Factor B protease

Abstract

Provided herein is an engineered protease of the S1A family that is specific to and capable of cleaving factor B. Also provided herein are methods for making and using such engineered proteases. The engineered proteases provided herein can be useful for treating diseases or conditions associated with dysregulation of the complement system by reducing complement activation through cleavage and inactivation of factor B.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 135,496, filed January 8, 2021, and U.S. Provisional Patent Application No. 63 / 221,108, filed July 13, 2021, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] The complement system includes the classical, alternative and lectin pathways and is tightly controlled by several regulatory factors and components. One such component is complement factor B (interchangeably referred to herein as CFB, factor B, FB), a serine protease proenzyme that circulates in the blood as a single-chain polypeptide. When factor B associates with an active form of C3, such as surface-bound C3b or fluid-phase C3(H2O), to form a proconvertase complex, factor B can subsequently be cleaved by factor D into two fragments, Ba and Bb. The cleavage site of factor B targeted by factor D contains the Arg234-Lys235 bond. The resulting Ba cleavage product is non-catalytic and is released from the complex, whereas the resulting Bb cleavage product is a catalytic serine protease that can then cleave C3 into C3a and C3b. This generation of C3b is part of the amplification loop of the complement system, allowing C3b to bind to another factor B to form C3bBb.

[0003] Factor B and its cleavage products regulate complement activation. Dysregulated complement is implicated in diseases involving the complement system, and thus there is a need for methods to regulate or inhibit specific points of regulation within the complement system, such as the generation of inactive factor B fragments. Provided herein are compositions and methods that address these needs. Summary of the Invention

[0004] Provided herein are engineered, non-naturally occurring chymotrypsin-like serine proteases. Also provided herein are methods of making and using such non-naturally occurring chymotrypsin-like serine proteases. The engineered proteases provided herein can be useful for treating diseases or conditions associated with dysregulation of the complement system or overactivation of complement.

[0005] Thus, in one aspect, provided herein is an engineered protease of the S1A serine protease family, the engineered protease being specific for and capable of cleaving factor B. More specifically, the engineered protease of the present disclosure comprises a modified chymase protease domain, a modified membrane-type serine protease 1 (MTSP-1) protease domain, a modified urokinase-type plasminogen activator (uPA) protease domain, or a modified kallikrein-related peptidase 5 (KLK5) protease domain, the engineered protease being capable of cleaving factor B. The modifications include one or more of substitution, addition, and deletion of one or more amino acid residues, and / or one or more of substitution, addition, and deletion of one or more domains of the chymotrypsin-like serine protease.

[0006] In some embodiments, the engineered protease is based on the MTSP-1 protease domain. In some embodiments, the engineered protease is not based on the MTSP-1 protease domain. In some embodiments, the engineered protease comprises one or more modifications with respect to the MTSP-1 protease domain comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the one or more modifications are selected from those set forth in Table 5A. In some embodiments, the one or more modifications are selected from the exemplary mutation sequence set forth in Table 5B. In some embodiments, the engineered protease comprises a modified membrane serine protease 1 (MTSP-1) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:7.

[0007] In some embodiments, the engineered protease is based on a uPA protease domain. In some embodiments, the engineered protease is not based on a uPA protease domain. In some embodiments, the engineered protease comprises one or more modifications with respect to a uPA protease domain comprising the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the one or more modifications are selected from those set forth in Table 3A. In some embodiments, the one or more modifications are selected from the exemplary mutation sequence set forth in Table 3B. In some embodiments, the engineered protease comprises a modified urokinase-type plasminogen activator (uPA) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:22.

[0008] In some embodiments, the engineered protease comprises one or more modifications with respect to a chymase protease domain comprising the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the one or more modifications are selected from those set forth in Table 7A. In some embodiments, the one or more modifications are selected from the exemplary mutation series set forth in Table 7B. In some embodiments, the engineered protease comprises a modified chymase protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:6.

[0009] In some embodiments, the engineered protease is based on the KLK5 protease domain, and optionally includes one or more amino acid modifications of SEQ ID NO: 23. In some embodiments, the engineered protease includes a modified kallikrein-related peptidase 5 (KLK5) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:23.

[0010] In some embodiments, cleavage of factor B with an engineered protease generates one or more functionally inactive fragments. In some embodiments, the one or more functionally inactive fragments are capable of reducing complement activation. In some embodiments, cleavage of factor B results in the generation of a factor B fragment with reduced functionality.

[0011] In some embodiments, factor B is a rodent factor B. In some embodiments, factor B is a non-human primate factor B. In some embodiments, the non-human primate is a cynomolgus monkey. In some embodiments, factor B is a human factor B. In some embodiments, factor B comprises the amino acid sequence shown in SEQ ID NO:1.

[0012] In some embodiments, cleavage of factor B occurs at a site that is not targeted by factor D. In some embodiments, cleavage at that site produces at least two fragments that are not Ba and Bb. In some embodiments, cleavage at that site results in reduced production of the factor B cleavage products Ba and Bb compared to cleavage by factor D.

[0013] In some embodiments, cleavage of factor B occurs at a site targeted by factor D. In some embodiments, the site targeted by factor D comprises QQKR / KIV (SEQ ID NO:9). In some embodiments, the factor B cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO:10), KNQKR / QKQ (SEQ ID NO:11), DVFY / QMI (SEQ ID NO:12), EGVD / AE (SEQ ID NO:13), DHKL / KSG (SEQ ID NO:14), TPW / SLA (SEQ ID NO:15), KVSEAD (SEQ ID NO:20), IRPSKG (SEQ ID NO:4), GGEKRD (SEQ ID NO:5), GKKEAG (SEQ ID NO:3), and DHKL / KSG (SEQ ID NO:21).

[0014] In some embodiments, the engineered protease is based on MTSP-1 or uPA (comprising an MTSP-1 or uPA protease domain) and the cleavage site in factor B comprises a sequence selected from the following: WEHR / KGT (SEQ ID NO: 10) and KNQKR / QKQ (SEQ ID NO: 11).

[0015] In some embodiments, the engineered protease is based on a chymase protease domain, in some embodiments, the engineered protease is based on a chymase protease domain and the cleavage site comprises a sequence selected from DVFY / QMI (SEQ ID NO: 12), EGVD / AE (SEQ ID NO: 13), DHKL / KSG (SEQ ID NO: 14), and TPW / SLA (SEQ ID NO: 15).

[0016] In some embodiments, cleavage of factor B results in the generation of a factor B fragment with reduced functionality or results in a factor B with reduced functionality. In some embodiments, the function of factor B or the factor B fragment is interaction with at least one complement component. In some embodiments, the function of factor B or the factor B fragment is interaction with hydrolyzed soluble C3. In some embodiments, the function of factor B or the factor B fragment is interaction with C3b. In some embodiments, the function of factor B or the factor B fragment is interaction with membrane-bound C3b.

[0017] In some embodiments, cleavage occurs when factor B is not bound to C3b.

[0018] In some embodiments, the cleavage activity towards non-factor B peptide substrates is about equal to or less than the cleavage activity towards the factor B site.

[0019] In some embodiments, the engineered protease has a cleavage potency of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, or about 1,900 M for factor B cleavage. -1 s -1 k cat / K m In some embodiments, the engineered protease has a cleavage efficiency of about 10 for factor B cleavage. 3 ~about 10 9 M -1 s -1 k cat / K m In some embodiments, the engineered protease has an EC 50 In some embodiments, the engineered protease has an EC 50 In some embodiments, the engineered protease has an EC 50 In some embodiments, the engineered protease has an EC 50 is about 1,000 to about 4,500 nM.

[0020] In some embodiments, the engineered protease has a plasma half-life in human plasma of greater than about 72 hours. In some embodiments, the engineered protease has a plasma half-life in human plasma of greater than about 120 hours. In some embodiments, the engineered protease has a plasma half-life in human plasma of about 7 days. In some embodiments, the catalytic activity is about 10% to about 50%, or about 90% to about 100%.

[0021] In some embodiments, the engineered protease has an increased half-life compared to the unmodified MTSP-1 protease domain. In some embodiments, the engineered protease has an increased bioavailability compared to the unmodified MTSP-1 protease domain. In some embodiments, the engineered protease has an increased half-life compared to the unmodified uPA protease domain. In some embodiments, the engineered protease has an increased bioavailability compared to the unmodified uPA protease domain. In some embodiments, the engineered protease has an increased half-life compared to the unmodified chymase protease domain. In some embodiments, the engineered protease has an increased bioavailability compared to the unmodified chymase protease domain. In some embodiments, the engineered protease has an increased bioavailability compared to the unmodified KLK5 protease domain.

[0022] In some embodiments, the engineered protease is non-immunogenic.

[0023] In some embodiments, the engineered protease is in a zymogen form. In some embodiments, the engineered protease is in an active form.

[0024] In some embodiments, the engineered protease further comprises a half-life extender. Exemplary half-life extenders include human serum albumin (HSA) and Fc (e.g., IgG1) fused to the engineered protease.

[0025] In another aspect, a method of inactivating factor B is provided, comprising contacting factor B with any of the engineered proteases of the present disclosure. In some embodiments, complement activation is inhibited. In some embodiments, the classical pathway of the complement pathway is inhibited. In some embodiments, the alternative pathway of the complement pathway is inhibited. In some embodiments, the lectin pathway of the complement pathway is inhibited.

[0026] In some embodiments, the method is in vitro. In some embodiments, the method is in vivo.

[0027] In another aspect, a method of treating a disease or condition in a subject in need thereof is provided, comprising administering to the subject any one of the engineered proteases of the present disclosure. In some embodiments, the disease or condition is associated with dysregulated complement. In some embodiments, the disease or condition is an inflammatory disease or condition. In some embodiments, the treatment is replacement therapy. In some embodiments, the treatment blocks complement activation. In some embodiments, the treatment modulates autoimmunity. In some embodiments, the disease or condition is a congenital complement deficiency. In some embodiments, the treatment is for endothelial cell injury or renal cell injury.

[0028] In some embodiments, the disease or condition is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplant ischemia and reperfusion (I / R) injury, antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), atypical hemolytic uremic syndrome (aHUS), membranous nephropathy (MN), and paroxysmal nocturnal hemoglobinuria (PNH). In some embodiments, the disease or condition is a regulatory protein deficiency. In some embodiments, the disease or condition is a secondary complement disorder. In some embodiments, the disease or condition is an immune-related disease or condition.

[0029] In some embodiments, the engineered protease is administered subcutaneously to the subject.

[0030] In some embodiments, the engineered proteases are activated in situ at the site of a dysregulated complement component.

[0031] In some embodiments, the engineered protease is provided in a liquid stable formulation.

[0032] In another aspect, a pharmaceutical composition is provided comprising any of the engineered proteases of the present disclosure, and optionally a pharma- ceutically acceptable carrier.

[0033] In some embodiments, the engineered protease is provided in a liquid stable formulation.

[0034] In some embodiments, the composition is formulated for subcutaneous administration. [Brief description of the drawings]

[0035] [Figure 1-A] FIG. 1 shows a schematic diagram of naturally occurring factor B showing the site at which factor B is cleaved by factor D (FD cleavage site; SEQ ID NOs: 13, 9, 20, residues 1-6 of SEQ ID NO: 12, SEQ ID NOs: 4, 5, 3, and residues 1-6 of SEQ ID NO: 11). [Figure 1-B]FIG. 2 shows two views of the protein structure of factor B indicating the various cleavage sites (SEQ ID NO: 13, 9, 11, 10, residues 1-6 of SEQ ID NO: 12 and SEQ ID NO: 14). [Figure 1-C] FIG. 2 shows two views of the protein structure of factor B indicating the various cleavage sites (SEQ ID NO: 13, 9, 11, 10, residues 1-6 of SEQ ID NO: 12 and SEQ ID NO: 14). [Figure 1-D] 1 shows a Coomassie gel showing an example of factor B cleavage by a chymase-based engineered protease. [Figure 1-E] 1 shows a graph showing an example of factor B cleavage by two chymase-based engineered proteases with low EC50. [Figure 2-A] Schematic diagrams of prochymase and mature chymase, respectively, are shown, with mature chymase providing an exemplary scaffold of the present disclosure. [Figure 2-B] Schematic diagrams of prochymase and mature chymase, respectively, are shown, with mature chymase providing an exemplary scaffold of the present disclosure. [Figure 3-A] Schematic diagrams of the extracellular portion of membrane serine protease 1 (MTSP-1) and the serine protease domain of MTSP-1, respectively. [Figure 3-B] Schematic diagrams of the extracellular portion of membrane serine protease 1 (MTSP-1) and the serine protease domain of MTSP-1, respectively. [Figure 3-C] 1 shows a schematic diagram of urokinase-type plasminogen activator (uPA or u-PA). [Figure 3-D] 1 shows a schematic diagram of urokinase-type plasminogen activator (uPA or u-PA). [Figure 4-A] 1 shows a graph depicting the stability of engineered chymase-based engineered proteases tested using peptide substrates. [Figure 4-B] 1 shows a graph depicting the stability of engineered chymase-based engineered proteases tested using peptide substrates. [Figure 4-C] 1 shows a graph depicting the stability of engineered chymase-based engineered proteases tested using peptide substrates. [Figure 4-D] 1 shows a graph depicting the stability of engineered chymase-based engineered proteases tested using peptide substrates. [Figure 4-E] 1 shows a graph depicting the stability of engineered chymase-based engineered proteases tested using peptide substrates. [Diagram 5] 1 shows a bar graph of the controls used in the Factor B add-back hemolysis assay. [Figure 6] 1 shows a graph depicting a standard curve of the results of a Factor B add-back hemolysis assay. [Figure 7-A] 1 shows a graph showing inhibition of hemolysis by KLK5 and chymase-based engineered protease C22S / F173Y / D175N / A226R. [Figure 7-B] 1 shows a graph depicting FB cleavage by two concentrations of KLK5 and chymase-based engineered protease C22S / F173Y / D175N / A226R. [Figure 8-A] 1 shows a Coomassie gel showing an example of factor B cleavage by KLK5 and the chymase-based engineered protease C22S / F173Y / D175N / A226R. [Figure 8-B] 1 shows a Coomassie gel showing an example of factor B cleavage by KLK5 and the chymase-based engineered protease C22S / F173Y / D175N / A226R. [Figure 9] 1 shows mass spectrometry (MS) data identifying a cleavage site at 234Arg within the QQKR / KIV (sequence number 9) cleavage site of factor B by KLK5 and a cleavage site at 221Asp within the EGVDAE (sequence number 13) cleavage site of factor B by the chymase-based engineered protease C22S / A226R. [Figure 10] FIG. 1 is a schematic diagram showing a general method for measuring complement activation and cytokine release from tissues of a mouse model of acute respiratory distress syndrome (ARDS) treated with chymase-based engineered proteases. [Figure 11-A]FIG. 1 shows pulmonary congestion index, body weight loss, and neutrophil-to-lymphocyte ratio in bronchoalveolar lavage fluid (BALF NLR), respectively, measured in a mouse model of acute respiratory distress syndrome (ARDS). [Figure 11-B] FIG. 1 shows pulmonary congestion index, body weight loss, and neutrophil-to-lymphocyte ratio in bronchoalveolar lavage fluid (BALF NLR), respectively, measured in a mouse model of acute respiratory distress syndrome (ARDS). [Figure 11-C] FIG. 1 shows pulmonary congestion index, body weight loss, and neutrophil-to-lymphocyte ratio in bronchoalveolar lavage fluid (BALF NLR), respectively, measured in a mouse model of acute respiratory distress syndrome (ARDS). [Figure 12] 1 shows the results of BALF and lung cytokine measurements from mouse tissues after treatment with chymase-based engineered proteases. [Figure 13] FIG. 1 is a schematic diagram showing a general method for measuring lung function in a mouse model of acute respiratory distress syndrome (ARDS) treated with a chymase-based engineered protease of the present disclosure. [Figure 14] 1 shows the results of plethysmography measurements and significant protection against pulmonary congestion upon administration of one of the chymase-based engineered proteases of the present disclosure. [Figure 15] 1 is an SDS-PAGE (reduced) showing expression, purification, and activation of the chymase-based engineered proteases of the present disclosure. [Figure 16] 1 is an SDS-PAGE showing expression of chymase-based engineered proteases in HEK293 cells. [Figure 17] FIG. 1 is an SDS-PAGE showing expression of chymase-based engineered proteases fused to human HSA or Fc in HEK293 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present disclosure provides compositions and methods useful for regulating the signal transduction and regulation of the complement system. Specifically, engineered proteases are provided herein that contain a protease domain of the chymotrypsin-like S1A serine protease family, and such engineered proteases of the present disclosure are specific for and can cleave factor B. These engineered proteases contain modified protease domains and are generated using the methods and sequences provided herein.

[0037] The engineered proteases of the present disclosure target factor B for cleavage and are interchangeably referred to as "complement factor B degrading factors" or "CFB degrading factors." Use of these engineered proteases can result in (1) cleavage of factor B into fragments that are neither Ba nor Bb, or (2) cleavage of factor B into Ba and Bb, which are functionally inactive. Without being bound by any theory or mechanism, the fragments Ba and Bb resulting from cleavage of factor B while it is not complexed with C3 or C3b are believed to be functionally inactive fragments that may further reduce or inhibit complement activation, act to limit increased complement activation, and / or limit / reduce amplification of the complement pathway. In either of these scenarios, these cleavage products may have functions that are not the native functions of Ba and Bb, or may be inactive fragments. As used herein, a "functionally inactive" fragment of factor B refers to a fragment of factor B that is a cleavage product that can reduce complement activation, limit the increase in complement activation, and / or limit / reduce the amplification of the complement pathway.

[0038] In some embodiments, the engineered protease targets factor B for cleavage at a cleavage site that is not targeted by factor D. In other embodiments, the engineered protease targets factor B at a site that is targeted by factor D before factor B associates with C3b to form a complex, thus preventing formation of the proconvertase complex.

[0039] The disclosure also provides methods of making and using such engineered proteases, for example, in the treatment of diseases or conditions associated with complement dysregulation, e.g., treating an overactive complement response.

[0040] Chymotrypsin-like serine proteases useful for regulating the complement system by targeting factor B Provided herein are engineered proteases that contain one or more modifications relative to naturally occurring chymotrypsin-like serine proteases. As used herein, an "engineered" protease of the present disclosure is a non-naturally occurring S1A family serine protease that contains one or more modifications relative to a wild-type or naturally occurring S1A family serine protease. As used herein, a "modification" to a naturally occurring S1A family chymotrypsin-like serine protease includes one or more of the following: deletion of one or more amino acid residues, deletion of one or more domains, substitution of one or more amino acid residues, insertion of one or more amino acid residues, insertion of one or more domains, and substitution of one or more domains.

[0041] In some embodiments, an engineered protease of the present disclosure comprises a non-naturally occurring serine protease domain of the S1A family, which domain comprises one or more modifications relative to a wild-type or naturally occurring serine protease domain of the S1A family. As used herein, a "modification" to a naturally occurring chymotrypsin-like serine protease domain of the S1A family includes one or more of the following: a deletion of one or more amino acid residues, a substitution of one or more amino acid residues, and an insertion of one or more amino acid residues.

[0042] The engineered proteases of the present disclosure comprise a non-naturally occurring serine protease domain of the S1A family. In some embodiments, the engineered proteases of the present disclosure comprise a non-naturally occurring serine protease domain of the S1A family and also comprise additional sequences and / or additional domains. In some embodiments, the engineered proteases of the present disclosure consist of a non-naturally occurring serine protease domain of the S1A family. In some embodiments, the engineered proteases of the present disclosure consist essentially of a non-naturally occurring serine protease domain of the S1A family and may include additional sequences useful for expression, stability, improved pharmacokinetics, subcutaneous delivery, tissue targeting, and the like.

[0043] As used herein, "naturally occurring chymotrypsin-like serine protease" of the S1A family refers to such a protease that exists in nature, even if it is not a wild-type sequence. In other words, the naturally occurring serine protease is not engineered. The naturally occurring serine protease may or may not have a signal sequence, may or may not have an activation peptide, and may be of any species.

[0044] The engineered proteases provided herein are designed to cleave factor B. In some embodiments, the engineered proteases provided herein target factor B at a non-factor D cleavage site. In some embodiments, the engineered proteases provided herein target factor B at a factor D cleavage site. In some embodiments, the engineered proteases provided herein can target factor B for cleavage while factor B is in a complex with C3. In some embodiments, the engineered proteases provided herein can target factor B for cleavage while factor B is in a complex with C3b. In some embodiments, the engineered proteases provided herein can target factor B for cleavage while factor B is circulating alone.

[0045] In some embodiments, the cleavage products resulting from cleavage of factor B with the engineered proteases provided herein may be functionally inactive fragments, as described above. In some embodiments, functionally inactive fragments have no naturally occurring physiological function. In some embodiments, functionally inactive fragments may perform a function, but not necessarily the same function as Ba and / or Bb.

[0046] In some embodiments, the engineered proteases provided herein can modulate the activity of the complement system by reducing the amount of factor B fragments Ba and Bb produced, thereby suppressing / inhibiting complement activation, limiting increased complement activation, and / or limiting / reducing amplification of the complement pathway.

[0047] In some embodiments, the engineered proteases provided herein are useful for administration to a subject in need thereof. As used herein, the terms "patient" or "subject" are used interchangeably and refer to mammals, including, but not limited to, humans and other primates (e.g., chimpanzees, cynomolgus monkeys, and other ape and monkey species), livestock (e.g., cows, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), and laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs). In an exemplary embodiment, the subject is a human.

[0048] Table 1 provides the amino acid sequence of human factor B targeted by the engineered proteases of the present disclosure.

[0049] Table 1 also provides amino acid sequences of exemplary chymotrypsin-like serine proteases and S1A family protease domains that can be utilized as scaffolds from which engineered proteases of the present disclosure are generated, including MTSP-1, uPA, chymase, and kallikrein-related peptidase 5 (KLK5). Thus, in some embodiments, the engineered proteases provided herein are based on MTSP-1, or a serine protease domain thereof. In some embodiments, the engineered proteases provided herein are based on uPA, or a serine protease domain thereof. In some embodiments, the engineered proteases provided herein are based on KLK5, or a serine protease domain thereof. In some embodiments, the engineered proteases provided herein are not based on chymase, or a serine protease domain thereof. In some embodiments, the engineered proteases provided herein are not based on MTSP-1, or a serine protease domain thereof. In some embodiments, the engineered proteases provided herein are not based on uPA or its serine protease domain. In some embodiments, the engineered proteases provided herein are not based on KLK5 or its serine protease domain. In some embodiments, the engineered proteases provided herein are not based on chymase or its serine protease domain.

[0050] Table 1 also includes the amino acid sequence of the mature chymotrypsin polypeptide in SEQ ID NO: 19. Note that the protease domain of the serine protease can be aligned with the protease domain of chymotrypsin such that the amino acid residues of the aligned proteases (e.g., MTSP-1, uPA, KLK5, or chymase) correspond to the amino acids of chymotrypsin and are provided with the numbering of chymotrypsin. This is generally referred to herein as chymotrypsin numbering, and the numbering and corresponding positions of the aligned proteases can be determined by one of skill in the art. Standard nomenclature (e.g., notation used for additions or deletions or residues) useful for chymotrypsin numbering can also be determined by one of skill in the art. Residues present in the aligned proteases (e.g., MTSP-1, uPA, KLK5, or chymase) that are not present in chymotrypsin are provided in lowercase notation. As an example, using the chymotrypsin numbering key for the uPA protease domain (Table 2), modified S37dP (chymotrypsin numbering) translates to S184P in conventional amino acid sequence notation. In Table 1, the signal or leader sequence is underlined and the cleavage site sequence is bolded. In some instances, the disclosure and claims include reference to conventional amino acid numbering and / or chymotrypsin-based numbering and are specified accordingly.

[0051] The chymase in Table 1 is mast cell chymase, the sequence of which can be found at https: / / www.uniprot.org / uniprot / P23946.

[0052] [Table 1-1]

[0053] [Table 1-2]

[0054] [Table 1-3]

[0055] [Table 1-4]

[0056] Figure 1A shows a schematic diagram of naturally occurring factor B showing various cleavage site sequences, including the site where factor B is cleaved by factor D into Ba and Bb (FD cleavage site). Ba is composed of three complement control protein (CCP) domains and a linker. Bb is composed of a von Willebrand factor type A (VWA) domain and a serine protease (SP) domain. Figures 1B-1C show two views of the protein structure of factor B showing the various cleavage sites.

[0057] In some embodiments, the present disclosure provides engineered proteases that cleave factor B, but at a cleavage site that is not targeted by factor D, i.e., a cleavage site that is not an FD cleavage site. In other embodiments, the present disclosure provides engineered proteases that cleave factor B at a cleavage site that is targeted by factor D. As contemplated herein, an FD cleavage site is a site that is the amino acid sequence of QQKR / KIV (SEQ ID NO: 9). In other embodiments, the present disclosure also provides engineered proteases that can cleave factor B at the cleavage site that is targeted by factor D (SEQ ID NO: 9) before factor B forms a complex with C3b, but without being bound by any theory or mechanism, it is anticipated that such cleavage can result in a fragment that does not increase complement activity.

[0058] Various exemplary cleavage sites of factor B that can be targeted by the engineered proteases of the present disclosure are shown in the schematic diagram of Figure 1 and include, but are not limited to, QQKR / KIV (SEQ ID NO: 9), WEHR / KGT (SEQ ID NO: 10), KNQKR / QKQ (SEQ ID NO: 11), DVFY / QMI (SEQ ID NO: 12), EGVD / AE (SEQ ID NO: 13), DHKL / KSG (SEQ ID NO: 14), TPW / SLA (SEQ ID NO: 15), KVSEAD (SEQ ID NO: 20), IRPSKG (SEQ ID NO: 4), GGEKRD (SEQ ID NO: 5), GKKEAG (SEQ ID NO: 3), and DHKL / KSG (SEQ ID NO: 21). In some exemplary cleavage sites, a slash is used to represent the cleavage site. However, cleavage at these sites is not limited to such.

[0059] Figure 1D shows a Coomassie gel showing an example of factor B cleavage by chymase-based engineered proteases. Two lots of each engineered protease set were used, and each lot demonstrated the ability of the engineered protease to cleave factor B. Figure 1E shows a low EC 50 1 shows a graph showing an example of factor B cleavage by two chymase-based engineered proteases having the following structure: These results are discussed in more detail in Example 1 below.

[0060] The amino acid sequence of wild-type human factor B is shown in Table 1 below and represented by SEQ ID NO:1. As shown in Table 1, in some embodiments, a site comprising the amino acid sequence KVSEAD (SEQ ID NO:2) may be targeted as a cleavage site by an engineered protease of the present disclosure. In some embodiments, an engineered protease that can target the sequence of SEQ ID NO:2 for cleavage is based on chymase. In some embodiments, a site comprising the amino acid sequence GKKEAG (SEQ ID NO:3) may be targeted as a cleavage site by an engineered protease of the present disclosure. In some embodiments, an engineered protease that can target the sequence of SEQ ID NO:3 for cleavage is based on MTSP-1 or uPA. In some embodiments, a site comprising the amino acid sequence IRPSKG (SEQ ID NO:4) and / or GGEKRD (SEQ ID NO:5) may be targeted as a cleavage site by an engineered protease of the present disclosure, and in some embodiments, such engineered proteases are constructed on an MTSP-1-based scaffold.

[0061] 2A-2B show schematic diagrams of prochymase and mature chymase, respectively. Prochymase comprises a chymase domain and an activation peptide, and mature chymase is produced when the signal peptide and activation peptide are cleaved at the cleavage site shown in FIG. 2A. The mature chymase shown in FIG. 2B can be utilized as a scaffold for the generation of engineered proteases of the present disclosure. The amino acid sequence of the wild-type mature chymase protease domain is shown in Table 1 and is set forth in SEQ ID NO:6.

[0062] 3A-3B show schematic diagrams of the extracellular portion of membrane serine protease 1 (MTSP-1) and the serine protease domain of MTSP-1, respectively. As shown in FIG. 3B, the MTSP-1 serine protease domain can be utilized as a scaffold for the generation of engineered proteases of the present disclosure. The amino acid sequence of the naturally occurring MTSP-1 protease domain is shown in Table 1, SEQ ID NO: 7.

[0063] 3C-3D show schematic diagrams of urokinase-type plasminogen activator (uPA or u-PA). FIG. 3C shows a schematic diagram of uPA zymogen and FIG. 3D shows a schematic diagram of mature two-chain uPA. Mature uPA polypeptide is generated by proteolytic cleavage. As shown in FIG. 3D, the uPA serine protease domain can be utilized as a scaffold for the generation of engineered proteases of the present disclosure. In wild-type uPA, the protease domain is linked to the "A" chain by a disulfide bridge (shown in FIG. 3D). In engineered uPA-based proteases provided herein that include a uPA serine protease domain, there can be a C to S substitution (C122S as indicated by chymotrypsin numbering in SEQ ID NO: 22) to reduce aggregation.

[0064] In some embodiments, provided herein are engineered proteases in which the serine protease is specific for factor B at a site that is not targeted by factor D. In other embodiments, provided herein are engineered proteases in which the serine protease is specific for factor B at a site that is targeted by factor D. In some embodiments, cleavage of factor B by an engineered protease provided herein at a site that is targeted by factor D or at a site that is not targeted by factor D results in reduced complement activation. In some embodiments, cleavage at the site generates at least two functionally inactive fragments. In some embodiments, cleavage of factor B by an engineered protease provided herein at a site that is targeted by factor D or at a site that is not targeted by factor D generates one or more functionally inactive fragments. In some embodiments, cleavage of factor B by an engineered protease provided herein at a site that is targeted by factor D or at a site that is not targeted by factor D results in reduced function of factor B. In some embodiments, cleavage of factor B by an engineered protease provided herein at a site targeted by factor D or at a site not targeted by factor D results in a reduction of the factor B cleavage products Ba and Bb.

[0065] In some embodiments, factor B targeted by engineered proteases of the present disclosure can be of any species. In some embodiments, factor B is primate factor B. In some embodiments, factor B is human factor B. In some embodiments, human factor B comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, primate factor B is non-human primate factor B. In some embodiments, the non-human primate is a cynomolgus monkey. In some embodiments, factor B is rodent factor B, e.g., rat or mouse factor B.

[0066] In some embodiments, the engineered proteases provided herein are specific for factor B at a site not targeted by factor D, wherein the site targeted by factor D comprises the amino acid sequence QQKR / KIV (SEQ ID NO: 9). In some embodiments, the site on factor B not targeted by factor D comprises a sequence selected from the group consisting of WEHR / KGT (SEQ ID NO: 10), KNQKR / QKQ (SEQ ID NO: 11), DVFY / QMI (SEQ ID NO: 12), EGVD / AE (SEQ ID NO: 13), DHKL / KSG (SEQ ID NO: 14), TPW / SLA (SEQ ID NO: 15), KVSEAD (SEQ ID NO: 20), IRPSKG (SEQ ID NO: 4), GGEKRD (SEQ ID NO: 5), GKKEAG (SEQ ID NO: 3), DHKL / KSG (SEQ ID NO: 21), and WEHR / KGT (SEQ ID NO: 10).

[0067] In other embodiments, the engineered proteases provided herein are specific for factor B at the site targeted by factor D, wherein the site targeted by factor D comprises the amino acid sequence QQKR / KIV (SEQ ID NO:9).

[0068] In some embodiments, the engineered proteases provided herein are based on chymotrypsin-like serine proteases of the S1A family, including, but not limited to, membrane-type serine protease 1 (MTSP-1), urokinase-type plasminogen activator (uPA), KLK5, and chymase. The engineered proteases of the present disclosure comprise modified protease domains based on the scaffolds of serine proteases such as MTSP-1, uPA, KLK5, or chymase.

[0069] uPA-based engineered proteases In some embodiments, the engineered protease is based on uPA, e.g., based on the uPA serine protease domain, hi some embodiments, such engineered proteases are specific for factor B at a site not targeted by factor D, e.g., the cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO: 10) and KNQKR / QKQ (SEQ ID NO: 11).

[0070] In some embodiments, the uPA-based engineered protease comprises one or more modifications with respect to uPA comprising the amino acid sequence set forth in SEQ ID NO:8.

[0071] In some embodiments, the uPA-based engineered protease comprises one or more modifications with respect to a uPA protease domain comprising the amino acid sequence set forth in SEQ ID NO:22.

[0072] Modifications to uPA or the uPA protease domain can be referred to by numbering the residues of the uPA protease domain according to the chymotrypsin numbering system. The corresponding chymotrypsin numbers for the uPA protease domain of SEQ ID NO:22 are shown in Table 2 (corresponding to amino acid positions 159-411 of uPA shown in SEQ ID NO:8).

[0073] Table 2 provides four columns for each amino acid. Column 1 lists the conventional amino acid sequence numbering of SEQ ID NO:22, the uPA protease domain. Column 2 lists the conventional amino acid sequence numbering of residues 159-411 of SEQ ID NO:8, the uPA protease domain. Column 3 provides the amino acid single letter abbreviations. Column 4 provides the corresponding chymotrypsin numbering under each amino acid single letter abbreviation. Residues present in the protease domain that are not present in chymotrypsin are represented by the last letter of the notation. For example, residues in chymotrypsin that are part of the loop with amino acid 60 based on the chymotrypsin numbering that are inserted into engineered uPA are designated D60a, Y60b, P60c.

[0074] Table 2 provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the uPA protease domain. In subsequent tables and throughout this disclosure, modifications to the uPA protease domain are referred to either by the chymotrypsin numbering or the conventional amino acid numbering. Where a particular modification is provided only in chymotrypsin numbering designation, one of skill in the art will understand how to refer to Table 2 and make the necessary conversions to understand the modification in conventional amino acid terms, and vice versa.

[0075] [Table 2-1]

[0076] [Table 2-2]

[0077] [Table 2-3]

[0078] [Table 2-4]

[0079] The uPA-based engineered proteases of the present disclosure include at least one modification of the serine protease domain of uPA. As described above, the modification can be any one or more of the following: deletion of one or more amino acid residues, deletion of one or more domains, substitution of one or more amino acid residues, insertion of one or more amino acid residues, insertion of one or more domains, and substitution of one or more domains. Table 3A provides exemplary modifications to the serine protease domain of uPA. As an example, Table 3A provides three columns, the first column provides the modifications using chymotrypsin numbering. The second column provides the conventional amino acid sequence numbering for SEQ ID NO:8, and the third column provides the amino acid sequence numbering for SEQ ID NO:22.

[0080] The engineered proteases can be generated by use of any one or more of the exemplary modifications provided in Table 3 A. Thus, the uPA-based engineered proteases of the present disclosure may include any one or more of the modifications provided in Table 3A.

[0081] In some embodiments, the modification is at any one or more positions corresponding to positions G18, R36, S37, V38, Y40, D60, A96, L97, A98, H99, C122, Y151, V159, A184, Q192, R217, K224 using chymotrypsin numbering. By way of example, the modification to G18E is a substitution of E at a position corresponding to position 18 of the uPA serine protease domain using chymotrypsin numbering. By way of example, the modification D97delinsEG indicates the deletion of D at position 97 and the insertion of EG in its place using chymotrypsin numbering. By way of example, the modification L97b_H99del indicates the deletion of residues L97b to H99 using chymotrypsin numbering.

[0082] [Table 3-1]

[0083] [Table 3-2]

[0084] [Table 3-3]

[0085] Provided in Table 3B are exemplary modifications of the present disclosure (referred to herein as the mutation column). Accordingly, provided herein are uPA-based engineered proteases that include one or more modifications provided in Table 3A. Such exemplary engineered proteases may be capable of cleaving factor B or may exhibit other cleavage activity. The second column provides exemplary combinations of modifications using conventional numbering with respect to SEQ ID NO:22.

[0086] [Table 4-1]

[0087] [Table 4-2]

[0088] In some embodiments, a uPA-based engineered protease of the present disclosure comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:8.

[0089] In some embodiments, a uPA-based engineered protease of the present disclosure comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:22.

[0090] In some embodiments, a uPA-based engineered protease of the present disclosure comprises a protease domain that comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:22.

[0091] MTSP-1-based engineered proteases In some embodiments, the engineered protease is based on MTSP-1, e.g., based on a modified MTSP-1 serine protease domain, hi some embodiments, such an engineered protease is specific for factor B at a site not targeted by factor D, and the cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO: 10) and KNQKR / QKQ (SEQ ID NO: 11).

[0092] In some embodiments, the engineered MTSP-1 protease comprises one or more modifications with respect to MTSP-1 comprising the amino acid sequence set forth in SEQ ID NO:18.

[0093] In some embodiments, the MTSP-1 based engineered protease comprises one or more modifications with respect to the MTSP-1 protease domain comprising the amino acid sequence set forth in SEQ ID NO:7.

[0094] Modifications to MTSP-1 or the MTSP-1 protease domain can be referred to by numbering the residues of the MTSP-1 protease domain according to the chymotrypsin numbering system. Table 4 shows the corresponding chymotrypsin numbering for the MTSP-1 protease domain of SEQ ID NO: 7 (corresponding to amino acid positions 615-855 of MTSP-1 shown in SEQ ID NO: 18).

[0095] Table 4 provides four columns for each amino acid. Column 1 lists the conventional amino acid sequence numbering of SEQ ID NO:7, the MTSP-1 protease domain. Column 2 lists the conventional amino acid sequence numbering of residues 615-855 of SEQ ID NO:18, the MTSP-1 protease domain. Column 3 provides the amino acid single letter abbreviations. Column 4 provides the corresponding chymotrypsin numbering under each amino acid single letter abbreviation. Residues present in the protease domain that are not present in chymotrypsin are represented by the last letter of the notation. For example, the residues in chymotrypsin that are part of the loop with amino acid 60 based on the chymotrypsin numbering that are inserted into engineered MTSP-1 are referred to as D60a and R60c.

[0096] Table 4 provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the MTSP-1 protease domain. In subsequent tables and throughout this disclosure, modifications to the MTSP-1 protease domain are referred to using either the chymotrypsin numbering or the conventional amino acid numbering. Where a particular modification is provided solely in chymotrypsin numbering designation, one of skill in the art will understand how to refer to Table 4 and make the necessary conversions to understand the modification in conventional amino acid terms, and vice versa.

[0097] [Table 5-1]

[0098] [Table 5-2]

[0099] [Table 5-3]

[0100] The MTSP-1 based engineered proteases of the present disclosure include at least one modification of the serine protease domain of MTSP-1. As described above, the modification can be any one or more of the following: deletion of one or more amino acid residues, deletion of one or more domains, substitution of one or more amino acid residues, insertion of one or more amino acid residues, insertion of one or more domains, and substitution of one or more domains. Table 5A provides exemplary modifications to the serine protease domain of MTSP-1. As an example, Table 5A provides three columns, the first column provides the modifications using chymotrypsin numbering. The second column provides the conventional amino acid sequence numbering for SEQ ID NO: 18, and the third column provides the amino acid sequence numbering for SEQ ID NO: 7.

[0101] Modifications to MTSP-1 or the MTSP-1 protease domain can be referred to by numbering the residues of the MTSP-1 protease domain according to the chymotrypsin numbering system. Table 4 shows the corresponding chymotrypsin numbering for the MTSP-1 protease domain of SEQ ID NO: 7 (corresponding to amino acid positions 615-855 of MTSP-1 shown in SEQ ID NO: 18).

[0102] The engineered proteases can be generated by use of any one or more of the exemplary modifications provided in Table 5 A. Thus, the MTSP-1 based engineered proteases of the present disclosure may include any one or more of the modifications provided in Table 5A.

[0103] In some embodiments, the modification is at any one or more positions corresponding to positions D23, I41, L70, A77, F94, D96, F97, T98, F99, K110, C122, D125, Y146, Q175, V183, Q192, A204, D217, and K224 using chymotrypsin numbering. By way of example, a modification to F99S in MTSP-1 is a substitution modification at a position corresponding to position 99 of the MTSP-1 serine protease domain using chymotrypsin numbering.

[0104] [Table 6-1]

[0105] [Table 6-2]

[0106] Provided in Table 5B are exemplary modifications (mutation sequences) of the present disclosure. Accordingly, provided herein are MTSP-1 based engineered proteases that include one or more of the mutation sequences provided in Table 5B. Such exemplary engineered proteases may be capable of cleaving factor B or may exhibit other cleavage activity. Residues noted in brackets, such as C

[17] and C

[19] , refer to residues that are part of a chain in the zymogen form of the protease that is subsequently cleaved and does not remain in the mature protease.

[0107] [Table 7-1]

[0108] [Table 7-2]

[0109] In some embodiments, the MTSP-1 based engineered protease of the disclosure comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:18.

[0110] In some embodiments, the MTSP-1 based engineered proteases of the disclosure comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:7.

[0111] In some embodiments, the MTSP-1 based engineered proteases of the present disclosure comprise a protease domain that comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:7.

[0112] Chymase-based engineered proteases In some embodiments, the engineered protease is based on chymase, e.g., based on a modified chymase serine protease domain. In some embodiments, such an engineered protease is specific for factor B at a site not targeted by factor D, and the cleavage site comprises a sequence selected from DVFY / QMI (SEQ ID NO: 12), EGVD / AE (SEQ ID NO: 13), DHKL / KSG (SEQ ID NO: 14), and TPW / SLA (SEQ ID NO: 15).

[0113] In some embodiments, the chymase-based engineered protease comprises one or more modifications with respect to the chymase protease domain comprising the amino acid sequence set forth in SEQ ID NO:6.

[0114] Modifications to chymase or the chymase protease domain can be referred to by the numbering of the residues in the chymase protease domain according to the chymotrypsin numbering system. The corresponding chymotrypsin numbers for amino acid positions 1-226 of the chymase protease domain of SEQ ID NO:6 are shown in Table 6.

[0115] Table 6 provides three columns for each amino acid. The first column lists the conventional amino acid sequence numbering of SEQ ID NO:6, which is the chymase protease domain. The second column provides the amino acid single letter abbreviation. The third column provides the corresponding chymotrypsin numbering of the chymase protease domain under each amino acid single letter abbreviation. Residues present in the protease domain that are not present in chymotrypsin are represented by the last letter of the notation. For example, the residues in chymotrypsin at amino acid 36 based on the chymotrypsin numbering that are inserted into the engineered chymase are designated V36a, S36b, and N36c.

[0116] Table 6 provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the chymase protease domain. In the tables that follow and throughout this disclosure, modifications to the chymase protease domain are referred to using either the chymotrypsin numbering or the conventional amino acid numbering. Where a particular modification is provided only in chymotrypsin numbering designation, one of skill in the art will understand how to refer to Table 6 and make the necessary conversions to understand the modification in conventional amino acid terms, and vice versa.

[0117] [Table 8-1]

[0118] [Table 8-2]

[0119] [Table 8-3]

[0120] The chymase-based engineered protease of the present disclosure comprises at least one modification of the serine protease domain of chymase. As described above, the modification can be any one or more of the following: deletion of one or more amino acid residues, deletion of one or more domains, substitution of one or more amino acid residues, insertion of one or more amino acid residues, insertion of one or more domains, and substitution of one or more domains. Table 7A provides exemplary modifications to the serine protease domain of chymase. As an example, Table 7A provides two columns, the first column provides modifications using chymotrypsin numbering. The second column provides conventional amino acid sequence numbering for SEQ ID NO:6.

[0121] The engineered proteases can be generated by using any one or more of the exemplary modifications provided in Table 7A. Thus, the chymase-based engineered proteases of the present disclosure may include any one or more of the modifications provided in Table 7A.

[0122] In some embodiments, the modification is at any one or more positions corresponding to positions C22, S36, P38, G43, R49, K87, K93, I103, L114, L116, F123, V138, F173, D175, S189, A190, F191, K192, L199, V213, G216, A220, A226, F228 using chymotrypsin numbering. By way of example, a modification to C22S of chymase is a substitution modification at a position corresponding to position 22 of the chymase serine protease domain using chymotrypsin numbering.

[0123] [Table 9-1]

[0124] [Table 9-2]

[0125] [Table 9-3]

[0126] [Table 9-4]

[0127] [Table 9-5]

[0128] Table 7B provides exemplary modifications (mutation sequences) of the present disclosure. Accordingly, chymase-based engineered proteases are provided herein that include one or more modifications (mutation sequences) provided in Table 7A. Such exemplary engineered proteases may be capable of cleaving factor B or may exhibit other cleavage activities. By way of example, engineered chymase-based engineered proteases include proteases having exemplary combinations of modifications, based on the numbering of chymotrypsin: C22S / P38Q / K40M / F41R / V138I / F173Y / D175R / A190S / V213A / S218V / A226R and C22S / P38Q / K40M / F41H / V138I / F173Y / D175R / A190S / V213A / S218V / A226R.

[0129] [Table 10-1]

[0130] [Table 10-2]

[0131] [Table 10-3]

[0132] [Table 10-4]

[0133] [Table 10-5]

[0134] [Table 10-6]

[0135] [Table 10-7]

[0136] [Table 10-8]

[0137] In some embodiments, the chymase-based engineered protease of the present disclosure comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:6.

[0138] In some embodiments, the chymase-based engineered protease of the present disclosure comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:6.

[0139] In some embodiments, the chymase-based engineered protease of the present disclosure comprises a protease domain that comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:6.

[0140] KLK5-based engineered proteases In some embodiments, the engineered protease is based on KLK5. In some embodiments, the engineered protease is based on KLK5 and is specific for factor B at the site targeted by factor D, and the cleavage site comprises the amino acid sequence QQKR / KIV (SEQ ID NO:9).

[0141] In some embodiments, the KLK5-based engineered protease is based on KLK5 comprising the amino acid sequence set forth in SEQ ID NO:23. Residues in or modifications to KLK5 can be referred to by numbering the residues of KLK5 according to the numbering of chymotrypsin. The numbering scheme of chymotrypsin for amino acid positions 45-271 of KLK5 SEQ ID NO:28 as set forth in SEQ ID NO:23 is provided in Table 7C. Table 7C lists amino acid residues 45-271 of SEQ ID NO:23 above the single-letter amino acid designation and lists the corresponding numbering of chymotrypsin below each single-letter amino acid designation. Residues present in the protease that are not present in chymotrypsin are represented by the last letter of the designation. For example, the residues in chymotrypsin at amino acid 36 according to the numbering of chymotrypsin that are inserted into the engineered KLK5 are referred to as 36a, 36b, 36c.

[0142] Table 7C provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the KLK5 protease domain. In subsequent tables and throughout this disclosure, modifications to the KLK5 protease domain are referred to using either the chymotrypsin numbering or the conventional amino acid numbering. Where a particular modification is provided solely in chymotrypsin numbering designation, one of skill in the art will understand how to refer to Table 7C and make the necessary conversions to understand the modification in conventional amino acid terms, and vice versa.

[0143] [Table 11-1]

[0144] [Table 11-2]

[0145] [Table 11-3]

[0146] It should be understood that the engineered proteases of the present disclosure are not limited to those shown in the table above. Such modifications may increase the half-life, bioavailability, or other characteristics of the serine protease.

[0147] Fusion proteins The engineered proteases can be further modified, for example, they can include the fusion (addition) of another component or domain. Examples of such components or domains include, but are not limited to, half-life extenders, and activation peptides or activation signals. For example, components that can increase the half-life or bioavailability of the engineered proteases of the present disclosure can be added. Half-life extenders can include, but are not limited to, Fc (e.g., IgG1, IgG2, IgG3, or IgG4), affibodies, PEG, and albumins such as human serum albumin (HSA).

[0148] Such addition of additional components can be added, for example, by PASylation®. Such addition can extend the half-life or bioavailability of the engineered protease compared to a serine protease without the half-life extender. In some embodiments, the addition of a half-life extender or other similar component can also improve or modify any one or more properties of the engineered protease, including, but not limited to, stability, bioavailability, serum half-life, shelf life, transportability, and immunogenicity.

[0149] Thus, in some embodiments, the engineered proteases provided herein can further comprise a half-life extender. In some embodiments, the half-life extender is an addition at the N-terminus of the engineered protease. In some embodiments, the half-life extender is an addition at the C-terminus of the engineered protease. In some embodiments, the half-life extender is added directly to the serine protease. In some embodiments, the half-life extender is added to the serine protease via a linker or two or more linkers. In some embodiments, the half-life extender is Fc, and is a human wild-type Fc domain or a variant thereof. In some embodiments, the half-life extender is albumin, e.g., human serum albumin, or a variant thereof.

[0150] In some embodiments, the engineered proteases provided herein may include two or more half-life extenders. In some embodiments, each of the half-life extenders is an addition at the N-terminus of the serine protease. In some embodiments, each of the half-life extenders is an addition at the C-terminus of the serine protease. In some embodiments, one half-life extender is an addition at the N-terminus of the engineered protease and the other half-life extender is an addition at the C-terminus of the engineered protease. In some embodiments, the half-life extender is Fc and is a human wild-type Fc domain or a variant thereof. In some embodiments, the half-life extender is albumin, e.g., human serum albumin, or a variant thereof.

[0151] In an exemplary embodiment, the chymase-based engineered protease of the present disclosure is fused to a wild-type Fc domain or a variant thereof. In an exemplary embodiment, the chymase-based engineered protease of the present disclosure is fused to human serum albumin or a variant thereof.

[0152] In an exemplary embodiment, the uPA-based engineered protease of the present disclosure is fused to a wild-type Fc domain or a variant thereof. In an exemplary embodiment, the uPA-based engineered protease of the present disclosure is fused to human serum albumin or a variant thereof.

[0153] In an exemplary embodiment, the MTSP-1 based engineered protease of the present disclosure is fused to a wild-type Fc domain or a variant thereof. In an exemplary embodiment, the MTSP-1 based engineered protease of the present disclosure is fused to human serum albumin or a variant thereof.

[0154] In an exemplary embodiment, the KLK5-based engineered protease of the present disclosure is fused to a wild-type Fc domain or a variant thereof. In an exemplary embodiment, the KLK5-based engineered protease of the present disclosure is fused to human serum albumin or a variant thereof.

[0155] The fusion protein can also include an activation sequence such that the resulting fusion protein containing the engineered protease of the disclosure is in an active form, such as a two-chain form. The activation sequence contains, or can be modified to contain, a cysteine ​​that can form a disulfide bond with a free Cys, such as C122, in the modified u-PA polypeptide, such that upon activation, the resulting activated polypeptide contains two chains. Exemplary activation sequences include the enterokinase activation sequence and the furin activation sequence, and modified versions thereof.

[0156] Engineered protease activity In some embodiments, the engineered proteases of the present disclosure cleave factor B at sites that are not targeted by factor D or that are targeted by factor D, and cleavage at such sites results in a decrease in function of factor B or a factor B fragment. In some embodiments, the function of factor B or a factor B fragment is interaction with at least one complement component. In some embodiments, the function of factor B or a factor B fragment is interaction with hydrolyzed soluble C3. In some embodiments, the function of factor B or a factor B fragment is interaction with C3b. In some embodiments, the C3b is membrane-bound C3b. In some embodiments, cleavage at non-factor D sites occurs when factor B is not bound to C3b. In some embodiments, cleavage at factor D sites occurs when factor B is not bound to C3b. In some embodiments, cleavage at non-factor D sites occurs when factor B is bound to C3b (i.e., complexed with C3b).

[0157] In some embodiments, the engineered proteases provided herein are also capable of cleaving factor B while being capable of cleaving other peptide substrates that are not factor B. In some embodiments, the cleavage activity towards the non-factor B peptide substrates is about equal to or less than the cleavage activity towards the factor B site.

[0158] In some embodiments, the engineered proteases provided herein have a cleavage potency of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, or about 1,900 M for factor B cleavage. -1 s -1 K cat / K m In some embodiments, K cat / K m is approximately 10e 8In some embodiments, the engineered proteases provided herein have a cleavage rate of about 10 3 ~about 10 6 M -1 s -1 K cat / K m has.

[0159] In some embodiments, the engineered proteases provided herein have an EC for factor B cleavage of about 1 nM to about 20 nM. 50 In some embodiments, the engineered proteases provided herein have an EC for factor B of less than about 20 nM. 50 In some embodiments, the engineered proteases provided herein have an EC for factor B cleavage of less than about 1 nM. 50 In some embodiments, the engineered proteases provided herein have an EC for factor B cleavage of about 5 nM to about 100 nM. 50 In some embodiments, the engineered proteases provided herein have an EC for cleavage of factor B of about 20, about 25, or about 60 nM. 50 In some embodiments, the EC for cleavage of factor B is 50 is about 20 nM. In some embodiments, the EC 50 In some embodiments, the engineered proteases provided herein have an EC for factor B cleavage of about 1,000 nM to about 4,500 nM. 50 In some embodiments, the engineered proteases provided herein have an EC for factor B cleavage of about 1,000 nM, or about 2,000 nM, or about 3,000 nM, or about 4,000 nM, or about 5,000 nM. 50 has.

[0160] In some embodiments, the engineered proteases provided herein have a catalytic lifetime in human plasma of greater than about 72 hours. In some embodiments, the engineered proteases provided herein have a catalytic lifetime in human plasma of about 120 hours or more. In some embodiments, the engineered proteases provided herein have a catalytic lifetime in human plasma of about 120 hours or more and are useful for chronic indications. In some embodiments, the engineered proteases provided herein have a catalytic lifetime in human plasma of about 24 hours. In some embodiments, the engineered proteases provided herein have a catalytic lifetime in human plasma of about 24 hours or more and are useful for acute indications.

[0161] In some embodiments, the engineered proteases provided herein have catalytic activity for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In some embodiments, the catalytic activity is about 10% to about 85% of the initially measured activity after about 7 days.

[0162] In some embodiments, the engineered proteases provided herein have an increased half-life compared to unmodified MTSP-1 or MTSP-1 protease domain. In some embodiments, the engineered proteases provided herein have an increased bioavailability compared to unmodified MTSP-1 or MTSP-1 protease domain. In some embodiments, the engineered proteases provided herein have an increased half-life compared to unmodified uPA or uPA protease domain. In some embodiments, the engineered proteases provided herein have an increased bioavailability compared to unmodified uPA or uPA protease domain. In some embodiments, the engineered proteases provided herein have an increased half-life compared to unmodified chymase or chymase protease domain. In some embodiments, the engineered proteases provided herein have an increased bioavailability compared to unmodified chymase or chymase protease domain. In some embodiments, the engineered proteases provided herein have an increased half-life compared to an unmodified KLK5 or KLK5 protease domain, hi some embodiments, the engineered proteases provided herein have an increased bioavailability compared to an unmodified KLK5 or KLK5 protease domain.

[0163] In some embodiments, the engineered proteases provided herein are non-immunogenic.

[0164] In some embodiments, the engineered proteases provided herein are in zymogen form. As used herein, zymogen form refers to the full-length protease prior to cleavage into the mature form. In some embodiments, the engineered proteases provided herein are in an active form, also referred to as the mature form. In some embodiments, the zymogen form may be activated to the mature form in vivo (in situ) upon administration. In some embodiments, the zymogen form is activated ex vivo (in vitro), for example, prior to administration of the engineered protease.

[0165] In some embodiments, the engineered protease is in an activated form. In some embodiments, the engineered protease is activated by an enzyme, such as enterokinase. In some embodiments, the chymase-based engineered protease of the present disclosure is activated by enterokinase. In some embodiments, the engineered protease is activated during recombinant production in a host cell. In some embodiments, activation by an enzyme during production in a host cell is by overexpression of an enzyme, such as enterokinase. In some embodiments, the engineered protease is activated after production and secretion by the host cell, optionally in the medium.

[0166] Use of engineered proteases The engineered proteases of the present disclosure may be used to regulate the complement system.

[0167] In some embodiments, the engineered proteases of the present disclosure can regulate the classical complement pathway. In some embodiments, the engineered proteases of the present disclosure can regulate the alternative complement pathway. In some embodiments, the engineered proteases of the present disclosure can regulate the lectin complement pathway. In some embodiments, the engineered proteases of the present disclosure can reduce amplification of the complement system.

[0168] In some embodiments, the engineered proteases of the present disclosure are capable of reducing the function of factor B or a factor B fragment. As discussed herein, in some embodiments, the engineered proteases of the present disclosure are capable of reducing the production of factor B fragments Ba and / or Bb, or producing factor B fragments Ba and / or Bb that are functionally inactive.

[0169] Provided herein are methods of inactivating factor B, comprising contacting factor B with any of the engineered proteases disclosed herein. In some embodiments, such methods are used to inhibit complement activation. In some embodiments, the classical pathway of the complement pathway is inhibited. In some embodiments, the alternative pathway of the complement pathway is inhibited. In some embodiments, the lectin pathway of the complement pathway is inhibited.

[0170] In some embodiments, the method is in vitro. In some embodiments, the method is in vivo.

[0171] The engineered proteases of the present disclosure may be used for therapy in a subject. Thus, provided herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject any one of the engineered proteases of the present disclosure. In some embodiments, the disease or condition is associated with dysregulated complement, and thus in some embodiments, the disease or condition involves complement dysregulation. In some embodiments, the treatment is replacement therapy. In some embodiments, the treatment blocks complement activation. In some embodiments, the treatment modulates autoimmunity. In some embodiments, the treatment is for endothelial cell injury or renal cell injury.

[0172] In some exemplary embodiments, the disease or condition is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplant ischemia and reperfusion (I / R) injury, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), sepsis, acute respiratory distress syndrome (ARDS), SARS-associated coronavirus (SARS-CoV), atypical hemolytic uremic syndrome (aHUS), membranous nephropathy (MN), and paroxysmal nocturnal hemoglobinuria (PNH).

[0173] In some embodiments, the engineered proteases provided herein are useful for treating inflammatory diseases or conditions. In some embodiments, the engineered proteases provided herein are capable of reducing inflammatory cytokines. In some exemplary embodiments, the engineered proteases provided herein are effective in reducing the inflammatory cytokines IL-2 and IL-6, and the chemokine CXCL9, and are useful for treating diseases such as ARDS.

[0174] In some embodiments, the engineered protease is administered subcutaneously to a subject. In some embodiments, the engineered protease is activated in situ at the site of a dysregulated complement component or at the site of a dysregulated pathophysiology. In some embodiments, the engineered protease is provided in a liquid stable formulation. In vivo administration of the engineered protease can be intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, intrathecal, intraarterial, intraventricular, intranasal, transmucosal, implanted, or by inhalation. In some embodiments, the engineered proteases provided herein are administered using a mechanical device.

[0175] Pharmaceutical Compositions The present disclosure also provides a pharmaceutical composition comprising any one of the engineered proteases disclosed herein, and optionally a pharma- ceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical compositions may be formulated to be compatible with their intended route of administration. In some embodiments, the pharmaceutical compositions of the present disclosure are suitable for administration to human subjects or other non-human primates.

[0176] Kits and manufactured products The present disclosure also provides a kit or article of manufacture comprising any one of the engineered proteases disclosed herein, or any pharmaceutical composition disclosed herein. In some embodiments, the kit may further comprise instructional materials for practicing any of the methods disclosed herein. In some embodiments, the kit may further comprise a sterile container or vial for holding the fusion construct and / or pharmaceutical composition disclosed herein. In some embodiments, the kit may further comprise a sterile delivery device for administering the fusion construct and / or pharmaceutical composition disclosed herein. In some embodiments, the article of manufacture comprises any pharmaceutical composition of the present disclosure.

[0177] Production of engineered proteases Methods and compositions for producing engineered proteases are provided herein. Accordingly, nucleic acids and vectors encoding any of the engineered proteases of the present disclosure are provided herein. Cells containing one or more nucleic acids encoding the engineered proteases of the present disclosure are also provided.

[0178] The engineered proteases provided herein can be cloned or isolated using any available method known in the art for cloning and isolating nucleic acid molecules, including PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening and activity-based screening.

[0179] Methods are provided herein for producing engineered proteases of the disclosure using expression systems, for example, engineered proteases of the disclosure may be expressed in bacteria (e.g., E. coli), yeast, insect, or mammalian cells (e.g., CHO cells, HEK cells). In certain embodiments, transformation of a host cell with a recombinant DNA molecule incorporating an isolated engineered protease protein gene, cDNA, or synthetic DNA sequence allows for the generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by growing the transformants, isolating the recombinant DNA molecule from the transformants, and, if necessary, recovering the inserted gene from the isolated recombinant DNA. In some embodiments, the engineered protease is active when produced. In some embodiments, the engineered protease needs to be activated when produced. In such embodiments, the engineered protease is engineered in an activated state; the method includes producing the zymogen form of the engineered protease in a bacterial, yeast, or mammalian host system, followed by activation.

[0180] Administration of engineered proteases In some embodiments, provided herein are methods for administering an engineered protease of the present disclosure by delivery of a vector / nucleic acid encoding the engineered protease. In some embodiments, the method comprises administration of a recombinant vector. In some embodiments, provided herein are engineered proteases for use in gene expression therapy using non-viral vectors. In other embodiments, provided herein are engineered proteases for use in gene expression therapy using viral vectors. In some embodiments, cells are engineered to express the engineered protease, for example, by integrating a nucleic acid encoding the engineered protease into a genomic location such that it is operably linked to a regulatory sequence or is located operably linked to a regulatory sequence at the genomic location. In some embodiments, such cells are then administered locally or systemically to a subject, such as a subject in need of treatment.

[0181] For example, nucleic acid molecules encoding engineered proteases can be isolated using methods for amplification of nucleic acids, including polymerase chain reaction (PCR). Nucleic acid-containing materials can be used as starting materials from which engineered protease-encoding nucleic acid molecules can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts (e.g., from liver), liquid samples (e.g., blood, serum, saliva), samples from healthy and / or diseased subjects can be used in amplification methods. Nucleic acid libraries can also be used as a source of starting material. Primers can be designed to amplify and modify molecules encoding engineered proteases. For example, primers can be designed based on the expressed sequence from which the engineered protease is generated.

[0182] Additional nucleotide sequences can be linked to the engineered protease-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector (e.g., a protein expression vector or a vector designed for amplification of a core protein-encoding DNA sequence). In addition, additional nucleotide sequences specifying functional DNA elements can be operably linked to the engineered protease-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to promote intracellular protein expression, and secretory sequences designed to promote protein secretion. Additional nucleotide sequences, such as sequences specifying protein binding regions, can also be linked to the engineered protease-encoding nucleic acid molecule. Such regions include, but are not limited to, sequences that facilitate uptake of the engineered protease into specific target cells, or sequences that otherwise enhance the pharmacokinetics of the synthetic gene.

[0183] Enumerated Embodiments The present disclosure provides the following set of non-limiting enumerated embodiments.

[0184] Set I Embodiment I-1. An engineered protease of the S1A serine protease family that is specific for and capable of cleaving factor B.

[0185] Embodiment I-2. The engineered protease according to embodiment I-1, wherein cleavage of factor B by the engineered protease generates one or more functionally inactive fragments.

[0186] Embodiment I-3. The engineered protease according to any of embodiments I-2, wherein the one or more functionally inactive fragments are capable of reducing complement activation.

[0187] Embodiment I-4. The engineered protease according to any one of embodiments 1 to 3, wherein cleavage of factor B results in the production of a factor B fragment with reduced functionality or results in factor B with reduced functionality.

[0188] Embodiment I-5. The engineered protease of any one of embodiments 1-4, wherein the Factor B is rodent Factor B.

[0189] Embodiment I-6. The engineered protease according to any one of embodiments 1 to 4, wherein the factor B is a non-human primate factor B.

[0190] Embodiment I-7. The engineered protease of embodiments I-6, wherein the non-human primate is a cynomolgus monkey.

[0191] Embodiment I-8. The engineered protease according to any one of embodiments 1 to 4, wherein the factor B is human factor B.

[0192] Embodiment I-9. The engineered protease according to embodiment I-8, wherein Factor B comprises the amino acid sequence set forth in SEQ ID NO:1.

[0193] Embodiment I-10. The engineered protease of any one of embodiments 1-9, wherein cleavage of Factor B occurs at a site that is not targeted by Factor D.

[0194] Embodiment I-11. The engineered protease of embodiment I-10, wherein cleavage at the site generates at least two fragments that are not Ba and Bb.

[0195] Embodiment I-12. The engineered protease of any one of embodiments 1-11, wherein cleavage at the site results in reduced production of Factor B cleavage products Ba and Bb compared to cleavage by Factor D.

[0196] Embodiment I-13. The engineered protease of any one of embodiments 1-9, wherein cleavage of Factor B occurs at a site targeted by Factor D.

[0197] Embodiment I-14. The engineered protease according to any one of embodiments 1 to 13, wherein the site targeted by factor D comprises QQKR / KIV (SEQ ID NO: 9).

[0198] Embodiment I-15. The engineered protease according to embodiment I-10, wherein the factor B cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO:10), KNQKR / QKQ (SEQ ID NO:11), DVFY / QMI (SEQ ID NO:12), EGVD / AE (SEQ ID NO:13), DHKL / KSG (SEQ ID NO:14), TPW / SLA (SEQ ID NO:15), KVSEAD (SEQ ID NO:20), IRPSKG (SEQ ID NO:4), GGEKRD (SEQ ID NO:5), GKKEAG (SEQ ID NO:3), and DHKL / KSG (SEQ ID NO:21).

[0199] Embodiment I-16. The engineered protease according to any one of embodiments 1 to 15, wherein the engineered protease is based on a chymotrypsin-like serine protease selected from the group consisting of membrane-type serine protease 1 (MTSP-1), urokinase-type plasminogen activator (uPA), chymase, and kallikrein-related peptidase 5 (KLK5).

[0200] Embodiment I-17. The engineered protease of embodiment I-16, wherein the engineering of the engineered protease comprises one or more modifications selected from the group consisting of substitutions, additions, and deletions of amino acid residues, and substitutions, additions, and deletions of domains of chymotrypsin-like serine proteases.

[0201] Embodiment I-18. The engineered protease according to any one of embodiments 16 to 17, wherein the engineered protease is based on MTSP-1 or uPA and the cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO: 10) and KNQKR / QKQ (SEQ ID NO: 11).

[0202] Embodiment I-19. An engineered protease according to any one of embodiments 1 to 18, based on MTSP-1.

[0203] Embodiment I-20. An engineered protease according to any one of embodiments 1 to 18, which is not based on MTSP-1.

[0204] Embodiment I-21. The engineered protease of embodiment I-19, comprising one or more modifications with respect to MTSP-1 comprising the amino acid sequence shown in SEQ ID NO:7, wherein the residues are numbered according to the numbering of chymotrypsin.

[0205] Embodiment I-22. An engineered protease according to any one of embodiments 1 to 18, which is based on uPA.

[0206] Embodiment I-23. An engineered protease according to any one of embodiments 1 to 18 which is not based on uPA.

[0207] Embodiment I-24. The engineered protease of embodiment I-22, comprising one or more modifications with respect to uPA comprising the amino acid sequence shown in SEQ ID NO:8, wherein the residues are numbered according to the numbering of chymotrypsin.

[0208] Embodiment I-25. An engineered protease according to any one of embodiments 1 to 17, which is based on chymase.

[0209] Embodiment I-26. The engineered protease according to embodiment I-25, wherein the engineered protease is based on chymase and the cleavage site comprises a sequence selected from DVFY / QMI (SEQ ID NO: 12), EGVD / AE (SEQ ID NO: 13), DHKL / KSG (SEQ ID NO: 14), and TPW / SLA (SEQ ID NO: 15).

[0210] Embodiment I-27. An engineered protease according to any one of embodiments I-1 to I-17, based on KLK5.

[0211] Embodiment I-28. The engineered protease according to embodiment I-25, comprising one or more modifications with respect to chymase comprising the amino acid sequence shown in SEQ ID NO:6, wherein the residues are numbered according to the numbering of chymotrypsin.

[0212] Embodiment I-29. The engineered protease of embodiment I-19, wherein the one or more modifications are at one or more positions corresponding to one or more positions selected from D23, I41, L70, A77, F94, D96, F97, T98, F99, K110, C122, D125, Y146, Q175, V183, Q192, A204, D217, and K224 in MTSP-1 comprising the sequence of amino acids set forth in SEQ ID NO:7, wherein the residues are numbered according to the numbering of chymotrypsin.

[0213] Embodiment I-30. The engineered protease of embodiment I-22, wherein the one or more modifications are at one or more positions corresponding to one or more positions selected from G18, R36, S37, V38, Y40, D60, A96, L97, A98, H99, C122, Y151, V159, A184, Q192, R217, K224 in uPA comprising the sequence of amino acids set forth in SEQ ID NO:8, wherein the residues are numbered according to the numbering of chymotrypsin.

[0214] Embodiment I-31. The engineered protease according to embodiment I-25, wherein the one or more modifications are at one or more positions corresponding to one or more positions selected from C22, S36, P38, G43, R49, K87, K93, I103, L114, L116, F123, V138, F173, D175, S189, A190, F191, K192, L199, V213, G216, A220, A226, F228 in a chymase comprising a sequence of amino acids set forth in SEQ ID NO: 6, wherein the residues are numbered according to the numbering of chymotrypsin.

[0215] Embodiment I-32. The engineered protease according to any one of embodiments 4 to 31, wherein the function of the factor B or factor B fragment is interaction with at least one complement component.

[0216] Embodiment I-33. The engineered protease according to any one of embodiments 4 to 31, wherein the function of the factor B or factor B fragment is interaction with hydrolyzed soluble C3.

[0217] Embodiment I-34. The engineered protease according to any one of embodiments 4 to 33, wherein the function of factor B or the factor B fragment is interaction with C3b.

[0218] Embodiment I-35. The engineered protease according to any one of embodiments 4 to 34, wherein the function of factor B or factor B fragment is interaction with membrane-bound C3b.

[0219] Embodiment I-36. The engineered protease of any one of embodiments 1-35, wherein cleavage occurs when Factor B is not bound to C3b.

[0220] Embodiment I-37. The engineered protease of any one of embodiments 1-36, wherein the cleavage activity towards non-factor B peptide substrates is about equal to or less than the cleavage activity towards the factor B site.

[0221] Embodiment I-38. The engineered protease of any one of embodiments 1-36 having a kcat / Km for Factor B cleavage of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1.700, about 1,800, or about 1,900 M-1s-1.

[0222] Embodiment I-39. The engineered protease of any one of embodiments 1-38, having a kcat / Km of about 103 to about 109 M-1s-1 for Factor B cleavage.

[0223] Embodiment I-40. The engineered protease of any one of embodiments 1-39, wherein the EC50 for Factor B is less than about 20 nM.

[0224] Embodiment I-41. The engineered protease of any one of embodiments 1-40, wherein the EC50 for Factor B is less than about 1 nM.

[0225] Embodiment I-42. The engineered protease of any one of embodiments 1-39, wherein the EC50 for Factor B is about 20, about 25, or about 60 nM.

[0226] Embodiment I-43. The engineered protease of any one of embodiments 1-39, having an EC50 for cleaving Factor B of about 1,000 to about 4,500 nM.

[0227] Embodiment I-44. The engineered protease of any one of embodiments 1-43, having a plasma half-life in human plasma of greater than about 72 hours.

[0228] Embodiment I-45. The engineered protease of any one of embodiments 1-44, wherein the engineered protease has a plasma half-life in human plasma of greater than about 120 hours.

[0229] Embodiment I-46. The engineered protease of any one of embodiments 1-45, having a plasma half-life of about 7 days in human plasma.

[0230] Embodiment I-47. The engineered protease of embodiment I-46, wherein the catalytic activity is between about 10% and about 50%, or between about 90% and about 100%.

[0231] Embodiment I-48. The engineered protease of embodiment I-29, having an increased half-life compared to unmodified MTSP-1.

[0232] Embodiment I-49. The engineered protease of any one of embodiments I-29 and I-48, having increased bioavailability compared to unmodified MTSP-1.

[0233] Embodiment I-50. The engineered protease of embodiment I-30, which has an increased half-life compared to unmodified uPA.

[0234] Embodiment I-51. The engineered protease of any one of embodiments I-30 and I-50, which has increased bioavailability compared to unmodified uPA.

[0235] Embodiment I-52. The engineered protease of embodiment I-31, having an increased half-life compared to unmodified chymase.

[0236] Embodiment I-53. The engineered protease according to any one of embodiments I-31 and I-52, having increased bioavailability compared to unmodified chymase.

[0237] Embodiment I-54. The engineered protease according to any one of embodiments I-1 to I-53, which is non-immunogenic.

[0238] Embodiment I-55. The engineered protease according to any one of embodiments set I 1-54, which is in the zymogen form.

[0239] Embodiment I-56. The engineered protease according to any one of the embodiments set I 1-54, which is in an activated form.

[0240] Embodiment I-57. The engineered protease of any one of embodiments set I 1-56, further comprising a half-life extender.

[0241] Embodiment I-58. A method for inactivating factor B comprising contacting factor B with any of the engineered proteases according to any of embodiments I-1 to I-57.

[0242] Embodiment I-59. The method of embodiment I-58, wherein complement activation is inhibited.

[0243] Embodiment I-60. The method of embodiment I-59, wherein the classical pathway of the complement pathway is inhibited.

[0244] Embodiment I-61. The method of any one of embodiments 59-60, wherein the alternative pathway of the complement pathway is inhibited.

[0245] Embodiment I-62. The method of any one of embodiments 59-61, wherein the lectin pathway of the complement pathway is inhibited.

[0246] Embodiment I-63. The method of any one of embodiments set I 58-62, wherein the method is in vitro.

[0247] Embodiment I-64. The method of any one of embodiments set I 58-62, wherein the method is in vivo.

[0248] Embodiment I-65. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject any one of the engineered proteases described in any one of embodiments 1-57.

[0249] Embodiment I-66. The method of embodiment I-65, wherein the disease or condition is associated with dysregulated complement.

[0250] Embodiment I-67. The method of any one of embodiments set I 65-66, wherein the disease or condition is an inflammatory disease or condition.

[0251] Embodiment I-68. The method of any one of embodiments 65-67, wherein the treatment is replacement therapy.

[0252] Embodiment I-69. The method of any one of embodiments set I 65-68, wherein the treatment blocks complement activation.

[0253] Embodiment I-70. The method of any one of embodiments I65-69, wherein the treatment modulates autoimmunity.

[0254] Embodiment I-71. The method of any one of embodiments set I 65-70, wherein the disease or condition is a congenital complement deficiency.

[0255] Embodiment I-72. The method of any one of embodiments set I 65-71, wherein the treatment is for endothelial cell injury or renal cell injury.

[0256] Embodiment I-73. The method of any one of embodiments set I 65-72, wherein the disease or condition is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplant ischemia and reperfusion (I / R) injury, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), sepsis, acute respiratory distress syndrome (ARDS), SARS-associated coronavirus (SARS-CoV), atypical hemolytic uremic syndrome (aHUS), membranous nephropathy (MN), and paroxysmal nocturnal hemoglobinuria (PNH).

[0257] Embodiment I-74. The method of any one of embodiments set I 65-73, wherein the disease or condition is a regulatory protein deficiency.

[0258] Embodiment I-75. The method of any one of embodiments set I 65-73, wherein the disease or condition is a secondary complement disorder.

[0259] Embodiment I-76. The method of any one of embodiments 65-73, wherein the disease or condition is an immune-related disease or condition.

[0260] Embodiment I-77. The method of any one of embodiments set I 65-76, wherein the engineered protease is administered subcutaneously to the subject.

[0261] Embodiment I-78. The method of embodiment I-77, wherein the engineered protease is activated in situ at the site of a dysregulated complement component.

[0262] Embodiment I-79. The method of any one of embodiments set I 65-78, wherein the engineered protease is provided in a liquid stable formulation.

[0263] Embodiment I-80. A pharmaceutical composition comprising any of the engineered proteases described in any one of embodiments set I 1-57, and optionally a pharma- ceutically acceptable carrier.

[0264] Embodiment I-81. The pharmaceutical composition of embodiment I-80, wherein the engineered protease is provided in a liquid stable formulation.

[0265] Embodiment I-82. A pharmaceutical composition according to any one of embodiments set I 80-81, formulated for subcutaneous administration.

[0266] Set II Embodiment II-1. An engineered protease comprising a modified chymase protease domain, a modified membrane-type serine protease 1 (MTSP-1) protease domain, a modified urokinase-type plasminogen activator (uPA) protease domain, or a modified kallikrein-related peptidase 5 (KLK5) protease domain, capable of cleaving factor B.

[0267] Embodiment II-2. The engineered protease according to embodiment II-1, wherein cleavage of factor B by the engineered protease generates one or more functionally inactive fragments.

[0268] Embodiment II-3. The engineered protease according to embodiment II-2, wherein the one or more functionally inactive fragments are capable of reducing complement activation.

[0269] Embodiment II-4. The engineered protease according to any one of embodiments II-1 to II-3, wherein cleavage of factor B results in the generation of a factor B fragment with reduced functionality.

[0270] Embodiment II-5. The engineered protease according to any one of embodiments II-1 to II-4, wherein factor B is a non-human primate factor B.

[0271] Embodiment II-6. The engineered protease of embodiment II-5, wherein the non-human primate is a cynomolgus monkey.

[0272] Embodiment II-7. The engineered protease according to any one of embodiments II-1 to II-4, wherein factor B is human factor B.

[0273] Embodiment II-8. The engineered protease according to embodiment II-7, wherein Factor B comprises the amino acid sequence set forth in SEQ ID NO:1.

[0274] Embodiment II-9. The engineered protease according to any one of embodiments II-1 to II-8, wherein cleavage of Factor B occurs at a site that is not targeted by Factor D.

[0275] Embodiment II-10. The engineered protease according to embodiment II-9, wherein cleavage at a site not targeted by Factor D generates at least two fragments which are not Ba and Bb.

[0276] Embodiment II-11. The engineered protease according to any one of embodiments II-1 to II-10, wherein cleavage of Factor B results in reduced production of the Factor B cleavage products Ba and Bb compared to cleavage by Factor D.

[0277] Embodiment II-12. The engineered protease according to any one of embodiments II-1 to II-8, wherein cleavage of Factor B occurs at the site targeted by Factor D.

[0278] Embodiment II-13. The engineered protease according to embodiment II-12, wherein the factor B cleavage site targeted by factor D comprises QQKR / KIV (SEQ ID NO: 9).

[0279] Embodiment II-14. The engineered protease according to embodiment II-9, wherein the factor B cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO:10), KNQKR / QKQ (SEQ ID NO:11), DVFY / QMI (SEQ ID NO:12), EGVD / AE (SEQ ID NO:13), DHKL / KSG (SEQ ID NO:14), TPW / SLA (SEQ ID NO:15), KVSEAD (SEQ ID NO:20), IRPSKG (SEQ ID NO:4), GGEKRD (SEQ ID NO:5), GKKEAG (SEQ ID NO:3), and DHKL / KSG (SEQ ID NO:21).

[0280] Embodiment II-15. The engineered protease of embodiment II-9, wherein the factor B cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO:10) and KNQKR / QKQ (SEQ ID NO:11), and the engineered protease comprises a modified MTSP-1 protease domain or a modified uPA protease domain.

[0281] Embodiment II-16. An engineered protease according to any one of embodiments II-1 to II-15, comprising a modified MTSP-1 protease domain.

[0282] Embodiment II-17. The engineered protease according to any one of embodiments II-1 to II-15, which does not comprise a modified MTSP-1 protease domain.

[0283] Embodiment II-18. The engineered protease according to embodiment II-16, comprising one or more modifications with respect to the MTSP-1 protease domain comprising the amino acid sequence set forth in SEQ ID NO:7.

[0284] Embodiment II-19. The engineered protease according to embodiment II-18, wherein the modification is one or more of a substitution, an addition, and a deletion of one or more amino acid residues.

[0285] Embodiment II-20. The engineered protease according to embodiment II-16, wherein the one or more modifications are at one or more positions corresponding to one or more positions selected from D622, I640, L678, A686, F703, D705, F706, T707, F708, K719, C731, D734, Y755, Q783, V791, Q802, A814, D828, and K835 in the MTSP-1 protease domain comprising the sequence of amino acids set forth in SEQ ID NO: 18.

[0286] Embodiment II-21. The engineered protease according to embodiment II-16, wherein the one or more modifications are selected from those shown in Table 5A.

[0287] Embodiment II-22. The engineered protease according to embodiment II-16, wherein the one or more modifications are selected from the exemplary mutation series shown in Table 5B.

[0288] Embodiment II-23. The engineered protease of any one of embodiments II-1 to II-15, comprising a modified uPA protease domain.

[0289] Embodiment II-24. The engineered protease according to any one of embodiments II-1 to II-15, which does not comprise a modified uPA protease domain.

[0290] Embodiment II-25. The engineered protease of embodiment II-23, comprising one or more modifications with respect to the uPA protease domain comprising the amino acid sequence set forth in SEQ ID NO:8.

[0291] Embodiment II-26. The engineered protease according to embodiment II-25, wherein the modification is one or more of a substitution, an addition, and a deletion of one or more amino acid residues.

[0292] Embodiment II-27. The engineered protease according to embodiment II-23, wherein the one or more modifications are at one or more positions corresponding to one or more positions selected from S37, D60, L97, G161, R179, H180, V185, Y187, I207, A247, D248, A251, H252, C279, Y308, V316, A343, Q353, R378, K385 in a uPA protease domain comprising the sequence of amino acids set forth in SEQ ID NO:8.

[0293] Embodiment II-28. The engineered protease according to embodiment II-23, wherein the one or more modifications are selected from those shown in Table 3A.

[0294] Embodiment II-29. The engineered protease according to embodiment II-23, wherein the one or more modifications are selected from the exemplary mutation series shown in Table 3B.

[0295] Embodiment II-30. An engineered protease according to any one of embodiments II-1 to II-19, comprising a modified chymase protease domain.

[0296] Embodiment II-31. The engineered protease according to any one of embodiments II-1 to II-19, which does not comprise a modified chymase protease domain.

[0297] Embodiment II-32. The engineered protease of embodiment II-30, wherein the engineered protease comprises a modified chymase protease domain and the cleavage site comprises a sequence selected from DVFY / QMI (SEQ ID NO: 12), EGVD / AE (SEQ ID NO: 13), DHKL / KSG (SEQ ID NO: 14), and TPW / SLA (SEQ ID NO: 15).

[0298] Embodiment II-33. The engineered protease of embodiment II-30, comprising one or more modifications with respect to the chymase protease domain comprising the amino acid sequence set forth in SEQ ID NO:6.

[0299] Embodiment II-34. The engineered protease according to embodiment II-33, wherein the modification is one or more of a substitution, an addition, and a deletion of one or more amino acid residues.

[0300] Embodiment II-35. The engineered protease according to embodiment II-30, wherein the one or more modifications are at one or more positions corresponding to one or more positions selected from S36 C7, V21, P26, G31, R37, K74, K80, I90, L101, L103, F110, V125, F157, D159, S176, A177, F178, K179, L186, V196, G199, A203, A207, F209 in the Chymase protease domain comprising the sequence of amino acids set forth in SEQ ID NO:6.

[0301] Embodiment II-36. The engineered protease according to embodiment II-30, wherein the one or more modifications are selected from those shown in Table 7A.

[0302] Embodiment II-37. The engineered protease according to embodiment II-30, wherein the one or more modifications are selected from the exemplary mutation series shown in Table 7B.

[0303] Embodiment II-38. An engineered protease according to any one of embodiments II-1 to II-19, comprising a modified KLK5 protease domain and optionally comprising one or more amino acid modifications of SEQ ID NO:23.

[0304] Embodiment II-39. The engineered protease according to any one of embodiments II-1 to II-19, which does not comprise a modified KLK5 protease domain.

[0305] For factor B cleavage, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, or about 1,900 M -1 s -1 k cat / K m The engineered protease according to any one of embodiments II-1 to II-39, having the following structure:

[0306] For factor B cleavage, about 10 3 ~about 10 9 M -1 s -1 k cat / K m The engineered protease according to any one of embodiments II-1 to II-40, having the following structure:

[0307] EC against factor B 50 The engineered protease of any one of embodiments II-1 to II-41, wherein the affinity of the engineered protease is less than about 20 nM.

[0308] EC against factor B 50 The engineered protease of any one of embodiments II-1 to II-42, wherein the affinity of the protease is less than about 1 nM.

[0309] EC against factor B 50 The engineered protease according to any one of embodiments II-1 to II-41, wherein the IgE concentration is about 20, about 25, or about 60 nM.

[0310] EC for cleaving factor B 50 The engineered protease according to any one of embodiments II-1 to II-41, wherein the concentration of the engineered protease is from about 1,000 to about 4,500 nM.

[0311] Embodiment II-46. The engineered protease of any one of embodiments II-1 to II-45, having a plasma half-life of greater than about 72 hours in human plasma.

[0312] Embodiment II-47. The engineered protease of any one of embodiments II-1 to II-46, having a plasma half-life in human plasma of greater than about 120 hours.

[0313] Embodiment II-48. The engineered protease according to any one of embodiments II-1 to II-47, having a plasma half-life of about 7 days in human plasma.

[0314] Embodiment II-49. The engineered protease of embodiment II-48, wherein the catalytic activity is between about 10% and about 50%, or between about 90% and about 100%.

[0315] Embodiment II-50. The engineered protease of embodiment II-16, which has an increased half-life compared to a protease comprising an unmodified MTSP-1 protease domain.

[0316] Embodiment II-51. The engineered protease of embodiment II-16, which has increased bioavailability compared to a protease comprising an unmodified MTSP-1 protease domain.

[0317] Embodiment II-52. The engineered protease of embodiment II-23, which has an increased half-life compared to a protease comprising an unmodified uPA protease domain.

[0318] Embodiment II-53. The engineered protease of embodiment II-23, which has increased bioavailability compared to a protease comprising an unmodified uPA protease domain.

[0319] Embodiment II-54. The engineered protease of embodiment II-30, having an increased half-life compared to a protease comprising an unmodified chymase protease domain.

[0320] Embodiment II-55. The engineered protease of embodiment II-30, having increased bioavailability compared to a protease comprising an unmodified chymase protease domain.

[0321] Embodiment II-56. The engineered protease according to any one of embodiments II-1 to II-55, which is non-immunogenic.

[0322] Embodiment II-57. The engineered protease according to any one of embodiments II-1 to II-56, which is in the zymogen form.

[0323] Embodiment II-58. The engineered protease according to any one of embodiments II-1 to II-56, which is in an active form.

[0324] Embodiment II-59. The engineered protease according to any one of embodiments II-1 to II-58, which is fused to a moiety that increases the half-life of the engineered protease.

[0325] Embodiment II-60. The engineered protease according to embodiment II-59, wherein the component is an Fc domain.

[0326] Embodiment II-61. The engineered protease according to embodiment II-59, wherein the component is human serum albumin.

[0327] Embodiment II-62. The engineered protease of any one of embodiments II-1 to II-15, comprising a modified chymase protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:6.

[0328] Embodiment II-63. The engineered protease of embodiment II-62, wherein the modified chymase protease domain of SEQ ID NO:6 comprises one of the mutations in Table 7B.

[0329] Embodiment II-64. The engineered protease of any one of embodiments II-1 to II-15, comprising a Modified Membrane Serine Protease 1 (MTSP-1) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:7.

[0330] Embodiment II-65. The engineered protease of embodiment II-64, wherein the modified MTSP-1 protease domain of SEQ ID NO:7 comprises one of the mutation sequences in Table 5B.

[0331] Embodiment II-66. The engineered protease of any one of embodiments II-1 to II-15, comprising a modified urokinase-type plasminogen activator (uPA) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:22.

[0332] Embodiment II-67. The engineered protease of embodiment II-66, wherein the modified uPA protease domain of SEQ ID NO:22 comprises one of the mutations in the sequence of Table 3B.

[0333] Embodiment II-68. The engineered protease of embodiment II-1, comprising a modified kallikrein-related peptidase 5 (KLK5) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:23.

[0334] Embodiment II-69. A method for inactivating factor B, comprising contacting factor B with any of the engineered proteases according to any of embodiments II-1 to II-68.

[0335] Embodiment II-70. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject any one of the engineered proteases described in any one of embodiments II-1 to II-68.

[0336] Embodiment II-71. The method of embodiment II-70, wherein the disease or condition is associated with dysregulated complement.

[0337] Embodiment II-72. The method of any one of embodiments II-70 to II-71, wherein the disease or condition is an inflammatory disease or condition.

[0338] Embodiment II-73. The method according to any one of embodiments II-70 to II-72, wherein the treatment is replacement therapy.

[0339] Embodiment II-74. The method of any one of embodiments II-70 to II-73, wherein the treatment blocks complement activation.

[0340] Embodiment II-75. The method according to any one of embodiments II-70 to II-74, wherein the treatment modulates autoimmunity.

[0341] Embodiment II-76. The method of any one of embodiments II-70 to II-75, wherein the disease or condition is a congenital complement deficiency.

[0342] Embodiment II-77. The method according to any one of embodiments II-70 to II-76, wherein the treatment is for endothelial cell injury or renal cell injury.

[0343] Embodiment II-78. The method of any one of embodiments II-70 to II-77, wherein the disease or condition is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplant ischemia and reperfusion (I / R) injury, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), sepsis, acute respiratory distress syndrome (ARDS), SARS-associated coronavirus (SARS-CoV), atypical hemolytic uremic syndrome (aHUS), membranous nephropathy (MN) and paroxysmal nocturnal hemoglobinuria (PNH).

[0344] Embodiment II-79. The method of any one of embodiments II-70 to II-78, wherein the disease or condition is a regulatory protein deficiency.

[0345] The method of any one of embodiments II-70 to II-78, wherein the disease or condition is a secondary complement disorder.

[0346] The method of any one of embodiments II-70 to II-78, wherein the disease or condition is an immune-related disease or condition.

[0347] The method of any one of embodiments II-70 to II-81, wherein the engineered protease is administered subcutaneously to the subject.

[0348] Embodiment II-83. The method of embodiment II-82, wherein the engineered protease is activated in situ at the site of the dysregulated complement component.

[0349] Embodiment II-84. The method of any one of embodiments II-70 to II-83, wherein the engineered protease is provided in a liquid stable formulation.

[0350] Embodiment II-85. A pharmaceutical composition comprising any of the engineered proteases according to any one of embodiments II-1 to II-68, and optionally a pharma- ceutically acceptable carrier.

[0351] Embodiment II-86. The pharmaceutical composition of embodiment II-85, wherein the engineered protease is provided in a liquid stable formulation.

[0352] Embodiment II-87. A pharmaceutical composition according to any one of embodiments II-85 to II-86, which is formulated for subcutaneous administration. EXAMPLES

[0353] Example 1: Expression, purification and initial characterization of a chymotrypsin-like serine protease Expression and purification of MTSP-1 and uPA-based proteases Small-scale expression and purification of engineered MTSP-1 and uPA-based proteases were performed. Briefly, MTSP-1 and uPA were each expressed as zymogens in E. coli strains in the BL21-Gold(DE3) bacterial host, isolated from inclusion bodies, denatured, refolded by rapid dilution, dialyzed, and subsequently activated on an immobilized trypsin column. The active protein was then purified on an anion exchange column. Similarly, large-scale refolding and purification of MTSP-1 was also performed. MTSP-1 and uPA were solubilized, refolded, and purified on an anion exchange column. Endotoxins were then removed from MTSP-1 and uPA.

[0354] Expression and purification of chymase-based proteases Small scale expression and purification of chymase was performed. Briefly, chymase was expressed as a zymogen in inclusion bodies in E. coli strains with BL21 Gold (DE3) bacterial host. Insoluble chymase was isolated from the inclusion bodies, denatured in the presence of a reducing agent, refolded by rapid dilution, and dialyzed. The zymogen form of the protein was purified using 4 mL of Fast Flow SP beads packed into a BioRADd column using gravity and activated overnight with enterokinase. Native chymase was then purified from the zymogen using cation exchange chromatography using a step elution method.

[0355] Factor B cleavage by chymase-based engineered proteases Factor B cleavage by chymase-based engineered proteases was tested and verified by Coomassie gel. Briefly, digestion reactions were prepared with 2.0 μM human factor B (Complement Technologies) in 20 μL of buffer (50 mM Tris pH 7.4 / 50 mM NaCl / 0.01% Tween 20). Different concentrations of chymase-based engineered proteases were added to factor B (3000 nM at the top, diluted 1:2, 10 steps, including 0.0 nM) and incubated at 37 °C for 1 h. After digestion, 15 μL of the reaction was transferred to a 96-well plate containing 1.5 μL of 0.2 N HCL to stop the chymase digestion. After quenching, the reaction was prepared for SDS-PAGE. 20 μL of the reaction mixture was loaded per well of a 4-12% Bis-Tris Criterion gel. Densitometric analysis of factor B cleavage and EC 50 was calculated. FIG. 1D shows a Coomassie gel showing an example of factor B cleavage by the chymase-based engineered proteases tested. Table 8A lists the chymase-based engineered proteases tested in FIG. 1D. All references to engineered proteases in the table are given in chymotrypsin numbering. See Table 6 for the chymotrypsin numbering key of the modified protease domains of chymase. Two lots of each engineered protease set were used, and each lot demonstrated the ability of the engineered protease to cleave factor B. Lot 1 of each set showed visible amounts of zymogen that were not titratable but were likely activated or somewhat active against factor B. These results also show that the engineered proteases are very efficient in cleaving factor B. The engineered proteases tested are shown in Table 8A below.

[0356] Figure 1E shows the low EC calculated from ELISA measurements. 50FIG. 1 shows a graph showing an example of factor B cleavage by two chymase-based engineered proteases with the following structure: All references to engineered proteases in this example are given with chymotrypsin numbering. See Table 6 for the chymotrypsin numbering key of the modified protease domains of chymase. Briefly, digestion reactions were prepared with 2.0 μM human factor B (Complement Technologies) in 20 μL of buffer (50 mM Tris pH 7.4 / 50 mM NaCl / 0.01% Tween 20). Different concentrations of two chymase-based engineered proteases were used: C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R and C22S / P38Q / K40M / F41R / V138I / F173Y / D175R / A190S / V213A / S218V / A226R. These were each provided at 3000 nM and diluted 1:2 to obtain 1500, 750, 375, 188, 94, 47, 23, 12, 6, and 3 nM, plus a 0.0 nM control. These were each added to 2 μM factor B and incubated at 37° C. for 1 hour. After digestion, 15 μL of the reaction was transferred to a 96-well plate containing 1.5 μL of 0.2 N HCL. After quenching, the reaction was prepared for Factor B ELISA.

[0357] A factor B standard curve was generated at 800, 533.3, 355.5, 237.0, 158.0, 105.3, 70.2, 46.8, 31.2, 20.8, 0.0 pM in 1% BSA-PBST. A 384-well plate was coated with a monoclonal antibody against human factor B fragment (Quidel) at 2 μg / ml in carbonate buffer (25 μL / well). After blocking with 1% BSA-PBST (100 μL / well) for 1 h at room temperature, the digest (chymase+FB) was diluted to 800 pM and standards were diluted 1:1.5 from 800 pM in blocking buffer (25 μL / well). The plate was agitated for 30 min at room temperature.

[0358] A biotinylated monoclonal antibody against human factor Bb fragment (Quidel) was then added at 0.125 μg / ml to detect bound FB. Streptavidin-HRP was then diluted 1:200 in blocking buffer (25 μL / well) and the plate was agitated for 30 minutes at room temperature. The plate was developed with ELISABright (50 μL / well) for 1 minute at room temperature and read on an EnVision plate reader. Table 8B below shows the calculated EC for the two engineered proteases. 50 Figure 1E shows that 30-42 nM of the tested chymase-based engineered proteases were required to inhibit 50% of factor B.

[0359] [Table 12]

[0360] [Table 13]

[0361] Example 2: Protein characterization and active site titration of chymase-based engineered proteases Selected chymase-based engineered proteases were tested to measure the activity of these proteases by using an active site titration method based on reaction with inhibitory serpin baits, followed by HPLC analysis to quantify the active fraction. All references to engineered proteases in this example, including Table 9, are given in chymotrypsin numbering. See Table 6 for the chymotrypsin numbering key of the engineered protease domains of chymase. HPLC analysis detects a shift in the measured peak in the presence of serpin bait, indicating that the protease is bound to the bait and therefore active. Briefly, a working concentration of 24 μM serpin bait was prepared from a stock solution along with 50 μM low molecular weight heparin for antithrombin (AT)-based baits. Protease solutions were prepared by using 30 μL of 5 μM working solution. The reaction mixtures were incubated at 37° C. for 2 hours and each reaction mixture (protease alone or protease with bait) was analyzed by HPLC using standard protocols. All protease alone samples were run first, followed by samples of protease with serpin bait. A summary of the results for the proteases tested is summarized below in Table 9. cat / K m was measured using EGVD / AE-QF (SEQ ID NO: 52), and the second-order reaction rate constant K * app was measured using EGVD / AE-ACT (SEQ ID NO: 53). These results were obtained by measuring the protease activity after purification, and most of the chymase-based engineered proteases tested showed activity between 60% and 120%.

[0362] [Table 14-1]

[0363] [Table 14-2]

[0364] Example 3: Serine protease stability studies using factor B cleavage and a selection of serine proteases Cleavage of factor B by various engineered proteases was evaluated. Engineered chymase, MTSP-1, and uPA-based proteases were tested. All references to engineered proteases in this example, including Tables 10A, 10B, 11A, and 11B, are indicated with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of engineered proteases can be found in Tables 2, 4, and 6 for uPA, MTSP-1, and chymase, respectively. These experiments were also performed to evaluate the stability of non-naturally occurring chymase in various media, including cynomolgus monkey vitreous humor, human plasma, and phosphate-buffered saline (PBS). Briefly, factor B was diluted in assay buffer and incubated with naturally occurring (wild-type) and non-naturally occurring (engineered) proteases in vitreous humor, plasma, or PBS at 37° C., and the reaction was quenched with HCl at various time points. Time points tested for chymase included T=0 using 6 hours preincubation, T=2 hours using 4 hours preincubation, T=4 hours using 2 hours preincubation, and T=6 hours using 0 hours preincubation. Samples were assayed using ELISA or Alpha Screen for human C3 cleavage using standard protocols.

[0365] A summary of the results is presented in Tables 10A-10B below, showing the various proteases tested, the concentration of protease used (nM), and the EC 50Table 10 presents human factor B cleavage by various non-naturally occurring chymase-based engineered proteases tested in 80% human plasma compared to chymase containing the mutation sequence C122S. Blank cells indicate that no testing was performed for the particular protease mutation sequence. These results generally show that selected chymase-based engineered proteases, such as C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R and C22S / P38Q / K40M / F41R / V138I / F173Y / D175R / A190S / V213A / S218V / A226R, exhibit high factor B cleavage activity.

[0366] Table 10B shows human factor B cleavage by various engineered chymase, MTSP-1, or uPA-based proteases tested in mouse or human plasma. Briefly, for ELISA analysis of factor B cleavage, anti-Ba was used and Bb was detected with anti-Bb. A dynamic range of 25-1600 pM was used. Half-lives in 50% human plasma were also calculated for various chymase-based engineered proteases.

[0367] These results showed that proteases engineered from uPA-based scaffolds did not exhibit efficient factor B cleavage, proteases engineered from MTSP-1-based scaffolds showed factor B cleavage, but only for two candidates with an EC of less than 50 nM. Proteases engineered from chymase-based scaffolds showed an EC of less than 50 nM. 50 This value indicated the most efficient cleavage of factor B.

[0368] [Table 15-1]

[0369] [Table 15-2]

[0370] [Table 15-3]

[0371] [Table 15-4]

[0372] [Table 15-5]

[0373] [Table 15-6]

[0374] [Table 15-7]

[0375] [Table 15-8]

[0376] [Table 15-9]

[0377] [Table 16]

[0378] Additionally, various chymase-based engineered proteases were tested to assess their baseline stability by testing the effect of indirect and MTSP buffers (+ / - EDTA) on the chymase-based engineered proteases. The peptide substrate EGVDAE-QF (SEQ ID NO: 52) was used in these experiments.

[0379] Figures 4A-4E show graphs depicting the stability of five different chymase proteases tested with peptide substrates in PBS at 37° C. Table 11A summarizes the data shown in Figures 4A-4E and lists the engineered proteases tested.

[0380] [Table 17]

[0381] As shown, these proteases were more stable in indirect buffers with a pH of 7.4. Wild-type or naturally occurring chymase showed loss of activity in all buffers, and EDTA had no apparent effect on stability.

[0382] Second-order rate constants (k*) were then measured for the inhibition of various non-naturally occurring chymase-based proteases by plasma for extrapolation of half-life in 100% plasma. Briefly, a baseline chymase with modification C22S / A226R was used. The concentrations of baseline chymase and tested chymase-based engineered proteases listed in Table 11B below were used at 50 nM, and another tested chymase C22S / L99R / F173Y / K192R / S218L / A226R was used at 25 nM. For baseline chymase, the time course of protease activity in the presence of plasma was measured at 37°C using 4.2 uM CPQ2-ITLLSA-K(5FAM)-K-PEG8-K(biotin)-NH2 (SEQ ID NO: 17) / 21 uM neutravidin. A summary of the results is shown in Table 11B below.

[0383] [Table 18]

[0384] Example 4: Factor B add-back hemolysis assay Hemolytic assays were performed to evaluate the hemolytic activity of various lots of factor B, lots obtained from CompTech, compared to in-house expressed lots. Briefly, factor B-depleted human serum was used and plasma purified or recombinant human factor B was used for add-back. Rabbit red blood cells (RBCs) or chicken RBCs were washed in GVB / Mg / EGTA and the depleted serum was spiked with factor B. RBC lysis was then monitored with CVF / FD / human factor B convertase. These were analyzed to generate standard curves from which factor B lots were compared. Figures 5-6 show bar graphs of the controls used in the hemolytic assay and the standard curves measured from the engineered proteases tested from the factor B add-back hemolytic assay, respectively. Table 12 below summarizes the data from the MTSP-1 and uPA-based engineered proteases. Figure 6 also shows that the MTSP-1 based engineered protease F97E / K224N / F99L / D217I / C122S / C

[17] S / C

[19] S can inhibit hemolysis. The engineered protease was tested at two concentrations (samples 1 and 2). All references to engineered proteases in this example are given with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of the engineered proteases can be found in Tables 2, 4 and 6 for uPA, MTSP-1 and chymase, respectively.

[0385] [Table 19]

[0386] Example 5: Peptide cleavage assay (k cat / K M ) Peptide cleavage assays were performed to determine the kappa activity of various engineered proteases of the present disclosure based on MTSP-1, uPA, or chymase. cat / K mAll references to engineered proteases in this example are given in chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of the engineered proteases can be found in Tables 2, 4, and 6 for uPA, MTSP-1, and chymase, respectively. Cleavage of various substrates was assessed to determine k for protease activity. cat / K m In short, k cat / K m Values ​​of V as a function of substrate concentration o The linearity of the kinetics was determined from the slope of the linear portion of the plot. In general, the following conditions were used: protease concentration of 50 nM, substrate concentration up to 20 μM and 1.5-fold serial dilutions, and a temperature of 30° C. For calculations, a semi-automated Michaelis-Menten kinetic protocol with quadruplicate measurements was used.

[0387] For chymase-based engineered proteases, the following quenched fluorescent (QF) peptide substrates were used: TQ2-KDVFYQMKK-Lys(5FAM)-NH2 (SEQ ID NO: 29), and TQ2-KDVFYQMKK-Lys(5FAM) (SEQ ID NO: 30). For MTSP-1 and uPA-based engineered proteases, the following peptide substrates were used: 5FAM-EQQKRKIVL-K(QXL520)-NH2 (SEQ ID NO: 31), CPQ2-PEQQKR-K(5FAM)-NH2 (SEQ ID NO: 32), TQ2-GEQQKRKIVL-Lys(5FAM)-NH2 (SEQ ID NO: 33), Ac-QQKR-ACC (SEQ ID NO: 34). Table 13A below shows the various substrates used to test for protease activity at specific cleavage site sequences, and Table 13B below shows the EGVDAE QF (SEQ ID NO: 52) substrate k based on the various proteases tested. cat / K m (M -1 s -1 ) is shown.

[0388] [Table 20]

[0389] [Table 21]

[0390] The results of the peptide cleavage assays are presented below in Tables 14A-14B.

[0391] [Table 22-1]

[0392] [Table 22-2]

[0393] [Table 23-1]

[0394] [Table 23-2]

[0395] [Table 24]

[0396] [Table 25]

[0397] [Table 26-1]

[0398] [Table 26-2]

[0399] [Table 27]

[0400] Example 6: Inhibition studies on engineered proteases Inhibition studies were performed with chymase-based engineered proteases. Various serpins capable of inhibiting protease activity were used to test whether they could be used for suitable inhibition of the tested chymase-based engineered proteases, allowing the selection of engineered proteases resistant to inhibition in plasma. A summary of the serpin inhibition studies is provided in Table 15 below. All references to engineered proteases in this example are given with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of engineered proteases can be found in Tables 2, 4 and 6 for uPA, MTSP-1 and chymase, respectively.

[0401] [Table 28]

[0402] Example 6: Factor B cleavage and activity of KLK5 protease FIG. 7A shows a graph showing hemolysis inhibition by KLK5 and compared to the chymase-based engineered protease C22S / F173Y / D175N / A226R. All references to engineered proteases in this example are given with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of the engineered proteases can be found in Tables 2, 4 and 6 for uPA, MTSP-1 and chymase, respectively. Briefly, digestion reactions were prepared with 4.0 μM human factor B (Complement Technologies) in 20 μL of buffer (50 mM Tris pH 7.4 / 50 mM NaCl / 0.01% Tween 20). Different concentrations of KLK5 protease or chymase-based engineered protease C22S / F173Y / D175N / A226R (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39, as well as 0.0 nM control) were added to 4 μM factor B and incubated for 1 h at 37° C. After digestion, 6 μL of the reaction was evaluated in a hemolysis assay.

[0403] The factor B digestion reaction was diluted with 20 μL of GVB (Complement Technologies), 10 mM MgCl2, and 8 mM EGTA (GVB / Mg / EGTA). 45 μL of human factor B depleted serum (Complement Technologies) was then added to 5 μL of human factor B digest to obtain a final volume of 90% serum. In parallel, 50 μL of rabbit red blood cells (Colorado Serum Co.) were diluted into 950 μL of GVB / Mg / EGTA and mixed gently. After spinning at 2000 RPM for 5 minutes at 4° C., the rabbit cells were resuspended in 1 mL of GVB / Mg / EGTA. The washed rabbit cells were then incubated with the serum+factor B digestion mixture in GVB / Mg / EGTA buffer to obtain a final serum concentration of 15% and incubated at 37° C. for 1 hour with agitation. The reactions were then centrifuged at 2000 RPM for 5 minutes and 100 μL of the supernatant was transferred to a clear flat-bottom 96-well plate. Plate absorbance was read at 415 nm using a spectrophotometer and EC 50The results of the hemolysis assay are shown in FIG. 7A and demonstrate that the KLK5 protease was effective in inhibiting hemolysis.

[0404] FIG. 7B shows a graph depicting factor B cleavage by KLK5 and chymase-based engineered protease C22S / F173Y / D175N / A226R. Briefly, digestion reactions are prepared with 4.0 uM human factor B (Complement Technologies) in 20 μL of buffer (50 mM Tris pH 7.4 / 50 mM NaCl / 0.01% Tween 20). Different concentrations of KLK5 (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39, as well as a 0.0 nM control) are added to 4 μM factor B and incubated at 37° C. for 1 hour. After digestion, 15 μL of the reaction is transferred to a 96-well plate containing 1.5 μL of 0.2 N HCL. After quenching, the reactions are prepared for Factor B ELISA.

[0405] A factor B standard curve is generated at 800, 533.3, 355.5, 237.0, 158.0, 105.3, 70.2, 46.8, 31.2, 20.8, 0.0 pM in 1% BSA-PBST. A 384-well plate was coated with a monoclonal antibody against human factor Ba (#A225, Quidel) at 2 μg / mL in carbonate buffer (25 μL / well). After blocking with 1% BSA-PBST (100 μL / well) for 1 h at room temperature, the digest (chymase + factor B) was diluted to 800 pM and standards were diluted 1:1.5 from 800 pM in blocking buffer (25 μL / well). The plate was agitated for 30 min at room temperature.

[0406] Biotinylated monoclonal antibody against human factor Bb (Quidel) was then added at 0.125 μg / ml to detect bound factor B. Streptavidin-HRP was then diluted 1:200 in blocking buffer (25 μL / well) and the plate was agitated for 30 min at room temperature. The plate was developed with ELISABright (50 μL / well) for 1 min at room temperature and read on an EnVision plate reader. Figure 7B shows two independent experiments using different stocks of KLK5. These results indicate that the KLK5 protease is effective in cleaving factor B, with comparable activity at the various concentrations used.

[0407] Factor B cleavage by KLK5 was also assessed by Coomassie gel. Figures 8A-8B show Coomassie gels showing examples of factor B cleavage by KLK5 and the chymase-based engineered protease C22S / F173Y / D175N / A226R. Briefly, digestion reactions were prepared with 4.0 μM human factor B (Complement Technologies) in 20 μL of buffer (50 mM Tris pH 7.4 / 50 mM NaCl / 0.01% Tween 20). Different concentrations of KLK5 or chymase-based engineered proteases C22S / F173Y / D175N / A226R (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39, as well as 0.0 nM control) were added to 4 μM factor B and incubated at 37 °C for 1 h. After digestion, 15 μL of the reaction was transferred to a 96-well plate containing 1.5 μL of 0.2 N HCL. After quenching, the reaction was prepared for SDS-PAGE gel. 20 μL of the reaction mixture was loaded per well of a 4-12% Bis-Tris Criterion gel. Densitometric analysis of factor B cleavage was performed to determine EC 50 was calculated.

[0408] FIG. 9 shows the QQKR / KIV (SEQ ID NO: 9) cleavage site of factor B by KLK5. 234The cleavage site at Arg was identified within the EGVDAE (SEQ ID NO: 13) cleavage site of factor B by the chymase-based engineered protease C22S / A226R. 221 Mass spectrometry (MS) data identifying the cleavage site at Asp are shown. Briefly, human factor B (Comptech) was incubated at 2 μM with different concentrations of kallikrein 5 (KLK5, R&D Systems) or chymase-based engineered protease C22S / A226R at 10 nM or 100 pM in 20 mM Tris pH 8 buffer containing 16-O or 18-O water for 10 min to 1 h at 37 °C. The reaction was quenched with 20 μM of the inhibitor FFR-CMK for 30 min at room temperature. Half of the sample was further treated with Rapidgest / chymotrypsin and adjusted to pH 3 by addition of 1 mL of 1% TFA. TCEP (100 mM final concentration) was then added to reduce disulfides for 30 min at 37 °C. 12 mL of each sample was bound to a Ziptip (Millipore) and eluted with 15 mL of 80% ACN-0.1% TFA. After drying in a Speedvac, the samples were redissolved in 4 mL of 30% ACN-0.05% TFA. 0.35 mL of sample was loaded onto an OptiPlate with 0.45 mL of CHCA matrix (10 mg / mL) and analyzed by MALDI-MS (ABI 4700) in both linear (m / z 2-22k) and reflector (m / z 1500-5400) modes. When possible, tandem MS was performed on peptides of interest. Two large peptide fragments were detected at approximately 33 and 59 kDa. In summary, 100 pM KLK5 cleaves one of 38 Args in a 10-minute reaction and four of 38 Args in a 60-minute reaction (Arg175, Arg193, Arg730, and Arg739). At 10 nM, cleavage was detected at 18 of 38 Args in 10-minute and 60-minute reactions (Arg50, Arg74, Arg94, Arg175, Arg182, Arg193, Arg202, Arg203, Arg259, Arg381, Arg415, Arg658, Arg679, Arg708, Arg710, Arg730, Arg739). The earliest cleavage event was at Arg 730.

[0409] The reconstructed ion chromatograms shown in Figure 9 are of the reaction products from kallikrein-5 ("KLK"), chymase-based engineered protease C22S / A226R ("chymase-based"), and plasma-derived factor B ("Fb control"). These chromatograms demonstrate that chymase-based engineered protease C22S / A226R is 221 The peak at 3.8 min corresponds to intact chymase, the peak at 4.73 min corresponds to intact factor B (739 residues with four A2 glycans, MW 91802), and the peak at 4.63 min corresponds to residues 222 to 739 (MW 62827) containing two A2 glycans, indicating cleavage of factor B at Asp. 221 In contrast, KLK5 corresponds to cleavage at Asp. 234 Factor B is cleaved at Arg (peak at 4.69 min, MW 61437). No remaining intact factor B was present in this sample (top trace).

[0410] Example 7: Complement activation and cytokine release measured in a mouse model of acute respiratory distress syndrome Figure 10 is a schematic diagram showing a general method for measuring complement activation and cytokine release from tissues of a mouse model of acute respiratory distress syndrome (ARDS) treated with chymase-based engineered proteases. All references to engineered proteases in this example are indicated with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of engineered proteases can be found in Tables 2, 4 and 6 for uPA, MTSP-1 and chymase, respectively. Briefly, mice were injected with chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R, lung function was measured, and bronchoalveolar lavage fluid was measured after sacrifice. In Figure 10, the engineered protease is indicated as "protease".

[0411] After the adaptation period, each animal was weighed and randomly assigned to a treatment group based on body weight. On day 0 (0 hours), mice were anesthetized and received a single intratracheal instillation (IT) of lipopolysaccharide (LPS, Sigma) at a dose of 50 μg per mouse. Control mice received an instillation of sterile 0.9% saline (50 μl). All animals were monitored for general health and weight over the course of the disease. Respiratory function was measured by whole body plethysmography (WBP) in conscious mice at 0, 6, 24 and 48 hours after LPS IT. Three hours after LPS IT, mice were intravenously (IV) injected with chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R at 5 mg / kg or 6.5 mg / kg, and control animals were IV injected with vehicle (PBS). A subset of mice received a second IV injection of chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R at 5 mg / kg or 6.5 mg / kg 27 hours after LPS IT (i.e., 24 hours after the first IV injection). Mice were sacrificed 24 and 48 hours after LPS IT. To obtain plasma, blood was collected by facial puncture under anesthesia and collected in K2EDTA microtainer tubes. After centrifugation at 2,000×g for 10 min at 4° C., plasma was aliquoted (60 μl) and stored at −80° C. for future cytokine and complement analysis.

[0412] A tracheotomy was then performed to expose the lungs. The trachea was connected to a cannula, and while the left lung was clamped, 0.9 mL of cold PBS 1X, protease inhibitor 1X (SigmaFAST®) solution (3x300 μL) was injected to perform a bronchoalveolar lavage fluid (BALF) on the right lobe of the lung. The first aliquot (300 μL) was kept for BALF total and differential cell counts. The other two aliquots of 60 μL each were stored at -80°C for future complement and cytokine analysis. The right lung was immediately snap frozen and stored at -80°C for complement and cytokine analysis in lung homogenates. The lungs were homogenized in 1x PBS + 0.1% Triton X-100 with protease cocktail inhibitors to obtain a homogenate of 20 mg / 100 μL and centrifuged at 2,520 x g for 15 min at 4°C. The supernatants were then processed for cytokine analysis.

[0413] Three hours after injection of the chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R, plethysmography measurements showed a significant decrease in PenH values ​​suggesting improved lung function. However, this effect did not persist over time (24 and 48 h measurements). 48 h after LPS, animals receiving two doses of the chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R at 5–6.5 mg / kg showed a significant improvement in body weight, suggesting that this engineered protease reduced the severity of ARDS symptoms. When sacrificed 48 hours after LPS, animals receiving two doses of chymase-based engineered protease at 5-6.5 mg / kg showed a trend towards lower neutrophil-to-lymphocyte ratios in the BALF, indicating that inflammatory infiltrates are reduced by chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R. These results are shown in Figures 11A-11C. Figure 11A shows the pulmonary congestion index indicated by PenH values. Figure 11B shows the weight loss measured in the test animals. Figure 11C shows the BALF neutrophil-to-lymphocyte ratio (NLR) measured in the test animals. Data shown are mean + / - SEM, *p-values ​​are <0.05 using Student's T-test. These results show that the chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R is effective in improving respiratory function in a mouse model of ARDS. In Figures 11A-11C, this engineered protease is referred to as "protease."

[0414] Figures 12A-12D show the results of BALF and lung cytokine measurements from mouse tissues after treatment with chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R. In Figures 12A-12D, this engineered protease is referred to as "Protease". Briefly, BALF and lung tissues were collected as described above 24 hours after a single dose of chymase-based engineered protease C22S / P38Q / K40L / F41R / V138I / F173Y / D175N / A190S / V213A / S218V / A226R was administered intravenously at 5 mg / kg 3 hours after LPS. Tissues were processed for cytokine / chemokine quantification using a Mouse Cytokine Array / Chemokine Array 31-Plex (MD31) (Eve Technologies) measuring Eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p 40), IL-12(p70), IL-13, IL-15, IL-17A, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF. Samples were centrifuged before aliquoting and diluted 2-fold before performing the assay according to the Eve Technology protocol. IL-2, IL-6 and CXCL9 were significantly decreased in mouse bronchoalveolar fluid (BALF) and IL-6 was significantly decreased in lung tissue, indicating lower lung inflammation that may result in a subsequent decrease in chemoattraction of inflammatory cells to lung tissue. Values ​​are mean + / - SEM, *p<0.05, **p<0.01 using Student's t-test. These results indicate that this engineered protease is effective in reducing inflammatory cytokines IL-2 and IL-6, and the chemokine CXCL9 in a mouse model of ARDS.

[0415] Example 8: Activity evaluation and characterization of mammalian expressed chymase-based engineered proteases The ability of chymase-based engineered protease zymogens (C22S / P38Q / K40A / F41R / L99H / V138I / F173Y / D175N / A190S / V213A / S218I / A226R) (Table 7B), produced in a mammalian culture system, purified and activated, to cleave CFB was evaluated. All references to engineered proteases in this example are given with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of the engineered proteases can be found in Tables 2, 4 and 6 for uPA, MTSP-1 and chymase, respectively. Enzymatic cleavage of complement factor B (CFB, Complement Technologies catalog number A135) was evaluated by AlphaLISA assay. After 1 h of in vitro cleavage at 37 °C, the reaction was diluted and transferred to a multiwell plate for bead-based detection of CFB. For detection of CFB, anti-factor Ba antibody (Quidel catalog number A225) labeled with DIG (Biotium Mix N'Stain Kit, catalog number 92450) was paired with anti-DIG acceptor beads (Perkin Elmer catalog number AL113C) and biotinylated anti-factor Bb antibody (Quidel catalog number A712) was paired with streptavidin donor beads (Perkin Elmer, catalog number 6760002S). If uncleaved CFB remains, the acceptor and donor beads are brought together and upon laser excitation, singlet oxygen from the donor bead drives a chemiluminescent signal from the acceptor bead that activates a fluorophore contained in the same acceptor bead. Upon CFB cleavage with the engineered proteases tested in this example, the resulting Ba and Bb cleavage products no longer associate, resulting in a loss or reduction in signal. Cleavage of full-length CFB was quantified by linear regression against a CFB standard curve in the absence of chymase. Table 16 shows the results of CFB cleavage using the engineered proteases listed.

[0416] [Table 29]

[0417] The engineered proteases were also evaluated for inhibition of hemolysis in both a standard alternative pathway (AP) hemolysis assay as well as an enhanced version of the assay similar to that described in Example 4. Hemolysis inhibition by the low molecular weight factor B inhibitor LNP023 (Iptacopan, MedChemExpress Cat. No. HY-127105) was evaluated in the same experiment as a comparison. For the standard AP assay, various concentrations of C22S / P38Q / K40A / F41R / L99H / V138I / F173Y / D175N / A190S / V213A / S218I / A226R engineered proteases or LNP023 or relevant vehicle controls were premixed with 20% normal human serum (NHS) for 10 min at 37°C. Rabbit red blood cells (RBCs) were then added with alternative pathway buffer (Gelatin Veronal Buffer, GVB+Mg+EGTA, 0.1% gelatin, 5 mM veronal, 145 mM NaCl, 0.025% NaN3, pH 7.3, 10 mM MgCl2 and 8 mM EGTA) and incubated at 37°C for 30 minutes. Cells were then pelleted and supernatant OD was measured at 415 nm to assess lysis. For an enhanced version of the assay, the following adjustments were made: NHS was replaced with human factor B depleted serum (Complement Technologies catalog number A335), factor B purified from human serum was spiked back into the serum to a final concentration of 1.6 mM in the hemolysis assay, and preincubation of serum with drug was performed for 180 minutes at 37°C. Percent lysis was calculated using the following formula: [(OD of sample) / (OD of sample)]. 415 -OD 415 EDTA negative control) / (OD 415 Saline positive control - OD 415 EDTA)*100] (Table 17A and Table 17B). I C 50 was calculated by non-linear regression (Prism 9, log(inhibitor, bv) vs. response-4 parameter variable slope model) (Tables 18A and 18B).

[0418] [Table 30]

[0419] [Table 31]

[0420] [Table 32]

[0421] [Table 33]

[0422] Example 9: Pulmonary function measured in a mouse model of acute respiratory distress syndrome Figure 13 is a schematic diagram showing a general method for measuring lung function in a mouse model of acute respiratory distress syndrome (ARDS) treated with chymase-based engineered proteases of the present disclosure. All references to engineered proteases in this example are shown with chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of engineered proteases can be found in Tables 2, 4 and 6 for uPA, MTSP-1 and chymase, respectively. Briefly, mice received injections of chymase-based engineered protease C22S / P38Q / K40A / F41R / L99H / V138I / F173Y / D175N / A190S / V213A / S218I / A226R or gavage of LNP023 (AdooQ Biosciences A18905) and lung function was measured before treatment and 24 hours after LPS instillation.

[0423] After the adaptation period, each animal was weighed and randomly assigned to a treatment group based on body weight. On day 0 (0 hours), mice were anesthetized and received a single intratracheal instillation (IT) of lipopolysaccharide (LPS, Sigma) at a dose of 50 μg per mouse. All animals were monitored for general health and weight throughout the disease course. Respiratory function was measured by whole body plethysmography (WBP) in conscious mice at 0 and 24 hours after LPS IT. Immediately before LPS IT, mice were intravenously (IV) injected with the chymase-based engineered proteases listed above at 5.15 mg / kg. Negative control animals received an IV injection of vehicle (PBS). Active comparator animals received a 30 mg / kg dose of LNP023 administered orally. Mice were sacrificed 24 hours after LPS IT.

[0424] Twenty-four hours after administration of the chymase-based engineered proteases listed above or LNP023, plethysmography measurements showed significant protection against pulmonary congestion, suggesting protection of lung function with treatment. These results are shown in FIG. 14. The effect on pulmonary congestion index is shown by the fold change from baseline in PenH values. Data shown are mean + / - SEM, **p-values ​​<0.01 using one-way ANOVA with Dunnett's multiple comparison test.

[0425] Table 19 shows a comparison of dosage levels of the chymase-based engineered proteases of the examples with LNP023 dosage levels administered in vivo.

[0426] [Table 34]

[0427] Table 19, in combination with the protective effect of treatment on the pulmonary congestion index observed in Figure 14, demonstrates that the chymase-based engineered proteases tested in this example are as effective as the activity comparator LNP023 in protecting respiratory function in a mouse model of ARDS when administered at approximately 355-fold lower molar concentrations.

[0428] The results suggest efficient regulation of the engineered proteases at low concentrations, whereas small molecule therapeutics require higher concentrations and frequent dosing.

[0429] Example 10: Expression and purification of untagged chymase-based engineered proteases in mammalian expression systems HEK293 cells were transiently transfected with chymase-based engineered protease expression vectors, harvested, and clarified by depth filtration. All references to engineered proteases in this example are given in chymotrypsin numbering. The chymotrypsin numbering keys for the modified protease domains of engineered proteases are found in Tables 2, 4, and 6 for uPA, MTSP-1, and chymase, respectively. The culture harvest is diluted 1.5-fold with 25 mM Tris-HCl, pH 7.5 (CCS). The CCS is loaded onto a cation exchange column (Capto SP ImPres or similar) at 20 mL / min. The column is washed with 10 CV of 90% Buffer A (25 mM Tris-HCl, pH 7.5) + 10% Buffer B (containing 25 mM Tris-HCl, 1 M NaCl pH 7.5) at 20 mL / min. The recombinant engineered chymase is eluted from the column at 10 mL / min using a 40 CV linear gradient from 10% buffer B to 65% buffer B, and 5 mL peak fractions containing engineered chymase are collected, pooled and quantified by absorbance at 280 nm.

[0430] The chymase-based engineered protease was activated by incubation with enterokinase after adjusting the pooled fractions to 150 mM NaCl in buffer A and adding CaCl2 to 4 mM. Activation was initiated by the addition of enterokinase (EKmax, Invitrogen) and incubated overnight at 37°C. The engineered chymase protease was approximately 90% activated by this method and was further purified from non-activated chymase and enterokinase by cation exchange chromatography using the same procedure outlined above. The pooled fractions were formulated in PBS, 0.1% PS80 (pH 7.4) with a purity of >98% monomer, <2% HMWS and LMWS (Figure 15).

[0431] FIG. 15 is an SDS-PAGE (reduced) showing the expression, purification, and activation of the chymase-based engineered proteases of the present disclosure.

[0432] Example 11: Half-life extension (HE) and manufacturability strategies using HSA or IgG1 Fc domain (Fc) Nineteen chymase-based engineered proteases (Table 10) based on cleavage activity were transiently expressed and tested as zymogens in HEK293 cells. All references to engineered proteases in this example are given in chymotrypsin numbering. The chymotrypsin numbering key for the modified protease domains of engineered proteases can be found in Tables 2, 4, and 6 for uPA, MTSP-1, and chymase, respectively. Eight of the 19 were expressed as assessed by SDS-PAGE analysis of clarified tissue culture supernatants (Table 20, Figure 16). Human HSA or Fc was chosen as the C-terminal fusion partner of selected mutants transiently expressed in CHO-S or HEK293 cells for the purposes of extending solubility, chemical, and in vivo half-life. Both HSA and Fc tagged fusion proteins were expressed using the HEK293 transient expression system (Figure 17). HSA fusions rescued expression of engineered protease mutant sequence number 1 (MS#1), which was ranked highly based on specific activity in the original E. coli production screen.

[0433] [Table 35]

Claims

1. An engineered protease comprising a modified chymase protease domain, a modified membrane serine protease 1 (MTSP-1) protease domain, a modified urokinase-type plasminogen activator (uPA) protease domain, or a modified kallikrein-related peptidase 5 (KLK5) protease domain, which is capable of cleaving factor B. Claim 2: a) cleavage of factor B by the engineered protease generates one or more functionally inactive fragments, optionally wherein the one or more functionally inactive fragments are capable of reducing complement activation; and / or b) cleavage of factor B results in the generation of a factor B fragment with reduced function; 2. The engineered protease of claim 1.

3. a) the B factor is a non-human primate B factor, and optionally the non-human primate is a cynomolgus monkey; or b) said factor B is human factor B, optionally wherein said factor B comprises the amino acid sequence set forth in SEQ ID NO:1; 3. The engineered protease of claim 1 or 2.

4. Cleavage of factor B occurs at a site not targeted by factor D, and optionally: a) cleavage at said site not targeted by Factor D generates at least two fragments which are not Ba and Bb; b) the factor B cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO:10), KNQKR / QKQ (SEQ ID NO:11), DVFY / QMI (SEQ ID NO:12), EGVD / AE (SEQ ID NO:13), DHKL / KSG (SEQ ID NO:14), TPW / SLA (SEQ ID NO:15), KVSEAD (SEQ ID NO:20), IRPSKG (SEQ ID NO:4), GGEKRD (SEQ ID NO:5), GKKEAG (SEQ ID NO:3), and DHKL / KSG (SEQ ID NO:21); or c) the factor B cleavage site comprises a sequence selected from WEHR / KGT (SEQ ID NO:10) and KNQKR / QKQ (SEQ ID NO:11), and the engineered protease comprises a modified MTSP-1 protease domain or a modified uPA protease domain. The engineered protease of any one of claims 1 to 3.

5. 5. The engineered protease of any one of claims 1 to 4, wherein cleavage of Factor B results in reduced production of Factor B cleavage products Ba and Bb compared to cleavage by Factor D.

6. 4. The engineered protease of any one of claims 1 to 3, wherein cleavage of Factor B occurs at a site targeted by Factor D, and optionally the Factor B cleavage site targeted by Factor D comprises QQKR / KIV (SEQ ID NO:9).

7. A modified MTSP-1 protease domain, optionally comprising: a) comprises one or more modifications with respect to the MTSP-1 protease domain comprising the amino acid sequence set forth in SEQ ID NO:7; b) the one or more modifications are at one or more positions corresponding to one or more positions selected from D622, I640, L678, A686, F703, D705, F706, T707, F708, K719, C731, D734, Y755, Q783, V791, Q802, A814, D828, and K835 in an MTSP-1 protease domain comprising the sequence of amino acids set forth in SEQ ID NO:18; c) the one or more modifications are selected from those shown in Table 5A; or d) the one or more modifications are selected from the exemplary mutation sequence shown in Table 5B; The engineered protease of any one of claims 1 to 6.

8. does not contain a modified MTSP-1 protease domain, optionally wherein the modification is one or more of a substitution, addition, and deletion of one or more amino acid residues; The engineered protease of any one of claims 1 to 6.

9. A modified uPA protease domain, optionally comprising: a) comprising one or more modifications with respect to a uPA protease domain comprising the amino acid sequence set forth in SEQ ID NO:8, optionally wherein said modifications are one or more of substitution, addition, and deletion of one or more amino acid residues; b) the one or more modifications are at one or more positions corresponding to one or more positions selected from S37, D60, L97, G161, R179, H180, V185, Y187, I207, A247, D248, A251, H252, C279, Y308, V316, A343, Q353, R378, K385 in a uPA protease domain comprising the sequence of amino acids set forth in SEQ ID NO:8; c) the one or more modifications are selected from those shown in Table 3A; or d) the one or more modifications are selected from the exemplary mutation sequence shown in Table 3B; The engineered protease of any one of claims 1 to 6.

10. 7. The engineered protease of any one of claims 1 to 6, which does not contain a modified uPA protease domain.

11. A modified chymase protease domain, optionally comprising: a) the engineered protease comprises a modified chymase protease domain and the cleavage site comprises a sequence selected from DVFY / QMI (SEQ ID NO:12), EGVD / AE (SEQ ID NO:13), DHKL / KSG (SEQ ID NO:14), and TPW / SLA (SEQ ID NO:15); b) comprising one or more modifications with respect to the chymase protease domain comprising the amino acid sequence set forth in SEQ ID NO:6, optionally wherein said modifications are one or more of substitution, addition, and deletion of one or more amino acid residues; c) the one or more modifications are at one or more positions corresponding to one or more positions selected from S36 C7, V21, P26, G31, R37, K74, K80, I90, L101, L103, F110, V125, F157, D159, S176, A177, F178, K179, L186, V196, G199, A203, A207, F209 in the chymase protease domain comprising the sequence of amino acids set forth in SEQ ID NO:6; d) the one or more modifications are selected from those shown in Table 7A; or e) the one or more modifications are selected from the exemplary mutation sequence shown in Table 7B; The engineered protease of any one of claims 1 to 8.

12. 9. The engineered protease of any one of claims 1 to 8, which does not contain a modified chymase protease domain.

13. 9. The engineered protease of any one of claims 1 to 8, comprising a modified KLK5 protease domain and optionally comprising one or more amino acid modifications of SEQ ID NO:

23.

14. 9. The engineered protease of any one of claims 1 to 8, which does not contain a modified KLK5 protease domain.

15. For factor B cleavage, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, or about 1,900 M －１ s －１ No. ｃａｔ / K ｍ 15. The engineered protease of any one of claims 1 to 14, having the following structure:

16. For factor B cleavage, about 10 ３ ~about 10 ９ M －１ s －１ No. ｃａｔ / K ｍ and optionally having: a) an EC 50 for factor B of less than about 20 nM; b) an EC50 for factor B of about 20, about 25, or about 60 nM; or c) an EC 50 for cleaving factor B of about 1,000 to about 4,500 nM; 16. The engineered protease of any one of claims 1 to 15.

17. EC against factor B ５０ 17. The engineered protease of any one of claims 1 to 16, wherein the affinity of the protease is less than about 1 nM.

18. In human plasma: a) more than about 72 hours; b) greater than about 120 hours; and / or c) about 7 days, optionally with catalytic activity of about 10% to about 50%, or about 90% to about 100%; 18. The engineered protease of any one of claims 1 to 17, having a plasma half-life.

19. The method of claim 19, wherein the protease comprises: a) having an increased half-life compared to a protease containing an unmodified MTSP-1 protease domain; or b) have increased bioavailability compared to proteases containing the unmodified MTSP-1 protease domain; 8. The engineered protease of claim 7.

20. The method of claim 1, wherein the protease comprises: a) having an increased half-life compared to a protease containing an unmodified uPA protease domain; or b) have increased bioavailability compared to proteases containing the unmodified uPA protease domain; 10. The engineered protease of claim 9.

21. The method of claim 20, wherein the protease comprises: a) having an increased half-life compared to a protease containing an unmodified chymase protease domain; or 12. The engineered protease of claim 11, wherein b) the engineered protease has increased bioavailability compared to a protease containing an unmodified chymase protease domain.

22. 22. The engineered protease of any one of claims 1 to 21, which is non-immunogenic.

23. 23. The engineered protease of any one of claims 1 to 22, which is in a zymogen form.

24. 23. The engineered protease of any one of claims 1 to 22, which is in an active form.

25. fused to a component that extends the half-life of the engineered protease, and optionally comprising: a) the component is an Fc domain; or b) the component is human serum albumin; 25. The engineered protease of any one of claims 1 to 24.

26. 7. The engineered protease of any one of claims 1 to 6, comprising a modified chymase protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:6, and optionally said modified chymase protease domain of SEQ ID NO:6 comprises one of the mutation series in Table 7B.

27. 7. The engineered protease of any one of claims 1-6, comprising a modified membrane serine protease 1 (MTSP-1) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:7, and optionally said modified MTSP-1 protease domain of SEQ ID NO:7 comprises one of the mutation series in Table 5B.

28. 7. The engineered protease of any one of claims 1-6, comprising a modified urokinase-type plasminogen activator (uPA) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:22, optionally wherein said modified uPA protease domain of SEQ ID NO:22 comprises one of the mutation series in Table 3B.

29. 7. The engineered protease of any one of claims 1 to 6, comprising a modified kallikrein-related peptidase 5 (KLK5) protease domain having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 95% sequence identity to SEQ ID NO:

23.

30. 30. A method of inactivating factor B comprising contacting said factor B with any of the engineered proteases of any one of claims 1-29.

31. 30. A method of treating a disease or condition in a subject in need thereof, comprising administering to said subject any one of the engineered proteases of any one of claims 1-29, optionally wherein said disease or condition is associated with dysregulated complement.

32. The disease or condition is a) an inflammatory disease or condition; b) have a congenital complement deficiency; c) selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplant ischemia and reperfusion (I / R) injury, antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), sepsis, acute respiratory distress syndrome (ARDS), SARS-associated coronavirus (SARS-CoV), atypical hemolytic uremic syndrome (aHUS), membranous nephropathy (MN) and paroxysmal nocturnal hemoglobinuria (PNH); and / or d) a regulatory protein deficiency, a secondary complement disorder, or an immune-related disease or condition; 32. The method of claim 31 .

33. The process comprising: a) Replacement therapy; b) blocking complement activation; c) modulating autoimmunity; and / or d) for endothelial cell injury or renal cell injury; 33. The method according to any one of claims 31 to 32.

34. The method of claim 34, wherein: a) the engineered protease is administered subcutaneously to a subject, and optionally the engineered protease is activated in situ at the site of a dysregulated complement component; and / or b) the engineered protease is provided in a liquid stable formulation; 34. The method according to any one of claims 31 to 33.

35. 30. A pharmaceutical composition comprising any of the engineered proteases of any one of claims 1 to 29, and optionally a pharma- ceutically acceptable carrier, and optionally: a) the engineered protease is provided in a liquid stable formulation; and / or b) formulated for subcutaneous administration; Pharmaceutical compositions.

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