or reducing or eliminating immunogenicity, clearance or toxicity of the C1-INH protein. Such suitable domains or portions for the C1-INH fusion protein include, but are not limited to, Fc domains and albumin domains. Suitable fusion domains or moieties (e.g., Fc or albumin domains) may be linked, fused or attached directly or indirectly to the N-terminus, C-terminus or internally to the C1-INH protein. The following sections describe exemplary C1-INH fusion proteins that can be conjugated.

Fc domains

In some embodiments, a suitable C1-INH fusion protein contains an Fc domain or portion thereof that binds to the FcRn receptor. As a non-limiting example, suitable Fc domains may be derived from an immunoglobulin subclass, such as IgG. In some embodiments, suitable Fc domains are derived from IgG1, IgG2, IgG3, or IgG 4. In some embodiments, suitable Fc domains are derived from IgM, IgA, IgD, or IgE. Particularly suitable Fc domains include those derived from human or humanized antibodies. In some embodiments, a suitable Fc domain is a modified Fc portion, for example a modified human Fc portion.

The C1-inhibitor Fc fusion protein may exist as a dimer, as shown in figure 1.

In some embodiments, an Fc domain suitable for the present invention may comprise an amino acid sequence that is 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 wild-type human IgG1Fc domain:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)。

in some embodiments, a suitable Fc domain may include one or more mutations that reduce or eliminate complement activation and/or antibody-dependent cell-mediated cytotoxicity (ADCC) activity (also referred to as "effector function"). For example, a suitable Fc domain may include mutations corresponding to L234A and L235A (LALA) according to EU numbering of IgG 1. An exemplary human IgG1Fc domain with a LALA mutation (mutated residues underlined) is shown below:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。

in some embodiments, an Fc domain suitable for use in the invention comprises an amino acid sequence that is 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 retaining mutations corresponding to L234A and L235A (LALA) according to EU numbering of IgG 1.

It is contemplated that improving the binding between the Fc domain and the FcRn receptor results in a prolonged serum half-life. Thus, in some embodiments, a suitable Fc domain comprises one or more amino acid mutations that result in improved binding to FcRn. Various mutations within the Fc domain that affect improved binding to FcRn are known in the art and may be suitable for practicing the present invention. In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and/or Asn434 according to EU numbering of human IgG 1.

For example, suitable Fc domains may contain mutations of H433K (His433Lys) and/or N434F (Asn434 Phe). As a non-limiting example, a suitable Fc domain may contain the mutations H433K (His433Lys) and N434F (Asn434 Phe). Additional amino acid substitutions that may be included in the Fc domain include those described in, for example, U.S. patent nos. 6,277,375, 8,012,476, and 8,163,881, all of which are hereby incorporated by reference.

In some embodiments, Fc domains suitable for use in the present invention include amino acid sequences that are 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 IgG1Fc domain while retaining one or more mutations (underlined) corresponding to Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and/or Asn434 according to EU numbering of human IgG 1:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(SEQ ID NO:5)。

in some embodiments, Fc domains suitable for use in the invention include amino acid sequences that are 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 IgG1Fc domain while retaining mutations corresponding to L234A and L235A (LALA) of IgG1 and one or more mutations corresponding to Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and/or Asn434 according to EU numbering of human IgG1 (mutated residues underlined):

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(SEQ ID NO:6)。

in some embodiments, the invention uses an Fc domain derived from IgG 4. Without wishing to be bound by any theory, IgG4 was reported to have lower complement activity than WT IgG 1. Thus, in some embodiments, the invention uses a wild-type human IgG4 Fc domain. In some embodiments, an Fc domain suitable for the present invention is derived from human IgG4 having a mutation corresponding to the S228P substitution in the core hinge region sequence according to the EU index. This substitution is also known as S241P, according to Kabat et al (1987 Sequences of proteins. United States Department of Health and human Services, Washington DC.). Without wishing to be bound by any theory, it is thought that this substitution has the effect of making the sequence of the hinge region core identical to that of a wild-type IgGl or IgG2 isotype antibody, and results in the production of a homologous form of IgG4 antibody, thus eliminating dissociation and recombination of the heavy chains that often result in the production of heterodimeric IgG4 antibodies. In addition, IgG 4-derived Fc domains can be used for stability at high concentrations.

Thus, in some embodiments, an Fc domain suitable for the present invention comprises an amino acid sequence that is 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 wild-type human IgG4 Fc domain:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:9)。

in some embodiments, Fc domains suitable for use in the invention include amino acid sequences that are 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 IgG4 Fc domain while retaining mutations corresponding to S241P substitutions according to EU numbering (mutated residues are underlined):

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:10)。

in some embodiments, an Fc domain described herein can include a signal peptide. Exemplary signal peptides suitable for use in the present invention includeMETPAQLLFLLLLWLPDTTGAt least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the same amino acid sequence.

For example, a suitable Fc domain may have an amino acid sequence that is 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 Fc domain of human IgG1 with a signal peptide and has mutations that enhance binding to the FcRn receptor (signal peptide and mutated residues underlined):

METPAQLLFLLLLWLPDTTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(SEQ ID NO:7)。

in some embodiments, Fc domains suitable for use in the invention include amino acid sequences that are 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 Fc domain of human IgG1 with a signal peptide and that have both LALA and mutations that enhance binding to the FcRn receptor (mutated residues underlined):

METPAQLLFLLLLWLPDTTGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(SEQ ID NO:8)。

exemplary C1-INH-Fc fusion proteins

In particular embodiments, suitable C1-INH fusion proteins include a C1-INH polypeptide or domain and an Fc domain. In some embodiments, a suitable C1-INH fusion protein includes a linker that associates a C1-INH polypeptide or domain with an Fc domain. In certain embodiments, as shown in figure 2, the Fc portion may be fused directly to the N-terminal region of the full-length (1-478aa) and mature (98-478) C1-inhibitors. By way of non-limiting example, a suitable C1-INH Fc fusion protein may have an amino acid sequence as shown below:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:11)

or

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:12)

Or

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:13)

Or

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:14)

Or

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:32)

Or

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:33)

Or

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:15)

Or

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:16)。

In some embodiments, a suitable C1-INH Fc fusion protein has an amino acid sequence that is 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. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 32, or SEQ ID No. 33.

It is contemplated that the C1-INH-Fc fusion protein may be provided in a variety of configurations, including homodimeric or monomeric configurations. For example, a suitable homodimeric configuration can be designed such that the C-terminus of a fusion partner (e.g., C1-INH polypeptide plus a linker) is attached to the N-termini of the two Fc polypeptide chains. Suitable monomer configurations can be designed to fuse the C-terminus of a fusion partner (e.g., C1-INH polypeptide plus a linker) to an Fc dimer.

Monomeric (also referred to herein as monovalent) forms are useful for certain applications and routes of administration, e.g., subcutaneous administration. The monomer configuration may reduce steric hindrance, increase half-life, and/or may increase bioavailability.

Without wishing to be bound by any theory, it is contemplated that the monovalent form may be particularly useful for the C1-INH-Fc fusion construct, as C1-INH is a suicide inhibitor. Since it is a suicide inhibitor, binding of one C1-INH "arm" of the dimeric Fc fusion will result in increased clearance of the bound C1-INH fusion protein, even if the second arm remains unbound.

The advantages of Fc fusion proteins (both monomeric and dimeric) are: fc expression was found to occur at higher levels than expression of C1-INH alone. Comparison of the activity of the dimer C1-INH-Fc construct with C1-INH without Fc fusion assays have shown similar C1q binding activity. The inclusion of a linker was also tested and it was found that the linker did not affect the ability of the C1-INH-Fc fusion protein to bind its target.

Methods of making monomeric antibody fusion proteins include those described in the following references: for example, PCT publication nos. WO2011/063348, WO2012/020096, WO2013/138643, WO 2014087299; dumont, J.et al, monomer Fc Fusions: Impact on pharmaceutical and Biological Activity of protein Therapeutics, Biodrugs,20(3): 151-; ishino, T.et al, protein Structure and Folding, Half-life Extension of biological analysis by N-Glycosylation for the Engineering a monomer Fc Domain, J.biol.chem.,288:16529-16537(2013), the disclosures of which are hereby incorporated by reference.

Monovalent C1-inhibitors can be prepared by using a Fc-C1-containing plasmid co-transfected with a Fc-only plasmid. Alternatively, it can be prepared by using a dual promoter plasmid, where one promoter generates Fc-C1 and the other generates Fc. Monovalent Fc can also be prepared using bispecific techniques in which specific amino acids in the Fc hinge region are mutated to confer stability to the Fc region (e.g., Knob and hole (Knob and hole) techniques or other stabilizing mutations that drive the formation of monovalent C1).

Albumin domain

In some embodiments, a suitable C1-INH fusion protein contains an albumin domain. Albumin is a soluble monomeric protein that accounts for about half of serum proteins. Albumin is mainly used as a carrier protein for steroids, fatty acids and thyroid hormones, and plays a role in stabilizing extracellular fluid volume. Albumin has a globular unglycosylated serum protein with a molecular weight of 66,500. Albumin is synthesized in the liver as a prealbumin with an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product pro-albumin is then cleaved in the golgi vesicles to produce secreted albumin.

Albumin consists of three homeodomains (I-III) and each of them consists of two subdomains (a and B). The major region of ligand binding to human serum albumin is located in the cavity of subdomains IIA and IIIA, which are formed primarily from hydrophobic and positively charged residues and exhibit similar chemical properties. Human serum albumin has 585 amino acids and a molecular weight of 66,500 Da. These amino acids include 35 cysteines, all but one of which are involved in the formation of 17 stabilizing disulfide bonds.

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

Typically, the albumin fusion proteins of the invention are monomeric. In some embodiments, this feature may be superior to dimeric Fc fusion embodiments for the reasons described above with respect to monomeric Fc fusion embodiments.

In some embodiments, albumin polypeptides suitable for the invention include an amino acid sequence that is 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 wild-type human serum protein:

MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYKTTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASRAALGL(SEQ ID NO:17)。

in some embodiments, an albumin polypeptide suitable for the invention includes an amino acid sequence that is 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 protein:

METPAQLLFLLLLWLPDTTGVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASRAALGL(SEQ ID NO:20)。

joints or spacers

A suitable C1-INH polypeptide or domain may be linked directly or indirectly to an Fc domain or albumin domain in some embodiments a suitable C1-INH fusion protein contains a linker or spacer that joins the C1-INH polypeptide or domain and the Fc or albumin domain the amino acid linker or spacer is typically designed to be flexible or to interpose a structure (such as a α -helix) between the two protein moieties the linker or spacer may be relatively short or may be longer typically the 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, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20) amino acids in length the linker or spacer is equal to or longer in length than 2, 3, 4, 5, 6, 7, 10-40, 10-35, 10-30, 10-25, 10-20 amino acids in some embodiments the linker or the linker may contain at least 5, 10-100, 10-70, 10-20, 10-60-100, 10-5, 10-60-5, 10-20, 10-20, or more amino acid residues in length in embodiments, 10-70, or more embodiments the amino acid residues in embodiments, 10-60-70, 10-20, or more embodiments.

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

Other suitable linkers includeGAPGGGGGAAAAAGGGGGGAP(GAG linker, SEQ ID NO:34),GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP(GAG2 linker, SEQ ID NO:35) andGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP(GAG3 linker, SEQ ID NO: 36).

Suitable linkers or spacers also include those having an amino acid sequence that is 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 exemplary linkers described above (e.g., the GGG linker, GGSGGGGS ((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 can be found in US2012/0232021 filed 3/2/2012, the disclosure of which is hereby incorporated by reference in its entirety.

Typically, linkers are included that associate the C1-INH polypeptide or domain with the Fc or albumin domain without substantially affecting or reducing the ability of the C1-INH polypeptide or domain to bind to any of its cognate ligands (e.g., C1, etc.).

Glycosylation/glycan profile (profile) of C1-INH protein

According to the present invention, the C1-INH protein may be conjugated via glycan residues and/or amine groups. In particular, C1-INH proteins may be conjugated at glycan residues (e.g., sialic acid residues or galactose residues). Thus, C1-INH proteins suitable for conjugation according to the invention can be characterized by different glycan profiles (in particular sialic acid content). In some embodiments, the C1-INH protein has a glycosylation profile similar to that of plasma-derived C1-INH. In some embodiments, the C1-INH protein has a glycosylation profile that differs from plasma-derived C1-INH.

Without wishing to be bound by any theory, it is believed that glycan profiles, including glycan linkages as well as the shape and complexity of the branched structures, can affect in vivo clearance, bioavailability, and/or efficacy.

Generally, glycan profiles can be determined by enzymatic digestion and subsequent chromatographic analysis. Various enzymes can be used for enzymatic 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 suitable enzyme is neuraminidase. Glycans can be detected by chromatographic analysis. For example, glycans can be detected by high performance anion exchange chromatography (HPAE-PAD) or size exclusion High Performance Liquid Chromatography (HPLC) with pulsed amperometric detection. The number of glycans represented by each peak on the glycan profile can be calculated using a standard curve of glycans according to methods known in the art and disclosed herein.

In some embodiments, the C1-INH protein may be characterized by a glycan profile. The relative amount of glycans corresponding to each peak group can 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 profiling are known in the art and can be used in the practice of the present invention. In some embodiments, the C1-INH protein is characterized by a glycan profile comprising five or fewer peak groups selected from the group of peaks indicating: neutral, mono-, di-, tri-or tetra-sialylated C1-INH protein.

In some embodiments, the C1-INH protein has a glycosylation profile comprising at least one of: neutral glycan species, mono-sialylated species, di-sialylated species, tri-sialylated species and/or tetra-sialylated species. In some embodiments, the C1-INH protein has a glycosylation profile comprising a neutral glycan species, a mono-sialylated species, a di-sialylated species, a tri-sialylated species, and a tetra-sialylated species. In some embodiments, the C1-INH protein has a glycosylation profile comprising no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of neutral glycan species. In some embodiments, the C1-INH protein has a glycosylation profile comprising about 5% to about 30% neutral glycan species. In some embodiments, the C1-INH protein has a glycosylation profile comprising about 5% to about 25% neutral glycan species. In some embodiments, the C1-INH protein has a glycosylation profile comprising about 10% to about 20% of neutral glycan species. In some embodiments, the C1-INH protein comprises, on average, at least about 80% charged glycans per molecule (e.g., greater than about 85%, 90%, 95%, or 99% glycans per molecule). In some embodiments, the C1-INH protein has a glycosylation profile comprising about 10% to about 30% of mono-sialylated species. In some embodiments, the C1-INH protein has a glycosylation profile comprising about 30% to about 50% of disialylated species. In some embodiments, the C1-INH protein has a glycosylation profile comprising about 15% to about 35% trisialylated species. In some embodiments, the C1-INH protein has a glycosylation profile comprising about 5% to about 15% tetrasialylated species. In some embodiments, the C1-INH protein has a glycosylation profile comprising no more than 30% neutral glycan species, from about 20% to about 30% monosialylated glycan species, from about 30% to about 40% disialylated glycan species, from about 10% to about 20% trisialylated glycan species, and from about 5% to about 10% tetrasialylated glycan species.

In some embodiments, the C1-INH protein has a sialylation profile similar to that of plasma-derived C1-INH. In some embodiments, the C1-INH protein has a sialylation profile that is different from plasma-derived C1-INH. In some embodiments, the C1-INH protein has a sialylation profile that results in a half-life similar to or longer than that of plasma-derived C1-INH. In some embodiments, the C1-INH protein comprises an average of at least about 10, 11, 12, 13, or 14 sialylated glycan residues per molecule. In some embodiments, the C1-INH protein comprises an average of at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 sialylated glycan residues per molecule. In some embodiments, the C1-INH protein comprises an average of at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 sialylated glycan residues per molecule.

In some embodiments, the C1-INH protein contains less than about 20%, 15%, 10%, or 5% of one or more of mannose, α -galactose, N-glycolylneuraminic acid (NGNA), or oligomannose-type glycosylation in some embodiments, the C1-INH protein contains no more than about 20%, 15%, 10%, or 5% of one or more of mannose, α -galactose, N-glycolylneuraminic acid (NGNA), or oligomannose-type glycosylation.

In some embodiments, the C1-INH protein has a glycosylation profile that is non-immunogenic, in some embodiments, the C1-INH protein has a glycosylation profile that does not increase in serum clearance compared to plasma-derived human C1-INH in some embodiments, the C1-INH protein has a glycosylation profile that decreases in serum clearance compared to plasma-derived human C1-INH in some embodiments, the C1-INH protein has a glycosylation profile that decreases in serum clearance compared to kaempta α (contestat alfa).

Various methods of manipulating the glycosylation profile of a protein are known in the art. These methods, as well as other methods yet to be developed, are contemplated by the present invention. Methods of manipulating the glycosylation profile of the C1-INH proteins and polypeptides of the invention include in vitro, in situ, and in vivo methods. In some embodiments, the glycosylation profile of the expressed protein or polypeptide is altered by post-expression chemical modification of the expressed protein or polypeptide. In some embodiments, the cell culture conditions are manipulated to achieve expression of a protein having a desired glycosylation profile. These cell culture conditions include control of the production and culture processes, including the length of the culture, media supplements, and/or co-expression of genes to enhance glycosylation. The selection of host cells and particular clones of transfected host cells may also be used to enhance glycosylation. Some methods of enhancing glycosylation include purification methods to enrich for proteins or polypeptides with a desired glycosylation profile.

In some embodiments, cells engineered to express C1-INH protein may also be engineered to modify glycosylation, in particular to increase sialylation of the expressed C1-INH. For example, the cell can be engineered to express the heterologous enzyme in a glycosylation pathway (wild-type or mutant) to achieve the desired glycosylation (e.g., increase sialylation). In some embodiments, the cell can also be engineered to overexpress an endogenous enzyme to achieve a desired glycosylation (e.g., increase sialylation). In some embodiments, the cell is engineered to reduce or prevent (e.g., by an antisense construct) expression of an endogenous enzyme that would reduce, inhibit, or degrade sialylation.

The various glycosylation patterns/glycan profiles described herein, and in particular sialylation profiles or levels, may be applied in the case of a C1-INH domain or polypeptide alone or a fusion protein (e.g., C1-INH-Fc or C1-INH-albumin fusion protein). C1-INH proteins having the glycosylation pattern/glycan profile described herein, and in particular the sialylation profile or level, may be conjugated or unconjugated. It is contemplated that the desired glycosylation pattern/glycan profile (including the desired sialylation profile or level) may extend the in vivo half-life of the C1-INH protein. In particular, a desired glycosylation pattern/glycan profile (including a desired sialylation profile or level) in combination with an Fc or albumin fusion can achieve a desired in vivo half-life of the C1-INH proteins described herein even without conjugation. However, conjugation (e.g., pegylation) further extended the in vivo half-life of C1-INH proteins, including those with desired glycosylation patterns or sialylation levels.

PEGylation

According to the present invention, a chemical or biological moiety may be conjugated directly or indirectly to the C1-INH protein described herein. In particular, such moieties are polyethylene glycol (PEG) moieties, including but not limited to single or multiple (e.g., 2-4) PEG moieties. As used herein, the process of conjugation of a PEG moiety to a protein, either directly or indirectly, is referred to as pegylation. As described herein, PEGylation may result in an increase in the half-life of C1-INH.

Pegylation can be performed by any suitable reaction known in the art. Methods of making pegylated proteins may generally comprise: (a) reacting the polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions such that the polypeptide is attached to one or more PEG groups; (b) one or more reaction products are obtained. In general, the reaction conditions can be individually determined based on known parameters and desired results.

There are many PEG attachment methods available to those skilled in the art and are described in the following references: for example, EP 0401384; malik et al, exp. Hematol.,20: 1028-; EP 0154316; EP 0401384; WO 92/16221; and WO 95/34326. For example, the step of pegylating the therapeutic molecule described herein may be performed by an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule.

In some embodiments, the PEG moiety undergoing conjugation is activated PEG. For example, suitable PEG moieties may include aminooxy functional groups. In some embodiments, suitable PEG moieties may include hydrazide functional groups. In some embodiments, suitable PEG moieties may include maleimide or iodoacetamide functional groups. In some embodiments, suitable PEG moieties may include N-hydroxysuccinimide (NHS) esters. Thus, a PEG moiety may be conjugated to a C1-INH protein through an oxime, amide, hydrazone, thioether, or other type of bond.

In some embodiments, the PEG moiety may have a linear or branched structure. For example, a PEG moiety may include 2, 3, 4, or 5 arm branches. Suitable PEG-NHS moieties can include linear PEG-NHS 1K, linear PEG-NHS 2K, linear PEG-NHS5K, branched PEG-NHS5K, branched PEG-NHS 20K, or branched PEG-NHS 40K. As another example, a PEG-aminooxy moiety can include a straight or branched chain PEG-aminooxy 2K, PEG-aminooxy 5K, PEG-aminooxy 5K, PEG-aminooxy 10K, PEG-aminooxy 20K or PEG-aminooxy 40K.

In some embodiments, the PEG is conjugated to C1-INH through one or more amino acid residues of the C1-INH protein. See fig. 3.

In some embodiments, PEG is conjugated to C1-INH through one or more galactose residues of the C1-INH protein. In some embodiments, one or more galactose residues of the C1-INH protein are oxidized prior to conjugation of PEG to the galactose residues.

In some embodiments, PEG is conjugated to C1-INH through one or more sialic acid residues of the C1-INH protein. In some embodiments, one or more sialic acid residues of the C1-INH protein are oxidized prior to conjugation of PEG to the sialic acid residues.

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

According to the present invention, the C1-INH protein may be PEGylated at various levels. For example, the molar ratio of PEG to C1-INH may range from about 5:1 to 100:1, about 10:1 to 100:1, about 15:1 to 100:1, about 20:1 to 100:1, about 25:1 to 100:1, about 30:1 to 100:1, about 40:1 to 100:1, about 50:1 to 100:1, about 10:1 to 90:1, about 10:1 to 80:1, about 10:1 to 70:1, about 10:1 to 60:1, about 10:1 to 50:1, about 10:1 to 40:1, about 15:1 to 35:1, or 20:1 to 30: 1. In some embodiments, the molar ratio of PEG to C1-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 about 1:1 to about 1:50, about 1:1 to about 1:45, about 1:1 to about 1:40, about 1:1 to about 1:35, about 1:1 to about 1:30, about 1:1 to about 1:25, about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, or about 1:1 to about 1: 5.

Polysialic acid conjugation

Polysialic acid (PSA), also known as Colominic Acid (CA), is a naturally occurring polysaccharide, it is a homopolymer of N-acetylneuraminic acid having α (2 → 8) ketoglycosidic linkages, and contains vicinal diol groups at its non-reducing end, it is negatively charged, and is a natural component of the human body.

PSA consists of a polymer (usually a homopolymer) of N-acetylneuraminic acid. A secondary amino group usually carries an acetyl group, but it may also carry a hydroxyacetyl group. Possible substituents on the hydroxyl group include acetyl, lactyl, ethyl, sulfate and phosphate groups.

PSA and modified PSA (mPSA) typically comprise linear polymers consisting essentially of N-acetylneuraminic acid moieties linked by 2, 8-glycosidic linkages or 2, 9-glycosidic linkages or combinations of these (e.g., alternating 2, 8-linkages and 2, 9-linkages). in some embodiments, the glycosidic linkages of PSA and mPSA are α -2, 8. such PSA and mPSA are derived from polyacetylcerazine typical PSA and mPSA comprise at least 2, preferably at least 5, more preferably at least 10, and most preferably at least 20N-acetylneuraminic acid moieties.

When the PSA comprises moieties other than N-acetylneuraminic acid (e.g., in mPSA), they are preferably located at one or both ends of the polymer chain. Such "other" moieties may be moieties derived from a terminal N-acetylneuraminic acid moiety, for example by oxidation or reduction.

For example, WO 2001/087922 describes mPSA in which a non-reducing terminal N-acetylneuraminic acid unit is converted into an aldehyde group by reaction with sodium periodate. In addition, WO 2005/016974 describes mPSA in which a reducing terminal N-acetylneuraminic acid unit is reductively opened at the reducing terminal N-acetylneuraminic acid unit by reduction, thereby forming a vicinal diol group, which is subsequently converted into an aldehyde group by oxidation.

Different PSA derivatives can be prepared from oxidized PSA containing a single aldehyde group at the non-reducing end. The preparation of aminoxy PSAs is described, for example, in WO2012/166622, the contents of which are hereby incorporated by reference. PSA-NH2 containing terminal amino groups can be prepared by reacting NH₄Cl and PSA-SH containing a terminal thiol group are prepared by reductive amination of PSA-NH2 with 2-iminothiolane (Traut's reagent), both procedures being described in U.S. Pat. No. 3, 7,645,860, 2. According to US 7,875,708B 2, PSA hydrazides can be reacted with oxygenReacted PSA with hydrazine. PSA hydrazides can be prepared by the reaction of oxidized PSA with adipic acid dihydrazide (WO 2011/012850 a 2).

Polyacetylneuraminic acids (a subset of PSA) are homopolymers of N-acetylneuraminic acid (NANA) having α (2 → 8) ketoside linkages and are produced, inter alia, by specific strains of E.coli having the K1 antigen.

As used herein, "sialic acid moiety" includes sialic acid monomers or polymers ("polysaccharides") that are soluble in aqueous solutions or suspensions and have little or no negative impact (such as side effects) on a mammal after the PSA-coagulation protein conjugate is administered in a pharmaceutically effective amount. In one aspect, the polymers are characterized 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 the chain.

In some embodiments, the sialic acid moiety of the polysaccharide compound is highly hydrophilic, while in another embodiment, the entire compound is highly hydrophilic. Hydrophilicity is mainly conferred by the side-chain carboxyl groups of the sialic acid units as well as by the hydroxyl groups. The saccharide units may contain other functional groups such as amine, hydroxyl or sulfate groups or combinations thereof. These groups may be present in naturally occurring saccharide compounds or introduced into derivatized polysaccharide compounds.

Naturally occurring polymeric PSAs are available as polydisperse preparations that exhibit a broad size distribution (e.g., Sigma C-5762) and high Polydispersity (PD). Since polysaccharides are usually produced in bacteria that carry the inherent risk of co-purifying endotoxins, purification of long sialic acid polymer chains can increase the likelihood of increased endotoxin content. Short PSA molecules with 1-4 sialic acid units (Kang SH et al, Chem Commun.2000; 227-8; Ress DK and Linhardt RJ, Current Organic Synthesis.2004; 1:31-46) can also be prepared synthetically, thus minimizing the risk of high endotoxin levels. However, it is now possible to produce PSA preparations with narrow size distribution and low polydispersity and also free of endotoxins. In one aspect, the polysaccharide compounds particularly useful in the present disclosure are those produced by bacteria. Some of these naturally occurring polysaccharides are called glycolipids. In some embodiments, these polysaccharide compounds are substantially free of terminal galactose units.

In some embodiments, the PSA is conjugated to the C1-INH through one or more sialic acid residues of the C1-INH protein. In some embodiments, one or more sialic acid residues of the C1-INH protein are oxidized prior to conjugation of PSA to the sialic acid residues.

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

According to the present invention, the C1-INH protein can be conjugated to PSA at various levels. For example, the molar ratio of PSA to C1-INH may range from about 5:1 to 100:1, about 10:1 to 100:1, about 15:1 to 100:1, about 20:1 to 100:1, about 25:1 to 100:1, about 30:1 to 100:1, about 40:1 to 100:1, about 50:1 to 100:1, about 10:1 to 90:1, about 10:1 to 80:1, about 10:1 to 70:1, about 10:1 to 60:1, about 10:1 to 50:1, about 10:1 to 40:1, about 15:1 to 35:1, or 20:1 to 30: 1. In some embodiments, the molar ratio of PSA to C1-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 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 PSA to sialic acid is about 1:1 to about 1:50, about 1:1 to about 1:45, about 1:1 to about 1:40, about 1:1 to about 1:35, about 1:1 to about 1:30, about 1:1 to about 1:25, about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, or about 1:1 to about 1: 5.

Extended half-life

According to the present invention, conjugation (e.g., pegylation or PSA conjugation) extends the in vivo half-life of C1-INH. Typically, conjugated (e.g., pegylated or PSA conjugated) C1-INH has a longer half-life than unconjugated (e.g., ungegylated or non-PSA conjugated) C1-INH. In some embodiments, conjugated (e.g., pegylated or PSA conjugated) C1-INH has a half-life comparable to or greater than 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%, 90% -500%, 100% -500%, 110% -500%, 120% -500%, 80% -400%, 90% -300%, 100% -250%, 100% -200%, or 100% -150% of the half-life of the plasma derived C1-INH protein.

In some embodiments, the half-life of the conjugated (e.g., pegylated or PSA conjugated) C1-INH protein is 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 in vivo half-life of the conjugated (e.g., pegylated or PSA conjugated) C1-INH is or is longer 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, the in vivo half-life of the conjugated (e.g., pegylated or PSA conjugated) C1-INH protein is in the following range: 0.5 to 14 days, 0.5 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 2.5 to 10 days, 2.5 to 9 days, 2.5 to 8 days, 2.5 to 7 days, 2.5 to 6 days, 2.5 to 5 days, 2.5 to 4 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 3.5 to 10 days, 3 to 5 days, 3 to 4 days, 3.5 to 10 days, 3.5 to 5 days, 3.5 to 4 days, 3.5 to 9 days, 3 to 4 days, 3.5 to 4 days, 3 to 5 days, 3.5 to 4 days, 3 to 4 days, 3.5 to 4 days, 3 to 5 days, 3.5 days, 3 to 4 days, 3 to 5 days, 3.5 to 9 days, 3 to 5 days, 3 days, 3.5 days, 4 days, 3 to 5 days, 3, 4 to 5 days, 4.5 to 10 days, 4.5 to 9 days, 4.5 to 8 days, 4.5 to 7 days, 4.5 to 6 days, 4.5 to 5 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 5.5 to 10 days, 5.5 to 9 days, 5.5 to 8 days, 5.5 to 7 days, 6 to 10 days, 7 to 10 days, 8 to 10 days, 9 to 10 days, 10 to 11 days, 11 to 12 days, 12 to 13 days, 13 to 14 days.

Pharmaceutical composition

The present invention also provides pharmaceutical compositions containing conjugated C1-INH described herein and a physiologically acceptable carrier. The carrier and conjugated C1-INH protein are generally sterile and formulated to be suitable for a mode of administration.

Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, 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, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, and combinations thereof. If desired, the pharmaceutical formulations can be mixed with adjuvants (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and/or aromatic substances, etc.) which do not deleteriously react with the active compounds or interfere with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

Suitable pharmaceutical compositions or medicaments may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The compositions may also be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

The pharmaceutical composition or medicament may be formulated according to conventional procedures as a pharmaceutical composition suitable for administration to a human. For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. If necessary, the composition may further include a solubilizing agent and a local anesthetic to relieve pain at the injection site. Typically, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose/water. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.

The conjugated C1-INH described herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

A preferred formulation comprises 50mM NaPO4(pH 7.2), 50mM sorbitol, and 150mM glycine. The formulations may be liquid, or may be lyophilized and reconstituted prior to administration.

Route of administration

Conjugated C1-INH described herein (or compositions or agents containing conjugated C1-INH described herein) is administered by any suitable route. In some embodiments, conjugated C1-INH or pharmaceutical compositions containing the same are administered systemically. Systemic administration can be intravenous, intradermal, intracranial, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, oral, and/or transmucosal administration. In some embodiments, the conjugated C1-INH or pharmaceutical composition containing same is administered subcutaneously. As used herein, the term "subcutaneous tissue" is defined as a loose, irregular layer of connective tissue immediately beneath the skin. For example, subcutaneous administration may be performed by injecting the composition into an area including, but not limited to, the thigh, abdomen, buttocks, or scapula. In some embodiments, the conjugated C1-INH or pharmaceutical composition containing same is administered intravenously. In some embodiments, conjugated C1-INH or pharmaceutical compositions containing the same are administered orally. In some embodiments, the conjugated C1-INH or pharmaceutical composition containing same is administered intracranially. In some embodiments, conjugated C1-INH or pharmaceutical compositions containing the same are administered intrathecally. If desired, more than one route may be used simultaneously.

In some embodiments, conjugated C1-INH or a pharmaceutical composition containing the same is administered to a subject subcutaneously (i.e., beneath the skin). For such purpose, the formulation may be injected using a syringe. However, other devices for administration of the formulation are available, such as injection devices (e.g. injection-ease)^TMAnd Genject^TMA device); injection pen (e.g. GenPen)^TM) (ii) a Needleless devices (e.g. mediJecter)^TMAnd BioJector^TM) (ii) a And a subcutaneous patch delivery system. Accordingly, the present invention also provides a kit comprising: pharmaceutical compositions (e.g., in liquid and lyophilized forms) containing conjugated C1-INH, and injection devices (such as syringes). In some embodiments, the syringe is pre-loaded with a pharmaceutical composition comprising conjugated C1-INH. Wherein the pharmaceutical composition is lyophilized, and the kit further comprises a reconstitution buffer.

The present invention contemplates single administration as well as multiple administrations of a therapeutically effective amount of conjugated C1-INH described herein or a pharmaceutical composition containing the same. Depending on the nature, severity and extent of the subject's condition (e.g., hereditary angioedema), conjugated C1-INH or pharmaceutical compositions containing it may be administered at regular intervals. In some embodiments, a therapeutically effective amount of conjugated C1-INH or a pharmaceutical composition containing the same may be administered periodically (e.g., once per year, once every six months, once every five months, once every three months, twice a month (once every two months), once per month (once every month), once per week (once every two weeks), once per week, once per day, or continuously) at regular intervals.

In some embodiments, administration produces only a local effect in the individual, while in other embodiments, administration produces an effect, e.g., a systemic effect, throughout portions of the individual. Typically, administration results in delivery of conjugated C1-INH to one or more target tissues. In some embodiments, conjugated C1-INH is delivered to one or more target tissues including, but not limited to, heart, brain, skin, blood, spinal cord, striated muscle (e.g., skeletal muscle), smooth muscle, kidney, liver, lung, and/or spleen. In some embodiments, conjugated C1-INH is delivered to the heart. In some embodiments, conjugated C1-INH is delivered to the central nervous system, particularly the brain and/or spinal cord. In some embodiments, conjugated C1-INH is delivered to triceps, tibialis anterior, soleus, gastrocnemius, biceps, trapezius, deltoid, quadriceps, and/or diaphragm.

Dosage forms and dosing regimens

In some embodiments, the compositions are administered in a therapeutically effective amount and/or according to an administration regimen associated with a particular desired outcome (e.g., prevention of complement-mediated chronic diseases, such as HAE).

The particular dose or amount to be administered according to the present invention may depend, for example, on the nature and/or extent of the desired result, the details of the route of administration and/or the timing of administration, and/or one or more characteristics (e.g., weight, age, personal history, genetic characteristics, lifestyle parameters, severity of cardiac defect and/or level of risk of cardiac defect, etc., or combinations thereof). These dosages or amounts can be determined by one of ordinary skill. In some embodiments, the appropriate dose or amount is determined according to standard clinical techniques. Alternatively or additionally, in some embodiments, the appropriate dose or amount is determined by using one or more in vitro or in vivo assays to help identify the desired or optimal dosage range or amount to be administered.

In various embodiments, the conjugated C1-INH is administered in a therapeutically effective amount. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit (e.g., prevention, treatment, modulation, cure, prevention, and/or amelioration of an underlying disease or condition) to a subject. Generally, the amount of therapeutic agent (e.g., conjugated C1-INH) administered to a subject in need thereof will depend on the characteristics of the subject. These characteristics include the condition, disease severity, general health, age, sex and weight of the subject. One of ordinary skill in the art will be readily able to determine the appropriate dosage based on these and other relevant factors. In addition, objective and subjective determinations can optionally be employed to determine optimal dosage ranges. In some particular embodiments, the appropriate dose or amount to be administered may be inferred from a dose-response curve derived from an in vitro or animal model test system.

In some embodiments, the composition is provided as a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is or comprises a unit dose for administration according to an administration regimen associated with achieving a reduced incidence or risk of HAE attacks.

In some embodiments, a formulation comprising conjugated C1-INH described herein is administered as a single dose. In some embodiments, a formulation comprising conjugated C1-INH described herein is administered at regular intervals. As used herein, administration at "intervals" indicates that the therapeutically effective amount is administered periodically (as opposed to a single dose). The interval can be determined by standard clinical techniques. In some embodiments, a formulation comprising conjugated C1-INH described herein is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, daily, twice daily, or once every six hours. The administration interval for a single individual need not be a fixed interval, but may vary over time, depending on the needs of the individual.

A therapeutically effective amount is typically administered in a dosage regimen that may comprise multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and/or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration, in combination with other pharmaceutical agents. In addition, 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 particular pharmaceutical agent employed; the particular composition employed; the age, 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 particular C1-INH employed; the duration of the treatment; and similar factors well known in the medical arts.

As used herein, the term "bi-monthly" means administered once/two months (i.e., once every two months); the term "monthly" means administered once/month; the term "once in three weeks" means administered once/three weeks (i.e., once every three weeks); the term "biweekly" means once/two weeks (i.e., once every two weeks); the term "once per week" means administered once per week; and "once daily" means administered once/day.

In some embodiments, a formulation comprising conjugated C1-INH described herein is administered at regular intervals indefinitely. In some embodiments, a formulation comprising conjugated C1-INH described herein is administered at regular intervals over a defined period of time.

It is to be further understood that for any particular subject, specific 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 replacement therapy, and that the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the invention.

Combination therapy

In some embodiments, conjugated C1-INH is administered in combination with one or more known therapeutic agents currently used to treat complement-mediated diseases (e.g., corticosteroids). In some embodiments, the known therapeutic agent is administered according to its standard or approved dosing regimen and/or schedule. In some embodiments, the known therapeutic agents are administered according to a regimen that is altered compared to its standard or approved dosing regimen and/or schedule. In some embodiments, such an altered regimen differs from a standard or approved dosing regimen in that the amount of one or more unit doses is altered (e.g., decreased or increased), and/or the dosing is altered in frequency (e.g., one or more intervals between unit doses is expanded, resulting in a lower frequency, or intervals are decreased, resulting in a higher frequency).

Complement mediated disorders

Conjugated C1-INH and pharmaceutical compositions containing the same are useful for treating HAE and various other complement-mediated disorders.

In some embodiments, the conjugated proteins provided by the invention are suitable for acute episodes associated with complement-mediated disorders (e.g., NMOSD, AMR, or HAE events). These episodes may be long or short. In some embodiments, the disease or disorder is chronic. In some embodiments, the compositions and methods of the invention are used prophylactically. Exemplary complement-mediated diseases that can be treated using the compositions and methods disclosed herein include, but are not limited to, hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 Inflammatory Demyelinating Polyneuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

Examples

Other features, objects, and advantages of the invention will be apparent from the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the invention, are given by way of illustration and not of 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 of C1-INH

This example shows an exemplary method suitable for pegylation of C1-INH protein. Three different pegylation strategies were explored. Exemplary pegylation schemes are shown in fig. 3-5. These protocols are conjugation of PEG to sialic acid residues (sialic acid mediates [ SAM ] chemistry), conjugation of PEG to galactonic acid residues (galactose mediates [ GAM ] chemistry) and amine mediated PEG conjugation.

aminooxy-PEG was utilized in order to form a more stable oxime linkage. Pegylation was performed using a technique developed based on the method described in the following literature: park et al, Carbohydrate-Mediated Polyethylene conjugation of TSH improvements, Endocrinology, March 2013,154(3): 1373) 1383.

Exposed sialic acid residues on glycosylated proteins generally result in an increased half-life compared to proteins with fewer or no sialic acid residues, whereas terminal galactose residues on carbohydrate chains are known to cause receptor-mediated clearance and reduce the serum half-life of the protein. Therefore, initial efforts focused on GAM PEG conjugation to block receptor-mediated C1INH clearance. While all three of these methods appear promising, we surprisingly found that amine and SAM pegylation resulted in the greatest degree of C1-INH pegylation with minimal and acceptable loss of potency. In contrast, GAM pegylation was less efficient and had greater heterogeneity.

An initial in vivo PK study was performed to evaluate pegylated C1-INH. In particular, SAM5kDa and 40kDa PEGylated C1-INH were compared to amino PEGylated C1-INH in a rat PK study. Referring to FIG. 6 (panels A-C), PEGylated C1-INH was quantitated using antigen assay using C1-INH to draw a standard curve. Samples were also analyzed by Western blot to check for potential degradation. In humans at doses of 1mg/kg IV and 3mg/kgIn the range of 2-3 mg/kg. These studies indicate that the half-life of pegylated proteins may be increased 3-4 fold due to decreased clearance.

A further pharmacokinetic study of C1-INH-PEG was performed using 1mg/kg intravenous administration to male SD rats. These results are provided in table 1 below.

PEG is linear. PEG is branched chain

TABLE 1 pharmacokinetic parameters of C1-INH-PEG intravenously administered to male Sprague Dawley (SD) rats.

Furthermore, in NHP studies, subcutaneous bioavailability of about 30% to 40% was observed, an unexpected improvement over the unconjugated recombinant C1-INH protein.

Thus, pegylated C1-INH appears to have a half-life suitable for therapeutic use and sufficient bioavailability.

Example 2 exemplary PEGylation protocol

Process A

Purified C1-INH was dialyzed into 100mM sodium acetate pH 5.6. Periodate oxidation was performed at 4 ℃ for 30 minutes. The reaction was quenched with glycerol at 4 ℃ for 15 minutes. The oxidized C1-INH was dialyzed into acetate buffer. The material was then pegylated at 4 ℃ overnight and then quenched with glycine. Free PEG was removed by anion exchange. An exemplary schematic of process a is provided in fig. 7.

The C1-INH-PEG 40KDa prepared by Process A was further purified using the following method.

Approximately 1mg of 40kDa PEG amine conjugated to C1-INH was diluted 20-fold with sample dilution buffer (5 mM NaPO 4pH 7.00). The resulting solution exhibited a conductivity of 0.716 mS/cm. The sample was loaded onto a 10mL GigaCap Q (650) column XK 16. The flow rate of the whole process was 150 cm/h. The column was washed extensively with sample dilution buffer, and then the protein was eluted with a 10 column volume gradient to 500mM NaCl. Fractions of 2mL were collected and samples were analyzed by SDS-PAGE. The chromatographic results are shown in Table 11. The peak fractions were then pooled and dialyzed into formulation buffer (50mM phosphate (pH 7.1), 150mM glycine, 50mM sorbitol), concentrated to ≥ 1.0mg/ml, and quantified by absorbance at 280nm (Nano-drop).

Similar purifications were performed for the C1-INH-PEG 20kDa preparation and are shown in FIG. 12, and for the C1-INH-PEG5kDa preparation and are shown in FIG. 13.

All samples were quantified on a nano-drop instrument using the extinction coefficient and the molecular weight of the amino acid sequence derived from the protein. The results are shown in table 2 below:

table 2: quantification of samples from the C1-INH PEGylation procedure.

Process B

Purified C1-INH was exchanged into 100mM sodium acetate pH 5.6 by TFF buffer exchange. Periodate oxidation was performed for 30 minutes at room temperature. Periodate was provided in a 40x molar excess. C1-INH was present in the reaction up to 4 mg/mL. The reaction was quenched with glycerol at room temperature for 15 minutes. The material was then pegylated at room temperature overnight. PEG was provided in 100x molar excess. C1-INH was present in the reaction up to 2 mg/mL. Free PEG was removed by TFF buffer exchange. An exemplary schematic of process B is provided in fig. 8.

Other exemplary pegylation protocols suitable for pegylation of C1-INH are summarized in fig. 9A-E.

SAM Process-PEG 5K

In this process, about 200mL of octyl loading material (about 0.9mg/mL of C1-INH in Tris/ammonium sulfate solution) buffer was exchanged into 100mM sodium acetate pH 5.6 using a Pellicon XL, Biomax,30kDa (PES) TFF cassette at 10x diafiltration volume exchange. 40 μ M C1-INH (3.7mg/mL) was treated with 1.6mM sodium periodate (40X) with gentle stirring at room temperature for 30 min (50mL reaction in 100mM sodium acetate, pH 5.6). The reaction was quenched with 1.5% glycerol at room temperature for 15 minutes.

21.6 μ M C1-INH (2mg/ml) was treated with 2.16mM 5kDa-PEG (100X) at room temperature and stirred gently overnight (92.5ml reaction in 100mM sodium acetate, pH 5.6). The reaction was then quenched with 2.16mM glycine (100X) for 1 hour at room temperature.

Free PEG was removed by diafiltration by TFF into 50mM sodium phosphate, 150mM glycine and 50mM sorbitol at pH 7.1 using Pellicon XL, Biomax 100kDa MWCO (PES) TFF cassette at 10x diafiltration volume exchange. The product was then filter sterilized using 22uM, PES, Millipore sterifp filters. The IC50 for the pegylated samples is shown in fig. 10 (panels a and B) and fig. 20 (panels a-B).

The yields after the individual process steps are shown in table 3 below:

table 3: octyl loading pegylation step yield.

SAM Process-PEG straight chain 2K, 5K, branched chain 5K, 10K, 20K, 40K

The SAM procedure was also used to prepare C1-INH-PEG with the following classes of PEG: linear 2K, linear 5K, branched 10K, branched 20K, and branched 40K.

C1-INH PEGylated with PEG2K, 5K and 10K was purified using an Amicon centrifugal filter (30K cut-off). C1-INH PEGylated with PEG-aminoxy 20K or 40K was purified by AKTA system to remove free PEG. The characterization of C1-INH is shown in FIG. 18(A-E panels). The purity and potency of C1-INH-PEG prepared by the SAM process was determined and PK was evaluated in a rat model. These data are provided in fig. 19(a-C panels). Other characterization and IC50 values for the pegylated samples are shown in fig. 24 (panels a and B).

The SAM PEGylation conditions for the production of C1-INH-PEG are shown in Table 4 below.

TABLE 4 SAM PEGylation conditions for the production of C1-INH-PEG.

PEGylation by amine coupling procedure

C1-INH-PEG was also prepared using an amine coupling procedure. A schematic of an exemplary pegylation by an amino coupling process is depicted in fig. 21.

C1-INH PEGylated with PEG1K, linear 5K and branched 5K was purified by Amicon centrifugal filter (30K cut-off). Barium iodide staining was used to detect free PEG in the PEG5K fraction, and RP-HPLC was used to detect free PEG1K and PEG 2K. C1-INH PEGylated with NHS-PEG20K and 40K was purified by AKTA pure chromatography system. Characterization of the PEGylated C1-INH is shown in FIG. 22 (panels A-D).

The purity, potency and PK of C1-INH-PEG prepared by the amine coupling process were determined and assessed in a rat model. These data are provided in fig. 23(a-C panels).

The PEGylation conditions for the production of C1-INH-PEG by the amine coupling process are shown in Table 5 below.

TABLE 5 PEGylation conditions for the production of C1-INH-PEG by amine coupling procedure. Pegylation with 100X PEG20K had low conversion and was retreated with additional 40X PEG 20K.

Example 3: non-human primate PK study of PEGylated C1-INH administered IV

Non-human primates (NHPs) (cynomolgus monkeys) were divided into two groups and 30mg/kg of recombinant human C1-INH (rhC1-INH) or 5mg/kg of PEGylated rhC1-INH was administered intravenously. Exemplary results of this study are summarized in fig. 14 and table 6.

In NHPs, pegylated rhC1-INH showed 6-fold lower clearance and 3-fold longer terminal half-life compared to rhC 1-INH. A similar trend was also observed in the rat study, showing a 4-fold decrease in clearance and a 4-fold increase in half-life.

Table 6: NHP PK study results of PEGylated rhC 1INH and rhC 1INH

^aOne of the three monkeys in the study showed an increase in elimination rate after 408 hours and was excluded from PK calculations

Effect of PEG Loading on PK of NHP with IV administration of C1-INH

Further PK studies were performed on NHPs. NHPs received C1-INH-PEG administered IV at 5x, 10x, 20x and 40x loading. Exemplary results are shown in fig. 15.

Example 4: IV PK Studies with SC administration of PEGylated C1-INH NHP

NHPs were divided into two groups and 5mg/kg PEGylated C1-INH was administered intravenously or 10mg/kg PEGylated C1-INH was administered Subcutaneously (SC). The results of this study are summarized in fig. 16 and table 7. The functional activity of pegylated C1-INH was maintained throughout the time course of the study (SA ═ 4.8U/mg).

Significantly and unexpectedly, pegylated C1-INH exhibited 85% bioavailability in NHPs and a half-life comparable to that of IV administration. Preclinical data collected to date supports the possibility of once weekly or even less frequent dosing.

Table 7: IV and SC administration of PEGylated rhC1-INH in NHP

After SC administration in NHP, F58% for hrC1-inh

Example 5: oxidation/titration to test minimum PEG to maximize PK Curve

The DT-1215 titration assay used is an ELISA-based method that captures PEG-rC1-INH protein from serum samples with anti-PEG antibodies. This protein was then detected with a labeled anti-C1-INH protein. A standard curve was drawn using PEG-rC 1-INH. FIG. 17 depicts the results of DT-1215 titration analysis and sample specific activity. Further data are provided in tables 8 and 9.

Table 8: changes in specific activity observed at different levels of periodate treatment. As seen in table 9 (below), the change in periodate levels resulted in different ratios of PEG to C1 INH.

Table 9: half-life achieved at different PEG levels compared to unconjugated C1 INH.

Example 6: physical characterization of PEGylated C1-INH

Purity of the PEGylated preparation was analyzed using SEC and SEC-MAL CD spectra of 0.1mg/ml PEG-C1-INH protein was measured at 25 deg.C CD data was processed by AVIV and CDNN software No significant change was observed when the protein was PEGylated based on CD spectra and secondary structure analysis.27% helices and 30% β -folds were determined according to the C-terminal crystal structure of C1-INH (2 OAY).

Table 10: the data indicate that PEGylation does not alter the secondary structure of C1-INH.

The melting temperature (Tm) of PEGylated C1-INH was measured by NanoDSF. Pegylation was found not to significantly alter the thermal stability of C1-INH. The Tm of the 40KDa amino-pegylated C-INH was measured to be 2 ℃ higher than for the other conjugates tested. The data are provided in table 11.

Table 11: tm analysis of PEGylated C1-INH.

Nuclear Magnetic Resonance (NMR) was used to characterize the pegylation level. PEGylation on amines is very low, about 3 PEG moieties per C1-INH. Sialic acids can be highly pegylated to reach saturation with a 5K PEG reactant. 40K PEGylation on sialic acid reaches about 9 PEGs per molecule. The pegylation level was quantified at different periodate concentrations. The data are provided in table 12.

Table 12: NMR characterization of PEGylated C1-INH preparations.

Example 7: characterization of C1-INH-PSA

C1-INH was conjugated to polysialic acid (PSA) by a Sialic Acid Mediated (SAM) process. The purity and potency of C1-INH-PSA prepared by the SAM process was determined. These data are provided in fig. 24(a and B panels). The data indicate that while free PSA does not interfere with the potency assay itself, the PSA tested here, C1-INH potency, decreases by about 4-7 fold under C1INH conditions.

Using C1-INH-PSA, C1-INH-PEG andPEG PK studies were performed in rats. Data are provided in fig. 25(a-C panels).

Equivalents and ranges

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

Claims (99)

1. A composition comprising a conjugated C1 esterase inhibitor (C1-INH), said conjugated C1 esterase inhibitor comprising:

a C1-INH protein comprising at least one glycan residue; and

at least one polyethylene glycol (PEG) moiety,

wherein the at least one polyethylene glycol (PEG) moiety is covalently attached to the at least one glycan residue.

2. A composition comprising a conjugated C1 esterase inhibitor (C1-INH), said conjugated C1 esterase inhibitor comprising:

C1-INH protein comprising at least one polyethylene glycol (PEG) moiety; and is

Wherein the at least one PEG moiety is covalently linked to the C1-INH protein through an oxime bond.

3. The composition of claim 2, wherein the oxime bond is between the PEG moiety and a glycan residue or amine group of C1-INH.

4. The composition of any one of the preceding claims, wherein the glycan residue is a sialic acid residue or a galactose residue.

5. The composition of claim 4, wherein the glycan residue is a sialic acid residue.

6. The composition of any one of the preceding claims, wherein the C1-INH protein is recombinantly produced or plasma derived.

7. The composition of any one of the preceding claims, wherein the C1-INH protein comprises a C1-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.

8. The composition of any one of the preceding claims, wherein the C1-INH protein is a fusion protein.

9. The composition of claim 7, wherein the fusion protein comprises an Fc domain fused directly or indirectly to a C1-INH domain.

10. The composition of claim 8, wherein the Fc domain is derived from IgG 1.

11. The composition of claim 8 or 9, wherein the Fc domain comprises amino acid substitutions corresponding to L234A and L235A according to EU numbering.

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

13. The composition of any one of the preceding claims, wherein prior to pegylation, 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.

14. The composition of any one of the preceding claims, wherein prior to pegylation, the C1-INH protein has a glycosylation profile comprising about 5% to about 25% neutral glycan species.

15. The composition of any one of the preceding claims, 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.

16. The composition of any one of the preceding claims, wherein the C1-INH protein contains less than about 20%, 15%, 10%, or 5% of one or more of mannose, α -galactose, NGNA, or oligomannose-type glycosylation prior to pegylation.

17. The composition of any one of the preceding claims, wherein prior to pegylation, the C1-INH protein has a glycosylation profile comprising one or more of:

about 5% to about 30% of neutral glycan species;

from about 10% to about 30% of a monosialylated glycan species;

from about 30% to about 50% of a disialylated glycan species;

from about 15% to about 35% trisialylated glycan species; or

From about 5% to about 15% tetrasialylated glycan species.

18. The composition of any one of the preceding claims, wherein prior to pegylation, the C1-INH protein has a glycosylation profile comprising:

no more than 30% neutral glycan species;

from about 20% to about 30% of a monosialylated glycan species;

from about 30% to about 40% of a disialylated glycan species;

from about 10% to about 20% of trisialylated glycan species; and

from about 5% to about 10% tetrasialylated glycan species.

19. The composition of any one of the preceding claims, wherein 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.

20. The composition of any one of the preceding claims, 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 moles of sialic acid per mole of protein

21. The composition of any one of the preceding claims, wherein the PEG has a molecular weight of about 1KDa to 50KDa, about 1KDa to 40KDa, about 5KDa to 40KDa, about 1KDa to 30KDa, about 1KDa to 25KDa, about 1KDa to 20KDa, about 1KDa to 15KDa, about 1KDa to 10KDa, or about 1KDa to 5 KDa.

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

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

24. The composition of any one of the preceding claims, wherein the conjugated C1-INH has a half-life comparable to or longer than plasma-derived human C1-INH.

25. The composition of any one of the preceding claims, wherein the half-life of the conjugated C1-INH is in the range of 100% -500% of the half-life of the plasma-derived C1-INH.

26. The composition of any one of the preceding claims, 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.

27. The composition of any one of claims 1 to 14, 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.

28. The composition of any one of the preceding claims, wherein the specific activity of the conjugated C1-INH is in the range of 50-150% of the specific activity of plasma-derived human C-INH.

29. A method of preparing a conjugated C1 esterase inhibitor (C1-INH), the method comprising the steps of:

providing a C1-INH protein comprising at least one glycan residue and/or at least one amine group; and

providing a PEG moiety to form a bond under conditions that allow the PEG moiety to react with the at least one glycan residue and/or the at least one amine group, thereby preparing the conjugated C1-INH.

30. The method of claim 29, wherein the PEG moiety comprises PEG-CH₂-O-NH₂。

31. The method of claim 29 or 30, wherein the at least one glycan residue is a sialic acid residue.

32. The method of any one of claims 29 to 31, wherein the at least one glycan residue is a galactose residue.

33. The method of any one of claims 29 to 32, wherein the method further comprises the step of oxidizing the at least one glycan residue prior to reacting with the PEG moiety.

34. The method of claim 33, wherein the oxidizing step comprises periodate oxidation.

35. The method of claim 30, wherein the periodate oxidation is carried out at a periodate to C1-INH molar ratio of about 20:1 to about 50: 1.

36. The method of claim 35, wherein the molar ratio of periodate to PEG is from about 2.5 to about 40.

37. The method of any one of claims 29-36, wherein the molar ratio of PEG to C1-INH is about 25:1 to 100: 1.

38. The method according to any one of claims 29 to 37, wherein the method further comprises the step of purifying the conjugated C1-INH.

39. The method of claim 38, wherein the purifying step comprises one or more of anion exchange, tangential flow filtration diafiltration, and dialysis.

40. A conjugated C1 esterase inhibitor (C1-INH) prepared by the method of any one of claims 29 to 39.

41. A pharmaceutical composition comprising a conjugated C1 esterase inhibitor (C1-INH) according to any one of claims 1 to 28 and 40 and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of claim 41, wherein the composition is a liquid.

43. The pharmaceutical composition of claim 41, wherein the composition is lyophilized.

44. A kit comprising a pharmaceutical composition according to any one of claims 41 to 43 and

a syringe.

45. The kit of claim 44, wherein the syringe is pre-loaded with the pharmaceutical composition.

46. The kit of claim 44, wherein the pharmaceutical composition is lyophilized and the kit further comprises a reconstitution buffer.

47. A method of treating a complement-mediated disorder comprising administering to a subject in need of treatment a pharmaceutical composition according to any one of claims 41-47.

48. The method of claim 47, wherein the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 Inflammatory Demyelinating Polyneuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

49. Use of a composition comprising a conjugated C1 esterase inhibitor according to any of claims 1 to 28 and 40, for the manufacture of a medicament for treating a complement-mediated disorder.

50. The use of claim 49, wherein the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 Inflammatory Demyelinating Polyneuropathy (CIDP), myasthenia gravis, and/or multifocal motor neuropathy.

51. A composition comprising a conjugated C1 esterase inhibitor (C1-INH), said conjugated C1 esterase inhibitor comprising:

a C1-INH protein comprising at least one glycan residue;

at least one polysialic acid (PSA) moiety,

wherein the at least one polysialic acid (PSA) moiety is covalently linked to the at least one glycan residue.

52. A composition comprising a conjugated C1 esterase inhibitor (C1-INH), said conjugated C1 esterase inhibitor comprising

A C1-INH protein comprising at least one glycan residue; and

at least one polysialic acid (PSA) moiety,

wherein the at least one polysialic acid (PSA) moiety is covalently linked to the C1-INH protein via an oxime or hydrazone bond.

53. The composition of claim 52, wherein the at least one polysialic acid (PSA) moiety is covalently attached to the C1-INH protein via an oxime bond.

54. The composition of claim 52, wherein the at least one polysialic acid (PSA) moiety is covalently attached to the C1-INH protein through a hydrazone bond.

55. The composition of claim 52, wherein the oxime bond is between the PSA moiety and a glycan residue or amine group of C1-INH.

56. The composition of any one of claims 51-55, wherein the glycan residue is a sialic acid residue.

57. The composition of any one of claims 51 to 56, wherein said C1-INH protein is recombinantly produced or plasma derived.

58. The composition of any one of claims 51-57, wherein the C1-INH protein comprises a C1-INH domain having an amino acid sequence at least 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.

59. The composition of any one of claims 51 to 58, wherein the C1-INH protein is a fusion protein.

60. The composition of claim 58, wherein the fusion protein comprises an Fc domain fused directly or indirectly to a C1-INH domain.

61. The composition of claim 59, wherein the Fc domain is derived from IgG 1.

62. The composition of claim 59 or 60, wherein the Fc domain comprises amino acid substitutions corresponding to L234A and L235A according to EU numbering.

63. The composition of claim 59, wherein the fusion protein comprises an albumin domain fused directly or indirectly to a C1-INH domain.

64. The composition of any one of claims 51-63, wherein prior to PEGylation, the C1-INH protein has a glycosylation profile comprising no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of neutral glycan species.

65. The composition of any one of claims 51-64, wherein prior to PEGylation, the C1-INH protein has a glycosylation profile comprising about 5% to about 25% neutral glycan species.

66. The composition of any one of claims 51 to 65, 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.

67. The composition of any one of claims 51 to 66, wherein the C1-INH protein contains less than about 20%, 15%, 10%, or 5% of one or more of mannose, α -galactose, NGNA, or oligomannose-type glycosylation prior to conjugation to PSA.

68. The composition of any one of claims 51 to 67, wherein prior to conjugation to PSA, the C1-INH protein has a glycosylation profile comprising one or more of:

about 5% to about 30% of neutral glycan species;

from about 10% to about 30% of a monosialylated glycan species;

from about 30% to about 50% of a disialylated glycan species;

from about 15% to about 35% trisialylated glycan species; or

From about 5% to about 15% tetrasialylated glycan species.

69. The composition of any one of claims 51 to 68, wherein prior to conjugation to PSA, the C1-INH protein has a glycosylation profile comprising:

no more than 30% neutral glycan species;

from about 20% to about 30% of a monosialylated glycan species;

from about 30% to about 40% of a disialylated glycan species;

from about 10% to about 20% of trisialylated glycan species; and

from about 5% to about 10% tetrasialylated glycan species.

70. The composition of any one of claims 51 to 69, wherein 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.

71. The composition of any one of claims 51-69, 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 moles of sialic acid per mole of protein

72. The composition according to any one of claims 51 to 71, wherein the PSA has a molecular weight of about 1 to 50kDa, about 1 to 40kDa, about 5 to 40kDa, about 1 to 30kDa, about 1 to 25kDa, about 1 to 20kDa, about 1 to 15kDa, about 1 to 10kDa, or about 1 to 5 kDa.

73. The composition according to any one of claims 51 to 72, wherein the PSA has a molecular weight of about 1kDa, 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, or 50 kDa.

74. The composition of any one of claims 51 to 73, wherein the conjugated C1-INH has a PSA/C1-INH ratio of about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5.

75. The composition of any one of claims 51 to 73, wherein the conjugated C1-INH has a half-life comparable to or longer than plasma-derived human C1-INH.

76. The composition of any one of claims 51 to 73, wherein the half-life of C1-INH is in the range of 100% -500% of the half-life of the plasma-derived C1-INH.

77. The composition of any one of claims 51 to 76, 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.

78. The composition of any one of claims 51 to 76, 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.

79. The composition of any one of claims 51 to 76, wherein the specific activity of the conjugated C1-INH is in the range of 50% -150% of the specific activity of plasma-derived human C-INH.

80. A method of preparing a conjugated C1 esterase inhibitor (C1-INH), the method comprising the steps of:

providing a C1-INH protein comprising at least one glycan residue and/or at least one amine group; and

providing a polysialic acid (PSA) moiety to form a bond under conditions that allow the PSA moiety to react with the at least one glycan residue and/or the at least one amine group, thereby preparing a conjugated C1-INH.

81. The method of claim 80, wherein the at least one glycan residue is a sialic acid residue.

82. The method according to any one of claims 80 to 81 wherein the method further comprises the step of oxidizing the at least one glycan residue prior to reacting with the PSA moiety.

83. The method of claim 82, wherein the oxidizing step comprises periodate oxidation.

84. The method of claim 83, wherein the periodate oxidation is carried out at a periodate to C1-INH molar ratio of about 20:1 to about 50: 1.

85. The method of claim 84, wherein the molar ratio of periodate to PSA is from about 2.5 to about 40.

86. The process of any one of claims 80 to 85 wherein the molar ratio of PSA to C1-INH is about 25:1 to 100: 1.

87. The method according to any one of claims 80 to 86, wherein the method further comprises the step of purifying the conjugated C1-INH.

88. The method of claim 87, wherein the purifying step comprises one or more of anion exchange, tangential flow filtration diafiltration, and dialysis.

89. A conjugated C1 esterase inhibitor (C1-INH) prepared by the method of any one of claims 78 to 86.

90. A pharmaceutical composition comprising a conjugated C1 esterase inhibitor (C1-INH) according to any one of claims 51 to 79 and 89 and a pharmaceutically acceptable carrier.

91. The pharmaceutical composition of claim 90, wherein the composition is a liquid.

92. The pharmaceutical composition of claim 90, wherein the composition is lyophilized.

93. A kit comprising the pharmaceutical composition of any one of claims 90-92 and a syringe.

94. The kit of claim 93, wherein the syringe is pre-loaded with the pharmaceutical composition.

95. The kit of claim 93, wherein the pharmaceutical composition is lyophilized and the kit further comprises a reconstitution buffer.

96. A method of treating a complement-mediated disorder comprising administering to a subject in need of treatment a pharmaceutical composition according to any one of claims 90-92.

97. The method of claim 96, wherein the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 Inflammatory Demyelinating Polyneuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

98. Use of a composition comprising a conjugated C1 esterase inhibitor according to any of claims 51 to 79 and 89, for the manufacture of a medicament for treating a complement-mediated disorder.

99. The use of claim 98, wherein the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica lineage disease, 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 Inflammatory Demyelinating Polyneuropathy (CIDP), myasthenia gravis, and/or multifocal motor neuropathy.