CN117355324A - Factor B protease - Google Patents

Factor B protease Download PDF

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CN117355324A
CN117355324A CN202280017817.2A CN202280017817A CN117355324A CN 117355324 A CN117355324 A CN 117355324A CN 202280017817 A CN202280017817 A CN 202280017817A CN 117355324 A CN117355324 A CN 117355324A
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protease
engineered
factor
seq
engineered protease
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M·波普科夫
V·索罗斯
N·勒莫安
G·E·布劳斯
T·迈尔斯
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Vertex Pharmaceuticals Inc
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Vertex Pharmaceuticals Inc
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Priority claimed from PCT/US2022/011873 external-priority patent/WO2022150729A1/en
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Abstract

Provided herein are engineered proteases of the S1A family that are specific for and are capable of cleaving factor B. Also provided herein are methods of making and using such engineered proteases. The engineered proteases provided herein are useful for reducing complement activation by cleaving and inactivating factor B, thereby treating diseases or disorders associated with dysregulation of the complement system.

Description

Factor B protease
Cross Reference to Related Applications
The present application claims priority from U.S. provisional application 63/135,496, filed on 8 a 1 st 2021, and U.S. provisional application 63/221,108, filed on 13 a 7 th 2021, the contents of which are incorporated herein by reference in their entirety.
Background
The complement system includes the classical pathway, the alternative pathway, and the lectin pathway, and is tightly controlled by a variety of regulatory factors and components. One such component is complement factor B (interchangeably referred to herein as CFB, factor B, FB), which is a serine protease zymogen that circulates in the blood as a single chain polypeptide. When factor B is associated with an active form of C3 such as surface-bound C3B or fluid phase C3 (H 2 O) to form a pro-conversion complex, factor B may then be cleaved by factor D into two fragments Ba and Bb. Factor D targets the cleavage site of factor B including the Arg234-Lys235 bond. The resulting Ba cleavage product is non-catalytic and released from the complex, while the resulting Bb cleavage product is a catalytic serine protease, which can then cleave C3 into C3a and C3b. This production of C3B is part of the complement system amplification loop, allowing C3B to bind to another factor B to form C3bBb.
Factor B and its cleavage products regulate complement activation. Dysregulated complement is associated with diseases involving the complement system, and thus methods for modulating or inhibiting specific regulatory points within the complement system (such as the production of inactive factor B fragments) are needed. Compositions and methods that meet these needs are provided herein.
Disclosure of Invention
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 are useful for treating diseases or disorders associated with dysregulation of the complement system or excessive activation of complement.
Thus, in one aspect, provided herein is an engineered protease of the S1A serine protease family, wherein the engineered protease is specific for and capable of cleaving factor B. More specifically, the engineered proteases of the present disclosure comprise a modified chymotrypsin 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 (KLK 5) protease domain, wherein the engineered protease is capable of cleaving factor B. The modification comprises one or more of a substitution, addition, and deletion of one or more amino acid residues, and/or one or more of a substitution, addition, and deletion of one or more domains of a chymotrypsin-like serine protease.
In some embodiments, the engineered protease is based on an MTSP-1 protease domain. In some embodiments, the engineered protease is not based on an MTSP-1 protease domain. In some embodiments, the engineered protease comprises one or more modifications relative to a 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 shown in table 5A. In some embodiments, the one or more modifications are selected from those exemplary mutational strings shown 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.
In some embodiments, the engineered protease is based on the uPA protease domain. In some embodiments, the engineered protease is not based on the uPA protease domain. In some embodiments, the engineered protease comprises one or more modifications relative 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 shown in table 3A. In some embodiments, the one or more modifications are selected from those exemplary mutational strings shown 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.
In some embodiments, the engineered protease comprises one or more modifications relative to a chymotrypsin 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 shown in table 7A. In some embodiments, the one or more modifications are selected from those exemplary mutational strings shown 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.
In some embodiments, the engineered protease is based on the KLK5 protease domain, which optionally comprises one or more amino acid modifications of SEQ ID NO. 23. In some embodiments, the engineered protease comprises a modified kallikrein related peptidase 5 (KLK 5) 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.
In some embodiments, one or more functionally inactive fragments are generated by engineering protease cleavage factor B. 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 production of a reduced function factor B fragment.
In some embodiments, factor B is 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 human factor B. In some embodiments, factor B comprises the amino acid sequence shown as SEQ ID NO. 1.
In some embodiments, cleavage of factor B occurs at a site not targeted by factor D. In some embodiments, cleavage at this site results in at least two fragments that are not Ba and Bb. In some embodiments, cleavage at this site results in reduced production of factor B cleavage products Ba and Bb as compared to cleavage by factor D.
In some embodiments, cleavage of factor B occurs at the site targeted by factor D. In some embodiments, the factor D-targeted site comprises QQKR/KIV (SEQ ID NO: 9). In some embodiments, the factor B cleavage site 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) and DHKL/KSG (SEQ ID NO: 21).
In some embodiments, the engineered protease is based on MTSP-1 or uPA (including MTSP-1 or uPA protease domains), and the cleavage site in factor B comprises a sequence selected from the group consisting of WEHR/KGT (SEQ ID NO: 10) and KNQKR/QKQ (SEQ ID NO: 11).
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 the group consisting of 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).
In some embodiments, cleavage of factor B results in the production of a reduced function factor B fragment or results in reduced function factor B. In some embodiments, the function of factor B or a fragment of factor B is to interact with at least one complement component. In some embodiments, the function of factor B or a factor B fragment is to interact with hydrolyzed soluble C3. In some embodiments, the function of factor B or a fragment of factor B is to interact with C3B. In some embodiments, the function of factor B or a factor B fragment is to interact with membrane-bound C3B.
In some embodiments, cleavage occurs when factor B does not bind to C3B.
In some embodiments, the cleavage activity for a non-factor B peptide substrate is about equal to or less than the cleavage activity for a factor B site.
In some embodiments, the engineered protease has about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, 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 1,900M for factor B cleavage -1 s -1 K of (2) cat /K m . In some embodiments, the engineered protease has about 10 for factor B cleavage 3 To about 10 9 M -1 s -1 K of (2) cat /K m . In some embodiments, the engineered protease has an EC of less than about 20nM for factor B 50 . In some embodiments, the engineered protease has an EC of less than about 1nM for factor B 50 . In some embodiments, the engineered protease has an EC of about 20nM, about 25nM, or about 60nM for factor B 50 . In some embodiments, the engineered protease has an EC of about 1,000nM to about 4,500nM for cleavage factor B 50 。
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 from about 10% to about 50%, or from about 90% to about 100%.
In some embodiments, the engineered protease has an increased half-life as compared to the unmodified MTSP-1 protease domain. In some embodiments, the engineered protease has increased bioavailability compared to an 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 increased bioavailability compared to the unmodified uPA protease domain. In some embodiments, the engineered protease has an increased half-life as compared to the unmodified chymase. In some embodiments, the engineered protease has increased bioavailability compared to the unmodified chymotrypsin protease domain. In some embodiments, the engineered protease has increased bioavailability compared to the unmodified KLK5 protease domain.
In some embodiments, the engineered protease is non-immunogenic.
In some embodiments, the engineered protease is in the zymogen form. In some embodiments, the engineered protease is in an active form.
In some embodiments, the engineered protease further comprises a half-life extending factor. Exemplary half-life extending factors include Human Serum Albumin (HSA) and Fc (e.g., igG 1) fused to an engineered protease.
In another aspect, a method of inactivating factor B is provided, the method 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.
In some embodiments, the method is in vitro. In some embodiments, the method is in vivo.
In another aspect, there is provided a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject any of the engineered proteases of the present disclosure. In some embodiments, the disease or disorder is associated with dysregulated complement. In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the treatment is an alternative therapy. In some embodiments, the treatment blocks complement activation. In some embodiments, the treatment modulates autoimmunity. In some embodiments, the disease or disorder is congenital complement deficiency. In some embodiments, the treatment is for endothelial cell or renal cell injury.
In some embodiments, the disease or disorder is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplantation ischemia reperfusion (I/R) injury, anti-neutrophil cytoplasmic antibody (ANCA) -associated vasculitis (AAV), atypical hemolytic uremic syndrome (aHUS), membranous Nephropathy (MN), and paroxysmal sleep hemoglobinuria (PNH). In some embodiments, the disease or disorder is a control protein deficiency. In some embodiments, the disease or disorder is a secondary complement disorder. In some embodiments, the disease or disorder is an immune-related disease or disorder.
In some embodiments, the engineered protease is administered to the subject subcutaneously.
In some embodiments, the engineered protease is activated in situ at a site that modulates an aberrant complement component.
In some embodiments, the engineered protease is provided in a liquid stable formulation.
In another aspect, a pharmaceutical composition is provided comprising any of the engineered proteases of the present disclosure and optionally a pharmaceutically acceptable carrier.
In some embodiments, the engineered protease is provided in a liquid stable formulation.
In some embodiments, the composition is formulated for subcutaneous administration.
Drawings
FIG. 1A depicts a schematic representation of naturally occurring factor B showing the site of factor B cleavage by factor D (FD cleavage site; residues 1-6 of SEQ ID NO:13, 9, 20, SEQ ID NO:12, residues 1-6 of SEQ ID NO:4, 5, 3, and SEQ ID NO: 11).
FIGS. 1B and 1C depict two views of the protein structure of factor B showing different cleavage sites (SEQ ID NOS: 13, 9, 11, 10, residues 1-6 of SEQ ID NO:12, and SEQ ID NO: 14).
FIG. 1D depicts a Coomassie gel showing an example of factor B cleavage by chymase-based engineered proteases.
FIG. 1E depicts a display made with low EC 50 Is a graph of an example of factor B cleavage by chymase-based engineered proteases.
FIGS. 2A and 2B depict schematic diagrams of chymase precursors (pro-chymase) and mature chymase, respectively. Mature chymase provides an exemplary scaffold of the present disclosure.
FIGS. 3A and 3B depict schematic diagrams of the extracellular portion of membrane serine protease 1 (MTSP-1) and the serine protease domain of MTSP-1, respectively.
FIGS. 3C and 3D depict schematic diagrams of urokinase-type plasminogen activator (uPA or u-PA).
Fig. 4A, 4B, 4C, 4D and 4E depict graphs showing the stability of engineered chymase-based engineered proteases tested using peptide substrates.
Figure 5 depicts a bar graph of a control for factor B reverse addition hemolysis assay.
FIG. 6 depicts a graph showing a standard curve for factor B reverse addition hemolysis assay results.
FIG. 7A depicts a graph showing inhibition of hemolysis with KLK5 and chymase based engineered protease C22S/F173Y/D175N/A226R.
FIG. 7B depicts a graph showing FB cleavage with two concentrations of KLK5 and chymotrypsin-based engineered protease C22S/F173Y/D175N/A226R.
FIGS. 8A and 8B depict Coomassie gels showing examples of factor B cleavage with KLK5 and chymase based engineered protease C22S/F173Y/D175N/A226R.
FIG. 9 depicts Mass Spectrometry (MS) data identifying KLK5 vs. QQKR/KIV of factor B (SEQ ID NO: 9) cleavage sites 234 Cleavage site at Arg and within the cleavage site of EGVDAE (SEQ ID NO: 13) identifying chymase-based engineered protease C22S/A226R versus factor B 221 Cleavage site at Asp.
Fig. 10 is a schematic drawing depicting a general method for measuring complement activation and cytokine release in tissues of Acute Respiratory Distress Syndrome (ARDS) mouse model treated with chymase-based engineered proteases.
Fig. 11A, 11B and 11C depict the pulmonary congestion index, weight loss and neutrophil to lymphocyte ratio in bronchoalveolar lavage fluid (BALF NLR), respectively, measured in an Acute Respiratory Distress Syndrome (ARDS) mouse model.
Fig. 12A, 12B, 12C and 12D depict BALF and lung cytokine measurements from mouse tissues after treatment with chymase-based engineered proteases.
Fig. 13 is a schematic drawing depicting 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.
Fig. 14 depicts the results of plethysmographic measurements and significant protection of pulmonary congestion following administration of one of the chymase-based engineered proteases of the present disclosure.
FIG. 15 is SDS-PAGE (simplified) depicting expression, purification and activation of chymase-based engineered proteases of the present disclosure.
FIG. 16 is SDS-PAGE depicting expression of chymase-based engineered proteases in HEK293 cells.
FIG. 17 is SDS-PAGE depicting expression of chymase-based engineered proteases fused to human HSA or Fc in HEK293 cells.
Detailed Description
The present disclosure provides compositions and methods useful for modulating signaling and regulation of the complement system. In particular, provided herein are engineered proteases comprising the protease domain of the chymotrypsin-like S1A serine protease family, such engineered proteases of the disclosure being specific for and capable of cleaving factor B. These engineered proteases comprise modified protease domains and are produced using the methods and sequences provided herein.
The engineered proteases of the present disclosure target factor B for cleavage and are interchangeably referred to as "complement factor B degrading agents" or "CFB degrading agents. The use of these engineered proteases may (1) result in factor B cleavage into fragments that are neither Ba nor Bb, or may (2) result in factor B cleavage into Ba and Bb but are functionally inactive. Without being bound by any theory or mechanism, fragments Ba and Bb produced by cleavage of factor B without complexing factor B with C3 or C3B are believed to be functionally inactive fragments and may further reduce or inhibit complement activation, for limiting the increase in complement activation, and/or limit/reduce the amplification of the complement pathway. In either of these cases, these cleavage products may have a function other than the natural 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, can limit an increase in complement activation, and/or can limit/reduce amplification of the complement pathway.
In some embodiments, the protease is engineered to target factor B to cleave at a cleavage site that is not targeted by factor D. In other embodiments, the engineered protease targets factor B at the site targeted by factor D before factor B associates with C3B and forms a complex, thereby preventing formation of a converting zymogen complex.
The disclosure also provides methods of making and using such engineered proteases, e.g., to treat diseases or disorders associated with abnormal complement regulation, e.g., to treat overactive complement response.
Chymotrypsin-like serine proteases useful for modulating the complement system by targeting factor B
Provided herein are engineered proteases comprising one or more modifications relative to a naturally occurring chymotrypsin-like serine protease. As used herein, an "engineered" protease of the present disclosure is a non-naturally occurring serine protease of the S1A family and comprises one or more modifications relative to a wild-type or naturally occurring serine protease of the S1A family. As used herein, a "modification" to a naturally occurring chymotrypsin-like serine protease of the S1A family includes one or more of the following: a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion of one or more domains, and a substitution of one or more domains.
In some embodiments, the engineered proteases of the disclosure comprise a non-naturally occurring serine protease domain of the S1A family comprising one or more modifications relative to a wild-type or naturally occurring serine protease domain of the S1A family. As used herein, a "modification" of a naturally occurring chymotrypsin-like serine protease domain of the S1A family includes one or more of 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.
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 further 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 the non-naturally occurring serine protease domain of the S1A family, and may comprise additional sequences for expression, stability, improved pharmacokinetics, subcutaneous delivery, tissue targeting, and the like.
It is noted that as used herein, the "naturally occurring chymotrypsin-like serine protease" of the S1A family refers to such a protease that occurs in nature, even if it is not a wild-type sequence. In other words, naturally occurring serine proteases are not engineered. Naturally occurring serine proteases may or may not have a signal sequence, with or without an activating peptide, and may be of any species.
The engineered proteases provided herein are designed for cleavage of 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 the factor D cleavage site. In some embodiments, the engineered proteases provided herein can target factor B for cleavage, while factor B forms a complex with C3. In some embodiments, the engineered proteases provided herein can target factor B for cleavage, while factor B is complexed with C3B. In some embodiments, the engineered proteases provided herein can target factor B for cleavage, while factor B alone is present in the circulation.
In some embodiments, the cleavage products produced by the engineered protease cleavage factor B provided herein can be functionally inactive fragments, as discussed above. In some embodiments, the functionally inactive fragments do not have a naturally occurring physiological function. In some embodiments, the functionally inactive fragments may perform a function, but not necessarily the same function as Ba and/or Bb.
In some embodiments, the engineered proteases provided herein are capable of modulating the activity of the complement system by reducing the amount of factor B fragments Ba and Bb produced, thereby suppressing/inhibiting complement activation, limiting the increase in complement activation, and/or limiting/reducing the amplification of the complement pathway.
In some embodiments, the engineered proteases provided herein are useful for administration to a subject in need thereof. As used herein, the term "patient" or "subject" is used interchangeably to refer to mammals and includes, but is not limited to, humans and other primates (e.g., chimpanzees, cynomolgus monkeys and other apes and monkey species), farm animals (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.
Table 1 provides the amino acid sequences of human factor B targeted by the engineered proteases of the present disclosure.
Table 1 also provides amino acid sequences of exemplary chymotrypsin-like serine proteases and protease domains of the S1A family useful as scaffolds upon which to generate the engineered proteases of the present disclosure, including: MTSP-1, uPA, chymase and kallikrein related peptidase 5 (KLK 5). Thus, in some embodiments, the engineered proteases provided herein are based on MTSP-1 or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are based on uPA or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are based on KLK5 or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are not based on chymase or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are not based on MTSP-1 or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are not based on uPA or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are not based on KLK5 or serine protease domains thereof. In some embodiments, the engineered proteases provided herein are not based on chymase or serine protease domains thereof.
Table 1 also includes the amino acid sequence of the mature chymotrypsin polypeptide of SEQ ID NO. 19. It is noted that the protease domain of a 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 chymotrypsin-numbered amino acids 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 skilled in the art. Standard nomenclature for chymotrypsin numbering may also be determined by one skilled in the art, such as the notation for adding or deleting residues. Residues that are present in the aligned proteases (e.g., MTSP-1, uPA, KLK5, or chymase) but are not present in chymotrypsin are indicated in lowercase. For example, the uPA protease domain (table 2) is represented using chymotrypsin numbering keys, modifying S37dP (chymotrypsin numbering) to S184P in the conventional amino acid sequence notation. In table 1, the signal or leader sequence is underlined and the cleavage site sequence is in bold. In some cases, the disclosure and claims include references to conventional amino acid numbering and/or chymotrypsin-based numbering, and accordingly so identified.
The chymase of Table 1 is a large cell chymase, the sequence of which can be found in https:// www.uniprot.org/uniprot/P23946.
Table 1: human factor B and wild-type serine protease sequences
Fig. 1A depicts a schematic of naturally occurring factor B, showing various cleavage site sequences, including the site at which factor B is cleaved into Ba and Bb by factor D (FD cleavage site). Ba consists of three Complement Control Protein (CCP) domains and a linker. Bb consists of von Willebrand factor type A (VWA) domain and Serine Protease (SP) domain. FIGS. 1B-1C depict two factor B protein structural views showing various cleavage sites.
In some embodiments, the present disclosure provides engineered proteases that cleave factor B, which can be located at cleavage sites that are not targeted by factor D, i.e., not at FD cleavage sites. In other embodiments, the present disclosure provides engineered proteases that cleave factor B at a cleavage site targeted by factor D. As contemplated herein, the FD cleavage site is the site of 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 a cleavage site targeted by factor D (SEQ ID NO: 9) prior to formation of a complex of factor B with C3B, without being bound by any theory or mechanism, and such cleavage is expected to result in a fragment that does not increase complement activity.
Various exemplary cleavage sites for factor B that can be targeted by the engineered proteases of the present disclosure are shown on the schematic of fig. 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, diagonal lines are used to indicate cleavage sites. However, cleavage at these sites is not limited thereto.
FIG. 1D depicts a Coomassie gel showing an example of factor B cleavage by chymase-based engineered proteases. Two batches were used for each engineered protease group, and each batch showed the ability of the engineered protease to cleave factor B. FIG. 1E depicts a display made with low EC 50 Is a graph of an example of factor B cleavage by chymase-based engineered proteases. These results are discussed in further detail in example 1 below.
The amino acid sequence of wild-type human factor B is shown in Table 1 below 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, the engineered protease that can target the sequence of SEQ ID NO. 2 for cleavage is chymase-based. 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 disclosure. In some embodiments, the engineered protease that can target the sequence of SEQ ID NO. 3 for cleavage is based on MTSP-1 or uPA. In some embodiments, the site comprising the amino acid sequences IRPSKG (SEQ ID NO: 4) and/or GGEKRD (SEQ ID NO: 5) may be targeted as a cleavage site by the engineered proteases of the present disclosure, in some embodiments such engineered proteases are constructed on a MTSP-1 based scaffold.
FIGS. 2A-2B depict schematic diagrams of chymase precursors and mature chymase, respectively. Chymase precursors comprise a chymase domain and an activation peptide, and chymase is produced when the signal peptide and activation peptide are cleaved at the cleavage site shown in FIG. 2A. Mature chymase as shown in fig. 2B may be used as a scaffold to produce the engineered proteases of the present disclosure. The amino acid sequence of the wild-type mature chymase protease domain is shown in Table 1 by SEQ ID NO. 6.
FIGS. 3A-3B depict schematic diagrams of the extracellular portion of membrane serine protease 1 (MTSP-1) and the serine protease domain of MTSP-1, respectively. The MTSP-1 serine protease domain as shown in FIG. 3B can be used as a scaffold for the production of the engineered proteases of the present disclosure. The amino acid sequence of the protease domain of naturally occurring MTSP-1 is shown in SEQ ID NO. 7 of Table 1.
Fig. 3C to 3D depict schematic diagrams of urokinase-type plasminogen activator (uPA or u-PA). Fig. 3C depicts a schematic of a pro-uPA and fig. 3D depicts a schematic of a mature double-stranded uPA. Mature uPA polypeptides are produced by proteolytic cleavage. The uPA serine protease domain as shown in fig. 3D can be used as a scaffold to produce the engineered proteases of the present disclosure. In wild-type uPA, the protease domain is linked to the "a" chain by disulfide bridges (as shown in fig. 3D). In the uPA based engineered proteases provided herein that include a uPA serine protease domain, a C-to-S substitution (C122S as shown in chymotrypsin numbering of SEQ ID NO: 22) may be present to reduce aggregation.
In some embodiments, provided herein are engineered proteases, wherein serine proteases are specific for factor B at sites not targeted by factor D. In other embodiments, provided herein are engineered proteases, wherein serine proteases are specific for factor B at a site targeted by factor D. In some embodiments, cleavage of factor B by the engineered proteases provided herein at a site targeted by factor D or at a site not targeted by factor D results in a reduction in complement activation. In some embodiments, cleavage at the site results in at least two functionally inactive fragments. In some embodiments, cleavage of factor B by an engineered protease provided herein results in one or more functionally inactive fragments at a site targeted by factor D or at a site not targeted by factor D. In some embodiments, cleavage of factor B by the engineered proteases provided herein at a site targeted by factor D or at a site not targeted by factor D results in a decrease in function of factor B. In some embodiments, cleavage of factor B by the engineered proteases provided herein at a site targeted by factor D or at a site not targeted by factor D results in a reduction in factor B cleavage products Ba and Bb.
In some embodiments, the factor B targeted by the engineered proteases of the present disclosure may be 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 shown in SEQ ID NO. 1. In some embodiments, primate 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 rodent factor B, e.g., factor B of a rat or mouse.
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).
In other embodiments, the engineered proteases provided herein are specific for factor B at a site 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 engineered proteases provided herein are based on chymotrypsin-like serine proteases of the S1A family, including but not limited to membrane 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 scaffolds of serine proteases such as MTSP-1, uPA, KLK5 or chymase.
Engineered protease based on uPA
In some embodiments, the engineered protease is uPA-based, e.g., based on the uPA serine protease domain. In some embodiments, such engineered proteases are specific for factor B at a site not targeted by factor D, e.g., wherein the cleavage site comprises a sequence selected from WEHR/KGT (SEQ ID NO: 10) and KNQKR/QKQ (SEQ ID NO: 11).
In some embodiments, the uPA-based engineered protease comprises one or more modifications relative to a uPA comprising the amino acid sequence set forth in SEQ ID NO. 8.
In some embodiments, the uPA based engineered protease comprises one or more modifications relative to a uPA protease domain comprising the amino acid sequence set forth in SEQ ID NO. 22.
Modification of the uPA or uPA protease domain can be represented by numbering residues of the uPA protease domain according to chymotrypsin numbering. The corresponding chymotrypsin numbers for the uPA protease domain of SEQ ID NO. 22 (corresponding to amino acid positions 159-411 of uPA as shown in SEQ ID NO. 8) are shown in Table 2.
Table 2 provides four rows for each amino acid. The first line lists the conventional amino acid sequence numbers of SEQ ID NO. 22 (uPA protease domain). The second row lists the conventional amino acid sequence numbers for residues 159-411 of SEQ ID NO. 8 (uPA protease domain). The third row provides amino acid single letter abbreviations. The fourth line provides the corresponding chymotrypsin number under each amino acid single letter abbreviation. Residues present in the protease domain but not in chymotrypsin are indicated by letters at the end of the symbol. For example, the residues of chymotrypsin that are part of the loop with amino acid 60 based on chymotrypsin numbering are referred to as D60a, Y60b, P60c, which are inserted into engineered uPA.
Table 2 provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the uPA protease domain. In the tables and throughout the disclosure that follow, modifications to the uPA protease domain are indicated by chymotrypsin numbering or conventional amino acid numbering. If a particular modification is provided with only chymotrypsin numbering symbols, the skilled artisan will understand how to refer to table 2 and make the necessary conversions to understand the modifications in conventional amino acid terminology and vice versa.
Table 2: chymotrypsin numbering of uPA protease domains
The engineered uPA-based proteases of the present disclosure comprise at least one modification of the serine protease domain of uPA. As described above, the modification may be any one or more of the following: a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion of one or more domains, and a substitution of one or more domains. Table 3A provides exemplary modifications to the serine protease domain of uPA. For example, table 3A provides three columns, the first column providing modifications numbered using chymotrypsin; the second column provides the conventional amino acid sequence numbering relative to SEQ ID NO. 8; and the third column provides the amino acid sequence numbering relative to SEQ ID NO. 22.
The engineered proteases may be produced by using any one or more of the exemplary modifications provided in table 3A. Thus, the uPA-based engineered proteases of the present disclosure may comprise any one or more of the modifications provided in table 3A.
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. For example, the modification to G18E is to replace E at a position corresponding to position 18 of the uPA serine protease domain using chymotrypsin numbering. For example, modification D97delinsEG means using chymotrypsin numbering, deleting D at position 97, and inserting EG at its position. For example, modification l97b_h99del represents the deletion of residues from L97b to H99 using chymotrypsin numbering.
Table 3A: exemplary modifications to serine protease domain of uPA
Exemplary modifications of the present disclosure (referred to herein as mutation strings) are provided in table 3B. Accordingly, provided herein are uPA-based engineered proteases comprising one or more of the modifications provided in table 3A. Such exemplary engineered proteases are capable of cleaving factor B, or exhibit other cleavage activities. The second column provides exemplary combinations of modifications using conventional numbering relative to SEQ ID NO. 22.
Table 3B: exemplary uPA-based engineered proteases
In some embodiments, the uPA-based engineered proteases of the present disclosure bind to SEQ ID NO:8 has 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.
In some embodiments, the uPA-based engineered proteases of the present disclosure have 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.
In some embodiments, the uPA-based engineered proteases of the present disclosure comprise a 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.
Engineered MTSP-1-based proteases
In some embodiments, the engineered protease is based on MTSP-1, e.g., based on a modified MTSP-1 serine protease domain. In some embodiments, such engineered proteases are specific for factor B at a site not targeted by factor D, wherein the cleavage site comprises a sequence selected from WEHR/KGT (SEQ ID NO: 10) and KNQKR/QKQ (SEQ ID NO: 11).
In some embodiments, the engineered MTSP-1 protease comprises one or more modifications relative to MTSP-1 comprising the amino acid sequence set forth in SEQ ID NO. 18.
In some embodiments, an engineered MTSP-1-based protease comprises one or more modifications relative to an MTSP-1 protease domain comprising the amino acid sequence set forth in SEQ ID NO. 7.
Modifications to the MTSP-1 or MTSP-1 protease domains can be represented by numbering residues of the MTSP-1 protease domains according to chymotrypsin numbering. The corresponding chymotrypsin numbers for the MTSP-1 protease domain of SEQ ID NO. 7 (corresponding to amino acid positions 615-855 of MTSP-1 as shown in SEQ ID NO. 18) are shown in Table 4.
Table 4 provides four rows for each amino acid. The first line lists the conventional amino acid sequence numbers of SEQ ID NO. 7 (MTSP-1 protease domain). The second line sets forth the conventional amino acid sequence numbering of residues 615-855 of SEQ ID NO. 18 (MTSP-1 protease domain). The third row provides amino acid single letter abbreviations. The fourth line provides the corresponding chymotrypsin number under each amino acid single letter abbreviation. Residues present in the protease domain but not in chymotrypsin are indicated by letters at the end of the symbol. For example, residues of chymotrypsin that are part of a loop with amino acid 60 based on chymotrypsin numbering are referred to as D60a and R60c, which are inserted into engineered MTSP-1.
Table 4 provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the MTSP-1 protease domain. In the tables and throughout the disclosure that follow, modifications to the MTSP-1 protease domain are indicated by chymotrypsin numbering or using conventional amino acid numbering. If a particular modification is provided with chymotrypsin numbering symbols only, the skilled artisan will understand how to refer to table 4 and make the necessary conversions to understand the modifications in conventional amino acid terminology and vice versa.
Table 4: chymotrypsin numbering of MTSP-1 protease domains
The engineered MTSP-1-based proteases of the present disclosure comprise at least one modification of the serine protease domain of MTSP-1. As described above, the modification may be any one or more of the following: a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion of one or more domains, and a substitution of one or more domains. Table 5A provides exemplary modifications to the serine protease domain of MTSP-1. For example, table 5A provides three columns, the first column providing modifications numbered using chymotrypsin; the second column provides the conventional amino acid sequence numbering relative to SEQ ID NO. 18; and the third column provides the amino acid sequence numbering relative to SEQ ID NO. 7.
Modifications to the MTSP-1 or MTSP-1 protease domains can be represented by numbering residues of the MTSP-1 protease domains according to chymotrypsin numbering. The corresponding chymotrypsin numbers for the MTSP-1 protease domain of SEQ ID NO. 7 (corresponding to amino acid positions 615-855 of MTSP-1 as shown in SEQ ID NO. 18) are shown in Table 4.
The engineered proteases may be produced by using any one or more of the exemplary modifications provided in table 5A. Thus, the MTSP-1 based engineered proteases of the present disclosure may comprise any one or more of the modifications provided in table 5A.
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. For example, the modification of F99S of MTSP-1 is a substitution modification at a position corresponding to position 99 of the MTSP-1 serine protease domain using chymotrypsin numbering.
Table 5A: exemplary modifications to the serine protease domain of MTSP-1
Exemplary modifications (mutational strings) of the present disclosure are provided in table 5B. Accordingly, provided herein are MTSP-1 based engineered proteases comprising one or more of the mutant strings provided in table 5B. Such exemplary engineered proteases are capable of cleaving factor B, or exhibit other cleavage activities. Residues noted in brackets such as C17 and C19 refer to residues that are part of the chain of the protease in the zymogen form, which are subsequently cleaved and are not retained in the mature protease.
Table 5B: exemplary MTSP-1-based engineered proteases
In some embodiments, the engineered MTSP-1-based proteases of the present disclosure have 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.
In some embodiments, the engineered MTSP-1-based proteases of the present disclosure have 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.
In some embodiments, an engineered MTSP-1-based protease of the present disclosure comprises a 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.
Engineered proteases based on chymase
In some embodiments, the engineered protease is based on a chymase, e.g., based on a modified chymase serine protease domain. In some embodiments, such engineered proteases are specific for factor B at a site not targeted by factor D, wherein the cleavage site comprises a sequence selected from the group consisting of: 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).
In some embodiments, the chymase-based engineered protease comprises one or more modifications relative to a chymase protease domain comprising the amino acid sequence set forth in SEQ ID No. 6.
Modification of the chymase or chymase protease domain may be represented by numbering residues of the chymase protease domain according to chymotrypsin numbering. The corresponding chymotrypsin numbering for amino acid positions 1-226 of the chymotrypsin protease domain of SEQ ID NO. 6 is given in Table 6.
Table 6 provides three rows for each amino acid. The first line lists the conventional amino acid sequence numbers of SEQ ID NO. 6 (chymase protease domain). The second row provides amino acid single letter abbreviations. The third line provides the corresponding chymotrypsin numbering of the chymotrypsin protease domains under each amino acid single letter abbreviation. Residues present in the protease domain but not in chymotrypsin are indicated by letters at the end of the symbol. For example, the residues at amino acid 36 in chymotrypsin, based on chymotrypsin numbering, are referred to as V36a, S36b and N36c, which are inserted into the engineered chymase.
Table 6 provides the chymotrypsin numbering scheme and the conventional numbering scheme for its corresponding chymotrypsin protease domains. In the tables and throughout the disclosure that follow, modifications to the chymotrypsin protease domains are indicated by chymotrypsin numbering or using conventional amino acid numbering. If a particular modification is provided with only chymotrypsin numbering symbols, the skilled artisan will understand how to refer to table 6 and make the necessary conversions to understand the modifications in conventional amino acid terminology and vice versa.
Table 6: chymotrypsin numbering of chymotrypsin protease domains
The chymase-based engineered proteases of the present disclosure comprise at least one modification of the serine protease domain of the chymase. As described above, the modification may be any one or more of the following: a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion of one or more domains, and a substitution of one or more domains. Table 7A provides exemplary modifications to the serine protease domain of chymase. For example, table 7A provides two columns, the first column providing modifications numbered using chymotrypsin; the second column provides the conventional amino acid sequence numbering relative to SEQ ID NO. 6.
The engineered proteases may be produced 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 comprise any one or more of the modifications provided in table 7A.
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. For example, the 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.
Table 7A: exemplary modifications to serine protease domains of chymase
Exemplary modifications (mutational strings) of the present disclosure are provided in table 7B. Accordingly, provided herein are chymase-based engineered proteases comprising one or more of the modifications (mutation strings) provided in table 7A. Such exemplary engineered proteases are capable of cleaving factor B, or exhibit other cleavage activities. As an example, chymotrypsin-based numbering of engineered proteases based on engineered chymase includes proteases with the following exemplary modification combinations: C22S/P38Q/K40M/F41R/V138I/F173Y/D175R/A190S/V213A/S218V/A226R and
C22S/P38Q/K40M/F41H/V138I/F173Y/D175R/A190S/V213A/S218V/A226R。
Table 7B: exemplary chymase-based engineered proteases
In some embodiments, the chymase-based engineered proteases of the present disclosure have 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.
In some embodiments, the chymase-based engineered proteases of the present disclosure have 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.
In some embodiments, the chymase-based engineered proteases of the present disclosure comprise a 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.
Engineered protease based on KLK5
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 a site targeted by factor D, wherein the cleavage site comprises the amino acid sequence QQKR/KIV (SEQ ID NO: 9).
In some embodiments, the KLK 5-based engineered protease is based on KLK5 comprising the amino acid sequence set forth in SEQ ID NO. 23. Residues within KLK5 or modifications to KLK5 may be represented by numbering residues of KLK5 according to chymotrypsin numbering. Table 7C shows chymotrypsin numbering schemes for amino acid positions 45-271 of KLK5 SEQ ID NO:28 as shown in SEQ ID NO: 23. Table 7C lists amino acid residues 45-271 of SEQ ID NO. 23 above the single letter abbreviations for amino acids, and the corresponding chymotrypsin numbers below each single letter abbreviation for amino acids. Residues present in the protease but not in chymotrypsin are indicated by the letter at the end of the symbol. For example, the residue at amino acid 36 in chymotrypsin, based on chymotrypsin numbering, is referred to as 36a, 36b, 36c, which is inserted into engineered KLK5.
Table 7C provides the chymotrypsin numbering scheme and its corresponding conventional numbering scheme for the KLK5 protease domain. In the tables and throughout the disclosure that follow, modifications to the KLK5 protease domain are indicated by chymotrypsin numbering or using conventional amino acid numbering. If a particular modification is provided with only chymotrypsin numbering symbols, the skilled artisan will understand how to refer to table 7C and make the necessary conversions to understand the modifications in conventional amino acid terminology and vice versa.
Table 7C: chymotrypsin numbering of KLK5
It should be understood that the engineered proteases of the present disclosure are not limited to those shown in the tables above. Such modifications may increase the half-life, bioavailability, or other characteristics of the serine protease.
Fusion proteins
The engineered proteases may be further modified, e.g. they may comprise a fusion (addition) of another component or domain. Examples of such components or domains include, but are not limited to, half-life extending factors and activation peptides or activation signals. For example, components capable of increasing the half-life or bioavailability of the engineered proteases of the present disclosure may be added. Half-life extending factors may include, but are not limited to, fc (e.g., igG1, igG2, igG3, or IgG 4), affibodies, PEG, and albumin such as Human Serum Albumin (HSA).
Such addition of additional components can be accomplished, for example, byTo be added. Such additions may increase the half-life or bioavailability of the engineered protease compared to serine proteases that do not include half-life extending factors. In some embodiments, the addition of half-life extending factors or other similar components may also improve or alter any one or more characteristics of the engineered protease, including, but not limited to, stability, bioavailability, serum half-life, shelf life, transport capacity, and immunogenicity.
Thus, in some embodiments, the engineered proteases provided herein can further comprise a half-life extending factor. In some embodiments, the half-life extending factor is an addition at the N-terminus of the engineered protease. In some embodiments, the half-life extending factor is an addition at the C-terminus of the engineered protease. In some embodiments, the half-life extending factor is added directly to the serine protease. In some embodiments, the half-life extending factor is added to the serine protease via one or more linkers. In some embodiments, the half-life extending factor is Fc and is a human wild-type Fc domain or variant thereof. In some embodiments, the half-life extending factor is an albumin such as human serum albumin or a variant thereof.
In some embodiments, the engineered proteases provided herein can comprise more than one half-life extending factor. In some embodiments, each half-life extending factor is added at the N-terminus of the serine protease. In some embodiments, each half-life extending factor is added at the C-terminus of the serine protease. In some embodiments, one half-life extending factor is added at the N-terminus of the engineered protease and the other half-life extending factor is added at the C-terminus of the engineered protease. In some embodiments, the half-life extending factor is Fc and is a human wild-type Fc domain or variant thereof. In some embodiments, the half-life extending factor is an albumin such as human serum albumin or a variant thereof.
In exemplary embodiments, the chymase-based engineered proteases of the present disclosure are fused to a wild-type Fc domain or variant thereof. In exemplary embodiments, the chymase-based engineered proteases of the present disclosure are fused to human serum albumin or variants thereof.
In exemplary embodiments, the uPA-based engineered proteases of the present disclosure are fused to a wild-type Fc domain or variant thereof. In exemplary embodiments, the uPA-based engineered proteases of the present disclosure are fused to human serum albumin or variants thereof.
In exemplary embodiments, the MTSP-1 based engineered proteases of the present disclosure are fused to a wild-type Fc domain or variant thereof. In exemplary embodiments, the MTSP-1 based engineered proteases of the present disclosure are fused to human serum albumin or variants thereof.
In exemplary embodiments, the KLK 5-based engineered proteases of the disclosure are fused to a wild-type Fc domain or variant thereof. In exemplary embodiments, the KLK 5-based engineered protease of the disclosure is fused to human serum albumin or variants thereof.
The fusion protein may also comprise an activating sequence such that the resulting fusion protein comprising the engineered protease of the disclosure is in an active form, such as a double-stranded form. The activation sequence may contain or be modified to contain a cysteine which may form a disulfide bond with a free cysteine, such as C122, in the modified u-PA polypeptide, for example, whereby upon activation, the resulting activation polypeptide contains two chains. Exemplary activating sequences include enterokinase activating sequences and furin activating sequences, and modified forms thereof.
Activity of engineered proteases
In some embodiments, the engineered proteases of the disclosure cleave factor B at sites not targeted by factor D or at sites targeted by factor D, and cleavage at such sites results in reduced function of factor B or a factor B fragment. In some embodiments, the function of factor B or a fragment of factor B is to interact with at least one complement component. In some embodiments, the function of factor B or a factor B fragment is to interact with hydrolyzed soluble C3. In some embodiments, the function of factor B or a fragment of factor B is to interact with C3B. In some embodiments, C3b is a membrane bound C3b. In some embodiments, when factor B does not bind to C3B, cleavage occurs at a non-factor D site. In some embodiments, when factor B does not bind to C3B, cleavage occurs at the factor D site. In some embodiments, cleavage occurs at a non-factor D site when factor B binds to C3B (i.e., complexes with C3B).
In some embodiments, the engineered proteases provided herein can cleave other peptide substrates than factor B, while also being able to cleave factor B. In some embodiments, the cleavage activity of the non-factor B peptide substrate is about equal to or less than the cleavage activity of the factor B site.
In some embodiments, the engineered proteases provided herein have about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, 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 1,900M for factor B cleavage -1 s -1 K of (2) cat /K m . In some embodiments, K cat /K m Up to or greater than about 10e 8 . In some embodiments, the engineered proteases provided herein have about 10 for factor B cleavage 3 To about 10 6 M -1 s -1 K of (2) cat /K m 。
In some embodiments, the engineered proteases provided herein have an EC of about 1nM to about 20nM for factor B cleavage 50 . In some embodiments, the engineered proteases provided herein have an EC of less than about 20nM for factor B 50 . In some embodiments, the engineered proteases provided herein have an EC of less than about 1nM for factor B cleavage 50 . In some embodiments, the present The engineered proteases provided herein have an EC of about 5nM to about 100nM for factor B cleavage 50 . In some embodiments, the engineered proteases provided herein have an EC of about 20nM, about 25nM, or about 60nM for factor B cleavage 50 . In some embodiments, the factor B cleaved EC 50 About 20nM. In some embodiments, the factor B cleaved EC 50 About 50nM. In some embodiments, the engineered proteases provided herein have an EC of about 1,000nm to about 4,500nm for factor B cleavage 50 . In some embodiments, the engineered proteases provided herein have an EC of about 1,000nm, or about 2,000nm, or about 3,000nm, or about 4,000nm, or about 5,000nm for factor B cleavage 50 。
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 or greater than about 120 hours. In some embodiments, the engineered proteases provided herein have a catalytic lifetime of about 120 hours or more in human plasma and are useful for chronic indications. In some embodiments, the engineered proteases provided herein have a catalytic lifetime of about 24 hours in human plasma. In some embodiments, the engineered proteases provided herein have a catalytic lifetime of about 24 hours or more in human plasma and are useful for acute indications.
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 between about 10% and about 85% of the activity initially measured after about 7 days.
In some embodiments, the engineered proteases provided herein have increased half-lives compared to unmodified MTSP-1 or MTSP-1 protease domains. In some embodiments, the engineered proteases provided herein have increased bioavailability compared to an unmodified MTSP-1 or MTSP-1 protease domain. In some embodiments, the engineered proteases provided herein have an increased half-life compared to an unmodified uPA or uPA protease domain. In some embodiments, the engineered proteases provided herein have increased bioavailability compared to an unmodified uPA or uPA protease domain. In some embodiments, the engineered proteases provided herein have an increased half-life as compared to an unmodified chymase or chymase protease domain. In some embodiments, the engineered proteases provided herein have increased bioavailability compared to an 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. In some embodiments, the engineered proteases provided herein have increased bioavailability compared to unmodified KLK5 or KLK5 protease domains.
In some embodiments, the engineered proteases provided herein are non-immunogenic.
In some embodiments, the engineered proteases provided herein are in the zymogen form. As used herein, the 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 a mature form. In some embodiments, the zymogen form may be activated to a mature form in vivo (in situ) following administration. In some embodiments, the zymogen form is activated ex vivo (in vitro) prior to, for example, administration of the engineered protease.
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 the host cell. In some embodiments, the activation by an enzyme during production in the host cell is over-expression by an enzyme, such as enterokinase. In some embodiments, the engineered protease is activated after production and secretion by the host cell, optionally in a culture medium.
Use of engineered proteases
The engineered proteases of the present disclosure are useful for modulating the complement system.
In some embodiments, the engineered proteases of the present disclosure are capable of modulating the classical complement pathway. In some embodiments, the engineered proteases of the present disclosure are capable of modulating the alternative complement pathway. In some embodiments, the engineered proteases of the present disclosure are capable of modulating the lectin complement pathway. In some embodiments, the engineered proteases of the present disclosure are capable of reducing amplification of the complement system.
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 functionally inactive factor B fragments Ba and/or Bb.
Provided herein are methods of inactivating factor B comprising contacting factor B with any of the engineered proteases disclosed herein. In some embodiments, complement activation is inhibited using such methods. 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.
In some embodiments, the method is in vitro. In some embodiments, the method is in vivo.
The engineered proteases of the present disclosure are useful in the treatment of a subject. Accordingly, provided herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject any of the engineered proteases of the present disclosure. In some embodiments, the disease or disorder is associated with dysregulated complement, and thus, in some embodiments, the disease or disorder involves dysregulated complement. In some embodiments, the treatment is an alternative 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 or renal cell injury.
In some exemplary embodiments, the disease or disorder is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplantation ischemia reperfusion (I/R) injury, anti-neutrophil 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 sleep hemoglobinuria (PNH).
In some embodiments, the engineered proteases provided herein are useful for treating inflammatory diseases or disorders. In some embodiments, the engineered proteases provided herein are capable of reducing inflammatory cytokines. In some exemplary embodiments, the engineered proteases provided herein are capable of effectively reducing the inflammatory cytokines IL-2 and IL-6 and the chemokine CXCL9, and are useful for treating diseases such as ARDS.
In some embodiments, the engineered protease is administered to the subject subcutaneously. In some embodiments, the engineered protease is activated in situ at a site of a complement component that is dysregulated or at a pathophysiological site of dysregulation. In some embodiments, the engineered protease is provided in a liquid stable formulation. In vivo administration of the engineered protease may be by intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, intrathecal, intraarterial, intraventricular, intranasal, transmucosal, by implantation or by inhalation. In some embodiments, the engineered proteases provided herein are administered by a mechanical device.
Pharmaceutical composition
The present disclosure also provides pharmaceutical compositions comprising any of the engineered proteases disclosed herein, and optionally a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical composition may be formulated to be compatible with its intended route of administration. In some embodiments, the pharmaceutical compositions of the present disclosure are suitable for administration to a human subject or other non-human primate.
Kit and article of manufacture
The present disclosure also provides kits or articles of manufacture comprising any of the engineered proteases disclosed herein or any of the pharmaceutical compositions disclosed herein. In some embodiments, the kit may further comprise instructional materials for performing any of the methods disclosed herein. In some embodiments, the kit may further comprise a sterile container or vial for containing the fusion construct and/or pharmaceutical composition disclosed herein. In some embodiments, the kit may further comprise a sterile delivery device for administering the fusion constructs and/or pharmaceutical compositions disclosed herein. In some embodiments, the article of manufacture comprises any of the pharmaceutical compositions of the present disclosure.
Production of engineered proteases
Provided herein are methods and compositions for producing engineered proteases. Thus, provided herein are nucleic acids and vectors encoding any of the engineered proteases of the disclosure. Also provided are cells comprising one or more nucleic acids encoding the engineered proteases of the present disclosure.
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. Such methods include PCR amplification of nucleic acids and library screening, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening.
Provided herein are methods of using the expression systems to generate the engineered proteases of the present disclosure, e.g., the engineered proteases of the present disclosure can be expressed in bacteria (e.g., escherichia coli), yeast, insect, or mammalian cells (e.g., CHO cells, HEK cells). In particular embodiments, transformation of a host cell with a recombinant DNA molecule that integrates an isolated engineered protease protein gene, cDNA, or synthetic DNA sequence enables the production of multiple copies of the gene. Thus, by culturing the transformant, isolating the recombinant DNA molecule from the transformant and recovering the inserted gene from the isolated recombinant DNA, if necessary, a large amount of the gene can be obtained. In some embodiments, the engineered protease is active in production. In some embodiments, the engineered protease is required to be activated at the time of production. In such embodiments, the engineered protease is engineered in the activated state; the method comprises producing an engineered protease in the form of a zymogen in a bacterial, yeast or mammalian host system, followed by activation.
Administration of engineered proteases
In some embodiments, provided herein are methods of administering an engineered protease of the present disclosure by delivering a vector/nucleic acid encoding the engineered protease. In some embodiments, the method comprises administering a recombinant vector. In some embodiments, provided herein are engineered proteases for use in gene expression therapies using non-viral vectors. In other embodiments, provided herein are engineered proteases for use in gene expression therapies using viral vectors. In some embodiments, the cell is engineered to express the engineered protease, such as by integrating a nucleic acid encoding the engineered protease into a genomic location, i.e., operably linked to a regulatory sequence or such that it is placed operably linked to a regulatory sequence in 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.
Methods of amplifying nucleic acids may be used to isolate nucleic acid molecules encoding engineered proteases, including, for example, polymerase Chain Reaction (PCR) methods. Nucleic acid-containing materials can be used as starting materials from which nucleic acid molecules encoding the engineered proteases can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts (e.g., from the liver), fluid samples (e.g., blood, serum, saliva), samples from healthy and/or diseased subjects may be used in the amplification method. Nucleic acid libraries can also be used as a source of starting materials. Primers can be designed to amplify and modify molecules encoding the engineered proteases. For example, primers may be designed based on the expressed sequence that generates the engineered protease.
In order to clone the synthetic gene into a vector, such as a protein expression vector or a vector designed to amplify a DNA sequence encoding a core protein, additional nucleotide sequences may be ligated to the nucleic acid molecules encoding the engineered proteases, including linker sequences containing restriction endonuclease sites. Furthermore, additional nucleotide sequences specifying functional DNA elements are operably linked to the nucleic acid molecule encoding the engineered protease. Examples of such sequences include, but are not limited to, promoter sequences designed to promote expression of a protein in a cell and secretion sequences designed to promote secretion of a protein. Additional nucleotide sequences, such as sequences specifying protein binding regions, may also be linked to the nucleic acid molecule encoding the engineered protease. Such regions include, but are not limited to, sequences that facilitate uptake of the engineered protease into a particular target cell or otherwise enhance the pharmacokinetics of the synthetic gene.
Detailed description of the illustrated embodiments
The present disclosure provides the following non-limiting enumerated set of embodiments.
Group I
Embodiment I-1. An engineered protease of the S1A serine protease family, wherein the engineered protease is specific for and capable of cleaving factor B.
Embodiment I-2 the engineered protease according to embodiment I-1, wherein cleavage of factor B by the engineered protease results in one or more functionally inactive fragments.
Embodiment I-3 the engineered protease according to any one of embodiments I-2, wherein said one or more functionally inactive fragments are capable of reducing complement activation.
Embodiment I-4 the engineered protease according to any of embodiments 1-3, wherein cleavage of factor B results in the production of a reduced function factor B fragment or results in reduced function factor B.
Embodiment I-5 the engineered protease according to any one of embodiments 1-4, wherein said factor B is rodent factor B.
Embodiment I-6 the engineered protease of any one of embodiments 1-4, wherein the factor B is a non-human primate factor B.
Embodiment I-7. The engineered protease according to embodiment I-6, wherein the non-human primate is a cynomolgus monkey.
Embodiment I-8 the engineered protease according to any one of embodiments 1-4, wherein said factor B is human factor B.
Embodiment I-9. The engineered protease according to embodiment I-8, wherein said factor B comprises the amino acid sequence as shown in SEQ ID NO. 1.
Embodiment I-10 the engineered protease according to any of embodiments 1-9, wherein cleavage of factor B occurs at a site not targeted by factor D.
Embodiment I-11. The engineered protease according to embodiment I-10, wherein cleavage at said site results in at least two fragments that are not Ba and Bb.
Embodiment I-12. The engineered protease according to any of embodiments 1-11, wherein cleavage at said site results in reduced production of factor B cleavage products Ba and Bb compared to cleavage by factor D.
Embodiment I-13 the engineered protease according to any of embodiments 1-9, wherein cleavage of factor B occurs at the site targeted by factor D.
Embodiments I-14. The engineered protease according to any one of embodiments 1-13, wherein the site targeted by factor D comprises QQKR/KIV (SEQ ID NO: 9).
Embodiment I-15 the engineered protease according to embodiment I-10, wherein said factor B cleavage site 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) and DHKL/KSG (SEQ ID NO: 21).
The engineered protease according to any one of embodiments 1-15, wherein said engineered protease is based on a chymotrypsin-like serine protease selected from the group consisting of: membrane serine protease 1 (MTSP-1), urokinase type plasminogen activator (uPA), chymotrypsin and kallikrein related peptidase 5 (KLK 5).
Embodiment I-17 the engineered protease according to embodiment I-16, wherein the engineering of the engineered protease involves one or more modifications selected from the group consisting of: substitution, addition and deletion of amino acid residues of the chymotrypsin-like serine proteases and substitution, addition and deletion of domains.
The engineered protease according to any one of embodiments 16-17, wherein said engineered protease is based on MTSP-1 or uPA and said cleavage site comprises a sequence selected from the group consisting of seq id nos: WEHR/KGT (SEQ ID NO: 10) and KNQKR/QKQ (SEQ ID NO: 11).
Embodiments I-19 the engineered protease according to any one of embodiments 1-18, wherein said engineered protease is MTSP-1-based.
Embodiment I-20 the engineered protease according to any one of embodiments 1-18, wherein said engineered protease is not MTSP-1-based.
Embodiment I-21. The engineered protease according to embodiment I-19 comprises one or more modifications relative to MTSP-1 comprising the amino acid sequence set forth in SEQ ID NO. 7, wherein the residues are numbered by chymotrypsin numbering.
Embodiment I-22 the engineered protease according to any one of embodiments 1-18, wherein said engineered protease is upA-based.
Embodiment I-23 the engineered protease according to any one of embodiments 1-18, wherein said engineered protease is not uPA-based.
Embodiments I-24. The engineered protease according to embodiments I-22, comprising one or more modifications relative to uPA comprising the amino acid sequence shown in SEQ ID NO. 8, wherein the residues are numbered by chymotrypsin numbering.
Embodiments I-25 the engineered protease according to any one of embodiments 1-17, wherein said engineered protease is chymase-based.
Embodiments I-26. The engineered protease according to embodiments I-25, wherein the engineered protease is chymase-based and the cleavage site comprises a sequence selected from the group consisting of 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).
Embodiments I-27. The engineered protease according to any one of embodiments I-1-17, wherein the engineered protease is based on KLK5.
Embodiments I-28. The engineered protease according to embodiments I-25, comprising one or more modifications relative to chymase comprising the amino acid sequence as set forth in SEQ ID NO. 6, wherein the residues are numbered by chymotrypsin numbering.
The engineered protease according to embodiments I-29, wherein said one or more modifications are located at one or more positions corresponding to one or more positions selected from the group consisting of 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 amino acid sequence set forth in SEQ ID NO. 7, wherein the residues are numbered by chymotrypsin numbering.
The engineered protease according to embodiments I-30, wherein said one or more modifications are located 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 amino acid sequence shown in SEQ ID No. 8, wherein the residues are numbered by chymotrypsin numbering.
Embodiments I-31. The engineered protease according to embodiments I-25, wherein the one or more modifications are located at one or more positions corresponding to one or more positions selected from the group consisting of 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 the amino acid sequence shown in SEQ ID NO. 6, wherein the residues are numbered by chymotrypsin numbering.
Embodiments I-32. The engineered protease according to any one of embodiments 4-31, wherein the function of factor B or a fragment of factor B is to interact with at least one complement component.
Embodiments I-33. The engineered protease according to any one of embodiments 4-31, wherein the function of factor B or factor B fragment is to interact with hydrolyzed soluble C3.
Embodiments I-34 the engineered protease according to any one of embodiments 4-33, wherein the function of factor B or factor B fragment is to interact with C3B.
Embodiments I-35 the engineered protease according to any one of embodiments 4-34, wherein the function of factor B or factor B fragment is to interact with membrane bound C3B.
Embodiments I-36. The engineered protease according to any one of embodiments 1-35, wherein cleavage occurs when factor B does not bind to C3B.
Embodiments I-37 the engineered protease of any of embodiments 1-36, wherein the cleavage activity for a non-factor B peptide substrate is about equal to or less than the cleavage activity for a factor B site.
The engineered protease according to any one of embodiments 1-36, wherein the engineered protease has 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 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1.700, about 1,800, or about 1,900M-1 s-1.
Embodiment I-39 the engineered protease according to any one of embodiments 1-38, wherein said engineered protease has a kcat/Km for factor B cleavage of about 103 to about 109M-1 s-1.
Embodiment I-40 the engineered protease according to any one of embodiments 1-39, wherein said engineered protease has an EC50 of less than about 20nM for factor B.
Embodiment I-41 the engineered protease according to any one of embodiments 1-40, wherein said engineered protease has an EC50 of less than about 1nM for factor B.
Embodiment I-42. The engineered protease according to any one of embodiments 1-39, wherein the engineered protease has an EC50 of about 20nM, about 25nM or about 60nM for factor B.
Embodiment I-43 the engineered protease according to any one of embodiments 1-39, wherein said engineered protease has an EC50 of about 1,000nM to about 4,500nM for cleavage factor B.
Embodiment I-44 the engineered protease according to any one of embodiments 1-43, wherein said engineered protease has a plasma half-life in human plasma of more than about 72 hours.
Embodiment I-45 the engineered protease according to any one of embodiments 1-44, wherein said engineered protease has a plasma half-life in human plasma of more than about 120 hours.
Embodiments I-46. The engineered protease according to any one of embodiments 1-45, wherein said engineered protease has a plasma half-life in human plasma of about 7 days.
Embodiment I-47. The engineered protease of embodiment I-46, wherein the catalytic activity is about 10% to about 50%, or about 90% to about 100%.
Embodiment I-48. The engineered protease according to embodiment I-29, wherein said engineered protease has an increased half-life compared to unmodified MTSP-1.
Embodiments I-49 the engineered protease according to any one of embodiments I29 and I-48, wherein said engineered protease has increased bioavailability compared to unmodified MTSP-1.
Embodiment I-50. The engineered protease of embodiment I-30, wherein the engineered protease has an increased half-life compared to unmodified uPA.
Embodiment I-51. The engineered protease according to any one of embodiments I-30 and I-50, wherein the engineered protease has increased bioavailability compared to unmodified uPA.
Embodiment I-52. The engineered protease of embodiment I-31, wherein the engineered protease has an increased half-life compared to an unmodified chymase.
Embodiment I-53 the engineered protease according to any one of embodiments I-31 and I-52, wherein said engineered protease has increased bioavailability compared to an unmodified chymase.
Embodiment I-54 the engineered protease according to any one of embodiments I-1 to I-53, wherein said engineered protease is non-immunogenic.
Embodiments I-55. The engineered protease according to any one of embodiments groups I1-54, wherein the engineered protease is in the zymogen form.
Embodiments I-56. The engineered protease according to any one of embodiments groups I1-54, wherein the engineered protease is in an active form.
Embodiments I-57 the engineered protease according to any one of embodiments group I1-56, further comprising a half-life extending factor.
Embodiment I-58. A method of inactivating factor B comprising contacting the factor B with any one of the engineered proteases of embodiments I-1 to I-57.
Embodiments I-59. The method of embodiments I-58, wherein complement activation is inhibited.
Embodiment I-60. The method of embodiment I-59, wherein the classical pathway of the complement pathway is inhibited.
Embodiments I-61. The method of any of embodiments 59-60, wherein the alternative pathway of the complement pathway is inhibited.
Embodiments I-62. The method of any one of embodiments 59-61, wherein the lectin pathway of the complement pathway is inhibited.
Embodiments I-63. The method according to any one of embodiments group I58-62, wherein the method is in vitro.
Embodiments I-64. The method according to any one of embodiments group I58-62, wherein the method is in vivo.
Embodiments I-65. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an engineered protease of any one of embodiments 1-57.
Embodiment I-66. The method according to embodiment I-65, wherein the disease or disorder is associated with dysregulated complement.
Embodiments I-67. The method according to any one of embodiments groups I65-66, wherein the disease or disorder is an inflammatory disease or disorder.
Embodiments I-68 the method according to any one of embodiments 65-67, wherein the treatment is an alternative therapy.
Embodiments I-69. The method according to any one of embodiments group I65-68, wherein the treatment blocks complement activation.
Embodiments I-70. The method according to any of embodiments group I65-69, wherein the treatment modulates autoimmunity.
Embodiments I-71. The method according to any of embodiments group I65-70, wherein the disease or disorder is congenital complement deficiency.
Embodiments I-72. The method according to any one of embodiments group I65-71, wherein the treatment is for endothelial cell or renal cell injury.
The method according to any one of embodiments set I65-72, wherein the disease or disorder is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplantation ischemia reperfusion (I/R) injury, anti-neutrophil 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 sleep-related hemoglobinuria (PNH).
Embodiments I-74. The method according to any one of embodiments group I65-73, wherein the disease or disorder is a control protein deficiency.
Embodiments I-75. The method according to any one of embodiments group I65-73, wherein the disease or disorder is a secondary complement disorder.
Embodiments I-76. The method according to any of embodiments group I65-73, wherein the disease or disorder is an immune-related disease or disorder.
The method according to any one of embodiments set I65-76, wherein the engineered protease is administered subcutaneously to the subject.
Embodiments I-78. The method according to embodiments I-77, wherein the engineered protease is activated in situ at a site that modulates an aberrant complement component.
The method according to any one of embodiments group I65-78, wherein the engineered protease is provided in a liquid stable formulation.
Embodiments I-80. A pharmaceutical composition comprising any one of the engineered proteases of embodiments group I1-57, and optionally a pharmaceutically acceptable carrier.
Embodiment I-81. The pharmaceutical composition according to embodiment I-80 wherein the engineered protease is provided in a liquid stable formulation.
The pharmaceutical composition according to any one of embodiments group I80-81, wherein the composition is formulated for subcutaneous administration.
Group II
Embodiment II-1. An engineered protease comprising a modified chymotrypsin 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 (KLK 5) protease domain, wherein the engineered protease is capable of cleaving factor B.
Embodiment II-2 the engineered protease according to embodiment II-1, wherein cleavage of factor B by the engineered protease results in one or more functionally inactive fragments.
Embodiment II-3 the engineered protease according to embodiment II-2, wherein said one or more functionally inactive fragments are capable of reducing complement activation.
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 production of a functionally reduced factor B fragment.
Embodiment II-5 the engineered protease of any one of embodiments II-1 to II-4, wherein the factor B is a non-human primate factor B.
Embodiment II-6 the engineered protease of embodiment II-5, wherein the non-human primate is a cynomolgus monkey.
Embodiment II-7 the engineered protease according to any one of embodiments II-1 to II-4, wherein said factor B is human factor B.
Embodiment II-8 the engineered protease according to embodiment II-7, wherein said factor B comprises the amino acid sequence as shown in SEQ ID NO. 1.
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 not targeted by factor D.
Embodiment II-10. The engineered protease according to embodiment II-9, wherein cleavage at said site not targeted by factor D results in at least two fragments that are not Ba and Bb.
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 factor B cleavage products Ba and Bb compared to cleavage by factor D.
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.
Embodiment II-13 the engineered protease according to embodiment II-12, wherein said factor B cleavage site targeted by factor D comprises QQKR/KIV (SEQ ID NO: 9).
Embodiment II-14 the engineered protease according to embodiment II-9, wherein the factor B cleavage site 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) and DHKL/KSG (SEQ ID NO: 21).
Embodiment II-15 the engineered protease according to embodiment II-9, wherein said factor B cleavage site comprises a sequence selected from the group consisting of WEHR/KGT (SEQ ID NO: 10) and KNQKR/QKQ (SEQ ID NO: 11), and wherein said engineered protease comprises a modified MTSP-1 protease domain or a modified uPA protease domain.
Embodiment II-16 the engineered protease according to any one of embodiments II-1 to II-15, wherein said engineered protease comprises a modified MTSP-1 protease domain.
Embodiment II-17 the engineered protease according to any one of embodiments II-1 to II-15, wherein said engineered protease does not comprise a modified MTSP-1 protease domain.
Embodiment II-18. The engineered protease according to embodiment II-16, comprising one or more modifications relative to the MTSP-1 protease domain comprising the amino acid sequence as set forth in SEQ ID NO. 7.
Embodiment II-19 the engineered protease according to embodiment II-18, wherein said modification is one or more of substitution, addition and deletion of one or more amino acid residues.
Embodiment II-20. The engineered protease according to embodiment II-16, wherein said one or more modifications are located at one or more positions corresponding to one or more positions selected from the group consisting of 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 amino acid sequence set forth in SEQ ID NO. 18.
Embodiment II-21 the engineered protease according to embodiment II-16, wherein said one or more modifications are selected from those shown in Table 5A.
Embodiment II-22 the engineered protease according to embodiment II-16, wherein said one or more modifications are selected from those exemplary mutational strings shown in Table 5B.
Embodiment II-23 the engineered protease of any one of embodiments II-1 to II-15, wherein the engineered protease comprises a modified uPA protease domain.
Embodiment II-24 the engineered protease of any one of embodiments II-1 to II-15, wherein the engineered protease does not comprise a modified uPA protease domain.
Embodiments II-25. The engineered protease according to embodiments II-23, comprising one or more modifications relative to the uPA protease domain comprising the amino acid sequence shown in SEQ ID NO. 8.
Embodiment II-26. The engineered protease according to embodiment II-25, wherein said modification is one or more of substitution, addition and deletion of one or more amino acid residues.
Embodiments II-27. The engineered protease according to embodiments II-23, wherein the one or more modifications are located at one or more positions corresponding to one or more positions selected from the group consisting of S37, D60, L97, G161, R179, H180, V185, Y187, I207, A247, D248, A251, H252, C279, Y308, V316, A343, Q353, R378, K385 in the uPA protease domain comprising the amino acid sequence shown in SEQ ID NO 8.
Embodiments II-28. The engineered protease according to embodiments II-23, wherein the one or more modifications are selected from those shown in Table 3A.
Embodiments II-29. The engineered protease according to embodiments II-23, wherein the one or more modifications are selected from those exemplary mutational strings shown in Table 3B.
Embodiment II-30 the engineered protease of any one of embodiments II-1 to II-19, wherein the engineered protease comprises a modified chymase protease domain.
Embodiment II-31 the engineered protease of any one of embodiments II-1 to II-19, wherein the engineered protease does not comprise a modified chymase protease domain.
Embodiment II-32. The engineered protease according to embodiment II-30, wherein the engineered protease comprises a modified chymase protease domain and the cleavage site comprises a sequence selected from the group consisting of 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).
Embodiments II-33. The engineered protease according to embodiments II-30, comprising one or more modifications relative to a chymase protease domain comprising an amino acid sequence as set forth in SEQ ID NO. 6.
Embodiment II-34 the engineered protease according to embodiment II-33, wherein said modification is one or more of substitution, addition and deletion of one or more amino acid residues.
Embodiments II-35. The engineered protease according to embodiments II-30, wherein the one or more modifications are located at one or more positions corresponding to one or more positions selected from the group consisting of 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 amino acid sequence set forth in SEQ ID NO 6.
Embodiments II-36. The engineered protease according to embodiments II-30, wherein the one or more modifications are selected from those shown in Table 7A.
Embodiment II-37 the engineered protease according to embodiment II-30, wherein said one or more modifications are selected from those exemplary mutational strings shown in Table 7B.
Embodiment II-38 the engineered protease according to any one of embodiments II-1 to II-19, wherein said engineered protease comprises a modified KLK5 protease domain, optionally comprising one or more amino acid modifications of SEQ ID NO. 23.
Embodiment II-39 the engineered protease of any of embodiments II-1 to II-19, wherein the engineered protease does not comprise a modified KLK5 protease domain.
Embodiment II-40. The engineered protease of any one of embodiments II-1 to II-39, wherein the engineered protease has about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, 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 1,900M for factor B cleavage -1 s -1 K of (2) cat /K m 。
Embodiment II-41 the engineered protease of any one of embodiments II-1 to II-40, wherein the engineered protease has about 10 for factor B cleavage 3 To about 10 9 M -1 s -1 K of (2) cat /K m 。
Embodiment II-42 the engineered protease of any one of embodiments II-1 to II-41, wherein the engineered protease has an EC of less than about 20nM for factor B 50 。
Embodiment II-43 the engineered protease of any one of embodiments II-1 to II-42, wherein the engineered protease has an EC of less than about 1nM for factor B 50 。
Embodiment II-44 the engineered protease of any one of embodiments II-1 to II-41, wherein the engineered protease has an EC of about 20nM, about 25nM or about 60nM for factor B 50 。
Embodiment II-45 the engineered protease of any one of embodiments II-1 to II-41, wherein the engineered protease has an EC of about 1,000nM to about 4,500nM for cleavage factor B 50 。
Embodiment II-46 the engineered protease of any one of embodiments II-1 to II-45, wherein the engineered protease has a plasma half-life in human plasma of greater than about 72 hours.
Embodiment II-47. The engineered protease of any one of embodiments II-1 to II-46, wherein the engineered protease has a plasma half-life in human plasma of greater than about 120 hours.
Embodiment II-48 the engineered protease according to any one of embodiments II-1 to II-47, wherein said engineered protease has a plasma half-life in human plasma of about 7 days.
Embodiment II-49 the engineered protease of embodiment II-48, wherein the catalytic activity is about 10% to about 50%, or about 90% to about 100%.
Embodiment II-50. The engineered protease according to embodiment II-16, wherein said engineered protease has an increased half-life compared to a protease comprising an unmodified MTSP-1 protease domain.
Embodiment II-51. The engineered protease according to embodiment II-16, wherein said engineered protease has increased bioavailability compared to a protease comprising an unmodified MTSP-1 protease domain.
Embodiment II-52. The engineered protease according to embodiment II-23, wherein the engineered protease has an increased half-life compared to a protease comprising an unmodified uPA protease domain.
Embodiment II-53. The engineered protease according to embodiment II-23, wherein said engineered protease has increased bioavailability compared to a protease comprising an unmodified uPA protease domain.
Embodiment II-54 the engineered protease according to embodiment II-30, wherein said engineered protease has an increased half-life compared to a protease comprising an unmodified chymotrypsin protease domain.
Embodiment II-55. The engineered protease according to embodiment II-30, wherein the engineered protease has increased bioavailability compared to a protease comprising an unmodified chymotrypsin protease domain.
Embodiment II-56 the engineered protease of any one of embodiments II-1 to II-55, wherein the engineered protease is non-immunogenic.
Embodiment II-57 the engineered protease according to any one of embodiments II-1 to II-56, wherein the engineered protease is in the zymogen form.
Embodiment II-58 the engineered protease according to any one of embodiments II-1 to II-56, wherein the engineered protease is in active form.
Embodiment II-59 the engineered protease of any one of embodiments II-1 to II-58, wherein the engineered protease is fused to a component that extends the half-life of the engineered protease.
Embodiment II-60. The engineered protease according to embodiment II-59, wherein said component is an Fc domain.
Embodiment II-61 the engineered protease according to embodiment II-59, wherein said component is human serum albumin.
Embodiment II-62. The engineered protease according to 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.
Embodiment II-63. The engineered protease according to embodiment II-62, wherein said modified chymotrypsin protease domain of SEQ ID NO. 6 comprises one of the mutation strings of Table 7B.
Embodiment II-64 the engineered protease according to 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.
Embodiment II-65. The engineered protease according to embodiment II-64, wherein said modified MTSP-1 protease domain of SEQ ID NO. 7 comprises one of the mutation strings of Table 5B.
Embodiment II-66. The engineered protease according to 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.
Embodiment II-67. The engineered protease according to embodiment II-66, wherein said modified uPA protease domain of SEQ ID NO. 22 comprises one of the mutation strings of Table 3B.
Embodiment II-68. The engineered protease of embodiment II-1 comprising a modified kallikrein related peptidase 5 (KLK 5) 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.
Embodiment II-69. A method of inactivating factor B comprising contacting the factor B with any one of the engineered proteases of embodiments II-1 to II-68.
Embodiment II-70 a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an engineered protease according to any one of embodiments II-1 to II-68.
Embodiment II-71. The method of embodiment II-70 wherein the disease or disorder is associated with dysregulated complement.
Embodiment II-72 the method according to any one of embodiments II-70 to II-71, wherein the disease or disorder is an inflammatory disease or disorder.
Embodiment II-73. The method according to any of embodiments II-70 to II-72, wherein the treatment is an alternative therapy.
Embodiment II-74 the method of any one of embodiments II-70 to II-73, wherein the treatment blocks complement activation.
Embodiment II-75. The method according to any one of embodiments II-70 to II-74, wherein the treatment modulates autoimmunity.
Embodiment II-76 the method according to any of embodiments II-70 to II-75, wherein the disease or disorder is congenital complement deficiency.
Embodiment II-77 the method of any one of embodiments II-70 to II-76, wherein said treating is for endothelial cell or renal cell injury.
Embodiment II-78. The method according to any one of embodiments II-70 to II-77, wherein the disease or disorder is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplantation ischemia reperfusion (I/R) injury, anti-neutrophil 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 sleep hemoglobinuria (PNH).
Embodiment II-79 the method according to any one of embodiments II-70 to II-78, wherein the disease or disorder is a control protein deficiency.
Embodiment II-80. The method according to any of embodiments II-70 to II-78, wherein the disease or disorder is a secondary complement disorder.
Embodiment II-81 the method according to any one of embodiments II-70 to II-78 wherein the disease or disorder is an immune-related disease or disorder.
Embodiment II-82 the method of any one of embodiments II-70 to II-81, wherein the engineered protease is administered subcutaneously to the subject.
Embodiment II-83. The method of embodiment II-82 wherein the engineered protease is activated in situ at a site that modulates an aberrant complement component.
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.
Embodiment II-85. A pharmaceutical composition comprising an engineered protease according to any one of the engineered proteases of embodiments group II-1 to II-68, and optionally a pharmaceutically acceptable carrier.
Embodiments II-86 the pharmaceutical composition according to embodiments II-85, wherein the engineered protease is provided in a liquid stable formulation.
The pharmaceutical composition of any one of embodiments II-85 to II-86, wherein the composition is formulated for subcutaneous administration.
Examples
Example 1: chymotrypsinExpression and purification and initial characterization of enzyme-like serine proteases
Expression and purification of MTSP-1 and uPA based proteases
Small scale expression and purification of MTSP-1 and uPA based engineered proteases was performed. Briefly, MTSP-1 and uPA were each expressed as zymogens in E.coli strains of BL21-Gold (DE 3) bacterial hosts, isolated from inclusion bodies, denatured, refolded by rapid dilution, dialyzed and subsequently activated on immobilized trypsin columns. The active protein is then purified on an anion exchange column. Similarly, large scale refolding and purification of MTSP-1 was also performed. MTSP-1 and uPA were dissolved, refolded and purified on an anion exchange column. Next, endotoxin was removed from MTSP-1 and uPA.
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 of BL21 Gold (DE 3) bacterial hosts. Insoluble chymase is separated 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 4mL Fast Flow SP beads packed in BioRAD columns using gravity and activated with enterokinase overnight. The native chymase is then purified from the zymogen by cation exchange chromatography using a step elution method.
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. Mu.M human factor B (Complement Technologies) in 20. Mu.L buffer (50 mM Tris pH 7.4/50mM NaCl/0.01% Tween 20). Different concentrations of chymotrypsin-based engineered protease were added to factor B (top 3000nM,1:2 dilution, 10 steps, including 0.0 nM) and incubated for 1 hour at 37 ℃. After digestion, 15 μl of the reaction was transferred to a 96-well plate containing 1.5 μl of 0.2N HCL to stop chymase digestion.After quenching, the reaction was prepared for SDS-PAGE. mu.L of the reaction mixture was loaded into each well of a 4% -12% Bis-Tris Criterion gel. Optical density analysis of factor B cut was performed and EC was calculated 50 . FIG. 1D depicts a Coomassie gel showing an example of factor B cleavage by a test chymase-based engineered protease. Table 8A lists the chymase-based engineered proteases tested in fig. 1D. All references to engineered proteases in the table are described in chymotrypsin numbering. For chymotrypsin numbering keys for modified protease domains of chymotrypsin, see table 6. Two batches were used per engineered protease group, and each batch showed the ability of the engineered protease to cleave factor B. Batch 1 of each group shows a visible amount of zymogen, which is not titratable, but may be activated or have some activity for factor B. These results also indicate that the engineered protease is efficient in factor B cleavage. The engineered proteases tested are shown in table 8A below.
FIG. 1E depicts a graph showing that the low EC was calculated by having the ELISA measurements 50 Is a graph of an example of factor B cleavage by chymase-based engineered proteases. All references to engineered proteases in this example are described in chymotrypsin numbering. For chymotrypsin numbering keys for modified protease domains of chymotrypsin, see table 6. Briefly, digestion reactions were prepared with 2.0. Mu.M human factor B (Complement Technologies) in 20. Mu.L buffer (50 mM Tris pH 7.4/50mM NaCl/0.01% Tween 20). Two chymase-based engineered proteases were used at different concentrations: C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R and C22S/P38Q/K40M/F41R/V138I/F173Y/D175R/A190S/V213A/S218V/A226R. They were each provided at 3000nM and diluted 1:2 to obtain 1500nM, 750nM, 375nM, 188nM, 94nM, 47nM, 23nM, 12nM, 6nM and 3nM, additionally using a control of 0.0 nM. They were added to 2. Mu.M of factor B, respectively, and incubated at 37℃for 1 hour. After digestion, 15. Mu.L of the reaction was transferred to a 96-well plate containing 1.5. Mu.L of 0.2N HCL. After quenching, factor B was prepared for ELISA reactions.
Factor B standard curves were made with 800pM, 533.3pM, 355.5pM, 237.0pM, 158.0pM, 105.3pM, 70.2pM, 46.8pM, 31.2pM, 20.8pM, 0.0pM in 1% BSA-PBST. 384 well plates were coated with 2 μg/ml monoclonal antibody (Quidel) against human factor Ba fragment in carbonate buffer (25 μl/well). After blocking with 1% BSA-PBST (100. Mu.L/well) for 1 hour at room temperature, the digests (chymase+FB) were diluted to 800pM and standards were diluted 1:1.5 from 800pM into blocking buffer (25. Mu.L/well). The plate was stirred at room temperature for 30 minutes.
Biotinylated monoclonal antibody to 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. Mu.L/well) and the plates were stirred at room temperature for 30 min. Plates were developed with elisbright (50 μl/well) for 1 min at room temperature and read in an EnVision plate reader. Table 8B below shows calculated ECs for the two engineered proteases 50 . FIG. 1E shows that the tested chymase-based engineered protease is required to inhibit factor B by 50% in the range of 30nM to 42 nM.
Table 8A: chymase-based engineered proteases tested in FIG. 1D
Table 8B: EC50 for factor B cleavage by chymase-based engineered proteases
Example 2: protein characterization and active site titration of chymase-based engineered proteases
Testing selected chymase-based engineered proteases to measure the activity of these proteases by using an active site titration method based on reaction with an inhibitory serine protease inhibitor bait followed by analysis of the quantitative active fractions by HPLC. All references to engineered proteases in this example (including table 9) are described in chymotrypsin numbering. For chymotrypsin numbering keys for modified protease domains of chymotrypsin, see table 6. The shift in the measured peak was detected by HPLC analysis when the serine protease inhibitor decoy was present, indicating that the protease bound the decoy and was therefore active. Briefly, serpin baits with working concentrations of 24 μm were prepared from stock solutions using 50 μm of low molecular weight heparin for Antithrombin (AT) based baits. A protease solution was prepared using 30. Mu.L of 5. Mu.M working solution. The reaction mixtures were incubated at 37 ℃ for 2 hours and each reaction mixture (protease alone or protease with bait) was analyzed by HPLC using standard protocols. All protease-only samples were run first, then the protease was run with the serpin bait sample. The results of the proteases tested are summarized in table 9 below. Measurement of k using EGVD/AE-QF (SEQ ID NO: 52) cat /K m And measuring the secondary rate constant K using EGVD/AE-ACT (SEQ ID NO: 53) app . These results measure the protease activity after purification and most of the produced chymotrypsin-based engineered proteases tested showed between 60% -120% activity.
Table 9: active site titration summary of chymase-based engineered proteases
Example 3: stability studies and serine protease selection using factor B cleavage Selecting
Cleavage of factor B by various engineered proteases was evaluated. Engineered proteases based on chymase, MTSP-1 and uPA were tested. All references to engineered proteases in this example (including tables 10A, 10B, 11A and 11B) are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, 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 into assay buffer and incubated with naturally occurring (wild-type) and non-naturally occurring (engineered) proteases in vitreous, plasma or PBS at 37 ℃ and the reaction quenched with HCl at different time points. Time points for testing chymase included t=0 pre-incubated for 6 hours, t=2 hours pre-incubated for 4 hours, t=4 hours pre-incubated for 2 hours, and t=6 hours pre-incubated for 0 hours. Sample determination was performed on human C3 cleavage using standard protocols, with ELISA or alpha screening.
Summary of the results is shown in tables 10A-10B below, which illustrate various proteases tested, protease concentration used in nM and human factor B cleaved EC 50 . Table 10 shows human factor B cleavage by various non-naturally occurring chymase-based engineered proteases tested in 80% human plasma as compared to chymase comprising the mutant string C122S. The blank boxes indicate that no test was performed on a particular protease mutation string. These results generally indicate 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.
Table 10B shows human factor B cleavage by various chymase, MTSP-1 or uPA based engineered proteases tested in mouse or human plasma. For ELISA analysis of factor B cleavage, in brief, anti-Ba was used and Bb was detected with anti-Bb. A dynamic range of 25pM to 1600pM was used. The half-life of various chymase-based engineered proteases in 50% human plasma was also calculated.
These results indicate that the uPA-based scaffoldsThe engineered proteases did not show efficient factor B cleavage and the proteases engineered with MTSP-1 based scaffolds showed factor B cleavage, but only for two candidates below 50 nM. Proteases engineered with chymase-based scaffolds showed the most efficient factor B cleavage, EC thereof 50 The value was below 50nM.
50 Table 10A: EC of chymase-based engineered protease for cleavage factor B
50 Table 10B: MTSP-1, uPA and chymase based engineered protease cleavage assay for EC factor B
In addition, by testing the effect of indirect and MTSP buffers (+/-EDTA) on chymase-based engineered proteases, various chymase-based engineered proteases were tested to evaluate their baseline stability. The peptide substrate EGVDAE-QF (SEQ ID NO: 52) was used for these experiments.
Fig. 4A-4E depict graphs showing the stability of five different chymotrypsin proteases tested in PBS at 37 ℃ using peptide substrates. Table 11A summarizes the data shown in fig. 4A-4E and lists the engineered proteases tested.
Table 11A: activity of chymase-based engineered proteases tested with peptide substrates
As shown, these proteases were more stable in an indirect buffer at pH 7.4. Wild-type or naturally occurring chymase showed loss of activity in all buffers and EDTA had no significant effect on stability.
Next, the secondary rate constants (k) of plasma inhibition of various non-naturally occurring chymase-based proteases were measured to infer half-life in 100% plasma. Briefly, a baseline chymase with a modification of C22S/A226R was used. The baseline chymase and the engineered chymase-based protease tested are listed in Table 11B below at 50nM and the other chymase tested is used at 25nM C22S/L99R/F173Y/K192R/S218L/A226R. For baseline chymase, the time course of protease activity in the presence of plasma was measured at 37℃using 4.2. Mu.M CPQ2-ITLLSA-K (5 FAM) -K-PEG8-K (biotin) -NH2 (SEQ ID NO: 17)/21. Mu.M neutravidin. The results are summarized in table 11B below.
1/2 Table 11B: t of second order rate constant measurement and calculation
Example 4: factor B reverse addition hemolysis assay
A hemolysis assay was performed to evaluate the hemolytic activity of different batches of factor B, one batch obtained from CompTech and the other batch expressed internally. Briefly, factor B depleted human serum was used and plasma purified or recombinant human factor B was used for reverse addition. Rabbit Red Blood Cells (RBCs) or chicken RBCs were washed in GVB/Mg/EGTA and depleted serum was labeled with factor B. RBC lysis was then monitored with CVF/FD/human factor B convertase. These were analyzed to form a standard curve from which factor B batches were compared. Figures 5 to 6 depict bar graphs of controls for haemolysis assays and standard curves measured by the tested engineered protease from factor B reverse addition haemolysis assay, respectively. Table 12 below shows a summary of data for MTSP-1 and uPA based engineered proteases. FIG. 6 also shows that MTSP-1 based engineered protease F97E/K224N/F99L/D217I/C122S/C17S/C19S is capable of inhibiting hemolysis. The engineered protease was tested at two concentrations (samples 1 and 2). All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively.
Table 12: factor B reverse addition of hemolysis data summary
cat M Example 5: peptide cleavage assay (K/K)
Peptide cleavage assays were performed to evaluate k of various engineered proteases of the present disclosure based on MTSP-1, uPA or chymase cat /K m . All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. Evaluation of various kindsCleavage of substrate to determine k of protease Activity cat /K m . Briefly, k cat /K m The value is determined by V as a function of the substrate concentration o Slope determination of the linear portion of the graph. Generally, the following conditions are used: protease concentration was 50nM, substrate concentration was 20. Mu.M maximum, 1.5-fold serial dilutions at 30 ℃. For the calculation, a semi-automatic Michaelis Menten Kinetics protocol with quadruplicate measurements was used.
For chymase-based engineered proteases, the following Quenched Fluorescent (QF) peptide substrates were used: TQ2-KDVFYQMKK-Lys (5 FAM) -NH2 (SEQ ID NO: 29) and TQ2-KDVFYQMKK-Lys (5 FAM) (SEQ ID NO: 30). For the MTSP-1 and uPA based engineered proteases, the following peptide substrates were used, 5FAM-EQQKRKIVL-K (QXL 520) -GEQQKRKIVL 2 (SEQ ID NO: 31), CPQ 2-PEQKR-K (5 FAM) -NH2 (SEQ ID NO: 32), TQ 2-NH-Lys (5 FAM) -NH2 (SEQ ID NO: 33), ac-QQKR-ACC (SEQ ID NO: 34). Table 13A below shows various substrates used to test protease activity at specific cleavage site sequences, and Table 13B below shows EGVDAE QF (SEQ ID NO: 52) substrate k based on the various proteases tested cat /K m (M -1 s -1 )。
Table 13A: peptide substrates for various cleavage site sequences
cat M Table 13B: K/K of chymase-based engineered protease measured with EGVDAE-QF (SEQ ID NO: 52)
The results of the peptide cleavage assay are shown in tables 14A-14B below.
Table 14A: peptide cleavage assay data for uPA-based scaffolds
Table 14A: peptide cleavage assay data for uPA-based scaffolds (follow-up)
Table 14A: peptide cleavage assay data for uPA-based scaffolds (follow-up)
Table 14B: peptide cleavage assay data for MTSP-1-based and chymase-based scaffolds
Table 14B: peptide cleavage assay data for MTSP-1-based and chymase-based scaffolds (Programming)
Table 14B: peptide cleavage assay data for MTSP-1-based and chymase-based scaffolds (Programming)
Example 6: inhibition test of engineered proteases
Inhibition tests were performed using chymase-based engineered proteases. Various serine protease inhibitors capable of inhibiting protease activity were used to test whether they could be used to sufficiently inhibit the chymase-based engineered proteases tested, to enable selection of engineered proteases resistant to inhibition in plasma. A summary of serine protease inhibition assays is provided in table 15 below. All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively.
Table 15: summary of serine protease inhibitor inhibition
Example 6: factor B cleavage and KLK5 protease Activity
FIG. 7A depicts a graph showing inhibition of haemolysis of KLK5 and compared to chymase based engineered protease C22S/F173Y/D175N/A226R. All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. Briefly, digestion reactions were prepared with 4.0. Mu.M human factor B (Complement Technologies) in 20. Mu.L buffer (50 mM Tris pH 7.4/50mM NaCl/0.01% Tween 20). Different concentrations of KLK5 protease or chymotrypsin-based engineered protease C22S/F173Y/D175N/A226R (400 nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM and 0.39nM, control 0.0 nM) were added to 4. Mu.M factor B and incubated for 1 hour at 37 ℃. After digestion, 6 μl of the reaction was evaluated in a hemolysis assay.
Factor B digestion reactions were performed on 20. Mu.L GVB (Complement Technologies), 10mM MgCl 2 And 8mM EGTA (GVB/Mg/EGTA). Then 45 μl of human factor B depleted serum (Complement Technologies) was added to 5 μl of human factor B digest to obtain a final volume of 90% serum. At the same time, 50. Mu.L of rabbit erythrocytes (Colorado Serum Co.) were diluted into 950. Mu.L of GVB/Mg/EGTA and gently mixed. After 5 minutes of rotation at 2000RPM at 4℃rabbit cells were resuspended in 1ml GVB/Mg/EGTA. The washed rabbit cells were then incubated with a mixture of serum+factor B digests in GVB/Mg/EGTA buffer to obtain a final concentration of 15% serum and incubated for 1 hour at 37 ℃ with stirring. The reaction was then centrifuged at 2000RPM for 5 minutes and 100 μl of supernatant was transferred to a clear flat bottom 96 well plate. Plate absorbance was read at 415nm with a spectrophotometer and EC was calculated 50 . The results of the hemolysis assay are shown in fig. 7A, which demonstrates that KLK5 protease is effective in inhibiting hemolysis.
FIG. 7B depicts a graph showing factor B cleavage with KLK5 and chymase based engineered protease C22S/F173Y/D175N/A226R. Briefly, digestion reactions were prepared with 4.0. Mu.M human factor B (Complement Technologies) in 20. Mu.L buffer (50 mM Tris pH 7.4/50mM NaCl/0.01% Tween 20). Different concentrations of KLK5 (400 nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM and 0.39nM, controls 0.0 nM) were added to 4. Mu.M factor B and incubated for 1 hour at 37 ℃. After digestion, 15 μl of the reaction was transferred to a 96-well plate containing 1.5 μ L0.2N HCL. After quenching, factor B was prepared for ELISA reactions.
Factor B standard curves were made with 800pM, 533.3pM, 355.5pM, 237.0pM, 158.0pM, 105.3pM, 70.2pM, 46.8pM, 31.2pM, 20.8pM, 0.0pM in 1% BSA-PBST. 384-well plates were coated with 2 μg/mL of monoclonal antibody directed against human factor Ba (#a225, quidel) in carbonate buffer (25 μl/well). After blocking with 1% BSA-PBST (100. Mu.L/well) for 1 hour at room temperature, the digests (chymase+factor B) were diluted to 800pM and standards were diluted 1:1.5 from 800pM into blocking buffer (25. Mu.L/well). The plate was stirred at room temperature for 30 minutes.
Biotinylated monoclonal antibody (Quidel) against human factor Bb was then added at 0.125 μg/ml to detect bound factor B. streptavidin-HRP was then diluted 1:200 in blocking buffer (25. Mu.L/well) and the plates were stirred at room temperature for 30 min. Plates were developed with elisbright (50 μl/well) for 1 min at room temperature and read in an EnVision plate reader. Fig. 7B depicts two independent experiments using different KLK5 stock solutions. These results indicate that the KLK5 protease is able to cleave factor B effectively with considerable activity at the different concentrations used.
Factor B cleavage by KLK5 was also assessed by coomassie gel. FIGS. 8A-8B depict Coomassie gels showing examples of factor B cleavage with KLK5 and chymase based engineered protease C22S/F173Y/D175N/A226R. Briefly, digestion reactions were prepared with 4.0. Mu.M human factor B (Complement Technologies) in 20. Mu.L buffer (50 mM Tris pH 7.4/50mM NaCl/0.01% Tween 20). Different concentrations of KLK5 or chymotrypsin-based engineered proteases C22S/F173Y/D175N/A226R (400 nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM and 0.39nM, control 0.0 nM) were added to 4. Mu.M factor B and incubated for 1 hour at 37 ℃. After digestion, 15. Mu.L of the reaction was transferred to a 96-well plate containing 1.5. Mu.L of 0.2N HCL. Quenching of After extinction, the reaction was prepared for SDS-PAGE gel. mu.L of the reaction mixture was loaded into each well of a 4% -12% Bis-Tris Criterion gel. Optical density analysis of factor B cut was performed and EC was calculated 50 。
FIG. 9 depicts Mass Spectrometry (MS) data identifying KLK5 vs. QQKR/KIV of factor B (SEQ ID NO: 9) cleavage sites 234 Cleavage site at Arg and within the cleavage site of EGVDAE (SEQ ID NO: 13) identifying chymase-based engineered protease C22S/A226R versus factor B 221 Cleavage site at Asp. Briefly, human factor B (Comptech) was combined with varying concentrations of kallikrein 5 (KLK 5, R) at 2. Mu.M&D Systems) or chymase-based engineered protease C22S/A226R was incubated at 37℃for 10 min to 1 hr in 20mM Tris pH 8 buffer with 16-O or 18-O water at 10nM or 100 pM. The reaction was quenched with 20. Mu.M inhibitor FFR-CMK for 30 min at room temperature. Half of the sample was further treated with rapidest/chymotrypsin and adjusted to pH 3 by the addition of 1ml 1% TFA. TCEP (100 mM final concentration) was then added to reduce the disulfide at 37 ℃ for 30 minutes. 12mL of each sample was bound to Ziptip (Millipore) and eluted with 15mL of 80% ACN-0.1% TFA. After drying in Speedvac, the samples were redissolved in 4ml of 30% ACN-0.05% TFA. 0.35mL of the sample was loaded onto OptiPlate along with 0.45mL of CHCA matrix (10 mg/mL) and analyzed by MALDI-MS (ABI 4700) in linear (m/z 2-22 k) and reflective (m/z 1500-5400) modes. Tandem MS was performed on peptides of interest when possible. Two large peptide fragments were detected at about 33kDa and 59 kDa. In summary, 100pm KLK5 cleaved at 1 Arg of 38 args in 10 min reaction and at 4 Arg of 38 args in 60 min reaction (Arg 175, arg193, arg730, and Arg 739). Cleavage (Arg 50, arg74, arg94, arg175, arg182, arg193, arg202, arg203, arg259, arg381, arg415, arg658, arg679, arg708, arg710, arg730, arg 739) was detected in 18 Arg of 38 Arg in 10-min and 60-min reactions at 10 nM. The earliest cleavage event is at Arg 730.
FIG. 9 shows a reconstructed ion chromatogram from kallikrein-5 ("KLK"), chymotrypsin-based engineered protease C22S/A22Reaction products of 6R ("chymase-based") and plasma derived factor B ("Fb control"). These chromatograms indicate that chymase-based engineered protease C22S/A226R is found in 221 Cleavage factor B at Asp, wherein 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 221 Cleavage at Asp is residues 222-739 (MW 62827) containing two A2 glycans. In contrast, KLK5 in 234 Cleavage factor B at Arg (peak at 4.69 min, MW 61437). There was no remaining whole factor B (top trace) in this sample.
Example 7: complement activation and cytokine release measured in mice model of acute respiratory distress syndrome
Fig. 10 is a schematic drawing depicting a general method for measuring complement activation and cytokine release in tissues of Acute Respiratory Distress Syndrome (ARDS) mouse model treated with chymase-based engineered proteases. All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. Briefly, mice received an injection of chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R, pulmonary function was measured, and bronchoalveolar lavage was measured after sacrifice. In fig. 10, the engineered protease is referred to as "protease".
After the adaptation period, each animal was weighed and randomly assigned to treatment groups 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 a sterile 0.9% saline (50 μl) instillation. The general health and body weight of all animals were monitored during the course of the disease. Respiratory function was measured in awake mice by Whole Body Plethysmography (WBP) at 0, 6, 24, and 48 hours after LPS IT. Three (3) hours after LPS IT, mice received 5mg/kg or 6.5mg/kg of basalIntravenous (IV) injection of chymase engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R and control animals received IV injection of vehicle (PBS). 27 hours after LPS IT (i.e., 24 hours after the first IV injection), a subset of mice received a second IV injection of 5mg/kg or 6.5mg/kg of chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R. Mice were sacrificed 24 hours and 48 hours after LPS IT. To obtain plasma, blood was drawn under anesthesia by facial puncture and collected at K 2 EDTA microsampling tube. After centrifugation at 2,000Xg for 10 minutes at 4℃plasma was aliquoted (60. Mu.l) and stored at-80℃for future cytokine and complement analysis.
Next, a tracheotomy is performed to expose the lungs. The trachea was connected to the cannula and the left lung was clamped while 0.9ml cold PBS1X, protease inhibitor 1X was injectedSolutions (3×300 μl) were collected for bronchoalveolar lavage (BALF) on right lobes. The first aliquot (300 μl) was saved for BALF total and differential cell counts. Two additional aliquots of 60 μl were each stored at-80 ℃ for future complement and cytokine analysis. Right lungs were immediately flash frozen and stored at-80 ℃ for complement and cytokine analysis in lung homogenates. The lungs were homogenized in 1XPBS+0.1% Triton X-100 with protease inhibitor cocktail to obtain 20 mg/100. Mu.L homogenate and centrifuged at 2,520 Xg for 15 min at 4 ℃. The supernatant is then processed for cytokine analysis.
Three hours after injection with chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R, plethysmography measurements showed a significant decrease in PenH values, indicating an improvement in lung function. However, this effect does not last over time (24 hours and 48 hours measured). Later, 48 hours after LPS, animals receiving 2 doses of chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R at 5-6.5mg/kg showed a significant improvement in body weight, indicating that the engineered protease reduced the severity of ARDS symptoms. Animals receiving 2 doses of chymase-based engineered protease at 48 hours post-LPS were shown to have a lower tendency for neutrophil to lymphocyte ratios in BALF, indicating a reduced inflammatory infiltration with chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R. These results are shown in fig. 11A to 11C. Fig. 11A depicts the pulmonary congestion index shown by PenH values. Figure 11B depicts weight loss measured in the test animals. Fig. 11C depicts BALF neutrophil to lymphocyte ratio (NLR) measured in the test animals. Data shown are mean +/-SEM, and tested using Student's T, p values <0.05. These results indicate that chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R is effective in improving respiratory function in ARDS mouse models. In fig. 11A to 11C, the engineered protease is referred to as "protease".
FIGS. 12A-12D depict BALF and lung cytokine measurements of mouse tissue after treatment with chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R. In fig. 12A to 12D, the engineered protease is referred to as "protease". Briefly, BALF and lung tissue were collected similarly as described above, 24 hours after intravenous administration of a single dose of chymase-based engineered protease C22S/P38Q/K40L/F41R/V138I/F173Y/D175N/A190S/V213A/S218V/A226R at 5mg/kg 3 hours post LPS. Measurement of eosinophil chemokine (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 (p 70), 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 (Eve Technologies) using mouse cytokine array/chemokine array 31-Plex (MD 31). Samples were centrifuged prior to aliquoting and diluted 2-fold prior to measurement according to Eve Technology protocol. IL-2, IL-6 and CXCL9 are significantly reduced in mouse bronchoalveolar fluid (BALF) and IL-6 is significantly reduced in lung tissue, indicating lower lung inflammation, which can lead to a subsequent reduction in chemical attraction of inflammatory cells into lung tissue. Values are mean +/-SEM, and tested using Student's t, p <0.05, p <0.01. These results indicate that the engineered protease is effective in reducing the inflammatory cytokines IL-2 and IL-6 and the chemokine CXCL9 in the ARDS mouse model.
Example 8: assessment and characterization of the Activity 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) (produced in mammalian culture systems, purified and activated) to cleave CFB was evaluated. All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. Enzymatic cleavage of complement factor B (CFB, complement Technologies catalog No. a 135) was assessed via the AlphaLISA assay. After in vitro cleavage at 37 ℃ for 1 hour, the reagents were diluted and transferred into multiwell plates for bead-based CFB detection. To detect CFB, an anti-factor Ba antibody (Quidel catalog No. a 225) labeled with DIG (Biotium Mix N' staining kit, catalog No. 92450) was paired with an anti-DIG receptor bead (Perkin Elmer catalog No. AL 113C), and a biotinylated anti-factor Bb antibody (Quidel catalog No. a 712) was paired with a streptavidin donor bead (Perkin Elmer catalog No. 67670002S). If an uncleaved CFB remains, the acceptor and donor beads are clustered together and upon laser excitation, singlet oxygen from the donor beads drives a chemiluminescent signal from the acceptor beads that activates fluorophores contained in the same acceptor bead. Upon CFB cleavage by the engineered protease tested in this example, the resulting Ba and Bb cleavage products no longer associate and result in signal loss or reduction. The cleavage of full length CFB was quantified by linear regression with respect to CFB standard curve in the absence of chymase. Table 16 shows the results of cleavage of CFB using the listed engineered proteases.
Table 16: cleavage of Complement Factor B (CFB)
Inhibition of hemolysis by the engineered protease was also evaluated in a standard Alternative Pathway (AP) hemolysis assay and an enhanced version of the assay, similar to that described in example 4. The inhibition of hemolysis of the small molecule factor B inhibitor LNP023 (Iptacopan, medChemExpress catalog number HY-127105) was evaluated in the same experiment as a comparison. For standard AP assays, different concentrations of C22S/P38Q/K40A/F41R/L99H/V138I/F173Y/D175N/A190S/V213A/S218I/A226R engineered protease or LNP023 or related vehicle controls were pre-mixed with 20% Normal Human Serum (NHS) at 37℃for 10 minutes. Rabbit Red Blood Cells (RBC) were then combined with an alternative pathway buffer (gelatin Florida buffer, GVB+Mg+EGTA, 0.1% gelatin, 5mM Florida, 145mM NaCl, 0.025% NaN) 3 、pH 7.3、10mM MgCl 2 And 8mM EGTA) was added and incubated at 37℃for 30 min. Cells were then pelleted and the OD of the supernatant at 415nm was measured to assess lysis. For the enhancement version of the assay, the following adjustments were made: instead of NHS, human factor B depleted serum (Complement Technologies catalog No. a 335), factor B purified from human serum was spiked back into serum to a final concentration of 1.6mM in the hemolysis assay, and serum was pre-incubated with drug for 180 minutes at 37 ℃. The percent lysis was calculated using the formula: [ (OD of sample) 415 -OD 415 EDTA negative control)/(OD 415 Saline positive control-OD 415 EDTA)*100](Table 17A and Table 17B). Calculation of IC by nonlinear regression 50 (Prism 9, log (inhibitor, bv) and response-4 parameter variable slope model) (tables 18A and 18B).
Table 17A: inhibition in standard AP RBC hemolysis
Table 17B: enhanced inhibition in AP RBC hemolysis
Table 18A: inhibition in standard AP RBC hemolysis
Test article Engineered proteases LNP023
IC 50 (mM) 0.92 0.027
Table 18B: enhanced inhibition in AP RBC hemolysis
Test article Engineered proteases LNP023
IC 50 (mM) 0.27 2.6
Example 9: pulmonary function measured in a mouse acute respiratory distress syndrome model
Fig. 13 is a schematic drawing depicting 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 described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. Briefly, mice received chymase-based injections of engineered protease C22S/P38Q/K40A/F41R/L99H/V138I/F173Y/D175N/A190S/V213A/S218I/A226R or oral gavage of LNP023 (AdooQ Biosciences A18905) and lung function was measured 24 hours prior to treatment and after LPS instillation.
After the adaptation period, each animal was weighed and randomly assigned to treatment groups 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. The general health and body weight of all animals were monitored during the course of the disease. Respiratory function was measured in awake mice by Whole Body Plethysmography (WBP) 0 and 24 hours after LPS IT. Immediately prior to LPS IT, mice received an Intravenous (IV) injection of 5.15mg/kg of the chymase-based engineered protease listed above. Negative control animals received IV injection of vehicle (PBS). The moving comparison animals received LNP023 at a dose of 30mg/kg administered orally. Mice were sacrificed 24 hours after LPS IT.
Twenty-four hours after administration of the chymase-based engineered protease or LNP023 described above, the plethysmography measurements showed significant protection of pulmonary congestion, indicating protection of pulmonary function by treatment. These results are shown in fig. 14. The effect on pulmonary congestion index was demonstrated by fold change of PenH values from baseline. The data shown are mean +/-SEM, and one-way ANOVA with Dunnett multiple comparison test was used with p values <0.01.
Table 19 shows a comparison of dosage levels of chymase-based engineered proteases of the examples with LNP023 dosage levels administered in vivo.
TABLE 19
Table 19 in combination with the observed protective effect of treatment on pulmonary congestion index in fig. 14 shows that the chymase-based engineered protease tested in this example is as effective as the activity comparator LNP023 in protecting respiratory function in ARDS mouse model when administered at molar concentrations as low as about 1/355.
The results indicate effective modulation of engineered proteases at low concentrations, whereas small molecule therapeutics require higher concentrations and frequent dosing.
Example 10: expression and purification of unlabeled chymase-based engineered proteases in mammalian expression systems
HEK293 cells were transiently transfected with chymotrypsin-based engineered protease expression vectors, harvested and clarified by depth filtration. All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. The culture harvest was diluted 1.5-fold with 25mM Tris HCl,pH7.5 (CCS). CCS is loaded onto a cation exchange column (Capto spimepres or the like) at 20 mL/min. The column was washed with 10CV of 90% buffer A (25mM Tris HCL,pH7.5) +10% buffer B (containing 25mM Tris HCL,1MNaCl pH7.5) at 20 mL/min. The recombinant engineered chymase was eluted from the column at 10mL/min with a 40CV linear gradient of 10% buffer B to 65% buffer B, and 5mL peak fractions containing the engineered chymase were collected, pooled and quantified by absorbance at 280 nm.
After adjusting the pooled fractions to 150mM NaCl with buffer A and adding CaCl 2 After 4mM, chymase-based engineered proteases were activated by incubation with enterokinase. Enterokinase (EKmax, invitrogen) was added to trigger activation and incubated overnight at 37 ℃. About 90% of the engineered chymase is activated by this method and further purified from the unactivated chymase and enterokinase by cation exchange chromatography using the same methods described above. The pooled fractions were formulated in PBS, 0.1% PS80 (pH 7.4), greater than 98% monomer, and less than 2% HMWS and LMWS (FIG. 15).
FIG. 15 is SDS-PAGE (simplified) depicting expression, purification and activation of chymase-based engineered proteases of the present disclosure.
Example 11: half-life extension (HE) and manufacturability strategies using HSA or IgG1 Fc domain (Fc)
Nineteen chymase-based engineered proteases based on cleavage activity (table 10) were tested for transient expression as zymogens in HEK293 cells. All references to engineered proteases in this example are described in chymotrypsin numbering. Chymotrypsin numbering keys for modified protease domains of engineered proteases are shown in tables 2, 4 and 6 for uPA, MTSP-1 and chymotrypsin, respectively. Eight of nineteen were expressed as assessed by SDS-PAGE analysis of clarified tissue culture supernatants (Table 20, FIG. 16). Human HSA or Fc was chosen as the C-terminal fusion partner for the selected variants for transient expression in CHO-S or HEK293 cells with the aim of increasing solubility, chemical and in vivo half-life. HSA and Fc-tagged fusion proteins were expressed using HEK293 transient expression system (fig. 17). HSA fusion rescued the expression of engineered protease mutant cluster 1 (MS No. 1), which was ranked top based on specific activity in the initial e.coli production screen.
Table 20: exemplary chymase-based engineered protease factor B cleavage Activity

Claims (87)

1. An engineered protease comprising a modified chymotrypsin 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 (KLK 5) protease domain, wherein the engineered protease is capable of cleaving factor B.
2. The engineered protease of claim 1, wherein cleavage of factor B by the engineered protease results in one or more functionally inactive fragments.
3. The engineered protease of claim 2, wherein the one or more functionally inactive fragments are capable of reducing complement activation.
4. The engineered protease of any one of claims 1 to 3, wherein cleavage of factor B results in the production of a reduced function factor B fragment.
5. The engineered protease of any one of claims 1-4, wherein the factor B is a non-human primate factor B.
6. The engineered protease of claim 5, wherein the non-human primate is a cynomolgus monkey.
7. The engineered protease of any one of claims 1 to 4, wherein the factor B is human factor B.
8. The engineered protease according to claim 7, wherein said factor B comprises an amino acid sequence as shown in seq id No. 1.
9. The engineered protease of any one of claims 1 to 8, wherein cleavage of factor B occurs at a site not targeted by factor D.
10. The engineered protease of claim 9, wherein cleavage at the site not targeted by factor D produces at least two fragments that are not Ba and Bb.
11. The engineered protease according to any one of claims 1 to 10, wherein cleavage of factor B results in reduced production of factor B cleavage products Ba and Bb compared to cleavage by factor D.
12. The engineered protease of any one of claims 1 to 8, wherein cleavage of factor B occurs at a site targeted by factor D.
13. The engineered protease of claim 12, wherein factor B cleavage site targeted by factor D comprises QQKR/KIV (SEQ ID NO: 9).
14. The engineered protease of claim 9, wherein the factor B cleavage site 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) and DHKL/KSG (SEQ ID NO: 21).
15. The engineered protease according to claim 9, wherein factor B cleavage site comprises a sequence selected from the group consisting of WEHR/KGT (SEQ ID NO: 10) and KNQKR/QKQ (SEQ ID NO: 11), and wherein the engineered protease comprises a modified MTSP-1 protease domain or a modified uPA protease domain.
16. The engineered protease according to any one of claims 1 to 15, wherein said engineered protease comprises a modified MTSP-1 protease domain.
17. The engineered protease according to any one of claims 1 to 15, wherein said engineered protease does not comprise a modified MTSP-1 protease domain.
18. The engineered protease according to claim 16, comprising one or more modifications relative to the MTSP-1 protease domain comprising the amino acid sequence set forth in SEQ ID No. 7.
19. The engineered protease of claim 18, wherein the modification is one or more of substitution, addition, and deletion of one or more amino acid residues.
20. The engineered protease according to claim 16, wherein said one or more modifications are located at one or more positions corresponding to one or more positions selected from the group consisting of 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 amino acid sequence set forth in SEQ ID No. 18.
21. The engineered protease of claim 16, wherein the one or more modifications are selected from those shown in table 5A.
22. The engineered protease according to claim 16, wherein said one or more modifications are selected from those exemplary mutational strings set forth in table 5B.
23. The engineered protease of any one of claims 1 to 15, wherein the engineered protease comprises a modified uPA protease domain.
24. The engineered protease of any one of claims 1 to 15, wherein the engineered protease does not comprise a modified uPA protease domain.
25. The engineered protease of claim 23, comprising one or more modifications relative to a uPA protease domain comprising the amino acid sequence set forth in SEQ ID No. 8.
26. The engineered protease of claim 25, wherein the modification is one or more of substitution, addition, and deletion of one or more amino acid residues.
27. The engineered protease of claim 23, wherein the one or more modifications are located 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 the uPA protease domain comprising the amino acid sequence set forth in SEQ ID No. 8.
28. The engineered protease of claim 23, wherein the one or more modifications are selected from those shown in table 3A.
29. The engineered protease according to claim 23, wherein said one or more modifications are selected from those exemplary mutational strings set forth in table 3B.
30. The engineered protease of any one of claims 1 to 19, wherein the engineered protease comprises a modified chymase protease domain.
31. The engineered protease of any one of claims 1 to 19, wherein the engineered protease does not comprise a modified chymase protease domain.
32. The engineered protease according to claim 30, wherein said engineered protease comprises a modified chymase protease domain and said 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).
33. The engineered protease according to claim 30, comprising one or more modifications relative to a chymase protease domain comprising the amino acid sequence as set forth in SEQ ID No. 6.
34. The engineered protease of claim 33, wherein the modification is one or more of substitution, addition, and deletion of one or more amino acid residues.
35. The engineered protease of claim 30, wherein the one or more modifications are located 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 amino acid sequence set forth in SEQ ID No. 6.
36. The engineered protease of claim 30, wherein the one or more modifications are selected from those shown in table 7A.
37. The engineered protease according to claim 30, wherein said one or more modifications are selected from those exemplary mutational strings set forth in table 7B.
38. The engineered protease of any one of claims 1 to 19, wherein the engineered protease comprises a modified KLK5 protease domain, optionally comprising one or more amino acid modifications of SEQ ID No. 23.
39. The engineered protease of any one of claims 1 to 19, wherein the engineered protease does not comprise a modified KLK5 protease domain.
40. The engineered protease of any one of claims 1 to 39, wherein the engineered protease has about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, 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 1,900M for factor B cleavage -1 s -1 K of (2) cat /K m 。
41. The engineered protease of any one of claims 1 to 40, wherein the engineered protease has about 10 for factor B cleavage 3 To about 10 9 M -1 s -1 K of (2) cat /K m 。
42. The engineered protease of any one of claims 1 to 41, wherein the engineered protease has an EC of less than about 20nM for factor B 50 。
43. The engineered protease of any one of claims 1 to 42, wherein the engineered protease has an EC of less than about 1nM for factor B 50 。
44. The engineered protease of any one of claims 1 to 41, wherein the engineered protease has an EC of about 20nM, about 25nM, or about 60nM for factor B 50 。
45. The engineered protease of any one of claims 1 to 41, wherein the engineered protease has an EC of about 1,000nm to about 4,500nm for cleavage factor B 50 。
46. The engineered protease of any one of claims 1 to 45, wherein the engineered protease has a plasma half-life in human plasma of greater than about 72 hours.
47. The engineered protease of any one of claims 1 to 46, wherein the engineered protease has a plasma half-life in human plasma of greater than about 120 hours.
48. The engineered protease of any one of claims 1 to 47, wherein the engineered protease has a plasma half-life of about 7 days in human plasma.
49. The engineered protease of claim 48, wherein catalytic activity is about 10% to about 50%, or about 90% to about 100%.
50. The engineered protease according to claim 16, wherein said engineered protease has an increased half-life compared to a protease comprising an unmodified MTSP-1 protease domain.
51. The engineered protease according to claim 16, wherein said engineered protease has increased bioavailability compared to a protease comprising an unmodified MTSP-1 protease domain.
52. The engineered protease of claim 23, wherein the engineered protease has an increased half-life compared to a protease comprising an unmodified uPA protease domain.
53. The engineered protease of claim 23, wherein the engineered protease has increased bioavailability compared to a protease comprising an unmodified uPA protease domain.
54. The engineered protease of claim 30, wherein the engineered protease has an increased half-life as compared to a protease comprising an unmodified chymase protease domain.
55. The engineered protease of claim 30, wherein the engineered protease has increased bioavailability compared to a protease comprising an unmodified chymase protease domain.
56. The engineered protease of any one of claims 1 to 55, wherein the engineered protease is non-immunogenic.
57. The engineered protease of any one of claims 1 to 56, wherein the engineered protease is in zymogen form.
58. The engineered protease of any one of claims 1 to 56, wherein the engineered protease is in an active form.
59. The engineered protease of any one of claims 1 to 58, wherein the engineered protease is fused to a component that extends the half-life of the engineered protease.
60. The engineered protease of claim 59, wherein the component is an Fc domain.
61. The engineered protease of claim 59, wherein the component is human serum albumin.
62. The engineered protease of any one of claims 1 to 15, comprising a modified chymotrypsin 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.
63. The engineered protease of claim 62, wherein the modified chymase protease domain of SEQ ID No. 6 comprises one of the mutant strings of table 7B.
64. The engineered protease according to any one of claims 1 to 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.
65. The engineered protease according to claim 64, wherein said modified MTSP-1 protease domain of SEQ ID NO. 7 comprises one of the mutation strings of Table 5B.
66. The engineered protease of any one of claims 1 to 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.
67. The engineered protease of claim 66, wherein said modified uPA protease domain of SEQ ID No. 22 comprises one of the mutant strings of table 3B.
68. The engineered protease of any one of claims 1 to 15, comprising a modified kallikrein related peptidase 5 (KLK 5) 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.
69. A method of inactivating factor B, the method comprising contacting the factor B with an engineered protease of any one of claims 1-68.
70. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an engineered protease of any one of claims 1-68.
71. The method of claim 70, wherein the disease or disorder is associated with dysregulated complement.
72. The method of any one of claims 70-71, wherein the disease or disorder is an inflammatory disease or disorder.
73. The method of any one of claims 70-72, wherein the treatment is an alternative therapy.
74. The method of any one of claims 70-73, wherein the treatment blocks complement activation.
75. The method of any one of claims 70-74, wherein the treatment modulates autoimmunity.
76. The method of any one of claims 70-75, wherein the disease or disorder is congenital complement deficiency.
77. A method according to any one of claims 70 to 76, wherein the treatment is for endothelial cell or kidney cell injury.
78. The method of any one of claims 70-77, wherein the disease or disorder is selected from lupus nephritis, C3 glomerulopathy (C3G), primary IgA nephropathy, renal transplant ischemia reperfusion (I/R) injury, anti-neutrophil 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 sleep hemoglobinuria (PNH).
79. The method of any one of claims 70-78, wherein the disease or disorder is a control protein deficiency.
80. The method of any one of claims 70-78, wherein the disease or disorder is a secondary complement disorder.
81. The method of any one of claims 70-78, wherein the disease or disorder is an immune-related disease or disorder.
82. The method of any one of claims 70-81, wherein the engineered protease is administered subcutaneously to the subject.
83. The method of claim 82, wherein the engineered protease is activated in situ at a site of regulatory abnormality of a complement component.
84. The method of any one of claims 70-83, wherein the engineered protease is provided in a liquid stable formulation.
85. A pharmaceutical composition comprising an engineered protease of any one of claims 1-68, and optionally a pharmaceutically acceptable carrier.
86. The pharmaceutical composition of claim 85, wherein the engineered protease is provided in a liquid stable formulation.
87. The pharmaceutical composition of any one of claims 85-86, wherein the composition is formulated for subcutaneous administration.
CN202280017817.2A 2021-01-08 2022-01-10 Factor B protease Pending CN117355324A (en)

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US63/135,496 2021-01-08
US202163221108P 2021-07-13 2021-07-13
US63/221,108 2021-07-13
PCT/US2022/011873 WO2022150729A1 (en) 2021-01-08 2022-01-10 Factor b proteases

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