Tissue plasminogen activator antibodies and methods of use thereof

Abstract

The present invention provides tissue plasminogen activator antibody molecules and their uses. More specifically, the invention disclosed herein provides humanized antibody molecules that specifically bind to tissue plasminogen activator (TPA) and their use in treating TPA-induced bleeding, especially treating systemic bleeding such as cerebral hemorrhage after ischemic stroke or myocardial infarction or treating systemic bleeding after TPA treatment of pulmonary embolism, ischemic stroke or myocardial infarction.

Description

Background of the Invention

[0001] Field of the Invention

[0002] The present invention provides tissue plasminogen activator antibody molecules and their uses. More specifically, the presently disclosed invention provides humanized antibody molecules that specifically bind to tissue plasminogen activator (TPA) and their use in treating TPA-induced bleeding; in particular, treating systemic bleeding such as cerebral hemorrhage after ischemic stroke or myocardial infarction or using TPA to treat systemic bleeding after pulmonary embolism, ischemic stroke or myocardial infarction; or in patients in whom endogenous TPA is elevated due to reasons including, but not limited to, prolonged coronary artery bypass surgery, liver transplantation, severe or multiple trauma, heat stroke or near drowning.

[0003] Background Information

[0004] Tissue plasminogen activator (TPA or tPA) is the only effective medical treatment for ischemic stroke and it also reduces mortality in patients with acute myocardial infarction. (Donnan GA, Davis SM, Parsons MW, Ma H, Dewey HM, Howells DW. How to make better use of thrombolytic therapy in acute ischemic stroke. Nat Rev Neural. 2011; 7: 400-409). However, TPA treatment significantly increases the risk of severe or fatal bleeding. Intracranial bleeding after TPA therapy can be catastrophic, and approximately 1% of patients with stroke treated with TPA will experience severe disabling or fatal bleeding. (Saver JL. Haemorrhage after thrombolytic therapy for stroke: the clinically relevant number needed to harm. Stroke. 2007; 38: 2279-2283). In a study of 511 patients with ischemic stroke treated with TPA, up to 20% developed an acute deterioration in their mental status requiring emergency CT scans, revealing a 17% incidence of symptomatic intracranial hemorrhage (sICH), resulting in an 87.5% mortality rate, compared with 22.4% in patients without sICH (James B, Chang AD, McTaggart RA, et al. Predictors of symptomatic intracranial haemorrhage in patients with an ischaemic stroke with neurological deterioration after intravenous thrombolysis. J Neurol Neurosurg Psychiatry 2018;89:866-869).As with alteplase, similar ICH rates are seen with tenecteplase (approximately 6%) (Ronning OM, Logallo N, Thommessen B, et al., Tenecteplase versus alteplase between 3 and 4.5 hours in low nationalinstitutes of health stroke scale. Stroke 2019;50(2):498-500), and the rates were slightly better with tenecteplase relative to alteplase in a meta-analysis for the occurrence of any ICH: 9.6% versus 11.7% (Xu N, Chen Z, Zhao C, et al., Different doses of tenecteplase vs alteplase in thrombolysis therapy or acute ischemic stroke: evidence from randomized controlled trials). Drug Des Devel Ther 2018;12:2071-2084). Similarly, 0.9-1.0% of patients given TPA for myocardial infarction develop intracranial hemorrhage and more than 50% die. (Gurwitz JH, Gore JM, Goldberg RJ, et al., Risk for intracranial haemorrhage after tissue plasminogen activator treatment for acute myocardial infarction. Participants in the National Registry of Myocardial Infarction 2. Ann Intern Med. 1998;129:597-604). Although bleeding complications are common in older adults, children face significant bleeding risks from TPA.(GuptaAA, Leaker M, Andrew M, et al., Safety and outcomes of thrombolysis with tissue plasminogen activator for treatment of intravascular thrombosis in children. J Pediatr. 2001; 139: 682-688). Fear of bleeding complications has undermined the therapeutic administration of TPA in patients who may benefit. (Saver JL. Haemorrhage after thrombolytic therapy for stroke: the clinically relevant number needed to harm. Stroke. 2007; 38: 2279-2283). A recent review of thrombolytic therapy in patients with pulmonary embolism (PE) presented "real-world" major bleeding and ICH rates of > 21% and 3.3%, respectively. Further, they concluded that this type of treatment should only be used in patients with PE who have an unstable cardiovascular status because of these bleeding rates. (Eberle H, Lyn R, Knight T, et al., Clinical update on thrombolytic use in pulmonary embolism: A focus on intermediate-risk patients. Am J Health-Syst Pharm 2018;75:1275-85.) Therefore, the lack of specific antidotes for TPA or tenecteplase limits access to these agents for the vast majority of PE patients.

[0005] Once TPA-induced bleeding occurs, there is no specific TPA inhibitor or antidote that can be used to treat bleeding. In an effort to restore coagulation, patients are usually given cryoprecipitate, fresh frozen plasma, and platelets in the absence of conclusive evidence of efficacy. (Morgenstern LB, Hemphill JC, 3rd, Anderson C et al., Guidelines for the management of spontaneous intracerebral haemorrhage: a guideline for healthcare professionals from the American Heart Association / American Stroke Association. Stroke. 201041: 2108-2129). Antifibrinolytic drugs such as tranexamic acid, ε-aminocaproic acid, aprotinin, and new plasmin inhibitors have also been used, but to a limited extent. Unfortunately, these agents not only inhibit the plasminogen (Pg) activation system, but also interfere with other molecular pathways. For example, aprotinin affects plasmin activity and the kallikrein system and has been associated with severe allergic reactions. (Munoz JI, Birkmeyer NJ, Birkmeyer JD, O'Connor GT, Dacey LJ. Is epsilon-aminocaproic acid as effective as aprotinin in reducing bleeding with cardiac surgery a meta-analysis. Circulation. 1999; 99: 81-89).

[0006] The mechanisms responsible for TPA bleeding remain relatively poorly understood. By comparison with streptokinase, TPA activation of Pg is significantly amplified by fibrin and this discriminating property of TPA is predicted to increase fibrinolysis without increasing bleeding complications. However, excessive generation of plasmin by TPA may degrade circulating coagulation factors, which affects coagulopathy and can enhance bleeding in vivo. TPA is a multidomain molecule that acts through catalytic and non-catalytic interactions. There is experimental evidence that non-catalytic effects of TPA (e.g., those that do not cause plasminogen activation) cause blood-brain barrier disruption and are responsible for some of the neurotoxic effects of TPA. As such, it is unclear whether TPA-induced cerebral hemorrhage requires the catalytic activity of TPA. TPA therapy is beneficial in the treatment of ischemic stroke and myocardial infarction, but in some patients, the therapy is complicated by severe or fatal bleeding in the brain or elsewhere. Fear of TPA-induced bleeding has limited the therapeutic use of TPA. In humans, TPA-induced bleeding and adverse outcomes are more frequent after prolonged local ischemia. Similarly, in experimental stroke, TPA reproducibly causes cerebral hemorrhage, blood-brain barrier disruption, and enhanced neuronal cell death following prolonged ischemia.

[0007] In non-thrombotic models of stroke, there is evidence that TPA can exert toxic effects through mechanisms that do not require plasminogen activation or affect fibrinolytic activity, such as PDGF-CC cleavage. (Su EJ, Fredriksson L, Geyer M, et al., Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke. Nat Med. 2008; 14: 731-737). In pathological conditions such as myocardial ischemia and stroke, the fibrinolytic activity of therapeutic TPA is enhanced by elevated levels of circulating fibrin fragments (e.g., D-dimers), which may enhance the bleeding process. (Barber M, Langhorne P, Rumley A, Lowe GD, Stott DJ. D-dimer predicts early clinical progression in ischemic stroke: confirmation using routine clinical assays. Stroke. 2006; 37: 1113-1115).

[0008] In international patent application PCT / US2014 / 012555 published as WO2014 / 116706A1, it is described that tissue plasminogen activator activates plasminogen by contributing to the fibrin-dependent mechanism in TPA treatment of post-ischemic stroke cerebral hemorrhage. SUMMARY OF THE INVENTION

[0010] This Summary of the Invention describes several embodiments of the presently disclosed subject matter and, in many cases, a list of variations and combinations of these embodiments. The Summary of the Invention merely exemplifies a variety and various embodiments. Reference to one or more representative features of a given embodiment is likewise exemplary. Such an embodiment may generally exist with or without the features mentioned; likewise, these features may be applicable to other embodiments of the presently disclosed subject matter, whether or not listed in the Summary of the Invention. To avoid excessive repetition, the Summary of the Invention does not list or suggest all possible combinations of such features.

[0011] The present invention solves these and other related needs, in particular by providing antibody molecules capable of inhibiting TPA-induced fibrinolysis. Tissue plasminogen activator activates plasminogen through a fibrin-dependent mechanism that contributes to systemic bleeding, particularly cerebral bleeding, or systemic bleeding after tissue plasminogen activator treatment, more particularly after TPA treatment of ischemic stroke. The antibody molecules of the present invention block this effect, thereby reducing TPA-induced bleeding, particularly systemic bleeding, more particularly cerebral bleeding, or systemic bleeding after tissue plasminogen activator treatment, more particularly after TPA treatment of ischemic stroke.

[0012] The present invention provides an antibody molecule that specifically binds to human TPA or a TPA mutant. The antibody molecule has a subnanomolar affinity for inhibiting fibrin-dependent plasminogen activation with an IC50 of <5 nM, and the amino acid sequence of the TPA mutant is at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2. The antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has a CDR1 selected from SEQ ID NOs: 3 and 4, a CDR2 selected from SEQ ID NOs: 5 and 6, and a CDR3 selected from SEQ ID NOs: 7 and 8, and the light chain variable domain has a CDR1 selected from SEQ ID NOs: 9 and 10, a CDR2 selected from SEQ ID NOs: 11 and 12, and a CDR3 selected from SEQ ID NOs: 13.

[0013] Typically, the antibody molecule selectively inhibits fibrin-promoted plasminogen activation. Typically, the antibody molecule inhibits human fibrin clot degradation while not affecting TPA amidolytic activity or fibrin-independent activation.

[0014] Typically, the antibody molecule is a purified or isolated antibody molecule.

[0015] The antibody molecule may be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, an antibody fragment or a monoclonal antibody, in particular a Fab, Fab′ or F(ab′)2 fragment, a single-chain antibody, in particular a single-chain variable fragment (scFv), a domain antibody, a nanobody, a diabody or a DARPin.

[0016] More specifically, the antibody molecule can be a humanized antibody or a humanized antibody fragment, in particular a Fab, Fab′ or F(ab′)2 fragment, a single-chain antibody, in particular a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody or a designed ankyrin repeat protein (DARPin).

[0017] In one aspect, the present invention provides a pharmaceutical composition comprising the antibody molecule of the present invention and a pharmaceutically acceptable carrier.

[0018] For use in therapy, the antibody molecule is incorporated into a pharmaceutical composition suitable for facilitating administration to an animal or human. Suitable formulations of the antibody molecule can be prepared by mixing the antibody molecule with a physiologically acceptable carrier, excipient or stabilizer, the suitable formulation being in a lyophilized form or otherwise a dry formulation or an aqueous solution or an aqueous or non-aqueous suspension form. The carrier, excipient, modulator or stabilizer is nontoxic at the dose used and the concentration used. They include buffer systems such as phosphates, citrates, acetates and other inorganic or organic acids and their salts; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyldimethylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as Polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosaccharides or polysaccharides and other sugars, including glucose, mannose, sucrose, trehalose, dextrin or dextran; complexing agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or ionic or nonionic surfactants such as TWEEN TM (Polysorbate), PLURONICS TM Or fatty acid esters, fatty acid ethers or sugar esters. The antibody preparation may also contain organic solvents such as ethanol or isopropanol. The excipient may also have the function of regulating release or release absorption.

[0019] In one aspect, the pharmaceutical composition comprises an antibody molecule of the invention in an aqueous buffer solution or a lyophilisate prepared from such a solution.

[0020] Suitable modes of administration are parenteral by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of administration such as by inhalation, transdermal, intranasal, buccal, oral may also be suitable.

[0021] In a further aspect, the invention provides an antibody molecule of the invention for use as a medicament.

[0022] In yet another aspect, the present invention provides an antibody molecule of the present invention for use in treating or preventing TPA-induced bleeding.

[0023] In one embodiment, the invention provides an antibody molecule of the invention for use in treating or preventing systemic bleeding, particularly cerebral hemorrhage or systemic bleeding after tissue plasminogen activator treatment, more particularly after TPA treatment of ischemic stroke.

[0024] In yet another aspect, the present invention provides a method for treating or preventing TPA-induced bleeding, the method comprising administering an effective amount of an antibody molecule of the present invention to a subject in need thereof.

[0025] In one embodiment, the present invention provides a method for treating or preventing systemic bleeding, especially cerebral hemorrhage or systemic bleeding after tissue plasminogen activator treatment, more specifically TPA treatment of ischemic stroke, comprising administering an effective amount of an antibody molecule of the present invention to a subject in need thereof.

[0026] In yet another aspect, the present invention provides a kit comprising the antibody molecule of the present invention or a pharmaceutical composition thereof.

[0027] In one aspect, the invention provides a method for producing an antibody molecule of the invention, the method comprising:

[0028] (a) providing a host cell comprising one or more nucleic acids encoding the antibody molecule in functional connection with expression control sequences,

[0029] (b) culturing the host cell, and

[0030] (c) recovering the antibody molecules from the cell culture.

[0031] In yet another aspect, the present invention provides a method for determining a molecule that can inhibit TPA-induced fibrinolysis of a human blood clot. The method comprises the steps of providing an antibody molecule of the present invention that specifically binds to TPA and inhibits TPA-induced fibrinolysis of a human blood clot, fixing the antibody molecule to a surface, providing TPA and introducing a substance that blocks the non-specific binding region of TPA to TPA, introducing a candidate molecule to TPA, introducing TPA to the antibody molecule, determining whether the candidate molecule has bound to a TPA epitope to which the antibody molecule has bound to TPA, and identifying any candidate molecule that binds to the epitope as a molecule that can inhibit TPA-induced fibrinolysis of a human blood clot.

[0032] Advantages of the presently disclosed subject matter will become apparent to one of ordinary skill in the art upon examination of the specification, drawings, and non-limiting examples contained in this document. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : PCR primers for amplification of mouse VK

[0035] Figure 2 : PCR primers for amplification of mouse VH

[0036] Figure 3 :Universal PCR primers and sequencing primers

[0037] Figure 4 :Protein sequence and DNA sequence of TPAi-1κ light chain variable region

[0038] Figure 5 : Protein and DNA sequences of the TPAi-1 heavy chain variable region

[0039] Figure 6 :TPAi-1κ light chain germline analysis

[0040] Figure 7 :TPAi-1 heavy chain germline analysis

[0041] Figure 8 : pHuG1 LIC vector

[0042] Fig. 9 : pHuK LIC vector

[0043] Fig.10 : pHuG1_Fab LIC vector

[0044] Fig.11 :TPAi-1κ light chain variable region (GenScript optimized)

[0045] Fig.12 :TPAi-1 heavy chain variable region (GenScript optimized)

[0046] Fig.13 : Binding of chimeric and murine TPAi-1 antibodies to tPA antigen

[0047] Fig.14 : Cloning and mutagenesis primers

[0048] Fig.15 : Binding of humanized TPAi-1 to human tPA

[0049] Fig.15A : Binding of humanized TPAi-1 to human tPA

[0050] Fig.16 : Thermal shift analysis of purified humanized candidate antibodies

[0051] Fig.17 : Aggregation analysis of humanized antibody candidates using DLS

[0052] Fig.18 : Non-specific protein-protein interactions (cross-interaction chromatography)

[0053] Fig.19 : Purified humanized antibody candidates evaluated for solubility

[0054] Fig. 20 :Freeze / thaw stress analysis of humanized antibody candidates

[0055] Fig.21 :Freeze / thaw stress analysis of humanized antibody candidates

[0056] Fig. 22 :Evaluation of serum stability of humanized antibody candidates

[0057] Fig.23 : Binding of TPAi-1 RHP / RKA Fab to human tPA

[0058] Fig.24 : Thermal transition analysis of purified TPAi-23 RHP / RKA Fab

[0059] Fig.25 :TPAi-1 RHP / RKA Fab aggregation analysis

[0060] Fig.26 :Non-specific protein-protein interactions of TPAi-1RHP / RKA Fab

[0061] Fig. 27 : Purified TPAi-1 RHP / RKA Fab evaluated for solubility

[0062] Fig.28:Freeze / thaw stress analysis of TPAi-1 RHP / RKA Fab

[0063] Fig.29 :TPAi-1 RHP / RKA Fab heat-induced stress analysis

[0064] Fig.30 : TPAi-1 RHP / RKA Fab serum stability evaluation

[0065] Fig.31 :Preparation of TPAi-1 RHP / RKA F(ab')2

[0066] Fig.32 :TPAi-1 RHP / RKA F(ab')2 aggregation analysis

[0067] Fig.33 : Binding of TPAi-1 RHP / RKA F(ab')2 to human tPA

[0068] Fig.34 : Binding and activity of chimeric TPAi-1 and humanized TPAi-1 (RHP / RKA) compared to murine TPAi-1.

[0069] Fig.35 :Dose response study of mouse TPAi-1, humanized TPAi-1(RHP / RKA), and TPAi-1(RHP / RKA) Fab in an in vitro human plasma clot lysis inhibition assay

[0070] Fig.36 : Tail bleeding (hemoglobin loss) as an indicator of peripheral or surgical bleeding

[0071] Fig.37 : Percentage of the cerebral hemisphere showing intracerebral hemorrhage

[0072] Fig.38 : Percentage of the cerebral hemisphere showing infarction

[0073] Fig.39 : Binding of chimeric TPAi-1, humanized TPAi-1(RHP / RKA), and TPAi-1(RHP / RKA) Fab to TPA mutant tenecteplase DETAILED DESCRIPTION OF THE INVENTION

[0075] Some of the polypeptide sequences disclosed herein are cross-referenced in Accession No. . Expressly incorporated by reference in Cross-referenced sequences in the database, The same applies to equivalent and related sequences present in or other public databases. Databases contain annotations associated with the sequences disclosed herein.

[0076] The present invention provides an antibody molecule that specifically binds to human TPA or a TPA mutant to inhibit the degradation of human fibrin clots, wherein the antibody has an IC 50 A subnanomolar affinity for inhibiting fibrin-dependent plasminogen activation of <5 nM, and wherein the amino acid sequence of the TPA mutant is at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2; wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has a CDR1 selected from SEQ ID NOs: 3 and 4, a CDR2 selected from SEQ ID NOs: 5 and 6, and a CDR3 selected from SEQ ID NOs: 7 and 8, and the light chain variable domain has a CDR1 selected from SEQ ID NOs: 9 and 10, a CDR2 selected from SEQ ID NOs: 11 and 12, and a CDR3 selected from SEQ ID NO: 13.

[0077] The present invention relates to providing and using antibody molecules that are specific inhibitors of fibrin-dependent Pg activation in TPA-induced bleeding, particularly systemic bleeding such as cerebral hemorrhage, systemic bleeding after tissue plasminogen activator treatment. More specifically, certain antibody molecules act as inhibitors and work synergistically to reduce plasminogen activation and fibrinolysis with greater potency than plasminogen activator inhibitor-I (PAI-I). In a thromboembolic stroke model, these inhibitors significantly reduce cerebral hemorrhage and surgical bleeding after administration of TPA.

[0078] The present invention provides an antibody molecule that specifically binds to human TPA or a TPA mutant to inhibit the degradation of human fibrin clots, wherein the antibody has an IC 50<5 nM of subnanomolar affinity for inhibiting fibrin-dependent plasminogen activation, and wherein the amino acid sequence of the TPA mutant is at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the antibody molecule does not affect TPA amidolytic activity or fibrin-independent activation. The amino acid sequence of the TPA mutant is at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2. The TPA mutant may therefore also have greater than 65%, e.g., 70%, 75%, 80%, 85%, 90%, 95%, etc. homology to these sequences. A non-limiting example of a TPA mutant is reteplase, which is a TPA deletion mutant having 67.7% of the residues present in full-length TPA. An alternative non-limiting example of a TPA mutant is tenecteplase, which is a TPA substitution mutant of a 527 amino acid glycoprotein formed by introducing the following modifications to human native tPA complementary DNA (cDNA): asparagine substitution for threonine 103 and glutamine substitution for asparagine 117, both substitutions within the kringle 1 domain, and tetraalanine substitutions at amino acids 296-299 in the protease domain. In some embodiments, the amino acid sequence of human TPA is SEQ ID NO: 1 or SEQ ID NO: 2.

[0079] One object of the present invention is to generate TPA-specific antibody molecules with subnanomolar dissociation constants (for a review on the definition and measurement of antibody-antigen affinity, see Neri et al., (1996). Trends in Biotechnol. 14, 465-470).

[0080] The present invention provides an antibody molecule that specifically binds to human TPA or a TPA mutant to inhibit the degradation of human fibrin clots, wherein the antibody has an IC 50 A subnanomolar affinity for inhibiting fibrin-dependent plasminogen activation of <5 nM, and wherein the amino acid sequence of the TPA mutant is at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2; wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has a CDR1 selected from SEQ ID NOs: 3 and 4, a CDR2 selected from SEQ ID NOs: 5 and 6, and a CDR3 selected from SEQ ID NOs: 7 and 8, and the light chain variable domain has a CDR1 selected from SEQ ID NOs: 9 and 10, a CDR2 selected from SEQ ID NOs: 11 and 12, and a CDR3 selected from SEQ ID NO: 13.

[0081] In one embodiment, the antibody molecule of the invention comprises a heavy chain variable domain of CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 7; and a light chain variable domain of CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 13.

[0082] In yet another embodiment, the antibody molecule of the invention comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 14 to 28, and a light chain variable domain selected from the group consisting of SEQ ID NOs: 29 and 30.

[0083] In yet another embodiment, the antibody molecule of the invention comprises a heavy chain variable domain selected from SEQ ID NOs: 14 to 28, and a light chain variable domain of SEQ ID NO:29.

[0084] In one embodiment, the antibody molecule of the invention comprises a heavy chain variable domain of SEQ ID NO: 14 and a light chain variable domain of SEQ ID No: 29, or a heavy chain variable domain of SEQ ID NO: 15 and a light chain variable domain of SEQ ID No: 29, or a heavy chain variable domain of SEQ ID NO: 14 and a light chain variable domain of SEQ ID No: 30, or a heavy chain variable domain of SEQ ID NO: 15 and a light chain variable domain of SEQ ID No: 30.

[0085] In one embodiment, an antibody molecule of the invention has a heavy chain comprising SEQ ID NO:40 or SEQ ID NO:41; and a light chain comprising SEQ ID NO:42.

[0086] The term "mutant" as used herein includes peptides whose sequences are substantially similar to those of TPA. It is known in the art that an amino acid sequence substantially similar to a reference peptide can be produced with a mutant peptide having no significant changes in physiological, chemical or functional properties compared to the reference peptide. In this case, the reference peptide and the mutant peptide will be considered as "substantially identical" polypeptides. Sequence identity is used to evaluate the similarity of two sequences; similarity is confirmed by calculating the percentage of identical residues when the two sequences are aligned for maximum correspondence between residue positions. Any known method can be used to calculate sequence identity; for example, computer software can be used to calculate sequence identity. In the absence of any limitation, sequence identity can be calculated using software such as BLAST-P, BLAST-N or FASTA-N or any other suitable software known in the art. Substantially identical sequences of the present invention may be at least 65% identical. In another example, substantially identical sequences may be at least 65, 70, 75, 80, 85, 90, 95 or 100% identical to the sequences described herein at the amino acid level.

[0087] Antibodies (also referred to as immunoglobulins, abbreviated as lg) are gamma globulins that can be found in the blood or other body fluids of vertebrates, and are used by the immune system to identify and neutralize foreign bodies, such as bacteria and viruses. They are generally composed of basic structural units-each having two large heavy chains and two small light chains-basic structural units, such as to form a monomer with one unit, a dimer with two units, or a pentamer with five units. Through non-covalent interactions, antibodies can be bound to other molecules or structures called antigens. This binding is specific in the following sense: antibodies will only bind to specific structures with high affinity. The unique part of the antigen recognized by the antibody is called an epitope or antigenic determinant. The part to which the antibody is bound to the epitope is sometimes called a paratope and resides in the so-called variable domains or variable regions (Fv) of the antibody. The variable domains include three so-called complementary determining regions (CDRs) separated by framework regions (FRs).

[0088] In the context of the present invention, reference to CDRs is based on the definitions of Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196:901-917) together with Kabat (EA Kabat, TT Wu, H. Bilofsky, M. Reid-Miller and H. Perry, Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983)).

[0089] Antibodies that can be used in medicine and technology have been developed. Therefore, in the context of the present invention, the term "antibody molecule" or "antibody" (used in this article in a synonymous manner) not only comprises antibodies as they exist in nature (e.g., comprising two light chains and two heavy chains), or only two heavy chains as in camelid species, but also additionally encompasses whole molecules comprising at least one paratope having a binding specificity for an antigen and a structural similarity to the variable domains of an immunoglobulin.

[0090] The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments, as long as they show the desired biological activity. The term "polyclonal antibody" as used herein refers to a group of antibody molecules having different amino acid sequences and can be obtained from the blood of vertebrates immunized with antigens by methods well known in the art. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting this population are substantially the same, with the exception of possible naturally occurring mutations. Monoclonal antibodies are highly specific, i.e., directed against a single antigenic site. For example, the monoclonal antibodies that can be used in the present invention can be prepared from hybrid cell lines (called hybridomas) by the hybridoma method described by Kohler et al., Nature 256: 495 (1975), which represent clones of specific B cells that produce antibodies fused with myeloma (B cell cancer) cells, or can be produced in bacterial cells, eukaryotic animal cells or plant cells using recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Alternatively, "monoclonal antibodies" can be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0091] For use in the human body, it is often necessary to reduce the immunogenicity of antibodies initially derived from other species such as mice. This can be done by constructing chimeric antibodies or by a process called "humanization". In this case, "chimeric antibodies" are understood to be a kind of antibody, which comprises a sequence portion (e.g., variable domains) derived from a species (e.g., mouse), which is fused to a sequence portion (e.g., constant domains) derived from a different species (e.g., people). "Humanized antibodies" are antibodies comprising variable domains initially derived from non-human species, in which certain amino acids have been mutated so that the overall sequence of the variable domains more realistically mimics the sequence of human variable domains. Antibody chimerization and humanization methods are known in the art (Billetta R, Lobuglio AF. "Chimeric antibodies". Int Rev Immunol. 1993; 10(2-3): 165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature: 332: 323).

[0092] However, the production and development of monoclonal antibody / monoclonal antibody fragment (mAbs / Fabs) candidate drug molecules are complex. Despite the fact that many of the techniques used in the production of mAb / Fab have been standardized, each Mab / Fab is unique because of its specific structure derived from the origin of its binding to a specific antigen target. In addition, compared to small molecules, its production and characterization are much more complicated because they are 200-1000 times larger, more complex in structure and highly sensitive to its production conditions (Kizhedath A, Wilkinson S and Glassey J.Applicability of predictive toxicologymethods for monoclonal antibody therapeutics: status Quo and scope (predictive toxicology methods for monoclonal antibody therapeutics: status and scope of application). Arch Toxicol 2017; 91: 1595-1612.). Another layer of complexity is added when unique changes are deemed necessary for each candidate molecule to optimize its pharmacodynamic and pharmacokinetic properties while reducing its potential toxicokinetic properties such as immunogenicity, off-target or immunostimulatory (cytokine storm) effects.

[0093] In addition, techniques for producing antibodies based on sequences derived from the human genome have been developed, for example by phage display or the use of transgenic animals (WO 90 / 05144; D. Marks, HR Hoogenboom, TP Bonnert, J. McCafferty, AO Griffiths and G. Winter (1991) "By-passing immunisation. Human antibodies from V-gene libraries displayed on phage." J. Mol. Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus, "Concepts in antibody phage display." Briefings in Functional Genomics and Proteomics 20021(2): 189-203; Lonberg ... N, Huszar D. "Human antibodies from transgenic mice". Int Rev Immunol. 1995; 13(1): 65-93.; Bruggemann M, Taussig MJ. "Production of human antibody repertoires in transgenic mice". Curr Opin Biotechnol. 1997 Aug; 8(4): 455-8). Such antibodies are "human antibodies" in the context of the present invention.

[0094] The "monoclonal antibodies" herein include those "chimeric antibodies" in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0095] As used herein, the term "antibody" (Ab) also includes antibody fragments. An "antibody fragment" is a portion of an intact antibody, preferably the antigen binding region or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), 2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Such fragments can be obtained by fragmenting immunoglobulins (e.g., by proteolytic digestion) or by recombinantly expressing such fragments. For example, immunoglobulin digestion can be achieved by routine techniques, such as using papain or pepsin (WO 94 / 29348) or the protein endoproteinase Lys-C (Kleemann et al., Anal. Chem. 80, 2001-2009, 2008). Papain or Lys-C digestion of antibodies generally produces two identical antigen-binding fragments, so-called "Fab" fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment produces F(ab')2. Methods for producing Fab molecules by recombinant expression in host cells are outlined in more detail below.

[0096] A variety of techniques have been developed for arranging the variable domains of immunoglobulins or molecules derived from such variable domains in different structural contexts. These should also be considered as "antibody molecules" of the present invention. Generally speaking, these antibody molecules are smaller in size than immunoglobulins, and may comprise a single amino acid chain or be composed of several amino acid chains. For example, a single-chain variable fragment (scFv) is a fusion of the variable regions of immunoglobulin heavy and light chains, connected together by a short linker that is usually serine (S) or glycine (G) (WO 88 / 01649; WO 91 / 17271; Huston et al. International Reviews of Immunology, Vol. 10, 1993, 195-217). "Single domain antibodies" or "nanobodies" include an antigen binding site in a single domain (WO 94 / 04678; WO 03 / 050531, Ward et al., Nature, 1989 Oct 12; 341(6242): 544-6; Revets et al., Expert Opin Biol Ther. 5(1): 111-24, 2005). One or more single domain antibodies with binding specificity to the same or different antigens can be linked together. Diabodies are bivalent antibody molecules consisting of two amino acid chains comprising two variable domains (WO 94 / 13804, Holliger et al., Proc Natl Acad Sci USA. 1993 Jul 15; 90(14): 6444-8). Other examples of antibody-like molecules are immunoglobulin superfamily antibodies (IgSF; Sriniva and Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). Alternatively, small modular immunopharmaceuticals (SMIPs) comprise an Fv domain connected to a single chain hinge and an effector domain lacking the constant domain CH1 (WO 02 / 056910).

[0097] Therefore, the antibody molecule of the present invention can be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, an antibody fragment, in particular a Fab, Fab′ or F(ab′)2 fragment, a single-chain antibody, in particular a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody or a designed ankyrin repeat protein (DARPin).

[0098] In one embodiment, the antibody molecule of the invention is a humanized antibody or a humanized antibody fragment, in particular a Fab, Fab′ or F(ab′)2 fragment, a single-chain antibody, in particular a single-chain variable fragment (scFv), a small modular immunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody or a designed ankyrin repeat protein (DARPin).

[0099] In yet another embodiment, the antibody molecule of the invention is a humanized antibody or a humanized antibody fragment, in particular a Fab, Fab' or F(ab')2 fragment.

[0100] The variable domains disclosed above can each be fused to an immunoglobulin constant domain, preferably a human immunoglobulin constant domain. Thus, the heavy chain variable domain can be fused to a CH1 domain (the so-called Fd fragment) and the light chain variable domain can be fused to a CL domain.

[0101] In one embodiment, the antibody molecule of the present invention is a Fab molecule, particularly a humanized Fab molecule, which Fab molecule has a Fd fragment comprising SEQ ID NO:31 or SEQ ID NO:32; and a light chain comprising SEQ ID NO:33.

[0102] Fab molecules can be produced from full-length antibody molecules by enzymatic cleavage, wherein the intact antibody is cut by enzymes such as papain, pepsin or ficin. The advantage of this method is that the platform process for robust and efficient fermentation and purification is applicable, which is easy to amplify and produce in high yield with the desired product quality. For purification, affinity chromatography using recombinant protein A resin can be used to separate Fab fragments from Fc (crystallized fragments) and residual intact antibodies. Protein A affinity chromatography is generally used to produce high purity.

[0103] Alternatively, nucleic acids encoding Fab constructs can be used to express such heavy and light chains in host cells such as E. coli, Pichia pastoris or mammalian cell lines (e.g., CHO, HEK293 or NSO). Methods for allowing these chains to correctly fold, associate and disulfide bond to synthesize functional Fab molecules comprising Fd fragments and light chains are known in the art (Burtet et al., J. Biochem. 2007, 142 (6), 665-669; Ning et al., Biochem. Mol. Biol. 2005, 38: 204-299; Quintero-Hernandez et al., Mol. Immunol. 2007, 44: 1307-1315; Willems et al. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2003; 786: 161-176).

[0104] In one embodiment, the antibody molecule of the invention is a scFv molecule.

[0105] The variable domains disclosed herein can be fused to each other via a suitable linker peptide, for example, selected from SEQ ID NO: 33, 34, 35 or 36. The construct can comprise these elements in order from N-terminus to C-terminus: (heavy chain variable domain)-(linker peptide)-(light chain variable domain) or (light chain variable domain)-(linker peptide)-(heavy chain variable domain).

[0106] In yet another embodiment, the antibody molecule of the invention is a scFv, wherein the heavy chain variable domain and the light chain variable domain are connected to each other via a linker peptide selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

[0107] In yet another embodiment, the antibody molecule of the invention comprises SEQ ID NO:38 or SEQ ID NO:39.

[0108] Methods are known in the art that allow for the recombinant expression of nucleic acids encoding scFv constructs in host cells such as E. coli, Pichia pastoris, or mammalian cell lines such as CHO or NSO to produce functional scFv molecules (see, e.g., Rippmann et al., Applied and Environmental Microbiology 1998, 64(12):4862-4869; Yamawaki et al., J. Biosci. Bioeng. 2007, 104(5):403-407; Sonoda et al., Protein Expr. Purif. 2010, 70(2):248-253).

[0109] The antibody molecules of the present invention can be fused (as fusion proteins) or otherwise connected (by covalent or non-covalent bonds) to other molecular entities that have a desired effect on the properties of the antibody molecules. For example, it may be desirable to improve the pharmacokinetic properties of the antibody molecule, or stability, for example, in body fluids such as blood, especially in the case of single-chain antibodies or domain antibodies. A variety of techniques have been developed in this regard, especially for extending the half-life of such molecules in circulation, such as pegylation (WO98 / 25971; WO 98 / 48837; WO 2004081026), fusing or otherwise covalently joining the antibody molecule to another antibody molecule with affinity for serum proteins such as albumin (WO 2004041865; WO 2004003019), or expressing the antibody molecule as a fusion protein with all or part of a serum protein (such as albumin or transferrin) (WO 01 / 79258).

[0110] In yet another aspect of the invention, the antibody molecule is capable of neutralizing the activity of the fibrinolytic drug. That is, when bound to the antibody molecule, TPA is no longer able to exert its fibrinolytic activity through plasminogen activation, or exerts this activity at a significantly reduced magnitude. Preferably, the fibrinolytic activity is reduced at least 2-fold, 5-fold, 10-fold or 100-fold upon antibody binding, as determined in a suitable activity assay (Longstaff C, Whitton CM. A proposed reference method for plasminogen activators that enables calculation of enzyme activities in SI units. J Thromb Haemost. 2004; 2: 1416-1421), and in particular coagulation assays sensitive to fibrin degradation factors, such as DD-dimer measurement (Gebhardt J, Kepa S, Hofer S, Koder S et al. Fibrinolysis in patients with mild-to-moderate bleeding tendency or unknown cause. Ann Hematol 2017; 96: 489-495).

[0111] To produce the antibody molecules of the present invention, the skilled person can choose from a variety of methods well known in the art (Norderhaug et al., J Immunol Methods 1997, 204(1):77-87; Kipriyanow and Le Gall, Molecular Biotechnology 26:39-60, 2004; Shukla et al., 2007, J. Chromatography B, 848(1):28-39).

[0112] As outlined above, human TPA and mutants are well known in the art. In this case, TPA includes TPA and mutants.

[0113] TPA-induced cerebral hemorrhage and systemic bleeding in vivo are blocked by TPA's potent synergistic inhibitor of fibrin-dependent plasminogen activation. This suggests that hemorrhage is related to TPA's fibrin-targeted mechanism of plasminogen activation and that targeted inhibitors of this process can serve as specific antidotes for TPA-related hemorrhage. TPA therapy is beneficial in the treatment of ischemic stroke and myocardial infarction, but in some patients, the therapy is complicated by severe or fatal hemorrhage in the brain or other parts. Fear of TPA-induced hemorrhage has limited the therapeutic use of TPA. In humans, TPA-induced hemorrhage and adverse outcomes are more frequent after prolonged local ischemia. Similarly, in experimental stroke, after prolonged local ischemia, TPA can reproducibly cause cerebral hemorrhage, blood-brain barrier damage, and enhanced neuronal cell death. In non-thrombotic models of stroke, there is evidence that TPA can exert toxic effects through mechanisms that do not require plasminogen activation or affect fibrinolytic activity, such as PDGF-CC cleavage (Su EJ, Fredriksson L, Geyer M, et al., Activation of PDGF-CC by tissueplasminogen activator impairs blood-brain barrier integrity during ischemic stroke. Nat Med. 2008; 14: 731-737). In pathological conditions such as myocardial ischemia and stroke, the fibrinolytic activity of therapeutic TPA is enhanced by elevated levels of circulating fibrin fragments (e.g., D-dimers), which may enhance the bleeding process. (Barber M, Langhorne P, Rumley A, Lowe GD, Stott DJ. D-dimer predicts early clinical progression in ischemic stroke: confirmation using routine clinical assays. Stroke. 2006; 37: 1113-1115).

[0114] In addition to the therapeutic use of TPA, elevated TPA levels have also been associated with excessive systemic bleeding. TPA-induced bleeding has been suspected in patients post-cardiopulmonary bypass. (Manji RA, Grocott HP, Leake J, et al., Seizures following cardiac surgery: the impact of tranexamic acid and other risk factors. Can J Anaesth. 2012; 59: 6-13). In a similar manner, high levels of circulating TPA have been associated with bleeding in disease conditions such as liver failure and transplantation. (Leiper K, Croll A, Booth NA, Moore NR, Sinclair T, Bennett B. Tissueplasminogen activator, plasminogen activator inhibitors, and activator-inhibitor complex in liver disease. J Clin Pathol. I994; 47: 214-217). Fibrinolytic inhibitors (e.g., tranexamic acid, ε-aminocaproic acid, aprotinin, etc.) reduce the risk of postoperative blood transfusion (Bayes-Genis A, Mateo J, Santalo M et al., D-Dimer is an early diagnostic marker of coronary ischemia in patients with chest pain). J. 2000; 140: 379-384). However, these agents have broad inhibitory effects on other pathways and associated toxicities. For example, tranexamic acid increases the risk of seizures following cardiac surgery. (Manji RA, Grocott HP, Leake J, et al., Seizures following cardiac surgery: the impact of tranexamic acid and other risk factors. Can J Anaesth. 2012; 59: 6-13). Broad inhibition of fibrinolysis can carry the risk of subsequent thrombotic seizures (such as stroke, thromboembolism). (Fergusson DA, Hebert PC, Mazer CD, et al., A comparison of aprotinin and lysine analogs in high-risk cardiac surgery. N Engl J Med. 2008; 358: 2319-2331). This concern is exacerbated by the unintended finding that aprotinin use increases mortality after cardiac surgery (supra).

[0115] Severe trauma or trauma resulting in multiple organ damage produces a hyperfibrinolytic state mediated by elevated levels of endogenous TPA (Cardenas JC, Matijevic N, Baer LA, Holcomb JB, Cotton BA, Wade CE. Elevated tissue plasminogen activator and reduced plasminogen activator inhibitor promote hyperfibrinolysis in trauma patients. Shock 2014; 41(6): 514-21). Therefore, inhibition of elevated endogenous TPA by antibody drugs may normalize fibrinolysis and prevent the coagulopathy seen in these conditions. Tranexamic acid has been studied in severely injured trauma patients with hyperfibrinolysis caused by elevated endogenous TPA, resulting in increased 6-hour survival but without affecting long-term survival (Khan M, Jehan F, Bulger EM, et al. Severely injured trauma patients with admission hyperfibrinoloysis: Is there a role of tranexamic acid? Findings from the PROPPR trial. J Trauma Acute Care Surg 2018;85(5):851-857).

[0116] The use of antifibrinolytics to treat TPA-induced bleeding remains very limited, probably because these drugs are known to interfere with other biochemical pathways. PAI-I or PAI-I mutants have been shown to inhibit TPA-induced bleeding after injury. However, in addition to inhibiting TPA, PAI-I inhibits uPA and several other proteases. Through its non-protease interactions with vitronectin, heparin, low-density lipoprotein receptor family members and other molecules, PAI-I has a 'pleiotropic' effect on a variety of other biological processes and has been implicated in the pathophysiology of several disease processes. Therefore, PAI-I has an inhibitory and non-inhibitory function in angiogenesis, apoptosis, cell migration and cancer.

[0117] In one aspect, the present invention provides a pharmaceutical composition comprising the antibody molecule of the present invention and a pharmaceutically acceptable carrier.

[0118] In a further aspect, the invention provides an antibody molecule of the invention for use as a medicament.

[0119] In yet another aspect, the present invention provides an antibody molecule of the present invention for use in treating or preventing TPA-induced bleeding.

[0120] As described above, elevated levels of TPA may result from exogenous or endogenous processes, i.e., specific administration of TPA or from the generation of elevated levels of TPA in vivo (e.g., after cardiopulmonary bypass). In this context, 'TPA-induced bleeding' includes bleeding induced by elevated levels of TPA resulting from exogenous or endogenous processes.

[0121] The antibody molecule inhibits fibrinolysis induced by TPA. In some embodiments, the antibody molecule inhibits the initiation of fibrinolysis. In some embodiments, the antibody molecule inhibits ongoing fibrinolysis.

[0122] In one embodiment, the invention provides an antibody molecule of the invention for use in treating or preventing systemic bleeding, in particular cerebral hemorrhage and systemic bleeding after tissue plasminogen activator treatment, more particularly after TPA treatment of ischemic stroke.

[0123] In an alternative embodiment, the invention provides an antibody molecule of the invention for use in treating or preventing systemic bleeding in a subject in which endogenous TPA is elevated due to conditions including, but not limited to, prolonged coronary artery bypass surgery, liver transplantation, severe or multiple trauma, heat stroke, and near drowning.

[0124] In yet another aspect, the present invention provides a method for treating or preventing TPA-induced bleeding, the method comprising administering an effective amount of an antibody molecule of the present invention to a subject in need thereof.

[0125] In one embodiment, the present invention provides a method for treating or preventing systemic bleeding, especially cerebral hemorrhage and systemic bleeding after tissue plasminogen activator treatment, more specifically TPA treatment of ischemic stroke, comprising administering an effective amount of an antibody molecule of the present invention to a subject in need thereof.

[0126] In an alternative embodiment, the present invention provides a method for treating or preventing systemic bleeding in a subject in which endogenous TPA is elevated due to conditions including, but not limited to, prolonged coronary artery bypass surgery, liver transplantation, severe or multiple trauma, heat stroke, and near drowning, the method comprising administering to a subject in need thereof an effective amount of an antibody molecule of the present invention.

[0127] In yet another aspect, the present invention provides a kit comprising the antibody molecule of the present invention or a pharmaceutical composition thereof.

[0128] In one embodiment, the kit comprises:

[0129] (a) an antibody of the present invention or a pharmaceutical composition thereof;

[0130] (b) containers; and

[0131] (c) Marker.

[0132] In one embodiment, the kit comprises an antibody of the invention or a pharmaceutical composition thereof and human tissue plasminogen activator (TPA) or a TPA mutant, wherein the amino acid sequence of the TPA mutant has at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0133] In yet another embodiment, human tissue plasminogen activator (TPA) or a TPA mutant is selected from alteplase ( rtPA), reteplase and tenecteplase ( TNK-tPA).

[0134] In yet another embodiment, the kit comprises:

[0135] (a) an antibody of the present invention or a pharmaceutical composition thereof;

[0136] (b) a pharmaceutical composition comprising alteplase ( rtPA), reteplase and tenecteplase ( human tissue plasminogen activator (TPA) or TPA mutants (TNK-tPA);

[0137] (c) containers; and

[0138] (d) Label.

[0139] In yet another embodiment, the kit comprises:

[0140] (a) a first pharmaceutical composition comprising alteplase ( rtPA), reteplase and tenecteplase ( human tissue plasminogen activator (TPA) or TPA mutants (TNK-tPA);

[0141] (b) a second pharmaceutical composition comprising an antibody of the invention;

[0142] (c) instructions for separately administering the first and second pharmaceutical compositions to a subject, wherein the first and second pharmaceutical compositions are contained in separate containers and administering the second pharmaceutical composition to a subject in need of treatment or prevention of systemic bleeding following TPA treatment.

[0143] Herein, a 'subject in need of treatment' is a subject exhibiting symptoms of severe bleeding and a 'subject in need of prevention' is a subject not exhibiting symptoms of severe bleeding but who is determined by the treating physician to be at high risk.

[0144] In one aspect, the invention provides a method for producing an antibody molecule of the invention, the method comprising:

[0145] (a) providing a host cell comprising one or more nucleic acids encoding the antibody molecule in functional connection with expression control sequences,

[0146] (b) culturing the host cell, and

[0147] (c) recovering the antibody molecules from the cell culture.

[0148] A "purified or isolated" antibody is an antibody or polypeptide that has been identified and separated and / or recovered from components of its natural environment. Impure components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Preferably, the antibody is purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence by use of a spinning cup sequenator or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present.

[0149] As used herein, the terms "treat," "treating," or "treatment" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or alleviating at least one physical parameter, including those that may not be perceptible to the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to regulating the disease or condition physically (e.g., stabilizing perceptible symptoms), physiologically (e.g., stabilizing a physical parameter), or both or physiologically. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or condition.

[0150] "Preventing" a condition or disorder means delaying or preventing the onset or reducing the severity of the condition or disorder as assessed by the presence or extent of one or more symptoms of the condition or disorder.

[0151] As used herein, the term "subject" refers to an animal. Generally, an animal is a mammal. A subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

[0152] As used herein, a subject is "in need of" treatment if the subject would benefit biologically, medically, or in terms of quality of life from such treatment.

[0153] As used herein, a subject in which 'endogenous TPA is elevated' refers to a subject in which the plasma concentration of endogenous TPA is elevated relative to baseline levels. The World Health Organization (WHO / BS / 07.2068, 2007) cites normal plasma levels of tPA as <10 ng / mL, with most values ​​reported being around 4 ng / mL. A 5- to 10-fold increase has been reported in subjects with hyperfibrinolysis (Chapman et al., Overwhelming tPA Release, not PAI-1 Degradation, is Responsible for Hyperfibrinolysis in Severely Injured Trauma Patients, J Trauma Acute Care Surg. 2016 Jan;80(1):16-25; Duque et al., Pathophysiological Response to Trauma-Induced Coagulopathy: A Comprehensive Review, 2019 Anesthesia & Analgesia: Oct 15, 2019 - Vol. Published Ahead of Print - Issue - p doi: 10.1213 / ANE.0000000000004478).

[0154] The compositions of the invention may contain an "effective amount" or a "therapeutically effective amount" or a "prophylactically effective amount" of the antibodies or antigen-binding portions of the invention. These terms are used interchangeably. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amount of the antibody or antibody portion may vary depending on factors such as the disease state, the age, sex, and weight of the subject, and the ability of the antibody or antibody portion to elicit a desired response in the subject. Dosage unit form as used herein refers to physically discrete units suitable as single dosages for mammalian subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect when combined with the required pharmaceutical carrier.

[0155] In yet another aspect, the present invention provides a method for determining a molecule that can inhibit TPA-induced fibrinolysis of human blood clots. The method comprises the steps of providing an antibody molecule of the present invention that specifically binds to TPA and inhibits TPA-induced fibrinolysis of human blood clots, fixing the antibody molecule to a surface, providing TPA and introducing a substance that blocks the non-specific binding region of TPA to TPA, introducing a candidate molecule to TPA, introducing TPA to the antibody molecule, determining whether the candidate molecule has been bound to a TPA epitope in which the antibody molecule has been bound to TPA, and identifying any candidate molecule bound to the epitope as a molecule that can inhibit TPA-induced fibrinolysis of human blood clots. In an example embodiment, the antibody molecule of the present invention is fixed in a well of a microtiter plate. Non-specific protein binding sites are blocked. A mixture of TPA and potential new inhibitor molecules pre-incubated together is added to the well containing the immobilized antibody molecule. After one hour, the well is washed and a polyclonal anti-TPA antibody coupled to peroxidase is added. After one hour, the well is washed and a peroxidase substrate TMB is added and A370 is monitored. Wells with reduced A370 contain molecules that compete with the antibody molecules of the invention for TPA binding and are therefore prime candidates as specific TPA inhibitors. This will be confirmed in detailed studies of TPA-induced lysis of human blood clots.

[0156] The following examples are intended to illustrate the present invention and should not be construed as limiting thereof.

[0157] Abbreviations used are those conventional in the art and, if not defined, the terms have their generally accepted meanings.

[0158] Abbreviations and acronyms used herein include the following:

[0159] Expi293 Human Embryonic Kidney (HEK293 High Density / Serum-free) Cells

[0160] bp base pair

[0161] ℃ Celsius

[0162] MEM Minimum Essential Medium

[0163] DLS Dynamic Light Scattering

[0164] DNA deoxyribonucleic acid

[0165] ELISA enzyme-linked immunosorbent assay

[0166] EC50 is the concentration of antibody that gives a half-maximal response.

[0167] ECD extracellular domain

[0168] g

[0169] HRP Horseradish Peroxidase

[0170] IgG Immunoglobulin-G

[0171] LIC ligase-independent cloning

[0172] min

[0173] MALS Multi-angle Light Scattering

[0174] nm nanometer

[0175] OD Optical density

[0176] PBS Phosphate buffered saline

[0177] PCR polymerase chain reaction

[0178] RT Room temperature

[0179] s seconds

[0180] SEC Size Exclusion Chromatography

[0181] TMB 3,3',5,5'-Tetramethylbenzidine

[0182] UV

[0183] VCI Vernier residues, canonical residues, and interface residues

[0184] VH immunoglobulin heavy chain variable region

[0185] VK immunoglobulin kappa light chain variable region

[0186] Nucleotides:

[0187] A Adenine C Cytosine G Guanine T Thymine K G or T (IUPAC convention) M A or C (IUPAC convention) R A or G (IUPAC convention) S C or G (IUPAC convention) V A or C or G (IUPAC convention) W A or T (IUPAC convention) Y C or T (IUPAC convention)

[0188] Amino Acids

[0189] Ala(A) Alanine Met(M) Methionine Cys(C) Cysteine Asn(N) Asparagine Asp(D) Aspartic acid Pro(P) Proline Glu(E) Glutamate Gln(Q) Glutamine Phe(F) Phenylalanine Arg(R) Arginine Gly(G) Glycine Ser(S) Serine His(H) Histidine Thr(T) Threonine Ile(I) Isoleucine Val(V) Valine Lys(K) Lysine Trp(W) Tryptophan Leu(L) Leucine Tyr(Y) Tyrosine

[0190] Material

[0191] Culture reagents

[0192] Product UK Supplier Catalogue Number

[0193] ExpiCHO Expression Medium Invitrogen A29100-01

[0194] Expi293 Expression Medium Invitrogen A1435102

[0195] Penicillin and Streptomycin Invitrogen 15140-148

[0196] I Invitrogen 11058021

[0197] SOC Invitrogen 15544-034

[0198] Trypan Blue BioRad 145-0013

[0199] ExpiFectamine CHO Kit 1L Invitrogen A29129

[0200] Expifectamine 293 Kit 1L Invitrogen A14524

[0201] Penicillin and Streptomycin Invitrogen 15140-148

[0202] I Invitrogen 11058021

[0203] SOC Invitrogen 15544-034

[0204] Trypan Blue BioRad 145-0013

[0205] ExpiFectamine CHO Kit 1L Invitrogen A29129

[0206] Expifectamine 293 Kit 1L Invitrogen A14524

[0207] Immunology and Molecular Biology Reagents

[0208] Product UK Supplier Catalogue Number

[0209] First Strand Synthesis Kit GE Life Sciences 27-9261-01

[0210] Agarose (UltraPure TM )Invitrogen 15510-027

[0211] Albumin bovine (BSA) Sigma A3294

[0212] Bam HI NEB R0136S

[0213] BfuA1 NEB R0701S

[0214] dNTP Solution Set NEB N0446S

[0215] Electrophoresis gel 2% agarose G521802

[0216] Electrophoresis gel 2% agarose double combination Invitrogen G6018-02

[0217] Goat anti-human IgG / Fc fragment specific antibody Stratech Scientific 109-005-098

[0218] Goat anti-human Fcγ specific) peroxidase conjugate Jackson 109-035-098

[0219] Goat anti-human kappa chain horseradish peroxidase conjugate Sigma A7164

[0220] Goat anti-mouse F(ab')2-peroxidase conjugate Jackson 115-036-006

[0221] HBS-EP+ Buffer 10×GE Healthcare BR100669

[0222] Human Antibody Capture Kit GE Healthcare BR-1008-39

[0223] Human IgG1 / κ antibody. Sigma I5154

[0224] Kanamycin Invitrogen 11815024

[0225] K-Blue HRP Substrate SkyBio 308176

[0226] Mouse Antibody Capture Kit GE Healthcare BR-1008-38

[0227] Oligonucleotides Sigma na

[0228] PBS tablets Sigma P4417

[0229] Phusion Flash High-Fidelity PCR Master Mix Scientific F-548S

[0230] PureYield Plasmid Maxi Kit Promega A2393

[0231] Q5 Site-Directed Mutagenesis Kit NEB E0554S

[0232] QIAGEN Gel Extraction Kit Qiagen 28704

[0233] QIA Spin Column Microprep Kit Qiagen 27106

[0234] QIAquick PCR Purification Kit (250) 28106

[0235] 100 bp DNA ladder NEB N0467S

[0236] 1kb DNA ladder NEB N0468S

[0237] QuikChange Lightning Site-Directed Mutagenesis Kit, 10Rxn. Agilent 210518

[0238] Red stop solution (for K Blue) SkyBio Ltd 301475

[0239] RNeasy Micro Kit Qiagen 74106

[0240] Sensor chip CM5 GE Healthcare BR100399

[0241] Subcloning Efficiency TM TOP10 TM Chemically competent E. coli Invitrogen 404003

[0242] SYBR Safe DNA Gel Stain Invitrogen S33102

[0243] SYPRO Orange

[0244] T4 DNA Polymerase NEB M0203S

[0245] 20Sigma P9416-100ML

[0246] 1.0 Antibody Sequencing

[0247] 1.1 Monoclonal Antibody Generation

[0248] C57BL / 6J mice (Jackson Laboratory, Bar Harbor, ME) were immunized with recombinant human TPA (Genentech), and spleen cells isolated from the immunized mice were subsequently fused with myeloma cells using conventional hybridoma technology (Nelson PN, Reynolds GM, Waldron EE, Ward E, Giannopoulos K, Murray PG. Monoclonal antibodies. Mol Pathol. 2000; 53: 111-117). Microplate ELISA assays were performed to screen positive clones as described below. Positive clones were further subcloned by limiting dilution to generate stable monoclonal antibodies (mAbs). The cells were cultured in a humidified atmosphere at 37°C with 5% CO 2 All cell cultures were maintained in DMEM medium supplemented with 5% fetal bovine serum, 2 mmol / L L-glutamine and 1% penicillin-streptomycin (Invitrogen) in a 95% air incubator. Mouse mAbs were purified from the culture medium using goat anti-mouse IgG agarose (Invitrogen) and further characterized using a mouse antibody isotyping kit (Zymed Laboratories).

[0249] 1.1.1 ELISA assay for detecting TPA mAb binding

[0250] In order to screen positive TPA binding clones, microplates were coated with 1-211 g / ml TPA in phosphate buffered saline (PBS, Invitrogen) at room temperature for one hour, followed by blocking with 1% bovine serum albumin (BSA, Invitrogen) in PBS for one hour. Afterwards, 2-5 μg / ml purified anti-TPA mAb in mouse serum, hybridoma cell culture supernatant or PBS solution was loaded and incubated for one hour. The bound mAb was detected by goat anti-mouse IgG (Santa Cruz Biotechnology Inc) conjugated with horseradish peroxidase (HRP) together with TMB substrate (3,3′,5,5′-tetramethylbenzidine (TMB) substrate, HRP conjugated kit (Fisher Scientific) activated by Pierce Plus. In some tests, 3 μg / ml human PAI-I was incubated in TPA-coated BSA-blocked wells before adding anti-TPA mAb to examine the binding between mAb and TPA-PAI-1 complex; complex formation was confirmed by detecting bound PAI-I with mouse anti-human PAI-I mAb. TPA binding constants and anti-TPA mAb binding constants were estimated by ELISA assays using saturation binding experiments. Briefly, 7.5 μg / mL of purified anti-TPA mAb in PBS was coated on microtiter plates for one hour at room temperature and then blocked with 1% BSA in PBS. Different concentrations of human TPA (0-4 μg / ml) were then loaded in human serum pre-quenched with 20 μM PPACK (d-Phe-Pro-Arg chloromethyl ketone (PPACK) (Calbiochem)) and 200 units of aprotinin, an inhibitor of kallikrein. Bound TPA was detected with HRP-conjugated mouse anti-human TPA polyclonal antibody followed by TMB substrate. The reaction was monitored at A370nm within the dynamic range of the microplate reader. The binding constant was calculated using Graphpad Prism software (La Jolla, CA). In order to test whether anti-TPA mAbs compete with each other for TPA binding, 211g / ml TPA was coated on a microplate. After blocking with 1% BSA in PBS, different concentrations of anti-TPA mAbs labeled with HRP were added. After washing, the bound mAb was detected with TMB substrate. In some wells, different concentrations of purified anti-TPA mAbs were added to a fixed amount of HRP-labeled anti-TPA mAbs to compete for the binding to the coated TPA. Based on the difference between the bound mAbs labeled with HRP in the absence and presence of purified anti-TPA mAbs, the percentage of inhibition of binding was calculated.

[0251] 1.2. Preparation of RNA from hybridoma cells:

[0252] Mouse hybridoma cells generated according to 1.1 and identified as TPAi-1 were pelleted and washed with PBS. The pellet was processed using the Qiagen RNeasy kit to isolate RNA following the manufacturer's protocol (section 1.2.1).

[0253] 1.2.1 RNeasy miniprep protocol for isolating total RNA (Qiagen)

[0254] 1. Disrupt cells by adding Buffer RLT. For pelleted cells, thoroughly loosen the cell pellet by shaking the tube. Add Buffer RLT (600 μl) and proceed to step 2. Note: Incomplete loosening of the cell pellet may result in inefficient lysis and reduced yield.

[0255] 2. Homogenize cells by passing the lysate through an 18-20 gauge needle fitted to an RNase-free syringe at least 5 times.

[0256] 3. Add 1 volume of 70% ethanol to the homogenized lysate and mix thoroughly by aspiration. Do not centrifuge. The lysate volume may be less than 350 μl or 600 μl due to losses during homogenization.

[0257] 4. Transfer up to 700 μl of sample, including any precipitate that may have formed, to an RNeasy spin column in a 2 ml collection tube. Gently close the tube cap and centrifuge at ≥ 8000 x g for 15 seconds. Discard the flow-through. Reuse the centrifuge tube from step 5.

[0258] 5. Add 700 μl of Buffer RW1 to the RNeasy column. Gently close the tube cap and centrifuge at ≥8000 x g for 15 seconds to wash the column membrane. Discard the flow-through. Reuse the centrifuge tube in step 6.

[0259] 6. Add 500 μl of Buffer RPE to the RNeasy column. Gently close the tube cap and centrifuge at ≥8000 x g for 15 seconds to wash the column membrane. Discard the flow-through. Reuse the centrifuge tube in step 7.

[0260] 7. Add another 500 μl of Buffer RPE to the RNeasy column. Gently close the cap and centrifuge at ≥8000 x g for 2 seconds to dry the RNeasy spin column membrane.

[0261] 8. Place the RNeasy spin column in a new 2 ml collection tube and discard the old collection tube along with the flow-through. Gently close the tube cap and centrifuge at full speed for 1 minute.

[0262] 9. For elution, transfer the RNeasy column to a new 1.5 ml collection tube. Add 30 μl of RNase-free water directly onto the RNeasy spin column membrane. Gently close the tube. Let it stand for 1 minute and then centrifuge at ≥8000 xg for 1 minute.

[0263] 1.3. First-strand cDNA synthesis

[0264] Following the manufacturer's protocol, reverse transcribe RNA (about 3 μg) to produce cDNA (1.3.1) using the GE Life Sciences First Strand cDNA Synthesis Kit. This step was repeated twice to produce 3 independent cDNA products (1st, 2nd and 3rd rounds) to detect and avoid reverse transcriptase-induced cDNA.

[0265] 1.3.1 First-strand cDNA synthesis protocol (GE Life Sciences)

[0266] 1. Place RNA sample in a microcentrifuge tube and add RNase-free water to make up the RNA to the appropriate volume (20 μL - 12x dilution, see Table A).

[0267] 2. Heat the RNA solution to 65°C for 10 minutes and then cool on ice. Gently pipette the first-strand cDNA reaction stock mixture to obtain a homogenous suspension. (BSA may precipitate in the mixture when stored; this precipitate will dissolve during incubation).

[0268] 3. Add the first-strand cDNA reaction stock mixture (11 μL) to a sterile 1.5 or 0.5 ml microcentrifuge tube. Add 1 μL of DTT solution, 1 μL (0.2 μg, 1:25 dilution) NotI-d(T)18 primer, and heat-denatured RNA to the tube. Pipette up and down several times to mix.

[0269] 4. Incubate at 37°C for 1 hour and heat inactivate the transcriptase at 94°C for 5 minutes.

[0270] Table A. Volumes of components in the first chain reaction

[0271] First chain reaction stock mixture Primers DTT RNA Final volume of first chain reaction 11μL 1μL 1μL 20μL 33μL

[0272] cDNA Purification: A simple protocol designed to remove contaminating first-strand cDNA primers that may interfere with subsequent PCR reactions.

[0273] 1. Add 99 μl Buffer QG (from Qiagen Gel Extraction Kit, Cat. No: 28704) and 33 μl IPA. Mix and add to QiaQuick Gel Extraction Column. Centrifuge and discard flow-through.

[0274] 2. Wash the column once with 500 μl Buffer QG. Discard the flow-through.

[0275] 3. Wash the column once with 750 μl Buffer PE. Discard the flow-through.

[0276] 4. Centrifuge the column to remove any residual ethanol and allow the column to dry.

[0277] 5. Elute cDNA with 50 μl of distilled water preheated to 65°C.

[0278] 1.4. cDNA Sequencing

[0279] cDNA was amplified by PCR in 3 separate reactions as described in 1.4.1. Using Phusion High Fidelity PCR Master Mix, κ light chain primers plus MKC ( Figure 1 ) or heavy chain primer plus MHCmix ( Figure 2 ) PCR amplification of immunoglobulin cDNA.

[0280] The result of each PCR reaction was a single amplification product, which was purified using the QIAquick PCR Purification Kit (1.4.2) and PCR amplified using M13 forward primer and M13-reverse primer ( Figure 3 ) and sequenced in two directions to obtain three independent sets of sequence information for each immunoglobulin chain.

[0281] 1.4.1 PCR cloning of mouse variable regions

[0282] Sterile water: Deionized distilled water was treated with DEPC (Sigma, D-5758) (final concentration 0.1%) overnight at room temperature. Autoclave at 115°C and 15 psi for 20 minutes.

[0283] PCR cloning primers (see Figure 1 and Figure 2 ): Prepare separate 10 μM stocks of MHV 1-12, MKV 1-11, MHC, and MKC primers in sterile water.

[0284] 5x TBE buffer: (0.45M Tris-borate, pH 8.3 10mM EDTA)

[0285] 10x TAE buffer: (0.4M Tris-acetate, pH 8.0, 10mM EDTA)

[0286] 1. Label 11 GeneAmp TM PCR reaction tubes are labeled MKV1-11 and 12 are labeled MHV1-12. For each reaction, add the following:

[0287] Kappa chain Heavy chain 9 μl sterile water 9 μl sterile water 12.5 μl Phusion master mix 12.5 μl Phusion master mix 1.25 μl 10 μM MKCv2 primer 1.25 μl 10 μM of one MHCv2 primer, e.g. MHCG1v2 1 μl first-strand reaction cDNA template 1 μl first-strand reaction cDNA template

[0288] 2. Add 1.25 μl of the appropriate MKV-v2 or MHV-v2 primer to each reaction. Load the reaction tubes into a DNA thermal cycler and cycle:

[0289] Phusion Phusion Flash 98℃ 5min 10sec 98℃ 20sec 1sec x30 cycles 60℃ 20sec 5sec 72℃ 20sec 6-7 seconds 72℃ 5min 1min 4℃ Keep Keep

[0290] 3. Run 5 μl of sample from each PCR reaction on a 2% (w / v) agarose / 1x TBE (or 1x TAE) gel containing 1x SYBR safe DNA stain to determine which lead primer produced the PCR product. Positive PCR-clones will be approximately 420-500 bp in size.

[0291] 4. For positive clones, perform PCR purification on the remaining sample using QIAGEN PCR purification kit and elute into 30-50 μl buffer EB. This buffer should be sent to an external contractor (e.g. GATC) for PCR fragment sequencing using M13 forward primer and M13 reverse primer.

[0292] 5. Repeat those PCR reactions that appear to amplify the full length variable domain gene using the other two first strand reaction preparations. It is critical to have three independent clones of each variable domain gene to eliminate PCR errors and RT errors.

[0293] 1.4.2 QIAquick PCR Purification Microcentrifugation and Vacuum Protocol (QIAGEN)

[0294] 1. All centrifugation steps were performed in a conventional tabletop microcentrifuge at 17,900 x g (13,000 rpm).

[0295] 2. Add 5 volumes of Buffer PBI to 1 volume of PCR reaction and mix. If the color of the mixture is orange or violet, add 10 μl of 3M Sodium Acetate, pH 5.0, and mix. The color of the mixture will turn yellow.

[0296] 3. Place the QIAquick column in the 2 ml collection tube provided or in the vacuum manifold.

[0297] 4. To bind DNA, apply sample to the QIAquick column and ▲ centrifuge for 30-60 seconds or ● apply vacuum to the manifold until all sample has passed through the column. ▲ Discard the flow-through and place the QIAquick column back into the same tube.

[0298] 5. To wash, add 0.75 ml Buffer PE to the QIAquick column and ▲ centrifuge for 30-60 seconds or ● apply vacuum. ▲ Discard the flow-through and place the QIAquick column back in the same tube.

[0299] 6. Centrifuge the column for 1 minute in the 2 ml collection tube (provided).

[0300] 7. Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.

[0301] 8. To elute the DNA, add 40-50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 minute.

[0302] VK and VH DNA sequences

[0303] Figure 4 and Figure 5 The consensus sequence of hybridoma TPAi-1 VK and the consensus DNA sequence of TPAi-1 VH are shown in .

[0304] 2.0 Generation of a chimeric form of the TPAi-1 antibody

[0305] VK and VH DNA sequences

[0306] Germline analysis of the TPAi-1 sequence revealed that the kappa light chain is murine VK1 with two somatic mutations, both in framework 1 ( Figure 6 ). The heavy chain is murine VH1. This shows low identity to this germline, has a fairly high number of somatic mutations and has an unexpectedly short CDR-1 not represented in published murine antibody germlines. ( Figure 7 ).

[0307] 2.2. Construction of chimeric expression vector

[0308] The construction of chimeric expression vectors involved cloning the amplified variable regions into IgG / κ vectors (pHuG1 and pHuK- Figure 8 and Fig. 9 ). Compatible overhangs were generated by digestion with BfuA1 (BspM1) vector (pCMV modification) and subsequent use of T4 DNA polymerase 3′-5′ exonuclease activity (+dATP).

[0309] The genes for TPAi-1 VH and VK were codon optimized for human sequences and synthesized by GenScript. Antibody sequences were amplified by PCR from GenScript constructs using primers ( Fig.11 and Fig.12), the primers contain the 3' end of the leader sequence (most of the sequence is present in the vector) - forward primer - or the start of the constant region (IgG1 or κ) - reverse primer, followed by the start of the variable region (in each direction) ( Figure 3 ). Complementary overhangs were generated in the PCR product by treatment with T4 DNA polymerase (+dTTP) (2.2.1). The vector and insert were incubated at room temperature and used to transform chemically competent TOP10 bacteria and plated on kanamycin plates.

[0310] Several clones were isolated and colony screening was performed by PCR using primers HCMVi and HuG1 LIC Rev for VH or primers HuK LIC Rev for VK ( Figure 3 ). A clone generating a PCR product of the correct size was selected, isolated plasmid DNA was miniprepped using a QIAGEN kit (2.2.2), and sequenced using the same primers.

[0311] 2.2.1 Generation of mAb expression vectors by LIC method

[0312] Insert preparation

[0313] 1. Generate LIC primers using the sequence.

[0314] 2. Use the LIC method to directly transfer the first-strand cDNA or synthetic DNA to the LIC expression vector ( Figure 8 , Fig. 9 and Fig.10 The variable domains were cloned using the vector shown in FIG.

[0315] 3. Set up PCR reaction:

[0316]

[0317]

[0318] Note: You must use a polymerase that produces blunt-ended PCR products in this step. Other polymerases that produce T-overhangs are not suitable.

[0319] 4. Loop:

[0320]

[0321] 5. Run 5 μL of PCR product on a gel to ensure the correct size product - should be approximately 350 bp.

[0322] 6. Perform PCR purification of the product using Qiagen PCR purification kit to remove nucleotides and primers. Elute into 40 μL 10 mM Tris-HCl, pH 8.5 (Buffer EB).

[0323] 7. Treat the insert with T4 DNA polymerase:

[0324]

[0325] 8. Incubate at room temperature for 30 minutes and then inactivate the enzyme at 70°C for 20 minutes.

[0326] Vector preparation

[0327] 9. Digest the LIC vector with BfuAI by incubating at 50°C for 3 hours or overnight:

[0328]

[0329]

[0330] 10. After BfuAI digestion add: 2 μL Bam HI and incubate at 37°C for 2 hours.

[0331] 11. Treat the vector with T4 DNA polymerase as follows:

[0332]

[0333] 12. Incubate at room temperature for 30 minutes and then inactivate the enzyme at 70°C for 20 minutes.

[0334] clone

[0335] 13. Mix 2 μL of insert with 1 μL of vector at room temperature for 5 minutes. Add 1 μL of 25 mM EDTA, mix gently and let stand at room temperature for 5 minutes. Always perform vector-only transformation.

[0336] 14. Use the ligation mix to transform 50 μL of chemically competent Invitrogen TOP 10 bacteria following the manufacturer's instructions (2.2.1.1) and spread on 90 mm diameter LB agar plates containing kanamycin (50 μg / ml). Incubate overnight at 37°C.

[0337] Pick colonies from transformants

[0338] 15. PCR confirmation using Phusion PCR Master Mix:

[0339] Reagents Volume (for 20 μl final reaction volume) 2x Phusion PCR Master Mix 10uL HCMVi primers 1μL HuG1 / HuK LIC Primers 1μL dH20 Up to 20 μL DNA Dip a colony (a culture of the same colony grown on the same day)

[0340]

[0341]

[0342] 16. Electrophorese each PCR-reaction on a 2% agarose electrophoresis gel cassette and run for 15 minutes to determine the size of any PCR product bands on the gel.

[0343] 17. Grow a kanamycin starter culture overnight, miniprep the construct and sequence the DNA of at least two independent positive clones derived from the variable gene (using the same primers) to identify any possible errors due to the PCR-reaction itself.

[0344] 2.2.1.1 Top 10 Conversions TM Escherichia coli (Invitrogen protocol)

[0345] 1. Briefly centrifuge the vial containing the ligation reaction and place on ice.

[0346] 2. Thaw one 50 μL vial of One Shot cells on ice for each ligation / transformation.

[0347] 3. Transfer 1 to 5 μL of each ligation reaction directly into the competent cell vial and mix by gentle tapping.

[0348] Do not mix by pipetting up and down. The remaining ligation mix can be stored at -20 °C.

[0349] 4. Incubate the vial on ice for 30 minutes.

[0350] 5. Incubate in a 42°C water bath for exactly 30 seconds and then place on ice.

[0351] 6. Add 250 μL of pre-warmed SOC medium to each vial.

[0352] 7. Shake the vial in a shaker at 225 rpm at 37°C for exactly 1 hour.

[0353] 8. Spread 200 μL from each transformation vial onto separate labeled LB agar plates containing 500 μg / ml kanamycin.

[0354] 9. Invert the plate and incubate overnight at 37°C.

[0355] 2.2.2 Use Plasmid DNA miniprep isolation (Qiagen protocol)

[0356] 1. Resuspend the pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Make sure RNase A has been added to Buffer P1.

[0357] 2. Add 250 μL of Buffer P2 and gently invert the tube 4-6 times to mix.

[0358] 3. Add 350 μL of Buffer N3 and immediately but gently invert the tube 4-6 times. The solution should be cloudy.

[0359] 4. Centrifuge at 13,000 rpm (approximately 17,900 x g) for 10 minutes in a tabletop microcentrifuge. A compact white precipitate will form.

[0360] 5. Apply the supernatant from step 4 to a QIAprep spin column by pipetting.

[0361] 6. Centrifuge for 30-60 seconds. Discard the flow-through.

[0362] 7. Wash the column by adding 0.5 mL of Buffer PB and centrifuging for 30-60 seconds.

[0363] 8. Wash the column by adding 0.75 mL of Buffer PE and centrifuging for 30-60 seconds.

[0364] 9. Discard the flow-through and centrifuge for an additional 1 minute.

[0365] 10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 50 μL of Buffer EB (10 mM TrisHCl, pH 8.5) to the center of the QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.

[0366] 2.3. Generation of chimeric antibodies

[0367] ExpiCHO suspension cells grown in ExpiCHO expression medium and antibiotics were co-transfected with TPAi-1 VH.pHuG1 and TPAi-1 VK.pHuK (1 μg DNA each) using ExpiFectamine-CHO reagent (2.3.1). Cells were cultured in 1 ml growth medium for 7 days. Chimeric TPAi-1 antibody up to 220 μg / ml was measured in the conditioned medium by Octet using a protein G biosensor (2.3.2).

[0368] 2.3.1 ExpiCHO transfection in 1 ml transfection in 24-well plates

[0369] Material:

[0370] ExpiFectamine CHO Kit 1L (ThermoFisher Scientific Cat. No. A29129)

[0371] -ExpiFectamine CHO Reagent

[0372] -ExpiCHO Enhancer

[0373] -ExpiCHO feed

[0374] OptiPRO SFM (ThermoFisher Scientific Cat. No. 12309-050)

[0375] ExpiCHO Expression Medium (Cat. No. A29100-01)

[0376] plan:

[0377] 1. Subculture and expand ExpiCHO cells until the cells reach approximately 4-6x10 6 The density of viable cells / mL.

[0378] Day -1: Cell Splitting

[0379] 2. On the day before transfection (day -1), split the expiCHO culture to a final density of 3-4x10 6 viable cells / mL and allowed to grow overnight.

[0380] Day 0: Transfection

[0381] 3. Dilute cells to 6x10 6 viable cells / mL.

[0382] 4. Aliquot 0.9 mL of cells into each well of a 24-well plate for transfection.

[0383] 5. Preparation of ExpiFectamine / DNA complex:

[0384] a. For each well to be transfected, dilute the plasmid DNA by adding 1 μL DNA to a final volume of 50 μL OptiPro (1 ug plasmid DNA per mL culture volume to be transfected).

[0385] b. For each well to be transfected, dilute 4 μL of ExpiFectamine CHO reagent in 46 μL of OptiPro medium (no incubation time required).

[0386] c. Add diluted ExpiFectamine CHO to the diluted DNA and mix by gentle pipetting 3-4 times (incubate 1 to 5 minutes).

[0387] 6. Add 100 μL of the complex mixture to each well containing culture in a 24-well plate.

[0388] 7. Cover the board with a breathable cover.

[0389] 8. On a rotary shaker containing 8% CO2 The 24-well plates were incubated in a 37°C incubator (the recommended shaking speed of the shaker is 225 rpm and the orbital throw is 19 mm).

[0390] Day 1: 18-22 hours after transfection, add 6 ul ExpiCHO Enhance and 190 ul ExpiCHO Feed.

[0391] Harvesting: For the standard protocol: protein expression is generally intact and supernatants are ready for harvesting by day 7-8 post-transfection.

[0392] 2.3.2 Quantification of human antibodies by Octet using protein G biosensor

[0393] Material

[0394] 384-well slanted bottom plate (18-5080-Pack, ForteBio)

[0395] 96-well, PP, F-bottom, black plate (655209, Greiner)

[0396] Protein G Sensor (Pack), 18-5083

[0397] An appropriate diluent, ideally the same buffer or supernatant as your sample (Expi293 or ExpiCHO medium when testing neat supernatant)

[0398] Human IgG Standard:

[0399] MRCT Human IgG1 Isotype Control

[0400] 1. Turn on the Octet instrument (at least 40 minutes before starting analysis)

[0401] 2. Prepare 100 μl (enough for 2 x 40 μl) sample:

[0402] a. Isotype standards at 500, 250, 125, 62.5, 31.25, 15.6, 7.81, or 0 μg / ml using the diluent that matches your samples (e.g., if your samples are purified, clear culture medium or PBS)

[0403] b. Test (unknown) samples

[0404] 3. Open the software and select Mouse IgG quantitation (8CH_96W). Modify the protocol so that Plate 1 is a 384-well plate. Set up the plate layout, including additional wells for replicate samples, diluents (references), and controls (e.g., conditioning / expression controls)

[0405] 4. Pre-soak (≥10 min) the biosensor in 200 μl of dilute buffer

[0406] 5. 40 μl / sample was aliquoted into 384-well slanted bottom (product code: 18-5080) plates in duplicate. The plates were sealed and centrifuged in a benchtop centrifuge (1000 rpm, 2 minutes).

[0407] 6. Remove plate seal and insert plate. Verify / set software run parameters:

[0408] a. Save the target file

[0409] b. Set the temperature to 25°C (if the room has air conditioning, use 30°C)

[0410] c. Wait 10 minutes (600 seconds) before starting and shaking

[0411] 7. Start the experiment.

[0412] 8. To process the data, open the evaluation software. Select the wells to be used in the standard calculation and fit to the dose response - 5PL fit.

[0413] Biosensor Storage

[0414] At the end of the run, the biosensor will return to the source location.

[0415] To store the sensor for reuse:

[0416] Carefully remove the sensor from the tray

[0417] Soak in 15% sucrose for about 10 minutes (you can use the same 96-well black plate, but with different columns).

[0418] Allow to dry and store in storage tray.

[0419] 2.4 tPA Binding Activity of Chimeric Antibodies

[0420] The binding of the chimeric TPAi-1 antibody to human recombinant tPA (rh-tPA: abcam ab92637), mouse recombinant tPA (rm-tPA: abcam ab92715), and rat recombinant tPA (rr-tPA: abcam ab92596) was measured by ELISA and compared to the original mouse antibody (2.4.1). The chimeric and mouse TPAi-1 antibodies bound to rh-tPA with similar EC50 values ​​( Fig.12 ). Neither TPAi-1 antibody bound to rm-tPA or rr-tPA.

[0421] 2.4.1 tPA binding ELISA

[0422] 1. Coat each well of a 94-well MaxiSorp plate (Nunc) with 100 μL aliquots of 0.2 μg / mL human tPA (AbCam ab92637), murine tPA (AbCam ab92715), or rat tPA (AbCam ab92596) in PBS and incubate overnight at 4°C.

[0423] 2. Wash three times with PBS-T (0.1% Tween 20).

[0424] 3. Block the experimental plate and fresh plate (dilution plate) with 250 μL PBS / 0.2% BSA / 0.05% Tween20 per well and incubate at room temperature for 1 hour. Wash 3 times with PBS-T (0.1% Tween20).

[0425] 4. Using the dilution plate, add 200uL of antibody (diluted in PBS / 0.2% BSA / 0.05% Tween20 if necessary) to the wells in column 1; add 120μL of buffer (PBS / 0.2% BSA / 0.05% Tween20) to the other wells.

[0426] 5. Transfer 56 μL from column 1 to the adjacent well in column 2. 0.5 , 3.162x) dilutions are transferred to column 12. 100 μL per well is transferred from the dilution plate to the experimental plate.

[0427] 6. Incubate at room temperature for 1 hour. Wash each well 3 times with PBS-T.

[0428]

[0429] Add 100 μL to each well. Incubate for 1 hour at room temperature and repeat the wash step.

[0430] 8. Add 150 μL substrate (Enhanced KB) per well and incubate at room temperature for 10 minutes.

[0431] 9. Stop the reaction by adding 50 μl RED Stop Solution per well.

[0432] 10. Read the optical density at 650 nm.

[0433] 3.0. Design of TPAi-1 humanized antibody variants

[0434] 3.1. Human VH and VK cDNA databases

[0435] Protein sequences human and mouse immunoglobulins from the International Immunogenetics Database 2009 (Lefranc, 2015) and the Kabat database of Sequences of Proteins of Immunological Interest version 5 (last updated November 17, 1999) (Kabat et al., 1991) were used to compile a database of human immunoglobulin sequences in the (Kabat) alignment.

[0436] 3.2. Molecular model of TPAi-1

[0437] The mouse TPAi-1 antibody variable region homology model was calculated using the Discovery Studio 4.1 program running in automatic mode. The Accelrys antibody pdb structure database was analyzed by Blast to determine the sequence templates of the light chain variable region and the heavy chain variable region. These templates were used for potential models.

[0438] 3.3. Human frame selection

[0439] Humanization requires identification of appropriate human V regions. The sequence analysis program Gibbs was used to query the human VH and VK databases with the TPAi-1 VH and VK protein sequences using a variety of selection criteria. CDR residues (KABAT and IMGT definitions) were identified in the structure of the mouse TPAi-1 antibody using the program Discovery Studio (Accelrys). FW residues within the range.

[0440] Using a variety of selection criteria, human heavy chain donor candidates and human kappa light chain donor candidates were identified.

[0441] 3.4. Design of TPAi-1 human heavy chain

[0442] The initial design of the humanized version of TPAi-1 was to graft CDRs 1, 2 and 3 from TPAi-1 VH into the FW acceptor of potential heavy chain donor candidates. Potential sequences were assembled in silico.

[0443] 3.5. Design of TPAi-1 human kappa light chain

[0444] CDRs 1, 2, and 3 from TPAi-1 VK were grafted into the acceptor FW of a potential human kappa light chain donor candidate to generate a potential form of humanized TPAi-1.

[0445] 3.6. Remodeling of TPAi-1

[0446] Humanized TPAi-1 candidates were remodeled using various selection criteria, including mutations.

[0447] 4.0. Generation and characterization of humanized forms of TPAi-1

[0448] 4.1. Generation of humanized TPAi-1 antibody

[0449] The genes for the humanized TPAi-1 candidates were synthesized by GenScript and codon optimized for human sequences. The sequences were optimized by silent mutagenesis using GenScript proprietary software algorithms to use codons that are preferentially utilized and synthesized by human cells. The heavy chain constructs and the kappa light chain constructs were PCR amplified with specific primers for the expression vector + insert (as previously described for the chimeric form) and these constructs were inserted into pHuG1 and pHuK ( Figure 8 and Fig. 9 ) and used to transform TOP10 bacteria (2.2.1.1). Fig.14 The subsequent humanized variants were obtained by PCR mutagenesis (4.1.1) using the primers in . The clones were sequenced and expression plasmid DNA was prepared using a QIAGEN plasmid miniprep kit or a Qiagen plasmid large-scale preparation kit (4.1.2 and 4.1.3). The expression plasmid preparations encoding (humanized or chimeric) VH and VK were used to transfect ExpiCHO cells (2.3.1), cultured in serum-free medium for 5-7 days, and the conditioned medium containing secreted antibodies was thus harvested.

[0450] 4.1.1 QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene)

[0451] 1. Prepare the reaction as shown below:

[0452] 5 μL 10× reaction buffer

[0453] 0.12 μL (25 ng) heavy chain or kappa light chain template

[0454] 1.3 μL (125 ng) oligonucleotide mutagenesis forward primer

[0455] 1.3 μL (125 ng) oligonucleotide mutagenesis reverse primer

[0456] 1 μL dNTP mix

[0457] 1.5 μL QuikSolution reagent

[0458] ddH2O to a final volume of 50 μL

[0459] 1 μL QuikChange Lightning Enzyme

[0460] 2. Cycle each reaction using the cycling parameters outlined in the table below:

[0461]

[0462] 3. Add 2 μL Dpn I restriction enzyme

[0463] 4. Gently and thoroughly mix each reaction, microcentrifuge briefly, and then immediately incubate at 37°C for 5 minutes to digest the parental dsDNA.

[0464] 5. Transform 2 μL of Dpn I-treated DNA from each reaction into separate 45 μL (+ 2 μL β-ME) aliquots of XL10-Gold Super Competent Cells (see TOP10 TM coli transformation).

[0465] 6. Screen colonies using the Phusion method, miniprep DNA and sequence to verify the correct mutation.

[0466] 4.1.2 Use Plasmid DNA miniprep isolation (Qiagen protocol)

[0467] 1. Resuspend the pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Make sure RNase A has been added to Buffer P1.

[0468] 2. Add 250 μL of Buffer P2 and gently invert the tube 4-6 times to mix.

[0469] 3. Add 350 μL of Buffer N3 and immediately but gently invert the tube 4-6 times. The solution should be cloudy.

[0470] 4. Centrifuge at 13,000 rpm (approximately 17,900 x g) for 10 minutes in a tabletop microcentrifuge. A compact white precipitate will form.

[0471] 5. Apply the supernatant from step 4 to a QIAprep spin column by pipetting.

[0472] 6. Centrifuge for 30-60 seconds. Discard the flow-through.

[0473] 7. Wash the column by adding 0.5 mL of Buffer PB and centrifuging for 30-60 seconds.

[0474] 8. Wash the column by adding 0.75 mL of Buffer PE and centrifuging for 30-60 seconds.

[0475] 9. Discard the flow-through and centrifuge for an additional 1 minute.

[0476] 10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 50 μL of Buffer EB (10 mM TrisHCl, pH 8.5) to the center of the QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.

[0477] 4.1.3 Qiagen protocol for large-scale plasmid DNA preparation

[0478] 1. Pick a single colony from a freshly streaked selective plate and inoculate a 2-5 ml LB medium starter culture containing the appropriate selective antibiotic. Incubate at 37°C for about 8 hours with vigorous shaking (about 300 rpm). Use a tube or flask with a volume at least 4 times the culture volume.

[0479] 2. Dilute the starter culture 1 / 500 to 1 / 1000 into selective LB medium. For high copy plasmids, inoculate 25 ml or 100 ml of medium. For low copy plasmids, inoculate 100 ml or 500 ml of medium. Grow at 37°C for 12-16 hours with vigorous shaking. Use a flask or autoclave with a volume at least 4 times the volume of the culture. The culture should reach approximately 3-4x10 9 The cell density is 10 cells / ml, which generally corresponds to a pellet wet weight of approximately 3 g / L of culture medium.

[0480] 3. Harvest bacterial cells by centrifugation at 6000 x g for 15 min at 4 °C. GSA or GS3 or Beckman TM 6000 rpm in a JA-10 rotor. Remove all traces of supernatant by inverting the open centrifuge tube until all the medium has drained away.

[0481] 4. Resuspend the bacterial pellet in 4 ml or 10 ml of Buffer P1. For efficient lysis, it is important to use a container large enough to allow thorough mixing of the lysis buffer. Make sure that RNase A has been added to Buffer P1. The bacteria should be thoroughly resuspended by vortexing or pipetting up and down until no cell clumps remain.

[0482] 5. Add 4 ml or 10 ml of Buffer P2, mix gently but thoroughly by inverting 4-6 times, and incubate at room temperature for 5 minutes. Do not vortex to mix, as this will cause shearing of genomic DNA. The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 minutes.

[0483] 6. Add 4 ml or 10 ml of chilled Buffer P3, mix immediately but gently by inverting 4-6 times, and incubate on ice for 15 minutes or 20 minutes. Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After adding Buffer P3, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris, and SDS. The lysate should be mixed thoroughly to ensure even precipitation of potassium dodecyl sulfate. If the mixture still appears viscous and brownish, more mixing is required to completely neutralize the solution.

[0484] 7. Centrifuge at 20,000 x g for 30 minutes at 4°C. Remove the supernatant containing the plasmid DNA promptly. The sample should be mixed again before loading into the centrifuge. Centrifugation should be performed in non-glass (e.g., polypropylene) tubes. A centrifugal force of 20,000 x g corresponds to 12,000 rpm in a Beckman JA-17 rotor or 13,000 rpm in a Sorvall SS-34 rotor. The supernatant should be clear after centrifugation.

[0485] 8. Centrifuge the supernatant again at 20,000 x g for 15 minutes at 4°C. Remove the supernatant containing the plasmid DNA quickly. This second centrifugation step should be performed to avoid applying suspended or particulate material to the QIAGEN spin column. Suspended material (causing the sample to appear turbid) may clog the QIAGEN spin column and reduce or eliminate natural flow.

[0486] 9. Equilibrate the QIAGEN spin column 100 or QIAGEN spin column 500 by applying 4 ml or 10 ml of Buffer QBT and allow the column to drain by natural flow. Buffer flow will automatically begin due to the reduction in surface tension caused by the detergent present in the equilibration buffer. Allow the QIAGEN spin column to drain completely. The QIAGEN spin column can be left unattended, as buffer flow will stop when the meniscus reaches the upper frit in the column.

[0487] 10. Apply the supernatant from step 8 to the QIAGEN spin column and allow it to enter the resin by natural flow. The supernatant should be loaded onto the QIAGEN spin column quickly. If it is retained too long and becomes cloudy due to further protein precipitation, it is necessary to centrifuge or filter the supernatant again before loading to prevent clogging of the QIAGEN spin column.

[0488] 11. Wash the QIAGEN spin column with 2x10ml or 2x30ml Buffer QC. Allow Buffer QC to move through the QIAGEN spin column by natural flow. The first wash is sufficient to remove all impurities in most plasmid DNA preparations. The second wash is particularly necessary when using large culture volumes or bacterial strains that produce large amounts of sugars.

[0489] 12. Elute the DNA with 5 ml or 15 ml of Buffer QF. Collect the eluate in a 10 ml or 30 ml tube. Polycarbonate centrifuge tubes are not recommended because polycarbonate is not tolerant to the alcohol used in subsequent steps.

[0490] 13. Precipitate the DNA by adding 3.5 ml or 10.5 ml (0.7 volumes) of room temperature isopropanol to the eluted DNA. Mix and immediately centrifuge at 15,000 x g for 30 minutes at 4°C. Carefully decant the supernatant. All solutions should be at room temperature to minimize salt precipitation, but centrifugation is performed at 4°C to prevent overheating the sample. A centrifugal force of 15,000 x g corresponds to 9500 rpm in a Beckman JS-13 rotor and 11,000 rpm in a Sorvall SS-34 rotor. Alternatively, disposable conical bottom centrifuge tubes can be used for centrifugation at 5000 x g for 60 minutes at 4°C. The isopropanol precipitate has a glassy appearance.

[0491] Isopropanol precipitates are also loosely bound to the sides of the tube and care should be taken when removing the supernatant.

[0492] 14. Wash the DNA pellet with 2 ml or 5 ml of room temperature 70% ethanol and centrifuge at 15,000 x g for 10 minutes. Carefully decant the supernatant without disturbing the pellet.

[0493] 15. Allow the pellet to dry for 5-10 minutes and redissolve the DNA in an appropriate volume of buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5). Redissolve the DNA pellet by rinsing the tube walls to recover all of the DNA, especially if glass tubes have been used. Pipetting the DNA up and down to facilitate resuspension may cause shearing and should be avoided. Excessive drying of the pellet will make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline conditions; it does not dissolve easily in acidic buffers.

[0494] 4.2. Antibody expression

[0495] The concentration of IgG1κ antibody in the conditioned medium of ExpiCHO cells was measured by Octet using a Protein G biosensor (2.3.2).

[0496] Examples of humanized TPAi-1 antibodies are shown in Table 1.

[0497] Table 1: Humanized TPAi-1 Antibody

[0498] mAb identifiers Variable heavy chain sequence Variable light chain sequence TPAi-1 RHE / RKA SEQ ID No.14 SEQ ID No.29 TPAi-1 RHP / RKA SEQ ID No.15 SEQ ID No.29 TPAi-1 RHB / RKA SEQ ID No.16 SEQ ID No.29 TPAi-1 RHJ / RKA SEQ ID No.17 SEQ ID No.29 TPAi-1 RHK / RKA SEQ ID No.18 SEQ ID No.29 TPAi-1 RHL / RKA SEQ ID No.19 SEQ ID No.29 TPAi-1 RHM / RKA SEQ ID No.20 SEQ ID No.29 TPAi-1 RHN / RKA SEQ ID No.21 SEQ ID No.29 TPAi-1 RHO / RKA SEQ ID No.22 SEQ ID No.29 TPAi-1 RHQ / RKA SEQ ID No.23 SEQ ID No.29 TPAi-1 RHR / RKA SEQ ID No.24 SEQ ID No.29 TPAi-1 RHS / RKA SEQ ID No.25 SEQ ID No.29 TPAi-1 RHT / RKA SEQ ID No.26 SEQ ID No.29 TPAi-1 RHU / RKA SEQ ID No.27 SEQ ID No.29 TPAi-1 RHV / RKA SEQ ID No.28 SEQ ID No.29

[0499] 4.3. Antigen Binding of Humanized TPAi-1 Antibody

[0500] The binding activity to tPA antigen was measured by binding ELISA (2.4.1). Fig.15 and Fig.15A The data shown in show the binding potency of the humanized forms of TPAi-1 in Table 1.

[0501] 4.4. Determination of Tm (melting temperature) of humanized candidate antibodies

[0502] To determine the melting temperatures of antibodies TPAi-1 RHE / RKA and TPAi-1 RHP / RKA, these antibodies were tested in a thermal shift assay. The samples were incubated with a fluorescent dye (Sypro Orange) for 71 cycles with a 1°C increase per cycle in a qPCR thermocycler. Fig.16 The Tm values ​​of the two humanized antibodies are shown in . Both antibodies had a Tm of at least 70°C, indicating that they met the thermal stability requirements.

[0503] Aggregation analysis of humanized antibody candidates

[0504] Aggregation assessment of TPAi-1 RHE / RKA and TPAi-1 RHP / RKA antibodies was performed by dynamic light scattering (DLS). The antibodies were found to have a hydrodynamic radius and polydispersity consistent with monomers ( Fig.17 ). The data suggest that aggregation is not a problem in the antibody samples analyzed.

[0505] 4.6. Nonspecific protein-protein interactions (CIC)

[0506] Cross-interaction chromatography using large volumes of purified human polyclonal IgG is a technique to monitor nonspecific protein-protein interactions and can be used to distinguish soluble from insoluble antibodies.

[0507] An increased retention index (k') indicates a low tendency for self-interaction and low solubility. Both candidate antibodies showed a retention index below 0.05, indicating a low tendency for nonspecific interactions and good solubility ( Fig.18 ).

[0508] 4.7. Solubility of humanized antibody candidates

[0509] Purified candidate antibodies were concentrated using a solvent absorption concentrator (MWCO 7500 kDa) and concentrations were measured at scheduled time intervals. Two samples were concentrated to more than 40 mg / ml with no obvious precipitation ( Fig.19 ).

[0510] 4.8. Freeze / thaw stress analysis of candidate antibodies

[0511] Samples of purified RHE / RKA and RHP / RKA antibodies were subjected to 10 cycles of 15 minutes at -80°C followed by 15 minutes of thawing at room temperature. Control samples were kept at 4°C throughout. Samples were then analyzed for aggregation by SEC-MALS ( Fig. 20 ). The data showed that freeze / thaw did not cause aggregation in the antibodies tested.

[0512] 4.9. Heat-induced stress analysis of candidate antibodies

[0513] Samples of purified RHE / RKA and RHP / RKA antibodies were heat exposed at a) room temperature, b) 37°C and c) 50°C or kept at 4°C for 30 days. The samples were then analyzed for aggregation by SEC-MALS ( Fig.21 ). Incubation did not result in any aggregation. Overall, the data suggest that there is no aggregation problem in any of the humanized TPAi-1 antibody samples analyzed.

[0514] 4.10 Serum stability assessment of candidate antibodies

[0515] Purified samples of humanized antibodies TPAi-1 RHE / RKA and RHP / RKA (4.10.1) were incubated in mouse serum, human serum and cynomolgus monkey serum. The binding capacity of the antibodies after 29 days of incubation was measured by binding ELISA against human tPA. For each antibody, one ELISA plate compared the binding of the 4°C control sample with samples incubated at 37°C in PBS and human serum. The second ELISA plate compared the binding of the 4°C control sample with samples incubated at 37°C in mouse serum and cynomolgus monkey serum. Fig. 22 The curve and the EC obtained from this curve 50 The values ​​show that for both RHE / RKA and RHP / RKA, the binding capacities of the antibody samples incubated in various sera were similar to those of the 4°C control sample and the samples incubated in PBS at 37°C. Thus, the TPAi-1 RHE / RKA and RHP / RKA antibodies retained their binding capacity when incubated for 29 days in mouse serum, human serum, and cynomolgus monkey serum.

[0516] 4.10.1 Antibody serum stability assessment

[0517] Sample: 600 μl 0.4 mg / ml purified antibody (240 μg) in PBS

[0518] Test conditions: Mouse serum (SCD-808), human serum (S-123), and cynomolgus monkey serum (S-118) from Seralab

[0519] 1. Aliquot 150 μl of serum and PBS control in a round-bottom 96-well plate and add 50 μl of 0.4 mg / mL antibody solution in PBS to each serum type in triplicate (final concentration 100 μg / mL) in a tissue culture incubator (BSL-2). Reserve some antibody solution at 4°C for later use as a control.

[0520] Serum incubation plate layout

[0521] PBS Mouse serum human serum Cynomolgus monkey serum PBS Mouse serum human serum Cynomolgus monkey serum PBS Mouse serum human serum Cynomolgus monkey serum

[0522] 2. Seal the plate with ELISA plate sealer and incubate at 37°C.

[0523] 3. Take 30 μl samples under sterile conditions (BSL-2) on days 6, 13, 20 and 29 to avoid contamination. Freeze at -20°C until analysis.

[0524] 4. Analyze the longer incubation first (if no loss of binding is seen, no need to test additional columns).

[0525] 5. Dilute the samples appropriately and analyze antigen binding by generating an ELISA binding curve (5-fold dilution series) for each specimen (Section 8.13) using unincubated antibody as a control.

[0526] 5.0 Generation and Characterization of Humanized TPAi-1 Fab

[0527] Generation of TPAi-1 RHP / RKA Fab

[0528] DNA of the heavy chain variable region of TPAi-1 RHP / RKA was amplified from the IgG1 expression construct TPAi-1_RHP.pHuG1 using primers containing either the 3' end of the leader sequence (most of the sequence is present in the vector) - forward primer - or the start of the constant region (IgG1) - reverse primer followed by the start of the variable region (in each direction), Figure 3 Complementary overhangs were generated in the PCR product by T4 DNA polymerase + dTTP treatment (2.2.1). The pHuG1_Fab LIC vector and insert were incubated at room temperature and used to transform chemically competent TOP10 bacteria and plated on kanamycin plates. Several clones were isolated and colony screening was performed by PCR using primers HCMVi and E1_alpha_rev ( Figure 3). A clone that produced a PCR product of the correct size was selected, isolated plasmid DNA was prepared using a QIAGEN kit, and sequenced using the same primers, and HuG1 LICRev.TPAi-1_RHP.pHuG1_Fab and TPAi-1_RKA.pHuK expression plasmid preparations were used to transfect Expi293 cells (2.3.1). These cells were cultured in serum-free medium for 5-7 days, and the conditioned medium containing secreted antibodies was harvested.

[0529] 5.2.TPAi-1 RHP / RKA Fab Expression

[0530] The concentration of TPAi-1 RHP / RKA Fab in Expi293 cell conditioned media was measured by Octet at 102 μg / ml using an anti-human kappa chain reagent coated Streptavadin biosensor (5.2.1). Larger scale transfection and culture produced 72 mg of purified Fab from 1 L of conditioned media.

[0531] 4.10.1 Quantification of human Fab by Octet using anti-κ light chain coated sensors

[0532] A 15-minute loading step was performed using the reagent diluted to 5 μg / mL in HBS-P+ buffer and the CaptureSelect TM Biotin anti-LC-K (Hu) conjugate (13 kDa alpaca antibody fragment; 7103272100, ThermoScientific) was coated with streptavidin (SA) biosensor (18-5020). The coated biosensor was subjected to 3 regeneration cycles: 10 mM glycine pH 2.0 (15 seconds) / HBS-P+ buffer (15 seconds), soaked in 15% sucrose solution for 10 minutes and allowed to dry.

[0533] These sensors were used to quantify the concentration of Fab in supernatant samples by following the procedure section 2.3.2, but using the template file 'FAb quantitation (capture selection anti-κLC)'. A purified human Fab control was used as a standard.

[0534] 5.3. Antigen Binding of TPAi-1 RHP / RKA Fab

[0535] The binding activity of TPAi-1 RHP / RKA Fab to human tPA antigen was compared with that of purified TPAi-1 RHP / RKA antibody in a binding ELISA. Initial experiments used RHP / RKA Fab in Expi293 cell conditioned medium and showed dose-dependent binding of Fab to human tPA ( Fig.23). As would be expected when comparing a monovalent Fab to a bivalent antibody, the EC of the interaction 50 (1.149 nM) greater than the EC of intact RHP / RKA antibody 50 After purification of TPAi-1RHP / RKAFab from large-scale culture, its binding to human tPA was confirmed by ELISA ( Fig.23 ). In this assay, the plateaus of the two binding curves are more pronounced. The IgG binding curve reaches a plateau at the A650 level, approximately 1.4-fold higher than the plateau of the Fab curve, indicating that some but not all IgGs are bivalently bound.

[0536] 5.4. Determination of TPAi-1 RHP / RKA Fab Tm (melting temperature)

[0537] Thermal shift assay was used to determine the melting temperature of TPAi-1 RHP / RKA Fab (24). The Tm of TPAi-1 RHP / RKA Fab was 74°C and the Fab thus met the thermal stability requirement.

[0538] Aggregation analysis of TPAi-1RHP / RKA Fab

[0539] In an HPLC system, TPAi-1 RHP / RKA Fab was injected at 0.4 mL / min onto a size exclusion column and analyzed by multi-angle light scattering to determine absolute molar mass and to check aggregation (see Fig.25 ). The Fab showed no signs of aggregation and had an average molecular weight of 49.5 kDa, which is in the expected range for Fab monomers in this analytical setting. All samples were monodisperse (Mw / Mn < 1.05). Mass recovery was 100% (calculated mass vs. injected mass), indicating that protein recovery was good and the samples did not appear to adhere to the column or contain insoluble aggregates that would be retained by the guard column. Overall, the data suggest that aggregation is not an issue in the RHP / RKAFab samples.

[0540] 5.6. Nonspecific protein-protein interactions (CIC) of TPAi-1RHP / RKA Fab

[0541] Cross-interaction chromatography using large volumes of purified human polyclonal IgG is a technique to monitor nonspecific protein-protein interactions and can be used to distinguish soluble from insoluble antibodies. An elevated retention index (k') indicates self-interaction propensity and low solubility. TPAi-1 RHP / RKA Fab showed a retention index below 0.05, indicating low nonspecific interaction propensity and good solubility ( Fig.26 ).

[0542] Solubility of TPAi-1RHP / RKA Fab

[0543] Purified TPAi-1 RHP / RKAFab was concentrated using a solvent absorption concentrator (MWCO 7500 kDa) and the concentration was measured at scheduled time intervals. The sample was concentrated to more than 65 mg / ml with no obvious precipitation ( Fig. 27 ).

[0544] 5.8 Freeze / thaw stress analysis of TPAi-1 RHP / RKA Fab

[0545] Samples of purified RHP / RKA Fab were subjected to 10 cycles of 15 min at -80°C followed by 15 min thawing at room temperature. Control samples were kept at 4°C throughout. Samples were then analyzed for aggregation by SEC-MALS ( Fig.28 ). The data showed that freeze / thaw did not cause aggregation in the Fab.

[0546] 5.9 Thermally induced stress analysis of TPAi-1 RHP / RKA Fab

[0547] Samples of purified RHP / RKA Fab were heat exposed at a) room temperature, b) 37°C and c) 50°C or kept at 4°C for 30 days. The samples were then analyzed for aggregation by SEC-MALS ( Fig.29 ). Room temperature and 37°C incubations did not produce any aggregation, but there was 8.2% aggregation in the 50°C sample. According to QC standards, the 50°C sample should ideally contain less than 5% aggregates and strictly contain less than 10% aggregates compared to the unstressed sample. TPAi-1 RHP / RKA Fab did not contain less than 10% aggregates after heat-induced stress, but was more unstable than intact RHP / RKA IgG (see Section 4.9).

[0548] 5.10 Serum stability evaluation of TPAi-1 RHP / RKA Fab

[0549] Purified samples of TPAi-1 RHP / RKA Fab (4.10.1) were incubated in mouse serum, human serum and cynomolgus monkey serum. The binding capacity of the antibody after 29 days of incubation was measured by binding ELISA against human tPA ( Fig.30 ). Fig.30The straight line portion of the binding curves shows that the binding capacities of RHP / RKA Fab samples incubated in various sera are similar to those of the 4°C control sample and the samples incubated in PBS at 37°C. The presence of a significant amount of serum in the ELISA wells with higher Fab concentrations (11% serum at 300 nM Fab) appears to reduce the binding of the Fab samples. This reduction in the binding signal at the top of the curve results in an EC of 1.3 for Fab samples incubated with serum. 50 However, this does not affect the conclusion that TPAi-1 RHP / RKA Fab retains its binding capacity when incubated in mouse serum, human serum, and cynomolgus monkey serum for 29 days.

[0550] 6.0. Preparation and Characterization of Humanized TPAi-1 F(ab')2

[0551] Conversion of TPAi-1 RHP / RKA from full IgG to Fab format resulted in loss of binding affinity to human tPA in binding ELISA (Section 5.3; Fig.23 To determine whether the reduction in avidity was entirely due to the change from bivalent to monovalent binding or whether the loss of the Fc was also a factor, TPAi-1 RHP / RKAF(ab')2 was prepared and tested in an ELISA.

[0552] 6.1 Preparation of TPAi-1RHP / RKA F(ab')2

[0553] 5 mg of TPAi-1 RHP / RKA IgG1κ antibody was digested with pepsin and the sample was analyzed by SDS-PAGE to confirm digestion and the presence of F(ab') 2 Molecules of expected size (non-reduced MW approximately 110 kDa; Fig.31 .1). Then the F(ab') was purified by gel filtration. 2 ( Fig.31 .2), yielding 1.7 mg of purified protein. TPAi-1 RHP / RKA F(ab') was verified by SDS-PAGE 2 Purity and integrity of the preparation ( Fig.31 .3).

[0554] 6.2. TPAi-1RHP / RKA F(ab') 2 Aggregation analysis

[0555] In the HPLC system, TPAi-1 RHP / RKA F(ab') 2 Injection was loaded onto the size exclusion column at 0.4 mL / min and analyzed by multi-angle light scattering to determine the absolute molar mass and to check aggregation (see Fig.32 ). F(ab') 2 There was no sign of aggregation, and the average molecular weight was 102.6 kDa. All samples were monodisperse (Mw / Mn < 1.05). The mass recovery was 100% (calculated mass to injected mass), indicating that the protein was well recovered and the sample did not adhere to the column or contain insoluble aggregates that would be retained by the guard column. Overall, the data suggest that RHP / RKAF(ab') 2 There was no aggregation problem in the samples.

[0556] 6.3.TPAi-1 RHP / RKA F(ab') 2 Antigen binding

[0557] In a binding ELISA, purified TPAi-1 RHP / RKA F(ab') 2 Binding activity to human tPA antigen was compared with that of purified TPAi-1 RHP / RKA whole IgG1 antibody and RHP / RKA Fab. RHP / RKA whole IgG1 and F(ab') 2 The binding curves are very similar to those of 50 The values ​​were 0.475nM and 0.379nM ( Fig.33 In contrast, TPAi-1RHP / RKAFab had an EC of 1.675 nM. 50 EC values ​​for IgG1 and Fab 50 The values ​​are consistent with those seen in earlier experiments. There is a consistent 4-fold affinity difference between the two formats.

[0558] TPAi-1 RHP / RKA intact IgG and F(ab') 2 Comparative binding of the formats showed that removal of the IgG1 Fc region did not affect the affinity for human tPA. The lower affinity displayed by TPAi-1 RHP / RKA Fab is therefore entirely due to the change from bivalent to monovalent binding.

[0559] 7.0 Analysis of TPA activity

[0560] The amidolytic activity of TPA was investigated with 500 μM chromogenic substrate S2288. TPA activation of Pg was determined by monitoring the amidolytic activity of plasmin with 500 μM S2251. All experiments were performed at 37°C in Tris-NaCl buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) as previously described (Sazonova IY, McNamee RA, Houng AK, King SM, Hedstrom L, Reed GL. Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator. J Thromb Haemost. 2009; 7: 1321-1328). Pg was pretreated with aprotinin-agarose beads at 4°C for 4 hours to remove contaminating plasmin. In both assays, the absorbance at 405 nm (A405 nm) was recorded continuously. The amidolytic activity of TPA was determined from the initial slope of A405 nm over time. Based on the method described by Longstaff et al., Longstaff C, Whitton CM. A proposed reference method for plasminogen activators that enables calculation of enzyme activities in SI units. J Thromb Haemost. 2004; 2: 1416-1421, A405 nm / sec over the initial time range of the reaction was used when the net change in absorbance was less than 0.1 2 The activation rate of Pg by TPA relative to plasmin was calculated by comparing the changes in . In some experiments, anti-TPA mAbs or fibrin (Fn) fragments were incubated with TPA to study their effects on TPA activity or Pg activation.

[0561] 8.0 Fibrinolysis

[0562] In a test tube, 20 μl of human plasma (with trace amounts of 125 I-fibrinogen) and 5 μl of a mixture of thrombin and calcium solution (final concentration: 1 Ul / ml thrombin and 10 mM Ca 2+), human blood clots were formed. The blood clots were incubated at 37°C for one hour, followed by the addition of a total of 45 μl of different amounts of human TPA with or without anti-TPA mAb. At the sampling time, 10 μl of supernatant was collected and the radioactivity of this sample was monitored using a Cobra II γ counter (Perkin-Elmer-Packard BioScience, Waltham, MA). After γ counting, the samples were replaced in the test tubes. The percentage of fibrinolysis was determined by dividing the radioactivity in the supernatant by the initial radioactivity of the blood clot. The percentage of mAb inhibition of fibrinolysis was calculated by reference to the amount of fibrinolysis in the absence of mAb.

[0563] Both chimeric TPAi-1 and humanized TPAi-1 (TPAi-1 RHP / RKA) significantly inhibited human TPA-induced human blood clot lysis ( Fig.34 ).

[0564] The effect of TPAi-1 RHP / RKA Fab in inhibiting plasma clot lysis in a dose-response manner was also tested. Human plasma clots were formed by mixing together pooled fresh frozen (3.8% sodium citrate) human plasma (50 μl), calcium and thrombin (10 μl), t-PA (10 μl, 1.5 nM) and purified mouse, chimeric, humanized mAbs and humanized Fabs (0-140 nM). The lysis or lysis of the clot was continuously monitored at A405 nm at 37°C in a microtiter plate reader. The percentage inhibition of lysis was determined by comparing the absorbance readings at A405 nm at (turbidity) baseline and at 1 hour. Fig.35 As seen in Figure 2, approximately twice the amount of TPAi-1 RHP / RKA Fab was required to produce an inhibitory effect equivalent to that produced by TPAi-1 RHP / RKA. This is consistent with the fact that the Fab is monovalent and the mAb is bivalent.

[0565] 9.0 Thromboembolic stroke and hemorrhage in the middle cerebral artery of mice

[0566] Animal studies performed in Dr. Reed's laboratory were approved by the UT-Memphis Institutional Animal Care and Use Committee. C57BL / 6J adult mice (29 to 35 g, Jackson Lab, Bar Harbor ME) were anesthetized with a mixture of 1.5-2% isoflurane and oxygen administered throughout the study. Rectal temperature was maintained at 37°C with a thermostatically controlled heating pad. The left common carotid artery was isolated after a neck incision, and the external carotid, thyroid, and occipital arteries were ligated. Microvascular clamps were temporarily placed on the common carotid and internal carotid arteries. A small arteriotomy was performed on the external carotid artery to insert a retrograde thrombus containing an embolus. 125PES catheter with I-fibrinogen (approximately 5000 cpm / 2ul). The PES catheter containing the blood clot was inserted into the left external carotid artery and screwed into the internal cerebral artery until the origin of the middle cerebral artery (MCA). The thrombus was embolized in a volume of 100ul saline at a rate of 0.45ml / min. Continuous laser-Doppler monitoring was used to assess regional cerebral perfusion to ensure the adequacy of embolization (perfusion dropped to <20% of the pre-ischemic baseline). The right jugular vein was cannulated for drug administration. Mice received recombinant human TPA (rtPA) (10mg / kg at 2.5 hours of local ischemia) as a 20% bolus and 80% infusion over 30 minutes. Mice receiving mAb inhibitors were treated with stoichiometric doses of mouse monoclonal antibodies, chimeric monoclonal antibodies, and humanized monoclonal antibodies, which were given as intravenous boluses 30 or 60 minutes after TPA administration. Tail bleeding was assessed 20 minutes after TPA infusion and monitored for 30 minutes by measuring the time and amount of bleeding from the tail, wherein the tail was preheated in 3 mL saline at 37 ° C in a water bath as described for 5 minutes. Hemoglobin (Hgb) loss from tail bleeding was measured using Drabkin's test kit according to the manufacturer's data sheet (Sigma). Six hours after thromboembolism, animals were killed, brains were separated, cut into 2 mm coronal sections, and incubated for 30 minutes at room temperature in 2% triphenyltetrazolium chloride (Sigma, St.Louis, MO) solution. The stained slices were subsequently transferred to 4% formaldehyde for fixation. Images of four brain slices were collected with a digital camera. Hemispheric size, gross hemorrhage area, and infarct size were digitally analyzed using Image Pro Plus 6.2 software and a modified Swanson method (Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semi-automated method for measuring brain infarct volume. J Cereb Blood Flow Metab. 1990; 10: 290-293). The amount of clot lysis was determined by comparing the radioactivity of residual thrombus in the brain with that of the initial clot.

[0567] 9.1 Limiting the duration of r-tPA-induced plasminogen activation reduces brain damage and bleeding

[0568] The potent, specific effects of TPAi-1 allowed the investigation of whether the persistence of r-tPA-induced plasminogen activation is detrimental during prolonged cerebral ischemia in a thromboembolic model with relevance for human stroke transformation. Mice were randomized to receive placebo, murine TPAi-1, chimeric TPAi-1, or humanized TPAi-1 (TPAi-1 RHP / RKA) 30 or 60 minutes after r-tPA bolus therapy, which was given 2.5 hours after middle cerebral artery thromboembolism. In these mice, bleeding after tail transection (under anesthesia) was monitored as an indicator of arterial and venous surgical bleeding associated with persistent plasminogen activation. Mice given TPAi-1 30 or 60 minutes after the r-tPA bolus showed a significant reduction in tail bleeding (P<0.05) compared to mice receiving placebo. Fig.36 Treatment with chimeric TPAi-1 or humanized TPAi-1 (TPAi-1RHP / RKA) also significantly reduced bleeding when compared with placebo ( Fig.36 ).

[0569] r-tPA treatment within 2.5 hours after stroke onset was associated with significant intracerebral hemorrhage ( Fig.37 However, in mice that received murine TPAi-1 30 or 60 minutes after the bolus injection of human TPA, the size of the hemisphere was significantly reduced (measured as a percentage of the hemisphere) relative to placebo-treated mice. Similarly, mice treated with chimeric TPAi-1 or humanized TPAi-1 (TPAi-1 RHP / RKA) also showed a significant reduction in intracerebral hemorrhage when treated 30 minutes after the bolus injection of human TPA ( Fig.37 ).

[0570] Murine TPAi-1 (m) as well as chimeric (c) and humanized Mab (h) (TPAi-1 RHP / RKA) reduced the amount of intracerebral hemorrhage.

[0571] Treatment with mTPAi-1 30 or 60 minutes after initial r-tPA therapy significantly reduced cerebral infarction when compared to control mice. Cerebral infarction was also significantly reduced when mice were treated with mtPAi-1 or htPAi-1 (TPAi-1 RHP / RKA) 30 minutes after initial tPA therapy. Fig.38 ).

[0572] 10.0. Binding of Chimeric TPAi-1, Humanized TPAi-1(RHP / RKA) and TPAi-1(RHP / RKA) Fab to TPA Mutant Tenecteplase

[0573] Binding activity to the TPA mutant tenecteplase (TNK) was measured by binding ELISA (2.4.1). The binding of the Fab (TPAi-1RHP / PKA Fab) to tenecteplase was compared with the binding of the chimeric mAb TPAi-1 and the humanized mAb TPAi-1RHP / RKA and the control Fab ( Figure 4 .3), both were found to bind to TNK with high affinity, albeit with similar valency effects as seen with binding to recombinant human TPA (see 5.3 and Fig.23 The EC of chimeric mAb TPAi-1, humanized mAb TPAi-1RHP / RKA and Fab (TPAi-1 RHP / PKAFab) 50 The binding concentrations were 0.762, 0.633 and 1.306 nM, respectively. No binding was detected for the control Fab.

[0574] All publications, patents and patent applications herein are incorporated herein by reference to the same extent as if each individual publication and patent application were specifically and solely incorporated by reference. The foregoing detailed description has been given only for clarity of understanding and no unnecessary limitations should be understood therefrom, as modifications will be apparent to those skilled in the art. It is not acknowledged that any information provided herein is prior art or is relevant to the invention currently claimed, or that any publication specifically or implicitly cited is prior art. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the invention belongs.

[0575] Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention which follow generally from the principles of the invention and include such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and as may be applicable to the essential features hereinbefore described and which fall within the scope of the appended claims.

[0576] Sequence Listing

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Claims

1. An antibody molecule that specifically binds to human tissue plasminogen activator (TPA) or a TPA mutant to inhibit the degradation of human fibrin clots, wherein the antibody has an IC 50 <5 nM subnanomolar affinity for inhibiting fibrin-dependent plasminogen activation, and wherein the TPA mutant is tenecteplase; wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein in the heavy chain variable domain, CDR1 is SEQ ID NO: 3, CDR2 is SEQ ID NO: 5, and CDR3 is SEQ ID NO: 7, and in the light chain variable domain, CDR1 is SEQ ID NO: 9, CDR2 is SEQ ID NO: 11, and CDR3 is SEQ ID NO: 13; wherein the antibody molecule is a humanized antibody or a fragment of a humanized antibody.

2. The antibody molecule according to claim 1, comprising a heavy chain variable domain selected from SEQ ID NOs: 14 to 28 and a light chain variable domain selected from SEQ ID NOs: 29 and 30.

3. The antibody molecule according to claim 2, comprising a heavy chain variable domain selected from SEQ ID NOs: 14 to 28, and a light chain variable domain of SEQ ID NO:

29.

4. The antibody molecule according to claim 2, comprising a heavy chain variable domain of SEQ ID NO: 14 and a light chain variable domain of SEQ ID No: 29, or a heavy chain variable domain of SEQ ID NO: 15 and a light chain variable domain of SEQ ID No: 29, or a heavy chain variable domain of SEQ ID NO: 14 and a light chain variable domain of SEQ ID No: 30, or a heavy chain variable domain of SEQ ID NO: 15 and a light chain variable domain of SEQ ID No:

30.

5. The antibody molecule of claim 1, wherein the humanized antibody or humanized antibody fragment is a Fab, Fab' or F(ab')2 fragment, or a single chain variable fragment (scFv).

6. The antibody molecule of claim 5, wherein the humanized antibody or humanized antibody fragment is a Fab, Fab' or F(ab')2 fragment.

7. The antibody molecule according to claim 5, which is a scFv, wherein the heavy chain variable domain and the light chain variable domain are connected to each other via a linker peptide selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO:

37.

8. The antibody molecule according to claim 7, comprising SEQ ID NO:38 or SEQ ID NO:

39.

9. The antibody molecule of claim 1, having a heavy chain comprising SEQ ID NO:40 or SEQ ID NO:41; and a light chain comprising SEQ ID NO:

42. 10 . The antibody molecule according to claim 5 , which is a Fab molecule having a Fd fragment comprising SEQ ID NO: 31 or SEQ ID NO: 32; and a light chain comprising SEQ ID NO:

33.

11. An antibody molecule according to any one of claims 1 to 10 for use as a medicament.

12. A pharmaceutical composition comprising the antibody molecule according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

13. The antibody molecule according to any one of claims 1 to 10, for use in treating or preventing TPA-induced bleeding.

14. The antibody molecule according to claim 13, for use in treating or preventing systemic bleeding after TPA treatment.

15. Use of the antibody molecule according to any one of claims 1 to 10 for the preparation of a medicament for treating or preventing TPA-induced bleeding.

16. Use of the antibody molecule according to any one of claims 1 to 10 for the preparation of a medicament for treating or preventing systemic bleeding after TPA treatment.

17. A method for preparing an antibody molecule according to any one of claims 1 to 10, include: (a) providing a host cell comprising one or more nucleic acids encoding the antibody molecule in functional connection with expression control sequences, (b) culturing the host cell, and (c) recovering the antibody molecules from the cell culture.

18. A kit comprising the antibody according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 12.

19. A kit comprising: (a) the antibody according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 12; (b) containers; and (c) Marker.

20. The kit according to claim 18 or 19, comprising the antibody according to any one of claims 1 to 10 and human tissue plasminogen activator (TPA) or a TPA mutant, wherein the TPA mutant is tenecteplase.

21. The kit according to claim 20, wherein human tissue plasminogen activator (TPA) or TPA mutant is tenecteplase ( TNK-tPA).

22. The kit according to claim 21, comprising: (a) the antibody according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 12; (b) A pharmaceutical composition comprising human tissue plasminogen activator (TPA) or a TPA mutant, wherein the TPA mutant is tenecteplase ( TNK-tPA); (c) containers; and (d) Label.

23. The kit according to claim 21 or 22, comprising: (a) a first pharmaceutical composition comprising human tissue plasminogen activator (TPA) or a TPA mutant, wherein the TPA mutant is tenecteplase ( TNK-tPA); (b) a second pharmaceutical composition comprising the antibody according to any one of claims 1 to 10; (c) instructions for separately administering the first and second pharmaceutical compositions to a subject, wherein the first and second pharmaceutical compositions are contained in separate containers and administering the second pharmaceutical composition to a subject in need of treatment or prevention of systemic bleeding following TPA treatment.

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