Modified urokinase-type plasminogen activator polypeptides and methods of use - Patents.com

Abstract

Provided are u-PA polypeptides and fusion proteins containing u-PA polypeptides. The u-PA polypeptides have been modified to inhibit complement activation by altering their activity and / or specificity, thereby cleaving complement proteins, such as complement protein C3. Modified u-PA polypeptides and fusion proteins that inhibit complement activation can be used to treat diseases and conditions mediated by or in which complement activation plays a role. These disorders include ischemia-reperfusion injury, including myocardial infarction and stroke, sepsis, autoimmune diseases, diabetic retinopathy, age-related macular degeneration, transplanted organ rejection, inflammatory diseases, and diseases with an inflammatory component.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 786,302, filed December 28, 2018, entitled "MODIFIED UROKINASE-TYPE PLASMINOGEN ACTIVATOR POLYPEPTIDES AND METHODS OF USE," inventors Edwin L. Madison, Christopher Thanos, Mikhail Popkov, Vanessa Soros, and Kimberly Tipton, and applicant Catalyst Biosciences, Inc. Where permitted, the subject matter of this application is incorporated by reference in its entirety.

[0002] Incorporation by reference of electronically provided sequence listings An electronic version of the sequence listing is also submitted, the contents of which are incorporated by reference in their entirety. The electronic file was created on December 26, 2019, is 2,294 kilobytes in size, and is titled 4940seqPC1.txt.

[0003] FIELD OF THE INVENTION Provided are modified u-PA polypeptides that inhibit complement activation by cleaving complement proteins. This inhibition allows the modified u-PA polypeptides to be used in the treatment of diseases and conditions that are mediated by complement or in which complement activation plays a role. These diseases and conditions include, but are not limited to, ophthalmologic indications, including macular degeneration, e.g., age-related macular degeneration (AMD) and Stargardt's disease; delayed graft function (DGF); ischemia-reperfusion injury, including myocardial infarction and stroke; sepsis; autoimmune diseases; inflammatory diseases; and diseases with an inflammatory component, including Alzheimer's disease and other neurodegenerative disorders. [Background technology]

[0004] background The complement (C) system is part of the immune system and plays a role in eliminating invading pathogens and initiating inflammatory responses. The complement system in humans and other mammals encompasses more than 30 soluble and membrane-bound proteins that participate in a coordinated series of reactions that result in complement activation. The blood complement system has a wide array of functions associated with a wide range of host defense mechanisms, including antimicrobial and antiviral effects. Products derived from activation of C elements include the nonself-recognition molecules C3b, C4b, and C5b, and the anaphylatoxins C3a, C4a, and C5a, which affect various cellular immune responses. These anaphylatoxins also function as pro-inflammatory agents.

[0005] The complement system is composed of an array of enzymes, non-enzyme proteins, and receptors. Complement activation occurs through one of three primary modes known as the "classical," "alternative," and "lectin" pathways (see Figure 1). Complement is typically activated or triggered by one of these three pathways, which converge on C3 activation, as shown in Figure 1. In a fourth complement-activation mechanism, called the intrinsic pathway, serine proteases associated with the coagulation / fibrinolysis cascade directly activate the complement system through cleavage of C3 or C5, independent of the classical, alternative, and lectin pathways.

[0006] These pathways can be distinguished by the process that initiates complement activation. The classical pathway is initiated by antibody-antigen complexes or aggregated forms of immunoglobulin; the alternative pathway is initiated by recognition of structures on microbial or cell surfaces; and the antibody-independent lectin pathway is initiated by the binding of mannan-binding lectin (MBL, also known as mannose-binding protein) to carbohydrates, such as those displayed on the surface of bacteria or viruses. Activation of the cascade leads to the generation of complexes involved in proteolysis or cell lysis, as well as peptides involved in opsonization, anaphylaxis, and chemotaxis.

[0007] The complement cascade, a central component of animal immune responses, is an irreversible cascade. Numerous protein cofactors regulate the process. Inappropriate regulation, typically inappropriate activation, of the process may be an aspect of or occur in various disorders involving inappropriate inflammatory immune responses, such as those observed in acute and chronic inflammatory diseases and other conditions involving inappropriate immune responses. These diseases and disorders include autoimmune diseases, such as rheumatoid arthritis and lupus, cardiac disorders, and other inflammatory diseases, such as sepsis and ischemia-reperfusion injury. Summary of the Invention [Problem to be solved by the invention]

[0008] Due to the involvement of the complement pathway in various diseases and conditions, components of the complement pathway are targets for therapeutic intervention, particularly pathway inhibition. Examples of such therapies include synthetic and natural small molecule therapies, antibody inhibitors, and recombinant soluble forms of membrane complement regulators. Strategies for preparing such therapies are limited. The short half-life of small molecules in vivo and the need for constant infusion to maintain complement inhibition limit their role, particularly in chronic diseases. Therapeutic antibodies can induce an immune response in the subject, which can complicate treatment, particularly treatments designed to modulate the immune response. Thus, there is a need for therapies for the treatment of complement-mediated diseases and diseases in which complement activation plays a role. These diseases include acute inflammatory diseases and chronic inflammatory diseases. Therefore, it is an object of the present specification to provide therapies that target activation of the complement cascade and to provide methods of disease therapy and treatment. [Means for solving the problem]

[0009] Modified urokinase-type plasminogen activator (u-PA) polypeptides are provided that contain amino acid insertions, deletions, and / or substitutions within the protease domain (the protease domain may include a substitution of a free Cys with Ser to reduce / eliminate aggregation) that result in increased cleavage activity against the complement protein C3 compared to the wild-type u-PA protease domain. Modified u-PA polypeptides include the protease domain, e.g., the full-length activated protease, its zymogen form, fusion proteins containing the modified u-PA polypeptide, and fusion partners that confer pharmacological properties or activities. The modified u-PA polypeptides and fusion proteins containing the modified u-PA polypeptides inhibit complement activation when in their active forms. In particular, these polypeptides and fusion proteins inhibit or eliminate C3 activity by cleaving C3.

[0010] The modifications described herein, including amino acid deletions, substitutions, and insertions, are within the protease domain. The modified u-PA polypeptides include or are the protease domain. The modified u-PA polypeptides may further include post-translational modifications and other modifications beyond the primary amino acid sequence, such as conjugation or binding to other polypeptides and moieties, which alter properties such as serum half-life and resistance to endogenous proteases. Such modifications include, but are not limited to, binding to albumin, binding to a multimerization domain, and pegylation. Thus, modified u-PA polypeptides can be modified by pegylation, albumination, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. Modifications include replacing a free cysteine ​​in the zymogen, such as C122 according to the chymotrypsin numbering system, with serine or alanine to reduce aggregation, particularly when expressed in vitro. This substitution may or may not be present and is not necessarily included in the polypeptide to be pegylated or expressed in vivo.

[0011] The modified u-PA polypeptides inactivate the complement protein C3 by cleaving it. The modified u-PA polypeptides inhibit complement activation by cleaving C3. For example, they inhibit complement activation by cleaving C3 at a site within the activation site to inactivate or inhibit C3 activity. The modified u-PA polypeptides provided herein are selected and designed to cleave within QHARASHLG, specifically where P1-P1' is RA (QHAR↓ASHL; see SEQ ID NO: 47, residues 737-744; cleavage is between residues 740 and 741). As a result, the modified u-PA polypeptides can be used as therapeutics to treat disorders, diseases, and / or conditions in which complement activation plays a role, such that inhibition of C3 activity can treat the disorder, disease, and / or condition. The modified u-PA polypeptides can also have reduced activity with respect to natural substrates, such as plasminogen, compared to wild-type u-PA or to those that only have a substitution corresponding to C122S in the chymotrypsin numbering system.

[0012] The diseases and conditions targeted by the modified u-PA polypeptides include any C3-mediated disease and condition, or complement-mediated or complement-associated disease and condition. These include ophthalmic disorders such as age-related macular degeneration (AMD) and diabetic retinopathy, as well as organ rejection, such as delayed graft growth factor (DGF), and other diseases, disorders, and conditions that can be treated by inhibiting complement activation. AMD is treated by administration into the vitreous humor, for example, by intravitreal injection, or by intraretinal or subretinal injection, and DGF is treated by intravenous or other systemic administration. The modified u-PA polypeptides and fusion proteins can be further modified, for example, by pegylation, to enhance, improve, or impart desired pharmacological properties, including extended half-life and / or reduced immunogenicity. Examples of other diseases and conditions include rheumatoid arthritis (RA), eye diseases, membranoproliferative glomerulonephritis (MPGN), multiple sclerosis (MS), myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex (IC)-mediated acute inflammatory tissue injury, Alzheimer's disease (AD), ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), and complement 3 glomerulopathy (C3G).

[0013] Unmodified u-PA polypeptides include precursor forms, mature forms, catalytic domains, and catalytically active forms thereof, as well as fusion proteins, such as those described in Examples 14-16. Exemplary unmodified u-PA polypeptides are those having the sequences set forth in SEQ ID NOS: 1-6. Included among unmodified u-PA polypeptides are those in which the free cysteine ​​in the catalytic domain (corresponding to C122 according to the chymotrypsin numbering) has been replaced with another amino acid, e.g., S or A, particularly S, which does not alter catalytic activity but reduces aggregation of the polypeptide. It is understood that all modified u-PA polypeptides may contain a substitution (usually S) at the residue corresponding to C122 according to the chymotrypsin numbering.

[0014] Provided herein are modified urokinase-type plasminogen activator (u-PA) polypeptides that have increased activity / specificity for complement proteins, compared to an unmodified active form of the u-PA polypeptide, by containing one or more amino acid modifications selected from substitutions corresponding to R35Q, H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI, and H99Q, and conservative amino acid modifications thereof, wherein the amino acid modifications are selected from substitutions, insertions, and deletions; the corresponding residues can be determined by alignment with the mature form of u-PA; The modified u-PA polypeptides inhibit or reduce complement activation by cleaving complement proteins, compared to unmodified u-PA polypeptides that do not contain the amino acid modifications; residues are numbered according to the chymotrypsin numbering system; unmodified u-PA polypeptides include the sequences set forth in any of SEQ ID NOS: 1-6 (wild-type human full-length u-PA, wild-type protease (catalytic) domain u-PA, wild-type mature u-PA, full-length u-PA with a substitution corresponding to C122S, protease domain u-PA with a substitution corresponding to C122S, and mature u-PA with a substitution corresponding to C122S), and catalytically active fragments thereof containing the amino acid substitutions. Conservative modifications are selected from R35Y, W, F, or N; H37R, Q, E, W, or F; V41K; D60aS; T97aD, L, or V; L97bG or S; and H99N (according to the chymotrypsin numbering system).

[0015] In particular, these modified urokinase-type plasminogen activator (uPA) polypeptides include those containing one or more amino acid modifications selected from substitutions corresponding to R35Q, H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI, and H99Q.

[0016] The modified u-PA polypeptides have reduced activity and / or specificity for cleaving substrate sequences in plasminogen. The complement protein for which the polypeptides have increased specificity / activity is C3, and cleavage inactivates C3. An example of a cleavage site is within the active site of C3. Some modified u-PA polypeptides have increased activity for cleaving C3 that is at least three-fold greater than the unmodified u-PA polypeptide of SEQ ID NO: 5 (protease domain with a C122S substitution).

[0017] Modified u-PA polypeptides include those containing the substitution H37Y, e.g., the substitution H37Y / V38E. Modified u-PA polypeptides include those containing the substitutions R35Y / H37K or R35Q / H37K, e.g., those containing the substitutions R35Y / H37K / V38E or R35Q / H37K / V38E.

[0018] Also provided are modified u-PA polypeptides containing the substitutions L97bA and / or R35Q and / or H99Q and / or D60aP and / or T97aI, including those described above.

[0019] The modified u-PA polypeptides may further comprise an amino acid substitution equivalent to T39Y, T39W, T39F, e.g., T39Y, or a conservative substitution selected from T39M or T39L. Other modified u-PA polypeptides comprise or further comprise the amino acid substitutions R35Q / H37Y and / or V38E / V41R / Y149R.

[0020] Other modified u-PA polypeptides include those containing the modification V41R, e.g., the modification V38E / V41R, and further including substitutions at one or more of positions R35, H37, and V38. These include modified u-PA polypeptides in which the substitution at V38 is E, e.g., R35Y / H37S / V38E / V41R, H37Y / V38E, and modified u-PA polypeptides containing other combinations of residues that contribute to C3 cleavage and / or stability, e.g., in body fluids.

[0021] The modified u-PA polypeptides provided herein exhibit an ED for the inactivating cleavage of C3. 50 is 100 nM, 50 nM, 30 nM, or 25 nM or less in an in vitro assay. Examples of these are listed in Table 14. 50 or less than 100 nM, or 50 ED 100 nM or less than 50 nM, as shown in Table 14. 50 ED 30 nM or less, as shown in Table 14. 50 ED is less than 25 nM. 50 Examples of assays to assess ED 50 One method involves incubating the substrate complement protein human C3 with various concentrations of each modified protease at 37°C for 1 hour to determine the EDTA of the modified u-PA polypeptide. In particular, the modified u-PA polypeptide exhibited an EDTA of 50 nM or less. 50 Anything that cleaves C3.

[0022] An unmodified u-PA polypeptide may consist of the amino acid sequence set forth in any of SEQ ID NOS: 1-6 or may contain additional modifications, including additional insertions and deletions. Any of the substitutions, insertions, or deletions herein may be contained within an unmodified u-PA polypeptide, e.g., within the protease domain, particularly the protease domain of SEQ ID NOS: 5. A modified u-PA polypeptide may have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a polypeptide of any of SEQ ID NOS: 1-6, or a catalytically active portion thereof. A modified u-PA polypeptide may contain one or up to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or seventeen amino acid substitutions, insertions, or deletions compared to an unmodified u-PA polypeptide of any of SEQ ID NOS: 1-6, or a catalytically active portion thereof.

[0023] Thus, provided are modified u-PA polypeptides containing the modifications V41R, or H37Y, or L97bA, or R35Q, or H99Q, or D60aP, or T97aI, or combinations thereof. Any modified u-PA polypeptide may further contain an amino acid substitution corresponding to T39Y, T39W, or T39F, or a conservative substitution thereof selected from T39M or T39L. In particular, any modified u-PA polypeptide may further contain the amino acid substitution T39Y, for example, the combination T39Y / V41R, and up to 12 or 13 additional modifications, or may contain C122S. Any modified u-PA polypeptide may further contain the amino acid substitution V38E, or may further contain one or more of the amino acid modifications R35Q, Y60bQ, and / or Y149R. Any modified u-PA polypeptide may further contain the amino acid modification R37aE or R37aS. Thus, the modified u-PA polypeptides provided herein may contain the substitutions R35Q / H37Y / T39Y / V41R or R35Q / H37Y / T39Y / V41R / C122S. Any of the modified u-PA polypeptides may also contain the substitution equivalent to H99Q.

[0024] Modified u-PA polypeptides provided herein include those containing the amino acid modifications R35Q / H37Y / T39Y / V41R / L97bA / H99Q / C122S or R35Q / H37Y / T39Y / V41R / L97bA / H99Q, or T39Y / V41R / Y60bQ / L97bA / H99Q or T39Y / V41R / Y60bQ / L97bA / H99Q / C122S or T39Y / V41R / D60aP / L97bA / H99Q / C122S or T39Y / V41R / D60aP / L97bA / H99Q / C122S. Also provided herein are modified u-PA polypeptides that contain amino acid modifications corresponding to Y40Q / V41L / L97bA / C122S or Y40Q / V41R / L97bA / C122S or Y40Q / V41L / L97bA or Y40Q / V41R / L97bA or R37aS / V41R / L97bG / H99Q or R37aS / V41R / L97bG / H99Q / C122S or T39Y / V41L / L97bA / H99Q / C122S or T39Y / V41R / L97bA / H99Q / C122S.

[0025] Modified u-PA polypeptides include those modified: R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / Y149R It contains:

[0026] Provided are modified u-PA polypeptides containing amino acid modifications, the modifications being: H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aA / Y60bP / T97aI / L97bA / H99Q / C122S / Y149R or R35L / H37D / R37aS / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R or R35M / H37G / R37aD / V38E / T39W / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37G / R37aP / V38E / T39Y / V41R / D60aP / Y60bE / T97aI / L97bA / H99Q / C122S / Y149R or R35A / H37G / R37aE / V38E / T39F / V41R / D60aE / Y60bP / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37S / R37aE / V38E / T39Y / V41R / D60aP / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37T / R37aP / V38E / T39Y / V41R / D60aE / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37G / R37aE / V38E / T39H / V41R / D60aP / Y60bA / T97aI / L97bA / H99Q / C122S / Y149R or R35W / H37D / R37aS / V38E / T39Y / V41R / D60aE / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37G / R37aE / V38E / T39Y / V41R / D60aP / Y60bT / T97aI / L97bA / H99Q / C122S / Y149R or R35W / H37P / R37aN / V38E / T39Y / V41R / D60aP / Y60bL / D97T / T97aE / L97bG / A98S / H99L / C122S or R35W / H37P / R37aN / V38E / T39Y / V41K / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L / Q192A or R35Y / H37V / R37aW / V38E / T39Y / V41R / D60aP / Y60bE / T97aI / L97bA / H99Q / C122S / Y151L / Q192T or R35W / H37P / R37aN / V38E / T39Y / V41K / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L / Q192T or Included are polypeptides each having no substitution at C122. Examples of these modified u-PA polypeptides are those containing the modifications R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R.

[0027] Examples of these polypeptides include those set forth in any of SEQ ID NOS: 8-44 and 987, e.g., 21 and 39-44, as well as precursor and full-length modified u-PA polypeptides containing the catalytically active portions thereof. It is also understood that in any of the modified u-PA polypeptides provided herein, the Cys at residue 122 according to the chymotrypsin numbering system may be substituted with Ser or may remain as Cys. Cys is retained for embodiments intended to use polypeptides, including fusion proteins containing the modified u-PA protease domain in a two-chain form, in which the free Cys forms a disulfide bond with another free Cys in the polypeptide or in which the Cys is modified, e.g., by pegylation. In all embodiments described herein, position 122 may be Cys or Ser. Those skilled in the art will be able to select the appropriate residue depending on the intended use.

[0028] Unmodified u-PA polypeptides include the protease domain of any of SEQ ID NOs: 1-6, or a catalytically active portion thereof, and include or contain only the protease domain of SEQ ID NO: 2 or SEQ ID NO: 5.

[0029] The modified u-PA polypeptides may contain additional modifications, including post-translational modifications, modifications that introduce or remove glycosylation sites, and modifications such as attachment or conjugation to polymers, e.g., PEG, to extend serum half-life and / or reduce immunogenicity. In particular, any or all of the modified u-PA polypeptides described herein and provided herein may be PEGylated. Fusion proteins containing the modified u-PA polypeptides provided herein, e.g., fusions with an Fc domain, or targeting agents specific for target cells or target antigens, are also provided.

[0030] Some modified u-PA polypeptides and fusion proteins provided herein have a stability of greater than 50% or 80% after 7 days of incubation in PBS or in body fluids, such as aqueous or serum, and some modified u-PA polypeptides, when in an active form, have a 100-fold or greater reduction in activity against plasmin compared to the corresponding form of the unmodified u-PA polypeptide.

[0031] Also provided herein are modified u-PA polypeptides and fusion proteins that are capable of inactivating cleavage of C3. 50 is 100 nM or less, or 50 nM or less, or 30 nM or less, or 25 nM or less, or 15 nM or less, or 10 nM or less in an in vitro assay, such as any of those exemplified in the Examples herein. These describe a number of mutation strings and provide EDs for modified u-PA polypeptide protease domains. 50 ED assessed as described in Example 2 50Examples of polypeptides that contain or are the protease domains set forth in Table 14 include polypeptides with an ED of 100 nM or less, less than 50 nM, less than 30 nM, less than 25 nM, less than 15 nM, or less than 10 nM. 50 Some modified u-PA polypeptides and fusion polypeptides having the following may be used as the protease domain or in longer u-PA forms and / or in the fusion proteins described herein.

[0032] Provided are conjugated proteins, including fusion proteins containing a modified u-PA polypeptide fused to a non-protease polypeptide or portion thereof, or a catalytically active portion of any of the modified u-PA polypeptides, including non-protease polypeptides, such as those containing a multimerization domain, e.g., an Fc domain, a polypeptide that increases serum stability, e.g., albumin, or a protein transduction domain (PTD).

[0033] As discussed above, all modifications can be to the unmodified polypeptide, and catalytically active portions thereof, whose sequence is set forth in any of SEQ ID NOs: 1-6. Included among the polypeptides is one in which the unmodified polypeptide has the sequence set forth in SEQ ID NO: 5 (protease domain with a C122S substitution).

[0034] Also provided are fusion proteins containing the modified u-PA polypeptides provided herein and additional polypeptides, such as serum albumin, a multimerization domain, a signal sequence, and other transport sequences and tags to facilitate expression and isolation.The fusion protein can also include an activation sequence to activate the u-PA portion.The active form of the fusion protein is generated by expression and removal of the signal sequence and any other processing and transport signals to produce an active fusion protein that cleaves C3.The active form of the fusion protein can include two-chain activated forms and even dimers, such as those resulting from the inclusion of a multimerization domain.

[0035] Fusion proteins include those containing modified u-PA polypeptides, or catalytically active portions of modified u-PA polypeptides, such as those in Table 14, fused to non-protease polypeptides or portions thereof. Fusion proteins may also include activation sequences, as well as signal sequences and other transport signals prior to processing. Non-protease polypeptides include, but are not limited to, any known to those skilled in the art that confer a desired pharmaceutical activity or property, including multimerization domains, e.g., F c The fusion protein may also include an activation sequence, such as a natural u-PA activation sequence or a furin activation sequence. Examples of furin activation sequences include those that are or contain QSGQKTLRRRKR (SEQ ID NO: 996), QCGQKTLRRRKR (SEQ ID NO: 995), or QSGQKTLRRKR (SEQ ID NO: 1044), or a furin activation site having at least 98% sequence identity thereto.

[0036] For example, a fusion protein comprising any of the modified u-PA polypeptides described or provided herein also includes a signal sequence and the modified u-PA polypeptide or a catalytically active portion thereof prior to processing or activation. Examples of signal sequences that encode secretion of the fusion protein include signal sequences from Il-2, u-PA, or IgGκ.

[0037] The fusion protein may also include a fusion partner, such as a multimerization domain, or a polypeptide that extends serum half-life, or one that imparts other desired pharmacological properties or activities. Examples of these include albumin, an Fc domain, a single-chain antibody or other antigen-binding fragment of an antibody, or a hyaluronic acid-binding domain (HABD). Exemplary fusion partners include, but are not limited to, tumor necrosis factor-stimulated gene-6 (TSG-6); HSA, IgG Fc, an antibody or its antigen-binding fragment, such as an anti-type II collagen antibody scFv fragment, or an anti-VEGFR antibody or its fragment.

[0038] The fusion protein may also include an activation sequence so that the resulting fusion protein containing u-PA is in an active form, e.g., a two-chain form. The activation sequence may contain, or be modified to contain, a cysteine ​​that forms a disulfide bond with a free Cys, e.g., C122, in the modified u-PA polypeptide, thereby activating the resulting activated polypeptide so that it comprises two chains. Exemplary activation sequences are the u-PA activation sequence and the furin activation sequence, as well as modified forms thereof, e.g., an activation sequence having a sequence set forth in any of SEQ ID NOs: 995-998, 1041, and 1044, or a sequence having at least 98% or 99% sequence identity thereto.

[0039] Exemplary fusion proteins include any that contain an activation sequence, a modified u-PA polypeptide, and HSA, such as those containing the amino acid sequence set forth in any of SEQ ID NOs: 1014, 1015, 1016, 1019, and 1040, or modified forms thereof (containing modifications within the sequence of the u-PA portion) with at least 95%, 96%, 97%, 98%, or 99% sequence identity. For use in treatment methods, fusion proteins usually do not contain a signal sequence. For use in gene therapy, the nucleic acid may encode a signal sequence.

[0040] Provided are such fusion proteins, such as those containing the amino acid sequence set forth in any of SEQ ID NOS: 1004-1019 and 1034-1040, or any having at least 95%, 96%, 97%, 98%, or 99% sequence identity (containing modifications within the sequence of the u-PA portion). Exemplary fusion proteins include those having the amino acid sequence set forth in SEQ ID NOS: 1015 or 1019. In particular, the signal sequence is removed prior to use, upon in vivo expression, or when produced in vitro. These include two-chain activated forms containing an A chain and a B chain. For example, a fusion protein in which the B chain begins at residue IIGG of the modified u-PA polypeptide and terminates at the C-terminus of the fusion protein, such as those containing a modified u-PA polypeptide and HSA, or those containing the amino acid sequence set forth in any of SEQ ID NOS: 1005, 1011, 1014, 1015, and 1036, but lacking the signal sequence. An example of an activated form of a fusion protein is a fusion protein containing an A chain from residues 21-178 and a B chain from residue 179 to the C-terminus of the protein, with a disulfide bond between residues 168-299. It is also understood that these include fusion proteins with at least 95%, 96%, 97%, 98%, or 99% sequence identity (containing modifications within the sequence of the u-PA portion). For example, provided is a fusion protein containing an A chain and a B chain, where the A chain consists of residues 21-178 of SEQ ID NO: 1015 and the B chain consists of residues 179-1022; the A chain and the B chain are linked via a disulfide bridge between C168 and C299 of SEQ ID NO: 1015.

[0041] Other fusion proteins provided herein may be processed to produce F c The antibody contains a multimerization domain so as to form multimers, such as dimers, formed through the interaction of complementary multimerization domains, such as domains.

[0042] Also provided are combinations, which can be packaged as a kit, containing a first composition containing a modified u-PA polypeptide (in all embodiments, including a fusion protein, particularly an activated form, or multiple thereof) and a second composition containing one or more second agents for treating a complement-mediated disease or disorder. The one or more second agents may be, for example, anti-inflammatory agents or anticoagulants. Examples of such agents include anti-inflammatory agents selected from any one or more of nonsteroidal anti-inflammatory drugs (NSAIDs), antimetabolites, corticosteroids, analgesics, cytotoxic agents, pro-inflammatory cytokine inhibitors, anti-inflammatory cytokines, B cell targeting agents, T antigen targeting compounds, adhesion molecule blockers, chemokine receptor antagonists, kinase inhibitors, PPAR-γ (gamma) ligands, complement inhibitors, heparin, warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate, argatroban, lepirudin, bivalirudin, and ximelagatran.

[0043] Provided are nucleic acid molecules encoding any of the modified u-PA polypeptides and fusion proteins provided herein. Also provided are vectors containing such nucleic acid molecules and encoding the modified u-PA polypeptides. Vectors include prokaryotic and eukaryotic vectors, including mammalian and insect vectors, such as baculovirus vectors, yeast vectors, such as Pichia and Saccharomyces, and viral vectors, such as herpes simplex virus vectors, vaccinia virus vectors, AAV vectors, adenovirus vectors, or retrovirus vectors. The vectors may be expression vectors for gene therapy, for producing modified u-PA polypeptides, and / or vectors such as adenoviruses and AAV viruses, particularly those directed to a tissue of interest, such as the liver or eye.

[0044] Provided are methods for producing modified u-PA polypeptides by growing cells containing a vector or nucleic acid encoding a modified u-PA polypeptide or fusion protein under conditions in which the vector is expressed, and, as appropriate, isolating or recovering the expressed modified u-PA polypeptide.

[0045] Also provided are isolated cells and cell cultures containing the nucleic acid molecules or vectors. The cells may be non-human cells or human cell cultures, but do not include any zygotes or cells that will develop into humans. Cells include mammalian cells and bacterial cells, including, but not limited to, bacterial cells such as Escherichia coli (E. coli), CHO, Balb / 3T3, HeLa, MT2, mouse NS0, BHK, insect cells, yeast cells, and other cells routinely used for recombinant expression of polypeptides. Methods for producing modified u-PA polypeptides include growing cells under conditions in which the encoded modified u-PA polypeptide is expressed and, if appropriate, isolating or purifying the modified u-PA polypeptide. Typically, modified u-PA polypeptides and their conjugates, e.g., fusion proteins, are produced in cells that glycosylate proteins. The isolated modified u-PA polypeptides may be further modified, for example, by pegylation.

[0046] Also provided are pharmaceutical compositions containing the modified u-PA polypeptides and fusion proteins and / or nucleic acids and / or vectors. Provided are uses of the pharmaceutical compositions, nucleic acids, or modified u-PA polypeptides for treating diseases or disorders mediated by complement activation or in which complement activation plays a role in the etiology or underlying pathogenesis of the disease or disorder by inhibiting complement activation. In particular, provided are uses of nucleic acid molecules and / or vectors for gene therapy to treat diseases, disorders, and conditions mediated by or involving complement activation, where inhibition of complement activation results in treatment or amelioration of the disease or condition. Also provided are methods for treating diseases or conditions mediated by or involving complement activation by administering vectors or nucleic acid molecules. In particular, the diseases, disorders, and conditions are those for which inactivation of C3, which inhibits or reduces complement activation, results in treatment.

[0047] Complement-mediated diseases, disorders, or conditions, or diseases, disorders, and conditions in which complement activation plays a role in the etiology or underlying pathogenesis, include, but are not limited to, any inflammatory disorder, sepsis, rheumatoid arthritis (RA), eye or ophthalmic diseases, cardiovascular disorders, membranoproliferative glomerulonephritis (MPGN), multiple sclerosis (MS), myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex (IC)-mediated acute inflammatory tissue injury, Alzheimer's disease (AD), ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), complement 3 glomerulopathy (C3G), and transplanted organ rejection, particularly late-onset transplanted organ rejection. Specific diseases and disorders include eye or ophthalmic diseases, such as macular degeneration or diabetic retinopathy, or inflammation caused by transplanted organs. Diseases, disorders, and conditions include age-related macular degeneration (AMD) and delayed graft function (DGF).

[0048] A method for inhibiting complement activation is provided. The method is carried out by contacting a modified u-PA polypeptide with complement protein C3, thereby cleaving complement protein C3 so that complement activation is reduced or inhibited. The contacting may be carried out in vitro by administering the modified u-PA polypeptide to a subject in whom complement inactivation or reduction is desired, but is usually carried out in vivo. Administration can be systemic, such as parenteral administration, including intravenous administration, or local administration by contacting affected tissue, such as the eye. Administration to the eye can be via eye drops, by linking the modified u-PA polypeptide to a protein transduction domain, or by intravitreal, intraretinal, or subretinal injection, or other such methods. Administration for diseases and conditions, such as DGF, can be carried out via intravenous administration. Other methods include subcutaneous and transdermal administration.

[0049] Methods and uses include treating any disease, disorder, or condition in which inhibition of complement activation results in the alleviation of inflammatory symptoms associated with a complement-mediated disease or disorder selected from inflammatory disorders, neurodegenerative disorders, ophthalmologic disorders, and cardiovascular disorders, including, but not limited to, inflammatory diseases, conditions, and disorders, sepsis, rheumatoid arthritis (RA), ophthalmologic disorders, membranoproliferative glomerulonephritis (MPGN), multiple sclerosis (MS), myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex (IC)-mediated acute inflammatory tissue injury, atypical hemolytic uremic syndrome (aHUS), complement 3 glomerulopathy (C3G), Alzheimer's disease (AD), ophthalmologic disorders such as AMD and diabetic retinopathy, and ischemia-reperfusion injury. Ischemia-reperfusion injury can include or be caused by an event or procedure selected from myocardial infarction (MI), stroke, angioplasty, coronary artery bypass graft, cardiopulmonary bypass (CPB), and hemodialysis, or a treatment of a subject. Treatment with modified u-PA polypeptide is performed before the treatment of the subject. Treatment includes organ transplantation. The disease, disorder, or condition includes an ophthalmic condition, or is an eye disease, or rejection or inflammation caused by a transplanted organ, such as diabetic retinopathy or macular degeneration. In particular, a method for treating age-related macular degeneration (AMD) is provided, which is the same as the method for treating delayed graft function (DGF). Treatment can be performed intravenously, subcutaneously, or locally, for example, by injection of modified u-PA polypeptide into the eye. Included are intravitreal, intraretinal, or subretinal injections to promote transduction into the vitreous humor, or linking the modified u-PA polypeptide to a protein transduction domain. The modified u-PA polypeptides can be linked or conjugated to moieties that target the polypeptide to a particular organ or tissue, or that extend serum half-life or reduce immunogenicity, for example, pegylation and / or conjugation to the Fc domain or to an antibody or antigen-binding portion thereof.

[0050] Therefore, provided is a method for treating a subject suffering from a complement-mediated disorder or condition, or a disorder or condition in which complement activation plays a role, by administering the modified u-PA polypeptide provided herein. Also provided is such use of the modified u-PA polypeptide and fusion protein provided herein. The modified u-PA polypeptide and fusion protein can provide treatment or be used for such treatment, because they inhibit or reduce complement activation by cleaving complement protein C3. Inhibition of complement activation results in the alleviation of inflammatory symptoms associated with complement-mediated disorders, diseases, or conditions, including inflammatory responses, and results in the alleviation of inflammatory symptoms associated with complement-mediated disorders, diseases, or conditions, selected from inflammatory disorders, neurodegenerative disorders, and cardiovascular disorders. These include ophthalmological conditions, such as diabetic retinopathy and macular degeneration, as well as late-onset organ rejection, such as DGF.

[0051] Dosages for use and methods, as well as single-dose formulations, are provided herein. A skilled physician can empirically determine the single dosage, for example, in the range of 0.1 mg to 1 mg for local administration, and 0.1 mg to 10, 15, 20, 30 mg, or more for systemic administration, e.g., intravenous administration. The specific dosage depends on the specific disorder, disease, or condition, the subject being treated, the stage of the disease, disorder, or condition, the route of administration, the regimen, and other such parameters. The dosage can be repeated daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days, at least every other week, at least every 2 weeks, every 3 weeks, every 4 weeks, or at longer intervals. The specific regimen and dosage depend, for example, on the disorder being treated, the mode of administration, and the subject's specifics, such as body weight. Such determination is within the skill of a skilled physician.

[0052] Also provided are methods, uses, and combinations, as well as modified u-PA polypeptides and fusion proteins, including those containing the modifications V41R or V41L, particularly V41R, e.g., V41I, or R and V38E, and H37Y / V38E. Examples of such modified u-PA polypeptides are those containing the modifications Y40Q / V41R / L97bA, or Y40Q / V41L / L97BA, or R37aS / V41R / L97bG / H99Q, or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / Y149R. The modifications are within any unmodified u-PA polypeptide, including those set forth in any of SEQ ID NOS: 1-6, and catalytically active portions thereof, which contain the residue corresponding to V41. Examples of such modified u-PA polypeptides are modified u-PA polypeptides comprising the sequence of amino acid residues set forth in SEQ ID NOS: 21 or 987, or in any of SEQ ID NOS: 40-44, where the modification is not at C122 according to the chymotrypsin numbering, and catalytically active portions thereof, and modified forms thereof, such as pegylated forms, and fusion proteins and modified forms thereof.

[0053] Also provided are methods for treating disorders, such as DGF, by intravenously administering modified u-PA polypeptides or fusion proteins (in activated form) described herein, including modified u-PA polypeptides comprising the amino acid residue sequence set forth in any of SEQ ID NOS: 21 and 40-44, and modified forms thereof, such as pegylated forms. A single dosage can be determined empirically by a skilled physician, and includes single dosages ranging from 0.1 mg to 1 mg. Dosages depend on the subject and the severity or stage of the disease or disorder, such as DGF. Treatments may be repeated multiple times, for example, two, three, or four times daily, and may be repeated once daily, daily, every two days, every three days, every four days, every five days, every six days, weekly, twice monthly, or monthly. The modified u-PA polypeptides may contain substitutions / insertions (R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R according to chymotrypsin numbering; and R20Q / H22Y / R23E / V27E / T28Y / V30R / D50P / Y51Q / T91I / L92A / H94Q / C121S / Y148R according to mature numbering). Examples are modified u-PA polypeptides containing the protease domain set forth in SEQ ID NO: 21, or catalytically active portions thereof, or full-length or precursor forms containing the protease domain, and modified forms thereof, such as pegylated forms and fusion proteins. Administration can be by any suitable method, including intravenous, subcutaneous, transdermal, topical, intramuscular, oral, and other systemic routes of administration. Typically, the administered form of the modified u-PA polypeptides provided herein will be the activated form, which, depending on the protein component (see, e.g., Example 15), will usually be the two-chain form.

[0054] The methods described herein, as described above and below, include the method of treating ophthalmic disorders or eye disorders by administering the modified u-PA polypeptide provided herein and any of its modified forms, such as pegylated forms and fusion proteins, such as those containing protein transduction domains, to the eye.Ophthalmic disorders, diseases, or conditions associated with complement activation include diabetic retinopathy and macular degeneration, such as AMD.Dosage is as described above, and includes a single dose of 0.1 mg to 1 mg. Modified u-PA polypeptides include those with substitutions R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / Y149R, Y40 These include those containing Q / V41L / L97bA / C122S or Y40Q / V41R / L97bA / C122S or Y40Q / V41L / L97bA or Y40Q / V41R / L97bA, as well as those containing the amino acid residue sequences set forth in any of SEQ ID NOS: 21 and 40-44, and catalytically active portions thereof, and modified forms thereof. Treatment can be repeated multiple times, for example, once daily. Use of modified u-PA polypeptides and modified forms thereof for treating AMD or DGF is provided. Modified u-PA polypeptides can include any of those described herein, including those with substitutions R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R 7aI / L97bA / H99Q / Y149R or Y40Q / V41L / L97bA / C122S or Y40Q / V41R / L97bA / C122S or Y40Q / V41L / L97bA or Y40Q / V41R / L97bA, and modified forms thereof that are pegylated or are fusion proteins as described herein. [Brief explanation of the drawings]

[0055] [Figure 1] Figure 1 provides an overview of the classical, lectin, and alternative complement pathways and the activation of the terminal complement complex, the membrane attack complex (MAC). The diagram depicts many of the more than 30 proteins involved in the complement cascade, their actions within the cascade, and, where applicable, their convergence points between complement pathways. For example, the three pathways converge upon the generation of C3 convertase, which cleaves C3 to form C5 convertase, leading to the formation of the MAC complex. The diagram also depicts the generation of many of the complement cleavage products. [Figure 2] 2A-2B are schematic diagrams of N-terminal u-PA fusion proteins. FIG. 2A is a schematic diagram of an N-terminal u-PA fusion protein containing a fusion partner (i.e., Fc) N-terminal to the u-PA catalytic domain. An exemplary N-terminal fusion protein is shown in SEQ ID NO: 1004, which contains a human immunoglobulin light chain kappa (κ) signal sequence, Fc (fusion partner), AGS (linker), a u-PA activation sequence, and a modified u-PA catalytic domain. FIG. 2B is a schematic diagram of an N-terminal wild-type protein that does not contain a fusion partner. An exemplary N-terminal wild-type protein is shown in SEQ ID NO: 1005, which contains a human immunoglobulin light chain kappa (κ) signal sequence, the N-terminus of u-PA, a u-PA activation sequence, and a modified u-PA catalytic domain. [Figure 3]3A-3C are schematic diagrams of C-terminal u-PA fusion proteins. FIG. 3A is a schematic diagram of a C-terminal u-PA fusion protein in which the fusion protein contains a fusion partner C-terminal to the u-PA catalytic domain lacking the activation sequence N-terminal to the u-PA catalytic domain. An exemplary C-terminal fusion protein is shown in SEQ ID NO: 1006, which contains the human IL2 signal sequence (hIL2SP), a modified u-PA catalytic domain, a linker, and Fc (the fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1007, which contains the human IL2 signal sequence (hIL2SP), a modified u-PA catalytic domain, a linker, and HSA (human serum albumin as the fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1008, which contains the human IL2 signal sequence (hIL2SP), a modified u-PA catalytic domain, a linker, and an scFV (C2scFv) binding collagen II (the fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1009, which contains the human IL2 signal sequence (hIL2SP), a modified u-PA catalytic domain, a linker, and HABD (hyaluronic acid binding domain) (fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1012, which contains the human IL2 signal sequence (hIL2SP), a wild-type u-PA catalytic domain, a linker, and Fc (fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1013, which contains the human IL2 signal sequence (hIL2SP), a wild-type u-PA catalytic domain, a linker, and HSA (fusion partner). Figure 3B is a schematic diagram of a C-terminal u-PA fusion protein containing a fusion partner (i.e., Fc or HSA) C-terminal to the u-PA catalytic domain. An exemplary C-terminal fusion protein is shown in SEQ ID NO: 1010, which contains a human immunoglobulin light chain kappa (κ) signal sequence, a furin activation site, a modified u-PA catalytic domain, a linker, and Fc (the fusion partner).Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1016, which contains a human immunoglobulin light chain kappa (κ) signal sequence, a furin activation sequence, a modified u-PA catalytic domain, a linker, and HSA (the fusion partner). Figure 3C is a schematic diagram of a u-PA fusion protein containing a fusion partner (i.e., Fc or HSA) C-terminal to the u-PA catalytic domain and a fusion partner (i.e., the wild-type N-terminus of u-PA) N-terminal to the u-PA catalytic domain. An exemplary fusion protein is shown in SEQ ID NO: 1011, which contains a human immunoglobulin light chain kappa (κ) signal sequence, a u-PA N-terminal domain, a modified u-PA catalytic domain, a linker, and Fc (the fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1014, which contains a human immunoglobulin light chain kappa (κ) signal sequence, the N-terminal region of u-PA, a furin activation site, a modified u-PA catalytic domain, a linker, and HSA (the fusion partner). Another exemplary C-terminal fusion protein is shown in SEQ ID NO: 1015, which contains the human immunoglobulin light chain kappa (κ) signal sequence, the N-terminal region of u-PA, the u-PA activation sequence, a modified u-PA catalytic domain, a linker, and HSA (the fusion partner). [Figure 4]Figures 4A-4H are schematic diagrams of activated forms of fusion proteins, where SPD refers to the serine protease domain (the modified u-PA polypeptide protease domain provided herein) and the u-PA N-terminus generally refers to residues 1-178 of u-PA or any modified form thereof. Figure 4A is a schematic diagram of the fusion protein of SEQ ID NO: 1010, which contains an Fc domain at the C-terminus of the u-PA protease domain (SEQ ID NO: 21) and a furin activation sequence, with disulfide bonds between the Fc domains forming dimers. Figure 4B is a schematic diagram of the fusion protein of SEQ ID NO: 1011, which contains an Fc domain at the C-terminus of the u-PA protease domain (SEQ ID NO: 987), and the N-terminus of u-PA and the u-PA activation sequence at the N-terminus of the protein, with disulfide bonds between the Fc domains forming dimers. Figure 4C is a schematic diagram of the fusion protein shown in SEQ ID NO: 1036, which contains an Fc domain at the C-terminus of the u-PA protease domain (SEQ ID NO: 987) and a furin activation sequence at the N-terminus of u-PA and the N-terminus of the fusion protein, and disulfide bonds between the Fc domains form a dimer. Figure 4D is a schematic diagram of the fusion protein shown in SEQ ID NO: 1014, which contains HSA at the C-terminus of the u-PA protease domain (SEQ ID NO: 987) and a furin activation sequence at the N-terminus of u-PA and the N-terminus of the fusion protein. Figure 4E is a schematic diagram of the fusion protein shown in SEQ ID NO: 1015, which contains HSA at the C-terminus of the u-PA protease domain (SEQ ID NO: 987) and a u-PA activation sequence at the N-terminus of u-PA and the N-terminus of the fusion protein. Figure 4F is a schematic diagram of the fusion protein shown in SEQ ID NO: 1016, which contains HSA at the C-terminus of the u-PA protease domain (SEQ ID NO: 21) and a furin activation sequence at the N-terminus of the protease domain. Figure 4F is a schematic diagram of the fusion protein shown in SEQ ID NO: 1017, which contains HSA at the C-terminus of the u-PA protease domain (SEQ ID NO: 21) and a SUMO activation sequence at the N-terminus of the protease domain.Figure 4H is a schematic diagram of the fusion protein shown in SEQ ID NO: 1018, which contains an Fc domain C-terminal to the u-PA protease domain (SEQ ID NO: 21) and a SUMO-activating sequence N-terminal to u-PA and the protease domain, with disulfide bonds between the Fc domains forming a dimer. DETAILED DESCRIPTION OF THE INVENTION

[0056] Overview A.Definition Structure and function of Bu-PA 1. Serine proteases 2. Structure 3. Function / activity C. Complement inhibition by targeting C3 1. Complement protein C3 and its role in complement initiation a. classical pathway b. Alternative pathway C lectin pathway D. Complement-mediated effector functions i. Complement-mediated lysis: membrane attack complex ii. Inflammation iii.Chemotaxis iv. Opsonization v. Activation of humoral immune responses 2. Structure and Function of C3 a.C3a b.C3b C.C3b inhibitors D. Modified u-PA polypeptides that cleave C3 1. Exemplary Modified u-PA Polypeptides 2. Further modifications Reduced immunogenicity b. Fc domain c. Conjugation to polymers d. Protein transduction domain E. Assays for assessing or monitoring u-PA activity in relation to complement-mediated functions 1. Methods for assessing u-PA activity in relation to the function of complement protein C3 Protein detection i.SDS-PAGE analysis ii. Enzyme immunoassay iii. Radial immunodiffusion (RID) b. Hemolytic assay c. Methods for determining cleavage sites 2. Methods for assessing wild-type u-PA activity A cleavage of plasminogen b. Plasminogen activation assay cu-PA-uPAR binding assay d.C3 cutting ACC-AGR+ELISA Evaluating specificity using peptide libraries 3. Specificity 4.Disease Models F. Methods for Producing Nucleic Acids Encoding the Modified U-PA Polypeptides 1. Isolation or preparation of nucleic acids encoding u-PA polypeptides 2. Generation of Mutant or Modified Nucleic Acids and Encoded Polypeptides 3. Vectors and Cells 4. Expression A prokaryotic cell b. Yeast cells C. Insects and insect cells d. Mammalian Expression e.Plants 5. Purification 6. Additional Modifications a.PEGylation b. Fusion Proteins and Other Conjugates 7. Nucleic acid molecules G. Compositions, Formulations and Administration 1. Administration of modified u-PA polypeptides 2. Administration of nucleic acids encoding modified u-PA polypeptides (gene therapy) H. Therapeutic Uses and Methods of Treatment 1. Diseases mediated by complement activation Rheumatoid arthritis b. Sepsis C. multiple sclerosis d. Alzheimer's disease e. Ischemic reperfusion injury f. Eye disorders Age-related macular degeneration (AMD) g. Organ transplantation and delayed graft function (DGF) 2. Therapeutic Use a. Immune-mediated inflammatory diseases b. Neurodegenerative disease c.Cardiovascular disease d. Age-related macular degeneration (AMD) e. Organ transplant Delayed Graft Function (DGF) 3. Combination therapy I. Working Example

[0057] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, patent publications, and publications, GENBANK sequences, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise specified. In the event of multiple definitions for terms herein, those in that section prevail. When a URL or other such identifier or address is referenced, it is understood that such identifiers may change and specific information on the Internet may not be fixed, but that equivalent information is known and readily accessible, for example, by searching the Internet and / or appropriate databases. The reference demonstrates the availability and general public knowledge of such information.

[0058] Cleavage, as used herein, refers to the cleavage of a peptide bond by a protease. The protease cleavage site motif encompasses residues N-terminal and C-terminal to the scissile bond (the unprimed and primed sides each have a protease cleavage site, defined as ...P3-P2-P1-P1'-P2'-P3'..., with cleavage occurring between the P1 and P1' residues). In human C3, cleavage by C3 convertase occurs between residues R and S of C3 [see residues 746-751 of SEQ ID NO: 47 (cleavage in human C3 between residues 748 and 749)]: P3 P2 P1 P1' P2' P3' Leu Ala Arg ↓ Ser Asn Leu

[0059] Typically, the cleavage of a substrate in a biochemical pathway is either activation cleavage or inhibitory cleavage. Activation cleavage refers to the cleavage of a polypeptide from an inactive form to an active form. This includes, for example, the cleavage of a zymogen into an active enzyme. Activation cleavage is also the cleavage of a protein into one or more proteins that themselves have activity. For example, the complement system is an irreversible cascade of proteolytic cleavage events. Its completion results in the formation of multiple effector molecules that stimulate inflammation, promote antigen phagocytosis, and directly lyse some cells. Thus, the cleavage of C3 into C3a and C3b by C3 convertase is activation cleavage. In contrast, the modified u-PA polypeptides provided herein perform inhibitory cleavage of C3, for example, by cleavage at the active site.

[0060] As used herein, inhibitory cleavage or inactivating cleavage refers to the cleavage of a protein into one or more non-functional degradation products. Inhibitory cleavage results in a decrease or reduction in the activity of a protein. Typically, the reduction in protein activity reduces the pathway or process in which the protein is involved. In one example, inhibitory cleavage of any one or more complement proteins results in a simultaneous reduction or inhibition of any one or more of the classical pathway, the lectin pathway, or the functional alternative pathway of complement. To be inhibitory, cleavage reduces activity by at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more compared to the native form of the protein. The percentage of protein cleavage required for cleavage to be inhibitory varies between proteins but can be determined by assaying for the activity of the protein.

[0061] As used herein, "complement activation" refers to the activation of the complement pathway. For example, complement activation refers to an increase in any one or more functions or activities of the complement pathway by proteases or an increase in the activity of any protein in the complement pathway. Complement activation can result in complement-mediated cell lysis or cell or tissue destruction. Inappropriate complement activation against host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases, and is also responsible for or associated with many pathologies related to bioincompatibility. It is understood that activation can refer to the increase in existing activity and the induction of new activity. Complement activation can occur in vitro or in vivo. Exemplary functions of complement that can be assayed and described herein include hemolytic assays and assays measuring any one or more complement effector molecules, such as by SDS PAGE followed by Western blot or Coomassie brilliant blue staining, or by ELISA. In some embodiments, complement activation is inhibited by a protease, such as a protease described herein, by 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or more compared to the activity of complement in the absence of the protease.

[0062] As used herein, "inhibition of complement activation" or "complement inactivation" refers to the reduction or decrease of any one or more complement-mediated functions or activities of the complement pathway, or the activity of any of the proteins in the pathway, by a protease. Complement functions or activities can occur in vitro or in vivo. Exemplary functions of complement that can be assayed and described herein include hemolytic assays and assays that measure any one or more of complement effector molecules, such as by SDS PAGE followed by Western blot or Coomassie brilliant blue staining, or by ELISA. Proteases can inhibit complement activation by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In other embodiments, complement activation is inhibited by the protease by 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or more compared to the activity of complement in the absence of the protease.

[0063] As used herein, a "complement protein" or "complement component" is a protein of the complement system that functions in both the host's defense against infection and in the inflammatory process. Complement proteins include those that function in the classical pathway, the alternative pathway, and the lectin pathway. Complement proteins include proteases that participate in the complement pathways.

[0064] As used herein, complement proteins include any "cleavage products" (also referred to as "fragments") formed upon activation of the complement cascade. Also included are inactive or altered forms of complement proteins, such as iC3b and C3a-desArg. Thus, complement proteins include, but are not limited to: C1q, C1r, C1s, C2, C3, C3a, C3b, C3c, C3dg, C3g, C3d, C3f, iC3, C3a-desArg, C4, C4a, C4b, iC4, C4a-desArg, C5, C5a, C5a-des-Arg, C6, C7, C8, C9, MASP-1, MASP-2, MBL, Factor B, Factor D, Factor H, Factor I, CR1, CR2, CR3, CR4, properdin, C1Inh, C4bp, MCP, DAF, CD59 (MIRL), clusterin, and HRF, as well as allelic and species variants of any complement protein.

[0065] As used herein, a "native" form of a complement protein is one that can be isolated from an organism, such as a vertebrate, in the absence of complement activation and has not been deliberately modified by man in the laboratory. Examples of native complement proteins include C1q, C1r, C1s, C2, C3, C4, factor B, factor D, properdin, C5, C6, C7, C6, and C9.

[0066] Typically, "native complement proteins" are inactive and become active upon activation. Activation may require activation cleavage, maturation cleavage, and / or complex formation with other proteins. Exceptions to this are factor I and factor D, which are enzymatically active in their native forms. In some cases, activation of native complement proteins occurs after protein cleavage. For example, complement zymogens, such as C3, are themselves proteases that are activated by protease cleavage, such that cleavage of C3 by the C3 convertase C4b2b generates the active fragments C3a and C3b. In another example, cleavage of an inactive native complement protein results in a change in the structural stability of the protein, leading to protein activation. For example, C3 contains an internal thioester bond that is stable within the native protein, but can become highly reactive and activated after structural changes resulting from protein cleavage. Therefore, the cleavage products of C3 are biologically active. C3 activation can also occur spontaneously in the absence of cleavage. This is the spontaneous conversion of a thioester bond within native C3, the initiating event of the alternative complement pathway. In other instances, activation of native complement proteins occurs after the release of complexed regulatory molecules that inhibit the activity of otherwise active native complement proteins. For example, C1inh binds to and inactivates C1s and C1r unless complexed with C1q.

[0067] As used herein, "maturation cleavage" is a general term that refers to any cleavage required for zymogen activation. Such cleavages include those that result in a conformational change that results in activity (i.e., an activation cleavage). They also include cleavages that expose critical binding sites, expose steric hindrance, or remove or move inhibitory segments.

[0068] As used herein, "altered forms" of complement proteins refer to complement proteins that exist in non-native forms resulting from modifications of their molecular structure. For example, C3 reaction of the thioester with water can occur in the absence of convertase cleavage to give a hydrolyzed, inactive form of C3 called iC3. In another example, anaphylatoxins, including C3a, C5a, and C4a, can be desarginated by carboxypeptidase N to more stable, less active forms.

[0069] As used herein, a "fragment" or "cleavage product" of a complement protein refers to a region or segment of a complement protein that contains a portion of the polypeptide sequence of the native complement protein. Complement protein fragments typically arise after activation of the complement cascade. Fragments typically result from proteolytic cleavage of a native complement protein. For example, complement protein C3 is enzymatically cleaved by C3 convertase to produce two fragments: C3a, which constitutes the N-terminal portion of C3; and C3b, which constitutes the C-terminal portion and contains a serine protease site. Complement protein fragments also result from proteolytic cleavage of another fragment of a complement protein. For example, C3b, a fragment generated from cleavage of C3, is cleaved by factor I to produce fragments iC3b and C3f. Complement protein cleavage products typically are biologically active products that function as cleaved effector molecules of the complement system. Therefore, complement protein fragments or portions include complement protein cleavage products, as well as portions of proteins that retain or exhibit at least one activity of a complement protein.

[0070] As used herein, "cleaved effector molecule" or "cleaved effector protein" refers to an active cleavage product generated as a result of the triggered enzymatic cascade of the complement system. The cleaved effector molecule, fragment, or cleavage product derived from complement activation can contribute to one or more complement-mediated functions or activities, including opsonization, anaphylaxis, cell lysis, and inflammation. Examples of cleaved or effector molecules include, but are not limited to, C3a, C3b, C4a, C4b, C5a, C5b-9, and Bb. Through their participation in the cascade, cleaved effector molecules of the complement system exhibit activities that include stimulating inflammation, promoting antigen phagocytosis, and directly lysing some cells. Complement cleavage products promote or participate in the activation of the complement pathway.

[0071] As used herein, "anaphylatoxin" refers to a cleaved effector protein that triggers degranulation or release of substances from mast cells or basophils, which are involved in inflammatory responses, particularly as part of defense against parasites. Excessive degranulation can lead to allergic reactions. Examples of anaphylatoxins include C3a, C4a, and C5a. Anaphylatoxins also indirectly mediate smooth muscle cell attacks (e.g., bronchospasm), increased capillary permeability, and chemotaxis.

[0072] As used herein, "chemotaxis" refers to the receptor-mediated migration of leukocytes toward a chemoattractant, typically in the direction of increasing its concentration, e.g., in the direction of increasing concentrations of an anaphylatoxin.

[0073] As used herein, "opsonization" refers to the modification of the surface of a pathogen or other particle so that it can be ingested by phagocytes. Proteins that bind to or modify the surface of a pathogen are called opsonins. Antibodies and complement proteins opsonize extracellular bacteria for uptake and destruction by phagocytes, such as neutrophils and macrophages.

[0074] As used herein, "cytolysis" refers to the breaking open of a cell by disruption of its wall or membrane. Hemolysis of red blood cells is a measure of cell lysis.

[0075] As used herein, "complement protein C3" or "C3" refers to complement protein C3 of the complement system, which functions in both host defense against infection and inflammatory processes. Human complement protein C3 is a 1663 amino acid single-chain preproprotein or zymogen set forth in SEQ ID NO:47, which contains a 22-amino acid signal peptide (amino acids 1-22 of SEQ ID NO:47) and a tetra-arginine sequence (amino acids 678-671 of SEQ ID NO:47) that is removed by a furin-like enzyme to yield a mature two-chain protein containing a beta chain (amino acids 23-667 of SEQ ID NO:47) and an alpha chain (amino acids 672-1663 of SEQ ID NO:47) linked by a disulfide bond between residues C559 and C816. Complement protein C3 is further activated by proteolytic cleavage by C3 convertase (C4b2b or C3bBb) between amino acids 748 and 749 of SEQ ID NO:47, generating the anaphylatoxin C3a and opsonin C3b.

[0076] As used herein, "zymogen" refers to a protein that is activated by proteolytic cleavage (including maturation cleavage, e.g., activation cleavage) and / or complex formation with other proteins and / or cofactors. A zymogen is an inactive precursor of a protein. Such precursors are usually, but not necessarily, larger than the active form. With respect to u-PA or complement protein C3, zymogens are converted to active enzymes by specific cleavage, including catalytic and autocatalytic cleavage, or by binding of an activating cofactor to generate the active enzyme. Thus, a zymogen is an enzymatically inactive protein that is converted to a proteolytic enzyme by the action of an activator. Cleavage can be performed autocatalytically. Some complement proteins are zymogens; they are inactive but are cleaved and activated upon initiation of the complement system after infection. Zymogens are usually inactive and can be converted to mature active polypeptides by catalytic or autocatalytic cleavage of the proregion from the zymogen.

[0077] As used herein, "proregion," "propeptide," or "prosequence" refers to a region or segment of a protein that is cleaved to produce the mature protein. This can include segments that function to inhibit enzymatic activity by masking the catalytic machinery and preventing the formation of catalytic intermediates (i.e., by sterically blocking the substrate binding site). A proregion is a sequence of amino acids located at the amino terminus of a biologically active mature polypeptide and can be as few as a few amino acids or can be a multi-domain structure.

[0078] As used herein, "activation sequence" refers to a sequence of amino acids within a zymogen that is the site required for activation or maturation cleavage to form an active protease. Activation sequence cleavage can be catalyzed autocatalytically or by activating a partner. Activation cleavage is a type of maturation cleavage in which a conformational change required for activity occurs. This is the classical activation pathway for serine proteases, where the cleavage generates a new N-terminus that interacts with conserved regions of the catalytic machinery, such as catalytic residues, to induce the conformational change required for activity. Activation can result in the generation of multi-chain forms of the protease. In some cases, a single-chain form of the protease can exhibit proteolytic activity.

[0079] As used herein, "domain" refers to a portion of a molecule, e.g., a protein or encoding nucleic acid, that is structurally and / or functionally distinct and identifiable from other portions of the molecule. An exemplary polypeptide domain is a portion of a polypeptide that can form an independently folding structure within the polypeptide, composed of one or more structural motifs (e.g., a combination of alpha helices and / or beta strands connected by loop regions), and / or that is recognized by a specific functional activity, e.g., enzymatic activity, dimerization, or substrate binding. A polypeptide can have one or more, typically multiple, distinct domains. For example, a polypeptide can have one or more structural domains and one or more functional domains. A single polypeptide domain can be distinguished based on structure and function. A domain can encompass a contiguous linear sequence of amino acids. Alternatively, a domain can encompass multiple non-contiguous amino acid portions that are non-contiguous along the linear sequence of amino acids of the polypeptide. Typically, a polypeptide contains multiple domains. For example, serine proteases can be characterized based on the sequence of their protease domains. Those skilled in the art are familiar with polypeptide domains and can identify them by their structural and / or functional homology with other such domains.For illustrative purposes herein, definitions are provided, but it is understood that it is well within the skill of the art to recognize specific domains by name.If necessary, suitable software can be used to identify domains.

[0080] As used herein, a "structural region" of a polypeptide is a region of the polypeptide that contains at least one structural domain.

[0081] As used herein, a "protease domain" is the catalytically active portion of a protease. Reference to the protease domain of a protease includes any single-chain, two-chain, and multi-chain forms of the protein. The protease domain of a protein contains all of the essential properties of the protein required for its proteolytic activity, e.g., its catalytic center.

[0082] As used herein, a "catalytically active portion" or "catalytically active domain" of a protease, e.g., a u-PA polypeptide, refers to the protease domain, or any fragment or portion thereof that retains protease activity. For example, a catalytically active portion of a u-PA polypeptide can be the u-PA protease domain, including an isolated single-chain or activated two-chain form of the protease domain. Significantly, at least in vitro, the protease, and single-chain forms of its catalytic domain or proteolytically active portion (typically a C-terminal truncation), exhibit protease activity.

[0083] As used herein, a "nucleic acid encoding a protease domain or catalytically active portion of a protease" refers to a nucleic acid that encodes only the recited single chain protease domain or active portion thereof, and does not encode other contiguous portions of the protease as a contiguous sequence.

[0084] As used herein, a description of a polypeptide consisting essentially of a protease domain means that only a portion of the polypeptide is the protease domain or a catalytically active portion thereof. The polypeptide may also contain additional, typically non-protease-derived, sequences of amino acids.

[0085] As used herein, the term "active site of a protease" refers to the substrate binding site where catalysis of the substrate occurs. The structure and chemistry of the active site allow for substrate recognition and binding, and subsequent hydrolysis and cleavage of scissile bonds within the substrate. The active site of a protease contains amino acids that contribute to the catalytic mechanism of peptide cleavage, e.g., amino acids Gln His Ala Arg Ala Ser His Leu (active site of C3; residues 737-744 of SEQ ID NO: 47), and amino acids that contribute to substrate sequence recognition, e.g., amino acids that contribute to long substrate binding specificity. For example, cleavage within the active site of C3 can inhibit its activity, e.g., QHAR ↓ASHL (residues 737-744 of SEQ ID NO: 47) P4 P3 P2 P1 ↓P1' P2' P3' P4' is.

[0086] As used herein, a "substrate recognition site" or "cleavage sequence" refers to a sequence recognized by the active site of a protease that is cleaved by the protease. Typically, cleavage sequences for serine proteases are six residues long, matching the long substrate specificity of many proteases, but may be longer or shorter depending on the protease. Typically, for example, for serine proteases, the cleavage sequence is composed of P1-P4 and P1'-P4' amino acids in the substrate, with cleavage occurring after the P1 position. Typically, cleavage sequences for serine proteases are six residues long, matching the long substrate specificity of many proteases, but may be longer or shorter depending on the protease.

[0087] As used herein, "target substrate" refers to a substrate cleaved by a protease. Typically, a target substrate is specifically cleaved by a protease at its substrate recognition site. At a minimum, the target substrate includes the amino acids that form the cleavage sequence. A target substrate may also include a peptide containing the cleavage sequence and any other amino acids. A full-length protein, allelic variant, isoform, or any portion thereof containing the cleavage sequence recognized by a protease is a target substrate for that protease. For example, for purposes herein in which complement inactivation is intended, a target substrate is complement protein C3, or any portion or fragment thereof, containing the cleavage sequence recognized by a u-PA polypeptide. Such a target substrate may be a purified protein or may be present in a mixture, such as an in vitro mixture or an in vivo mixture. Mixtures may include, for example, blood or serum, or other tissue fluids. In addition, target substrates include peptides or proteins containing additional moieties that do not affect cleavage of the substrate by the protease. For example, a target substrate may include a four-amino acid peptide or a full-length protein chemically linked to a fluorescent moiety. The protease may be modified to exhibit greater substrate specificity for the target substrate.

[0088] As used herein, "u-PA" or "uPA" or "u-PA polypeptide" refers to any u-PA polypeptide, including, but not limited to, recombinantly produced polypeptides, synthetically produced polypeptides, and u-PA polypeptides extracted or isolated from cells or tissues, including, but not limited to, liver and blood. Other names used interchangeably for u-PA include urokinase and urinary plasminogen activator, urokinase plasminogen activator, urinary-type plasminogen activator, and urokinase-type plasminogen activator. u-PA includes related polypeptides from various species, including, but not limited to, animals of human and non-human origin. Human u-PA includes u-PA, allelic variants, isoforms, synthetic molecules derived from nucleic acids, proteins isolated from human tissues and cells, and modified forms thereof. Exemplary unmodified human u-PA polypeptides include, but are not limited to, unmodified, wild-type, naturally occurring mature u-PA polypeptide (SEQ ID NO: 3), unmodified, wild-type precursor u-PA polypeptides including a propeptide and / or signal peptide (e.g., the u-PA polypeptide set forth in SEQ ID NO: 1), and a protease domain (e.g., the u-PA protease domain set forth in SEQ ID NO: 2). Those skilled in the art will recognize that the reference position of the mature u-PA polypeptide (SEQ ID NO: 3) differs by 20 amino acid residues when compared to the precursor u-PA polypeptide (SEQ ID NO: 1), which is a u-PA polypeptide containing a signal peptide sequence. Thus, the first amino acid residue of SEQ ID NO: 3 "corresponds to" the 21st (21st) amino acid residue of SEQ ID NO: 1.

[0089] The description of "u-PA" encompasses activated, or two-chain, forms of u-PA polypeptides containing an N-terminal A chain (amino acids 1-158 of SEQ ID NO:3) and a C-terminal B chain (amino acids 159-411 of SEQ ID NO:3) linked by a disulfide bond between residues 148C and 279C (corresponding to the mature u-PA polypeptide set forth in SEQ ID NO:3). The two-chain form or high molecular weight (HMW) u-PA is formed from the mature u-PA polypeptide (e.g., set forth in SEQ ID NO:3) by proteolytic cleavage after amino acid residue Lys158 and before residue Ile159. Proteolytic cleavage can be carried out, for example, by plasmin, kallikrein, cathepsin B, matriptase, and nerve growth factor gamma. The u-PA polypeptides provided herein may be further modified, for example, by chemical or post-translational modification. Such modifications include, but are not limited to, glycosylation, pegylation, albumination, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0090] u-PA includes u-PA from any species, including humans and non-human species. u-PA polypeptides of non-human origin include, but are not limited to, murine, canine, rabbit, avian, bovine, ovine, porcine, and other primate u-PA polypeptides. Exemplary u-PA polypeptides of non-human origin include those derived from mouse (Mus musculus, SEQ ID NO: 52), rat (Rattus norvegicus, SEQ ID NO: 53), cow (Bos taurus, SEQ ID NO: 54), pig (Sus scrofa, SEQ ID NO: 55), rabbit (Oryctolagus cuniculus, SEQ ID NO: 56), chicken (Gallus gallus, SEQ ID NO: 57), yellow baboon (Papio cynocephalus, SEQ ID NO: 58), Sumatran orangutan (Pongo abelii, SEQ ID NO: 59), dog (Canis lupus, SEQ ID NO: 60), sheep (Ovis aries, SEQ ID NO: 61), marmoset (Callithrix jacchus, SEQ ID NO: 62), rhesus monkey (Macaca mulatta, SEQ ID NO: 63), northern white-cheeked gibbon (Nomascus leucogenys, SEQ ID NO: 64), and chimpanzee (Pan troglodytes, SEQ ID NO: 65). troglodytes, SEQ ID NO: 65).

[0091] Reference to u-PA polypeptides also includes single- or two-chain forms, their active truncated forms, precursor and mature u-PA polypeptides, and isolated protease domains, including allelic and species variants, variants encoded by splice variants, and other variants, including polypeptides having at least, or at least about, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the precursor polypeptide set forth in SEQ ID NO: 1, or its mature form (SEQ ID NO: 3), or its protease domain (SEQ ID NO: 2). u-PA polypeptides include, but are not limited to, tissue-specific isoforms and allelic variants thereof, synthetic molecules prepared by translation of nucleic acids, proteins produced synthetically, including chemical synthesis, e.g., by recombinant methods, and ligation of shorter polypeptides, proteins isolated from human and non-human tissues and cells, chimeric u-PA polypeptides, and modified forms thereof. u-PA polypeptides also include fragments or portions of u-PA that are of sufficient length to retain at least one activity of the full-length mature polypeptide (upon activation, if necessary), or that contain a region suitable for doing so. In one example, the portion of u-PA is a protease domain, such as the protease domain set forth in SEQ ID NO:2 (which corresponds to amino acids 179-431 of the u-PA sequence set forth in SEQ ID NO:1). u-PA polypeptides also include those that contain chemical or post-translational modifications, as well as those that do not contain chemical or post-translational modifications.Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, HESylation (half-life extension by coupling of drug molecules to biodegradable hydroxyethyl starch (HES)), PASylation (conjugation of pharmacologically active compounds, such as proteins, peptides, and low molecular weight drugs, via genetic fusion or chemical coupling to naturally disordered biosynthetic polymers composed of the small L-amino acids Pro, Ala, and / or Ser), and other polypeptide modifications known in the art.

[0092] As used herein, "u-PA protease" or "u-PA protease domain" refers to any u-PA polypeptide, including, but not limited to, recombinantly produced polypeptides, synthetically produced polypeptides, and u-PA polypeptides extracted or isolated from cells or tissues, including, but not limited to, liver and blood. u-PA proteases include related polypeptides from various species, including, but not limited to, animals of human and non-human origin. Human u-PA proteases or u-PA protease domains include u-PA, allelic variants, isoforms, synthetic molecules derived from nucleic acids, proteins isolated from human tissues and cells, and modified forms thereof. Exemplary reference human u-PA protease domains include, but are not limited to, the unmodified and wild-type u-PA protease domain (SEQ ID NO: 2) and other protease domains (e.g., the u-PA protease domain set forth in SEQ ID NO: 5). Those skilled in the art will recognize that the reference position of the u-PA protease domain (SEQ ID NO:2) differs by 178 amino acid residues when compared to the mature u-PA polypeptide (SEQ ID NO:1), which is the u-PA polypeptide containing the full-length WT sequence. Thus, the first amino acid residue of SEQ ID NO:2 "corresponds to" the 179th (179th) amino acid residue of SEQ ID NO:1.

[0093] As used herein, "modification" refers to a modification of the amino acid sequence of a polypeptide or the sequence of nucleotides in a nucleic acid molecule, including deletion, insertion, and substitution of amino acids or nucleotides, respectively. Methods for modifying polypeptides are routine for those skilled in the art, for example, by using recombinant DNA methodology. There is a difference between a modification to the amino acid sequence of a polypeptide and a modification of the polypeptide. The former refers to insertion, deletion, and substitution (replacement or substitution) of amino acids; the latter refers to a modification of the polypeptide, such as post-translational modification of the protein, pegylation, and other modifications that change the properties and / or activity.

[0094] As used herein, "substitution" refers to the replacement of one or more nucleotides or amino acids in a native, target, wild-type, or other nucleic acid or polypeptide sequence with other nucleotides or amino acids without changing the length of the molecule (described in terms of the number of residues). Thus, one or more substitutions within a molecule do not change the number of amino acid residues or nucleotides in the molecule. Amino acid substitutions can be expressed in terms of the number of amino acid residues along the length of the polypeptide sequence, relative to a particular polypeptide. For example, a modified polypeptide having an amino acid modification at position 35 of the amino acid sequence (replacement of arginine (Arg; R) with glutamine (Gln; Q)) can be represented as R35Q, Arg35Gln, or 35Q. Simply, R35 can be used to indicate that the modified amino acid at position 35 is arginine.

[0095] As used herein, "modified u-PA" or "modified u-PA polypeptide" refers to a u-PA protease that exhibits altered activity, e.g., altered substrate specificity, compared to the unmodified form. Such proteases contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more modifications (i.e., amino acid changes) that alter the activity, e.g., substrate specificity, or selectivity of the u-PA protease for cleaving complement protein C3, compared to wild-type u-PA. The modified u-PA may be a full-length u-PA protease or a portion thereof, e.g., the protease domain of u-PA, so long as the modified u-PA protease contains modifications within regions that alter the activity or substrate specificity of the protease and the protease is proteolytically active. The modified u-PA protease or modified u-PA protease domain may also contain other modifications within regions that do not affect the substrate specificity of the protease. Thus, modified u-PA polypeptides typically have 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the corresponding sequence of amino acids in a wild-type u-PA polypeptide. The modified full-length u-PA polypeptide, or a catalytically active portion thereof, or a protease domain thereof, of the modified u-PA polypeptide may comprise a polypeptide that is a fusion protein, so long as the fusion protein has target specificity.

[0096] As used herein, chymotrypsin numbering refers to the amino acid numbering of the mature chymotrypsin polypeptide of SEQ ID NO: 76. Alignment of the protease domain of another protease, such as the protease domain of u-PA, can be performed with chymotrypsin. In such instances, amino acids of the u-PA polypeptide that correspond to amino acids of chymotrypsin are given the chymotrypsin amino acid numbering. Corresponding positions can be determined by alignment by one of skill in the art using manual alignment or by using one of the many available alignment programs (e.g., BLASTP). Corresponding positions may also be based on structural alignment, for example, by using computer-simulated alignment of protein structures. A statement that an amino acid of a polypeptide corresponds to an amino acid in a disclosed sequence refers to an amino acid identified by alignment of the polypeptide with the disclosed sequence using a standard alignment algorithm, such as the GAP algorithm, to maximize identity or homology (when conserved amino acids are aligned). The corresponding chymotrypsin numbers for amino acid positions 159-411 of the u-PA polypeptide set forth in SEQ ID NO: 3 are listed in Table 1. Amino acid positions relative to the sequence set forth in SEQ ID NO: 3 are in standard font, the amino acid residue at that position is in bold, and the corresponding chymotrypsin number is in italics. For example, by aligning the serine protease domain of u-PA (SEQ ID NO: 2) with mature chymotrypsin, the isoleucine (I) at position 159 in u-PA is given the chymotrypsin numbering of I16. Subsequent amino acids are numbered accordingly. In one example, the phenylalanine (F) at amino acid position 173 of mature u-PA (SEQ ID NO: 3) corresponds to amino acid position F30 based on the chymotrypsin numbering system. When a residue is present in the protease but not in chymotrypsin, the amino acid residue is given a letter designation.For example, based on chymotrypsin numbering, the portion of the loop having amino acid 60, but which is inserted into the u-PA sequence relative to chymotrypsin, is designated, for example, as D60a, Y60b, or P60c, which correspond to D208, Y209, and P210, respectively, according to the numbering relative to the mature u-PA sequence shown in SEQ ID NO:3.

[0097] Table 1. Chymotrypsin numbering of u-PA [Table 1-1] [Table 1-2]

[0098] As used herein, k cat is the catalytic activity of the enzyme; k cat The unit is seconds -1 k cat The reciprocal of k is the time required by an enzyme molecule to "turn over" one substrate molecule; cat k measures the number of substrate molecules turned over per enzyme molecule per second. cat is sometimes called the turnover number. cat The turnover number (also called the metabolic turnover number) is the maximum number of chemical transformations of substrate molecules per second that a single catalytic site can carry out for a given enzyme. It is the maximum rate of reaction (V) when all enzyme catalytic sites are saturated with substrate. max )

[0099] As used herein, specificity for a target substrate refers to the preference for cleavage of the target substrate by a protease compared to other substrates, referred to as non-target substrates. Specificity is determined by the specificity constant (k cat / K m ), which is a measure of the affinity of the protease for the substrate and the efficiency of the enzyme. cat / K m is a measure of enzyme efficiency; k cat (rapid turnover) or K m A small value of k (high affinity for the substrate) cat / K m Make it bigger.

[0100] As used herein, the specificity constant for cleavage is (k cat / K m ) where K m is the Michaelis-Menten constant ([S] is V max half of k cat is V max / [E T ] and E T is the final enzyme concentration. The parameter k cat , K. m , and k cat / K m is calculated by plotting the inverse of the substrate concentration against the inverse of the rate of substrate cleavage using the Lineweaver-Burk equation [1 / rate = (K m / V max )(1 / [S])+1 / V max ;where V max =[E T ]k cat ]. Any method for determining the rate of increase in cleavage over time in the presence of various concentrations of substrate can be used to calculate the specificity constant. For example, the substrate is linked to a fluorescent moiety that is released upon cleavage by the protease. By determining the rate of cleavage at various enzyme concentrations, k cat can be determined for a particular protease. The specificity constant can be used to determine the preference of a protease for one target substrate over another.

[0101] As used herein, substrate specificity refers to the preference of a protease for one target substrate over another. Substrate specificity can be measured as a ratio of specificity constants.

[0102] As used herein, a substrate specificity ratio is a ratio of specificity constants that can be used to compare the specificity of two or more proteases, or of a protease for two or more substrates. For example, the substrate specificity of a protease for a competing substrate, or of a competing protease for a substrate, can be expressed as k cat / K m For example, if the specificity constant is 2 × 10 for the target substrate, 6 M -1 sec -1 and 2 × 10 for non-target substrates. 4 M -1 sec -1 Using the specificity constants derived above, a protease has a substrate specificity ratio of 100 for a target substrate.

[0103] As used herein, target substrate preference or substrate specificity can be expressed as a substrate specificity ratio. The specific value of the ratio reflecting preference is a function of the substrate and protease in question. A substrate specificity ratio greater than 1 represents a preference for the target substrate, while a substrate specificity ratio less than 1 represents a preference for a non-target substrate. Typically, a ratio of at least 1, or about 1, reflects a sufficient difference to consider the protease a candidate therapeutic agent.

[0104] As used herein, altered specificity refers to a change in the substrate specificity of the engineered protease compared to the starting wild-type protease. Typically, the change in specificity reflects a change in the preference of the engineered protease for a target substrate compared to the wild-type substrate of the protease (referred to herein as a non-target substrate). Typically, the engineered u-PA proteases provided herein exhibit increased substrate specificity for complement protein C3 compared to the substrate specificity of the wild-type u-PA protease. For example, an engineered protease with a substrate specificity ratio for a target substrate over a non-target substrate of 100 exhibits a 10-fold increase in specificity compared to a scaffold protease with a substrate specificity ratio of 10. In another example, an engineered protease with a ratio of 1 exhibits a 10-fold increase in substrate specificity compared to a substrate specificity ratio of 0.1. To exhibit increased specificity compared to the scaffold protease, the modified protease has 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or more greater substrate specificity for any one or more of the complement proteins.

[0105] As used herein, "selectivity" can be used interchangeably with specificity when referring to the ability of a protease to selectively cleave one target substrate from a mixture of competing substrates. The increased selectivity of a protease for a target substrate compared to any one or more other target substrates can be determined, for example, by comparing the specificity constants of the cleavage of the target substrates by the protease. For example, if the specificity constant of the cleavage of a protease is 2×10 for the target substrate, the selectivity of the protease can be determined by comparing the specificity constants of the cleavage of the target substrate by the protease. 6 M -1 sec -1 and 2 × 10 for any one or more other substrates. 4 M -1 sec -1 If so, the protease is more selective for the target substrate.

[0106] As used herein, "activity" or "functional activity" of a polypeptide, e.g., a protease, refers to any activity exhibited by the polypeptide. Such activity can be determined experimentally. Exemplary activities include, but are not limited to, the ability to interact with a biomolecule, e.g., through substrate binding, DNA binding, or dimerization; enzymatic activity, e.g., kinase activity or proteolytic activity. For proteases (including protease fragments), activity includes, but is not limited to, the ability to specifically bind to a particular substrate; substrate binding affinity and / or specificity (e.g., high or low affinity and / or specificity); effector function, e.g., the ability to promote inhibition, neutralization, cleavage, or clearance of a substrate (e.g., a protein, i.e., C3); and in vivo activity, e.g., the ability to promote protein cleavage or clearance. Activity can be assessed in vitro or in vivo using recognized assays, such as ELISA, flow cytometry, surface plasmon resonance, or equivalent assays for measuring on- or off-rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, and binding assays. For example, for a protease, such as a modified u-PA protease, activity can be assessed by measuring substrate protein cleavage, turnover, residual activity, stability, and / or levels in vitro and / or in vivo. Results of such in vitro assays showing that a polypeptide exhibits activity can be correlated to the polypeptide's activity in vivo, and in vivo activity can be referred to as therapeutic activity or biological activity. The activity of the modified polypeptide can be at any percentage level of the activity of the unmodified polypeptide, including, but not limited to, at or about 1% of activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more of the activity compared to the unmodified polypeptide.Assays to determine the function or activity of modified (or mutant) proteases are well known in the art.

[0107] Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind to or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and ability to specifically bind to a polypeptide receptor or ligand.

[0108] As used herein, functional activity of complement proteins refers to complement-mediated functions, including, but not limited to, anaphylaxis, opsonization, chemotaxis, or cytolysis. Exemplary assays for testing complement activity include hemolysis of red blood cells and detection of complement effector molecules, for example, by ELISA or SDS-PAGE.

[0109] As used herein, catalytic activity or cleavage activity refers to the activity of a protease as assessed in an in vitro proteolysis assay that detects proteolysis of a selected substrate. Cleavage activity can be measured by assessing the catalytic efficiency of the protease.

[0110] As used herein, activity directed toward a target substrate refers to cleavage activity and / or functional activity, or other measurements reflecting the activity of a protease toward or directed toward a target substrate. The functional activity of a protease toward a complement protein target substrate can be measured by assessing the IC50 in a complement assay, such as erythrocyte lysis, or other assays known to those skilled in the art for assessing complement activity or provided herein. Cleavage activity can be measured by assessing the catalytic efficiency of the protease. For purposes herein, activity is increased if the protease exhibits greater proteolysis or cleavage of the target substrate compared to the absence of the protease and / or modulates (i.e., activates or inhibits) the functional activity of a complement protein.

[0111] As used herein, "increased activity" with respect to a modified u-PA polypeptide means that the modified u-PA polypeptide exhibits greater activity compared to an unmodified u-PA polypeptide that does not contain the amino acid substitution when tested under the same conditions. For example, the modified u-PA polypeptide exhibits at least, or at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more of the activity of an unmodified or reference u-PA polypeptide.

[0112] As used herein, the term "same" when used in reference to antibody binding affinity refers to the EC 50 , association constant (Ka), or dissociation constant (Kd) is within about 1-100 times or 1-10 times that of the reference antibody (1-100 times greater affinity, or 1-100 times less affinity than the reference antibody, or any number or range or value within such range).

[0113] As used herein, "binding activity" refers to a characteristic of a molecule, e.g., a polypeptide, with respect to whether or how it binds to one or more binding partners. Binding activity includes the ability to bind to a binding partner, the affinity (e.g., high affinity) with which it binds to the binding partner, the strength of binding to the binding partner, and / or the specificity with which it binds to the binding partner.

[0114] As used herein, EC, also referred to as apparent Kd, 50 is the concentration of protease (e.g., nM) at which 50% of maximal activity is observed for a fixed amount of substrate (e.g., the concentration of modified u-PA polypeptide required to cleave 50% of the available hC3). Typically, EC 50 The EC value was determined from a sigmoidal dose-response curve. 50is the concentration at the inflection point. A high protease affinity for the substrate indicates a low EC 50 The lower the affinity, the higher the EC 50 Affinity constants can be determined by standard kinetic methodologies for protease reactions, such as immunoassays, e.g., ELISA, followed by curve-fitting analysis.

[0115] As used herein, "affinity constant" refers to the binding constant (Ka) used to measure the affinity or molecular binding strength between a protease and a substrate. The higher the affinity constant, the greater the affinity of the protease for the substrate. Affinity constants are expressed in units of reciprocal molar concentration (i.e., M -1) and can be calculated from the rate constant for the binding-dissociation reaction measured by standard kinetic methodologies for protease-substrate reactions (e.g., immunoassays, surface plasmon resonance, or other kinetic interaction assays known in the art). The binding affinity of a protease can also be expressed as a dissociation constant, or Kd. The dissociation constant is the reciprocal of the binding constant, Kd = 1 / Ka. Therefore, the affinity constant can also be expressed in terms of Kd. Affinity constants can be determined by standard kinetic methodologies for protease reactions, such as immunoassays, surface plasmon resonance (SPR) [Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599], isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art [see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989)]. Instrumentation and methods for real-time detection and monitoring of binding kinetics are known and commercially available [e.g., BIAcore 2000, BIAcore AB, Uppsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335].

[0116] Methods for calculating affinity are well known and include, for example, those exemplified herein, such as EC 50 There are methods to calculate the EC value or the association / dissociation constant. 50 With respect to a protease, high binding affinity is when the protease has an EC50 activity of less than about 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, or less. 50 High binding affinity means that the antibody specifically binds to the target protein at a concentration of 10 -6M or less, e.g., 10 -7 M, 10 -8 M, 10 -10 M, 10 -11 M or 10 -12 A high binding affinity can be characterized by an equilibrium dissociation constant (Kd) of about 10 M or less. In terms of the equilibrium binding constant (Ka), a high binding affinity is typically about 10 6 M -1 That's about 10 7 M -1 That's about 10 8 M -1 or more, or about 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , or 10 12 M -1 Affinity can be estimated experimentally, or it can be determined comparatively, e.g., by comparing the affinities of two or more antibodies for a particular antigen, e.g., by calculating the pairwise ratio of the affinities of the antibodies being tested. For example, such affinities can be readily determined using conventional techniques, e.g., ELISA; equilibrium dialysis; surface plasmon resonance; radioimmunoassay using a radiolabeled target antigen; or by another method known to those of skill in the art. Affinity data can be calculated, e.g., by the method of Scatchard et al., Ann NY Acad. Sci., 51:660 (1949), or by the formula: y = ((AD) / (1 + ((x / C)^B))) + D, where A is the minimum asymptote, B is the slope factor, and C is the inflection point (EC 50 ) where D is the maximum asymptote] can be analyzed by curve fitting analysis using a four-parameter logistic nonlinear regression model.

[0117] As used herein, "ED 50" is the dose (e.g., mg / kg or nM) of a protease (e.g., modified u-PA) that produces a defined result (e.g., cleavage of complement protein C3) in 50% of the total population (e.g., the total amount of C3 present in a sample).

[0118] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore system (GE Healthcare Life Sciences).

[0119] As used herein, a human protein is one encoded by a nucleic acid molecule, e.g., DNA, present in the human genome, including all allelic variants and conservative variations thereof. A variant or modification of a protein is a human protein if the modification is based on the wild-type or prominent sequence of the human protein.

[0120] As used herein, naturally occurring α-amino acid residues are those 20 α-amino acid residues found in nature that, in humans, are incorporated into proteins by specific recognition of tRNA molecules charged with their cognate mRNA codons.

[0121] As used herein, a non-naturally occurring amino acid refers to an amino acid that is not genetically encoded.

[0122] As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs thereof typically linked together by a phosphodiester bond. The term "nucleic acid" also includes nucleic acid analogs, such as peptide nucleic acids (PNAs), phosphorothioate DNA, and other such analogs and derivatives, or combinations thereof. Nucleic acids also include DNA and RNA derivatives containing nucleotide analogs or "backbone" linkages other than phosphodiester linkages, e.g., phosphotriester linkages, phosphoramidate linkages, phosphorothioate linkages, thioester linkages, or peptide linkages (peptide nucleic acids). The term also includes single-stranded (sense or antisense) and double-stranded nucleic acids, as well as equivalents, derivatives, variants, and analogs of RNA or DNA formed from nucleotide analogs. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For RNA, the uracil base is uridine. Nucleic acid can be single-stranded or double-stranded. When referring to a probe or primer, which may be labeled with a detectable label, such as a fluorescent or radioactive label, a single-stranded molecule is intended. Such molecules are typically of a length such that the target is statistically unique, or of low copy number (typically less than 5, usually less than 3) for probing or priming a library. Usually, a probe or primer contains at least 14, 16, or 30 consecutive nucleotides of a sequence that is complementary or identical to the gene of interest. Probes and primers can be 10, 20, 30, 50, 100, or more nucleotides in length.

[0123] As used herein, an "isolated nucleic acid molecule" is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, e.g., a cDNA molecule, is substantially free of other cellular materials or culture media when produced by recombinant techniques, or is substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include the isolated nucleic acid molecules encoding the provided u-PA proteases.

[0124] As used herein, "synthetic," for example with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0125] As used herein, a "polypeptide" refers to two or more amino acids covalently linked together. The terms "polypeptide" and "protein" are used interchangeably herein.

[0126] As used herein, "peptide" refers to a polypeptide that is from 2 to about 40 amino acids in length.

[0127] As used herein, the amino acids that occur in the various sequences of amino acids provided herein are identified according to their known three-letter or one-letter abbreviations (Table 2). The nucleotides that occur in the various nucleic acid fragments are designated by the standard single-letter designations used routinely in the art.

[0128] As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. Polypeptides contain two or more amino acids. For purposes herein, amino acids include the 20 naturally occurring amino acids (Table 2), non-naturally occurring amino acids, and amino acid analogs (i.e., amino acids in which the α-carbon has a side chain). As used herein, the amino acids that occur in the various amino acid sequences of the polypeptides appearing herein are identified according to their well-known three-letter or one-letter abbreviations (see Table 2). Nucleotides that occur in the various nucleic acid molecules and fragments are designated by standard single-letter designations routinely used in the art.

[0129] As used herein, "amino acid residue" refers to an amino acid formed by chemical digestion (hydrolysis) of a polypeptide and its peptide bonds. The amino acid residues described herein are presumed to be in the "L" isomeric form. Residues in the "D" isomeric form (designated as such) can be substituted for any L-amino acid residue, so long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In accordance with standard polypeptide nomenclature as described in J. Biol. Chem., 243: 3557-3559 (1968) and adopted at 37 CFR §§ 1.821-1.822, abbreviations for amino acid residues are set forth in Table 2:

[0130] Table 2 - Correspondence table [Table 2]

[0131] All amino acid residue sequences represented by formulas herein are in the conventional left-to-right orientation toward the amino or carboxyl terminus. The phrase "amino acid residue" includes amino acids set forth in the Table of Correspondence (Table 2), modified amino acids, unnatural amino acids, and unusual amino acids. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, or to the amino-terminal group, e.g., NH2, or to the carboxyl-terminal group, e.g., COOH.

[0132] As used herein, "naturally occurring amino acids" refers to the 20 L-amino acids that occur in polypeptides. As used herein, naturally occurring α-amino acid residues are those 20 α-amino acid residues found in nature that are incorporated into proteins in humans by specific recognition of tRNA molecules charged with their cognate mRNA codons.

[0133] As used herein, "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid, but that has been structurally modified to mimic the structure and reactivity of the natural amino acid. Thus, examples of non-naturally occurring amino acids include amino acids or analogs of amino acids other than the 20 naturally occurring amino acids, including, but not limited to, D-stereoisomers of amino acids. Exemplary unnatural amino acids are known to those of skill in the art and include, but are not limited to, para-acetylphenylalanine, para-azidophenylalanine, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidine acid (piperidinic acid), and the like. acid) (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hy 1), allo-hydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), allo-isoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn). Exemplary unnatural amino acids are described herein and known to those of skill in the art.

[0134] As used herein, an isokinetic mixture is one in which the molar ratio of amino acids has been adjusted based on reported reaction rates [see, e.g., Ostresh et al. (1994) Biopolymers 34:1681].

[0135] As used herein, a DNA construct is a single- or double-stranded, linear or circular DNA molecule that contains segments of DNA combined and arranged in a manner not found in nature. DNA constructs exist as a result of human manipulation and include clones and other copies of engineered molecules.

[0136] As used herein, a DNA segment is a portion of a larger DNA molecule having a defined attribute. For example, a DNA segment encoding a defined polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read in the 5' to 3' direction, encodes the sequence of amino acids of the defined polypeptide.

[0137] As used herein, the term ortholog refers to a polypeptide or protein from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences between orthologs are the result of speciation.

[0138] As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term nucleotide is used for single- and double-stranded molecules, where the context allows. When used in reference to a double-stranded molecule, the term is used to indicate the total length and is understood to be equivalent to the term base pair. Those skilled in the art will understand that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends may be staggered; therefore, not all nucleotides within a double-stranded polynucleotide molecule may be paired. Such unpaired ends are typically no more than 20 nucleotides in length.

[0139] As used herein, sequence alignment refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences related by 50% or more identity are aligned. An aligned sequence set refers to two or more sequences aligned at corresponding positions and may include aligning sequences derived from RNA, such as ESTs and other cDNAs, aligned with genomic DNA sequences. Related or variant polypeptide or nucleic acid molecules can be aligned by any method known to those of skill in the art. Such methods typically maximize matches, for example, by using manual alignment, by using one of the many available alignment programs (e.g., BLASTP), or by other methods known to those of skill in the art. By aligning polypeptide or nucleic acid sequences, one of skill in the art can identify similar portions or positions using conserved and identical amino acid residues as a guide. Furthermore, one of skill in the art can find corresponding amino acid or nucleotide residues between human and non-human sequences using conserved amino acid or nucleotide residues as a guide. Corresponding positions may also be based on structural alignment, for example, by using computer-simulated alignments of protein structures. In other instances, corresponding regions can be identified. One skilled in the art can also find corresponding amino acid residues between human and non-human sequences using conserved amino acid residues as a guide.

[0140] As used herein, "sequence identity" refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test polypeptide or polynucleotide and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is typically determined by alignment to identify identical residues. Alignment can be local or global. Matches, mismatches, and gaps can be identified between compared sequences. A gap is a null amino acid or nucleotide inserted between residues of aligned sequences so that identical or similar characters are aligned. Typically, internal and terminal gaps can exist. Sequence identity can be determined by considering gaps as the number of identical residues divided by the length of the shortest sequence × 100. When gap penalties are used, sequence identity can be determined without penalty for terminal gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined without considering gaps as the number of identical positions divided by the length of the total aligned sequences × 100.

[0141] As used herein, "at a position corresponding to," or a description that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (e.g., as shown in the sequence listing) refers to a nucleotide or amino acid position identified by alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. For purposes herein, alignment of a u-PA sequence is with the amino acid sequence of the protease domain of human u-PA shown in SEQ ID NO: 2 or 5, particularly the reference human u-PA of SEQ ID NO: 5. By aligning the sequences, one skilled in the art can identify corresponding residues, e.g., using conserved and identical amino acid residues as a guide. Generally, to identify corresponding positions, amino acid sequences are aligned to obtain the highest order match [see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo et al. (1988) SIAM J Applied Math 48:1073].Alternatively, one skilled in the art can identify corresponding residues by numbering them by chymotrypsin number. For closely related sequences, no computer algorithm is required; they can be aligned visually.

[0142] As used herein, a "global alignment" is an alignment in which two sequences are aligned from beginning to end, with each character in each sequence aligned only once. Alignments are generated regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on a "global alignment" means that 50% of the residues are the same in the complete alignment of two compared sequences, each 100 nucleotides long. It is also understood that a global alignment can be used to determine sequence identity even when the aligned sequences are not the same length. Differences at the ends of the sequences are taken into account when determining sequence identity unless "no penalty for end gaps" is selected. Global alignments are typically used for sequences that share significant similarity over most of their entire length. An exemplary algorithm for performing a global alignment is the Needleman-Wunsch algorithm [Needleman et al. (1970) J. Mol. Biol. 48: 443]. Exemplary programs for performing global alignments are publicly available and include the Global Sequence Alignment Tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and the program available at deepc2.psi.iastate.edu / aat / align / align.html.

[0143] As used herein, a "local alignment" is an alignment of two sequences, but only aligns portions of the sequences that share similarity or identity. Thus, a local alignment determines whether a subsegment of one sequence exists within the other sequence. If there is no similarity, no alignment is returned. Local alignment algorithms include BLAST and the Smith-Waterman algorithm [Adv. Appl. Math. 2: 482 (1981)]. For example, 50% sequence identity based on a "local alignment" means that, in a complete sequence alignment of two compared sequences of any length, 50% of the residues in a 100-nucleotide-long region of similarity or identity are the same in the region of similarity or identity.

[0144] For the purposes herein, sequence identity can be determined by standard alignment algorithm programs, using the default gap penalty established by each supplier.The default parameters for GAP program can include: (1) the unary comparison matrix (containing a value of 1 for identity and 0 for non-identity) described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745; (2) a penalty of 3.0 per gap, and an additional penalty of 0.10 per symbol in each gap; and (3) no penalty for terminal gaps. Whether any two nucleic acid molecules or any two polypeptides have nucleotide or amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" (or other similar variations that describe percent identity) can be determined using known computer algorithms based on local or global alignments (see, e.g., wikipedia.org / wiki / Sequence_alignment_software, which provides links to dozens of known, publicly available alignment databases and programs).Typically, for purposes herein, sequence identity is determined using a computer algorithm based on global alignment, for example, the Needleman-Wunsch Global Sequence Alignment tool available from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign [implementing the Huang and Miller algorithm by William Pearson (Adv. Appl. Math. (1991) 12:337-357)]; and the program from Xiaoqui Huang available at deepc2.psi.iastate.edu / aat / align / align.html. Typically, when comparing nucleotide sequences herein, alignment with a penalty for terminal gaps is used. Local alignment can also be used when the sequences to be compared are substantially the same length.

[0145] The term "identity" as used herein refers to a comparison or alignment between a test polypeptide or polynucleotide and a reference polypeptide or polynucleotide. In a non-limiting example, "at least 90% identical to" refers to a percent identity of 90% to 100% with respect to a reference polypeptide or polynucleotide. For illustrative purposes, assuming a test polypeptide or polynucleotide length of 100 amino acids or nucleotides is compared, identity at a level of 90% or higher indicates that 10% (i.e., 10 out of 100) or less of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from those in the reference polypeptide. Similar comparisons can be made between test polynucleotides and reference polynucleotides. Such differences can be represented as point mutations randomly distributed throughout the entire length of the amino acid sequence, or can be clustered within one or more positions of varying lengths, up to a maximum allowable amount of, for example, 10 / 100 amino acid differences (approximately 90% identity). Differences can also be due to deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. At homology or identity levels greater than about 85-90%, depending on the length of the sequences being compared, results can be independent of the program and gap parameter set; such high levels of identity can often be easily assessed without relying on software.

[0146] As used herein, a disulfide bond (also referred to as an S-S bond or disulfide bridge) is a single covalent bond resulting from the coupling of thiol groups. Disulfide bonds within proteins are formed between the thiol groups of cysteine ​​residues to stabilize interactions between polypeptide domains.

[0147] As used herein, "coupled" or "conjugated" means attached via a covalent or non-covalent interaction. The conjugates provided herein contain a modified u-PA polypeptide protease domain (referred to as "SPD"; see, e.g., Figure 4 ) and all or part of the remaining u-PA polypeptide, linked directly or via a linker to another moiety (e.g., a polypeptide that confers a property (e.g., increased serum half-life) (e.g., human serum albumin (HSA)), facilitates expression or purification (e.g., SUMO, his-SUMO, TSG-6), or targets the protein to a receptor (e.g., an antibody that binds to a receptor)). The polypeptides can be linked directly or via a polypeptide linker (usually short, about 4-20 amino acids, e.g., a combination of Ser and Gly residues). Conjugates containing polypeptides are typically fusion proteins. Conjugates also include modified u-PA polypeptides in which amino acid residues are linked to moieties, such as PEG moieties, glycosylation moieties, and other such moieties.

[0148] As used herein, a "primer" refers to a nucleic acid molecule that can serve as an initiation point for template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerizing agent, such as DNA polymerase, RNA polymerase, or reverse transcriptase) in an appropriate buffer at an appropriate temperature. Those skilled in the art will understand that certain nucleic acid molecules can function as "primers" as well as "probes." However, primers have a 3' hydroxyl group for extension. Primers can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3' and 5' RACE, in situ PCR, ligation-mediated PCR, and other amplification protocols.

[0149] As used herein, a "primer" refers to an oligonucleotide containing two or more deoxyribonucleotides or ribonucleotides (typically more than three) from which the synthesis of a primer extension product can be initiated. Experimental conditions that result in synthesis include the presence of nucleoside triphosphates and an agent for polymerization and extension, such as a DNA polymerase, and an appropriate buffer, temperature, and pH.

[0150] As used herein, a primer pair refers to a set of primers that includes a 5' (upstream) primer that hybridizes to the 5' end of a sequence to be amplified (e.g., by PCR), and a 3' (downstream) primer that hybridizes to the complement of the 3' end of the sequence to be amplified.

[0151] As used herein, "specifically hybridize" refers to the annealing of a nucleic acid molecule (e.g., an oligonucleotide) to a target nucleic acid molecule through complementary base pairing. Those skilled in the art are familiar with in vitro and in vivo parameters that affect specific hybridization, such as the length and composition of a specific molecule. Furthermore, parameters particularly relevant to in vitro hybridization include annealing and washing temperature, buffer composition, and salt concentration. Exemplary washing conditions for removing nonspecifically bound nucleic acid molecules at high stringency are 0.1xSSPE, 0.1% SDS, and 65°C, and those at medium stringency are 0.2xSSPE, 0.1% SDS, and 50°C. Equivalent stringency conditions are known in the art. Those skilled in the art can easily adjust these parameters to achieve specific hybridization of a nucleic acid molecule to a target nucleic acid molecule suitable for a particular application.

[0152] As used herein, substantially identical to a product means sufficiently similar that the property of interest is sufficiently unchanged so that a substantially identical product can be used in place of the product.

[0153] It is also understood that the terms "substantially identical" or "similar" as used herein vary with context as understood by one of ordinary skill in the relevant art.

[0154] As used herein, a wild-type form of a polypeptide or nucleic acid molecule is the form encoded by a gene or the form encoded by a coding sequence encoded by a gene. Typically, a wild-type form of a gene or the molecule encoded thereby does not contain mutations or other modifications that alter function or structure. The term wild-type also encompasses forms with allelic variations that occur between species. As used herein, a predominant form of a polypeptide or nucleic acid molecule refers to the form of the molecule that is the major form produced from a gene. The "predominant form" varies depending on the source. For example, different cell or tissue types can produce different forms of a polypeptide, e.g., by alternative splicing and / or alternative protein processing. In each cell or tissue type, a different polypeptide may be the "predominant form."

[0155] As used herein, an allelic variant or allelic variation refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation can arise naturally through mutation, resulting in phenotypic polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. The term "allelic variant" is also used herein to refer to proteins encoded by allelic variants of a gene. Typically, a reference form of a gene encodes a wild-type and / or predominant form of a polypeptide from a population or a single reference member of a species. Typically, an allelic variant, including an interspecies variant, has at least 80%, 90%, or more amino acid identity with a wild-type and / or predominant form from the same species; the degree of identity depends on the gene, regardless of whether the comparison is between or within species. Typically, intraspecies allelic variants are at least, or at least about 80%, 85%, 90%, or 95% or more identical to the wild-type and / or predominant form, including at least, or at least about 96%, 97%, 98%, or 99% or more identical to the wild-type and / or predominant form of the polypeptide.

[0156] As used herein, "allele," which is used interchangeably with "allelic variant," refers to an alternative form of a gene or a portion thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for that gene. Alleles of a particular gene may differ from each other by a single nucleotide or several nucleotides, and may include nucleotide substitutions, deletions, and insertions. Alleles of a gene may also be forms of a gene that contain mutations.

[0157] As used herein, species variants refer to the variants in polypeptides between different species, including different mammalian species such as mice and humans. Usually, species variants have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity. Corresponding residues between species variants can be determined by comparing sequences and aligning them to maximize the number of matching nucleotides or residues, for example, so that the identity between sequences is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Then, the position of interest is given the number assigned in the reference nucleic acid molecule. Alignment can be performed manually or visually, especially when the sequence identity is greater than 80%.

[0158] As used herein, splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that gives rise to multiple mRNA forms.

[0159] As used herein, modifications of the primary amino acid sequence of a polypeptide or the sequence of nucleotides in a nucleic acid molecule include the deletion, insertion, and substitution of amino acids and nucleotides, respectively.This includes, in contrast to modifications of the polypeptide itself, post-translational modifications such as glycosylation, farnesylation, pegylation, and fusion, for example, fusion with other polypeptides, such as albumination, to change properties such as serum half-life, fusion with albumin, such as human serum albumin, and other such modifications to polypeptides.Therefore, reference to modifications of amino acid sequence refers to insertion, deletion, substitution, and combinations thereof.Modification of a polypeptide refers to modifications made to a polypeptide that do not change its sequence.

[0160] For purposes herein, amino acid substitutions, deletions, and / or insertions may be made in any u-PA polypeptide or catalytically active fragment thereof, provided that the resulting protein exhibits protease or other activity (or, if desired, such changes may be made to eliminate activity). Modifications can be made by making conservative as well as non-conservative amino acid substitutions. For example, amino acid substitutions can be made that alter the properties of the protein in a desired or advantageous way. In one embodiment, mutations can be made that prevent degradation of the polypeptide. Many proteases cleave after basic residues, such as R and K; to eliminate such cleavage, basic residues are substituted with non-basic residues. The interaction of a protease with an inhibitor can be blocked by making a non-conservative change at the site of interaction of the inhibitor with the protease, while retaining catalytic activity. Other activities can also be altered. For example, receptor binding can be altered without altering catalytic activity.

[0161] Contemplated amino acid substitutions include conservative substitutions, such as those shown in Table 3, which do not abolish proteolytic activity. Substitutions that alter the properties of the proteins described herein, such as removal of cleavage sites and other such sites, are also contemplated; such substitutions are typically non-conservative but can be readily made by one of ordinary skill in the art.

[0162] As used herein, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule.Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide will not substantially change biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224).Such substitutions can be made according to the following Table 3:

[0163] [Table 3]

[0164] Other substitutions are also permissible and can be determined empirically or in accord with known conservative substitutions.

[0165] As used herein, the term promoter refers to a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding region of genes.

[0166] As used herein, an isolated or purified polypeptide or protein, or biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from the cells of the tissue from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. A preparation can be determined to be substantially free if it appears free of readily detectable impurities, as determined by standard analytical methods, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), used by those skilled in the art to assess purity, or if it is sufficiently pure that further purification does not detectably alter the physicochemical properties of the material, such as enzymatic and biological activity. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such cases, further purification may enhance the specific activity of the compound.

[0167] The term substantially free of cellular material includes preparations of protein in which the protein is isolated or separated from cellular components of cells from which it is recombinantly produced. In one embodiment, the term substantially free of cellular material includes preparations of protease protein that contain less than about 30% (by dry weight) non-protease protein (also referred to herein as contaminating proteins), typically less than about 20% non-protease protein, 10% non-protease protein, or less than about 5% non-protease protein. When the protease protein or an active portion thereof is recombinantly produced, it is also substantially free of culture medium, i.e., culture medium represents no more than about 20%, 10%, or 5% of the volume of the protease protein preparation.

[0168] As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of protease proteins in which the protein is separated from chemical precursors or other chemicals involved in the synthesis of the protein. The term includes preparations of protease proteins having no more than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protease chemicals or components.

[0169] As used herein, production by recombinant means by using recombinant DNA methods refers to the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.

[0170] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, a method for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay to quantify the polypeptide in a cell lysate.

[0171] As used herein, a "host cell" is a cell used to receive, maintain, reproduce, and / or propagate a vector. Host cells can also be used to express a polypeptide encoded by a vector. Nucleic acids contained in a vector are replicated when the host cell divides, thereby amplifying the nucleic acid.

[0172] As used herein, a "vector" or "plasmid" refers to a replicable nucleic acid capable of expressing one or more heterologous proteins when the vector is transformed into an appropriate host cell. Reference to a vector includes separate elements used to introduce a heterologous nucleic acid into a cell for expression or replication. Reference to a vector also includes vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, typically by restriction digestion and ligation. Reference to a vector also includes vectors containing a nucleic acid encoding a protease, e.g., modified u-PA. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but can be designed to result in integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art. Vectors also include "viral vectors" or "viral vectors." Viral vectors are engineered viruses that are operably linked to foreign genes to carry (as a vehicle or shuttle) the foreign genes into cells.

[0173] As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, e.g., promoter regions, capable of effecting expression of DNA fragments. Such additional segments may include promoter and terminator sequences, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, e.g., a plasmid, phage, recombinant virus, or other vector, that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells, as well as those that remain episomal or that integrate into the host cell genome.

[0174] As used herein, vector also includes "viral vectors." Viral vectors are engineered viruses that are operably linked to foreign genes, carrying the foreign genes into cells (as vehicles or shuttles).

[0175] As used herein, adenovirus refers to any of a group of DNA-containing viruses that cause conjunctivitis and upper respiratory tract infections in humans.As used herein, naked DNA refers to histone-free DNA that can be used for vaccines and gene therapy.Naked DNA is the genetic material that is transferred from cell to cell during gene transfer process called transformation.In transformation, purified or naked DNA is accepted by recipient cells, and gives recipient cells new characteristics or phenotypes.

[0176] As used herein, "operably linked" with respect to a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide may be operably linked to a nucleic acid encoding a polypeptide such that the nucleic acids can be transcribed and translated to express a functional fusion protein, with the leader peptide providing for secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) may be operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acids may be transcribed as a single mRNA transcript, but translation of the mRNA transcript may express one of two polypeptides. For example, an amber stop codon may be positioned between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter is operably linked to a nucleic acid encoding a polypeptide such that the promoter is capable of regulating or mediating transcription of the nucleic acid.

[0177] As used herein, "primary sequence" refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.

[0178] As used herein, a protein binding sequence refers to a protein or peptide sequence that can specifically bind to other proteins or peptide sequences, usually to a set of proteins or peptide sequences, or to a particular protein or peptide sequence.

[0179] As used herein, a "tag" or "epitope tag" typically refers to a sequence of amino acids added to the N- or C-terminus of a polypeptide, such as u-PA, provided herein. The inclusion of a tag fused to a polypeptide can facilitate the purification and / or detection of the polypeptide. Typically, a tag or tag polypeptide refers to a polypeptide having enough residues to provide an epitope recognized by an antibody, or that may be useful for detection or purification, but is short enough so as not to interfere with the activity of the linked polypeptide. Tag polypeptides are typically sufficiently unique that a specifically binding antibody does not substantially cross-react with the epitope within the linked polypeptide. Epitope-tagged proteins can be affinity purified using highly specific antibodies raised against the tag.

[0180] Suitable tag polypeptides usually have at least 5 or 6 amino acid residues, often about 8-50 amino acid residues, and typically 9-30 residues. Tags can be attached to one or more proteins to enable detection or recovery of the proteins from a sample or mixture. Such tags are well known and can be easily synthesized and designed. Exemplary tag polypeptides include those used for affinity purification, such as Small Ubiquitin-like Modifier (SUMO) tag, FLAG tag, His tag, influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5 [Field et al. (1988) Mol. Cell. Biol. 8:2159-2165]; c-myc tag and its 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies [see, e.g., Evan et al. (1985) Molecular and Cellular Biology 5:3610-3616]; and herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al. (1990) Protein Engineering 3:547-553]. Antibodies used to detect epitope-tagged antibodies are typically referred to herein as secondary antibodies.

[0181] As used herein, a metal binding sequence refers to a protein or peptide sequence that can specifically bind to a metal ion in general, to a set of metal ions, or to a particular metal ion.

[0182] As used herein, the term assessing is intended to include quantitative and qualitative determinations in the sense of obtaining an absolute value for the activity of a protease or domain thereof present in a sample, as well as obtaining an index, ratio, percentage, visual, or other value indicative of the level of activity. Assessment may be direct or indirect, and the actual chemical species detected need not necessarily be the proteolytic product itself, but may be, for example, a derivative thereof or some further substance. Examples include detection of cleavage products of complement proteins, e.g., SDS-PAGE, and protein staining with Coomassie Blue.

[0183] As used herein, biological activity refers to the in vivo activity or physiological response of a compound that occurs upon in vivo administration of a compound, composition, or other mixture. Thus, biological activity encompasses the therapeutic effects and pharmaceutical activities of such compounds, compositions, and mixtures. Biological activity can be observed in an in vitro system designed to test or use such activity. Thus, for purposes herein, the biological activity of a protease is the catalytic activity by which a polypeptide is hydrolyzed.

[0184] As used herein, the term "equivalent," when referring to two sequences of nucleic acids, means that the two sequences in question encode the same sequence of amino acids or equivalent proteins. When used in reference to two proteins or peptides, the term "equivalent" means that the two proteins or peptides have substantially the same amino acid sequence, with only amino acid substitutions (e.g., conservative changes such as those shown in Table 3 above) that do not substantially alter the activity or function of the protein or peptide. When the term "equivalent" refers to a property, the property need not be present to the same degree (e.g., two peptides may exhibit different rates of the same type of enzymatic activity), but the activity is usually substantially the same. When referring to two nucleotide sequences, the term "complementary" means that the two sequences of nucleotides can hybridize, typically with less than 25%, 15%, or 5% mismatch between opposing nucleotides. If necessary, the percentage of complementarity will be specified. Typically, the two molecules are selected to hybridize under high stringency conditions.

[0185] As used herein, an agent that modulates the activity of a protein or the expression of a gene or nucleic acid decreases, increases, or otherwise alters, or in some manner upregulates or downregulates, the activity of a protein or otherwise alters the expression of a nucleic acid in a cell.

[0186] As used herein, a "chimeric protein" or "fusion protein" protease refers to a polypeptide operably linked to a different polypeptide. The chimeric or fusion proteins provided herein can include one or more proteases or portions thereof, such as a single-chain protease domain thereof, and one or more other polypeptides for any one or more of: transcriptional / translational control signals, signal sequences, localization tags, purification tags, portions of immunoglobulin G domains, and / or targeting agents. These chimeric or fusion proteins include those produced by recombinant means as fusion proteins, those produced by chemical means, such as chemical coupling, for example, by coupling to sulfhydryl groups, and those produced by any other method whereby at least one protease or portion thereof is linked directly or indirectly to another polypeptide via a linker.

[0187] As used herein, operably linked when referring to a fusion protein refers to a protease polypeptide and a non-protease polypeptide that are fused in-frame to each other. The non-protease polypeptide can be fused to the N-terminus or C-terminus of the protease polypeptide.

[0188] As used herein, a targeting agent is any moiety, such as a protein or an effective portion thereof, that achieves specific binding of the conjugate to a cell surface receptor, and, in some cases, can internalize the bound conjugate or portion thereof. A targeting agent can also facilitate or facilitate, for example, affinity isolation or purification of the conjugate; attachment of the conjugate to a surface; or detection of the conjugate or a complex containing the conjugate.

[0189] As used herein, "linker" refers to a short sequence of amino acids that connects two polypeptides (or nucleic acids encoding such polypeptides). A "peptide linker" refers to a short sequence of amino acids that connects two polypeptide sequences. An example of a polypeptide linker is the linker that connects two antibody chains in a synthetic antibody fragment, such as an scFv fragment. Linkers are well known, and any known linker can be used in the described methods. An example of a polypeptide linker is a linker with several Glu or Lys residues dispersed throughout to increase solubility (Gly-Ser). n Other exemplary linkers are described herein; any of these and other known linkers can be used in the compositions and methods described.

[0190] As used herein, a derivative or analog of a molecule refers to a portion derived from the molecule or a modified version of the molecule.

[0191] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from a cause or condition, including, but not limited to, infection, acquired condition, genetic condition, condition related to environmental exposure and human behavior, and condition characterized by a definable symptom.Disease or disorder includes clinically diagnosed disease and disturbance in the normal state of an organism that is not diagnosed as a clinical disease.Diseases and disorders of interest herein include those involving complement activation, those mediated by complement activation, and those in which complement activation plays a role in etiology or pathology.Diseases and disorders of interest herein include those characterized by complement activation (e.g., age-related macular degeneration and delayed development of transplant renal function).

[0192] As used herein, macular degeneration occurs when a small central portion of the retina (known as the macula) deteriorates. There are two types of AMD: dry (atrophic) and wet (neovascular or exudative). Most AMD begins as the dry form, and in 10-20% of individuals, it progresses to the wet form. Age-related macular degeneration is always bilateral (i.e., occurs in both eyes), but does not necessarily progress at the same pace in both eyes.

[0193] As used herein, age-related macular degeneration (AMD) is an inflammatory disease that causes visual impairment and blindness in elderly people.The proteins of the complement system are central to the development of this disease.The local inflammation and systemic inflammation in AMD are mediated by the deregulated action of other pathways of the complement system.

[0194] As used herein, delayed graft function (DGF) is a manifestation of acute kidney injury (AKI) that is uniquely attributable to the transplant process. It occurs after transplant surgery. Delayed graft function (DGF) is a common complication, often defined as the need for dialysis during the first post-transplant week. Intrinsic renal synthesis of the third complement component, C3 (C3), contributes to acute rejection by initiating a T cell-mediated response. For example, in brain-dead donors, local renal C3 levels are higher at the time of procurement and are inversely related to renal function 14 days after transplantation.

[0195] As used herein, a complement-mediated disease or disorder refers to any disorder in which one or more complement proteins play a role due to the absence or presence of complement proteins or complement-related proteins, or the activation or inactivation of complement or complement-related proteins. In some embodiments, the complement-mediated disorder is caused by a deficiency of a complement protein. In other embodiments described herein, the complement-mediated disorder is caused by the activation or excessive activation of a complement protein. Also, the complement-mediated disorder is caused by the presence of one or more complement proteins and / or the continuous activation of the complement pathway.

[0196] As used herein, "macular degeneration-related disorder" refers to any of several conditions in which the retinal macula becomes degenerated or dysfunctional (e.g., as a result of reduced macular cell proliferation, increased death or rearrangement of macular cells (e.g., RPE cells), loss of normal biological function, or a combination of these events). Macular degeneration results in loss of cellular structural integrity and / or loss of normal macular extracellular matrix and / or macular cellular function. Examples of macular degeneration-related disorders include age-related macular degeneration (AMD), geographic atrophy (GA), North Carolina macular dystrophy, Sorsby fundus dystrophy, Stargardt disease, pattern dystrophy, Best disease, dominant drusen, and malattia leventinese (radial drusen). Macular degeneration-related disorders also encompass additional extramacular changes that occur before or after macular dysfunction and / or deterioration. Thus, the term "macular degeneration-related disorder" also broadly includes any condition that alters or damages the integrity or function of the macula (e.g., damage to the RPE or Bruch's membrane). For example, the term encompasses retinal detachment, chorioretinal degeneration, retinal degeneration, photoreceptor degeneration, RPE degeneration, mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies, and cone degenerations.

[0197] The macular degeneration-related disorders described herein include macular degeneration, such as AMD macular degeneration. The macular degeneration-related disorders include disorders that are treated with anti-VEGF treatment, such as anti-VEGF antibodies, or laser treatment or implantable scopes.

[0198] As used herein, "treating" a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated or remain static following treatment. Thus, treatment encompasses prophylaxis, therapy, and / or cure. Prevention refers to the prevention of potential disease and / or the prevention of worsening of disease symptoms or progression. Treatment also encompasses any pharmaceutical use of modified u-PA polypeptides and compositions provided herein.

[0199] As used herein, "prevention" or "prophylaxis" refers to a method in which the risk or likelihood of developing a disease or condition is reduced.

[0200] As used herein, "therapeutic agent," "therapeutic regimen," "radioprotectant," or "chemotherapeutic agent" refers to conventional drugs and drug therapies known to those skilled in the art, including vaccines. Radiotherapeutic agents are well known in the art.

[0201] As used herein, "treatment" means any manner in which the symptoms of a condition, disorder, or disease are ameliorated or otherwise beneficially altered. Treatment also includes any pharmaceutical use of the compositions herein.

[0202] As used herein, "amelioration of symptoms" of a particular disease or disorder by treatment, e.g., by administration of a pharmaceutical composition or other therapeutic agent, refers to any reduction, whether permanent or temporary, long-lasting or transient, of symptoms that may be attributable to or associated with the administration of the composition or therapeutic agent.

[0203] As used herein, a "pharmaceutically active agent" includes, but is not limited to, any therapeutic or bioactive agent, including, for example, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic agents, including small molecule drugs and therapeutic proteins.

[0204] As used herein, an "effective amount" of a compound or composition for treating a particular disease is an amount sufficient to ameliorate or in some manner alleviate symptoms associated with the disease. Such an amount may be administered as a single dosage or may be administered according to a regimen to be effective. The amount may cure the disease, but is typically administered to ameliorate the symptoms of the disease. Typically, repeated administrations are required to achieve the desired alleviation of symptoms.

[0205] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a drug, compound, material, or composition containing at least enough of a compound to produce a therapeutic effect after administration to a subject. This is therefore the amount necessary to prevent, cure, ameliorate, suppress, or partially suppress the symptoms of a disease or disorder.

[0206] As used herein, a "therapeutic effect" means an effect resulting from treatment of a subject that alters, typically improves or ameliorates, the symptoms of a disease or condition, or cures the disease or condition.

[0207] As used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to the amount of a drug, compound, material, or composition containing a compound that, when administered to a subject, has the intended prophylactic effect, for example, the effect of preventing or delaying the onset or recurrence of a disease or symptom, the effect of reducing the likelihood of the onset or recurrence of a disease or symptom, or the effect of reducing the incidence of a viral infection. A complete prophylactic effect does not necessarily occur by administration of a single dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0208] As used herein, "administration of a non-complement protease," such as a modified u-PA protease, refers to any method of contacting a non-complement protease with its substrate. Administration can be performed in vivo, ex vivo, or in vitro. For example, for ex vivo administration, a bodily fluid, such as blood, is removed from a subject and contacted with a modified non-complement protease, such as a modified u-PA protease, outside the body. For in vivo administration, a modified non-complement protease, such as a modified u-PA protease, can be introduced into the body, for example, by local, topical, systemic, and / or other introduction routes. In vitro administration includes methods such as cell culture methods.

[0209] As used herein, "unit dosage form" refers to physically discrete units suitable for human and animal subjects and individually packaged as known in the art.

[0210] As used herein, a "patient" or "subject" to be treated includes humans and non-human animals. Mammals include primates, such as humans, chimpanzees, gorillas, and monkeys; domesticated animals, such as dogs, horses, cats, pigs, goats, and cows; and rodents, such as mice, rats, hamsters, and gerbils.

[0211] As used herein, a "combination" refers to any association between two or more items. The association can be spatial or refer to the use of two or more items for a common purpose. A combination can be two or more separate items, such as two compositions or two collections, a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are usually functionally related.

[0212] As used herein, a "composition" refers to any mixture of two or more products or compounds (e.g., drugs, modulators, regulators, etc.), which may be a solution, suspension, liquid, powder, paste, aqueous or non-aqueous formulation, or any combination thereof.

[0213] As used herein, stabilizer refers to a compound added to the formulation to protect the antibody or conjugate, for example, under the conditions (e.g., temperature) in which the formulation herein is stored or used.Therefore, examples include agents that prevent the protein from decomposing from other elements in the composition.Examples of such agents include amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors or substrates, and other agents described herein.

[0214] As used herein, "fluid" refers to any composition that can flow. Thus, fluids encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0215] As used herein, an "article of manufacture" is a product that is manufactured and sold. As used throughout this application, the term is intended to encompass therapeutic agents that contain a modified u-PA polypeptide or nucleic acid molecule in the same or separate packaging.

[0216] As used herein, a "kit" refers to a packaged combination that may include reagents and other products and / or components for practicing a method using the elements of the combination. For example, kits are provided that contain a modified protease polypeptide, such as a modified u-PA protease provided herein, or a nucleic acid molecule provided herein, as well as other items for purposes including, but not limited to, administration, diagnosis, and evaluation of biological activity or properties. Kits may also include instructions for use.

[0217] As used herein, "cellular extract" refers to a preparation or fraction made from lysed or disrupted cells.

[0218] As used herein, "animal" refers to any animal, including, but not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; poultry, such as chickens; ruminants, such as goats, cows, deer, sheep; porcines, such as pigs, and other animals. Non-human animals exclude humans as intended animals. The proteases provided herein are from all sources, animal, plant, prokaryotic, and fungal. Most proteases are of animal origin, including mammalian origin.

[0219] As used herein, a "single-dose" formulation refers to a formulation containing a single dose of a therapeutic agent for direct administration. Single-dose formulations typically do not contain any preservatives.

[0220] As used herein, a multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and is administered directly to provide several single doses of the therapeutic agent.Doses can be administered over minutes, hours, weeks, days, or months.Multi-dose formulations can allow for dose adjustment, dose pooling, and / or dose division.Because multi-dose formulations are used over a long period of time, they usually contain one or more preservatives to prevent microbial growth.

[0221] As used herein, "control" or "standard" refers to a sample that is substantially identical to the test sample, except that it is not treated with the test parameters or, if a plasma sample, may be derived from a normal volunteer not suffering from the condition of interest. A control may also be an internal control. For example, a control may be a sample with a known property or activity, such as a virus.

[0222] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more agents.

[0223] As used herein, the term "or" is used to mean "and / or" unless expressly stated to refer to alternatives only or if the alternatives are mutually exclusive.

[0224] As used herein, ranges and amounts can be expressed as "about" a particular value or range. Also, about includes the exact amount. Thus, "about 5 bases" means "about 5 bases," and even "5 bases."

[0225] As used herein, "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which said event or circumstance occurs and instances in which it does not occur. For example, a group that is optionally substituted means that the group is either unsubstituted or substituted.

[0226] Abbreviations for all protecting groups, amino acids, and other compounds used herein are their commonly used, recognized abbreviations, or those of the IUPAC-IUB Commission on Biochemical Nomenclature [see (1972) Biochem. 11:1726], unless otherwise specified. Follow.

[0227] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0228] BU-PA structure and function Urokinase-type plasminogen activator (u-PA, also known as urokinase or urinary plasminogen activator) is a serine protease that catalyzes the hydrolysis of plasminogen to plasmin. u-PA is found in urine, blood, semen, and many cancer tissues. It is involved in various biological processes related to the conversion of plasminogen to plasmin (itself a serine protease). Plasmin plays a role in various normal and pathological processes, including cell migration and tissue destruction through the cleavage of various molecules, including fibrin, fibronectin, proteoglycans, and laminin. u-PA is involved in wound healing, inflammatory cell migration, angiogenesis, and tissue remodeling during tumor cell invasion. u-PA also cleaves and activates other substrates, including, but not limited to, hepatocyte growth factor / scatter factor (HGF / SF), the latent form of membrane type 1 matrix metalloproteinase (MT-SP1), platelet-derived growth factor, and others.

[0229] Provided herein are modified urokinase-type plasminogen activator (u-PA) polypeptides that have been modified to cleave an inhibitory sequence within C3 such that activation of C3 into C3a and C3b fragments is inhibited. The activity / specificity of the modified u-PA polypeptides provided herein is greater than that of unmodified u-PA polypeptides, particularly any of SEQ ID NOS: 1-6, and / or exhibits greater activity and / or specificity or kappa activity. cat / k mThe modified u-PA polypeptides are such that they cleave C3 by cleavage. Furthermore, the modified u-PA polypeptides could reduce the activity or specificity, or both, of the unmodified u-PA polypeptides for the natural physiological substrate, plasminogen. Thus, the modified u-PA polypeptides provided herein inhibit complement activation in the complement pathway. Furthermore, the modified u-PA polypeptides exhibit increased selectivity for cleaving C3 compared to other u-PA substrates, such as plasminogen. Therefore, the modified u-PA polypeptides provided herein do not exhibit undesired cleavage activity against natural physiological u-PA substrates and therefore do not exhibit undesired side effects. In some embodiments, the modified u-PA polypeptide is a protease domain or single-chain form; in such cases, the free cysteine ​​(residue position 122 according to the chymotrypsin numbering system) is substituted with serine to reduce or eliminate aggregation upon protein preparation. In embodiments in which the modified u-PA polypeptide is full-length or in other forms that are activated by cleavage, the residue at position 122 (according to chymotrypsin numbering) is typically not substituted with S so that a disulfide bond can form to generate the two-chain activated polypeptide.

[0230] 1. Serine proteases Serine proteases (SPs), including secreted enzymes and enzymes sequestered within cytoplasmic organelles, have a variety of physiological roles, including blood coagulation, wound healing, digestion, the immune response, and tumor invasion and metastasis. For example, chymotrypsin, trypsin, and elastase function in the gastrointestinal tract; factor 10, factor 11, thrombin, and plasmin are involved in coagulation and wound healing; and C1r, C1s, and C3 convertases play roles in complement activation.

[0231] A class of cell surface proteins called type II transmembrane serine proteases are proteases that are membrane-anchored proteins via an extracellular domain. As cell surface proteins, they play a role in both intracellular signaling and mediating cell surface proteolytic events. Other serine proteases function membrane-bound as well; others are secreted. Many serine proteases exert their activity upon binding to cell surface receptors and therefore act at the cell surface. Cell surface proteolysis is a mechanism for the generation of biologically active proteins that mediate various cellular functions.

[0232] Serine proteases, including secreted transmembrane serine proteases, are involved in processes such as the onset and progression of neoplasia.Although the exact role of these proteases has not been fully described, serine proteases and their inhibitors are involved in the control of many intracellular and extracellular physiological processes, including cancer cell invasion and metastatic spread, and the degradative role in tumor angiogenesis, which is involved in tumor progression.Proteases are involved in the degradation and remodeling of extracellular matrix (ECM), and contribute to tissue remodeling, which is essential for cancer invasion and metastasis.The activity and / or expression of some proteases have been shown to be correlated with tumor progression and onset.

[0233] More than 20 families of serine proteases (representing S1–S27) have been identified and classified into six clans (SA, SB, SC, SE, SF, and SG) based on structural similarities and other functional evidence [Rawlings ND et al. (1994) Meth. Enzymol. 244: 19–61]. There are similarities in the reaction mechanisms of some serine peptidases. The chymotrypsin, subtilisin, and carboxypeptidase C clans share a catalytic triad of serine, aspartate, and histidine: serine serves as the nucleophile, aspartate as the electrophile, and histidine as the base. The geometric orientation of catalytic residues is similar across families despite the various protein folds. The linear arrangement of catalytic residues generally reflects clan relationships. For example, the catalytic triad is ordered as HDS in the chymotrypsinclane (SA), but as DHS in the subtilisinclane (SB), and as SDH in the carboxypeptidase clan (SC).

[0234] Examples of serine proteases of the chymotrypsin superfamily include tissue-type plasminogen activator (tPA), trypsin, trypsin-like proteases, chymotrypsin, plasmin, elastase, urokinase (or urinary-type plasminogen activator, u-PA), acrosin, activated protein C, C1 esterase, cathepsin G, chymase, and proteases of the blood coagulation cascade (including kallikrein, thrombin, and factors VIIa, IXa, Xa, XIa, and XIIa) [Barrett, AJ, In: Proteinase Inhibitors, Ed. Barrett, AJ, et al., Elsevier, Amsterdam, Pages 3-22 (1986); Strassburger, W. et al., (1983) FEBS Lett., 157: 219-223; Dayhoff, MO, Atlas of Protein Sequence and Structure, Vol. 5, National Biomedical Research Foundation, Silver Spring, Md. (1972); and Rosenberg, RD et al. (1986) Hosp. Prac., 21: 131-137].

[0235] The activity of proteases within the serine protease family is determined by a set of amino acid residues that form the active site. One of the residues is always serine; therefore, they are designated as serine proteases. For example, chymotrypsin, trypsin, and elastase share similar structures, and their active serine residue is at the same position (Ser195) in all three. Despite their similarities, they have different substrate specificities; they cleave different peptide bonds during protein digestion. For example, chymotrypsin prefers aromatic side chains on residues whose carbonyl carbon is part of the peptide bond to be cleaved. Trypsin prefers positively charged Lys or Arg residues at this position. Serine proteases vary significantly in the nature of their substrate recognition: some are highly specific (i.e., proteases involved in blood coagulation and the immune complement system); others are only partially specific (i.e., the mammalian digestive proteases trypsin and chymotrypsin); and some, such as subtilisins (bacterial proteases), are completely nonspecific. Despite these differences in specificity, the catalytic mechanisms of serine proteases are well conserved.

[0236] The mechanism of target protein cleavage by serine proteases is based on the nucleophilic attack of the target peptide bond by serine. Alternatively, cysteine, threonine, or a water molecule associated with aspartate or a metal can fulfill this role. Often, the nucleophilic character of the group is enhanced by the presence of histidine, which is held in a "proton acceptor state" by aspartate. The aligned side chains of serine, histidine, and aspartate constitute a catalytic triad common to most serine proteases. For example, the active site residues of chymotrypsin and serine proteases that are members of the same family as chymotrypsin, such as MTSP-1, are Asp102, His57, and Ser195.

[0237] The catalytic domains of all serine proteases in the chymotrypsin superfamily share sequence and structural homology, including 1) unique active site residues (e.g., Ser195, His57, and Asp102 in trypsin); 2) the oxyanion hole (e.g., Gly193, Asp194 in trypsin); and 3) conservation of cysteine ​​residues that form disulfide bridges in the structure [Hartley, BS, (1974) Symp. Soc. Gen. Microbiol., 24: 152-182]. The structural homology includes 1) a common fold characterized by two Greek key structures [Richardson, J. (1981) Adv. Prot. Chem., 34:167-339]; 2) common dynamics of catalytic residues; and 3) detailed conservation of structure within the core of the molecule [Stroud, RM (1974) Sci. Am., 231: 24-88].

[0238] Throughout the chymotrypsin family of serine proteases, backbone interactions between the substrate and the enzyme are completely conserved, but side chain interactions vary considerably. The identity of the amino acids comprising the S1-S4 pocket of the active site determines the substrate specificity of that particular pocket. Transplanting amino acids from one serine protease to another with the same fold alters the specificity of one toward the other. Typically, the amino acids comprising the S1-S4 pocket of a protease have side chains within 4-5 angstroms of the substrate. The interactions of these amino acids with the protease substrate are usually referred to as "first shell" interactions because they directly contact the substrate. However, there can be "second shell" and "third shell" interactions that ultimately position the first shell amino acids. The substrate-binding effectiveness of the first and second shells is primarily determined by the loops between the beta-barrel domains. Because these loops are not core elements of the protein, folding integrity is maintained while loop variants with novel substrate specificities can be selected during evolution to fill essential metabolic or regulatory niches at the molecular level. Typically, for serine proteases, the following amino acids within the primary sequence are determinants of specificity: 195, 102, 57 (catalytic triad); 189, 190, 191, 192, and 226 (S1); the loop between 57, 58, and 64, and 99 (S2); 192, 217, and 218 (S3); the loop between Cys168 and Cys180, 215, and 97-100 (S4); and 41 and 151 (S2') (based on chymotrypsin numbering), where amino acids within the S1 position affect P1 specificity, amino acids within the S2 position affect P2 specificity, amino acids within the S3 position affect P3 specificity, and amino acids within the S4 position affect P4 specificity. Position 189 within serine proteases is the residue that fills the bottom of the pocket that determines S1 specificity. The structural determinants for u-PA are set out in Table 4, with the protease domain for each of the named proteases aligned with that of chymotrypsin.The numbers under Cys168-Cys182, 60, and the Loop column heading indicate the amino acid number within the loop between the two amino acids. The yes / no designation under Cys191-Cys220 indicates whether a disulfide bridge is present in the protease. These regions are variable within the family of chymotrypsin-like serine proteases and represent their own structural determinants.

[0239] 2. Structure u-PA cDNAs have been cloned from numerous mammalian species. Exemplary u-PA precursor or prepro-urokinase polypeptides include, but are not limited to, u-PA polypeptides from human (encoded by SEQ ID NO: 1 and SEQ ID NO: 7), mouse (SEQ ID NO: 52), rat (SEQ ID NO: 53), bovine (SEQ ID NO: 54), porcine (SEQ ID NO: 55), rabbit (SEQ ID NO: 56), chicken (SEQ ID NO: 57), yellow baboon (SEQ ID NO: 58), Sumatran orangutan (SEQ ID NO: 59), dog (SEQ ID NO: 60), sheep (SEQ ID NO: 61), marmoset (SEQ ID NO: 62), rhesus macaque (SEQ ID NO: 63), northern white-cheeked gibbon (SEQ ID NO: 64), and chimpanzee (SEQ ID NO: 65). The mRNA transcript is typically translated to produce a precursor protein containing a 20-amino acid signal sequence at the N-terminus. After transport to the ER, the signal peptide is removed to yield the pro-urokinase polypeptide. Exemplary pro-urokinase polypeptides include, but are not limited to, u-PA polypeptides from human (SEQ ID NO: 3), mouse (SEQ ID NO: 66), rat (SEQ ID NO: 67), bovine (SEQ ID NO: 68), porcine (SEQ ID NO: 69), rabbit (SEQ ID NO: 70), chicken (SEQ ID NO: 71), yellow baboon (SEQ ID NO: 72), Sumatran orangutan (SEQ ID NO: 73), dog (SEQ ID NO: 74), and sheep (SEQ ID NO: 75). For example, human u-PA mRNA transcripts are typically translated to form a 431-amino acid precursor protein (SEQ ID NO: 1) containing a 20-amino acid signal sequence at the N-terminus: MetArgAlaLeuLeuAlaArgLeuLeuLeuCysValLeuValValSerAspSerLysGly (amino acid residues 1-20 of SEQ ID NO: 1). Thus, after transport to the ER and removal of the signal peptide, a 411-amino acid pro-urokinase polypeptide is produced, having the sequence of amino acids set forth in SEQ ID NO: 3. Pro-urokinase, described in more detail below, is a zymogen or proenzyme that is further processed by proteolytic cleavage to generate the two-chain mature u-PA polypeptide.Thus, for example, with reference to mature u-PA (SEQ ID NO: 3), wild-type chain-activated u-PA contains a first chain (A chain) (residues 1-158) disulfide-linked to residues 159-411 (B chain) via a disulfide bond between Cys148 (C97a chymotrypsin numbering) and Cys279 (C122 chymotrypsin numbering). Thus, in the modified u-PA polypeptides provided herein, when the protease domain is generated, it contains the substitution C122S, but when the activated form, which is a two-chain form, is generated, residue 122 (chymotrypsin numbering) is a C, and therefore typically forms a disulfide bond with another C within the activation sequence (see discussion below and Example 15).

[0240] Human precursor u-PA has the amino acid sequence set forth in SEQ ID NO:1 and is encoded by the nucleotide sequence set forth in SEQ ID NO:7. Human pro-u-PA, also referred to as mature u-PA, lacks a signal sequence and is set forth in SEQ ID NO:3. Two isoforms of human u-PA exist, generated by alternative splicing. Isoform 1 of human u-PA is the previously described standard form set forth in SEQ ID NO:1. In isoform 2 of human u-PA, amino acids 1-29 of SEQ ID NO:1 are replaced with amino acids 1-12 of SEQ ID NO:51, resulting in a protein containing 414 amino acids (set forth in SEQ ID NO:51). Allelic and other variants of human u-PA are known. For example, a uPA variant containing the amino acid modification V15L in the amino acid sequence set forth in SEQ ID NO:1 is known. In another example, a modified u-PA polypeptide containing the amino acid modification C299S (C122S according to chymotrypsin numbering) in the amino acid sequence set forth in SEQ ID NO:1 (corresponding to the amino acid sequence set forth in SEQ ID NO:4) is known. Further variants include those containing the amino acid modifications P121L, D130G, C131W, I194M, K211Q, G366c, and A410V in the mature u-PA shown in SEQ ID NO: 3 (corresponding to the amino acid modifications P141L, D150G, C151W, I214M, K231Q, G386C, and A430V in SEQ ID NO: 1).

[0241] The u-PA polypeptide is synthesized and secreted as a single-chain zymogen molecule (also called pro-urokinase or single-chain urokinase), which is converted to active two-chain u-PA by various proteases, including plasmin, kallikrein, cathepsin B, matriptase, and nerve growth factor-gamma. The cleavage that generates the two-chain form occurs between residues 158 and 159 (SEQ ID NO: 3) in the human pro-urokinase sequence (corresponding to amino acid residues 178 and 179 in SEQ ID NO: 1). The two resulting chains are linked by a disulfide bond between Cys148 and Cys279 to form the two-chain form of u-PA. The two-chain form of u-PA is also referred to as high molecular weight u-PA (HMW-u-PA). HMW-u-PA can be further processed to low molecular weight u-PA (LMW-u-PA) by cleavage of the A chain into a short chain A (A1, amino acids 136-157 of SEQ ID NO: 3) and an amino-terminal fragment. The 21-178 linked disulfide is linked to 179-411 via Cys corresponding to Cys148 and Cys279 (SEQ ID NO: 3).

[0242] Urokinase-type plasminogen activator (u-PA) is a classical serine protease containing a His-Asp-Ser catalytic triad that cleaves a unique Arg-Val bond in plasminogen to form plasmin. Plasmin can then cleave u-PA at Lys158-Ile159 of SEQ ID NO: 3 (corresponding to Lys15-Ile16 according to chymotrypsin numbering) to form the previously described two-chain form. The catalytic triad of human u-PA contains amino acids His204, Asp255, and Ser356 of SEQ ID NO: 3 (corresponding to His57, Asp102, and Ser195 according to chymotrypsin numbering). Residues Ser138 and Ser303 of human uPA, as shown in SEQ ID NO: 3, are phosphorylated [Franco et al. (1997) J Cell Biol 137:779-791]. Human u-PA contains O-linked glycosylation, e.g., fucosylation, at amino acid residue Thr18 of SEQ ID NO: 3 [Buko et al. (1991) Proc Natl Acad Sci USA 88:3992-3996], and N-linked glycosylation at amino acid residue Asn302 of SEQ ID NO: 3. Mature human u-PA contains intrachain disulfide bonds between residues C11-C19, C13-C31, C33-C42, C50-C131, C71-C113, C102-C126, C189-C205, C197-C268, C293-C362, C325-C341 and C352-C380 of SEQ ID NO: 3, and an interchain disulfide bond between residues C148-C279 of SEQ ID NO: 3.

[0243] The mature form of u-PA is a 411-residue protein (corresponding to amino acid residues 21-431 in the sequence of amino acids set forth in SEQ ID NO: 1, the precursor form containing a 20-amino acid signal peptide). u-PA contains three domains: a serine protease domain, a kringle domain, and a growth factor domain. In the mature form of human u-PA, amino acids 1-158 represent the N-terminal A chain, which contains the growth factor domain (amino acids 1-49), the kringle domain (amino acids 50-131), and the interdomain linker region (amino acids 132-158). Amino acids 159-411 represent the C-terminal serine protease domain or B chain. u-PA is synthesized and secreted as a single-chain zymogen molecule, which is converted to active two-chain u-PA by various proteases, including, for example, plasmin, kallikrein, cathepsin B, and nerve growth factor-gamma. Cleavage into the two-chain form occurs between residues 158 and 159 in the mature u-PA sequence (corresponding to amino acid residues 178 and 179 in SEQ ID NO: 3). The two resulting chains are held together by disulfide bonds, thereby forming the two-chain form of u-PA.

[0244] Urokinase-type plasminogen activator contains three domains: a serine protease domain, a kringle domain, and a growth factor domain. In the zymogen or proenzyme form of human u-PA, amino acids 1-158 of SEQ ID NO:3 represent the N-terminal A chain (or long A chain) containing the epidermal growth factor domain (amino acids 1-49), the kringle domain (amino acids 50-131), and the interdomain linker region (amino acids 132-158), while amino acids 159-411 represent the catalytically active C-terminal serine protease domain or B chain. The epidermal growth factor domain is responsible for binding of u-PA to the cell surface-anchored u-PA receptor (uPAR). In the extracellular matrix, u-PA is tethered to the cell membrane by binding to the u-PA receptor. LMW-u-PA is proteolytically active but does not bind to the u-PA receptor. The serine protease domain contains surface-exposed loops around residues 37, 60, 96, 110, 170, and 185 (according to the chymotrypsin numbering system). Upon activation or cleavage, the amino-terminal insertion into the hydrophobic binding cleft of the catalytic protease domain forms hydrophobic interactions, and a salt bridge to the side pocket of Asp194 stabilizes the substrate-binding pocket and oxyanion hole in the catalytically productive conformation. Asp194 (according to the chymotrypsin numbering system) participates in hydrogen bonding to the main-chain amino group of Gly142 and the main-chain carbonyl group of Lys143 [Blouse et al. (2009) J Biol Chem 284:4647-4657].The structural changes following cleavage encompass four disordered regions of the activation domain, including the activation loop (residues 16-21), the autolysis loop (residues 142-152), the oxyanion stabilization loop (residues 184-194), and the S1 entrance frame (residues 216-223) (all numbering follows chymotrypsin numbering) [see Blouse et al. (2009) J Biol Chem 284:4647-4657; Hedstrom (2002) Chem Rev 102:4501-4524; Huber and Bode (1978) Acc Chem Res 11:114-122; Madison et al. (1993) Science 262:419-421].

[0245] The structural determinants for u-PA are shown in Table 4 below, with numbering based on that of mature chymotrypsin. The numbers under the Cys168-Cys182 and 60 loop column headings indicate the number of amino acids within the loop between the two amino acids. A "yes" designation under the Cys191-Cys220 column heading indicates the presence of a disulfide bridge. These regions are variable within the family of chymotrypsin-like serine proteases and represent their own structural determinants. Modifications of u-PA polypeptides, such as altering any one or more of the amino acids within the S1-S4 pockets, affect the specificity or selectivity of the u-PA polypeptide for target substrates. Extended substrate specificity (P1–P4) reveals that u-PA has high specificity for cleavage after P1 Arg, selectivity for small amino acids at the P2 position, selectivity for small polar amino acids (Thr and Ser) at the P3 position, and no selectivity at the P4 position [Ke et al. (1997) J. Biol. Chem., 272:16603–16609; Harris et al. (2000) Proc Natl Acad Sci USA, 97:7754–7759].

[0246] [Table 4]

[0247] 3. Function / activity Urokinase-type plasminogen activator (u-PA) is a serine protease that catalyzes the hydrolysis of plasminogen to plasmin. Plasmin directly acts on the degradation of extracellular matrix proteins [Andreasen et al. (2000) Cell. Mol. Life Sci. 57: 25-40]. u-PA plays an important role in cell adhesion, migration, and invasion, tissue remodeling, and cancer [Blasi et al. (2002) Rev Mol Cell Biol 3:932; Andreasen et al. (2000) Cell. Mol. Life Sci. 57: 25-40; Mondino and Blasi (2004) Trends Immunol 25:450; Ploug (2003) Curr Pharm Des 9:1499]. Aberrant expression of u-PA has been associated with rheumatoid arthritis, allergic vasculitis, xeroderma pigmentosum, and the invasive potential of malignant tumors.

[0248] u-PA is regulated by binding to the high-affinity cell surface receptor uPAR. Binding of u-PA to uPAR increases the rate of plasminogen activation, enhancing extracellular matrix degradation and cell invasion. The binary complex formed between uPAR and u-PA interacts with membrane-bound plasminogen to form a higher-order activation complex that reduces the Km (i.e., the approximate kinetic rate constant of affinity for the substrate) for plasminogen activation [Bass et al. (2002) Biochem. Soc. Trans., 30: 189-194]. Binding of u-PA to uPAR protects the protease from inhibition by its cognate inhibitor, PAI-1. This is because single-chain u-PA normally present in plasma is not susceptible to inhibition by PAI-1, and therefore any active u-PA in plasma is inhibited by PAI-1. Receptor-bound active u-PA is fully available for inhibition by PAI-1. However, PAI-1 cannot access the binding activity molecules [Bass et al. (2002) Biochem. Soc. Trans., 30: 189-194]. As a result, u-PA functions primarily on the cell surface, and its function correlates with activation of plasmin-dependent pericellular proteolysis.

[0249] u-PA also cleaves hepatocyte growth factor / scatter factor (HGF / SF), the latent form of membrane type 1 matrix metalloproteinase (MT-SP1; matriptase), platelet-derived growth factor C (PDGF-C), platelet-derived growth factor D (PDGF-D), platelet-derived growth factor DD (PDGF-DD), and other proteins [see, e.g., Hurst et al. (2012) Biochem J 441:909-918; Ustach and Kim (2005) Mol Cell Biol 5:6279-6288; Ehnman et al. (2009) Oncogene 28(4):534-544]. Plasmin degrades fibrin clots, cleaving fibrin, fibronectin, thrombospondin, laminin, and von Willebrand factor, proteolyzing mediators of the complement system, and activating collagenase. Thus, plasmin is involved in thrombolysis or extracellular matrix degradation, and is associated with vascular disease and cancer. For example, elements of the plasminogen activation system have been observed to be highly expressed in malignant tumors. Hepatocyte growth factor / scatter factor regulates cell proliferation, cell motility, and morphogenesis through the binding of activated HGF to the HGF receptor c-Met, and its ability to stimulate mitogenesis, cell motility, and matrix invasion is associated with angiogenesis, tumorigenesis, and tissue regeneration. Platelet-derived growth factor regulates cell growth and division and plays a critical role in angiogenesis, which, when unregulated, is a hallmark of cancer. Once activated by proteolytic cleavage, PDGF binds to the PGDF receptor tyrosine kinase, leading to phosphorylation and several downstream signaling pathways involved in cancer. Due to the role of u-PA and the above-mentioned proteins in vascular disease, the u-PA polypeptides provided herein have been modified to reduce selectivity for these proteins. Due to the changes in specificity and activity, the modified u-PA polypeptides provided herein exhibit reduced or no activity, or substantially no activity, and increased activity against natural substrates compared to unmodified u-PA against complement protein C3.As a result, at therapeutic dosages, the modified u-PA polypeptides provided herein specifically inhibit complement activation with no or minimal side effects from cleavage of the natural u-PA target.

[0250] C. Complement inhibition by targeting C3 The modified u-PA polypeptides provided herein exhibit increased specificity and / or activity toward the inhibitory cleavage sequence within complement protein C3 compared to u-PA without the amino acid modification [e.g., wild-type human u-PA (see SEQ ID NO: 1 or 3)], or its catalytic or protease domain (see SEQ ID NO: 2), or the corresponding unmodified u-PA polypeptide containing the substitution C122S (according to chymotrypsin numbering). The substitution of S at residue 122 does not alter specificity or activity toward C3 but reduces aggregation. Because C3 is involved in the three initiation pathways of complement (see, e.g., Figure 1), targeting C3 through proteolytic inhibition provides a general and broad therapeutic target for inactivation of the complement cascade. Inactivating cleavage of C3 blocks the terminal activity of complement and the alternative pathway amplification loop. All three pathways converge on C3 (see, e.g., Figure 1). Due to their ability to inhibit complement activation, such modified u-PA polypeptides can be used to treat various diseases, conditions, and pathologies associated with complement activation, such as inflammatory responses and autoimmune diseases. Complement activation is associated with the progression of diseases and conditions by promoting local inflammation and damage to tissues, caused in part by the generation of effector molecules and membrane attack complexes. In one example, for example, in many autoimmune diseases, complement leads to tissue damage because it is activated under inappropriate conditions, for example, by antibodies against host tissues. In other situations, complement can be normally activated, for example, by sepsis, yet contribute to disease progression, for example, in respiratory distress syndrome. Pathologically, if not adequately controlled, complement can cause substantial damage to blood vessels (vasculitis), renal basement membranes and associated endothelial and epithelial cells (nephritis), joint synovium (arthritis), and red blood cells (hemolysis). The role of C3 in complement activation is discussed in more detail below.

[0251] 1. Complement protein C3 and its role in complement initiation The complement system encompasses more than 30 soluble and membrane-bound proteins that function not only in antibody-mediated immune responses but also in innate immune responses, recognizing and killing pathogens such as bacteria, virus-infected cells, and parasites. Complement activation is initiated on the surface of pathogens via three distinct pathways: the classical pathway, the alternative pathway, and the lectin pathway. Although these pathways differ in the components required for initiation, they ultimately generate the same set of effector molecules (e.g., C3 convertase) that cleave the complement protein C3 and trigger the formation of the membrane attack complex (MAC) (see, for example, Figure 1). Therefore, complement protein C3 is an attractive target for therapeutics, because modulation of C3 leads to the production of various opsonins, anaphylatoxins, and modulation of the MAC. Furthermore, naturally occurring complement inhibitor proteins, including factor H (FH), CR1, complement receptor Ig (CR1g), DAF, and MCP, inhibit C3 at the C3 level.

[0252] There are three (3) pathways of complement activation (see Figure 1, which illustrates these pathways). The complement pathways are distinct; each relies on different molecules and mechanisms for initiation. The pathways are similar in that they converge to generate the same set of effector molecules, namely, C3 convertases. In the classical and lectin pathways, C4b2b functions as the C3 convertase; in the alternative pathway, C3bBb is the C3 convertase (see Table 5). Cleavage of C3 generates C3b, which acts both as an opsonin and as the primary effector molecule of the complement system for subsequent complement reactions, and C3a, a peptide mediator of inflammation. Addition of C3b to each C3 convertase forms C5 convertase, which generates C5a and C5b. C5a, like C3a, is a peptide mediator of inflammation. C5b mediates the "late" events of complement activation, initiating the sequence of reactions culminating in the generation of the membrane attack complex (MAC). Although the three pathways produce various C3 and C5 convertases, all pathways generate cleavage products of C3 and C5 to form MAC. Additionally, C3 can be cleaved and activated by external proteases, such as lysosomal enzymes and elastase [Markiewski and Lambris (2007) Am J Pathology 171:715-727; Ricklin and Lambris (2007) Nat Biotechnol 25:1265-1275].

[0253] [Table 5]

[0254] a. classical pathway C1q is the first component of the classical complement pathway. C1q is a calcium-dependent binding protein related to the collectin family of proteins due to shared overall structural homology [Malhotra et al., (1994) Clin Exp Immunol. 97(2): 4-9; Holmskov et al. (1994) Immunol Today 15(2): 67-74]. Collectins are often referred to as pattern recognition molecules and typically function as opsonins that target pathogens for phagocytosis by immune cells. In contrast to conventional collectins, such as MBL, the carboxy-terminal globular recognition domain of C1q does not have lectin activity, but can function as a "charged" pattern recognition molecule due to the significant difference in the electrostatic surface potential of its globular domain [Gaboriaud et al. (2003) J. Biol. Chem. 278(47): 46974-46982].

[0255] C1q initiates the classical complement pathway in two distinct ways. First, the classical pathway is activated by the interaction of C1q with immune complexes (i.e., antigen-antibody complexes or aggregated IgG or IgM antibodies), so antibody-mediated humoral immune responses are associated with complement activation. When the Fab portion (variable region) of IgM or IgG binds to an antigen, the structure of the Fc (constant) region is altered, allowing C1q to bind. To be activated, C1q must bind to at least two Fc regions. However, C1q can also function in the innate or immediate immune response to infection by activating complement in the absence of antibodies. In addition to antibody initiation, complement activation is also achieved by the interaction of C1q with non-immune molecules, such as polyanions (bacterial lipopolysaccharides, DNA, and RNA), certain small polysaccharides, viral membranes, C-reactive protein (CRP), serum amyloid P element (SAP), and membrane elements of bacteria, fungi, and viruses.

[0256] C1q is part of the C1 complex, which contains a single C1q molecule bound to two molecules, the zymogens C1r and C1s. Binding of more than one C1q globular domain to a target surface (e.g., an agglutinated antibody or a pathogen) induces a conformational change in the (C1r:C1s) complex, which activates the C1r protease, cleaving C1s to generate an active serine protease. The activated C1s cleaves the subsequent complement components C4 and C2 to generate C4b and C2b, which combine to form the classical pathway C3 convertase. The C3 convertase cleaves C3 into C3b, which covalently attaches to pathogen surfaces and functions as an opsonin, and C3a, which stimulates inflammation. Some C3b molecules bind to the C4b2b complex to generate the classical cascade C5 convertase, C4b2b3b. Table 6 summarizes the proteins involved in the classical pathway of complement.

[0257] [Table 6]

[0258] b. Alternative pathway The alternative pathway is initiated by foreign pathogens in the absence of antibodies. Initiation of complement by the alternative pathway occurs through the spontaneous hydrolysis of C3 to C3b. Small amounts of C3b are always present in body fluids due to serum and tissue protease activity. Host autologous cells typically contain high levels of membrane sialic acid, which inactivates C3b upon binding, whereas bacteria contain low levels of external sialic acid, allowing C3b to bind without inactivating it. C3b on the surface of pathogens is recognized by the protease zymogen factor B. Factor B is cleaved by factor D. Factor D is the only activated protease of the complement system, circulating as an active enzyme rather than as a zymogen. However, because factor B is the only substrate for factor D, the presence of low levels of active protease in normal serum is generally safe for the host. Cleavage of factor B by factor D results in the active product Bb, which can combine with C3b to form C3bBb, the C3 convertase of the alternative pathway. Similar to the classical pathway, C3 convertase generates more C3b and C3a from C3. C3b covalently attaches to pathogen surfaces and functions as an opsonin, initiating the alternative pathway, while C3a stimulates inflammation. Some C3b combines with a complex to form C3bBb3b, the C5 convertase of the alternative pathway. C3bBb3b is stabilized by the plasma protein properdin or factor P, which binds to the surface of microorganisms and stabilizes the convertase. Table 7 summarizes the proteins involved in the alternative complement pathway.

[0259] [Table 7]

[0260] C lectin pathway The lectin pathway (also referred to as the MBL pathway) is initiated after the recognition and binding of pathogen-associated molecular patterns (PAMPs; i.e., carbohydrate moieties) by lectin proteins. Examples of lectin proteins that activate the lectin pathway of complement include mannose-binding lectin (MBL) and ficolins (i.e., L-ficolin, M-ficolin, and H-ficolin). As a member of the collectin family of proteins, MBL exists as an oligomer of identical polypeptide chain subunits, each containing a cysteine-rich, collagen-like, neck, and carbohydrate-recognition or lectin domain. MBL functions as a pattern recognition molecule that recognizes carbohydrate moieties, particularly neutral sugars such as mannose or N-acetylglucosamine (GlcNAc), on the surface of pathogens in a calcium-dependent manner via its globular lectin domain. MBL also functions as an opsonin, promoting phagocytosis of bacterial, viral, and fungal pathogens by phagocytes. Further initiators of the lectin pathway include the ficolins, including L-ficolin, M-ficolin, and H-ficolin (see, e.g., Liu et al. (2005) J Immunol. 175:3150-3156). Similar to MBL, the ficolins recognize carbohydrate moieties, such as N-acetylglucosamine and mannose structures.

[0261] Activation of the alternative pathway by MBL or ficolin is similar to activation of the classical pathway by C1q, whereby a single lectin molecule interacts with two protease zymogens. In the case of lectin proteins, the zymogens are MBL-related serine proteases, MASP-1 and MASP-2, which are closely homologous to the C1r and C1s zymogens of the classical pathway. Upon recognition of PAMPs by lectin proteins, for example, by binding to pathogen surfaces, MASP-1 and MASP-2 are activated to cleave C4 and C2 to form the MBL cascade C3 convertase. Subsequently, C3b binds to the complex to form the MBL cascade C5 convertase. MASP activation is involved not only in responses to microorganisms, but also in any response involving exposure to neutral sugars, including, but not limited to, tissue injury, such as that observed in organ transplants. Like the alternative cascades, the MBL cascade is activated independently of antibodies; like the classical cascade, the MBL cascade utilizes C4 and C2 to form C3 convertase. Table 8 summarizes the proteins involved in the lectin pathway of complement.

[0262] [Table 8]

[0263] D. Complement-mediated effector functions Regardless of which initiation pathway is used, the end result is the formation of activated fragments of complement proteins (e.g., C3a, C4a, and C5a anaphylatoxins, and the C5b-9 membrane attack complex), which serve as effector molecules that mediate diverse effector functions. Cellular recognition of complement effector molecules for initiating effector functions (e.g., chemotaxis and opsonization) is mediated by a diverse group of complement receptors. Complement receptors are distributed on a wide range of cell types, including erythrocytes, macrophages, B cells, neutrophils, and mast cells. When complement elements bind to receptors, the receptors initiate intracellular signaling cascades that result in cellular responses, such as stimulating bacterial phagocytosis and secreting inflammatory molecules from the cell. For example, complement receptors CR1 and CR2, which recognize C3b, C4b, and their products, are important for stimulating chemotaxis. CR3 (CD11b / CD18) and CR4 (CD11c / CD18) are integrins that are similarly important for phagocyte responses but also play a role in leukocyte adhesion and migration in response to iC3b. C5a and C3a receptors are G protein-coupled receptors that play a role in many of the proinflammatory functions of C5a and C3a anaphylatoxins. For example, the receptor for C3a, C3aR, is present on mast cells, eosinophils, neutrophils, basophils, and monocytes and is directly involved in the proinflammatory effects of C3a.

[0264] Thus, through complement receptors, these complement effector molecule fragments mediate several functions, including leukocyte chemotaxis, macrophage activation, vascular permeability, and cell lysis [Frank, M. and Fries, L. Complement. In Paul, W. (ed.) Fundamental Immunology, Raven Press, 1989]. A summary of some effector functions of complement products is listed in Table 9.

[0265] [Table 9]

[0266] i. Complement-mediated lysis: membrane attack complex The final step in all three complement cascade pathways is the formation of the membrane attack complex (MAC) (Figure 1). C5 can be cleaved into C5a and C5b by any of the C5 convertases. C5b combines with C6 and C7 in solution, and the C5b67 complex binds to pathogen lipid membranes via hydrophobic sites on C7. Several molecules of C8 and C9 (which also contain hydrophobic sites) bind to form the membrane attack complex, also known as C5b6789 or C5b-9. C5b-9 forms pores in the membrane that allow water and solutes to pass through, leading to osmotic lysis and cell death. If complement is activated on an antigen lacking a lipid membrane to which C5b67 can attach, the C5b67 complex can bind to nearby cells and initiate bystander lysis. A single MAC can lyse red blood cells, but unless multiple MACs are present, nucleated cells can endocytose the MACs and repair damage. Gram-negative bacteria with exposed outer membranes and enveloped viruses are generally susceptible to complement-mediated lysis. Gram-positive bacteria with a thick peptidoglycan layer protecting the plasma membrane, bacteria with a capsule or mucus layer surrounding the cell wall, or viruses without a lipid envelope are less susceptible. Similarly, MAC can be disrupted by proteins that bind to the complex before membrane insertion, such as streptococcal inhibitor of complement (SIC) and clusterin. Typically, MAC assists in the destruction of Gram-negative bacteria and human cells (virus-infected cells, tumor cells, etc.) that display foreign antigens by inducing their lysis and can also damage the envelope of enveloped viruses.

[0267] ii. Inflammation Inflammation is a process in which blood vessels dilate and become more permeable, allowing defense cells and chemicals to leave the blood and enter tissues. Complement activation leads to the formation of several proinflammatory mediators, such as C3a, C4a, and C5a. Intact anaphylatoxins in serum or plasma are rapidly converted by carboxypeptidase N to more stable, less active forms: C3a-desArg, C4a-desArg, or C5a-desArg. C3a, C4a, and C5a, and to a lesser extent their desArg derivatives, are potent bioactive polypeptides called anaphylatoxins because of their proinflammatory activity. Anaphylatoxins bind to receptors on various cell types, stimulating smooth muscle contraction, increasing vascular permeability, and activating mast cells to release inflammatory mediators. C5a, the most potent anaphylatoxin, acts primarily on leukocytes, particularly neutrophils. C5a stimulates leukocyte adhesion to blood vessel walls at sites of infection by increasing the expression of adhesion molecules, allowing leukocytes to be squeezed out of blood vessels and into tissues (a process called diarrhoea). C5a also stimulates neutrophils to generate reactive oxygen species for extracellular killing, proteolytic enzymes, and leukotrienes. C5a can also indirectly amplify the inflammatory process by inducing the production of chemokines, cytokines, and other proinflammatory mediators. C5a also interacts with mast cells, releasing vasodilators such as histamine, making blood vessels more permeable. C3a also interacts with leukocytes. Its primary effect on eosinophils suggests a role for C3a in allergic inflammation. C3a induces smooth muscle contraction, increases vascular permeability, and causes basophil degranulation and the release of histamine and other vasoactive substances. C2a can be converted to C2 kinin, which regulates blood pressure by dilating blood vessels.

[0268] Although not technically considered an anaphylatoxin, iC3b, an inactive derivative of C3b, functions to induce leukocyte adhesion to vascular endothelium and induce the production of the proinflammatory cytokine IL-1 through binding to its cell surface integrin receptors. C5b-9 also indirectly stimulates leukocyte adhesion, activation, and chemotaxis by inducing the expression of cell adhesion molecules, such as E-selectin, and by inducing interleukin-8 secretion [Bhole et al. (2003) Crit Care Med 31(1): 97-104]. C5b-9 also stimulates the release of secondary mediators that contribute to inflammation, such as prostaglandin E2, leukotriene B4, and thromboxane.

[0269] Conversion of human complement components C3 and C5 to produce their respective anaphylatoxin products has been implicated in certain naturally occurring pathological conditions, including autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, malignancies, myocardial infarction, Purtscher retinopathy, sepsis, and adult respiratory distress syndrome. Increased circulating levels of C3a and C5a have been detected in certain conditions associated with iatrogenic complement activation, such as cardiopulmonary bypass surgery, renal dialysis, and nylon fiber leukaphoresis.

[0270] iii.Chemotaxis Chemotaxis is the process by which cells are directed to migrate in response to chemicals in the environment. In the immune response, various chemokines direct the migration of cells, such as phagocytes, to sites of infection. For example, C5a is the primary chemotactic factor for circulating neutrophils, but it can also induce monocyte chemotaxis. Phagocytes migrate toward increasing concentrations of C5a and then attach, via the CR1 receptor, to C3b molecules attached to antigens. The chemotactic effects of C5a observed by basophils, eosinophils, neutrophils, and mononuclear phagocytes have been documented in the 10 -10 It is effective at concentrations as low as M.

[0271] iv. Opsonization An important function of complement is to facilitate the uptake and destruction of pathogens by phagocytes. This occurs through a process called opsonization, whereby complement elements bound to target bacteria interact with complement receptors on the surface of phagocytes, such as neutrophils or macrophages. In this instance, the complement effector molecules are called opsonins. Pathogen opsonization is the primary function of C3b and C4b. iC3b also functions as an opsonin. C3a and C5a increase the expression of C3b receptors on phagocytes, increasing their metabolic activity.

[0272] C3b, and to a lesser extent C4b, help remove harmful immune complexes from the body. C3b and C4b attach immune complexes to the CR1 receptor on red blood cells. The red blood cells then deliver the complexes to fixed macrophages in the spleen and liver for destruction. Immune complexes can lead to harmful type III hypersensitivity reactions.

[0273] v. Activation of humoral immune responses B cell activation requires ligation of the B cell receptor (BCR) by antigen. However, complement has been shown to play a role in lowering the threshold for B cell responses to antigens by up to 1,000-fold. This occurs through the binding of C3d or C3dg, a complement product generated from C3 degradation fragments, to the CR2 receptor on B-lymphocytes, where it can co-ligate with the BCR. Co-ligation occurs when an antigenic particle, such as an immune complex opsonized by C3d, binds to the CR2 receptor via C3d and to the BCR via the antigen. Co-ligation of antigen complexes can also occur when C3d binds to the antigen and enhances its uptake by antigen-presenting cells, such as dendritic cells, which can then present the antigen to B cells and enhance antibody responses. CR2-deficient mice exhibit defective B cell function, resulting in reduced levels of natural antibodies and impaired humoral immune responses.

[0274] 2. Structure and Function of C3 The mutant u-PA polypeptides provided herein inhibit complement by cleaving complement protein C3 or its proteolytic fragments. Human complement protein C3 (Uniprot Accession No. P01024) is a 1663 amino acid single-chain preproprotein having the amino acid sequence set forth in SEQ ID NO:47. The protein is encoded by a 41 kb gene (nucleotide sequence set forth in SEQ ID NO:46) located on chromosome 19. The preproprotein contains a 22 amino acid signal peptide (amino acids 1-22 of SEQ ID NO:47) and a tetra-arginine sequence (amino acids 678-681 of SEQ ID NO:47), which is removed by a furin-like enzyme to result in the formation of a mature two-chain protein containing a beta chain (amino acids 23-667 of SEQ ID NO:47) and an alpha chain (amino acids 672-1663 of SEQ ID NO:47) linked by an interchain disulfide bond between amino acid residues Cys559 and Cys816. The mature two-chain protein has the amino acid sequence set forth in SEQ ID NO:77.

[0275] During the complement cascade, complement protein C3 is further processed by proteolytic cleavage to form various C3 proteolytic fragments. As previously described, all three complement initiation pathways converge on the C3 convertases C4b2b and C3bBb. C3 convertase cleaves C3 between residues 748 and 749 of SEQ ID NO:47 (see Table 10 below) to generate the anaphylatoxin C3a (amino acids 672-748 of SEQ ID NO:47) and the opsonin C3b (C3b alpha' chain; amino acids 749-1663 of SEQ ID NO:47). C3a is involved in inflammation, and C3b forms the C5 convertase, ultimately resulting in the C5a anaphylatoxin and MAC. The mutant u-PA polypeptides provided herein inhibit complement and therefore do not cleave C3 at this GLAR cleavage site.

[0276] C3b has binding sites for various complement components, including C5, properdin (P), factors H, B, and I, complement receptor 1 (CR1), and membrane cofactor protein (MCP) [see Sahu and Lambris (2001) Immunological Reviews 180:35-48]. Binding of plasma protease factor I in the presence of cofactors H, CR1, and MCP results in inactivation of C3b, whereas binding of factors B and P in the presence of factor D results in amplification of C3 convertase and initiation of MAC. Factor I, in the presence of cofactors, cleaves C3b between residues 1303-1304, 1320-1321, and 954-955 of SEQ ID NO:47 (see Table 10 below) to generate fragments iC3b (amino acids 749-1303 of SEQ ID NO:47) and C3f (amino acids 1304-1320 of SEQ ID NO:47). Factor I then cleaves iC3b to generate C3c (C3c alpha' chain fragment 1; amino acids 749-954 of SEQ ID NO:47) and C3dg (amino acids 955-1303 of SEQ ID NO:47). The net result is that C3b is permanently inactivated [see Sahu and Lambris (2001) Immunological Reviews 180:35-48]. Because factor I inactivates C3b, the factor I cleavage site is a candidate for cleavage by the mutant u-PA polypeptides provided herein. Additional C3b proteolytic fragments include C3g (amino acids 955-1001 of SEQ ID NO:47), C3d (amino acids 1002-1303 of SEQ ID NO:47), and C3c alpha' chain fragment 2 (amino acids 1321-1663 of SEQ ID NO:47). The cleavage sequence within complement protein C3 is shown below in Table 10, which describes the P4-P1 residues, the amino acid residues at the cleavage site (the P1-P1' site), and the protease responsible for cleavage. The modified u-PA polypeptides provided herein do not cleave at these sites.

[0277] [Table 10]

[0278] a.C3a C3a (amino acids 672-748 of SEQ ID NO: 47) is an anaphylatoxin involved in inflammation, degranulation of basophils and mast cells, increased vascular permeability, smooth muscle contraction, and induction of suppressor T cells.

[0279] b.C3b C3b (amino acids 749-1663 of SEQ ID NO:47) has various roles in the complement cascade. C3b is an opsonin that promotes the uptake and destruction of pathogens by phagocytes. In addition, C3b combines with C3 convertase to generate C5 convertase, which activates complement protein C5 to generate C5a anaphylatoxin and C5b, which combine with C6, C7, C8, and C9 to form the membrane attack complex. Furthermore, as described in Section 1b above, C3b participates in the alternative pathway of complement initiation. C3b is regulated by the plasma protease complement regulatory protein factor I, which constitutively inactivates C3b by degrading it into various fragments, including iC3b, C3c, C3d, C3f, and C3dg.

[0280] C3b plays a key role in complement-mediated effector functions through its ability to bind to the C3 convertases C4b2b and C3bBb to generate the C5 convertases C4b2b3b and C3bBb3b. The C5 convertase cleaves the zymogen C5 into its active fragments, the C5a anaphylatoxin and C5b. C5a is involved in chemotaxis and inflammation, while C5b is involved in the formation of MAC.

[0281] C.C3b inhibitors C3b has binding sites for various complement components, including C5, properdin (P), factors H, B, and I, complement receptor 1 (CR1), and membrane cofactor protein (MCP) [see Sahu and Lambris (2001) Immunological Reviews 180:35-48]. Binding of the plasma protease factor I in the presence of cofactors H, CR1, and MCP results in inactivation of C3b, whereas binding of factors B and P in the presence of factor D results in amplification of C3 convertase and initiation of MAC. In the presence of cofactors, factor I cleaves C3b between residues 1303-1304, 1320-1321, and 954-955 of SEQ ID NO:47 to generate fragments iC3b (amino acids 749-1303 of SEQ ID NO:47) and C3f (amino acids 1304-1320 of SEQ ID NO:47). Although not technically considered an anaphylatoxin, iC3b, an inactive derivative of C3b, functions to induce leukocyte adhesion to vascular endothelium and induce the production of the proinflammatory cytokine IL-1 through binding to its cell surface integrin receptor. The iC3b protein functions as an opsonin. Factor I then cleaves iC3b to generate fragments C3c (C3c alpha' chain fragment 1; amino acids 749-954 of SEQ ID NO:47, and C3c alpha' chain fragment 2; amino acids 1321-1663 of SEQ ID NO:47) and C3dg (amino acids 955-1303 of SEQ ID NO:47). The net result is that C3b is permanently inactivated [see Sahu and Lambris (2001) Immunological Reviews 180:35-48]. C3dg can be further cleaved to generate fragments C3g (amino acids 955-1001 of SEQ ID NO: 47) and C3d (amino acids 1002-1303 of SEQ ID NO: 47).

[0282] D. Modified u-PA polypeptides that cleave C3 Modified or mutant urokinase-type plasminogen activator (u-PA) polypeptides are provided herein. Also provided are conjugates, such as fusion proteins, containing the modified u-PA polypeptides, whose resulting activated forms cleave C3. The modified u-PA polypeptides provided herein exhibit altered activity or properties compared to wild-type, native, or reference u-PA polypeptides. For example, the u-PA polypeptides provided herein contain modifications compared to wild-type, native, or reference u-PA polypeptides set forth in any of SEQ ID NOS: 1-6, or in polypeptides having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, particularly at least 95%, sequence identity to any of SEQ ID NOS: 1-6, such as the reference u-PA protease domain set forth in SEQ ID NOS: 5. The modified u-PA polypeptides provided herein include u-PA polypeptides that alter (inhibit) complement activation by effecting inhibitory cleavage of complement protein C3. Some modified u-PA polypeptides provided herein effect inhibitory cleavage of complement protein C3. The modified u-PA polypeptides may exhibit greater activity or specificity, K, than a corresponding form of u-PA that does not contain the modification (substitution, deletion, and / or insertion) or than a corresponding form of unmodified u-PA whose sequence is set forth in any of SEQ ID NOs: 1-6. cat / K m Also included are those that result in inhibitory cleavage of C3 by the amino acid modification. The modified u-PA polypeptides may also have reduced specificity and / or selectivity for substrates and targets cleaved or recognized by unmodified u-PA, including cleavage of plasminogen and / or binding to uPAR, compared to corresponding u-PA polypeptides that do not contain the amino acid modification.

[0283] The modified u-PA polypeptides provided herein inhibit or inactivate complement through inhibitory or inactivating cleavage of complement protein C3. The modified u-PA polypeptides provided herein inhibit or inactivate complement by cleaving complement protein C3 at a cleavage site that results in the inhibition or inactivation of C3. The inactivating or inhibitory cleavage of complement protein C3 can occur at any sequence in C3, so long as the resulting cleavage of C3 results in the inactivation or inhibition of activation of complement. Because the modified u-PA polypeptides provided herein inhibit complement activation, the modified u-PA polypeptides do not result in cleavage of the zymogen form of C3 to generate C3a and C3b activation fragments. Thus, the modified u-PA polypeptides provided herein do not cleave C3 between residues 748-749 of SEQ ID NO: 47, resulting in the generation of C3a and C3b. The inhibitory or inactivating cleavage site of complement protein C3 can be empirically determined or identified. If desired, the modified u-PA polypeptides can be tested for their ability to inhibit complement, as described below in Section E and illustrated in the Examples.

[0284] The modified u-PA polypeptides provided herein catalyze the inhibitory or inactivating cleavage of complement protein C3. They cleave complement protein C3 at any cleavage sequence, so long as the resulting C3 fragment is inactive or unable to activate complement-mediated effector functions. The modified u-PA polypeptides provided herein have altered (i.e., decreased) specificity and / or selectivity for u-PA's natural targets, including plasminogen and uPAR. In one example, the modified u-PA polypeptides provided herein have decreased specificity for cleaving plasminogen. In another example, the modified u-PA polypeptides provided herein have decreased selectivity for binding to uPAR. In some examples, the modified u-PA polypeptides provided herein have decreased specificity for cleaving plasminogen and decreased selectivity for binding to uPAR. In other examples, the modified u-PA polypeptides provided herein have increased specificity for cleaving complement protein C3 and decreased specificity for cleaving plasminogen. In other examples, the modified u-PA polypeptides provided herein have increased selectivity for complement protein C3 and decreased selectivity for plasminogen and / or uPAR.

[0285] The modified u-PA polypeptides provided herein and described in the Examples are, for example, isolated protease domains of u-PA. Smaller portions thereof that retain protease activity are also contemplated. The modified u-PA polypeptides provided herein are mutants of the protease domain of u-PA, particularly modified u-PA polypeptides in which a free Cys residue in the protease domain (i.e., one that does not form a disulfide bond with any other Cys residue in the protein) has been replaced with another amino acid substitution, preferably a conservative amino acid substitution or a substitution that does not eliminate activity, such as, for example, substitution with serine, and modified u-PA polypeptides in which a glycosylation site has been eliminated. Modified u-PA polypeptides with other conservative amino acid substitutions that retain catalytic activity are also contemplated (e.g., see Table 3 for exemplary amino acid substitutions).

[0286] The modified u-PA polypeptides provided herein contain one or more amino acid modifications such that they cleave complement protein C3 in a manner that results in complement inactivation or inhibition. The modifications can be single amino acid modifications, such as a single amino acid substitution (substitution), insertion, or deletion, or multiple amino acid modifications, such as multiple amino acid substitutions, insertions, or deletions. Exemplary modifications are amino acid substitutions, including single or multiple amino acid substitutions. The amino acid substitutions can be conservative substitutions as shown in Table 3 or non-conservative substitutions, such as any described herein. The modified u-PA polypeptides provided herein can contain at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more modified positions compared to u-PA polypeptides that do not contain the modifications.

[0287] The modifications described herein can be made in any u-PA polypeptide.For example, modifications may be made to a human u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or an allelic variant thereof; a mouse u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:52 or 66; a rat u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:53 or 67; a bovine u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:54 or 68; a porcine u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:55 or 69; a rabbit u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:56 or 70; a chicken u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:57 or 71; a yellow baboon u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:58 or 72; or a baboon u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:59 or 73. a Sumatran orangutan u-PA polypeptide having the sequence of amino acids set forth; a canine u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO: 60 or 74; an ovine u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO: 61 or 75; a marmoset u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO: 62; a rhesus monkey u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO: 63; a rhesus monkey u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO: 64 or a northern white-cheeked gibbon u-PA polypeptide having the sequence of amino acids set forth therein; and a chimpanzee u-PA polypeptide comprising or having the sequence of amino acids set forth in SEQ ID NO:65, or in sequence variants or catalytically active fragments exhibiting at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:1-6 and 52-75.

[0288] The modified u-PA polynucleotides provided herein can be modified in any region or domain of the modified u-PA polynucleotides provided herein, so long as the modified u-PA polynucleotide retains its ability to effect inactivating or inhibitory cleavage of complement protein C3. The modified u-PA polynucleotides provided herein can be single- or double-chain polypeptides, splice variants, allelic variants, isoforms, or catalytically active fragments thereof, such as the protease domain thereof. The u-PA polynucleotides provided herein can be full-length or truncated u-PA polypeptides. The modified u-PA polynucleotides provided herein can be the protease domain of u-PA or modified forms of the protease domain of u-PA. Also contemplated for use herein are zymogen, precursor, or mature forms of modified u-PA polypeptides, provided the u-PA polynucleotides retain their ability to effect inhibitory or activating cleavage of complement protein C3. Modifications in u-PA polynucleotides can also be made to u-PA polypeptides that also contain other modifications, including modifications of the primary sequence and modifications not within the primary sequence of the polypeptide. For example, the modifications described herein can be present in u-PA polypeptides that are fusion or chimeric polypeptides. The modified u-PA polypeptides provided herein also include polypeptides that are conjugated to polymers such as PEG reagents.

[0289] For purposes herein, references herein to positions and amino acids with respect to modifications, including amino acid substitution(s), are with respect to the u-PA polypeptide set forth in any of SEQ ID NOS: 1-6. It is within the level of ordinary skill in the art to make any of the modifications provided herein in another u-PA polypeptide by identifying the corresponding amino acid residue in another u-PA polypeptide, such as a variant thereof that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the u-PA polynucleotide set forth in any of SEQ ID NOS: 1-6 or the u-PA polypeptide set forth in any of SEQ ID NOS: 1-6. The corresponding position in another u-PA polypeptide can be identified by alignment of the u-PA polypeptide with a reference u-PA polypeptide set forth in any of SEQ ID NOS: 1-6. For purposes of modification (e.g., amino acid substitution), the corresponding amino acid residue can be any amino acid residue and need not be identical to the residue set forth in any of SEQ ID NOS: 1-6. Typically, a corresponding amino acid residue identified, for example, by alignment with a residue in SEQ ID NOS: 5 will be an amino acid residue that is identical to SEQ ID NOS: 5 or a conservative or semi-conservative amino acid residue thereto. It will also be understood that exemplary substitutions provided herein can be made with the corresponding residue in a u-PA polypeptide, such as the protease domain of u-PA, so long as the substitution is different from that present in an unmodified form of the u-PA polypeptide, such as the protease domain of u-PA. Based on this description and the descriptions elsewhere herein, it is within the level of ordinary skill in the art to generate modified u-PA polypeptides containing any one or more of the described mutations and test each for a property or activity as described herein.

[0290] The modified u-PA polypeptides provided herein alter complement activity through proteolytic inhibition or inactivation of complement protein C3. Furthermore, the modified u-PA polypeptides provided herein have reduced specificity for cleaving plasminogen and / or binding to uPAR. For example, the u-PA polypeptides provided herein exhibit less than 100% of the wild-type activity of the u-PA polypeptide for cleaving plasminogen, such as less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activity of a wild-type or reference u-PA polypeptide, such as a corresponding polypeptide that does not contain the amino acid modification, for cleaving plasminogen. In another example, the modified u-PA polypeptides provided herein exhibit less than 100% of the wild-type binding activity of the u-PA polypeptide to uPAR, such as less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activity of a wild-type or reference u-PA polypeptide, such as a corresponding polypeptide that does not contain the amino acid modification, for binding to uPAR.

[0291] Also provided herein are nucleic acid molecules encoding any of the modified u-PA polypeptides provided herein. In some instances, the encoding nucleic acid molecules can also be modified to contain a heterologous signal sequence to alter (e.g., increase) the expression and secretion of the polypeptide. The modified u-PA polypeptides and encoding nucleic acid molecules provided herein can be produced or isolated by any method known in the art, including isolation from natural sources, isolation of recombinantly produced proteins in cells, tissues, and organisms, by recombinant methods, methods involving in silico steps, synthetic methods, and methods known to those skilled in the art. The modified polypeptides and encoding nucleic acid molecules provided herein can be produced by standard recombinant DNA techniques known to those skilled in the art. Any method known in the art for producing any one or more amino acid mutations in a target protein can be used. Methods include standard site-directed or random mutagenesis of the encoding nucleic acid molecule or solid-phase polypeptide synthesis. For example, nucleic acid molecules encoding u-PA polypeptides can be subjected to mutagenesis, such as random mutagenesis of the encoding nucleic acid, error-prone PCR, site-directed mutagenesis, overlap PCR, gene shuffling, or other recombinant methods. The nucleic acid encoding the polypeptide can then be introduced into a host cell and heterologously expressed. Thus, nucleic acid molecules encoding any of the modified polypeptides provided herein are also provided herein. In some examples, the modified u-PA polypeptide is synthetically produced, for example, using solid-phase or solution-phase peptide synthesis. The nucleic acid molecule can be provided in a gene therapy vector, such as an AAV or adenovirus vector, for expression of the encoded modified u-PA polypeptide in vivo, for example, for intraocular or systemic administration. The encoded u-PA polypeptide can be a full-length polypeptide or a protease domain or other form that is active or can be activated.

[0292] The u-PA polypeptides provided herein have been modified to have increased specificity and / or selectivity for cleaving the inhibitory cleavage or inactivation cleavage sequence of complement protein C3. The u-PA polypeptides can be modified using any method known in the art for modifying proteins. These methods include site-directed mutagenesis and random mutagenesis. The biological function of modified u-PA polypeptides can be evaluated using assays for the biological function of complement activation, such as those provided herein and known in the art, to determine whether the modified u-PA polypeptide targets complement protein C3 for cleavage and inactivation. Exemplary methods for identifying u-PA polypeptides and modified u-PA polypeptides are provided herein.

[0293] 1. Exemplary Modified u-PA Polypeptides Provided herein are modified u-PA polypeptides that contain one or more amino acid modifications, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid modifications, in the u-PA polypeptide, such that complement is inhibited or inactivated, and that cleave the complement protein C3. The modifications are present in the primary amino acid sequence and include substitutions, deletions, and insertions of amino acid residues. The modifications alter the specificity / activity of the u-PA polypeptide when in its active form. The modified u-PA polypeptides herein are selected to recognize and cleave target sites in complement proteins, particularly C3, to inactivate it. The modified u-PA polypeptides herein can also be further modified and screened to have reduced specificity / activity toward in vivo substrates, such as plasminogen. The modified u-PA polypeptides herein can be selected and identified by any suitable protease screening method. The modified u-PA polypeptides herein were initially identified using the screening method described in U.S. Pat. No. 8,211,428, in which a library of modified proteases is reacted with cognate or other inhibitory serpins that have been modified to contain target sequences in the reactive site loop to capture modified proteases that cleave such targets.

[0294] The modified u-PA polypeptides provided herein have increased activity or specificity or K for the complement protein C3 at the site that inactivates C3. cat / K mand may have reduced activity or specificity for plasminogen and / or exhibit increased selectivity, specificity, and / or activity for the target site complement protein C3, whereby the modified u-PA polypeptide inactivates C3. The modified u-PA polypeptide exhibits increased activity for cleaving and inactivating C3 compared to wild-type or a corresponding form of the wild-type with the substitution C122S (according to chymotrypsin numbering). In particular, the protease domain of the modified u-PA polypeptide exhibits increased inactivating cleavage activity for C3 compared to the u-PA protease domain of SEQ ID NO: 5 (u-PA protease domain with C122S). The increase in activity can be 10%, 20%, 50%, 100%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, and more compared to unmodified u-PA.

[0295] The modified u-PA polypeptides may have reduced activity toward natural substrates, such as plasminogen. For example, the modified u-PA polypeptides may exhibit 0-99% of the u-PA activity toward plasminogen, and at least 0.5-fold, 1-fold, 2-fold, 3-fold, or more, of the u-PA activity toward cleaving and inactivating C3, of a wild-type or reference u-PA polypeptide, such as the u-PA polypeptide set forth in SEQ ID NO: 5. For example, the modified u-PA polypeptides provided herein exhibit less than about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of the u-PA activity of a corresponding polypeptide that does not contain the amino acid modification (e.g., amino acid substitution), e.g., a wild-type or reference u-PA polypeptide, such as the u-PA protease domain set forth in SEQ ID NO: 5.

[0296] For example, exemplary positions that may be modified by amino acid substitution or substitution include, but are not limited to, any of the positions corresponding to positions 173, 178, 179, 180, 181, 185, 186, 187, 188, 208, 209, 249, 250, 252, 306, 314 or 353 (corresponding to positions 30, 35, 36, 37, 37a, 38, 39, 40, 41, 60a, 60b, 97a, 97b, 99, 149, 157 or 192 according to chymotrypsin numbering) with respect to the sequence of amino acids set forth in SEQ ID NO:3. For example, the amino acid positions may be substitutions at positions corresponding to substitutions of phenylalanine (F) at one or more of positions 173, R178, R179, H180, R181, V185, T186, Y187, V188, D208, Y209, T249, L250, H252, Y306, M314 or Q353 with respect to the amino acid positions set forth in SEQ ID NO: 3 (corresponding to F30, R35, R36, H37, R37a, V38, T39, Y40, V41, D60a, Y60b, T97a, L97b, H99, Y149, M157 and Q192, respectively, according to chymotrypsin numbering).

[0297] Exemplary amino acid substitutions at any of the above positions are shown in Table 11. References to corresponding positions in Table 11 are relative to the positions shown in SEQ ID NO:3. (See also the Examples below.) It will be understood that substitutions can be made at corresponding positions in another u-PA polypeptide by alignment with the sequence shown in SEQ ID NO:3, whereby the corresponding positions are the aligned positions. For example, substitutions can be made in the u-PA protease domain with the sequence shown in SEQ ID NO:2, or in the reference u-PA protease domain with the sequence shown in SEQ ID NO:5. In some examples, amino acid substitutions may occur at corresponding positions in a u-PA polypeptide as set forth in SEQ ID NO: 5 or a variant thereof having at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, particularly 95% or more sequence identity thereto, so long as the resulting modified u-PA polypeptide exhibits altered (e.g., enhanced) specificity and / or altered selectivity for complement protein C3 compared to u-PA activity towards plasminogen. In one example, any one or more of the substitutions may be present in any of SEQ ID NOs: 1-6, so long as the resulting modified u-PA polypeptide exhibits altered (e.g., enhanced) specificity for complement protein C3 and / or altered selectivity for complement protein C3 compared to u-PA activity towards plasminogen.

[0298] [Table 11]

[0299] Exemplary amino acid modifications in the modified u-PA polypeptides provided herein include a tyrosine (Y) at a position corresponding to position 173 (30, according to chymotrypsin numbering); a W at a position corresponding to position 178 (35, according to chymotrypsin numbering); a Y at a position corresponding to position 178; a Q at a position corresponding to position 178; an H at a position corresponding to position 179 (36, according to chymotrypsin numbering); an E at a position corresponding to position 180 (37, according to chymotrypsin numbering); a P at a position corresponding to position 180; D at position 180; N at position 180; G at position 180; K at position 180; Y at position 180; Q at position 181 (37a according to chymotrypsin numbering); P at position 181; E at position 181; N at position 181; S at position 181; D at position 185 (38 according to chymotrypsin numbering); E at position 185; and Q at position 186 (39 according to chymotrypsin numbering). W at position 186; Y at position 186; F at position 186; H at position 187 (40 according to chymotrypsin numbering); F at position 187; Q at position 187; R at position 188 (41 according to chymotrypsin numbering); L at position 188; P at position 208; T at position 208 (60a according to chymotrypsin numbering); Q at position 209 (60b according to chymotrypsin numbering); H at position corresponding to position 209; S at position corresponding to position 209; A at position corresponding to position 209; T at position corresponding to position 209; L at position corresponding to position 209; E at position corresponding to position 249 (97a, according to chymotrypsin numbering); I at position corresponding to position 249; A at position corresponding to position 250 (97b, according to chymotrypsin numbering); G at position corresponding to position 250; Q at position corresponding to position 252 (99, according to chymotrypsin numbering); K at position corresponding to position 306 (149, according to chymotrypsin numbering);Substitutions include, but are not limited to, R at a position corresponding to position 306; K at a position corresponding to position 314 (157 according to chymotrypsin numbering); or H at a position corresponding to position 353 (192 according to chymotrypsin numbering), each with reference to the amino acid positions set forth in SEQ ID NO: 3. S at a position corresponding to position 279 (122S according to chymotrypsin numbering) replaces a free Cys, thereby reducing the tendency to aggregate.

[0300] Exemplary modified u-PA polypeptides containing two or more amino acid modifications are shown below in Table 12, and their activity to cleave C3 is set forth in Table 14. The SEQ ID NOs refer to exemplary u-PA protease domains containing the listed substitutions, including a substitution at C122S to reduce or eliminate aggregation. C122 is a free cysteine, which can result in cross-linking between protease polypeptides. It will be understood that the protease domains are exemplary, and that full-length and precursor molecules, as well as other catalytically active portions of the protease domain, full-length and precursor polypeptides, can contain the listed substitutions to form full-length, activated modified u-PA polypeptides and other forms.

[0301] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

[0302] 2. Further modifications Any of the modified u-PA polypeptides provided herein may contain any one or more additional modifications. Additional modifications may include, for example, any amino acid substitution, deletion, or insertion known in the art, typically any that increase specificity and / or alter selectivity for complement protein C3 compared to u-PA activity toward plasminogen. Modifications that alter any other activity of interest are also contemplated. Additional amino acid modifications of primary sequence have long been known [see, e.g., Wells (1990) Biochem 29:8509-8517]. Any modified u-PA polypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications to provide additional or altered activity.

[0303]

[0304] Modified u-PA polypeptides include those containing chemical or post-translational modifications. In some examples, the modified u-PA polypeptides provided herein contain neither chemical nor post-translational modifications. Chemical and post-translational modifications include, but are not limited to, pegylation, sialylation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, PASylation, HESylation, phosphorylation, conjugation to a multimerization domain such as Fc, and other polypeptide modifications known in the art. In addition to any one or more amino acid modifications provided herein, such as amino acid substitutions, insertions, deletions, and combinations thereof, the modified u-PA polypeptides provided herein can be conjugated or fused to any moiety using any method known in the art, including chemical and recombinant methods, provided that the resulting polypeptide retains the ability to cause inhibitory or inactivating cleavage of complement protein C3 when in its active form.

[0305] For example, in addition to any one or more amino acid modifications, such as amino acid substitutions, provided herein, the modified u-PA polypeptides provided herein can also contain other modifications, whether present or absent in the primary sequence of the polypeptide, including, but not limited to, carbohydrate moieties, polyethylene glycol (PEG) moieties, sialylation moieties, the Fc domain from immunoglobulin G, or any other domain or moiety. For example, such additional modifications can be made to increase the stability or serum half-life of the protein.

[0306] Reduced immunogenicity The modified u-PA polypeptides provided herein can be modified to have reduced immunogenicity. Reduced immunogenicity can be achieved by sequence changes that eliminate antigenic epitopes from the polypeptide or by post-translational modifications. Those skilled in the art are familiar with methods for identifying antigenic epitopes in polypeptides (see, for example, Liang et al. (2009) BMC Bioinformatics, 10:302; Yang et al. (2009) Rev. Med. Virol., 19:77-96). In some instances, one or more amino acids can be modified to remove or alter antigenic epitopes. In another example, altering the glycosylation of a protein can also affect immunogenicity. For example, altering the glycosylation of a polypeptide is contemplated as long as the polypeptide retains the ability to cause inhibitory or inactivating cleavage of complement protein C3. A glycosylation site can be removed by a single mutation. Glycosylation sites can be added by introducing a canonical sequence by insertion or single or multiple mutations, such as, for example, NXS(T) (where X is not proline). Glycosylation sites can also increase serum half-life.

[0307] b. Fc domain The modified u-PA polypeptide can be linked to the Fc region of an immunoglobulin polypeptide. Typically, such a fusion involves linking at least a functionally active hinge, C, or Fc region of the constant region of an immunoglobulin heavy chain. H 2 domain and C H For example, the full-length Fc sequence of IgG1 contains amino acids 99 to 330 of the sequence shown in SEQ ID NO:45 below. Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330

[0308] An exemplary Fc sequence for hIgG1 is shown in SEQ ID NO: 50: Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1 5 10 15 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230

[0309] The full-length Fc sequence of IgG1 includes a hinge sequence corresponding to amino acids 100-110 of SEQ ID NO: 45, a C sequence as shown in SEQ ID NO: 45, H 2 domain and C H It contains almost all of the complete sequences for the three domains.

[0310] Another exemplary Fc polypeptide is shown in PCT Publication No. 93 / 10151 and is a single-chain polypeptide (SEQ ID NO: 50) extending from the N-terminal hinge region to the natural C-terminus of the Fc region of a human IgG1 antibody. The exact site at which the linkage is made is not critical: particular sites are well known and can be selected to optimize the biological activity, secretion, or binding properties of the HABP polypeptide. For example, other exemplary Fc polypeptide sequences begin at amino acid C109 or P113 of the sequence shown in SEQ ID NO: 45 (see, e.g., U.S. Patent Application Publication No. 2006 / 0024298).

[0311] In addition to hIgG1 Fc, other Fc regions and other multimerization domains can also be used. For example, when effector functions mediated by Fc / FcγR interactions are minimized, fusions with IgG isotypes that poorly recruit complement or effector cells, such as IgG2 or IgG4 Fc, are contemplated. Furthermore, Fc fusions can be substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including, but not limited to, the IgG (including human subclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, and IgM classes of antibodies. A linker can be used to covalently link the Fc to another polypeptide to generate an Fc chimera.

[0312] Modified Fc domains are also well known. In some instances, an Fc region is modified so that it exhibits altered binding to FcR, resulting in altered (i.e., more or less) effector function than that of the Fc region of a wild-type immunoglobulin heavy chain. Thus, the modified Fc domain may have altered affinity, including, but not limited to, increased affinity, or low or no affinity for the Fc receptor. For example, different IgG subclasses have different affinities for FcγR, with IgG1 and IgG3 typically binding to the receptor substantially better than IgG2 and IgG4. Different FcγRs mediate different effector functions. FcγR1, FcγRIIa / c, and FcγRIIIa are positive regulators of immune complexes that trigger activation, characterized by intracellular domains with immunoreceptor tyrosine-based activation motifs (ITAMs). However, FcγRIIb has an immunotyrosine-based inhibitory motif (ITIM) and is therefore inhibitory. By altering the affinity of the Fc region for its receptor, the effector functions and / or pharmacokinetic properties associated with the Fc domain can be modulated. Modified Fc domains are known to those skilled in the art and have been described in the literature; see, e.g., U.S. Patent No. 5,457,035, U.S. Patent Application No. 2006 / 0024298, and International Patent Application Publication No. 2005 / 063816 for exemplary modifications.

[0313] The resulting chimeric polypeptides containing the Fc portion, and multimers formed therefrom, can be readily purified by affinity chromatography on protein A or protein G columns.

[0314] In another example, the modified u-PA polypeptide can be linked to human serum albumin (HSA), such as residues 25-608 of HSA, or a full length or portion thereof. [ka]

[0315] c. Conjugation to polymers In some examples, the modified u-PA polypeptides provided herein are conjugated to other polymers. The polymers can increase the size of the polypeptide, reducing renal clearance and thereby increasing half-life, or modify the structure of the polypeptide, increasing half-life or reducing immunogenicity. Exemplary polymers that can be conjugated to u-PA polypeptides include natural and synthetic homopolymers such as polyols (i.e., poly-OH), polyamines (i.e., poly-NH), and polycarboxylic acids (i.e., poly-COOH), as well as other heteropolymers, i.e., polymers containing one or more different coupling groups, such as hydroxyl and amino groups. Examples of suitable polymer molecules include polyalkylene oxides (PAOs), such as polyalkylene glycols, including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG), and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic anhydride, and polystyrene-co-maleic acid. Anhydrides, dextrans including carboxymethyl-dextran, heparin, homologous albumin, celluloses including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose, chitosan hydrolysates, starches such as hydroxyethyl starch and hydroxypropyl starch, glycogen, agaroses and their derivatives, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates and biopolymers.

[0316] Typically, the polymers are polyalkylene oxides (PAOs), such as polyethylene oxides, e.g., PEG, usually mPEG, which have few reactive groups capable of cross-linking. Typically, the polymers are non-toxic polymer molecules, such as (methoxy)polyethylene glycol (mPEG), that can be covalently conjugated to u-PA polypeptides (e.g., to binding groups on the protein surface) using relatively simple chemistry.

[0317] Suitable polymer molecules for attachment to u-PA polypeptides include, but are not limited to, polyethylene glycol (PEG) and PEG derivatives, such as methoxy-polyethylene glycols (mPEG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, and polyethylene oxide (PEO) [see, e.g., Roberts et al., Advanced Drug Delivery Review 2002, 54: 459-476; Harris and Zalipsky (eds.); "Poly(ethylene glycol), Chemistry and Biological Applications" ACS Symposium Series 680, 1997; Mehvar et al., J. Pharm. Pharmaceut. Sci., 3(1):125-136, 2000; Harris and Chess (2003) Nat Rev Drug Discov. 2(3):214-21; and Tsubery, J. Biol. Chem 279(37):38118-24, 2004]. Polymer molecules can typically have a molecular weight ranging from about 3 kDa to about 60 kDa. In some embodiments, polymer molecules conjugated to U-PA polypeptides provided herein have a molecular weight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 kDa, or greater than 60 kDa.

[0318] Methods for modifying polypeptides by covalently attaching (conjugating) PEG or PEG derivatives (i.e., "PEGylation") are well known in the art (see, e.g., U.S. Patent Application Publication No. 2006 / 0104968, U.S. Patent Nos. 5,672,662, 6,737,505, and U.S. Patent Application Publication No. 2004 / 0235734). Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see, e.g., Harris, Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiple PEG moieties to a single conjugation site (e.g., by use of branched PEGs, see, e.g., Veronese et al., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specific PEGylation and / or mono-PEGylation (see, e.g., Chapman et al., Nature Biotech. 17:780-783, 1999), and site-specific enzymatic PEGylation (see, e.g., Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002) [see also, e.g., Lu and Felix (1994) Int. J. Peptide Protein Res. 43:127-138; Lu and Felix (1993) Peptide Res. 6:142-6, 1993; Felix et al. (1995) Int. J. Peptide Res. 46:253-64; Benhar et al. (1994) J. Biol. Chem. 269:13398-404; Brumeanu et al. (1995) J Immunol. 154:3088-95; see also Caliceti et al. (2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt 2):3S-8S].Methods and techniques described in the art are capable of producing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 PEG or PEG derivatives attached to a single protein molecule (see, e.g., U.S. Patent Application Publication No. 2006 / 0104968).

[0319] Numerous reagents for PEGylation have been described in the art, including N-hydroxysuccinimidyl (NHS)-activated PEG, succinimidyl mPEG, mPEG 2-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester, homobifunctional PEG succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzoate ... Examples of suitable conjugates include, but are not limited to, benzotriazole carbonate, propionaldehyde PEG, mPEG butyraldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methyl ketone, mPEG "linkerless" maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridyl thioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinyl sulfone PEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see, e.g., Monfardini et al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J.Bioactive Compatible Polymers 12:197-207, 1997; US Patent No. 5,672,662; US Patent No. 5,932,462; US Patent No. 6,495,659; US Patent No. 6,737,505; US Patent No. 4,002,531; US ​​Patent No. 4,179,337; US Patent No. 5,122, No. 614; No. 5,183,550; No. 5,324,844; No. 5,446,090; No. 5,612,460; No. 5,643,575; No. 5,766,581; No. 5,795,569 ; Same No. 5,808,096; Same No. 5,900,461; Same No. 5,919,455; Same No. 5,985,263; Same No. 5,990,237; Same No. 6,113,906; Same No. 6,214,966; Same No. 6,258,351; 6,340,742; 6,413,507; 6,420,339; 6,437,025; 6,448,369; 6,461,802; 6,8 28,401; 6,858,736; U.S. Patent Application Publication Nos. 2001 / 0021763; 2001 / 0044526; 2001 / 0046481; 2002 / 0052430; 2002 / 0072573; 2002 / 0156047; 2003 / 0114647; 2003 / 0143596; 2003 / 0158333; 2003 / 0220447; See, e.g., 2004 / 0013637; 2004 / 0235734; 2005 / 000360; 2005 / 0114037; 2005 / 0171328; 2005 / 0209416; EP 01064951; EP 0822199; WO 00176640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925.

[0320] d. Protein transduction domain The modified u-PA polypeptides provided herein, such as fusion proteins containing antibodies or antigen-binding fragments thereof, can be linked and conjugated to a protein transduction domain (PTD) that increases retention of the antibody at a target site for therapy, such as a mucosal site, such as the eye. Any PTD can be used, so long as the PTD promotes binding to the surface of target cells at the treatment site (e.g., a mucosal site) and / or uptake of the modified u-PA polypeptide by target cells at the treatment site (e.g., a mucosal site, such as the eye).

[0321] Generally, PTDs comprise short cationic peptides that can bind to cell surfaces through electrostatic binding to the cell membrane and can be taken up by cells by membrane translocation [Kabouridis (2003) TRENDS Biotech 21(11) 498-503]. The provided PTDs generally interact with target cells via binding to glycosaminoglycans (GAGs), such as, for example, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate, keratin sulfate, or chondroitin sulfate and their derivatives.

[0322] A protein transduction domain can be of any length. Generally, the length of a PTD ranges from 5 or about 5 to 100 or about 100 amino acids in length. For example, the length of a PTD can range from 5 or about 5 to 25 or about 25 amino acids in length. In some examples, the PTD is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0323] A single PTD or multiple PTDs can be conjugated to a modified u-PA polypeptide. These can be advantageously used to treat eye diseases or ophthalmic diseases, such as diabetic retinopathy or macular degeneration, including AMD. For example, multiple copies of the same PTD (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, or higher multimers) or different PTDs can be conjugated to a modified u-PA polypeptide.

[0324] Several proteins and their peptide derivatives possess cellular internalization properties. Exemplary PTDs are known in the art, such as the human immunodeficiency virus 1 (HIV-1) TAT [SEQ ID NOS: 125-135; Ruben et al. (1989) J. Virol. 63:1-8], the herpesvirus tegument protein VP22 [SEQ ID NOS: 140; Elliott and O'Hare (1997) Cell 88:223-233], the homeotic protein of the Drosophila melanogaster Antennapedia (Antp) protein [Penetratin PTD; SEQ ID NOS: 112; Derossi et al. (1996) J. Biol. Chem. 271:18188-18193], protegrin 1 (PG-1) antimicrobial peptide SynB [e.g., SynB1 (SEQ ID NO: 121), SynB3 (SEQ ID NO: 122), and SynB4 (SEQ ID NO: 123); Kokryakov et al. (1993) FEBS Lett. 327:231-236], and Kaposi's fibroblast growth factor [SEQ ID NO: 105; Lin et al., (1995) J. Biol. Chem. 270-14255-14258], but are not limited to the PTDs listed in the following table, including PTDs derived from:

[0325] Other proteins and their peptide derivatives have been found to have similar cellular internalization properties. The carrier proteins derived from these proteins show little sequence homology with each other, and are all highly cationic and arginine- or lysine-rich. In fact, synthetic polyarginine peptides have been shown to be internalized with high levels of efficacy and can be selected for conjugation to the provided antibodies [Futaki et al. (2003) J. Mol. Recognit. 16:260-264; Suzuki et al. (2001) J. Biol. Chem. 276:5836-5840]. The PTD can also be selected from one or more synthetic PTDs, including, but not limited to, transportan [SEQ ID NO: 136; Pooga et al. (1988) FASEB J. 12:67-77; Pooga et al. (2001) FASEB J. 15:1451-1453], MAP [SEQ ID NO: 103; Oehlke et al. (1998) Biochim. Biophys. Acta. 1414:127-139], KALA [SEQ ID NO: 101; Wyman et al. (1997) Biochemistry 36:3008-3017], and other cationic peptides such as, for example, various β-cationic peptides [Akkarawongsa et al. (2008) Antimicrob. Agents and Chemother. 52(6):2120-2129]. Additional PTD peptides and mutant PTDs are also provided, for example, in U.S. Patent Application Publication Nos. 2005 / 0260756, 2006 / 0178297, 2006 / 0100134, 2006 / 0222657, 2007 / 0161595, 2007 / 0129305, European Patent Application Publication No. 1867661, PCT Application Publication Nos. 2000 / 062067, 2003 / 035892, 2007 / 097561, 2007 / 053512, and in Table 13 herein (infra). Any such PTD provided herein or known in the art can be conjugated to the provided therapeutic antibodies.

[0326] [Table 13-1] [Table 13-2] [Table 13-3]

[0327] In some instances, the PTD may be modified by substitution of a lysine or arginine with another basic amino acid, for example, substitution of a lysine with an arginine, or substitution of an arginine with a lysine.

[0328] E. Assays for assessing or monitoring u-PA activity in relation to complement-mediated functions The modified u-PA polypeptides provided herein exhibit altered specificity and / or selectivity for complement protein C3. Exemplary modified u-PA polypeptides specifically cleave complement protein C3, thereby altering complement activation. Furthermore, exemplary modified u-PA polypeptides provided herein have altered or reduced specificity and / or selectivity for cleaving natural substrates of u-PA, such as plasminogen, and binding to uPAR.

[0329] Various in vitro and in vivo assays can be used to monitor or screen u-PA polypeptides for their ability to cleave complement protein C3 and their effects on complement activation and complement-mediated diseases and disorders. Such assays are well known to those skilled in the art. Those skilled in the art can test specific u-PA polypeptides for cleavage of complement protein C3 and / or examine to assess any changes in the effect of u-PA on complement-mediated activity compared to the absence of protease. Several such assays are exemplified herein.

[0330] Exemplary in vitro and in vivo assays are provided herein for comparing the activity of modified u-PA polypeptides with respect to the function of complement protein C3. As discussed above, numerous assays, such as assays for measuring complement activation, are well known to those skilled in the art. Also provided herein are exemplary assays for determining the activity of modified u-PA polypeptides relative to wild-type u-PA activity, such as cleavage of plasminogen or binding to uPAR. Also provided are assays for determining the specificity of modified u-PA polypeptides relative to complement protein C3. Exemplary assays are described below.

[0331] 1. Methods for assessing u-PA activity in relation to the function of complement protein C3 The modified u-PA proteases can exhibit altered specificity and / or selectivity for any one or more complement proteins, thereby inactivating any one or more complement proteins, such as, for example, C3, compared to the corresponding full-length, scaffold, or wild-type forms of the modified u-PA proteases. The modified u-PA proteases retain their protease activity but can exhibit increased specificity and / or selectivity for any one or more complement proteins. Exemplary modified u-PA proteases specifically cleave any one or more complement proteins, such as, for example, C3, thereby altering the activity of the complement proteins. All such modified u-PA proteases with increased specificity and / or selectivity for any one or more complement proteins are candidate therapeutic agents.

[0332] If the modified u-PA protease exhibits increased specificity and / or selectivity for any one or more complement proteins, in vitro and in vivo assays can be used to monitor or screen the protease for its effect on complement-mediated function. Such assays are well known to those skilled in the art. For example, those skilled in the art can test the modified u-PA protease for cleavage of any one or more complement proteins, such as C3, and / or can test to assess any changes in the effect of the modified u-PA protease on complement-mediated activity compared to the absence of the modified u-PA protease. Several such assays are exemplified herein.

[0333] Exemplary in vitro and in vivo assays are provided herein for comparing the activity of modified u-PA proteases with the function of any one or more targeted complement proteins. Many of the assays are applicable to other proteases and modified proteases. As discussed above, assays for complement activity include, but are not limited to, assays that measure the production of any one or more activation products of complement activation, such as the C5b-9 MAC complex, and complement cleavage products, such as C4a, C5a, C3b, and C3d. Assays for measuring complement activation also include functional assays that measure the functional activity of specific components of the complement pathway, such as hemolytic assays used to measure activation of any one of the classical, lectin, or alternative pathways. Assays for assessing the effects of proteases and modified proteases on complement proteins and / or complement-mediated functions include, but are not limited to, SDS analysis followed by Western blot or Coomassie brilliant blue staining, enzyme immunoassays, and hemolytic assays. In one example, an in vitro assay can be performed using purified complement proteins. In another example, an in vivo assay can be performed by testing serum from mammalian or species, including human species, for functional activation of complement. Exemplary assays are described below.

[0334] In one example, in vitro assays can be performed using purified complement protein C3, as exemplified in Examples 2-4. In another example, in vitro assays can be performed in a physiologically relevant solution (i.e., vitreous humor), as exemplified in Example 5. In another example, in vitro assays can be performed using peptide libraries to assess cleavage specificity. In another example, assays can be performed to assess the normal function of modified u-PA polypeptides, i.e., activity against normal substrates. Various disease models known to those skilled in the art can be used to test the effectiveness of the u-PA polypeptides provided herein for various complement-mediated diseases and disorders.

[0335] Protein detection Protein detection is a means of measuring individual complement components in a sample. Complement proteins can be detected to directly assess the effect of u-PA polypeptide on the cleavage of complement protein C3, or complement proteins can be measured as a means of assessing complement activation. Complement protein C3 treated with or without u-PA polypeptide can be analyzed by any one or more assays, including SDS-PAGE followed by Coomassie staining or Western blot, enzyme immunoassay, immunohistochemistry, flow cytometry, nephelometry, agar gel diffusion, or radial immunodiffusion. Exemplary assays for protein detection are described below.

[0336] i.SDS-PAGE analysis Complement protein analysis in the presence or absence of increasing concentrations of u-PA polypeptide can be performed by analyzing the proteins on SDS-PAGE followed by detection of the proteins. In such examples, complement proteins can be detected by staining total proteins, for example, by Coomassie Brilliant Blue staining, silver staining, or any other method known to those skilled in the art, or by Western blotting using polyclonal or monoclonal antibodies specific for a particular protein. Typically, purified complement proteins, such as complement protein C3, can be incubated in the presence or absence of u-PA polypeptide. The treated complement proteins can be separated on an SDS-PAGE gel, followed by methods to detect the proteins in the gel, for example, by staining with Coomassie Brilliant Blue. The treated proteins can be compared with their cognate full-length proteins, and degradation products formed by protease cleavage of the proteins can be determined.

[0337] In another embodiment, samples, such as human serum or plasma, can be treated with or without u-PA polypeptides or collected after treating animals or humans with or without u-PA polypeptides. Samples treated with u-PA can be analyzed by SDS-PAGE, and specific complement proteins, such as C3, C5, or factor B, can be detected by Western blot using monoclonal or polyclonal antibodies against the proteins. Cleavage of complement proteins can be compared with samples not treated with u-PA polypeptides. Additionally, samples can be stimulated to initiate complement activation, for example, by incubation with IgG to stimulate activation of the classical pathway or LPS to stimulate activation of the alternative pathway. Samples can be separated by SDS-PAGE for detection of any one or more of the native complement proteins and compared with samples of proteins not treated with u-PA polypeptides to determine the presence or absence of cleavage products of specific proteins. In such examples, the cleaved effector molecules of natural complement proteins can also be analyzed by Western blot using monoclonal and polyclonal antibodies to evaluate the activation of one or more complement pathways. Examples of complement effector molecules can include, but are not limited to, C3a, C3d, iC3b, Bb, and C5-b9. For example, reduced expression of Bb in a sample can indicate that the u-PA polypeptide inhibits the activity of the alternative complement pathway. The cleavage products of the effector molecules can also be determined to evaluate the effect of increasing concentrations of the u-PA polypeptide on the cleavage of the complement effector molecules themselves.

[0338] ii. Enzyme immunoassay Enzyme immunoassays (EIA; also known as enzyme-linked immunosorbent assays or ELISAs) are assays used to measure the presence of proteins in a sample. Typically, protein measurement is an indirect measurement of the protein's binding to an antibody, which itself is chemically labeled with a detectable substrate, such as an enzyme or fluorescent compound. EIA assays can be used to measure the effect of u-PA polypeptides on complement activation by measuring for the presence of complement effector molecules generated after complement activation. In such examples, samples such as human serum or plasma can be pretreated with or without increasing concentrations of u-PA polypeptides and subsequently activated to induce complement activation by incubation with an initiator molecule, or collected after treatment of an animal or human with u-PA polypeptides. For example, the classical pathway can be activated by incubation with IgG, and the alternative pathway can be activated by incubation of the sample with LPS. Because the C4 / C2 cleavage activity of the lectin pathway is shared with the classical pathway, complement activation assays specific for the lectin pathway require that the classical pathway be inhibited. Inhibition of the classical pathway can be achieved using high ionic strength buffers, which inhibit the binding of C1q to immune complexes and disrupt the C1 complexes, while high ionic strength buffers do not affect the carbohydrate-binding activity of MBL. Thus, activation of the lectin pathway can be induced by incubation of a sample such as human serum or plasma with a mannan-coated surface in the presence of 1 M NaCl.

[0339] After activation, the sample can be quenched using Pefabloc (Roche) and the addition of EDTA to minimize continued activation of the pathway. Samples can be analyzed for the presence of complement effector molecules by EIA or ELISA assays. EIA and ELISA assays for measuring complement proteins are well known to those skilled in the art. Any complement activation product can be evaluated. Exemplary complement activation products for measuring complement activation include iC3b, Bb, C5b-9, C3a, C3a-desArg, and C5a-desArg. The activated complement pathway can be determined depending on the complement activation product measured. For example, measurement of Bb cleavage products is a characteristic marker of the alternative pathway.

[0340] In some instances, EIA can be combined with detection of cleaved complement proteins by analysis of protease-treated, complement-stimulated samples by SDS-PAGE, followed by Western blot analysis for identification of specific complement components. Using densitometry software, cleavage of complement products can be compared to cleaved full-length complement components throughout the assay, and the occurrence and percent cleavage of all major degradation products can be determined.

[0341] iii. Radial immunodiffusion (RID) Radial immunodiffusion (RID) is a technique that relies on the precipitation of immune complexes formed between antibodies that are incorporated into an agarose gel upon pouring. Antigens present in the test sample, resulting in a ring of precipitin, line the periphery of the sample well. The diameter of the precipitin ring is proportional to the concentration of antibody (or antigen) present in the test sample. By comparing the diameter of the test sample precipitin ring with that of a known standard, a relatively insensitive estimate of the concentration of a specific antibody or antigen can be achieved. RID can be used to measure the amount of complement proteins in a sample. For example, samples such as human serum or plasma can be treated with or without increasing concentrations of u-PA polypeptide. The protease-treated sample can be added to wells of an agarose gel engineered to incorporate polyclonal or monoclonal antibodies against any one of the complement proteins, including, but not limited to, C3, C5, C6, C7, C9, or factor B. After removal of non-precipitable proteins by exposure to 0.15 M NaCl, the precipitated protein rings can be assessed by staining with protein stains such as Coomassie brilliant blue or Crowles double stain.

[0342] b. Hemolytic assay Functional hemolytic assays provide information about complement function as a whole. This type of assay uses antibody-sensitized or non-antibody-sensitized sheep red blood cells. Hemolytic assays include total hemolytic complement assays (CH50), which measure the ability of the classical pathway and MAC to lyse sheep RBCs. Hemolytic assays rely on the sequential activation of classical pathway components (C1-C9) to lyse sheep red blood cells sensitized with an optimal amount of rabbit anti-sheep red blood cell antibody to create cellular antigen-antibody complexes. Hemolytic assays may also include alternative pathway CH50 assays (rabbit CH50 or APCH50), which measure the ability of the alternative pathway and MAC to lyse rabbit RBCs. One CH50 and / or APCH50 unit is defined as the amount or dilution of serum required to lyse 50% of the red blood cells in the test. Typically, to assess complement activation, samples such as human serum or plasma can be treated with or without increasing concentrations of u-PA polypeptide, or collected after animal or human treatment with or without u-PA polypeptide. The protease-treated sample can then be mixed with IgG-activated or sensitized sheep red blood cells. A water-only sample mixed with sheep red blood cells can serve as a total lysis control to accurately assess the percent lysis of the analyzed sample. The addition of 0.15 M NaCl to the sample can be added to stop the lysis reaction. Red blood cell lysis induced by activation of terminal components of the complement pathway can be assessed by measuring hemoglobin release. Measurements can be by optical density (OD) readings of the sample using a spectrophotometer at OD 415 nm.

[0343] In one embodiment, limiting dilution hemolytic assays can be used to measure the functional activity of specific components of either pathway. In these assays, a serum source is used that has an excess of all complement components but is deficient in the components being measured in the sample; i.e., the medium or serum source is complement-depleted with respect to the specific protein. Thus, the degree of hemolysis depends on the presence of the component being measured in the test sample. In these assays, purified complement proteins, such as any one of the natural complement proteins, including but not limited to C3, can be incubated in the presence or absence of increasing concentrations of u-PA polypeptides. The protease-treated purified complement proteins can then be mixed with complement-depleted medium or plasma and IgG-activated sheep red blood cells, and the hemolysis of the sample can be assessed as described above. In another embodiment, protease cleavage can be correlated with complement activation by assaying the protease-treated sample for hemolytic activity and subsequently analyzing the sample on an SDS-PAGE gel followed by staining with a protein stain, such as Coomassie blue. Protease-treated purified complement proteins can be assessed for cleavage, and the percentage of full-length complement components cleaved throughout the assay and the appearance of all major degradation products can be calculated. Alternatively, analysis of protease-treated complement proteins can be by Western blot.

[0344] An alternative to hemolytic assays, called liposome immunoassays (LIAs), can be used to assess classical pathway activation. LIAs (Waco Chemicals USA, Richmond, Va.) utilize dinitrophenyl phosphate (DNP)-coated liposomes containing the enzyme glucose-6-phosphate dehydrogenase. When serum is mixed with the liposomes and a substrate containing anti-DNP antibodies, glucose-6-phosphate, and nicotinamide adenine dinucleotide, the activated liposomes dissolve, resulting in an enzymatic colorimetric reaction that is proportional to total classical complement activity.

[0345] c. Methods for determining cleavage sites The cleavage sequence in complement protein C3 can be identified by any method known to those skilled in the art (see, e.g., U.S. Patent Application Publication No. 2004 / 0146938). In one example, the cleavage sequence is determined by incubating complement protein C3 with any of the modified u-PA polypeptides provided herein. After incubation with the u-PA polypeptide, the C3 protein can be separated by SDS-PAGE, and degradation products can be identified by staining with a protein dye such as Coomassie Brilliant Blue. The proteolytic fragments can be sequenced to determine the identity of the cleavage sequence. Once identified, activity can be assessed using a fluorogenic substrate designed based on the cleavage sequence of the desired target substrate, as described below.

[0346] 2. Methods for assessing wild-type u-PA activity The modified u-PA polypeptides provided herein have altered or reduced specificity for plasminogen and uPAR. The u-PA polypeptides can be tested to determine whether they retain catalytic efficiency and / or substrate specificity for their natural substrate, plasminogen. For example, cleavage of plasminogen can be assessed by incubating the u-PA polypeptide with plasminogen and detecting the cleavage product. In another example, cleavage of plasminogen can be assessed in vitro by measuring the cleavage of a fluorogenic substrate, such as a fluorogenic-tagged tetrapeptide of a peptide substrate, e.g., the fluorophore ACC (7-amino-4-carbamoyl-methylcoumarin) or AMC (7-amino-4-methylcoumarin), linked to the tetrapeptide substrate. In some examples, a plasminogen activation assay is used to determine the specificity of the u-PA polypeptides provided herein. In other examples, the ability of the u-PA polypeptides provided herein to bind to the u-PA receptor (uPAR) is determined.

[0347] A cleavage of plasminogen In one example, modified u-PA polypeptides can be assayed using individual fluorogenic peptide substrates corresponding to the desired cleavage sequences. For example, a method for assaying for a modified u-PA protease capable of cleaving any one or more of the desired cleavage sequences includes (a) contacting a peptide fluorogenic sample (containing the desired target cleavage sequences) with the protease in such a manner that, upon protease action, the fluorogenic moiety is released from the peptide substrate sequence, thereby producing a fluorescent moiety; and (b) observing whether the sample undergoes a detectable change in fluorescence, which is indicative of the presence of an enzymatically active protease in the sample. In such an example, the desired cleavage sequences are made into fluorogenic peptides by methods known in the art. In one embodiment, individual peptide cleavage sequences can be bound to a fluorogenic-tagged substrate, such as, for example, an ACC or AMC fluorogenic leaving group, and the release of the fluorogenic moiety can be determined as a measure of the specificity of the protease for the peptide cleavage sequences. The rate of increase in fluorescence of the target cleavage sequence can be measured, for example, by using a fluorescence spectrophotometer. The rate of increase in fluorescence can be measured over time. The Michaelis-Menten rate constant can be determined by standard kinetic methods. The rate constant k cat , K. m and k cat / K m can be calculated by plotting the inverse of the substrate concentration versus the inverse of the rate of substrate cleavage and using the Lein-Über-Burk equation (1 / rate = (K m / V max )(1 / [S])+1 / V max ;In the formula, V max =[ET]k cat The second-order rate constant or specificity constant (k cat / K m) is a measure of how well a substrate is cleaved by a particular protease. For example, an ACC or AMC-tetrapeptide, such as Ac-CPGR-AMC, can be made and incubated with a modified u-PA polypeptide provided herein, and the activity of the u-PA polypeptide can be assessed by assaying for release of a fluorogenic moiety. The choice of tetrapeptide depends on the desired cleavage sequence for the target and can be determined empirically.

[0348] In other embodiments, u-PA polypeptides can also be assayed to confirm that, in their active form, they cleave the desired sequence when presented in the context of the full-length protein. In one example, a purified target protein, i.e., plasminogen, can be incubated in the presence or absence of a selected u-PA polypeptide, and the cleavage event can be monitored by SDS-PAGE, followed by Coomassie Brilliant Blue staining of the protein, and analysis of the cleavage products using densitometry.

[0349] b. Plasminogen activation assay Any assay known to those skilled in the art can be used to determine whether u-PA polypeptide activates plasminogen. In one example, plasminogen activation can be determined by incubating in the presence of plasminogen and a detectable plasmin substrate, such as the chromogenic substrate HD-Val-Leu-Lys-p-nitroanalide (Chromogenix S-2251) or the fluorogenic substrate HD-Val-Leu-Lys-7-amido-4-methylcoumarin. Hydrolysis is then monitored by measuring absorbance at 405 nm or by detecting fluorescence using a fluorescent plate reader with an excitation wavelength of 390 nm and an emission wavelength of 480 nm. In another example, plasminogen activation is assessed while u-PA polypeptide is bound to uPAR. In such an example, u-PA polypeptide is first bound to uPAR on the surface of cells, such as U397 cells, and then plasminogen and a detectable plasmin substrate are added, and hydrolysis is measured as described above.

[0350] cu-PA-uPAR binding assay The binding of u-PA polypeptide to uPAR can be evaluated by any assay known to those skilled in the art for detecting protein-protein binding interactions, including but not limited to solid-phase binding assay, ELISA, surface plasmon resonance and FACS.In one example, ELISA can be used.Recombinant uPAR is immobilized on a microtiter plate, and u-PA polypeptide binding is evaluated by adding a reagent that specifically binds to u-PA, such as a u-PA-binding antibody.In another example, binding can be determined in a cell-based assay using a cell line that expresses u-PA receptor, such as U397 cells.U-PA polypeptide can be labeled with, for example, a chromogenic, fluorogenic or radioactive substrate to allow for binding detection.

[0351] d.C3 cutting The activity of modified uPA polypeptides can be assessed by cleavage of the substrate complement protein human C3 by measuring the amount of intact human C3 remaining after incubation with various concentrations of modified uPA protease. According to this assay, a signal is generated in the presence of intact human C3 and is lost upon C3 cleavage. In another example, a C3 activation assay is used to determine the specificity of the modified uPA polypeptides provided herein.

[0352] Purified C3 protein can be incubated with modified u-PA polypeptides, and the remaining level of undigested human C3 can be quantified by any assay known to those skilled in the art for assessing protein concentration, for example, using the Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen®; Perkin Elmer). The C3 / uPA polypeptide mixture is incubated with acceptor beads coated with α-mouse IgG, and after incubation, the α-hC3 mAb / acceptor bead mixture is incubated with biotinylated α-hC3 pAb. Streptavidin-coated donor beads are added to the mixture, and the "AlphaScreen" signal (excitation = 680 nm, emission = 570 nm) is then measured. This signal corresponds to the concentration of remaining C3 protein. The concentration of uPA polypeptide required to cleave 50% of the available hC3 (EC 50 ) can be calculated.

[0353] ACC-AGR+ELISA A method for evaluating the substrate specificity of modified u-PA polypeptides is provided herein. For example, a fluorogenic peptide substrate, such as 7-amino-4-methylcoumarin (AMC) or 7-amino-4-carbamoylmethylcoumarin (ACC), can be used to assay the activity of modified proteases, whereby upon protease action, a fluorogenic moiety is released from the peptide substrate, and the release of the fluorogenic moiety can be determined as a measure of the specificity of the protease for the peptide cleavage sequence. The rate of increase in fluorescence of the non-target substrate cleavage sequence or the target cleavage sequence can be measured, for example, by using a fluorescence spectrophotometer. The rate of increase in fluorescence can be measured over time. The Michaelis-Menten rate constant can be determined by standard kinetic methods. The rate constant k cat , K. m and k cat / K m can be calculated by plotting the inverse of the substrate concentration versus the inverse of the rate of substrate cleavage and using the Lein-Über-Burk equation (1 / rate = (K m / V max )(1 / [S])+1 / V max ;In the formula, V max =[ET]k cat The specificity constant (k cat / K m ) is a measure of how well a substrate is cleaved by a particular protease.

[0354] In one example, any one or more of the cleavage sequences of complement proteins can be determined and used as the desired target cleavage sequence, for example, any one or more of the C3 cleavage sequences. In another example, sequences corresponding to substrates of wild-type proteases can be used to assay for residual protease activity.

[0355] In further embodiments, full-length complement proteins can be used as target substrates to assay for protease specificity compared to the full-length natural target substrate of the protease. Furthermore, full-length complement proteins can be used to assess the correlation between substrate specificity and protease cleavage of the full-length target substrate versus the four amino acid P1-P4 substrate cleavage sequence contained within the target substrate. In one example, full-length C3 protein can be used as any one or more desired cleavage targets for a protease to assess specificity. In this example, cleavage of C3 by the modified protease can be compared to cleavage of another full-length substrate, or cleavage can be compared to the fluorogenic tetrapeptide cleavage sequence of C3. The specificity constant for cleavage of a full-length protein by a protease can be determined using gel densitometry to assess the densitometric change over time of the full-length target substrate band incubated in the presence of the protease.

[0356] In a further embodiment, the activity of modified u-PA polypeptides can be evaluated after prolonged incubation in cynomolgus monkey plasma or vitreous humor. In one example, residual protease activity is assayed using the fluorogenic substrate AGR-ACC (7-amino-4-carbamoylmethyl-coumarin) after incubation in 80% cynomolgus monkey vitreous humor. For example, the modified u-PA polypeptide of SEQ ID NO: 21 exhibits comparable ability to cleave the fluorogenic substrate AGR-ACC after 7 days of incubation in vitreous humor and PBS. In another example, the modified u-PA polypeptide of SEQ ID NO: 21 cleaves the fluorogenic substrate AGR-ACC at similar levels before and after 7 days of incubation in vitreous humor.

[0357] Evaluating specificity using peptide libraries Provided herein is a method for evaluating the substrate specificity of the resulting modified u-PA polypeptide using a peptide library coupled to a fluorogenic peptide. The modified u-PA polypeptide can be tested for P1-P4 substrate specificity at any given subsite using a peptide library coupled to a fluorogenic substrate (Harris et al., (2000) Proc. Natl. Acad. Sci. USA 97:7754; U.S. Patent Application Publication Nos. 2004 / 0175777; 2004 / 0146938). The use of a peptide library(ies) allows for the rapid and easy determination of proteolytic substrates. This strategy involves the use of a library of peptides in which one position of the library is held constant (i.e., the P1 position), while the remaining positions (i.e., P4-P2 and / or P1' and / or P2') are composed of all amino acid combinations used to prepare the library. For example, the use of combinatorial fluorogenic peptide substrate libraries, such as 7-amino-4-methylcoumarin (AMC) or 7-amino-4-carbamoylmethylcoumarin (ACC) fluorogenic peptide substrates, can be used to assay for the activity of modified proteases, whereby a fluorogenic moiety is released from the peptide substrate upon protease action. Those skilled in the art will appreciate that these methods offer a wide range of alternative library formats. In one example, proteases can be profiled using P1-diversity libraries. P1-diversity tetrapeptide libraries contain ACC or AMC-fluorogenic tetrapeptides in which the P1 position is systematically held constant, while the P2, P3, and P4 positions contain an equimolar mixture of any one or more of the 15 amino acids. Consideration of the specific limitations and determinations associated with any particular enzyme or substrate sequence specificity determination method is within the capabilities of those skilled in the art.

[0358] Those skilled in the art will appreciate that there are many methods for preparing peptides. In an exemplary embodiment, a substrate library is screened by attaching a fluorogenic-tagged substrate to a solid support. In one example, a fluorogenic leaving group ...

Claims

1. A modified urokinase-type plasminogen activator (u-PA) polypeptide, comprising amino acid modifications relative to an unmodified u-PA polypeptide, such that said modified u-PA polypeptide has increased cleavage activity and / or specificity in its active form for complement proteins compared to the unmodified active form of said u-PA polypeptide; the amino acid modification is selected from substitutions, insertions, and deletions within the primary sequence of the modified u-PA polypeptide; The modified u-PA polypeptide comprises R35Y / H37S / R37aP / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L, R35W / R36Q / H37S / V38P / T39Y / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L, F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / K82R / T97aI / L97bA / H99Q / K110aR / C122S / Y149R / M157K, F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K / K179R, F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / K92R / T97aI / L97bA / H99Q / C122S / Y149R / M157K, F30Y / R35V / R36H / H37G / V38E / T39W / Y40H / V41R / Y60bW / T97aI / L97bA / H99Q / C122S / Y149E / M157K, F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / K92S / T97aI / L97bA / H99Q / C122S / Y149R / M157K, F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / K61R / K62R / T97aI / L97bA / H99Q / C122S / Y149R / M157K, F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K / K179S, <h2 style=";text-align:left;direction:ltr">135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36T / H37S / V38 S / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157R / Q192Y、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V 38E / T39F / Y40F / V41R / K6 150 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> R35W / R36Q / H37S / V38T / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C12S / Y149R / Y151P / M157R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37Y / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> V38E / T39W / V41R / D60aW / Y 60bP / L97bG / H99L / C122S、 <h2 style=";text-align:left;direction:ltr"> 13562 aE / L97bA / H99Q / C122S / Y149R / Y151L / M157S / Q192H、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / R36H / H37W / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr">1356229 ... <h2 style=";text-align:left;direction:ltr"> 135622999 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V 38E / T39F / Y40F / V41R / K82 S / T97aI / L97bA / H99Q / K11 0aS / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / D60aP / Y 60bQ / L97bA / H99Q / C122S / T158A、 <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / R 37aQ / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37S / V38P / T9Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157R、 <h2 style=";text-align:left;direction:ltr">F30H / R35W / R36T / H37S / V38P / T39Y / V41R / Y60bN / T97aE / L97bA / H99Q / C12S / Y149R / Y151L / M157S、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37N / V3 8E / T39F / Y40F / V41R / K61E / 15 ... <h2 style=";text-align:left;direction:ltr"> R35W / R36Q / H37S / V38S / T39Y / V41R / Y60bN / T97aE I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35V / R36H / H37G / V38E / T9W / Y40H / V41R / Y60bA / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr">F30Y / R35W / R36S / H37S / V38 Q / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157S / Q192N、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / R37 aD / V38E / T39Y / Y40F / V41R / D60aE / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> R37aS / V38E / Y40V / V41R / H 99L / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bG / H99Q / C122S / Y151L / R217E、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / R36H / H37W / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35V / R36H / H37S / V38E / T39F / Y40H / V41R / Y60bS / T97aM / L97bA / H99Q / C122S / Y149W / M157K、 <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> N26D / F30Y / R35Y / R36H / H37E / V38E / T39F / Y40F / V41R / K61E / T97aI / L97bA / H99Q / R110dS / P114S / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / R 37aE / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr">I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / D60aP / Y60b Q / L97bA / H99Q / C122S / T158S / E167K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37D / V38E / T39W / Y40H / V41R / Y60bE / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37E / V 38E / T39F / Y40F / V41R / K61 E / T97aI / L97bA / H99Q / C122S / Y149R / M157K / T242A、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37P / R 37aQ / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / H99E / C122S / Y151L / R217E、 <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / T97a S / L97bR / H99E / C122S / Y1 51L / E175D / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr">F30Y / R35W / R36Q / H37S / V38 T / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157K / Q192T、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I117V / F30Y / R35W / R36H / H37E / V38E / T39W / Y40H / V41 10 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / H37W / V38D / Y40H / V41R / Y60 bN / L97bA / H99Q / C122S / Y149H / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37S / V38 E / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157T / Q192H、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 13555 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 43562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr">13562 I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40F / V41R / Y60bN / T97aE / L97 bA / H99Q / C122S / N145S / S146V / T1 47M / D148G / Y149Q / L150F / M157K、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> R35V / V38E / Y40Q / V41L / Y60bS / T 97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / L97bG / H99Q / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / T56S / D60aP / Y60bQ / L97bA / H99Q / C122S / T158S、 <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37W / V38D / V41R / L9 7bA / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / Y40H / Y60bN / T97aI / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr">F30Y / R35W / R36H / H37N / V38E / T39Y / Y40F / V41R / Y60bS / T97aE / L97bA / H9 9Q / C122S / Y149K / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36K / H37S / V38 D / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157R / Q192T、 <h2 style=";text-align:left;direction:ltr"> V41R / H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40P / V41L / L97bG / H 99L / C122S / Y151Q / R217E、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40L / V41R / H99L / C122S / Y151L / R217S、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / L97bG / H 99Q / C122S / Y151P / R217T、 <h2 style=";text-align:left;direction:ltr"> V38E / T39Y / V41R / D60aW / Y 60bP / L97bR / H99I / C122S、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37F / V38E / T39Y / Y40H / V41R / Y60bD / T97aV / L97bA / H99Q / C122S / Y149L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / S 37dP / V38E / T39W / Y40H / V4 1 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36K / H37E / V3 8E / T39W / Y40H / V41R / Y60bQ I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr">F30Y / R35W / R36H / H37D / V38E / T39Y / Y40H / V41R / Y60bS / T97aL / L97bA / H99Q / C122S / Y149L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37Y / R37 aE / V38E / T39Y / Y40F / V41R / D60aS / Y60bP / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I2445N / F30Y / R35W / R36H / H37E / V38E / T39W / Y40L / V41R / 5 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / H37W / V38D / Y40H / V41R / Y60bN / L97bA / H99Q / C122S / Y149H / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36A / H37E / V3 8E / T39W / Y40H / V41R / Y60bQ I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K / K223R / K224R、 <h2 style=";text-align:left;direction:ltr"> 13562 I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / D60aP / Y60bQ / P 60cS / L97bA / H99Q / C122S / I138V / E167K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr">1356229 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40F / V41L / L97b A / H99Q / C122S / Y151L / M157R、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40A / V41L / L97bG / H99Q / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> I24T / F30Y / R35W / R36H / H 37E / V38E / T39W / Y40H / V41 10 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40F / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30H / R35L / H37D / V38D / V41R / L97bA / H99Q / C122S / Y151L / M157K / R217E、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / R37 aE / V38E / T39Y / Y40F / V41R / D60aE / Y60bF / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / R36H / H37G / V38E / T39Y / Y40H / V41R / Y60bP / T97aE / L97bA / H99Q / C122S / Y149M / M157K、 <h2 style=";text-align:left;direction:ltr"> I17V / F30H / V38D / V41R / L97b A / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> R35V / Y40Q / V41L / Y60bS / T97a E / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr">F30Y / R35M / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> R35Q / V38D / V41R / L97bG / H99Q / C122S / Y151L、 <h2 style=";text-align:left;direction:ltr"> R37aS / V38E / Y40P / V41L / L97b G / H99Q / C122S / Y151Q / R217T、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / A96G / L97b A / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> 435, 436, 437, 438, 439, 440, 441, 442, 443, 4 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / V38E / T39Y / Y40F / V41R / Y60bQ / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / Y60bL / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / S146F / M157K / Q192H / K243Q、 <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37E / V3 8E / T39W / Y40H / V41R / Y60bQ / K61L / I65V / T97aE / L97bA / H99Q / C122S / Y149K / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> V41R / L97bR / H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R35V / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bP / T97aE / L97bA / H99Q / C122S / Y149E / M157K、 <h2 style=";text-align:left;direction:ltr">1356229 ... <h2 style=";text-align:left;direction:ltr"> R35S / V38D / V41R / L97bA / H99Q / C122S / Y151L、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / M157K / T158S、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / V38F / T39H / V41R / Y60bK / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / V38F / T39H / V41R / Y60bK / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / K61E / T97aI / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> R37aS / V38D / V41Q / L97bG / H99Q / C122S / Y151L / R217T、 <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H 99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35K / R36H / H37E / R37aK / V38E / T39F / Y40F / V41R / I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr">F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bG / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37E / V3 8A / T39W / Y40H / V41R / Y60bQ / K61D / I65V / T97aE / L97bA / H99Q / C122S / Y149K / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / T 56A / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37T / V38D / V41R / L9 7bA / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30H / R36L / V38E / V41R / K82R / L9 7bA / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> V38D / V41R / H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37Y / R37 aE / V38E / T39Y / Y40F / V41R / D60aE / Y60bA / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> R36S / V38E / Y40L / V41N / L97b G / H99Q / C122S / Y151L / R217T、 <h2 style=";text-align:left;direction:ltr"> 43560627 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / V38E / T39W / Y40H / V41R / Y60bQ / T97aE / L97bA / H99Q / C122S / Y149K / M157K、 <h2 style=";text-align:left;direction:ltr">13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37Q / V38E / T39H / Y40H / V41R / T97aE / L97bA / H99Q / C122S / Y149L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37Y / R 37aD / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H99Q / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> F350H / R35H / H37I / V38D / V41R / L97bA / H99Q / C122S / Y149W / Y151L / M157K / R217S、 <h2 style=";text-align:left;direction:ltr"> 43565676767 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 435, 4 ... <h2 style=";text-align:left;direction:ltr"> 435606 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / H99Q / C122S / Y151L / R217E、 <h2 style=";text-align:left;direction:ltr"> 1356266677 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> 14456 ... <h2 style=";text-align:left;direction:ltr"> F350Y / V38E / Y40H / V41R / T56A / L97bA / H99Q / C122S / M157K / K243M、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / V38D / V41R / L97b A / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr">I am sorry, I did not find this information. R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> R35V / R37aE / V38E / Y40Q / V41L / Y 60bS / T97aE / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / I138V / M157K、 <h2 style=";text-align:left;direction:ltr"> 435, 4 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / R36H / H37D / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40H / V41R / L97b A / H99Q / C122S / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> V38E / T39W / V41R / D60aP / Y 60bD / L97bA / H99L / C122S、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> R36S / V38E / Y40Q / V41R / L97b G / H99L / C122S / Y151P / R217E、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / Y60bL / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E、 <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / V38F / T39H / V41R / Y60bK / T97aS / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E、 <h2 style=";text-align:left;direction:ltr">F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / L97bA / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> F30H / R35H / V38D / V41R / K61E / L97bA / H99Q / C122S / Y151L / M157K / R206H、 <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40H / V41R / L 97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41L / L97bG / H99Q / C122S / Y151L / R217Q、 <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37W / V38D / V41R / D60aE / L97bA / H99Q / C122S / Y149L / Y151L / M157K / R217D、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35F / R36H / H37G / V38E / T39Y / Y40H / V41R / Y60bS / T97aD / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 43545 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> Y40H / V41Q / L97bG / H99Q / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 4356567676 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39F / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr">43567 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38D / Y40H / V41R / L97bA / H99L / C122S / F141L / M157K / T158A、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aA / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 435, 445, 445, 445 <h2 style=";text-align:left;direction:ltr"> F30H / R36H / V38D / V41R / T56A / L9 7bA / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35E / R36H / H37D / R 37aN / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / D60aP / Y60b L / L97bA / H99Q / C122S / Y149W、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37W / V38D / V41R / D60aE / Y60bS / L97bA / H99Q / C122S / Y149L / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38 E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151P / M157K / Q192H、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35M / R36H / H37D / R37 aD / V38E / T39Y / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / V38E / T39Y / Y40H / V41R / Y60bT / T97aD / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr">43567 ... <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> 43562 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40L / V41R / L97bA / H 99Q / C122S / Y151L / M157K / Q192H、 <h2 style=";text-align:left;direction:ltr"> F30H / V38D / Y40F / V41R / L97b A / H99Q / C122S / Y151L / M157F、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 12425 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / V38D / Y40H / V41R / N76S / L97bA / H99Q / C122S / M157K / K187E、 <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H 99Q / C122S / Y151L / M157S、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37G / V38E / T39W / Y40H / V41R / Y60bA / T97aE / L97bA / H99Q / C122S / Y149Q / M157K、 <h2 style=";text-align:left;direction:ltr"> 135 ... 100% <h2 style=";text-align:left;direction:ltr">H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / L97b R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / V38E / T39Y / Y40F / V41R / Y60bS / T97aE / L97bA / H9 9Q / C122S / Y149K / M157K、 <h2 style=";text-align:left;direction:ltr"> 135622999 <h2 style=";text-align:left;direction:ltr"> V38E / T39L / V41R / D60aN / Y 60bP / L97bG / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37A / V38E / T39Y / Y40H / V41R / Y60bQ / T97aV / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / R 37aP / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> H37T / R37aL / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L / Q192R、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / T39H / V41R / Y60bK / T97aS / L97b R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> 43545 ... <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr">F30Y / R36H / V38E / Y40H / V41R / T97aI / L9 7bA / H99Q / C122S / Y149N / L150V / M157K、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> R37aS / V38D / T39Y / Y40F / V41R / H99L / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / Y60bE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> Y40H / V41T / L97bG / H99Q / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> F30H / R35S / V38E / V41R / Y60bH / L 97bA / H99Q / C122S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. 10 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37N / V38E / T39Y / Y40H / V41R / Y60bQ / T97aV / L97bA / H99Q / C122S / Y149L / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40L / V41R / L97bA / H 99Q / C122S / Y151L / M157A / Q192Y、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40H / V41Q / L97bG / H99Q / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> R35V / R37aE / V38E / Y40Q / V41L / Y60b S / T97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H 99Q / C122S / Y151L / M157T、 <h2 style=";text-align:left;direction:ltr"> R35K / Y40Q / V41L / Y60bE / L 97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr">F30Y / R35V / R36H / H37T / V38E / T39A / Y40L / V41Q / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H99Q / C122S / Y151L / M157K / R217D、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / I65T / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36S / V38D / T39I / Y40H / V41R / L9 7bA / H99Q / C122S / S146P / M157K / T158S、 <h2 style=";text-align:left;direction:ltr"> R35V / R37aE / V38E / Y40Q / V41L / Y 60bS / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / Y60bL / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39F / Y40F / V41R / D60aS / Y60bP / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30H / G37bD / V38D / T39H / V41R / L97bA / H9 9Q / R110dH / C122S / Y151L / M157K / S240I、 <h2 style=";text-align:left;direction:ltr"> 1355 100% <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 43555555 <h2 style=";text-align:left;direction:ltr">F30Y / R36H / H37E / V38E / T39F / Y40H / V41R / Y60bN / T97aV / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / T97a S / L97bR / H99E / C122S / Y1 51L / E175D / Q192T / R217E、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / K61E / T97aI / L97bA / H99Q / C122S / Y149H / M157K / K187R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / K61E / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 43562 ... <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / T97aS / L97b R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / H37W / V38D / Y40H / V41R / Y60bN / L97bA / H99Q / C122S / Y149H / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / V38D / Y40H / V41R / L97bA / H99Q / C122S / Y149W / M157K / R217D、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / T97aE / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> 1356567 ... <h2 style=";text-align:left;direction:ltr"> 1356455 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aL / V38E / T39N / Y40F / V41R / D60aP / Y60bT / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35V / R36H / H37G / V38 E / T39L / Y40H / V41R / Y60bT / T97aI / L97bA / H99Q / C122S / Y149R / M157K / Q192T / R239H、 <h2 style=";text-align:left;direction:ltr">135 ... 100% <h2 style=";text-align:left;direction:ltr"> 4356 ... <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H99Q / C122S / Y149N / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> R35S / V38D / Y40H / V41L / Y 60bD / L97bG / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> R35V / R37aD / V38E / Y40Q / V41L / Y60b G / T97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> R36L / V38E / Y40Q / V41R / L97b G / H99Q / C122S / Y151P / R217S、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37D / V38E / T39W / Y40H / V41R / Y60bV / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / V38D / Y40H / V41R / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / V38D / Y40H / V41R / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41Q / D60aP / Y60b L / L97bA / H99Q / C122S / Y149W、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R 37aN / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr">4356567 ... <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / V38D / V41R / Y60bE / L97b A / H99Q / C122S / Y149N / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 1356567 ... <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 135 ... 100% <h2 style=";text-align:left;direction:ltr"> V38E / Y40H / V41T / L97bG / H99Q / C122S / R217T、 <h2 style=";text-align:left;direction:ltr"> V38D / V41Q / L97bG / H99Q / C122S / Y151L / R217T、 <h2 style=";text-align:left;direction:ltr"> 43555555 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37aE / V38E / T39I / Y40F / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149M / M157K、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / R37aH / V38E / Y40H / V41R / K61E / T97aI / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / V 38E / T39Y / Y40H / V41R / Y60 bD / T97aI / L97bA / H99Q / C1 22S / Y149R / M157K / Q192M、 <h2 style=";text-align:left;direction:ltr"> 43562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40H / V41R / L97b A / H99Q / C122S / Y149W / M157K、 <h2 style=";text-align:left;direction:ltr">F30Y / R35F / R36H / H37D / R37 aD / V38E / T39H / Y40F / V41R / D60aP / Y60bA / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37Y / V38D / V41R / D60aE / Y60bN / L97bA / H99Q / C122S / Y149R / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> L97bR / H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> 1445 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> R35K / V38D / Y40H / V41R / Y 60bS / L97bG / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / R36H / H37D / V38E / T39W / Y40H / V41R / Y60bA / T97aD / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / H37W / V38D / Y40H / V41R / Y6 0bN / L97bA / H99Q / C122S / Y149H / M157K、 <h2 style=";text-align:left;direction:ltr"> Y40H / V41Q / L97bG / H99Q / C122S、 <h2 style=";text-align:left;direction:ltr"> 435, 4 ... <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35A / R36H / H37T / R37aD / V38E / T39Y / Y40L / V41R / D60aP / Y60bE / T97aE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr">I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> R35V / R37aE / Y40Q / V41L / Y60bS / T 97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> 1356 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / Y60bL / L97bA / C122S、 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> F350Y / R35H / V38D / T39S / Y40L / V41R / L97bA / H99Q / C122S / T147S / M157K、 <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H99Q / C1 22S / Y151L / M157K / Q192H / R239H、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37M / R37aD / V 38E / T39G / Y40F / V41R / D60 I am sorry, I did not find this information. <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / L97bA / H99Q / C122S / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40L / V41Q / L97bA / H 99Q / C122S / Y151L / M157R / Q192Y、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / H37W / V38D / Y40H / V41R / Y6 0bN / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 135656777 <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41Q / L97bA / H 99Q / C122S / Y151L / M157R、 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 1356567 ... <h2 style=";text-align:left;direction:ltr">13556 ... <h2 style=";text-align:left;direction:ltr"> N26D / F30Y / V38D / Y40H / V41R / I65T / L97bA / H99Q / C122S / M126I / M157K、 <h2 style=";text-align:left;direction:ltr"> 1356229 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37T / V38D / V41R / L97bA / H9 9Q / C122S / Y149I / Y151L / M157K / R217D、 <h2 style=";text-align:left;direction:ltr"> F350H / V38D / L97bA / H99Q / C12S / Y151L / M157K、 <h2 style=";text-align:left;direction:ltr"> V38D / H99Q / C122S / Y151L / R217V、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35T / H37T / V38D / Y40H / V41R / D60aP / Y60bD / L97bA / H99Q / C122S / Y149T / M157K、 <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> R37aE / V38E / Y40Q / V41L / Y60bS / T 97aE / L97bA / H99Q / C122S / Y149R、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37E / V38E / T39Y / Y40H / V41R / Y60bN / T97aV / L97bA / H99Q / C122S / Y149K / M157K、 <h2 style=";text-align:left;direction:ltr"> V38D / V41Q / D60aP / Y60bS / T97aS / L97bR / H99 E / C122S / Y151L / E175D / Q192Y / R217E / K224R、 <h2 style=";text-align:left;direction:ltr"> F350H / R35Q / H37Q / V38D / V41R / A96D / L97bA / H99Q / C122S / Y149L / Y151L / M157K / R217D、 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / H37Y / V38D / Y40H / V41R / Y6 0bE / L97bA / H99Q / C122S / Y149R / M157K、 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38E / T39V / Y40H / V41R / Y60bH / T97aE / L97bA / H99Q / C122S / Y149S / M157K、 F30Y / V38D / Y40H / V41R / L97bA / H99Q / C122S, F30Y / R35V / R36H / H37S / V38E / T39F / Y40H / V41R / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R / M157K / Q192T, R35Q / H37Y / R37aE / V38E / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R, R35Q / H37Y / R37aE / V38E / T39Y / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R, R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / H99Q / C122S / Y149R, R35Q / H37T / R37aP / V38E / T39Y / V41R / D60aE / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R, V38D / V41Q / D60aH / Y60bS / T97aW / L97bR / H99E / C122S / Y151L / E175D / R217E / K224R, V38D / V41L / Y60bP / T97aM / L97bR / H99E / C122S / Y151L / E175D / R217E / K224R; or any of these polypeptides in which C122S is unmodified and is C122C (according to chymotrypsin numbering). containing a modification equivalent to any of the following: said modified u-PA polypeptide, when in an active form, inhibits or reduces complement activation by cleaving complement proteins; the complement protein is C3; Residues are numbered according to the chymotrypsin numbering system; the unmodified u-PA polypeptide consists of the sequence of amino acid residues set forth in any of SEQ ID NOs: 1-6; A modified u-PA polypeptide, wherein said modified u-PA polypeptide has at least 95% sequence identity to a polypeptide consisting of any of SEQ ID NOs: 1-6.

2. A modified urokinase-type plasminogen activator (u-PA) polypeptide, comprising one or more amino acid modifications in an unmodified u-PA polypeptide, whereby said modified u-PA polypeptide has increased cleavage activity and / or specificity for complement proteins compared to the unmodified active form of said u-PA polypeptide; the modified u-PA polypeptide inhibits or reduces complement activation by cleaving complement proteins; the complement protein is C3; Residues are numbered according to the chymotrypsin numbering system; the unmodified u-PA polypeptide consists of a sequence as set forth in any of SEQ ID NOs: 1-6; and 1. A modified u-PA polypeptide comprising modifications corresponding to any of the following (according to chymotrypsin numbering): Y40Q / V41L / L97bA / H99Q / C122S, Y40Q / V41L / L97bA / H99Q, Y40Q / V41L / L97bA / C122S, V40Q / V41L / L97bA, Y40Q / V41R / L97bA / C122S, and Y40Q / V41R / L97bA.

3. The modified u-PA polypeptide of claim 1 or 2, which has reduced activity towards plasminogen.

4. The modified u-PA polypeptide of any one of claims 1 to 3, wherein said unmodified u-PA polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2 or 5.

5. Amino acid modification R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aT / Y60bT / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aT / Y60bT / T97aI / L97bA / H99Q / Y149R The modified u-PA polypeptide of any one of claims 1, 3 and 4, comprising:

6. R35Y / H37S / R37aP / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L; R35W / R36Q / H37S / V38P / T39Y / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L; F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / K82R / T97aI / L97bA / H99Q / K110aR / C122S / Y149R / M157K; F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K / K179R; <h2 style=";text-align:left;direction:ltr">100% Working hours: 12:00 AM - 12:00 PM <h2 style=";text-align:left;direction:ltr"> F350Y / R35V / R36H / H37G / V38E / T39W / Y40H / V41R / Y60bW / T97aI / L97bA / H99Q / C122S / Y149E / M157K; <h2 style=";text-align:left;direction:ltr"> 11.11.2019 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V 38E / T39F / Y40F / V41R / K6 1 ... <h2 style=";text-align:left;direction:ltr"> 11.11.2019 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36T / H37S / V38 S / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157R / Q192Y; <h2 style=";text-align:left;direction:ltr"> 11.11.2019 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V 38E / T39F / Y40F / V41R / K6 150 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> R35W / R36Q / H37S / V38T / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C12S / Y149R / Y151P / M157R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37Y / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> V38E / T39W / V41R / D60aW / Y 60bP / L97bG / H99L / C122S; <h2 style=";text-align:left;direction:ltr">13562 aE / L97bA / H99Q / C122S / Y149R / Y151L / M157S / Q192H; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / R36H / H37W / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K / T158A; <h2 style=";text-align:left;direction:ltr"> 1356229999 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / V38E / T39Y / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> 13562999 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V 38E / T39F / Y40F / V41R / K82 S / T97aI / L97bA / H99Q / K11 0aS / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr">R35H / V38E / T39Y / V41R / D60aP / Y 60bQ / L97bA / H99Q / C122S / T158A; <h2 style=";text-align:left;direction:ltr"> 135622999 <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41Q / D60aH / Y60bS / T97aW / L97bR / H99E / C122S / Y151L / E175D / R217E / K224R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / R 37aQ / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37S / V38P / T9Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157R; <h2 style=";text-align:left;direction:ltr"> F30H / R35W / R36T / H37S / V38P / T39Y / V41R / Y60bN / T97aE / L97bA / H99Q / C12S / Y149R / Y151L / M157S; <h2 style=";text-align:left;direction:ltr"> F350Y / R35W / R36H / H37D / V38E / T39Y / Y40H / V41R / Y60bD / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37N / V3 8E / T39F / Y40F / V41R / K61E / 15 ... <h2 style=";text-align:left;direction:ltr"> R35W / R36Q / H37S / V38S / T39Y / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157S / Q192H; <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr">F350Y / R35V / R36H / H37G / V38E / T9W / Y40H / V41R / Y60bA / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> F350Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36S / H37S / V38 Q / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157S / Q192N; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / R37 aD / V38E / T39Y / Y40F / V41R / D60aE / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> R37aS / V38E / Y40V / V41R / H 99L / C122S / Y151L / R217V; <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bG / H99Q / C122S / Y151L / R217E; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R35V / R36H / H37D / V38E / T39W / Y40H / V41R / Y60bP / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / R36H / H37W / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr">F30Y / R35V / R36H / H37S / V38E / T39F / Y40H / V41R / Y60bS / T97aM / L97bA / H99Q / C122S / Y149W / M157K; <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> N26D / F30Y / R35Y / R36H / H37E / V38E / T39F / Y40F / V41R / K61E / T97aI / L97bA / H99Q / R110dS / P114S / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / R 37aE / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37P / V38E / T39Y / Y40F / V41R / Y60bS / T97aE / L97bA / H99Q / C122S / Y149K / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / D60aP / Y60b Q / L97bA / H99Q / C122S / T158S / E167K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R35Y / R36H / H37D / V38E / T39W / Y40H / V41R / Y60bE / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 100% Working hours: 12:00 AM - 12:00 PM <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37E / V 38E / T39F / Y40F / V41R / K61 E / T97aI / L97bA / H99Q / C122S / Y149R / M157K / T242A; <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / M157K / T158A; <h2 style=";text-align:left;direction:ltr">F30Y / R35Y / R36H / H37P / R 37aQ / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / H99E / C122S / Y151L / R217E; <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / T97a S / L97bR / H99E / C122S / Y1 51L / E175D / R217E / K224R; <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> 100% Working hours: 12:00 AM - 12:00 PM <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37S / V38 T / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157K / Q192T; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I117V / F30Y / R35W / R36H / H37E / V38E / T39W / Y40H / V41 10 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R35Q / H37W / V38D / Y40H / V41R / Y60 bN / L97bA / H99Q / C122S / Y149H / M157K / T158A; <h2 style=";text-align:left;direction:ltr"> 13555 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37S / V38 E / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157T / Q192H; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13555 <h2 style=";text-align:left;direction:ltr">F30Y / R35W / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 43562 ... <h2 style=";text-align:left;direction:ltr"> 11.11.2019 <h2 style=";text-align:left;direction:ltr"> F350H / R35W / R36H / H37S / V38E / T39Y / Y40M / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 13562 / L97bA / H99Q / C122S / Y149R / Y151L / M157R / Q192T; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / V38E / T39Y / Y40H / V41R / Y60bA / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40F / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / N145S / S146V / <h2 style=";text-align:left;direction:ltr"> T147M / D148G / Y149Q / L150F / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35I / R36H / H37D / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> R35V / V38E / Y40Q / V41L / Y60bS / T 97aE / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / L97bG / H99Q / C122S / R217T; <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / T56S / D60aP / Y60bQ / L97bA / H99Q / C122S / T158S; <h2 style=";text-align:left;direction:ltr">F30H / R35Q / H37W / V38D / V41R / L9 7bA / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / Y40H / Y60bN / T97aI / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> V38D / V41L / Y60bP / T97aM / L97bR / H99 E / C122S / Y151L / E175D / R217E / K224R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / V38E / T39F / Y40H / V41R / Y60bE / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37N / V38E / T39Y / Y40F / V41R / Y60bS / T97aE / L97bA / H9 9Q / C122S / Y149K / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36K / H37S / V38 D / T39Y / Y40L / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151L / M157R / Q192T; <h2 style=";text-align:left;direction:ltr"> V41R / H99Q / C122S / Y151L / R217V; <h2 style=";text-align:left;direction:ltr"> V38E / Y40P / V41L / L97bG / H 99L / C122S / Y151Q / R217E; <h2 style=";text-align:left;direction:ltr"> V38E / Y40L / V41R / H99L / C122S / Y151L / R217S; <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / L97bG / H 99Q / C122S / Y151P / R217T; <h2 style=";text-align:left;direction:ltr"> V38E / T39Y / V41R / D60aW / Y 60bP / L97bR / H99I / C122S; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / H37F / V38E / T39Y / Y40H / V41R / Y60bD / T97aV / L97bA / H99Q / C122S / Y149L / M157K; <h2 style=";text-align:left;direction:ltr">F30Y / R35W / R36H / H37E / S 37dP / V38E / T39W / Y40H / V4 1 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 1356222 <h2 style=";text-align:left;direction:ltr"> 43565676 <h2 style=";text-align:left;direction:ltr"> F350H / R35Q / R36H / H37Y / V38E / T39Y / Y40L / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36K / H37E / V3 8E / T39W / Y40H / V41R / Y60bQ I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr"> F350Y / R35W / R36H / H37D / V38E / T39Y / Y40H / V41R / Y60bS / T97aL / L97bA / H99Q / C122S / Y149L / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37Y / R37 aE / V38E / T39Y / Y40F / V41R / D60aS / Y60bP / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I2445N / F30Y / R35W / R36H / H37E / V38E / T39W / Y40L / V41R / 5 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / H37W / V38D / Y40H / V41R / Y60bN / L97bA / H99Q / C122S / Y149H / M157K / T158A; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36A / H37E / V3 8E / T39W / Y40H / V41R / Y60bQ I am sorry, I did not find anything. <h2 style=";text-align:left;direction:ltr">F350Y / R35Q / R36H / H37W / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / English: <h2 style=";text-align:left;direction:ltr"> 13562 aE / L97bA / H99Q / C122S / Y149R / Y151L / M157S / Q192T; <h2 style=";text-align:left;direction:ltr"> R35H / V38E / T39Y / V41R / D60aP / Y60bQ / P 60cS / L97bA / H99Q / C122S / I138V / E167K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> 13556 <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40F / V41L / L97b A / H99Q / C122S / Y151L / M157R; <h2 style=";text-align:left;direction:ltr"> V38E / Y40A / V41L / L97bG / H99Q / C122S / R217T; <h2 style=";text-align:left;direction:ltr"> I24T / F30Y / R35W / R36H / H 37E / V38E / T39W / Y40H / V41 10 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38E / T39Y / Y40F / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> I17V / F30Y / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / M157K / T158A; <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr">F30H / R35L / H37D / V38D / V41R / L97bA / H99Q / C122S / Y151L / M157K / R217E; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / R37 aE / V38E / T39Y / Y40F / V41R / D60aE / Y60bF / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F350Y / R35L / R36H / H37G / V38E / T39Y / Y40H / V41R / Y60bP / T97aE / L97bA / H99Q / C122S / Y149M / M157K; <h2 style=";text-align:left;direction:ltr"> I17V / F30H / V38D / V41R / L97b A / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35V / R36H / H37K / V38E / T39F / Y40H / V41R / Y60bN / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> R35V / Y40Q / V41L / Y60bS / T97a E / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35M / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> R35Q / V38D / V41R / L97bG / H99Q / C122S / Y151L; <h2 style=";text-align:left;direction:ltr"> R37aS / V38E / Y40P / V41L / L97b G / H99Q / C122S / Y151Q / R217T; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / A96G / L97b A / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> 100% <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / V38E / T39Y / Y40F / V41R / Y60bQ / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / Y60bL / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr"> 100% Working hours: 12:00 AM - 12:00 PM <h2 style=";text-align:left;direction:ltr">Y40Q / V41L / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37E / V3 8E / T39W / Y40H / V41R / Y60bQ / K61L / I65V / T97aE / L97bA / H99Q / C122S / Y149K / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> V41R / L97bR / H99Q / C122S / Y151L / R217V; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R35V / R36H / H37S / V38E / T39Y / Y40H / V41R / Y60bP / T97aE / L97bA / H99Q / C122S / Y149E / M157K; <h2 style=";text-align:left;direction:ltr"> 13560677 <h2 style=";text-align:left;direction:ltr"> R35S / V38D / V41R / L97bA / H99Q / C122S / Y151L; <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / M157K / T158S; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / V38F / T39H / V41R / Y60bK / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E / K224R; <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / V38F / T39H / V41R / Y60bK / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E; <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr">13556 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> 11.11.2019 <h2 style=";text-align:left;direction:ltr"> R37aS / V38D / V41Q / L97bG / H99Q / C122S / Y151L / R217T; <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H 99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35K / R36H / H37E / R37aK / V38E / T39F / Y40F / V41R / D60aP / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bG / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36Q / H37E / V3 8A / T39W / Y40H / V41R / Y60bQ / K61D / I65V / T97aE / L97bA / H99Q / C122S / Y149K / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / T 56A / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> 135556 ... <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37T / V38D / V41R / L9 7bA / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> F30H / R36L / V38E / V41R / K82R / L9 7bA / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> V38D / V41R / H99Q / C122S / Y151L / R217V; <h2 style=";text-align:left;direction:ltr">13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37Y / R37 aE / V38E / T39Y / Y40F / V41R / D60aE / Y60bA / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / R36H / H37E / V38E / T39N / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> R36S / V38E / Y40L / V41N / L97b G / H99Q / C122S / Y151L / R217T; <h2 style=";text-align:left;direction:ltr"> 4356062 <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37E / V38E / T39W / Y40H / V41R / Y60bQ / T97aE / L97bA / H99Q / C122S / Y149K / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R35W / R36H / H37Q / V38E / T39H / Y40H / V41R / T97aE / L97bA / H99Q / C122S / Y149L / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37Y / R 37aD / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H99Q / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> F350H / R35H / H37I / V38D / V41R / L97bA / H99Q / C122S / Y149W / Y151L / M157K / R217S; <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 13562299 <h2 style=";text-align:left;direction:ltr"> 435, 445, 445 <h2 style=";text-align:left;direction:ltr">13562 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / H99Q / C122S / Y151L / R217E; <h2 style=";text-align:left;direction:ltr"> 135626 ... <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> 14456 ... <h2 style=";text-align:left;direction:ltr"> 11.56.2019 <h2 style=";text-align:left;direction:ltr"> F350Y / R36H / R37aH / V38E / Y40H / V41R / K61E / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / V38D / V41R / L97b A / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> 43565676777 <h2 style=";text-align:left;direction:ltr"> I am sorry, I did not find this information. R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R; <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> R35V / R37aE / V38E / Y40Q / V41L / Y 60bS / T97aE / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / V38D / Y40H / V41R / L9 7bA / H99Q / C122S / I138V / M157K; <h2 style=";text-align:left;direction:ltr"> 435, 445, 445 <h2 style=";text-align:left;direction:ltr"> F30Y / R35H / R36H / H37D / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40H / V41R / L97b A / H99Q / C122S / M157K / T158A; <h2 style=";text-align:left;direction:ltr"> V38E / T39W / V41R / D60aP / Y 60bD / L97bA / H99L / C122S; <h2 style=";text-align:left;direction:ltr">F350Y / R36H / V38E / Y40H / V41R / I65T / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> V38D / V41R / L97bR / H99Q / C122S / Y151L / R217V; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 13562 <h2 style=";text-align:left;direction:ltr"> R36S / V38E / Y40Q / V41R / L97b G / H99L / C122S / Y151P / R217E; <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / Y60bL / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E; <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / V38F / T39H / V41R / Y60bK / T97aS / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Y / R36H / H37K / V38E / T39F / Y40F / V41R / T97aI / L97bA / H99Q / C122S / Y149R / M157K / K223S / K224Y; <h2 style=";text-align:left;direction:ltr"> Y40Q / V41L / L97bA / H99Q / C122S; <h2 style=";text-align:left;direction:ltr"> F30H / R35H / V38D / V41R / K61E / L97bA / H99Q / C122S / Y151L / M157K / R206H; <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40H / V41R / L 97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> 11.56.2019 <h2 style=";text-align:left;direction:ltr"> 135606 ... <h2 style=";text-align:left;direction:ltr"> V38D / V41L / L97bG / H99Q / C122S / Y151L / R217Q; <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37W / V38D / V41R / D60aE / L97bA / H99Q / C122S / Y149L / Y151L / M157K / R217D; <h2 style=";text-align:left;direction:ltr">F30Y / R35F / R36H / H37G / V38E / T39Y / Y40H / V41R / Y60bS / T97aD / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 435456 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R35I / R36H / H37E / V38E / T39Y / Y40H / V41R / Y60bS / T97aV / L97bA / H99Q / C122S / Y149L / M157K; <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> Y40H / V41Q / L97bG / H99Q / C122S / R217T; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39F / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> F30Y / R36H / V38D / Y40H / V41R / L97bA / H99L / C122S / F141L / M157K / T158A; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aA / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F30Y / R35Q / R36H / H37G / R37 aE / V38E / T39Y / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 435, 445, 445 <h2 style=";text-align:left;direction:ltr"> F30H / R36H / V38D / V41R / T56A / L9 7bA / H99Q / C122S / Y151L / M157K; <h2 style=";text-align:left;direction:ltr">F30Y / R35E / R36H / H37D / R 37aN / V38E / T39Y / Y40F / V4 1 ... <h2 style=";text-align:left;direction:ltr"> V38E / Y40Q / V41L / D60aP / Y60b L / L97bA / H99Q / C122S / Y149W; <h2 style=";text-align:left;direction:ltr"> F350Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / M157K; <h2 style=";text-align:left;direction:ltr"> F30H / R35Q / H37W / V38D / V41R / D60aE / Y60bS / L97bA / H99Q / C122S / Y149L / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37S / V38 E / T39Y / Y40H / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / Y151P / M157K / Q192H; <h2 style=";text-align:left;direction:ltr"> F30Y / R35M / R36H / H37D / R37 aD / V38E / T39Y / Y40F / V41R / D60aP / Y60bS / T97aE / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> F350Y / R35W / R36H / H37D / V38E / T39Y / Y40H / V41R / Y60bT / T97aD / L97bA / H99Q / C122S / Y149R / M157K; <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 4356567 ... <h2 style=";text-align:left;direction:ltr"> 435, 445, 445 <h2 style=";text-align:left;direction:ltr"> 13562 ... <h2 style=";text-align:left;direction:ltr"> 135 ... <h2 style=";text-align:left;direction:ltr"> F30Y / V38D / Y40L / V41R / L97bA / H 99Q / C122S / Y151L / M157K / Q192H; <h2 style=";text-align:left;direction:ltr"> F30H / V38D / Y40F / V41R / L97b A / H99Q / C122S / Y151L / M157F; <h2 style=";text-align:left;direction:ltr">H37M / R37aD / V38E / T39A / V41R / D60aP / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R; <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H99Q / Y151L / M157K; <h2 style=";text-align:left;direction:ltr"> 12425 ... <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35L / V38D / Y40H / V41R / N76S / L97bA / H99Q / C122S / M157K / K187E; <h2 style=";text-align:left;direction:ltr"> F30H / V38D / V41R / L97bA / H 99Q / C122S / Y151L / M157S; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F350Y / R36H / H37G / V38E / T39W / Y40H / V41R / Y60bA / T97aE / L97bA / H99Q / C122S / Y149Q / M157K; <h2 style=";text-align:left;direction:ltr"> 135 ... 100% <h2 style=";text-align:left;direction:ltr"> H37G / R37aD / G37bD / V38F / T39H / V41R / Y60bK / L97b R / H99E / C122S / Y151L / E1 75D / Q192T / R217E / K224R; <h2 style=";text-align:left;direction:ltr"> 435656767 <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> F30Y / R35W / R36H / H37D / V38E / T39Y / Y40F / V41R / Y60bS / T97aE / L97bA / H9 9Q / C122S / Y149K / M157K; <h2 style=";text-align:left;direction:ltr"> 13556 ... <h2 style=";text-align:left;direction:ltr"> V38E / T39L / V41R / D60aN / Y 60bP / L97bG / H99Q / C122S; F30Y / R36H / H37A / V38E / T39Y / Y40H / V41R / Y60bQ / T97aV / L97bA / H99Q / C122S / Y149R / M157K; F30Y / R35W / R36H / H37E / R37aP / V38E / T39Y / Y40F / V41R / Y60bN / T97aE / L97bA / H99Q / C122S / Y149Q / M157K; H37T / R37aL / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L / Q192R; H37G / R37aD / V38F / T39H / V41R / Y60bK / T97aS / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E / K224R; F30Y / R35W / R36H / H37S / V38E / Y40H / Y60bN / T97aE / L97bA / H99Q / C122S / Y149R / M157K; V38D / T39W / Y40L / V41R / T97aL / L97bA / H99Q / C122S; H37G / R37aD / G37bD / T39H / V41R / Y60bK / T97aS / L97bR / H99E / C122S / Y151L / E175D / Q192T / R217E / K224R; T39Y / V41R / L97bA / H99Q / C122S; V38D / T39L / Y40L / V41R / T97aW / L97bA / H99Q / C122S; F30Y / R36H / V38E / Y40H / V41R / T97aI / L97bA / H99Q / C122S / Y149N / L150V / M157K; R35S / V38D / L97bA / H99Q / C122S / Y151L / M157Y; R37aS / V38D / T39Y / Y40F / V41R / H99L / C122S / R217T; Y40Q / V41L / Y60bE / L97bA / H99Q / C122S / Y149R; and Y40H / V41T / L97bG / H99Q / C122S / R217T; and Any of these polypeptides in which C122S is unmodified and is C122C (according to chymotrypsin numbering) The modified u-PA polypeptide of claim 1, wherein the unmodified polypeptide consists of any of SEQ ID NOs: 1-6.

7. The amino acid modifications R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aT / Y60bT / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aT / Y60bT / T97aI / L97bA / H99Q / Y149R The modified u-PA polypeptide of any one of claims 1 and 3 to 6, comprising:

8. Amino acid modifications: R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Y / H37S / R37aP / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L; or R35Q / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / L97bA / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / H99Q / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / C122S / Y149R; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S; or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aA / Y60bP / T97aI / L97bA / H99Q / C122S / Y149R; or R35L / H37D / R37aS / V38E / T39Y / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R; or R35M / H37G / R37aD / V38E / T39W / V41R / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37G / R37aP / V38E / T39Y / V41R / D60aP / Y60bE / T97aI / L97bA / H99Q / C122S / Y149R; or R35A / H37G / R37aE / V38E / T39F / V41R / D60aE / Y60bP / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37S / R37aE / V38E / T39Y / V41R / D60aP / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37T / R37aP / V38E / T39Y / V41R / D60aE / Y60bD / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37G / R37aE / V38E / T39H / V41R / D60aP / Y60bA / T97aI / L97bA / H99Q / C122S / Y149R; or R35W / H37D / R37aS / V38E / T39Y / V41R / D60aE / Y60bS / T97aI / L97bA / H99Q / C122S / Y149R; or R35Q / H37G / R37aE / V38E / T39Y / V41R / D60aP / Y60bT / T97aI / L97bA / H99Q / C122S / Y149R; or R35W / H37P / R37aN / V38E / T39Y / V41R / D60aP / Y60bL / D97T / T97aE / L97bG / A98S / H99L / C122S; or R35W / H37P / R37aN / V38E / T39Y / V41K / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L / Q192A; or R35Y / H37V / R37aW / V38E / T39Y / V41R / D60aP / Y60bE / T97aI / L97bA / H99Q / C122S / Y151L / Q192T; or 7. The modified u-PA polypeptide of any one of claims 1 and 3 to 6, comprising R35W / H37P / R37aN / V38E / T39Y / V41K / D60aP / Y60bD / T97aI / L97bA / H99Q / C122S / Y151L / Q192T; or any of these polypeptides in which C122S is C122C (according to chymotrypsin numbering).

9. 9. The modified u-PA polypeptide of any one of claims 1 and 3 to 8, comprising the amino acid modifications R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / C122S / Y149R or R35Q / H37Y / R37aE / V38E / T39Y / V41R / D60aP / Y60bQ / T97aI / L97bA / H99Q / Y149R, wherein said unmodified u-PA polypeptide consists of the protease domain as set forth in SEQ ID NO:2 or SEQ ID NO:5, or consists of the mature unmodified u-PA polypeptide as set forth in SEQ ID NO:3 or SEQ ID NO:

6.

10. SEQ ID NOs: 8-15, 18-40, 42-44, 171, 176, 184, 198, 199, 216-219, 221-228, 230-233, 240, 242, 243, 249, 260-265, 267, 268, 270-273, 275-292, 299, 300, 307, 311-313, 315, 324-326, 329, 330, 335, 337-341, 346-351, 356-360, 364, 365, 368-374, 376, 377, 381, 382, ​​387, 389, 390, 398-404, 417, 436, 446, 466, 469, 475, 481, 484-487, 494-496, 498, 500, 503-511, 513-520, 523, 534, 535, 538, 541-545, 547, 550-553, 555, 557, 561, 562, 564, 565, 576, 584, 586-587, 5 91, 592, 594, 595, 597, 598, 600, 601, 603-608, 611-625, 627-629, 633, 645, 647-648, 650, 651, 653, 656-658, 661, 663-690, 692-722, 724-729, 731-754, 792-802, 807, 818, 820-822, 827, 829-836, 839, 841, 849, 855, 868-879, 88 7. The modified u-PA polypeptide of any one of claims 1 to 6, comprising the sequence of amino acid residues as shown in any of: 1-882, 884, 886, 900-904, 924-926, 932, 934-940, 942-944, 946-951, 953-961, 963, 972, 973, 975, and 977-987, or any of these sequences except that the residue at position C122 is C (Cys).

11. 11. The modified u-PA polypeptide of any one of claims 1 and 3 to 10, comprising the sequence of amino acid residues set out in SEQ ID NO: 21 or 987, or the sequence of amino acid residues set out in SEQ ID NO: 21 or 987 except that the residue at position C122 is C (Cys).

12. 11. The modified u-PA polypeptide of any one of claims 1, 3 to 7, 9 and 10, comprising the sequence of amino acid residues set out in SEQ ID NO: 18 or the sequence of amino acid residues set out in SEQ ID NO: 18 except that the residue at position C122 is C (Cys).

13. The modified u-PA polypeptide of any one of claims 1 to 12, which cleaves within residues QHARASHLG (residues 737-745) of human C3 (SEQ ID NO: 47).

14. The modified u-PA polypeptide of any one of claims 1 to 13, which is conjugated directly or via a linker to another moiety or polymer.

15. The modified u-PA polypeptide of claim 14 which is a fusion protein.

16. The modified u-PA polypeptide of claim 14 or 15, which is conjugated to a moiety and thereby pegylated.

17. The modified u-PA polypeptide of any one of claims 1 to 16, which is linked to a polymer or moiety that enhances serum half-life or reduces immunogenicity, or both.

18. The modified u-PA polypeptide of claim 17, which is directly or indirectly linked to serum albumin.

19. The modified u-PA polypeptide of claim 18, wherein the serum albumin is human serum albumin (HSA) and the HSA comprises the sequence set forth in SEQ ID NO:991, or a form having at least 90% or at least 95% sequence identity thereto.

20. The modified u-PA polypeptide of claim 14, wherein the moiety or polymer comprises a multimerization domain.

21. The modified u-PA polypeptide of claim 20, wherein the multimerization domain is an Fc domain comprising the sequence shown in SEQ ID NO:50 or SEQ ID NO:992, or a form having at least 95% sequence identity thereto.

22. The modified u-PA polypeptide of any one of claims 14 to 21, wherein the polymer is directly linked to the modified u-PA polypeptide or the polymer is linked to the modified u-PA polypeptide via a peptide linker.

23. The modified u-PA polypeptide of claim 22, wherein the polymer is linked via a linker comprising Gly and / or Ser.

24. The linker is (GS) ｎ or (G)n(S)n or (GGS)n or (SGG)n or (GGSSGG)n or (AGS)n, where n is 1-5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or is a linker containing up to 25 residues containing one or more of S, G, E, and K.

25. 24. The modified u-PA polypeptide of any one of claims 14 to 23, comprising a sequence of amino acid residues as set forth in any of SEQ ID NOs: 1001 to 1003 and 1024 to 1029, and multimers thereof, and sequences having at least 99% sequence identity thereto.

26. A fusion protein comprising a modified u-PA polypeptide according to any one of claims 1 to 14, or a catalytically active portion of a modified u-PA polypeptide, fused to a non-protease polypeptide or portion thereof.

27. 27. The fusion protein of claim 26, wherein the non-protease polypeptide is a multimerization domain or a protein transduction domain (PTD).

28. A fusion protein according to claim 26 or 27, comprising a heterologous activation sequence or a u-PA activation sequence.

29. The fusion protein of claim 28, wherein the activation sequence comprises a cysteine ​​and the modified u-PA polypeptide comprises a free cysteine, such that upon activation, the resulting activated polypeptide comprises two chains.

30. The fusion protein of claim 28 or 29, wherein the activation sequence is a u-PA activation sequence or a furin activation sequence.

31. The fusion protein of claim 30, wherein the activation sequence is a furin activation sequence comprising QSGQKTLRRRKR (SEQ ID NO: 996) or QCGQKTLRRRKR (SEQ ID NO: 995) or QSGQKTLRRKR (SEQ ID NO: 1044), or a furin activation sequence having at least 98% sequence identity thereto.

32. A fusion protein comprising a modified u-PA polypeptide or a catalytically active portion thereof according to any one of claims 1 to 25, comprising a signal sequence and said modified u-PA polypeptide or a catalytically active portion thereof.

33. The fusion protein of claim 32, wherein the signal sequence is derived from Il-2, u-PA, or IgGκ.

34. 34. The fusion protein of claim 32 or 33, further comprising a fusion partner.

35. 35. The fusion protein of claim 34, wherein the fusion partner is albumin, or an Fc domain, or a single chain antibody, or other antigen-binding fragment of an antibody, or a hyaluronic acid binding domain (HABD).

36. The fusion partner is a HABD that is tumor necrosis factor stimulated gene-6 (TSG-6), or is human serum albumin (HSA), or is IgG F ｃ 36. The fusion protein of claim 35, which is an anti-type II collagen antibody scFv fragment, or an anti-VEGFR antibody or fragment thereof.

37. A fusion protein according to any one of claims 32 to 36, comprising an activation sequence.

38. The fusion protein of claim 37, wherein the activation sequence comprises a cysteine ​​and the modified u-PA polypeptide comprises a free cysteine, such that upon activation, the resulting activated polypeptide comprises two chains.

39. The fusion protein of claim 37 or 38, wherein the activation sequence is a u-PA activation sequence or a furin activation sequence.

40. The fusion protein of any one of claims 37 to 39, wherein the activation sequence has a sequence set forth in any one of SEQ ID NOs: 995-998, 1041, and 1044, or a sequence having at least 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 995-998, 1041, and 1044.

41. The fusion protein of any one of claims 32 to 40, comprising an activation sequence, a modified u-PA polypeptide, and HSA.

42. The fusion protein according to any one of claims 32 to 41, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1006, 1007, 1009, and 1010, or a sequence having at least 95% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1006, 1007, 1009, or 1010.

43. The fusion protein according to any one of claims 32 to 39, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1004-1019 and 1034-1040, or a sequence having at least 95% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1004-1019 and 1034-1040.

44. 33. The fusion protein of claim 32, comprising an amino acid sequence set forth in SEQ ID NO: 1015 or 1019, or a sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1015 or 1019.

45. A fusion protein according to any one of claims 42 to 44, which does not have a signal sequence or which, upon expression, removes said signal sequence.

46. A fusion protein which is a two-chain activated form, chains A and B, of the fusion protein of any one of claims 26 to 45.

47. The fusion protein according to claim 46, comprising a sequence of amino acids as set forth in any one of SEQ ID NOs: 1005, 1011, 1014, 1015, 1016, 1019, and 1036, but lacking said signal sequence; or comprising a sequence having at least 95% sequence identity to a sequence of amino acids as set forth in any one of SEQ ID NOs: 1005, 1011, 1014, 1015, 1016, 1019, and 1036, but lacking said signal sequence; or comprising a sequence having at least 95% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 1005, 1011, 1014-1016, 1019, and 1036, but lacking said signal sequence.

48. The fusion protein of claim 46, wherein the B chain begins at residue IIGG of the modified u-PA polypeptide and ends at the C-terminus of the fusion protein.

49. A fusion protein according to any one of claims 46 to 48, comprising a modified u-PA polypeptide and HSA.

50. 50. The fusion protein of any one of claims 46 to 49, comprising an A chain from residues 21 to 178 and a B chain from residue 179 to the C-terminus of the protein, with a disulfide bond between residues 168 to 299.

51. 51. The fusion protein of any one of claims 26 to 50, which is a two-chain activated fusion protein, comprising an A chain and a B chain, said A chain consisting of residues 21-178 of SEQ ID NO: 1015 and said B chain consisting of residues 179-1022 of SEQ ID NO: 1015; said A chain and B chain are linked via a disulfide bridge between C168 and C299 of SEQ ID NO: 1015.

52. A fusion protein according to any one of claims 26 to 51, which dimerises via the interaction of complementary multimerisation domains, the fusion protein comprising the multimerisation domain.

53. The multimerization domain is F ｃ 53. The fusion protein of claim 52 which is a domain.

54. A nucleic acid molecule comprising a sequence of nucleotides encoding a modified u-PA polypeptide according to any one of claims 1 to 25 or a fusion protein according to any one of claims 26 to 53.

55. A vector comprising the nucleic acid molecule of claim 54.

56. 56. The vector of claim 55 which is an expression vector or a gene therapy vector.

57. 56. The vector of claim 55, which is a herpes virus simplex vector, or a vaccinia virus vector, or an adenovirus vector, or an adeno-associated virus vector, or a retrovirus vector, or an insect vector.

58. 58. An isolated cell or cell culture comprising a nucleic acid molecule according to claim 54 or a vector according to any one of claims 55 to 57.

59. A pharmaceutical composition comprising a modified u-PA polypeptide according to any one of claims 1 to 25, or a fusion protein according to any one of claims 26 to 53 in a pharma- ceutically acceptable medium.

60. 60. The pharmaceutical composition of claim 59, wherein the modified u-PA polypeptide or fusion protein is in two-chain activated form.

61. The pharmaceutical composition of claim 59 or 60, wherein the modified u-PA polypeptide or fusion protein comprises the protease domain of SEQ ID NO:21 or SEQ ID NO:

987.

62. The pharmaceutical composition of any one of claims 59 to 61, wherein the modified u-PA polypeptide or fusion protein has the sequence shown in SEQ ID NO: 1015 or 1019.

63. A modified u-PA polypeptide according to any one of claims 1 to 25, or a fusion protein according to any one of claims 26 to 53, or a nucleic acid molecule according to claim 54, or a vector according to any one of claims 55 to 57, or a pharmaceutical composition according to any one of claims 59 to 62, for use in treating a disease or condition mediated by or in which complement activation is involved, wherein inhibition of complement activation results in treatment or amelioration of the disease or condition.

64. 64. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of claim 63, wherein said disease or condition is selected from inflammatory diseases and conditions.

65. 65. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of claim 63 or 64, wherein the disease or condition is selected from complement 3 glomerulopathy (C3G), atypical hemolytic uremic syndrome (aHUS), sepsis, rheumatoid arthritis (RA), cardiovascular disease, membranoproliferative diseases and conditions, ophthalmic diseases or disorders, membranoproliferative glomerulonephritis (MPGN), multiple sclerosis (MS), myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex (IC)-mediated acute inflammatory tissue injury, Alzheimer's disease (AD), transplanted organ rejection, and ischemia-reperfusion injury.

66. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of claim 63 or 64, wherein the disease or condition is an ocular or ophthalmic disease, or rejection or inflammation due to a transplanted organ.

67. 67. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of claim 66, wherein said disease or condition is diabetic retinopathy or macular degeneration.

68. 68. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of claim 67, wherein said macular degeneration is age-related macular degeneration (AMD).

69. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of any one of claims 64 to 66, wherein said disease or condition is delayed graft function (DGF).

70. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition of any one of claims 63 to 69, wherein the modified u-PA polypeptide or fusion protein comprises the protease domain of SEQ ID NO:21 or SEQ ID NO:

987.

71. 70. The modified u-PA polypeptide, or fusion protein, or nucleic acid molecule, or vector, or pharmaceutical composition according to any one of claims 63 to 69, wherein the modified u-PA polypeptide or fusion protein comprises the sequence set forth in SEQ ID NO: 1015 or 1019.

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