Engineered plasmin molecules and related compositions for treating thrombosis

Abstract

The present invention provides engineered plasmin variants and related compositions that are resistant to inhibition of plasmin protease activities by plasmin inhibitors, which can be used as direct-acting thrombolytic agents in the treatment of thrombosis and thrombotic disorders.

Description

Attorney Docket No.: TSRI 2242.1PC ENGINEERED PLASMIN MOLECULES AND RELATED COMPOSITIONS FOR TREATING THROMBOSIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 63 / 643,473 (filed May 7, 2024; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under HL118114 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] This application includes by incorporation of reference a sequence listing in the XML format, 2242_1PC_Sequence Listing, which was created on April 14, 2025 and contains 21 KB in content. BACKGROUND OF THE INVENTION

[0004] Hemostasis is a marvel of intricate enzyme control. The activities of numerous enzymes and their inhibitors involved in coagulation (clot formation) and thrombolysis (clot breakdown) are precisely regulated to maintain equilibrium in the circulatory system. Disruptions of this balance that lead to clot formation inside a vessel can ultimately result in ischemia, heart attack, and stroke– major causes of human disease and death.

[0005] In contrast to Nature's sophisticated control over hemostasis, the current clinical paradigm for thrombolytic therapy (clot breakdown) is rather heavy-handed. Plasminogen activator (PA) enzymes, such as tissue plasminogen activator (tPA), are enzymes that cleave plasminogen (inactive zymogen) to generate plasmin (active serine protease), which is the key enzyme for breaking down fibrin mesh in blood clots.Plasminogen activators must be administered at relatively high concentrations in patients to account for their short half-lives (<5 min for tPA), resulting in activation of almost the entire plasmin pool in the blood and complete depletion of the endogenous levels of various inhibitors in the thrombolytic pathway. As a result of intentionally throwing the hemostatic system out of balance, the runaway pool of active plasmin is no longer subject to inhibitory regulation creating potential adverse effects that can be fatal. Despite being approved in the USA decades ago for treating heart attack (1987) and acute ischemic stroke (1996), use of tPA remains controversial due to its considerable risks, especially bleeding and intracranial hemorrhage. Newer analogs of tPA, such as tenecteplase, have not solved these issues.

[0006] Therefore, there is an essential and unmet need for a new generation of clot buster drugs that do not substantially disrupt the natural blood hemostasis. The present invention is directed to addressing this and other pressing needs in the art. SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides engineered plasmin variants that contain an inserted polypeptide or peptide epitope in the catalytic domain of plasmin. The insertion blocks inhibition of plasmin protease activity by a plasmin inhibitor but does not interfere with fibrin binding face of plasmin. In some embodiments, the polypeptide or peptide epitope is inserted after amino acid position 600, 625, 712 or 785, with the amino acid numbering being based on the sequence of human plasminogen with UniProt ID P00747 (SEQ ID NO:1). In some of these embodiments, the 2 amino acid residues immediately following the insertion position in the plasmin sequence are removed. In some preferred embodiments, the inserted epitope is capable of binding to a cognate antibody, thereby providing steric hindrance to plasmin binding by a plasmin inhibitor, e.g., α2AP.

[0008] In some engineered plasmin variants of the invention, the inserted peptide epitope is derived from human podoplanin and contains the amino acid sequence shown in in SEQ ID NO:7. Some engineered plasmin variants of the invention contains the amino acid sequence as set forth in any one of SEQ ID NOs:3-6, or a conservatively modified variant thereof. In some embodiments, the inserted polypeptide is an antibody moiety. In some of these embodiments, the antibody moiety is a scFv that specifically binds to fibrin. In various embodiments, the engineered plasmin variant is derived orconstructed from δ-plasmin. In some embodiments, the engineered plasmin variant blocks inhibition of plasmin activity by plasmin inhibitor α2AP or α2-macroglobulin.

[0009] In a related aspect, the invention provides plasmin compositions that degrade fibrin clots and resist inhibition of plasmin protease activity by a plasmin inhibitor. These plasmin compositions contain an engineered plasmin and an antibody moiety. The engineered plasmin is a plasmin variant containing an inserted polypeptide or peptide epitope in the catalytic domain, and the antibody moiety specifically binds the inserted polypeptide or peptide epitope. In some embodiments, the polypeptide or peptide epitope is inserted after amino acid position 600, 625, 712 or 785 in the plasmin sequence, with the amino acid numbering being based on human plasminogen (SEQ ID NO:1). In some embodiments, the 2 amino acid residues immediately following the insertion position in the plasmin sequence are removed.

[0010] In some plasmin compositions of the invention, the employed antibody moiety is a human antibody. In some embodiments, the employed antibody moiety is an Fab. In some embodiments, the employed antibody moiety is a bispecific molecule that also specifically binds to a target protein of interest (e.g., fibrin). In some embodiments, the employed antibody moiety contains HCDR1-3 and LCDR1-3 sequences as set forth in SEQ ID NOs:12-17, respectively. In some plasmin compositions of the invention, the plasmin variant is physically bound to the antibody moiety. In some of these embodiments, the plasmin variant is covalently linked to the antibody moiety. In some plasmin compositions of the invention, the inserted peptide epitope contains the amino acid sequence shown in SEQ ID NO:7, and the antibody moiety is an Fab that contains HCDR1-3 and LCDR1-3 sequences as set forth in SEQ ID NOs:12-17. In another aspect, the invention provides polynucleotide sequences that encode the engineered plasmin variants described herein.

[0011] In another related aspect, the invention provides methods for treating or preventing thrombosis in a subject. These methods entail administering to the subject in need thereof an effective amount of an engineered plasmin variant or a plasmin composition described herein. In various embodiments, the subject to be treated can be a human or a non-human mammal. In some embodiments, the subject to be treated suffers from or is at risk of developing a thrombotic disorder. In various embodiments, the thrombotic disorder to be treated is stroke, heart attack, peripheral vascular disease, superficial venous thrombosis, deep vein thrombosis (DVT) or pulmonary embolism.

[0012] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. Schematic overview of the approach for controlling interactions between plasmin and its inhibitor, α2AP. Normally, plasmin is rapidly and irreversibly inhibited by α2AP in circulation (half-life <1 second), preventing IV administration of plasmin. Inserting an epitope loop at appropriate sites in δ-plasmin resulted in plasmin variants such that antibody binding sterically blocks interactions with α2AP, while not impairing plasmin enzymatic activity to break down blood clots.

[0014] Figure 2. Structure-based design of δ-plasmin (δ-Pln) insertion mutants. These models show our design rationale for blocking α2AP approach to the surface of the δ-Pln enzymes through binding of an antibody / Fab to the inserted epitope loop. (A) Left: structure of the plasmin-α2AP Michaelis complex, derived from homology modeling and MD simulations4. Middle: a model of the NZ-1 Fab antibody5bound to AlphaFold model of δ-Pln-627. Right: overlay of the Pln-α2AP and Pln-Fab complexes, showing that α2AP and Fab would occupy the same space on the surface of the engineered δ-Pln variants. (B) Based on structural modeling, four surface-exposed loops were chosen for insertion sites of the epitope peptide in δ-plasmin. The images show an overlay of the AlphaFold structures of the four insertion mutants. The inserted epitope loop in each structure is highlighted. The view is taken from the region of space where α2AP would occupy / approach. (C) Alternate view of the overlaid insertion mutants, with α2AP binding surface shown as a transparent surface. (D) Close-up view, showing that epitope loops of δ-Pln-602 and δ-Pln-627 directly clash with α2AP in the α2AP-δ-plasmin complex. (E) Close-up view of epitope loops in δ-Pln-714 and δ-Pln- 787. Studies are underway to determine crystal structures of these engineered enzymes in complex with Fab will help refine and advance the structure-based design efforts.

[0015] Figure 3. Enzymatic characterization of δ-Pln constructs with and without Fab complexation. The plots on the left, showing initial rates of substrate conversion vs. substrate (in the absence of Fab), were used to determine Vmax, KM, and kcat for the enzymes. Plots on the right show the cleavage of fluorogenic peptide substrate by 125 nM enzyme at 37 C in the presence and absence of excess Fab.

[0016] Figure 4. Functional characterization of δ-plasmin variants with and without Fab complexation in presence of excess α2AP. The schematic diagram describes the experimental design. δ-plasminogen variants were activated with urokinase and incubated with various concentrations of Fab, then added to wells containing excess α2AP and fluorogenic peptide substrate. The fluorescence plots show proteolysis of substrate (1 mM) as mediated by the variants (125 nM).

[0017] Figure 5. Reinstating α2AP inhibition using an epitope peptide. (A) Competitive binding of Fab by an epitope peptide removes the steric impediment imposed by Fab and reinstates the ability for α2AP to inhibit the enzyme. (B) Sequence of the linear PA epitope peptide synthesized for preliminary studies (SEQ ID NO:7). (C) The linear epitope peptide competed with Fab binding to δ-Pln-627, reinstating α2AP inhibition in a concentration-dependent fashion.

[0018] Figure 6. Degradation of whole blood clots by δ-plasmin-Fab complexes. (A) To generate clots, blood was pipetted along the wall of wells in a 96-well plate, and clots were formed by adding thrombin and incubating at 37 °C for 4 hr (625 nM assay) or 8 hr (1 mM assay). (B) The plots show time courses of clot degradation in the presence of 2 equiv Fab relative to enzyme. Error bars represent SD of triplicate assays using different clots generated from the same human donor.

[0019] Figure 7. Design of a bispecific antibody to simultaneously afford clot targeting and block inhibition of plasmin by α2AP. One of the antibody combining sites will bind to the podoplanin epitope to block α2AP inhibition of the δ-Plasmin variants, while the other binding site will target fibrin to provide enhanced fibrin affinity / targeting.

[0020] Figure 8. Human plasma stability (in vitro) of d-Pln and XPln-627. Enzymes (100 nM) were incubated in pooled human plasma for various times in the absence or presence of Fab (400 nM). At specific times, fluorogenic peptide substrate was added (100 mM) and residual plasmin activity was determined. Controls confirmed that the substrate is not hydrolyzed by other plasma proteases. DETAILED DESCRIPTION I Overview

[0021] Plasmin is the key enzyme ultimately responsible for directly dissolving blood clots. Wild-type plasmin cannot be administered systemically because it israpidly and irreversibly inhibited by endogenous plasmin inhibitors such as α2- antiplasmin (α2AP). Nevertheless, there are ample precedents and compelling rationale for the use of plasmin as a therapeutic. For example, when plasmin is administered via catheter directly to a clot, preclinical studies and a phase 2 clinical trial completed in 2019 have established that plasmin has a superior safety profile and equal or better efficacy than tPA. Moreover, as the direct fibrin cleaving agent, plasmin does not require the presence of plasminogen on the clot, so plasmin would be effective even in chronically or completely occluded vessels that have low concentrations of plasminogen. Also, administration of plasmin itself would enable better control of plasmin levels in the blood versus treatments involving PAs, which induce a protein cascade to generate overwhelming levels of plasmin as an end product.

[0022] The present invention provides engineered plasmin variants and related combinations with antibodies that can be used as direct-acting thrombolytic agents without disrupting the delicate balance of hemostasis. As detailed below, this is in contrast to conventional thrombosis therapies that rely on the use of high concentrations of plasminogen activating enzymes, which activate plasmin excessively and deplete endogenous plasmin inhibitors. The invention is predicated in part on the studies undertaken by the invention to develop engineered plasmin variants and their complexes with Fab antibodies that resist inhibition by α2AP without impairing their fibrinolytic enzymatic activity, which can enable the first direct-acting thrombolytic enzymes that can be administered systemically. The inventive efforts were aimed at rendering plasmin suitable for systemic administration by engineering variants that resist inhibition by α2AP, with a built-in safety mechanism to turn off the thrombolytic activity if and when deemed desirable or necessary. As demonstrated herein, the engineered plasmin variants contain an inserted short epitope loop into δ-plasmin such that binding of a corresponding antibody to the epitope blocks inhibition by α2AP (Figure 1). As a specific exemplification, a plasmin variant bound by an Fab antibody to the inserted epitope was able to degrade whole blood clots (Figure 6) and also to substantially prolong plasmin enzymatic activity (Figure 8).

[0023] The invention represents novel treatment regimens for thrombotic diseases or disorders that are superior to current thrombolytic therapies that primarily rely on plasminogen activators (PAs). Therapies with PAs have many shortcomings, including poor pharmacokinetic profiles and causing disruption of hemostasis, which can lead tovarious adverse effects such as neurotoxicity and fatal bleeding complications. In general, PAs have short half-lives in vivo (e.g., tPA half-life being less than 5 min), which requires administration of large bolus doses, sometimes followed by prolonged infusion. This results a sudden rise in its blood concentration (e.g., by nearly 1,000-fold for recombinant tPA), causing a systemic rise in Pln activity, depletion of plasminogen and inhibitor proteins in the hemostatic system, and a corresponding drastic decrease in fibrinogen levels. These changes result in significantly reduced blood clotting capacity and substantial risk of intracranial hemorrhages that can be fatal. While there have been efforts to use plasmin as a direct-acting thrombolytic agent attempted by the skilled artisans, it has not been possible to administer plasmin systemically. Instead, the efforts in developing direct-acting thrombolytic agents were limited to catheter-directed therapy.

[0024] In contrast, the present invention provides a more controlled approach for thrombolytic therapy using engineered variants of plasmin as systemically administered, direct-acting thrombolytic enzymes. The approach overcomes the various shortcomings of thrombolytic therapies currently available or under development. Binding of an antibody to a plasmin variant improves the pharmacokinetic profile of the enzyme by reducing the clearance rate from plasma. Administration of an engineered plasmin variant that is resistant to plasmin inhibitors (e.g., α2AP) would enable better control of plasmin activity in the blood versus treatments involving PAs that induce a protein cascade to generate plasmin as an end product. The novel therapies of the invention allow for turning off the engineered plasmin activity after the blood clot is dissolved, if deemed necessary. While using a plasmin variant as a direct-acting thrombolytic agent will result in fewer bleeding side effects relative to PA enzymes, this additional level of built-in safety control over plasmin activity offers the possibility of shutting down the induced thrombolytic process in cases of bleeding complications (which is not possible with therapies based on PA enzymes), and would provide pre- determined control over the duration of fibrinolytic therapy. Importantly, this control could be guided by clinical parameters of thrombolytic success, such as reperfusion achieved as measured by, e.g., ultrasound.

[0025] Unless otherwise specified herein, the engineered plasmin variants and their complexes with antibodies, the encoding polynucleotides, expression vectors and host cells, as well as the related therapeutic applications, can all be generated or performedin accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos.4,965,343, and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol.57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention. II. Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), OxfordUniversity Press (4thed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0027] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, "an Env-derived trimer" can refer to both single or plural Env-derived trimer molecules, and can be considered equivalent to the phrase "at least one Env-derived trimer."

[0028] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0029] Epitope refers to an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0030] Effective amount of a therapeutic agent that is sufficient to generate a desired response, such as reducing or eliminating a sign or symptom of a condition or disease, such as a thrombotic disorder. For instance, this can be the amount necessary to prevent clot formation in the blood vessels or to break already formed clots. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in circulatory system) that has been shown to bethrombolytic in vitro. In some embodiments, an "effective amount" is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of thrombosis. In some embodiments, an effective amount is an amount that prevents one or more signs or symptoms of thrombosis from developing.

[0031] As used herein, a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein. The unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence. As used herein, proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell). For example, the amino acid sequence of plasmin and the amino acid sequence of podoplanin are not normally found joined together via a peptide bond.

[0032] "Heterologous", when used with reference to two polypeptides, indicates that the two are not found in the same cell or microorganism in nature. Allelic variations or naturally-occurring mutational events do not give rise to a heterologous biomolecule or sequence as defined herein. A "heterologous" region of a vector construct is an identifiable segment of polynucleotide within a larger polynucleotide molecule that is not found in association with the larger molecule in nature. When the heterologous region encodes a mammalian gene, the gene will usually be flanked by polynucleotide that does not flank the mammalian genomic polynucleotide in the genome of the source organism. As specific examples, the plasmin sequences are heterologous to the inserted peptide epitope or polypeptide in the engineered plasmin variant constructs of the invention.

[0033] “Linkage” refers to means of operably or functionally connecting two biomolecules (e.g., polypeptides or polynucleotides encoding two polypeptides), including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding. "Fused" refers to linkage by covalent bonding. A "linker" or "spacer" refers to a molecule or group of molecules that connects two biomolecules, and serves to place the two molecules in a preferred configuration with minimal steric hindrance.

[0034] As used herein, a pharmaceutical composition refers to a composition that contains an engineered plasmin variant described herein or its combination with acognate antibody that can treat or prevent the development of thrombosis upon being administered to a subject in need thereof.

[0035] Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0036] Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity / similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch, J. Mol. Biol.48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237- 44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio.24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0037] Plasmin, an activated serine protease and a key component of the fibrinolysis cascade, is generated from precursor protein plasminogen. Mature human plasminogen (i.e., without N-terminal signal peptide) is a 91 kDa zymogen containing 791 amino acids, produced in-vivo by the liver, and is heavily glycosylated in its circulating form. Cleavage of plasminogen at Arg561-Val562produces plasmin, which contains five kringle (K) domains (K1-K5) and a serine protease domain (or catalyticdomain is located at the C-terminus of plasmin. The kringle domains contain triple disulfide-linked peptide regions composed of 80 to 90 amino acid residues each, and a serine protease domain (Val562-Asn791). The kringle domains contain lysine-binding sites (LBSs) that play essential roles in binding to ligands with lysine residues at their C-termini, such as partially degraded fibrin or plasminogen-binding cell surface proteins. Plasmin consists of two polypeptide chains connected by two disulfide bonds. The heavy (A) chain (MW ~65,000) originates from the amino-terminal portion of plasminogen and contains the kringle (K) domains (binding sites). The light (B) chain (MW ~25,000) originates from the carboxy-terminus and contains the catalytic domain (active site). Plasmin’s protease activity is rapidly neutralized in plasma by the circulating inhibitor proteins α2-antiplasmin (α2AP), C1-inhibitor, and macroglobulin.

[0038] δ-plasmin is a truncated non-glycosylated recombinant plasmin variant, which is capable of being expressed and purified from E. coli. See, e.g., Hunt et al., Thromb. Haemost.2008, 100:413-9. This mutated plasmin eliminates four of the five kringle domains present on native plasmin, retaining only K1 that is fused directly to the unmodified catalytic domain of plasmin. It has been demonstrated that δ-plasmin exhibits similar enzymatic characteristics to full length plasmin despite its heavily mutated form.

[0039] As used herein, a signal sequence or “signal peptide” refers to the N- terminal portion of a secretory or membrane protein that assists it across the membrane of the rough endoplasmic reticulum, where it is synthesized, but is cleaved from the protein even before the synthesis of the protein is complete.

[0040] The term "subject" refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.

[0041] The term “treating” or “alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., stroke), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent themanifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. III. Engineered plasmin variants resisting inhibition by plasmin inhibitors

[0042] Plasmin is a serine protease that acts to dissolve fibrin blood clots. Plasmin is released as a zymogen called plasminogen (PLG) from the liver into the systemic circulation. Once in circulation, plasminogen adopts a closed, activation-resistant conformation. Upon binding to clots, or to the cell surface, plasminogen adopts an open form that can be converted into active plasmin by a variety of enzymes, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and factor XII (Hageman factor). Fibrin is a cofactor for plasminogen activation by tissue plasminogen activator. As molecular contact of fibrin strands is limited to the nodular bodies, open channels are available for plasminogen and plasminogen activators to enter. There they both adsorb on to the fibrin and plasminogen is activated in situ. Plasmin is therefore produced where it is needed and where it is protected from attack by plasmin inhibitors. As fibrin dissolves, plasmin is released into the circulation and is rapidly consumed by the inhibitor.

[0043] The present invention provides engineered plasmin variants that block inhibition of plasmin protease activity by plasmin inhibitors such as α2AP or α2- macroglobulin (α2M). Typically, the plasmin variants contain an inserted peptide epitope or polypeptide at a position in the catalytic domain of plasmin that is essential for the inhibitor to bind to plasmin. Other than providing steric hindrance to the inhibitor’s binding to plasmin, the inserted peptide epitope or polypeptide does not distort the structure of plasmin or disturb its enzyme activity. Any plasmin proteins known in the art can be used to generate the plasmin variants described herein. In some preferred embodiments, the employed plasmin molecule is derived from a human plasminogen. In humans, the plasmin protein in the zymogen form of plasminogen is encoded by the plg gene. Human plasminogen sequence and its recombinant expression have been well documented. See, e.g., Petersen et al., J. Biol. Chem.265: 6104-6111, 1990; Browne et al., Fibrinolysis 5: 257-260, 1991; and Law et al., Cell Reports 1: 185- 190, 2012. An exemplary full length human plasminogen sequence, including a 19-aa N-terminal signal peptide, is shown in SEQ ID NO:1 (Accession No. CAA28831.1; UniProt ID P00747). Natural variants of human plasminogen can also be employed toconstruct the engineered plasmin molecules of the invention. Examples of known plasminogen variants include variants K38E, L147P, R235H, V374F, R532H, S591P, A620T, and G751R.

[0044] Human plasminogen (SEQ ID NO:1): MEHKEVVLLLLLFLKSGQG- EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQ CVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQ KWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDIL ECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKN YCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRG NVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCH TTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSS TTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDP SVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRA TTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTT NPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVS LRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQE IEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGET QGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDS GGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0045] In some preferred embodiments, the plasmin molecule used for generating the engineered plasmin variants of the invention is δ-plasmin as exemplified herein. δ- plasmin is a truncated non-glycosylated recombinant plasmin variant, which contains only one of the five kringle domains of native plasmin (K1) and the catalytic domain. Sequence of wildtype δ-plasmin is shown below.

[0046] Amino acid sequence of δ-plasmin (SEQ ID NO:2):

[0047] MGKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSP ATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECAAPSFDCGKPQ VEPKKCPGRVVGGCVAHPHSWPWQVSLRT600- RF - GMHFCGGTLISPEWVLTAAHCLE625- KS- PRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIP ACLPSPNYVVADRTECFITGWGETQGTFG712-AG- LLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCF EKDKYILQGVTSWGLGCA785-RP- NKPGVYVRVSRFVTWIEGVMRNN

[0048] The polypeptide or peptide epitope can be inserted into plasmin catalytic domain (aka protease domain) at any site that will lead to steric hindrance to binding by plasmin inhibitors such as α2AP and macroglobulin. The site for insertion needs not be sequentially close to the essential residues or epitopes involved in plasmin / inhibitorinteraction, since the three-dimensional folding of the protease chain brings sequentially distant residues into spatial proximity. In general, the spatial relationships between the insertion site and the essential residues in plasmin / inhibitor interaction may be inferred by any appropriate method that are known to those skilled in the art. In some embodiments, the insertion site in plasmin sequence can be readily determined based on the well characterized structural components for binding by plasmin inhibitors, e.g., via three-dimensional structure modeling and analysis. As exemplified herein, utilizing crystal structures and molecular models of plasmin and its inhibitors (e.g., α2AP), the inventors selected insertion sites in plasmin catalytic domain where binding of an antibody could block approach of the inhibitor to plasmin. More specifically, the selected sites are in loop regions of the protease domain that are expected to block the inhibitor binding but do not interfere with the fibrin binding face of the enzyme, e.g., positions T600or E625as demonstrated herein.

[0049] In some embodiments, the insertion is at a location that will block binding of a plasmin inhibitor (e.g., α2AP) to the active site of plasmin. The active site of plasmin catalytic domain and the key residues have been extensively examined and characterized in the art. See, e.g., Ponting et al., Blood Coagul. Fibrinolysis.3: 605- 614, 1992; Law et al., Curr. Opin. Struct. Biol.23: 836-841, 2013; Castellino et al., Thromb. Haemost.93: 647-654, 2005; Law et al., Blood Advances 1: 766-771, 2017; Swedberg et al., Biochemistry 50: 8454-8462, 2011; Tsuda et al., Processes 9: 329, 2021; and Wu et al., Blood Adv.3: 729-733, 2019. For example, the catalytic triad of the active site of human plasmin contains residues His603, Asp646and Ser741. To construct the plasmin variants of the invention, the polypeptide or peptide epitope can be inserted at or around any one of these residues in the 3-dimentional structure.

[0050] Some engineered plasmin variants contain an inserted polypeptide that prevents binding of a plasmin inhibitor (e.g., α2AP) to the catalytic domain of plasmin, which can block binding by plasmin inhibitors without requiring the binding of a cognate antibody. In some preferred embodiments, an antibody fragment can be inserted into the catalytic domain to provide steric hindrance to the binding by the plasmin inhibitor. In some of these embodiments, the inserted antibody fragment can specifically bind to target molecule in addition to its function of preventing binding by a plasmin inhibitor. For example, a scFv fragment recognizing fibrin can be inserted into plasmin at one or more of the desired positions in the catalytic domain.Alternatively, a scFv targeting fibrin can be used to substitute for the kringle domains in plasmin, which resists α2AP inhibition (due to removal of the kringle domains and also provided clot targeting (due to the fused scFv), as exemplified. Engineered plasmin variants constructed in accordance with any of these designs enable targeted delivery of thrombolytic activity to blood clots.

[0051] Insertion of a polypeptide or peptide epitope to plasmin at desired positions in the catalytic domain can be performed in accordance with standard techniques well known in the art or the specific protocols exemplified herein. The method for generating the plasmin variants of the invention is not subject to any particular limitation. In some preferred embodiments, the plasmin variants of the invention are generated in accordance with the routinely practiced recombination methods. Such techniques are described, e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). As detailed below, the plasmin variant can be produced by inserting a polynucleotide (e.g., DNA) encoding the plasmin variant into a suitable expression system.

[0052] Some specific exemplifications for producing plasmin variants of the invention are discussed in detail in the Examples below. Typically, the methods involve inserting into a plasmin encoding polynucleotide sequence a second polynucleotide sequence (e.g., a cDNA sequence) encoding a peptide epitope or a polypeptide (e.g., a scFv antibody fragment) in frame at a desired position, through ligation or overlap extension PCR. In some embodiments, the inserted motif (e.g., a peptide) is directly linked at its N- and C- termini to the flanking plasmin sequences. In some other embodiments, linkers or spacers may be employed at the N- and C- termini of the inserted molecule (e.g., a scFv) for embedding into the plasmin sequence. This is to ensure proper folding and maintain the biological activities of the fusion partners. IV. Combinations of plasmin variants and cognate antibodies

[0053] The invention provides pharmaceutical compositions or combinations that contain an engineered plasmin variant described herein and a cognate antibody that binds to the inserted peptide or polypeptide in the plasmin variant. In some embodiments, the engineered plasmin variants of the invention contain an inserted peptide epitope, and require a cognate antibody that specifically binds to the peptideepitope in order to fully exert steric hindrance to the inhibitor’s binding to plasmin. In these embodiments, the employed antibody preferably binds to the inserted peptide epitope with a high affinity, e.g., with a KDthat is less than 1 x 10-8, less than 5.0 x 10-9, less than 1 x 10-9, less than 5.0 x 10-10, less than 2.5 x 10-10, or even lower. In some preferred embodiments, the employed antibody binds to the inserted peptide epitope with a KDthat is less than 1 x 10-10, less than 7.5 x 10-11, less than 5.0 x 10-11, less than 2.5 x 10-11, less than 1.0 x 10-11, or even lower.

[0054] Antibodies in the plasmin variant combinations include intact antibodies, antibody fragments or antigen-binding fragments (e.g., Fab fragments exemplified herein). Examples of antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a VHor VLdomain (see, e.g., Ward et al., Nature 341:544-546, 1989); and (vii) an isolated complementarity determining region (CDR) as a linear or cyclic peptide.

[0055] Suitable antibody fragments (or “antigen-binding fragments”) also include single chain antibodies. The term "single chain antibody" refers to a polypeptide comprising a VHdomain and a VLdomain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and / or carboxyl-termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable region fragment (scFv) is a single-chain antibody. Compared to the VL and VH domains of the Fv fragment which are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules.

[0056] In some embodiments, the employed cognate antibody that specifically binds to the inserted peptide epitope in the plasmin variant is a bispecific antibody. In addition to blocking inhibition of a plasmin inhibitor by binding to the inserted peptideepitope in the plasmin variant, the bispecific antibody can also target the complex to a desired site of interest (e.g., by binding to fibrin in blood clot) via its second binding arm. Bispecific molecules that target both the inserted peptide motif in the plasmin variant and a target antigen (e.g., fibrin in blood clots) can be readily generated in accordance with methods routinely practice in the art. See, e.g., Bos et al., Biotherapy 1992, 5, 187-199; Kurokawa et al., Thromb. Res. Suppl.1990, 10, 83-89; Thakur et al., Blood Rev.2018, 32, 339-347; Brinkmann et al., MAbs 2017, 9, 182-212; and Cho et al., Sci. Transl. Med.2021, 13, eabj5413. One specific example of bispecific antibodies that bind to both fibrin and the podoplanin epitope, utilizing the well-known anti-fibrin antibody “59D8”, is exemplified herein.

[0057] Several peptide epitopes and cognate binding antibodies can be readily employed in the engineered plasmin variants of the invention. In some embodiments, the inserted peptide is a human podoplanin derived epitope. As exemplified herein, this 12-residue peptide sequence, GVAMPGAEDDVV (SEQ ID NO:7), which corresponds to residues 40–51 of human podoplanin, can be paired with an antibody moiety that is derived from a rat anti-podoplanin molecule (“NZ1”) to provide the desired inhibition of plasmin binding by plasmin inhibitor α2AP. Four examples of engineered δ-plasmin variants containing such an inserted peptide epitope are shown in SEQ ID NOs:3-6, respectively. While termed ∆-Plg 602, ∆-Plg 627, ∆-Plg 714 and ∆-Plg 787 (aka δ-Pln- 602, δ-Pln-627, δ-Pln-714 and δ-Pln-787, respectively) herein, the insertion sites in these 4 δ-plasmin variants are actually after residue T600, E625, G712and A785, respectively. In these δ-plasmin variants, the two residues immediately after the insertion site in the plasmin sequence (i.e., R601F602, K626S627, A713G714, and R786P787respectively) are removed. Removal of the two residues is intended to provide appropriate space for the inserted epitope loop to occupy. Unless otherwise indicated, the amino acid numbering of δ-plasmin and other plasmin variants described herein are based on the full sequence of the plasminogen precursor protein. A prototype human plasminogen sequence is set forth in SEQ ID NO:1 (Accession No. CAA28831.1; Uniprot ID P00747). In some of these embodiments, the employed antibody is a human Fab molecule that is derived from the rat podoplanin antibody. Its heavy chain and light chain sequences are set forth in SEQ ID NOs:8 and 9, respectively. As demonstrated herein, this human Fab antibody binds to the inserted peptide motif with high affinity and slow dissociation.

[0058] ∆-Plg 602 (SEQ ID NO:3): MGKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGL EENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECAAPSFDCGKPQVEPKKCPG RVVGGCVAHPHSWPWQVSLRTGVAMPGAEDDVVGMHFCGGTLISPEWVLTAA HCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVI TDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNR YEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLG CARPNKPGVYVRVSRFVTWIEGVMRNN

[0059] ∆-Plg 627 (SEQ ID NO:4): MGKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGL EENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECAAPSFDCGKPQVEPKKCPG RVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEGVAMPG AEDDVVPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVI TDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNR YEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLG CARPNKPGVYVRVSRFVTWIEGVMRNN

[0060] ∆-Plg 714 (SEQ ID NO:5): MGKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGL EENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECAAPSFDCGKPQVEPKKCPG RVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSS YKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPS PNYVVADRTECFITGWGETQGTFGGVAMPGAEDDVVLLKEAQLPVIENKVCNR YEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLG CARPNKPGVYVRVSRFVTWIEGVMRNN

[0061] ∆-Plg 787 (SEQ ID NO:6): MGKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGL EENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECAAPSFDCGKPQVEPKKCPG RVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSS YKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPS PNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQS TELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCAGVAMPGAE DDVVNKPGVYVRVSRFVTWIEGVMRNN

[0062] Heavy chain of the podoplanin peptide-binding hFab (SEQ ID NO:8): The variable region (SEQ ID NO:10) is underlined, with the 3 CDRs (HCDRs1-3) (SEQ ID NOs:12-14, respectively) italicized. METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGRSLKLSCAASGFTFSNYG MAWVRQTPTKGLEWIASISAGGDKTYYGDSVKGRFSISRDNAKTTHYLQMDSLR SEDTATYYCAKTSRVYFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC

[0063] Light chain of the podoplanin peptide-binding hFab (SEQ ID NO:9): The variable region (SEQ ID NO:11) is underlined, with the 3 CDRs (LCDRs 1-3) (SEQ ID NOs:15-17, respectively) italicized. METDTLLLWVLLLWVPGSTGQFVLTQPNSVSTNLGSTVKLSCKRSTGNIGSNYV NWYQQHEGRSPTTMIYRDDKRPDGVPDRFSGSIDRSSNSALLTINNVQTEDEAD YFCHSYSSGIVFGGGTKLTIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTQGTTSVTKSFNRGEC

[0064] In addition to the exemplified human podoplanin peptide and the cognate human Fab antibody, other peptide and antibody pairs with strong binding affinities can also be employed in the practice of the invention. Examples of such peptide / antibody pairs include, e.g., HA epitope and the 4B2 antibody, FLAG epitope and the M2 antibody, and the HER2 epitope and the Herceptin antibody. See, e.g., Ranawakage et al., Sci Rep.2019; 9: 6895; Roosild et al., Acta Crystallogr Sect F Struct Biol Cryst Commun.2006; 62(Pt 9): 835-9; and Wang et al., Antibodies (Basel).2019; 8(1): 25.

[0065] In some embodiments, the combinations of the invention contain a plasmin variant and a cognate antibody as two separate non-interacting molecules. In some other combinations of the invention, the plasmin variant is physically associated with the cognate antibody. In some of these embodiments, the two molecules are non- covalently bound. In some other embodiments, the antibody is covalently conjugated to the plasmin variant. A physically connected plasmin-antibody complex could provide a permanent blockage of inhibition of plasmin activity by the plasmin inhibitor. This could be advantageous if the enzyme variant needs to function over a relatively longer time period. For covalent linkage, the antibody is preferably conjugated to the plasmin variant in a site-specific manner. Conjugation of an antibody molecule to a polypeptide sequence can be achieved with routinely practiced methods that are well known in the art. In some embodiments, site-specific conjugation of the antibody to the plasmin variant can be achieved via sortase-mediated enzymatic ligation. In some other embodiments, recombinant engineering and incorporated selenocysteine (e.g., as described in U.S. Patent 8,916,159 issued on December 23, 2014) can be used to for the conjugation. Other methods of antibody conjugation can include covalent coupling to native or engineered lysine side-chain amines or cysteine side-chain thiols in the plasmin variant. See, e.g., Wu et al., Nat. Biotechnol, 23: 1137-1146 (2005).V. Vectors and host cells for expressing engineered plasmin variants

[0066] Other than the engineered plasmin variants and their combinations with cognate antibodies described above, the invention also provides polynucleotide sequences that encode the engineered plasmin variants described herein, expression vectors that harbor the polynucleotide sequences, as well as host cells that harbor the polynucleotides or expression constructs. Polynucleotides or nucleic acids of the invention encompass deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences. They include, without limitation, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. The nucleic acids of the invention may be single- stranded, or partially or completely double-stranded (duplex). Duplex nucleic acids may be homoduplex or heteroduplex.

[0067] Expression constructs (i.e., expression vectors) of the invention contain polynucleotide sequences encoding an engineered plasmin variant described herein, as well as various structural components that are operably linked to the coding sequences. The operably linked structural components permit the transcription and translation of the encoding polynucleotide sequences. These components include a promoter and optionally also include an enhancer element or elements permitting the strong expression of the linked sequences. In addition to promoter / enhancer elements, expression vectors of the invention may further contain a suitable terminator, a polyadenylation sequence (e.g., the SV40 or Ad5E1b poly(A) sequence), and translational enhancer sequences (e.g., those from Adenovirus VA RNAs). Further, the vectors may encode a signal sequence directing the plasmin variant to a particular cellular compartment or, alternatively, may encode a signal directing secretion of the plasmin variant.

[0068] Vectors expressing the plasmin variants of the invention can be viral vectors, e.g., retroviral vectors or adenoviral vectors. Any viral vector that permits the introduction and expression of sequences encoding the plasmin variants or variants thereof in cells is acceptable for use in the methods of the invention. These include, e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, and Semiliki forest viral (alphaviral) vectors. The selection of a particular vector depends upon the intended use of the plasmin variants. For example, the selected vector must be capable of driving expression of the plasmin variant in the desired celltype, whether that cell type be prokaryotic or eukaryotic. Many vectors contain sequences allowing both prokaryotic vector replication and eukaryotic expression of operably linked gene sequences. Vectors useful for the invention may be autonomously replicating, that is, the vector exists extrachromosomally and its replication is not necessarily directly linked to the replication of the host cell's genome. Alternatively, the replication of the vector may be linked to the replication of the host's chromosomal DNA, for example, the vector may be integrated into the chromosome of the host cell as achieved by retroviral vectors and in stably transfected cell lines.

[0069] The polynucleotides and related vectors of the invention can be readily generated with standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfecting, transient gene expression and obtaining stable transfected cell lines are described in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res.19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0070] Host cells of the invention can be any cell into which recombinant vectors encoding a plasmin variant of the invention may be introduced and wherein the vectors are permitted to drive the expression of the plasmin variant is useful for the invention. Depending on the specific vector used for expressing the plasmin variant, various known cells or cell lines can be employed in the practice of the invention. Host cells for expressing the engineered plasmin variants can be, for example, a eukaryotic cell, or a prokaryotic cell, such as an animal cell, a plant cell, a bacterium, or a yeast. Host cells expressing the plasmin variants of the invention may be primary cultured cells, for example, primary human fibroblasts or keratinocytes, or may be an established cell line, such as NIH3T3, HEK293, HEK293T HeLa, MDCK, WI38, or CHO cells. A variety of expression vector / host systems are suitable for expressing the plasmin variants of the invention. Examples include, e.g., microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

[0071] The skilled artisans can readily establish and maintain a chosen host cell type in culture that expresses an engineered plasmin variant of the invention. Many specific examples of suitable cell lines that can be used in expressing the plasmin variants are described in the art. See, e.g., Smith et al., 1983., J. Virol 46:584; Engelhard, et al., 1994, Proc. Natl. Acad. Sci.91:3224; Logan and Shenk, 1984, Proc Natl Acad Sci, 81:3655; Scharf, et al., 1994, Results Probl. Cell Differ., 20:125; Bittner et al., 1987, Methods in Enzymol.153:516; Van Heeke & Schuster, 1989, J Biol Chem 264:5503; Grant et al., 1987, Methods in Enzymology 153:516; Brisson et al., 1984, Nature 310:511; Takamatsu et al., 1987, EMBO J 6:307; Coruzzi et al., 1984, EMBO J 3:1671; Broglie et al., 1984, Science, 224:838; Winter J and Sinibaldi R M, 1991, Results Probl Cell Differ., 17:85; Hobbs S or Murry L E in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill New York N.Y., pp 191-196 or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York, pp 421-463. The plasmin variant-expressing vectors may be introduced to selected host cells by any of a number of suitable methods known to those skilled in the art.

[0072] For the introduction of plasmin variant-encoding vectors to mammalian cells, the method used will depend upon the form of the vector. For plasmid vectors, DNA encoding the plasmin variant sequences may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation. These methods are detailed, for example, in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies offering reagents and methods for lipofection include Bio-Rad Laboratories, CLONTECH, GlenResearch, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0073] For long-term, high-yield production of the engineered plasmin variants, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the plasmin variant-encoding sequences controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. The selectable marker in the recombinant vector confers resistance to the selection and allows cells to stably integrate the vector into their chromosomes. Commonly used selectable markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene, 30: 147, 1984). Through appropriate selections, the transfected cells can contain integrated copies of the plasmin variant encoding sequence. VI. Pharmaceutical compositions and therapeutic applications

[0074] In another aspect, the invention provides pharmaceutical compositions and related therapeutic methods of using the engineered plasmin variants and their combinations with cognate antibodies as described herein. In some embodiments, the engineered plasmin variants or their combinations with cognate antibodies can be used for preventing and treating thrombosis and thrombosis related disorders. In the practice of the therapeutic methods of the invention, the subjects in need of prevention or treatment of thrombosis is administered (e.g., systemically) an engineered plasmin variant or a composition containing the plasmin variant and a cognate antibody. In some embodiments, the administered pharmaceutical composition contains (1) an engineered plasmin variant having an inserted peptide epitope and (2) an antibody moiety (e.g., an Fab fragment antibody) that specifically binds to the peptide epitope, which leads to steric hindrance to plasmin binding by plasmin inhibitors such as α2AP.

[0075] Thrombosis occurs when blood clots block the blood vessels. There are 2 main types of thrombosis: venous thrombosis and arterial thrombosis. Venous thrombosis is when the blood clot blocks a vein. Veins carry blood from the body back into the heart. Arterial thrombosis is when the blood clot blocks an artery. There are a number of disorders that are associated with or caused by thrombosis, or thromboticdisorders as referred to herein. Thrombotic disorders are distinguished by the location of the clot and the severity of the condition. Notable examples of thrombotic disorders include heart attack (or myocardial infarction), when the heart muscle tissue is deprived of oxygenated blood by a blockage; acute ischemic stroke, which occurs when a clot causes a loss of blood supply to the brain tissues and results in a loss of some neurological function; transient ischemic attack (or TIA), which has the same causes as an acute ischemic stroke when a clot impedes blood flow to the brain or spine cord, but is a transient neurological event; deep vein thrombosis, which occurs when a clot forms in a deep vein in the thigh, leg or pelvic muscle; pulmonary embolism, which is caused by a blockage of one or more arteries in the lungs; and phlebitis (or superficial phlebitis), which is caused by a clot that occurs in a superficial vein.

[0076] In the therapeutic applications of the invention, the engineered plasmin variant or its combination with a cognate antibody is typically provided as a pharmaceutical composition. The pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation. In general, the composition additionally includes one or more pharmaceutically acceptable carriers or vehicles and, optionally, other therapeutic ingredients. Various pharmaceutically acceptable additives can also be used in the compositions. Thus, some of the pharmaceutical compositions of the invention are vaccine compositions. For vaccine compositions, appropriate adjuvants can be additionally included. Examples of suitable adjuvants include, e.g., aluminum hydroxide, lecithin, AS01, Freund's adjuvant, MPLTMand IL-12. In some embodiments, the engineered plasmin variants or their combinations with cognate antibodies can be formulated as a controlled-release or time-release formulation. This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel. The various pharmaceutical compositions can be prepared in accordance with standard procedures well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 19thEd., Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos.4,652,441 and 4,917,893; U.S. Pat. Nos.4,677,191 and 4,728,721; and U.S. Pat. No.4,675,189.

[0077] Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example,intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. In general, the pharmaceutical composition is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate one or more symptom(s) associated with formation of thrombosis or development of a thrombotic disorder. For therapeutic applications, the compositions should contain a therapeutically effective amount of the engineered plasmin variant or its combination with a cognate antibody described herein. For prophylactic applications, the compositions should contain a prophylactically effective amount of the engineered plasmin variant or its combination with a cognate antibody described herein. The appropriate amount of the engineered plasmin variant or its combination with a cognate antibody can be determined based on the severity, age of the subject, and other personal attributes of the specific subject (e.g., the general state of the subject's health and the general condition of the subject's cardiovascular system). Determination of effective dosages can be additionally guided with animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject.

[0078] For prophylactic applications, the pharmaceutical composition is provided in advance of any symptom. The prophylactic administration of the pharmaceutical compositions serves to prevent or ameliorate any subsequent development of thrombosis. Thus, in some embodiments, a subject to be treated is one who has, or is at risk of developing, thrombosis or a thrombotic disorder, for example because of previous vein injury, lack of movement, and overweight. Following administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject can be monitored for symptoms associated with thrombosis. For therapeutic applications, the pharmaceutical composition is provided at or after the onset of a symptom of thrombosis or a thrombotic disorders. The pharmaceutical composition can thus be provided as soon as a subject is diagnosed with thrombosis or a thrombotic disorder. In various embodiments, the subject to be treated with the therapeutic compositions of the invention include one who has just suffered a heart attack (or myocardial infarction), an acute ischemic stroke, a transient ischemic attack (or TIA), a deep vein thrombosis, a pulmonary embolism, or phlebitis (or superficial phlebitis).

[0079] As noted above, some therapeutic applications of the invention involve the use of a pharmaceutical composition that contains (1) an engineered plasmin variant having an inserted peptide epitope and (2) a cognate antibody (e.g., an Fab molecule) that specifically binds to the inserted epitope, thereby providing steric hindrance to plasmin binding by plasmin inhibitors. In some of these embodiments, the subject may be further administered a competing agent that is also specifically recognized by the antibody bound to the engineered plasmin variant. This can be beneficial if, any time during the treatment process after administration of the pharmaceutical composition, the subject is at risk or shows signs of developing bleeding complications. This process can also provide control over the duration of thrombolytic activity for a pre-determined time or as guided by clinical parameters of thrombolytic success, (e.g., reperfusion achieved as measured by ultrasound). By displacing the antibody from the plasmin variant, the steric hindrance to plasmin binding by plasmin inhibitors is removed. As a result of restored plasmin inhibition by endogenous inhibitors such as α2AP, bleeding and intracranial hemorrhage due to excessive thrombolytic activities can be prevented or stopped. In some embodiments, the competing agent to displace the bound antibody contains a peptide sequence that is the same as or substantially identical to the inserted peptide epitope in the engineered plasmin variant. For example, the administered competing agent can be a linear or cyclic peptide containing the same sequence (e.g., SEQ ID NO:7) as that of the inserted peptide epitope in the engineered plasmin variant, as exemplified herein (see, e.g., Example 8).

[0080] For preventing or treating thrombosis, the engineered plasmin variants or their combinations with cognate antibodies of the invention can be used in combination with other known treatments for thrombosis. The latter treatments include blood- thinning anticoagulation medications (e.g., warfarin), which are commonly used to prevent blood clots from forming or getting bigger. Additionally, there are thrombolytic medications which can break up existing clots. Examples include streptokinase, alteplase, reteplase, Tenecteplase, urokinase, prourokinase, and anistreplase (APSAC). The subject may also receive catheter-directed treatments, such as percutaneous transcatheter treatment.

[0081] The engineered plasmin variants or their combinations with cognate antibodies of the invention can be provided as components of a kit. Optionally, such a kit includes additional components including packaging, instructions and various otherreagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents. An optional instruction sheet can be additionally provided in the kits. EXAMPLES

[0082] The following examples are offered to illustrate, but not to limit the present invention. Example 1. α2AP mechanism of inhibition and design rationale

[0083] Plasmin is a typical serine protease. Its mechanism of action initially involves attack by the active site Ser on the scissile amide bond of its substrate to form an acyl-enzyme intermediate and accompanying release of the C-terminal substrate fragment. Next, attack by a water molecule hydrolyzes the acyl-enzyme intermediate to complete the catalytic cycle. α2AP, like all protease inhibitors in the serpin class, has evolved to exploit the natural catalytic mechanism of plasmin18,56. The reactive center loop (RCL) of α2AP binds to the plasmin active site in an initial reversible step (Michaelis complex), and is then cleaved to generate the corresponding acyl-enzyme intermediate. However, following this cleavage event, α2AP undergoes a rapid structural rearrangement from the 'stressed' to 'relaxed' conformation (S to R transition). This rearrangement twists and partially denatures the protease, pulling the active site residues out of alignment, and prevents catalyzed hydrolysis of the acyl-enzyme intermediate. Essentially irreversible covalent inhibition of plasmin by α2AP is the end result. Example 2. Design rationale for engineering α2AP resistant plasmin variants.

[0084] Our approach to overcoming α2AP inhibition involves a structure- and mechanism-based rationale to engineer plasmin variants that are resistant to inhibition by α2AP and other general inhibitors such as a2-macroglobulin (Fig.1). Based on mechanistic considerations, we envisioned two possible strategies to prevent inhibition of plasmin by α2AP. The first is to sterically block the approach of α2AP (a protein of 50 kDa) to the plasmin active site. The second is to sterically prevent the α2AP structural rearrangement that distorts the plasmin structure, such that the α2AP acyl-enzyme intermediate can be hydrolyzed (as in the typical plasmin substrate turnover) to render α2AP ineffective as a covalent suicide inhibitor of the engineered plasmin. In our view, the former strategy is more desirable because it would not deplete the α2AP pool in plasma, whereas the second strategy, while workable, could lead to α2AP hydrolysis and depletion in vivo. Example 3. Structural model guided design of engineered δ-plasmin enzymes

[0085] Guided by available crystal structures and molecular models of plasmin57,58, α2AP59, and the plasmin-α2AP complex4, we identified four sites in the catalytic domain of plasmin where binding of an antibody could potentially block approach of α2AP to the enzyme (Fig.2). We targeted insertion sites that were in loop regions of the protease domain and that were expected to block binding of α2AP but not interfere with the fibrin binding face of the enzyme. We used AlphaFold to generate predicted structures of the four insertion variants (Fig.2). This analysis led to the selection of four insertion sites: δ-Pln-602, δ-Pln-627, δ-Pln-714, and δ-Pln-787 (the numbering refers to the site of loop insertion in the plasmin amino acid sequence). Example 4. Antibody-antigen selection and design of human Fab

[0086] For the epitope sequence in our design, we chose to use the PA tag5,60,61. The PA epitope is a 12-residue peptide sequence corresponding to residues 40–51 of human podoplanin, which was reported to bind a rat monoclonal antibody dubbed 'NZ- 1' with remarkably high affinity (KD~ 0.4 nM; ~2 orders of magnitude higher affinity than FLAG tag and its corresponding antibodies) and very slow dissociation kinetics (kd~ 6x10-6s-1)5,60. Previous reports have disclosed the crystal structure of the PA epitope- NZ-1 complex and have indicated that the PA epitope can be inserted into different proteins while retaining high affinity for the antibody5,60. The PA epitope was chosen primarily for two reasons. First, the high affinity and especially the slow dissociation was thought to be necessary for our purposes, because α2AP inhibition of plasmin is irreversible and could eventually overcome the reversible binding of an antibody to epitope if the antibody dissociation rate was too high. Second, with an eye toward future therapeutic use in humans, we desired an epitope sequence derived from a human protein (like the PA tag) because it is not expected to cause immunogenic responses. Anti-podoplanin antibodies, such as NZ-1, are under advanced development as cancertherapeutics and have shown no acute toxicity in dogs or monkeys62,63, suggesting that off-target binding of the Fab from our complexes to low levels of endogenous podoplanin will not pose a safety risk.

[0087] However, instead of using the commercial rat NZ-1 antibody for our studies, we decided to design and express the corresponding Fab with human heavy and light chains, with the help of colleague and collaborator, Prof. Ian Wilson (Scripps Research). We reasoned that designing the corresponding human Fab (hFab) from the start would be the best choice to circumvent future immunogenicity issues. Also, use of a human Fab is expected to facilitate structural studies, because the CKappa region employed contains a two amino acid deletion and several mutations in the FG loop, which aid in crystallization (crystallization of the δ-plasmin-hFab complexes are underway). Accordingly, light (human CL) and heavy (human CH1) chains were cloned into separate phCMV3 plasmids with an N-terminal murine Igkappa signal peptide. The light and heavy chain plasmids were then co-transfected into Expi293F cells in a 1:1 (w:w) ratio. After one week the media was harvested, and Fab purified by affinity chromatography. The Fab antibody was then further purified by size exclusion chromatography and characterized by mass spectrometry. Example 5. Expression of δ-Plasmin constructs and biophysical characterization of hFab binding

[0088] We recombinantly expressed and purified wild-type δ-plasmin along with the four PA epitope insertion mutants (δ-Pln-602, δ-Pln-627, δ-Pln-714, and δ-Pln-787) using reported procedures20,22. The WT δ-plasmin gene was cloned into a pET-28 b vector. The DNA sequences corresponding to the epitope were inserted at chosen sites using SOEing PCR and confirmed by sequencing. Plasmids were then transformed into BL21 (DE3) E. coli for expression. The transformed E. coli were brought to an exponential phase and the expression was induced with IPTG for 16 hours. The inclusion bodies containing δ-plasmin enzymes were harvested from the cell lysate and refolded by rapidly shifting the pH from 10.5 to 8 (See cited reference22). The refolded δ-plasmins were purified by affinity chromatography and characterized by SDS-PAGE and mass spectrometry.

[0089] hFab affinity for each of the plasminogen constructs was determined with biolayer interferometry (BLI) using an OctetRed96e (Sartorius) and Fab2G biosensors.Each of the δ-plasminogen variants containing the PA epitope bound the Fab with high affinity. Observed KD values were in the low / sub-nanomolar range, e.g., KD = 0.9±0.1 nM for δ-Pln-602, 2.1±0.1 nM for δ-Pln-627, 2.0±0.1 nM for δ-Pln-714, and 0.5±0.1 nM for δ-Pln-787 (Table 1). These affinity values for the Fab correspond well to reported binding constants for the NZ-1 antibody to the PA epitope5,60,61. Table 1: Enzymology and inhibition data for the engineered δ-plasmin variants in the presence and absence of Fab.

[0090] In Table 1, enzyme kinetics were determined using the fluorogenic substrate Ac-Lys-Gln-Trp-Lys-coumarin (concentrations of 5 mM–1,000 mM) with δ- plasmin concentrations of 5 nM and Fab concentrations of 100 nM. ki refers to the second order rate constant for inhibition of the enzyme by α2AP, using α2AP ranging from 5–50 nM, and substrate of 100 mM for all enzymes except δ-Pln-787, which used 1000 mM substrate. Example 6. Functional characterizations of δ-plasmin mutants and their Fab complexes

[0091] Functional characterizations of δ-plasmin mutants and their Fab complexes. With the δ-plasminogen insertion variants in hand, we first characterized their proteolytic activity using a fluorogenic peptide substrate (Fig.3). The δ-plasminogen proteins were incubated with urokinase at room temperature to bring about proteolytic conversion of the zymogens to active δ-plasmin enzymes. We verified by gel electrophoresis and mass spectrometry that incubation of the δ-plasminogen proteins with urokinase under these conditions resulted in the expected cleavage into two chains of >90% of the zymogen. To characterize the enzymatic properties of the four δ-plasmin variants, we synthesized a fluorescent peptide substrate, Ac-Lys-Gln-Trp-Lys- coumarin, based on reported plasmin substrate preferences64,65. Cleavage of the peptide substrate by δ-plasmin causes release of the coumarin moiety with a concomitant dramatic increase in fluorescence at 460 nm (excitation at 360 nm)66. We verified that urokinase does not cleave this substrate peptide (catalytic amounts of urokinase used to activate the δ-plasminogen remain present in the enzymatic assays). Enzymatic characterization with the fluorogenic substrate revealed that two of the four insertion mutants (δ-Pln-602 and δ-Pln-627) had kcat / KM values within 3-fold of wild type δ- plasmin, while the δ-Pln-714 and δ-Pln-787 mutants were much less active (Table 1 and Fig.3). Binding of Fab had little effect on turnover of the fluorogenic substrate for δ- Pln-627 and δ-Pln-787, whereas substrate conversion was considerably reduced upon Fab binding to δ-Pln-602 and δ-Pln-714 (Table 1 and Fig.3). Example 7. α2AP inhibition is blocked in Fab complexes of δ-Pln-627 and δ-Pln- 602

[0092] We next established if the δ-plasmin-Fab complexes would block α2AP from inhibiting the enzymes. Each δ-plasmin variant was briefly incubated with Fab ranging from sub-stoichiometric to excess concentrations (from 0.25 equiv to 2 equiv of Fab / δ-plasmin). The δ-plasmin-Fab mixture was then added to a well in a microtiter plate containing the fluorogenic peptide substrate and 2 equiv of α2AP relative to δ- plasmin. Proteolytic conversion of the fluorogenic substrate was monitored as a function of time (Fig.4). For wild-type δ-plasmin, which lacks the PA epitope and is not expected to bind Fab, turnover of the substrate was greatly reduced in the presence of α2AP, as expected due to rapid covalent inhibition by α2AP (Fig.4).

[0093] The δ-plasmin variants exhibited distinct behaviors in the presence of α2AP. δ-Pln-627 showed increasing resistance to α2AP inhibition with increasing ratios of Fab (Fig.4), as would be expected if Fab was sterically blocking inhibition by α2AP as intended. In the case where Fab was present in 2-fold excess, the substrate turnover for the δ-Pln-627-Fab complex was nearly identical to that of the reaction lacking α2AP. A different variant, δ-Pln-602, appeared to be more resistant to α2AP inhibition upon the addition of Fab (Fig.4). This finding is consistent with our structural modeling suggesting that epitope insertion at the 602 position can directly interfere with binding of α2AP (Fig.2). Interestingly, the concentration dependence of Fab blockingα2AP inhibition was less pronounced for δ-Pln-602 compared to the δ-Pln-627 variant. We are currently investigating if the changes engineered into the surface of δ-Pln-602 (epitope loop insertion) has blocked the conformational rearrangement of the α2AP acyl-enzyme intermediate, thus leading to increased hydrolysis of α2AP that is reflected in its loss of inhibitory function against this enzyme. In contrast to the above constructs, the δ-Pln-714 variant is not a promising enzyme for our purposes. Like the wild-type enzyme, δ-Pln-714 did not cleave the fluorogenic substrate under any Fab concentration in the presence of α2AP (Fig.4). This indicates that Fab binding at the 714 position does not block α2AP inhibition. More rigorous characterization of these enzymes, such as determining KIvalues for α2AP inhibition of the δ-plasmin-Fab complexes, is currently underway. It will also be invaluable to examine the X-ray crystallographic structures of these complexes. Example 8. Displacement of Fab from δ-plasmin construct using an epitope peptide

[0094] Our design offers a simple built-in safety catch to orthogonally control activity of the engineered plasmin using endogenous α2AP. This can be used to shut down the thrombolytic process if necessary in cases of bleeding complications, and would provide pre-determined control over the duration of fibrinolytic therapy. Importantly, this control could be guided by clinical parameters of thrombolytic success, such as reperfusion achieved as measured by ultrasound. The mechanism we have investigated employs an epitope peptide to competitively displace the Fab from the epitope loop in δ-plasmin (Fig.5). Competitive displacement of Fab from the plasmin construct would remove the steric impediment imposed by the Fab and reinstate the ability for endogenous α2AP to inhibit the enzyme. A linear peptide corresponding to the PA epitope has been used to elute PA-tagged proteins from a NZ-1 sepharose affinity resin60and has been crystallized in complex with the NZ-1 antibody5, supporting that the linear peptide could suffice for displacing the Fab. Indeed, we synthesized a linear PA epitope peptide and found that it could successfully compete with Fab in vitro to reinstate inhibitory function of α2AP against the δ-Pln-627 variant (Fig.5, C). Example 9. Degrading of whole blood clots by Fab complexes with plasmin variants

[0095] This example demonstrates that Fab complexes of δ-Pln-627 and δ-Pln-602 degrade whole blood clots under conditions, whereas wild type δ-plasmin is ineffective. We next characterized the activity of the δ-Pln-627-Fab and δ-Pln-602-Fab complexes in the more physiological context of whole blood clots using a halo clot assay6,67. These studies confirmed that both enzyme constructs in complex with Fab can cleave fibrin in the context of blood clots and can function in a setting involving endogenous concentrations of α2AP, a2-macroglobulin, and other factors present in blood. We first assessed the enzyme complexes at a concentration of 625 nM, which is lower than the physiological concentration of α2AP in plasma (~70 mg / L or 1.4 mM in the plasma)56. Wild-type δ-plasmin did not cause any breakdown of the clot, as would be expected due to its rapid inhibition by α2AP present in plasma. In contrast, the Fab complexes of δ- Pln-627 and δ-Pln-602 were both highly effective in dissolving the clot (Fig.6). Under conditions with a higher concentration of enzyme (1 mM), δ-Pln-627 and δ-Pln-602 variants completely degraded the clot whereas wild-type δ-plasmin again failed to cause any observed breakdown (Fig.6). These preliminary results provide a strong validation for the potential of this approach for developing plasmin variants as direct-acting thrombolytic agents. Example 10. Crystal structure of δ-Pln-787-Fab complex

[0096] Obtaining high-resolution structures of the plasmin-Fab complexes is an important aspect of our approach because it facilitates functional optimizations of the enzyme variants. We have obtained a 2.8 Å crystal structure of the δ-Pln-787 variant complexed to Fab. Crystals for the δ-Pln-787-Fab complex were grown at 20 C using the hanging drop method from a solution containing 0.085M sodium citrate, pH 5.6, 0.17M ammonium acetate, 15% glycerol, and 25.5% Peg4000. In the δ-Pln-787 structure we obtained, the entire epitope loop bound to Fab is clearly visible in the electron density 2Fo-Fc map. Whereas the AlphaFold prediction of the δ-Pln-787 structure was accurate for the overall folds of the kringle-1 and protease domains, the AlphaFold predicted structure of the epitope loop region did not match well with the actual observed orientation / conformation in the crystal structure. Moreover, the solved δ-Pln-787 structure clarified our understanding of why this enzyme is activated by urokinase relatively slowly, since the epitope extends out from the surface partiallycovering the urokinase cleavage site, probably interfering with urokinase binding / cleavage. Example 11. Use of bispecific antibodies to simultaneously block inhibition by α2AP and provide clot targeting

[0097] δ-Plasmin possesses inherent fibrin targeting ability due to the kringle-1 domain. However, it stands to reason that higher fibrin affinity, or targeting to other components of clots (e.g., platelets or Von Willebrand factor), could afford improved properties in a thrombolytic enzyme. Indeed, among the improvements to tPA during the development of tenecteplase were mutations that provided higher fibrin affinity and a corresponding increased plasma half-life29. The development of fibrin-binding antibodies has enjoyed a rich history42. Following the development of mAbs that targeted fibrin specifically over fibrinogen (Bode et al., 1985), various miniaturized fibrin-binding Ab fragments including Fab were described. Notable applications have included contrast / imaging of clots and targeted delivery of PA enzymes as thrombolytic agents (Bode et al., 1985; Bode et al., 1989; Kurokawa et al., 1990; Runge et al., 1991; Bos and Nieuwenhuizen, 1992; Lippi et al., 2013; Wang et al., 2014). The most widely studied fibrin-specific antibodies are termed 59D8 and T2G1S; both of these antibodies have been used in human clinical studies for imaging blood clots42. It should also be noted that the modularity of our design can also be used to readily employ bispecific antibodies targeting other binding sites (such as Von Willebrand factor, endothelial lumen, alveoli, microvascular occlusions (Ruppert et al., Bioconjug Chem 2002;13:804-811; Murciano et al., Blood 2003;101:3977-3984; de Maat et al., Blood 2022;139:597-607)). The ability to exchange modules and change target specificity could open up applications of engineered plasmin for treatment of clots for which currently there is no effective thrombolytic therapy available.

[0098] Illustrated in Figure 7 is a bispecific antibody (BsAb) that binds to the podoplanin epitope to block α2AP with one antigen binding domain, and binds to fibrin with the other antigen binding domain. This bispecific molecule uses the 59D8 antibody sequence to provide enhanced fibrin affinity. Notably, such a design should imbue the δ-Plasmin variants with higher fibrin affinity and specificity than natural, wild-type plasmin.

[0099] Anti-fibrin antibody (59D8) sequences:59D8_Heavy chain (SEQ ID NO:18) QVQLQQSGGDLVKPGGSLKLSCAASGFSFSSYGMSWVRQTPDKRLEWVASISS GGRHTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYFCARQEGDYDD WGQGTTVTVSS 59D8_Light chain (SEQ ID NO:19) DIELTQSPLTLSVIIGQPASISCKSSQSLLYSDGTTYLNWLLQRPGQSPKRLIYLV SKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLE LK Example 12. Design of plasmin variants in which the kringle domains are replaced with scFv antibody to provide clot targeting

[0100] δ-Plasmin represents a simplified and fully functional mutant of plasmin that lacks kringle domains 2-5 compared to wild-type plasmin, but retains kringle-1, which affords fibrin targeting. However, the kringle-1 domain also plays a role in binding of α2AP to plasmin. Accordingly, removal of the kringle-1 domain yields an enzyme (called microplasmin) that is inhibited by α2AP around 100-fold more slowly compared to wild-type plasmin or δ-Plasmin, but which also lacks specific fibrin binding22. Previous human clinical trials with microplasmin have reported minimal or no clinical benefits in treatment of peripheral arterial occlusion, which was due to the lack of binding to the clot resulting in washing away from the site of occlusion (clinical trial NCT00123305).

[0101] We designed variants of microplasmin in which a scFv corresponding to the fibrin targeting 59D8 antibody is fused to the N-terminus of the protease enzyme. This construct retains the fibrin cleaving activity of microplasmin, possess fibrin targeting imparted by the 59D8 scFv domain, and lack fast inhibition by α2AP since there is no kringle-1 domain. Moreover, the insertion of an epitope sequence or polypeptide at appropriate sites, as described herein, into these scFv-microplasmin fusion constructs could further block inhibition of the enzyme. We anticipate that epitope insertion, Fab binding, and the accompanying functional consequences (blockage of inhibition by α2AP) will behave similarly in microplasmin compared to δ-Plasmin, since the protease domains are identical. The ability to exchange the scFv domain to change target specificity opens up applications of engineered plasmin for treatment of clots for which currently there is no effective thrombolytic therapy available. Example 13 Fab binding to engineered δ-Pln variant enhanced plasmin stability

[0102] To examine whether Fab binding to the inserted PA epitope in the engineered Pln variant, we performed in vitro studies to compare Pln-627.Fab complex with the same variant enzyme without Fab or the parent δ-Pln enzyme with / without Fab. The results are shown in Figure 8. As expected, absence or presence of the Fab antibody does not affect stability of the parent δ-Pln enzyme since it does not contain an inserted PA epitope for the antibody to bind. In contrast, addition of the Fab antibody substantially prolonged enzymatic activity (e.g., due to greater stability) of the engineered δ-Pln-627 plasmin variant in human plasma.

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[0104] The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof.

[0105] It is further noted that all publications, sequence accession numbers, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. An engineered plasmin variant, comprising an inserted polypeptide or peptide epitope in the catalytic domain of plasmin that blocks inhibition of plasmin protease activity by a plasmin inhibitor but does not interfere with fibrin binding face of plasmin.

2. The engineered plasmin variant of claim 1, wherein the polypeptide or peptide epitope is inserted after amino acid position 625, 600, or 785; wherein the amino acid numbering is based on the sequence of human plasminogen with UniProt ID P00747 (SEQ ID NO:1).

3. The engineered plasmin variant of claim 2, wherein the 2 amino acid residues immediately following the insertion position in the plasmin sequence are removed.

4. The engineered plasmin variant of claim 1, wherein the inserted epitope is capable of binding to a cognate antibody, thereby providing steric hindrance to plasmin binding by the plasmin inhibitor.

5. The engineered plasmin variant of claim 1, wherein the inserted peptide epitope comprises SEQ ID NO:

7.

6. The engineered plasmin variant of claim 5, comprising an amino acid sequence as set forth in any one of SEQ ID NOs:3-6, or a conservatively modified variant thereof.

7. The engineered plasmin variant of claim 1, wherein the inserted polypeptide is an antibody moiety.

8. The engineered plasmin variant of claim 7, wherein the antibody moiety is a scFv that specifically binds to fibrin.

9. The engineered plasmin variant of claim 1, which is a variant of δ- plasmin.

10. The engineered plasmin variant of claim 1, wherein the plasmin inhibitor is α2AP or α2-macroglobulin.

11. A plasmin composition that degrades fibrin clots and resists inhibition of plasmin protease activity by a plasmin inhibitor, comprising an engineered plasmin and an antibody moiety, wherein the engineered plasmin is a plasmin variant containing an inserted polypeptide or peptide epitope in the catalytic domain, and the antibody moiety specifically binds the inserted polypeptide or peptide epitope.

12. The plasmin composition of claim 11, wherein the polypeptide or peptide epitope is inserted after amino acid position 625, 600, or 785; wherein the amino acid numbering is based on human plasminogen (SEQ ID NO:1).

13. The plasmin composition of claim 12, wherein 2 amino acid residues immediately following the insertion position in the plasmin sequence are removed.

14. The plasmin composition of claim 11, wherein the antibody moiety is a human antibody.

15. The plasmin composition of claim 11, wherein the antibody moiety is an Fab.

16. The plasmin composition of claim 11, wherein the antibody moiety is a bispecific molecule that also specifically binds to a target protein.

17. The plasmin composition of claim 16, wherein the target protein is fibrin.

18. The plasmin composition of claim 11, wherein the antibody comprises HCDR1-3 and LCDR1-3 sequences as set forth in SEQ ID NOs:12-17, respectively.

19. The plasmin composition of claim 11, wherein the plasmin variant is a variant of δ-plasmin.

20. The plasmin composition of claim 11, wherein the plasmin variant is physically bound to the antibody moiety.

21. The plasmin composition of claim 11, wherein the plasmin variant is covalently linked to the antibody moiety.

22. The plasmin composition of claim 11, wherein the inserted peptide epitope comprises SEQ ID NO:7, and the antibody moiety is an Fab.

23. A polynucleotide sequence encoding the engineered plasmin variant of claim 1.

24. A method for treating or preventing thrombosis in a subject, comprising administering to the subject in need thereof an effective amount of the engineered plasmin variant of claim 1 or the plasmin composition of claim 11.

25. The method of claim 24, wherein the subject is a human or a non- human mammal.

26. The method of claim 24, wherein the subject suffers from or is at risk of developing a thrombotic disorder.

27. The method of claim 26, wherein the thrombotic disorder is stroke, heart attack, peripheral vascular disease, superficial venous thrombosis, deep vein thrombosis (DVT) or pulmonary embolism.

28. The method of claim 24, wherein the subject is administered with a plasmin composition that comprises the engineered plasmin variant and an antibody that specifically binds to the inserted peptide epitope.

29. The method of claim 28, wherein the antibody is an Fab.

30. The method of claim 28, further comprising displacing the antibody from the engineered plasmin variant by administering to the subject an agent that is specifically recognized by the antibody, thereby to prevent or stop bleeding complications in the subject.

31. The method of claim 30, wherein the agent comprises a peptide sequence that is substantially identical to the inserted peptide epitope in the engineered plasmin variant.

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