Chimeric molecules comprising an Anti-fibrin antibody and an antithrombotic molecule

Abstract

Described herein are chimeric molecules comprising (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more antithrombotic molecule(s) and uses thereof.

Description

[0001] Chimeric molecules comprising an anti-fibrin antibody and an antithrombotic molecule

[0002] FIELD OF THE INVENTION

[0003] The invention pertains to the field of medicine, in particular immunotherapy.

[0004] BACKGROUND OF THE INVENTION

[0005] Venous thromboembolism (VTE) is a common vascular disorder encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE). VTE is the third leading cardiovascular cause of death, behind stoke and myocardial infarction. The onset of VTE usually starts on venous valves in large veins mainly of the lower limbs due to an impairment of blood flow, linked to at least one of the three factors of Virchow's Triad: stasis, a hypercoagulable state and endothelial dysfunction or injury. VTE is a multifactorial disease including genetic and acquired risk factors, such as inflammatory diseases, a sedentary lifestyle, diabetes, obesity, and hormonal contraception. Venous thrombi can be characterized based on their composition, essentially rich in fibrin and red blood cells. When the thrombus occludes a vessel, it leads to ischemia and hypoxia of the downstream tissues, inducing necrosis. When a portion of a thrombus breaks off, it can result in PE: the dissident portion migrates towards the lungs, taking up residence in a pulmonary artery.

[0006] Antithrombotic drugs, such as anticoagulant and thrombolytic drugs are the cornerstone of thrombosis treatment. Anticoagulant drugs directly or indirectly inhibit thrombin generation, which impacts fibrin synthesis, increasing the clot permeability and fostering fibrinolysis. There are two different classes of anticoagulants: 1) parenteral and 2) oral anticoagulants. Parenteral anticoagulants can inhibit directly thrombin (such as Argatroban) or indirectly by enhancing the activity of antithrombin III (such as Heparin (UFH or LMWH), Fondaparinux, Danaparoid). Direct oral anticoagulants (DOACs) inhibit coagulation factors such as thrombin (Dabigatran) or factor Xa (Rivaroxaban). Vitamin K antagonists such as warfarin inhibit vitamin K, which impacts all vitamin K dependant coagulation factors. In the first three months of treatment, patients are mostly treated the first days with DOACs or heparins, followed by either vitamin K antagonists or DOACs. On the other hand, thrombolytics break up clots by converting plasminogen to plasmin to induce fibrinolysis. This allows blood and oxygen to reperfuse the area, limiting tissue damage. Thrombolytics are more effective if initiated as soon as possible after the event (such as a heart attack, stroke, or pulmonary embolism (PE)).

[0007] However, the most frequent complication of antithrombotic treatment is haemorrhage, which is associated with substantial morbidity and mortality for the patient, and is a significant financial burden on society. There is thus a need of developing a more efficient antithrombotic drug for acute settings with lower side effects, in particular a low bleeding risk, for proposing new safe treatments of thrombose associated disorders and diseases, such as PE or VTE.

[0008] The invention seeks to meet this need.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors developed a chimeric molecule that comprises an antibody targeting fibrin fused to an antithrombotic molecule. The chimeric molecule implies multiple advantages:

[0011] Targeting fibrin, which is not present physiologically in the blood, increases the specificity of the chimeric molecule for the thrombus. Targeting the thrombus allows to inject a small amount of the antithrombotic agent, which implies a low systemic dose in the circulation, while the agent concentration will increase in and around the thrombus, thanks to the targeting capacity of the chimeric molecule. Lowering the injected dose decreases antithrombotics-related side effects such as bleeding, thus reducing the risk of haemorrhage. When the antithrombotic agent is a thrombolytic molecule, in particular alteplase or a fragment thereof, its coupling to an anti-fibrin antibody, as proposed by the invention, should deteriorate its ability to pass through the blood-brain barrier (BBB), lowering its neurotoxicity in case of ischemic stroke. This could help to reduce the neurotoxicity of some thrombolytics for ischemic stroke patients.

[0012] Because of its large size over an antithrombotic used alone, the chimeric molecule is more stable in the body, allowing bolus injections instead of perfusions.

[0013] In a first aspect, the invention concerns a chimeric molecule comprising:

[0014] (i) an anti-fibrin antibody or an antigen binding fragment thereof; and

[0015] (ii) one or more antithrombotic molecule(s), said antithrombotic molecule being a protein, a polypeptide or a peptide; wherein the C-terminal end of the antithrombotic molecule is covalently linked to the N-terminal end of the heavy and / or light chain of the anti-fibrin antibody or antigen binding fragment thereof.

[0016] Preferably, the anti-fibrin antibody or an antigen binding fragment thereof comprises:

[0017] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0018] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion.

[0019] Particularly, the anti-fibrin antibody or an antigen binding fragment thereof comprises or consists of (a) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 85% sequence identity thereto and (b) a light chain variable domain (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 8 or a mutant thereof having at least 85% sequence identity thereto, .wherein the amino acid variations are outside of the CDRs.

[0020] Preferably, the antithrombotic molecule is an anticoagulant molecule, preferably selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any variant or fragment thereof.

[0021] Even more preferably, the antithrombotic molecule is hirudin, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having at least 85 % sequence identity thereto.

[0022] Alternatively, the antithrombotic molecule is a thrombolytic molecule, preferably a plasminogen activator selected from the group consisting of alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK- tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof.

[0023] Preferably, the thrombolytic molecule is a alteplase's serine protease comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof having at least 85 % sequence identity thereto.

[0024] Typically, the chimeric molecule comprises one or more antithrombotic molecule(s) that are anticoagulant molecules and / or thrombolytic molecules, and wherein: - the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules, preferably hirudin, and the N-terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several thrombolytic molecules, preferably alteplase or a fragment thereof, or vice-versa; or

[0025] - the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules, preferably hirudin, and to one or several thrombolytic molecules, preferably alteplase or a fragment thereof; or

[0026] - the N-terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules, preferably hirudin, and to one or several thrombolytic molecule(s), preferably alteplase or a fragment thereof.

[0027] Preferably, the antithrombotic molecule is covalently linked to the anti-fibrin antibody or antigen binding fragment thereof by a non-cleavable peptide linker, more preferably comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 27-44.

[0028] Additionally or alternatively, the chimeric molecule comprises one or more peptide signal, said peptide signal being in N-terminus of the antithrombotic molecule and comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 9 if the antithrombotic molecule is covalently linked to the N- terminus of the heavy chain variable domain or in SEQ ID NQ:10 if the antithrombotic molecule is covalently linker to the N-terminus of the light chain variable domain, or any variant of SEQ ID NO 9 or 10 comprising one, two or three amino acid modifications selected from substitution, addition or deletion or any combination thereof.

[0029] In some embodiments, the chimeric molecule of the invention comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting :

[0030] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more hirudin molecule, preferably comprising or consisting of SEQ ID NO: 15; wherein the C-terminal end of the hirudin molecule is covalently linked to the N-terminal end of the heavy chain of the anti-fi brin antibody or antigen binding fragment thereof, preferably by a peptide linker, said peptide linker preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 27-44, more preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30.

[0031] In some other embodiments, the chimeric molecule of the invention comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting :

[0032] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0033] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more molecule of alteplase's serine protease, preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 19; wherein the C-terminal end of the hirudin molecule is covalently linked to the N-terminal end of the light chain of the anti-fibrin antibody or antigen binding fragment thereof, preferably by a peptide linker, said peptide linker preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 27-44, more preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30.

[0034] In a second aspect, the invention also concerns an isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding the chimeric molecule of the invention. The invention also relates to a vector, comprising the nucleic acid or group of nucleic acid molecules of the invention. The invention also envisions a host cell, comprising the vector, the nucleic acid or group of nucleic acid molecules of the invention.

[0035] In a third aspect, the invention concerns a method for producing the chimeric molecule of the invention, comprising a step of culturing a host cell of the invention and optionally a step of isolating the chimeric molecule.

[0036] In a fourth aspect, the invention relates to a pharmaceutical composition comprising the chimeric molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell of the invention.

[0037] Typically, the invention concerns a pharmaceutical composition comprising the chimeric molecule of the invention, wherein the pharmaceutical composition comprises: a) at least two chimeric molecules wherein a first chimeric molecule comprises one or more anticoagulant molecules, preferably hirudin, fused to the anti-fibrin antibody or antigen binding fragment thereof, and a second chimeric molecule comprises a thrombolytic agent, preferably alteplase or a fragment thereof fused to the anti-fibrin antibody or antigen binding fragment thereof; and / or b) one or more additional therapeutic agent, preferably an antithrombotic agent selected from (i) a thrombolytic agent selected from the group consisting of tissue-type plasminogen activator (t- PA), alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof; (ii) an anticoagulant agent selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any variant or fragment thereof, Argatroban, Heparin, UFH, LMWH, Fondaparinux, Danaparoid, Direct oral anticoagulants (DOACs) such as Dabigatran and Rivaroxaban, and Vitamin K antagonists such as warfarin and (iii) any combination thereof.

[0038] In a fifth aspect, the invention concerns the pharmaceutical composition according to the invention for use as a medicament, in particular for use in the prevention or treatment of thrombosis or a disease or disorder associated with thrombosis.

[0039] It also relates to the use of the chimeric molecule or pharmaceutical composition of the invention in the manufacture of a medicament for treating thrombosis or a disease or disorder associated with thrombosis in a subject in need thereof. It also concerns a method for treating thrombosis or a disease or disorder associated with thrombosis in a subject in need thereof, comprising administering a therapeutic effective amount of the chimeric molecule or of the pharmaceutical composition according to the invention.

[0040] The thrombosis envisioned herein is preferably venous or arterial thrombosis. The disease or disorder associated with thrombosis is preferably selected from the group consisting of ischemic stroke, myocardial infarction, venous thromboembolism, deep vein thrombosis, pulmonary embolism and peripheral arterial occlusion.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] Definitions

[0043] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.

[0044] By "amino acid change" or "amino acid modification" is meant herein a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitution, insertion and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acids sequence, generally specified with the one letter codes for the amino acids. The first amino acid in the amino acids sequence (i.e., starting from the N terminus) should be considered as having position 1.

[0045] For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables: Table A - Amino Acid Residue

[0046] Table B - Alternative Conservative Amino Acid Residue Substitution Groups

[0047] Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues

[0048] As used herein, the "sequence identity" between two sequences is described by the parameter "sequence identity", "sequence similarity" or "sequence homology". For purposes of the present invention, the "percentage identity" between two sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods in the art, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle" for pairwise sequence alignment of proteins providing by EMBL-EBI and available on: www.ebi.ac. uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, for example using default settings: (I) Matrix : BLOSUM62, (ii) Gap open : 10, (iii) gap extend : 0.5, (iv) output format : pair, (v) end gap penalty : false, (vi) end gap open : 10, (vii) end gap extend : 0.5.

[0049] Alternatively, Sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with the default settings.

[0050] The terms "derive from" and "derived from" as used herein refers to a compound having a structure derived from the structure of a parent compound or protein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar properties, activities and utilities as the claimed compounds.

[0051] As used herein, the terms "pharmacokinetics" and "PK" are used interchangeably and refer to the fate of compounds, substances or drugs administered to a living organism. Pharmacokinetics particularly comprise the ADME or LADME scheme, which stands for Liberation (i.e., the release of a substance from a composition), Absorption (i.e., the entrance of the substance in blood circulation), Distribution (i.e., dispersion or dissemination of the substance through the body) Metabolism (i.e., transformation or degradation of the substance) and Excretion (i.e., the removal or clearance of the substance from the organism). The two phases of metabolism and excretion can also be grouped together under the title elimination. Different pharmacokinetics parameters can be monitored by the man skilled in the art, such as elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma cleared of the drug per unit time), Cmax (Maximum serum concentration), and Drug exposure (determined by Area under the curve, see Scheff et al, Pharm Res. 2011 May;28(5):1081-9) among others.

[0052] As used herein, the terms "disorder" or "disease" refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease e.g., an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to immune and / or inflammatory diseases that affect animals and / or humans, such as cancer.

[0053] As used herein, the term "isolated" indicates that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an "isolated" antibody is one which has been identified and separated and / or recovered from a component of its natural environment.

[0054] The term "and / or" as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.

[0055] The term "a" or "an" can refer to one of or a plurality of the elements it modifies (e.g., "a reagent" can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.

[0056] The term "about" as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to + / - 10% (e.g., + / - 0.5%, + / -1 %, + / -1 -5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / -9.5%). The use of the term "about" at the beginning of a string of values modifies each of the values (i.e., "about 1, 2 and 3" refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).

[0057] As used herein, the term "consist essentially of" refers to those elements required for a given embodiment. This term indicates the inclusion of any recited characteristics and permits the optional presence of elements that do not materially affect nor change the characteristics or functions of said embodiment. Preferably, in the context of a chimeric molecule, it refers to a molecule that comprises the recited elements (e.g., antigen binding domain(s), immunoactive molecule(s) and peptide I inker(s)), and optionally includes other elements that do not particular interfere with the structure or function of the molecule, such as peptide spacers.

[0058] The term "at least one" means "one or more" or "one or several". For instance, it refers to one, two, three or more.

[0059] Chimeric molecule

[0060] In a first aspect, the invention concerns a chimeric molecule comprising or consisting of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more anticoagulant or thrombolytic molecule, wherein the C-terminal end of the anticoagulant or thrombolytic molecule is covalently linked to the N-terminal end of the heavy or light chain of the anti-fibrin antibody or antigen binding fragment thereof.

[0061] As used herein, the terms "chimeric molecule", « multifunctional molecule » or "polyfunctional molecule" are interchangeable and refer to conjugated or chimeric drugs from two or more entities, molecules or drugs having different biological and / or pharmacological activities. It particularly refers to a fusion molecule in which an antibody or a fragment thereof and an antithrombotic molecule are linked to each other, preferably via an appropriate linker. Typically the chimeric molecule is a fusion protein.

[0062] Particularly, the chimeric molecule of the invention is an Antibody-drug conjugate (ADC).

[0063] In some aspects, the chimeric molecule is a bifunctional molecule, in which a first function is hold by the antithrombotic molecule and a second function is hold by the antigen binding domain(s) of the anti-fibrin antibody or antigen biding fragment thereof.

[0064] In some aspects, the chimeric molecule is a trifunctional molecule, in which a first function is hold by an anticoagulant molecule, a second function is hold by an thrombolytic molecule and a third function is hold by the antigen binding domain(s) of the anti-fibrin antibody or antigen biding fragment thereof.

[0065] In some aspects, the chimeric molecule comprises between 1 and 15, between 1 and 10, between 1 and 6 or between 1 and 4 antithrombotic molecule(s). Preferably, the chimeric molecule comprises at least 2 antithrombotic molecule(s). In some aspects, the chimeric molecule comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 antithrombotic molecule(s), preferably 2 antithrombotic molecule(s).

[0066] Preferably, when the chimeric molecule comprises more than two antithrombotic molecules, the antithrombotic molecules are typically arranged in series and are preferably linked together by a peptide linker. Alternatively, the chimeric molecule comprises a single antithrombotic molecule linked to each of the N-terminal end of the VH and / or VL domain(s) of the anti-fibrin antibody or antigen biding fragment thereof.

[0067] Preferably, when the chimeric molecule comprises more than one antithrombotic molecule linked to the VH and / or VL domain, the antithrombotic molecules are typically arranged in series and are preferably linked together by a peptide linker.

[0068] Additionally or alternatively, the one or more antithrombotic molecules are only linked to the N- terminus of the VH domain of the anti-fibrin antibody.

[0069] Alternatively, the one or more antithrombotic molecules are only linked to the N-terminus of the VH domain of the anti-fibrin antibody.

[0070] Alternatively, the one or more antithrombotic molecules are linked to the N-terminus of the VH and VL domain of the anti-fibrin antibody.

[0071] In some aspects, the chimeric molecule comprises two different antithrombotic molecules, in particular one or more anticoagulant molecule and one or more thrombolytic molecule. In some aspects, the chimeric molecule comprises between 1 and 15, between 1 and 10, between 1 and 6 or between 1 and 4 anticoagulant molecules and between 1 and 15, between 1 and 10, between 1 and 6 or between 1 and 4 thrombolytic molecule(s). Preferably, the chimeric molecule comprises at least 2 anticoagulant molecule(s) and at least 2 thrombolytic molecule(s), preferably one in N-terminus of each of the VH. In some aspects, the chimeric molecule comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 anticoagulant molecule(s) and 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 thrombolytic molecule(s).

[0072] The number of anticoagulant molecule(s) and thrombolytic molecule(s) may be the same or different.

[0073] In some aspects, the chimeric molecule is asymmetrical. For example, the chimeric molecule comprises a first VH or VL domain linked to one or more anticoagulant molecule(s) and a second VH or VL domain linked to one or more thrombolytic molecule(s).

[0074] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fi brin antibody or an antigen binding fragment thereof and (ii) one or more anticoagulant or thrombolytic molecule, wherein the N- terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules and the N-terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several thrombolytic molecule(s), or vice-versa. Preferably, the thrombolytic molecule(s) and the anticoagulant molecule(s) are linked to the anti-fibrin antibody by a peptide linker.

[0075] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more anticoagulant or thrombolytic molecule, wherein the N- terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules and to one or several thrombolytic molecule(s). The chimeric molecule may comprise from N-terminus to C-terminus : one or several anticoagulant molecules, one or several thrombolytic molecule(s) and the heavy chain variable domain (VH) of the anti-fibrin antibody or antigen binding fragment thereof. Alternatively, the chimeric molecule comprises from N-terminus to C-terminus : one or several thrombolytic molecule(s); one or several anticoagulant molecule(s), and heavy chain variable domain (VH) of the anti-fibrin antibody or antigen binding fragment thereof. Preferably, in these embodiments, the thrombolytic molecule(s) and the anticoagulant molecule(s) are linked together by a peptide linker. Additionally, the thrombolytic molecule(s) and the anticoagulant molecule(s) are preferably linked to the anti-fibrin antibody by a peptide linker.

[0076] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more anticoagulant or thrombolytic molecule, wherein the N- terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules and to one or several thrombolytic molecule(s). The chimeric molecule may comprise from N-terminus to C-terminus : one or several anticoagulant molecules, one or several thrombolytic molecule(s) and the light chain variable domain (VL) of the anti-fibrin antibody or antigen binding fragment thereof. Alternatively, the chimeric molecule comprises from N-terminus to C-terminus : one or several thrombolytic molecule(s); one or several anticoagulant molecules, and the light chain variable domain (VL) of the anti-fibrin antibody or antigen binding fragment thereof. Preferably, in these embodiments, the thrombolytic molecule(s) and the anticoagulant molecule(s) are linked together by a peptide linker. Additionally, the thrombolytic molecule(s) and the anticoagulant molecule(s) are preferably linked to the anti-fibrin antibody by a peptide linker.

[0077] The anti-fibrin antibody and antigen biding fragments and the antithrombotic molecules envisioned herein are more particularly disclosed here below, and apply to any one of the chimeric molecules described herein. Examples of specific chimeric molecules are also provided.

[0078] Anti-fibrin antibody and antigen binding fragment thereof

[0079] The chimeric molecule of the invention comprises an anti-fibrin antibody or an antigen binding fragment thereof.

[0080] As use herein, the terms "fibrin" or "Factor la" refers to a fibrous, non-globular protein involved in the clotting of blood. It is formed from fibrinogen, a soluble plasma protein, through the action of the enzyme thrombin. Fibrinogen, composed of fibrinogen alpha (FGA), fibrinogen beta (FGB) and fibrinogen gamma (FGG), polymerize to form an insoluble fibrin matrix. Fibrin creates a mesh-like structure that, along with platelets, forms a stable blood clot, helping to stop bleeding. Excessive, overexpression or inappropriate fibrin formation can lead to thrombosis, where clots form inside blood vessels. Preferably, the fibrin is human fibrin, in particular such as described under the Uniprot reference P02671 (Fibrinogen alpha chain) P02675 (Fibrinogen beta chain) and P02679 (Fibrinogen gamma chain). Alternatively, the fibrin is murine fibrin, in particular such as described under the Uniprot reference E9PV24 (Fibrinogen alpha chain), Q8K0E8 (Fibrinogen beta chain) and Q8VCM7 (Fibrinogen gamma chain). In a preferred aspect, the anti-fibrin antibody or antigen binding fragment thereof is able to bind / cross-react with human fibrin and murine fibrin.

[0081] With regard to the "binding" capacity of the antigen binding domain, the terms "bind" or "binding" refer to antibodies including antigen binding fragments thereof and derivatives that recognize and contact another peptide, polypeptide, protein or molecule (e.g., fibrin).

[0082] As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding domain. Unless specifically noted otherwise, the term "antibody" includes intact or full immunoglobulins and "antigen binding fragment" and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site (e.g., that binds to fibrin), including glycosylation variants of antibodies. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2) or subclass. Preferably, the antibody is or derive from an IgG. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CHI" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341- 447 according to the EU index as in Kabat.

[0083] An "antibody heavy chain" as used herein, refers to the larger of the two types of polypeptide chains present in antibody conformations. The CDRs of the antibody heavy chain are typically referred to as "HCDR1", "HCDR2" and "HCDR3". The framework regions of the antibody heavy chain are typically referred to as "HFR1", "HFR2", "HFR3" and "HFR4". An antibody heavy chain variable domain (VH) is typically structured as follows : HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0084] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations; K and X light chains refer to the two major antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as "LCDR1", "LCDR2" and "LCDR3". The framework regions of the antibody light chain are typically referred to as "LFR1", "LFR2", "LFR3" and "LFR4". An antibody light chain variable domain (VL) is typically structured as follows : LFR1- LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0085] In some aspects, the antigen binding domain comprised in the chimeric molecule according to the invention is an anti-fibrin antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-fibrin antibody or antigen binding fragment thereof.

[0086] As used herein, the term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., chimeric antibodies that contain minimal sequence derived from a non-human antibody). A "humanized form" of an antibody, e.g., a non- human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. Additional framework region modifications may be made within the human framework sequences. Preferably humanized antibody has a T20 humanness score greater than 80%, 85% or 90%. "Humanness" of an antibody can for example be measured using the T20 score analyzer to quantify the humanness of the variable region of antibodies as described in Gao S H, Huang K, Tu H, Adler A S. BMC Biotechnology. 2013: 13:55 or via a web-based tool to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com.

[0087] By "chimeric antibody" is meant an antibody made by combining genetic material from a nonhuman source, preferably such as a mouse, with genetic material from a human being. Such antibody derives from both human and non-human antibodies linked by a chimeric region. Chimeric antibodies generally comprise constant domains from human and variable domains (typically CDRs) from another mammalian species, reducing the risk of a reaction to foreign antibodies from a non-human animal when they are used in therapeutic treatments.

[0088] In some embodiments, the anti-fibrin antibody is a chimeric antibody comprising or consisting of a human or humanized backbone and murine variable domains, preferably murine CDRs. Preferably, the chimeric antibody comprises human IgGl constant domains and murine variable heavy chain and light chains.

[0089] In some aspects, the anti-fibrin antibody or antigen binding domain thereof is recombinant.

[0090] As used herein, the term "recombinant antibody" refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0091] Preferably, the anti-fibrin antibody is a monoclonal antibody.

[0092] In some aspects, the anti-fibrin antibody is selected from the group consisting of monoclonal antibodies 59D8, C22A, T2G1, MH1 and GC4, preferably such as described in W01994009034, Rosebrough et al. Radiology 1987; 162: 575-577, L.C. Knight, A.H. Maurer, LA. Ammar et al., Radiology, 1989; 173:163-169, L.C. Knight, A.H. Maurer, LA. Ammar et al., J. Nucl. Med. 1988; 29:494-502, S.S.L. Harwig, J.F. Harwig, R.E. Coleman and MJ. Welch, Thromb. Res., 1975; 6: 375-386, S. DeNardo, H. Bogren and G. DeNardo, Am. J. Roentgenol. 1985; 145: 1045-1052, A. Alavi et al. Radiology, 1990; 175:79-85, P. deFaucal et al. J. Nucl. Med. 1991; 32:785-791, the contents of all of which are incorporated herein by reference. Preferably, the chimeric molecule comprises anti-fibrin antibody that is 59D8 or an antigen binding fragment thereof. Said antibody is particularly described in Hui, KY., et al. (1983). Science. 222 (4628); 1129-1132 and in Naik et al, Journal of Controlled Release 101, 20025, 35-45, the content of which is incorporated herein by reference (in particular the antibody sequences).

[0093] In some preferred aspects, the chimeric molecule comprises an anti-fibrin antibody that is a humanized version of the antibody 59D8 or an antigen binding fragment thereof.

[0094] In some aspects, the anti-fibrin antibody or an antigen binding fragment thereof comprises:

[0095] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, or a variant thereof having at least 90%, 95%, 97% or 99% sequence identity thereto, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 or a variant thereof having at least 90%, 95%, 97% or 99% sequence identity thereto and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 or a variant thereof having at least 90%, 95%, 97% or 99% sequence identity thereto; and

[0096] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, or a variant thereof having at least 90%, 95%, 97% or 99% sequence identity thereto, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 or a variant thereof having at least 90%, 95%, 97% or 99% sequence identity thereto, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 or a variant thereof having at least 90%, 95%, 97% or 99% sequence identity thereto.

[0097] Preferably, the CDRs have been determined by the IMGT method.

[0098] Preferably, the amino acid difference(s) / mutation(s) are outside of the CDRs (i.e., are in the FRs).

[0099] In some aspects, the anti-fibrin antibody or an antigen binding fragment thereof comprises:

[0100] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0101] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion.

[0102] In some aspects, the anti-fibrin antibody or an antigen binding fragment thereof comprises:

[0103] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3, and

[0104] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion.

[0105] In some aspects the anti-fibrin antibody or antigen binding fragment comprises or consists of (a) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto and (b) a light chain variable domain (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto. Preferably, the amino acid differences / mutations are outside of the CDRs (i.e., are in the FRs). In another aspect, the a nti-fi brin antibody or antigen binding fragment comprises or consists essentially of:

[0106] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, said modifications being in the framework region(s);

[0107] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 8 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, said modifications being in the framework region(s).

[0108] In some aspects the anti-fibrin antibody or antigen binding fragment comprises or consists of (a) a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto and (b) a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto. Preferably, the amino acid differences / mutations are outside of the CDRs (i.e., are in the FRs).

[0109] In another aspect, the anti-fibrin antibody or antigen binding fragment comprises or consists essentially of:

[0110] (a) a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 11; or a variant thereof having one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, said modifications being in the framework region(s);

[0111] (b) a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 12, or a variant thereof having one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, said modifications being in the framework region(s).

[0112] Is some aspects, the chimeric molecule comprises an antigen-binding domain of the anti-fibrin antibody described herein.

[0113] As used herein, an "antibody binding fragment", "antigen-binding fragment" or "antigen-binding domain" of an antibody means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for a particular antigen (e.g., fibrin), such fragment preferably exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding full / four- chain antibody. Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof.

[0114] Preferably, the antigen binding domain is selected from the list consisting of Fab, Fab', F(ab')2, Fv, scFv, scFab and CrossMAb. Preferably, the antigen binding domain is selected from the list consisting of Fab, Fab', F(ab')2, Fv, scFab and CrossMAb. Particularly, the antigen binding domain is not a scFv.

[0115] In some aspects, the antigen binding domain is derived from an antibody selected from the group consisting of monoclonal antibodies 59D8, C22A,T2G1, MHl and GC4, preferably such as described in W01994009034.

[0116] Preferably, the anti-fibrin antibody is a full length antibody comprising antigen binding domains or CDRs of an antibody selected from the group consisting of monoclonal antibodies 59D8, C22A,T2G1, MHl and GC4, preferably such as described in W01994009034.

[0117] In some aspects, the chimeric molecule comprises a Fc domain. The Fc domain can be from a heavy chain constant domain of an immunoglobulin heavy chain, for example, IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the chimeric molecule and / or the anti-fibrin antibody comprises an IgGl heavy chain, preferably an IgGl Fc domain.

[0118] In some aspects, the chimeric molecule comprises a human or humanized Fc domain, preferably a humanized IgGl Fc domain, more preferably a human IgGl Fc domain.

[0119] As used herein, the terms "fragment crystallizable region" "Fc region" or "Fc domain" are interchangeable and refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors. The Fc region or domain is typically composed of two domains, optionally identical, derived from the second and third constant domains of the antibody's two heavy chains (i.e., CH2 and CH3 domains). Portion of the Fc domain refers to the CH2 and / or CH3 domain(s). Optionally, the Fc region or domain may optionally comprise all or a portion of the hinge region between CHI and CH2. Accordingly, the Fc domain may comprise a hinge, a CH2 domain and a CH3 domain.

[0120] As envisioned herein, the Fc domain preferably comprises a first Fc chain and a second Fc chain that are complementary and are able to dimerize. In certain embodiments, the first Fc chain comprises an amino acid sequence that differs from that of the second Fc chain in one or more amino acid addition, deletion or substitution. Such Fc first and second Fc chain form a "heterodimeric Fc". In other embodiments, the first and second Fc chains comprise the same amino acid sequence and form a "homodimeric Fc".

[0121] Heterodimeric Fc domains can be made by altering the amino acid sequence of each monomer. The heterodimeric Fc domains rely on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and / or allow for ease of purification of heterodimers over the homodimers. Heterodimeric Fc domain can include steric variants (e.g., the "knobs and holes" or "skew" variants described below and the "charge pairs" variants described below) as well as "pi variants", which allows purification of homodimers away from heterodimers. WO2014 / 145806 discloses useful mechanisms for heterodimerization include "knobs and holes", "electrostatic steering" or "charge pairs", pi variants, and general additional Fc variants. See also, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, Merchant et al., Nature Biotech. 16:677 (1998), Gunasekaran et al., J. Biol. Chem. 285(25): 19637 (2010), and US 2012 / 0149876 hereby incorporated by reference in its entirety. In a preferred aspect, the Fc « hole » comprises the substitutions T366S, L368A, Y407V and Y349C and the Fc "knob" chain comprises the substitutions T366W and S354C.

[0122] As envisioned herein, the use of heterodimeric Fc domain allows the construction of a chimeric molecule that comprises an anti-fibrin antibody that is not symmetrical. In such embodiments, the N- terminus of the first VH (or VL) is linked to one or several anticoagulant molecules, and the N-terminus of the second VH (or VL) is linked to one or several thrombolytic molecules. Alternatively, the N- terminus of the first VH (or VL) is linked to one or several antithrombotic molecules, and the N-terminus of the second VH (or VL) is devoid of antithrombotic molecules.

[0123] Antithrombic agent

[0124] The chimeric molecules of the invention comprise one or more antithrombic molecule(s).

[0125] As used herein, the terms "antithrombotic molecule", "antithrombotic agent", «anti-thrombic molecule», "anti-thrombi molecule", "anti-thrombus molecule" and « anti-blood clotting molecule » are used interchangeably and refer to a drug that prevents or reduces the formation of blood clots (thrombi) and / or prevents or reduces the risk of thrombosis. As used herein, the antithrombotic molecule is a peptide, protein or polypeptide, so that the chimeric molecule typically is or comprises a fusion protein.

[0126] In some aspects, the term "antithrombotic molecule" encompassed any analogue, mutant or fragment of the antithrombic molecule(s) disclosed herein, said analogue, mutant or fragment retaining the antithrombotic activity of the full length, native or wild type antithrombic molecule(s). Preferably, the analogue, fragment or mutant retain the antithrombotic activity by at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% in comparison to the full length, native or wild type antithrombic molecule. Antithrombotic activity of a molecule can be assessed by any method known in the art, such as Global thrombosis test (GTT), coagulation tests including measurement of prothrombin time (PT) and / or activated partial thromboplastin time (aPTT). The aPTT method is particularly used, as described in the Example section of this application.

[0127] In some aspects, the one or more antithrombotic molecule is a recombinant protein, peptide or polypeptide. The term "recombinant" as used with respect to a peptide, protein or polypeptide means a peptide, protein or polypeptide produced by expression of a recombinant polynucleotide, in particular through recombinant DNA technology rather than being directly isolated from a natural source. "Recombinant" when referring to a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. It typically refers to a peptide, protein, or polypeptide that is produced by genetically engineered organisms in which the DNA coding for the desired molecule has been introduced using recombinant DNA technology. This process typically allows for the large-scale production of these molecules with high purity and consistency.

[0128] In some aspects, the antithrombotic molecule is an anticoagulant molecule and / or a thrombolytic molecule. molecules

[0129] In some aspects, the antithrombotic molecule is an anticoagulant molecule. As used herein, the term "anticoagulant molecule" refers to any polypeptide, protein or peptide, whether naturally occurring, recombinant or synthetic, that inhibits the coagulation or clotting of blood. Anticoagulant molecules work through various mechanisms to prevent the formation, extension, or propagation of blood clots (thrombi).

[0130] In some aspects, the anticoagulant molecule is selected from the group consisting of direct thrombin inhibitors, direct factor Xa inhibitors, vitamin K antagonists and any variants or fragments thereof.

[0131] Preferably, the anticoagulant molecule is a direct thrombin inhibitor. As used herein, a "direct thrombin inhibitor" (DTI) is a type of anticoagulant molecule that directly binds to and inhibits the activity of thrombin (e.g., factor Ila). By blocking thrombin's ability to convert fibrinogen to fibrin, DTIs effectively prevent the formation of blood clots.

[0132] In some preferred aspects, the antithrombotic molecule is a direct thrombin inhibitor selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any combination thereof. In some preferred aspects, the antithrombotic molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15-18 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0133] Preferably, the antithrombotic molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15-18 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination thereof. In some particular aspects, the chimeric molecule comprises a mutant or fragment of hirudin, bivalirudin, lepirudin and desirudin. Preferably, such mutant or fragment is function conservative, i.e., has similar or identical biological functions / properties, in particular anticoagulant properties.

[0134] In some aspects, the chimeric molecule of the invention comprises an analogue, mutant or a fragment of any of the anticoagulant molecule(s) disclosed herein, preferably hirudin, said an analogue, mutant or fragment retaining the anticoagulant activity of the full length, native or wild type anticoagulant molecule(s), preferably of a wild-type hirudin. Preferably, the fragment or mutant retain the anticoagulant activity by at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% in comparison to the full length, native or wild type anticoagulant molecule(s). Typically, the anticoagulant activity of a molecule can be assessed / measured by any techniques known to the man skilled in the art, such as coagulation time assays such as Activated Partial Thromboplastin Time (aPTT) assay, Prothrombin Time (PT) assay or by the Lee-White method, (see for example as described in Kamide et al., Polymer Journal, Vol. 15, No. 4, pp 309-321 (1983)).

[0135] In some preferred aspects, the antithrombotic molecule is hirudin or any analogue, variant or fragment thereof.

[0136] As used herein, the term "hirudin" refers to an anticoagulant peptide or protein that has been described to be one of the most potent and specific natural inhibitors of thrombin. Hirudin can be extracted from leeches such as Hirudo medicinalis, typically according to Markwardt's method (Z. Phys. U. Chem. 388, 147, 1957), or produced by recombinant techniques. In some preferred aspects, the antithrombotic molecule is a recombinant hirudin.

[0137] In some aspects, the chimeric molecule comprises one or more molecule of hirudin, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0138] Alternatively, the chimeric molecule comprises one or more molecule of hirudin, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination thereof. Preferably, the chimeric molecule comprises one or more hirudin molecule comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 15.

[0139] In some particular aspects, the term "hirudin" is understood to comprise hirudin variants or mutants, in particular such as HV1, HV2 and HV3 (or PA) or desulphatohirudin variant, for example as described in EP0225633, EP0367713 WO 88 / 03493, WO199119734, WQ9201712, WO198504418, WQ20210965, US5650301, and in Scharf et al., (1989) FEBS Lett 255: 105, the content of which are incorporated herein by reference. Typically, the variants differ from each other by a number of amino acids, for example at the N-terminal sequence which is Val-Val-Tyr for HV1, lle-Thr-Tyr for HV2 and PA and Thr- Tyr for "des-(Val)2-hirudin". Based on NMR studies, HV1 is composed of an N-terminal core domain with a protruding "finger" (residues 31-36), and an acidic terminal loop (Clore et al., EMBO Journal 6, 529, 1987). Hirudin variants typically have an accumulation of hydrophobic amino acids at the N- terminus and an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tyr 63) present as sulphate monoester and three disulphide bridges.

[0140] In some aspects, the hirudin variant is as described in Zhang and Lan, Biotechnol Genet Eng Rev. 2018 Oct;34(2):261-280, the content of which is incorporated herein by reference.

[0141] Preferably, hirudin is hirudin HV2.

[0142] Analogues or derivatives of hirudin have been produced by chemical modification or through recombinant DNA techniques. For example, European Patent Publication 273,800 discloses a hirudin analogue having the putative native asparagine in position 4 substituted with lysine, arginine or histidine and the native tyrosine in position 63 substituted with glutamine or asparagine.

[0143] Regarding hirudin fragments, U.S. Patent No. 4,767,742 discloses hirudin shortened at the aminoterminus by up to two amino acids and at the carboxy-terminus by up to 17 amino acids, as well as desulfated derivatives of hirudin. Hirudin fragments typically include Hirudin Fragment 55-65 and Hirudin Fragment 54-65.

[0144] In some preferred aspects, the antithrombotic molecule is bivalirudin or any analogue, variant or fragment thereof.

[0145] In some aspects, the chimeric molecule comprises one or more molecule of bivalirudin, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0146] Preferably, the chimeric molecule comprises one or more molecule of bivalirudin, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination thereof. Preferably, the chimeric molecule comprises one or more bivalirudin molecule comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 16.

[0147] In some preferred aspects, the antithrombotic molecule is lepirudin or any analogue, variant or fragment thereof. In some aspects, the chimeric molecule comprises one or more molecule of lepirudin comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0148] Alternatively, the chimeric molecule comprises one or more molecule of lepirudin comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination thereof. Preferably, the chimeric molecule comprises one or more lepirudin molecule comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 17.

[0149] In some preferred aspects, the antithrombotic molecule is desirudin or any analogue, variant or fragment thereof.

[0150] In some aspects, the chimeric molecule comprises one or more molecule of desirudin comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0151] Alternatively, the chimeric molecule comprises one or more molecule of desirudin comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination thereof. Preferably, the chimeric molecule comprises one or more desirudin molecule comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 18.

[0152] Thrombolytic molecules

[0153] In some aspects, the antithrombotic molecule is a thrombolytic molecule. As used herein, the term "thrombolytic molecule" refers to any polypeptide, protein or peptide, whether naturally occurring, recombinant or synthetic, that can break down or dissolve fibrin meshwork and / or blood clots (thrombi). These molecules typically facilitate the conversion of plasminogen to plasmin.

[0154] Preferably, the thrombolytic molecule is a plasminogen activator (PA), particularly a direct or indirect plasminogen activator. As used herein, the term "plasminogen activator", "PA", "tissue-type plasminogen activator" and "t-pa" are used interchangeably and refers to a peptide, protein or polypeptide that is responsible for the activation of plasminogen to plasmin. Typically, plasminogen activators (PAs) comprises or consists of proteases, especially serine proteases, that convert plasminogen to plasmin. In some aspects, the thrombolytic molecule comprises or consists of a serine protease. Preferably, the thrombolytic molecule is a plasminogen activator selected from the group consisting of alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof.

[0155] In some aspects the thrombolytic molecule is a recombinant plasminogen activator.

[0156] As used herein, the terms "recombinant plasminogen activator", "recombinant tissue-type plasminogen activator" and "r-PA" are used interchangeably and refer to a plasminogen activator produced by recombinant techniques. Recombinant proteins are artificially engineered or synthesized proteins. Recombinant proteins can have the same amino acid sequence as the naturally occurring wild-type protein, or have modifications introduced to alter the sequence for desired characteristics such as solubility, stability, increased efficiency, increased enzymatic activity, reduced toxicity or higher production yields compared to the native or wild type protein. Recombinant protein production is well described in the art, such as in Tripathi NK, Shrivastava A. Front Bioeng Biotechnol. 2019 Dec 20;7:420.

[0157] In some aspects, the recombinant plasminogen activator has an increased resistance towards plasminogen activator inhibitors such as PAI-1.

[0158] Preferably, the recombinant tissue plasminogen activator is selected from the group consisting of alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof.

[0159] In some aspects, the antithrombotic molecule is alteplase.

[0160] Preferably, the chimeric molecule of the invention comprises one or more molecule of alteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 20 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0161] Alternatively, the chimeric molecule of the invention comprises one or more molecule of alteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 20 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0162] In some aspects, the antithrombotic molecule is reteplase.

[0163] Preferably, the chimeric molecule of the invention comprises one or more molecule of reteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 21 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0164] Alternatively, the chimeric molecule of the invention comprises one or more molecule of reteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 21 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0165] In some aspects, the antithrombotic molecule is tenecteplase.

[0166] Preferably, the chimeric molecule of the invention comprises one or more molecule of tenecteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 22 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0167] Alternatively, the chimeric molecule of the invention comprises one or more molecule of tenecteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 22 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0168] In some aspects, the antithrombotic molecule is anistreplase.

[0169] Preferably, the chimeric molecule of the invention comprises one or more molecule of anistreplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0170] Alternatively, the chimeric molecule of the invention comprises one or more molecule of anistreplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0171] In some aspects, the antithrombotic molecule is desmoteplase.

[0172] Preferably, the chimeric molecule of the invention comprises one or more molecule of desmoteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 26 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0173] Alternatively, the chimeric molecule of the invention comprises one or more molecule of desmoteplase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 26 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0174] In some aspects, the antithrombotic molecule is a urokinase.

[0175] Preferably, the chimeric molecule of the invention comprises one or more molecule of urokinase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto. T1

[0176] Alternatively, the chimeric molecule of the invention comprises one or more molecule of urokinase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0177] In some aspects, the antithrombotic molecule is a streptokinase

[0178] Preferably, the chimeric molecule of the invention comprises one or more molecule of streptokinase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 24 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0179] Alternatively, the chimeric molecule of the invention comprises one or more molecule of streptokinase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 24 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0180] In some aspects, the antithrombotic molecule is a staphylokinase.

[0181] Preferably, the chimeric molecule of the invention comprises one or more molecule of staphylokinase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 51 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0182] Alternatively, the chimeric molecule of the invention comprises one or more molecule of streptokinase comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 51 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0183] In some aspects, the thrombolytic molecule is selected from the group consisting of tissue-type plasminogen activator (t-PA), alteplase (rt-PA), reteplase (r-PA), tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any combination thereof.

[0184] In some aspects, the chimeric molecule comprises one or more antithrombotic molecule comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 20-26 and 51 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0185] In some aspects, the chimeric molecule comprises one or more antithrombotic molecule comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 20-26 and 51 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0186] In some particular aspects, the chimeric molecule comprises a mutant or fragment of a plasminogen activator. The term "plasminogen activator" is understood to comprise t-pa variants or mutants, in particular having one or more amino acid mutations, preferably substitution(s) at amino acid position(s) 117, 146, 184 and / or 448, for example such as described in WO8904368 and CN107760660, the content of which are incorporated herein by reference. In some aspects, the t-PA molecule is a t-PA fragment for example such as disclosed in EP0234051, the content of which being incorporated by reference. Preferably, the t-PA fragment comprises or consists essentially of the proteolytic portion of the t-PA full length molecule, preferably of the kringle 2 and the proteolytic portion of the t-PA full length molecule.

[0187] Preferably, the plasminogen activator mutant or fragment is function conservative, i.e., has similar or identical biological functions / properties in comparison to the native or wild-type protein, in particular proteolytic and / or thrombolytic properties.

[0188] In particular, the chimeric molecule of the invention comprises a mutant or a fragment of any of the thrombolytic molecules disclosed herein, preferably a PA, said mutant or fragment retaining the thrombolytic activity of the full length, native or wild type thrombolytic molecule(s), preferably of a wild-type PA. Preferably, the fragment or mutant retain the thrombolytic activity by at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% in comparison to the full length, native or wild type thrombolytic molecule(s).

[0189] In particular, the chimeric molecule of the invention comprises a mutant or a fragment of any of the thrombolytic molecule(s) disclosed herein, preferably a PA, said mutant or fragment retaining the proteolytic activity of the full length, native or wild type thrombolytic molecule(s), preferably of a wildtype PA. Preferably, the fragment or mutant retain the proteolytic activity by at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% in comparison to the full length, native or wild type thrombolytic molecule(s).

[0190] Typically, the thrombolytic or proteolytic activity of a molecule can be assessed / measured by any techniques known to the man skilled in the art, such as Spectrophotometric Analysis of Thrombolytic Activity (SATA) Assay, fibrin plate assay, thromboelastography, fluorogenic assay, microfluidic models or clot lysis assay.

[0191] In some particular aspect, the thrombolytic molecule is a fragment of a plasminogen activator, said fragment comprising or consisting of a proteolytic fragment (e.g., comprising or consisting of an amino acid sequence encoding a protease domain, preferably a serine protease domain (SPD)). Preferably, said fragment comprises or consists of the protease catalytic domain of the native or wild-type protein.

[0192] Preferably, the fragment is a proteolytic fragment of alteplase (rt-PA), reteplase (r-PA), tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa) or staphylokinase, preferably of alteplase (rt-PA), reteplase (r-PA), tenecteplase (TNK-tPA), anistreplase or desmoteplase. In some aspects, the thrombolytic molecule is a protease or serine protease fragment / domain of a plasminogen activator, preferably comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 46-50 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto. In some aspects, the thrombolytic molecule is a protease or serine protease fragment of a plasminogen activator comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 46-50 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof.

[0193] In some preferred aspects, the thrombolytic molecule is an alteplase fragment, preferably comprising or consisting of alteplase's serine protease domain.

[0194] Preferably, the chimeric molecule of the invention comprises an alteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 19 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0195] Preferably, the chimeric molecule of the invention comprises an alteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 19 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof.

[0196] In some aspects, the thrombolytic molecule is a reteplase fragment, preferably comprising or consisting of reteplase's serine protease domain.

[0197] Preferably, the chimeric molecule of the invention comprises a reteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 46 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0198] Preferably, the chimeric molecule of the invention comprises a reteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 46 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof.

[0199] In some aspects, the thrombolytic molecule is a tenecteplase fragment, preferably comprising or consisting of tenecteplase's serine protease domain.

[0200] Preferably, the chimeric molecule of the invention comprises a tenecteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 47 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto. Preferably, the chimeric molecule of the invention comprises a tenecteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 47 or a variant thereof comprising 1,

[0201] 1, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof.

[0202] In some aspects, the thrombolytic molecule is a anistreplase fragment, preferably comprising or consisting of anistreplase's serine protease domain.

[0203] Preferably, the chimeric molecule of the invention comprises anistreplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 48 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0204] Preferably, the chimeric molecule of the invention comprises anistreplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 48 or a variant thereof comprising 1,

[0205] 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof.

[0206] In some aspects, the thrombolytic molecule is a desmoteplase fragment, preferably comprising or consisting of desmoteplase's serine protease domain.

[0207] Preferably, the chimeric molecule of the invention comprises a desmoteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 49 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0208] Preferably, the chimeric molecule of the invention comprises a desmoteplase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 49 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof.

[0209] In some aspects, the thrombolytic molecule is an urokinase fragment, preferably comprising or consisting of urokinase's serine protease domain.

[0210] Preferably, the chimeric molecule of the invention comprises a urokinase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 50 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0211] Preferably, the chimeric molecule of the invention comprises a urokinase fragment comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 50 or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, deletion and addition and any combination(s) thereof. In some aspects, the antithrombotic molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 19-26 and 46-51 or a variant thereof having at least 85 %, 90%, 95%, 97% or 99% sequence identity thereto.

[0212] In some aspects, the antithrombotic molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 19-26 and 46-51 or a variant thereof having 1, 2, 3, 4 or 5 amino acid modifications selected from the group consisting of substitution, addition, deletion and any combination thereof.

[0213] Peptide Linkers

[0214] In the chimeric molecules provided herein, the antithrombotic molecule is preferably covalently linked to the anti-fibrin antibody or fragment thereof by an appropriate peptide linker.

[0215] As used herein the terms "covalently linked", "coupled", "conjugated", "covalently bound" are used interchangeably and refer to a bond that involves the sharing of electrons to form electron pairs between atoms between two moieties, e.g., an antithrombotic molecule and a peptide linker. In the context of the invention, covalent links are not reversible / not cleavable.

[0216] Preferably, the peptide linker is not cleavable or does not contain any protease cleavage site. As used herein, the term "non-cleavable peptide linker" or "uncleavable linker" refers to an amino acid sequence that is designed to remain intact without being cleaved or broken down under certain conditions, such as physiological conditions (e.g. pH), enzymatic or chemical cleavage. Such peptide linkers typically ensure that the linked molecules (e.g., an antibody or an antigen binding fragment thereof and an antithrombotic agent such as disclosed herein) remain connected throughout the intended application, whether it is in vivo or in vitro.

[0217] Preferably, the one or more peptide linker(s) is a flexible linker or a rigid linker.

[0218] As used herein, a "flexible linker" or "flexible peptide linker" refers to a sequence of amino acids or nucleotides that connect two functional domains or regions within a biomolecule. This linker is typically characterized by its structural flexibility, allowing the connected domains to fold and / or move independently of each other, facilitating conformational changes or interactions with other molecules. A flexible linker can enable the individual domains of fusion construct to maintain their structural and functional integrity. The purpose of incorporating a flexible linker in a biomolecular structure is generally to provide freedom of movement and spatial separation between the molecules.

[0219] Examples of flexible linkers are Glycine-Serine linker, Glycine-Proline linker, Proline-Rich linker, (GGGS)n linker (e.g., SEQ ID NO: 27), (GGGGS)n linker (e.g., SEQ ID NO: 28), wherein n indicates the number of repeats of the motif and is an integer selected from 1-10, GEGKSSGSGSESKAS linker (SEQ ID NO: 34), and SPNSASHSGSAPQTSSAPGSQ linker ((SEQ ID NO: 35). In some embodiments, the linker has an amino acid sequence comprising or consisting of a sequence selected from the group consisting of (GGGGS)3(SEQ ID NO: 30), (GGGGS)4(SEQ ID NO: 31), (GGGGS)2(SEQ ID NO: 29), GGGGS (SEQ ID NO: 33) GEGKSSGSGSESKAS(SEQ ID NO: 34) and SPNSASHSGSAPQTSSAPGSQ (SEQ ID NO: 35) linker. Preferably, the flexible linker comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 30.

[0220] In comparison to a flexible linker, a "rigid linker' or "rigid peptide linker" refers to a sequence of amino acids or nucleotides that provides limited flexibility and maintains a more rigid structure. The purpose of a rigid linker is often to restrict the movement or maintain a specific orientation between two functional domains or regions within a biomolecule. Rigid linkers are commonly used in protein engineering to influence the relative positioning of connected domains.

[0221] Examples of rigid linkers are a-Helical Linkers such as (EAAAK)n linker (e.g., SEQ ID NO: 36), preferably (EAAAR)n linker (e.g., SEQ ID NO: 38), A(EAAAK)n (e.g., SEQ ID NO: 39), and coiled-coil linker such as (ALA)n or (AALAA)n (e.g., SEQ ID NO: 40), -Sheet linkers such as (GAGAGA)n (e.g., SEQ ID NO: 41), KLAKLAKKLAKLAK (e.g., SEQ ID NO: 42) and AEAAAKEAAAKA (e.g., SEQ ID NO: 43), (APPPAP)n (e.g., SEQ ID NO: 44), wherein n indicates the number of repeats of the motif and is an integer selected from 1- 10. Preferably, the rigid linker comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 37.

[0222] In some aspects, the antithrombotic molecule is covalently linked to the anti-fi brin antibody or antigen binding fragment thereof by a non-cleavable peptide linker, preferably comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 27-44.

[0223] Preferably, the antithrombotic molecule is covalently linked to the anti-fibrin antibody or antigen binding fragment thereof by a non-cleavable peptide linker comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30 or 37, preferably is SEQ ID NO: 30.

[0224] Optionally, the linker may be short or long in term of nucleic acid or amino acid sequence. Preferably, the linker has 3-30 nucleic acids or amino acids residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acid residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 nucleic acid residues.

[0225] In some aspects, the chimeric molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 peptide linker(s), typically depending on the number of antithrombotic molecules linked to the anti-fibrin antibody or antigen binding fragment thereof. The peptide linkers can be identical or different. For example, one or more peptide linker can be a rigid linker and one or more peptide linker can be a flexible linker. Alternatively, the one or more peptide linker can be flexible but of different sequence or length. Typically, the peptide linkers may have the same length or they can have a length slightly different, for instance having a difference in length of at most 10 to 20%, preferably 10% to 15%, in number of amino acids.

[0226] In some aspects, the chimeric molecule comprises one or more peptide linker(s) that link i) the antithrombotic molecule(s) to the anti-fibrin antibody or antigen binding fragment thereof and / or ii) the antithrombotic molecule(s) with each other.

[0227] The peptide linker between i) the antithrombotic molecule(s) and the anti-fibrin antibody or antigen binding fragment and ii) the antithrombotic molecules can be the same or different.

[0228] In some aspects, the chimeric molecule comprises a peptide signal, preferably in N-terminus of the heavy chain or light chain of the anti-fibrin antibody or antigen binding fragment thereof.

[0229] Peptide signal for enhancing / optimizing antibodies production are described in the art, such as in Haryadi et al., PLoS One. 2015 Feb 23;10(2):e0116878 and Ling et al., Front. Immunol., Sec. B Cell Biology, Volume 11 - 2020., the content (in particular the peptide sequences) of which is incorporated herein by reference.

[0230] Preferably, the peptide signal in N-terminus of the heavy chain comprises or consists of the amino acid sequence as set forth is SEQ ID NO : 9 or any variant thereof comprising one, two or three amino acid modifications selected from substitution, addition or deletion or any combination thereof.

[0231] Preferably, the peptide signal in N-terminus of the light chain comprises or consists of the amino acid sequence as set forth is SEQ ID NO : 10 or any variant thereof comprising one, two or three amino acid modifications selected from substitution, addition or deletion or any combination thereof.

[0232] Preferably, the peptide signal linked in N-terminus of the antithrombotic molecule comprises or consists of the amino acid sequence as set forth is SEQ ID NO : 9 or 10, depending on the chain of the antibody to which the antithrombotic molecule is connected (i.e., SEQ ID NO:9 if the antithrombotic molecule is in N-terminus of the VH, SEQ ID NQ:10 if the antithrombotic molecule is in N-terminus of the VL), or any variant thereof comprising one, two or three amino acid modifications selected from substitution, addition or deletion or any combination thereof.

[0233] In some embodiments, the chimeric molecule comprises one or more peptide signal, said peptide signal being in N-terminus of the antithrombotic molecule and comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 9 if the antithrombotic molecule is covalently linked to the N- terminus of the heavy chain variable domain or in SEQ ID NO:10 if the antithrombotic molecule is covalently linker to the N-terminus of the light chain variable domain, or any variant of SEQ ID NO 9 or 10 comprising one, two or three amino acid modifications selected from substitution, addition or deletion or any combination thereof.

[0234] Typically, the chimeric molecule comprises 1, 2, 3 or 4 peptide signals. Preferably, the chimeric molecule comprises a peptide signal in N-terminus of each polypeptide chain. For example, when the chimeric molecule is a full length antibody, the chimeric molecule comprise a peptide signal on both the light chains and heavy chains (i.e., 4 peptide signal in total). Alternatively, the peptide signal may only be present on the polypeptide chain bearing the antithrombotic molecule and absent on the other polypeptide chain. Therefore, when only the light or heavy chain is linked to an antithrombotic agent, the chimeric molecule comprises only 2 peptide signals (i.e., one on each heavy or light chain, in N- terminus of the antithrombotic agent linked to said heavy or light chain).

[0235] Examples of chimeric molecules

[0236] Below are provided Examples of chimeric molecules of the invention.

[0237] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fi brin antibody or an antigen binding fragment thereof and (ii) one or more one or more anticoagulant molecule selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any combination thereof, preferably hirudin, wherein the C-terminal end of the one or several anticoagulant molecule(s) is covalently linked to the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker.

[0238] In some aspects, the chimeric molecule comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising :

[0239] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more anticoagulant molecule selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any combination thereof, preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 15-18; wherein the C-terminal end of the one or several anticoagulant molecule(s) is covalently linked to the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker, in particular comprising or consisting of an amino acid sequence as described in SEQ ID NO : 27-44, preferably a flexible peptide linker, preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 27-35, even more preferably a flexible peptide linker of SEQ ID NO: 30.

[0240] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0241] In some aspects, the chimeric molecule comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting :

[0242] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0243] - a light-chain variable domain (VL) comprising a light chain complementary determining region

[0244] (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more hirudin molecule, preferably comprising or consisting of SEQ ID NO: 15; wherein the C-terminal end of the one or several hirudin molecule(s) is covalently linked to the N- terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker such as described in SEQ ID NO : 27-44, preferably in SEQ ID NO: 27- 35, even more preferably of SEQ ID NO: 30.

[0245] Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the hirudin molecule and / or light-chain variable domain, preferably of SEQ ID NO 9 or 10, respectively.

[0246] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0247] In some aspects, the chimeric molecule comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting of:

[0248] (a) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 85% sequence identity thereto and (b) a light chain variable domain (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 85% sequence identity thereto. b) one or more hirudin molecule, preferably comprising or consisting of SEQ ID NO: 15; wherein the C-terminal end of the one or several hirudin molecule(s) is covalently linked to the N- terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker such as described in SEQ ID NO : 27-44, preferably in SEQ ID NO: 27- 35, even more preferably of SEQ ID NO: 30.

[0249] Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the hirudin molecule and / or light-chain variable domain, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9 or 10, respectively.

[0250] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains. In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more antithrombotic molecule, wherein the chimeric molecule comprises : a) A first polypeptide comprising or consisting of from N- to C- terminal end : optionally a signal peptide - an anti-thrombotic molecule - a peptide linker - a heavy chain variable domain and constants domain of an anti-fibrin antibody. b) A second polypeptide comprising or consisting of from N- to C- terminal end: optionally a signal peptide - a light chain variable domain and a constant domain of an anti-fibrin antibody.

[0251] Preferably, the first and second polypeptides are duplicated so as to obtain a full antibody structure (i.e., having two heavy chains and two light chains).

[0252] Alternatively, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more antithrombotic molecule, wherein the chimeric molecule comprises : a) A first polypeptide comprising or consisting of from N- to C- terminal end : optionally a signal peptide - an anti-thrombotic molecule - a peptide linker - a light chain variable domain and a constant domain of an anti-fibrin antibody. b) A second polypeptide comprising or consisting of from N- to C- terminal end: optionally a signal peptide - a heavy chain variable domain and constant domains of an anti-fibrin antibody.

[0253] Preferably, the first and second polypeptides are duplicated so as to obtain a full antibody structure(i.e., having two heavy chains and two light chains).

[0254] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more hirudin molecule, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9, one or more hirudin molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 15, a peptide linker, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 30, a heavy chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 11; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 10, and a light chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 12.

[0255] Preferably, the first and second polypeptides are duplicated so as to obtain a full antibody structure. In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and one or more hirudin molecule, wherein the chimeric molecule comprises or consists of : a first polypeptide comprising or consisting of SEQ ID NO: 12 a second polypeptide comprising or consisting of SEQ ID NO: 13. Preferably, the first and second polypeptides are duplicated (i.e., so that the chimeric molecule comprises twice the first and second polypeptides) so as to obtain a full antibody structure.

[0256] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and one or more hirudin molecule, wherein the chimeric molecule comprises or consists of : a first polypeptide comprising or consisting of SEQ ID NO: 53 a second polypeptide comprising or consisting of SEQ ID NO: 13. Preferably, the first and second polypeptides are duplicated (i.e., so that the chimeric molecule comprises twice the first and second polypeptides) so as to obtain a full antibody structure.

[0257] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more thrombolytic molecule, wherein the N-terminal end of the heavy or light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several molecules of tissue-type plasminogen activator, preferably by a peptide linker.

[0258] In some aspects, the chimeric molecule comprises or consists of a) an anti-fibrin antibody or an antigen binding fragment thereof comprising :

[0259] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0260] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more thrombolytic molecule, preferably a plasminogen activator, even more preferably selected from the group consisting of alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK- tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof, preferably a serine protease domain thereof, even more preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 19- 26 and 46-51; wherein the C-terminal end of the one or several thrombolytic molecule(s) is covalently linked to the N-terminal end of the heavy or light chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker such as described herein, preferably a flexible or rigid peptide linker.

[0261] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0262] Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the heavy or light-chain variable domain or in N-terminal of the thrombolytic molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9 or 10, respectively.

[0263] In some aspects, the chimeric molecule comprises or consists of a) an anti-fibrin antibody or an antigen binding fragment thereof comprising :

[0264] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0265] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more serine protease domain of a plasminogen activator, preferably comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 46- 50; wherein the C-terminal end of the one or several thrombolytic molecule(s) is covalently linked to the N-terminal end of the heavy or light chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker such as described herein, preferably a flexible or rigid peptide linker preferably by a peptide linker such as described in SEQ ID NO : 27-44.

[0266] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0267] Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the heavy or light-chain variable domain or in N-terminal of the thrombolytic molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9 or 10, respectively.

[0268] In some aspects, the chimeric molecule comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising :

[0269] - a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and

[0270] - a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more plasminogen activator molecule, said plasminogen activator being alteplase or a fragment thereof, preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 19; wherein the C-terminal end of the one or several molecule(s) is covalently linked to the N-terminal end of the heavy or light chain of the anti-fibrin antibody or an antigen binding fragment thereof, preferably by a peptide linker such as described in SEQ ID NO : 27-44, preferably in SEQ ID NO: 27- 35, even more preferably of SEQ ID NO: 30.

[0271] Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the plasminogen activator molecule and / or light or heavy chain variable domain, preferably as set forth in SEQ ID NO:9 or 10, respectively.

[0272] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0273] In some aspects, the chimeric molecule comprises or consists of : an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting of: (i) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 85% sequence identity thereto and (b) a light chain variable domain (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 85% sequence identity thereto.; one or several molecule(s) consisting of the serine protease domain of alteplase, preferably comprising or consisting of SEQ ID NO: 19 or a variant thereof having at least 85 % sequence identity thereto; wherein the C-terminal end of the one or several molecule(s) of alteplase is covalently linked to the N-terminal end of heavy chain variable domain (VH) of the anti-fibrin antibody, preferably by a peptide linker such as described in SEQ ID NO : 27-44, preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30. Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the hirudin molecule and / or light-chain variable domain, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9 or 10, respectively.

[0274] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0275] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more molecule consisting of alteplase's serine protease domain, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9, one or more molecule consisting of alteplase's serine protease domain, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 19, a peptide linker, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 30 or 37, a heavy chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 11; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 10, and a light chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 12.

[0276] Preferably, the first and second polypeptides are duplicated so as to obtain a full antibody structure.

[0277] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more consisting of alteplase's serine protease domain, wherein the chimeric molecule comprises a first polypeptide comprising or consisting of SEQ ID NO: 12 and a second polypeptide comprising or consisting of SEQ ID NO: 45. Preferably, the first and second polypeptides are duplicated (i.e., so that the chimeric molecule comprises twice the first and second polypeptides) so as to obtain a full antibody structure.

[0278] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more consisting of alteplase's serine protease domain, wherein the chimeric molecule comprises a first polypeptide comprising or consisting of SEQ ID NO: 53 and a second polypeptide comprising or consisting of SEQ ID NO: 54. Preferably, the first and second polypeptides are duplicated (i.e., so that the chimeric molecule comprises twice the first and second polypeptides) so as to obtain a full antibody structure.

[0279] In some aspects, the chimeric molecule comprises or consists of : an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting of: (i) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7 , or a variant thereof having at least 85% sequence identity thereto and (b) a light chain variable domain (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 85% sequence identity thereto.; one or more molecule(s) consisting of alteplase's serine protease domain, preferably comprising or consisting of SEQ ID NO: 19 or a variant thereof having at least 85 % sequence identity thereto; wherein the C-terminal end of the one or more molecule(s) of alteplase is covalently linked to the N-terminal end of the light chain variable domain (VL) of the anti-fibrin antibody, preferably by a peptide linker such as described in in SEQ ID NO : 27-44, preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30.

[0280] Optionally, the chimeric molecule comprises a signal peptide in N-terminal of the hirudin molecule and / or light-chain variable domain, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9 or 10, respectively.

[0281] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0282] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more molecule(s) consisting of alteplase's serine protease domain, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9, and a heavy chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 11; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 10, one or more t-pa molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 19, a peptide linker, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 30 or 37, and a light chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 12.

[0283] Preferably, the first and second polypeptides are duplicated so as to obtain a full antibody structure. In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more molecule consisting of alteplase's serine protease domain, wherein the chimeric molecule comprises a first polypeptide comprising or consisting of SEQ ID NO: 11 and a second polypeptide comprising or consisting of SEQ ID NO: 14. Preferably, the first and second polypeptides are duplicated (i.e., so that the chimeric molecule comprises twice the first and second polypeptides) so as to obtain a full antibody structure.

[0284] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more molecule consisting of alteplase's serine protease domain, wherein the chimeric molecule comprises a first polypeptide comprising or consisting of SEQ ID NO: 52 and a second polypeptide comprising or consisting of SEQ ID NO: 14. Preferably, the first and second polypeptides are duplicated (i.e., so that the chimeric molecule comprises twice the first and second polypeptides) so as to obtain a full antibody structure.

[0285] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or several anticoagulant molecule, wherein the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to the C-terminal end of one or several hirudin molecules, and the N-terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several thrombolytic molecule, preferably a tissue-type plasminogen activator, or vice-versa.

[0286] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or several anticoagulant molecule, preferably hirudin, wherein the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to the C-terminal end of one or several hirudin molecules, and the N- terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several thrombolytic molecule, preferably alteplase or a fragment thereof, or vice-versa.

[0287] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody or a fragment thereof, (ii) one or more anticoagulant molecule and (iii) one or more thrombolytic molecule, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:9, one or more anticoagulant molecule(s), preferably hirudin, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 15, optionally a peptide linker, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30, a heavy chain variable domain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 11,; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 10, one or more thrombolytic agent, preferably alteplase or a fragment thereof, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 19, optionally a peptide linker, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30 or 37, and a light chain variable domain (VL) of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 12.

[0288] Preferably, such chimeric molecule further comprises light chain (CL) et heavy chain (CH1+CH2+CH3) constant domains, even more preferably IgGl constant domains.

[0289] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody or a fragment thereof, (ii) one or more anticoagulant molecule and (iii) one or more thrombolytic molecule, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 9, one or more anticoagulant molecule(s), preferably hirudin, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 15, optionally a peptide linker, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30, a heavy chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 11; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 10, one or more thrombolytic agent, preferably t-pa, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 19, optionally a peptide linker, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30, and a light chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 12.

[0290] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more hirudin molecule, wherein the chimeric molecule comprises : a) a first chain comprising or consisting of SEQ ID NO: 13; and b) a second chain comprising or consisting of SEQ ID NO: 14.

[0291] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more anticoagulant or thrombolytic molecule, wherein the N- terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to the C-terminal end of one or several anticoagulant molecules, and to one or several thrombolytic molecule(s).

[0292] In some aspects, the chimeric molecule comprises or consists of (i) an anti-fibrin antibody or an antigen binding fragment thereof and (ii) one or more anticoagulant or thrombolytic molecule, wherein the N- terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to the C-terminal end of one or several anticoagulant molecules and to one or several thrombolytic molecule(s).

[0293] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more hirudin molecule, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9, one or more hirudin molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 15, optionally a peptide linker, one or more molecule of alteplase or fragment thereof, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 19, a peptide linker, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 30, a heavy chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 11; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 10 and a light chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 12.

[0294] Preferably, the first and second polypeptides are duplicated (i.e. each are twice present) so as to obtain a full conventional antibody structure.

[0295] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody, (ii) one or more anticoagulant molecule and (iii) one or more thrombolytic molecule, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, one or more anticoagulant molecule(s), preferably hirudin, optionally a peptide linker, a variable heavy chain domain (VH) of an anti-fibrin antibody and a first Fc domain; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, one or more thrombolytic agent, preferably alteplase or a fragment thereof, optionally a peptide linker, a heavy chain variable domain (VH) of an anti-fibrin antibody, and a second Fc domain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and c) a third and fourth polypeptides comprising or consisting of from N- to C- terminal end : optionally a peptide signal and a light chain of an anti-fibrin antibody.

[0296] Preferably, the first Fc chain and the complementary second Fc chain form an heterodimeric Fc domain, such as based on the "knobs into holes" technology.

[0297] In some aspects, the chimeric molecule comprises or consists of an anti-fibrin antibody and (ii) one or more hirudin molecule, wherein the chimeric molecule comprises : a) a first polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 9, one or more hirudin molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 15, a peptide linker, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 30, a heavy chain variable domain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 7 and a first IgGl Fc domain; and b) a second polypeptide comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 10, one or more plasminogen activator molecule, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 19, a peptide linker, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 30 or 37, a heavy chain variable of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 7 and a second IgGl Fc domain complementary to the first IgGl Fc chain, the first and second Fc chain forming together a Fc domain; c) a third and fourth polypeptides comprising or consisting of from N- to C- terminal end : optionally a peptide signal, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 10, a light chain of an anti-fibrin antibody, preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 12. Preferably, the first Fc chain and the complementary second Fc chain form an heterodimeric Fc domain, such as based on the "knobs into holes" technology.

[0298] Nucleic acid molecules encoding the chimeric molecules of the present invention. Expression Vectors and Host Cells comprising such

[0299] Nucleic acid sequence

[0300] The invention also relates to an isolated nucleic acid molecule encoding the chimeric molecule as defined above or to a group of isolated nucleic acid molecules encoding the chimeric molecule as defined above. Nucleic acid encoding the chimeric molecule disclosed herein can be amplified by any techniques known in the art, such as Polymerase Chain Reaction. Such nucleic acids may be readily isolated and sequenced using conventional procedures known to the man skilled in the art.

[0301] In some aspects, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise or consists of:

[0302] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more antithrombotic molecule(s) ii) optionally a peptide linker, iii) a heavy chain of an anti-fibrin antibody;

[0303] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of the light chain of the anti-fibrin antibody.

[0304] Alternatively, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise or consists of:

[0305] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more antithrombotic molecule(s) ii) optionally a peptide linker, iii) a light chain of an anti-fibrin antibody;

[0306] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of the heavy chain of the anti-fibrin antibody.

[0307] In a particular aspect, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise:

[0308] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more anticoagulant agent(s), preferably hirudin, ii) optionally a peptide linker, iii) a VH or heavy chain of an anti-fibrin antibody;

[0309] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of the light chain of the anti-fibrin antibody. In a particular aspect, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise:

[0310] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more thrombolytic agent(s), preferably tissue-type plasminogen activator ii) optionally a peptide linker, iii) a heavy chain of an anti-fibrin antibody;

[0311] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of the light chain of the anti-fibrin antibody.

[0312] In a particular aspect, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise:

[0313] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more anticoagulant agent(s), preferably hirudin, ii) optionally a peptide linker, iii) a light chain of an anti-fibrin antibody;

[0314] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of the heavy chain of the anti-fibrin antibody.

[0315] In a particular aspect, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise:

[0316] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more thrombolytic agent(s), preferably tissue-type plasminogen activator ii) optionally a peptide linker, iii) a light chain of an anti-fibrin antibody;

[0317] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of the heavy chain of the anti-fibrin antibody.

[0318] In a particular aspect, the nucleic acid molecules encoding the chimeric molecule as defined herein comprise:

[0319] - a first nucleic acid encoding a polypeptide comprising or consisting of, from N to C-terminus i) one or more anticoagulant agent(s), preferably hirudin, ii) optionally a peptide linker, iii) a heavy chain of an anti-fibrin antibody;

[0320] - a second nucleic acid molecule encoding a polypeptide comprising or consisting of, from N to C- terminus i) one or more thrombolytic agent(s), preferably tissue-type plasminogen activator ii) optionally a peptide linker, iii) the light chain of the anti-fibrin antibody.

[0321] In one embodiment, the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule. Vectors

[0322] In another aspect, the invention relates to a vector comprising the nucleic acid molecule or the group of nucleic acid molecules as defined above.

[0323] As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids and artificial chromosomes. In general, engineered vectors comprise an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.

[0324] The nucleic acid molecule encoding the chimeric molecule can be cloned into a vector by those skilled in the art, and then transformed into host cells. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The methods known to the artisans in the art can be used to construct an expression vector containing the nucleic acid sequence of the chimeric molecule and appropriate regulatory components for transcription / translation.

[0325] Accordingly, the present invention also provides a recombinant vector, which comprises a nucleic acid molecule or group on nucleic acid molecules encoding the chimeric molecule according to the present invention. In one preferred aspect, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug-resistance gene for screening. An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants.

[0326] In one embodiment, both the heavy and light chains coding sequences of the chimeric molecule are included in one expression vector. Each of the heavy chain coding sequence and the light chain coding sequence may be in operable linkage to a suitable promoter. Alternatively, expression of both the heavy chain and the light chain may be driven by the same promoter. In another embodiment, each of the heavy and light chains of the chimeric molecule is cloned in to an individual vector. In the latter case, the expression vectors encoding the heavy and light chains can be co-transfected into one host cell for expression of both chains, which can be assembled to form intact antibodies either in vivo or in vitro. Alternatively, the expression vector encoding the heavy chain and that encoding the light chain can be introduced into different host cells for expression each of the heavy and light chains, which can then be purified and assembled to form intact antibodies in vitro.

[0327] Techniques for introducing vectors into eukaryotic cells and techniques for selecting stable transformants using a dominant selectable marker are described by Sambrook, Ausubel, Bebbington, "Expression of Antibody Genes in Nonlymphoid Mammalian Cells," in METHODS: A companion to methods in enzymology 136 (1991), and in Murray et al., Gene transfer and expression protocols (Humana Press 1991).

[0328] Host cells

[0329] In another aspect, the invention relates to a host cell comprising a vector or a nucleic acid molecule or group of nucleic acid molecules as defined above, for example for chimeric molecule production purposes.

[0330] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been recipient of vectors and / or nucleic acid molecules encoding the chimeric molecule of the invention. The term "host cell" is also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.

[0331] Suitable hosts cells are especially eukaryotic hosts cells which provide suitable post-translational modifications such as glycosylation. Preferably, the eukaryotic host cell is a mammalian cell such as BHK cells, 293 cells, CHO cells, NSO cells and COS cells, preferably CHO cells.

[0332] Then host cells stably or transiently express the chimeric molecule according to the present invention. Such expression methods are known by the man skilled in the art. Transient expression is typically achieved using various transfection techniques (e.g., lipofection, electroporation, calcium phosphate transfection and PEI transfection).

[0333] Preparation of chimeric molecules

[0334] The invention further relates to a nucleic acid encoding a chimeric molecule as disclosed above, a vector, preferably an expression vector, comprising the nucleic acid of the invention, a genetically engineered host cell transformed with the vector of the invention or directly with the sequence encoding the recombinant chimeric molecule, and a method for producing the chimeric molecule of the invention by recombinant techniques.

[0335] To produce a chimeric molecule according to the invention, in particular by mammalian cells, nucleic acid sequences or group of nucleic acid sequences coding for the chimeric molecule are cloned into one or more expression vectors. Such vectors are generally used to transfect mammalian cells. General techniques for producing molecules comprising antibody sequences are described in Coligan et al. (eds.), Current protocols in immunology, at pp. 10.19.1-10.19.11 (Wiley Interscience 1992) and in "Antibody engineering: a practical guide" from W. H. Freeman and Company (1992), the contents of which are hereby incorporated by reference

[0336] A method of production of the chimeric molecule is also provided herein. The method comprises culturing a host cell comprising a nucleic acid encoding the chimeric molecule or a vector as provided above, under conditions suitable for its expression, and optionally recovering the chimeric molecule from the host cell (or host cell culture medium). Particularly, for recombinant production of a chimeric molecule, nucleic acid molecule(s) encoding a chimeric molecule, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. The molecules or chimeric molecules are then isolated and / or purified by any methods known in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, supercentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and the combination thereof. As described, for example, by Coligan, chimeric molecule isolation techniques may particularly include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography and ion exchange chromatography. Protein A preferably is used to isolate the molecules or chimeric molecules of the invention.

[0337] Preferably, the chimeric molecule is a polypeptide or protein produced as a recombinant protein.

[0338] Generally, the production method comprises the steps of:

[0339] (1) transfecting or transforming appropriate host cells (e.g. CHO cells) with the polynucleotide(s) or group of nucleic acid molecules encoding the recombinant chimeric molecule of the invention or the vector containing the polynucleotide(s) or group of nucleic acid molecules;

[0340] (2) culturing the host cells in an appropriate medium; and

[0341] (3) optionally isolating or purifying the chimeric molecule from the medium and / or host cells.

[0342] Pharmaceutical Compositions

[0343] The present invention also relates to a pharmaceutical composition comprising the chimeric molecule(s) as described herein, the isolated nucleic acid molecule, the group of isolated nucleic acid molecules, the vector and / or the host cells as described hereabove, preferably as the active ingredient or compound. As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active agents, such as comprising a chimeric molecule according to the invention, with optional other components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism, preferably to a patient.

[0344] Preferably, the pharmaceutical composition is a combination of the active agent, e.g., the chimeric molecule, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. The formulations can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers, excipients, salts, anti-oxidant and / or stabilizers which do not deleteriously interact with the chimeric molecule of the invention, nucleic acid, vector and / or host cell of the invention and does not impart any undesired toxicological effects. .An "acceptable vehicle" or "acceptable carrier" as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0345] To facilitate administration, the chimeric molecule as described herein can be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005). Compositions of the present invention can be in a form suitable for any conventional route of administration or use, such as a topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration. Preferably, the pharmaceutical composition is suitable for intravenous administration.

[0346] The pharmaceutical composition may be prepared by mixing a chimeric molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, anti-oxidant, and / or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, anti-oxidant, and / or stabilizers are well known in the art and have been for example described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0347] It will be understood by one skilled in the art that the formulations of the invention is preferably isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or icefreezing type osmometer. Pharmaceutical composition of the invention typically is sterile and stable under the conditions of manufacture and storage. Prevention of presence of microorganisms may be ensured both by sterilization procedures and / or by the inclusion of various antibacterial and / or antifungal agents.

[0348] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.

[0349] The form of the pharmaceutical compositions, the route of administration and the dose of administration of the pharmaceutical composition or the chimeric molecule according to the invention can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, size, sex, and / or general physical condition. The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.

[0350] In some examples, the pharmaceutical composition comprises the chimeric molecules at a concentration within a range of 0.05 mg / ml to about 50 mg / ml. In some examples, the pharmaceutical composition comprises the chimeric molecules at a therapeutically effective dose comprised in a range of 0.5 to 25 mg / kg.

[0351] Combined therapy

[0352] The chimeric molecule according to the invention can be combined with some other potential strategies or with agents in clinical development or already on the market.

[0353] Accordingly, also provided herein are combined therapies with any of the chimeric molecules or a pharmaceutical composition comprising such, as described herein and a suitable second agent, for the treatment of a disease or disorder. Preferably, the second agent is an additional antithrombotic agent, preferably an anticoagulant or thrombolytic agent.

[0354] In an aspect, the chimeric molecule and the second agent can be present in a unique pharmaceutical composition as described above. Alternatively, the terms "combination therapy" or "combined therapy", as used herein, embrace administration of these two agents (e.g., a chimeric molecule as described herein and an additional or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route. The agents can be administered by the same route or by different routes. Preferably, the pharmaceutical composition of the invention further comprises an additional therapeutic agent.

[0355] Preferably, the pharmaceutical composition comprises: a) at least two chimeric molecules wherein a first chimeric molecule comprises one or more anticoagulant molecules, preferably hirudin, fused to the anti-fibrin antibody or antigen binding fragment thereof, and a second chimeric molecule comprises a thrombolytic agent, preferably rt- pa or a fragment thereof, fused to the anti-fibrin antibody or antigen binding fragment thereof; and / or b) one or more additional therapeutic agent, preferably an antithrombotic agent selected from (i) a thrombolytic agent selected from the group consisting of tissue-type plasminogen activator (t- PA), alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof ; (ii) an anticoagulant agent selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any variant or fragment thereof, Argatroban, Heparin, UFH, LMWH, Fondaparinux, Danaparoid, Direct oral anticoagulants (DOACs) such as Dabigatran and Rivaroxaban, and Vitamin K antagonists such as warfarin and (iii) any combination thereof.

[0356] In some aspects, the pharmaceutical composition or the combined therapy comprises or consists of at least two different chimeric molecules of the invention. Preferably, the pharmaceutical composition or the combined therapy comprises or consists of a first chimeric molecule comprising an anticoagulant agent, preferably hirudin, and a second chimeric molecule comprising a thrombolytic agent, preferably rt-pa or a fragment thereof. Additionnally, the pharmaceutical composition or the combined therapy may comprises an additional antithrombotic agent, such as an anticoagulant or thrombolytic agent described herein.

[0357] In some aspects, the pharmaceutical composition or the combined therapy comprises the chimeric molecule of the invention and a thrombolytic agent, even more preferably a thrombolytic agent selected from the group consisting of recombinant tissue-type plasminogen activator (t-PA), such as alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any combination thereof.

[0358] In some aspects, the pharmaceutical composition or the combined therapy comprises a chimeric molecule comprising or consisting of an anti-fibrin antibody covalently linked to an anticoagulant agent, preferably hirudin and an additional therapeutic agent, preferably a thrombolytic agent, even more preferably a thrombolytic agent selected from the group consisting of a recombinant tissue-type plasminogen activator (t-PA), such as alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any combination thereof.

[0359] In some aspects, the pharmaceutical composition or the combined therapy comprises a chimeric molecule comprising or consisting of an anti-fibrin antibody covalently linked to a thrombolytic agent, preferably alteplase or a fragment thereof, and an additional therapeutic agent, preferably a thrombolytic agent, even more preferably a recombinant tissue-type plasminogen activator (t-PA) selected from the group consisting of alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any combination thereof.

[0360] In some aspects, the pharmaceutical composition or the combined therapy comprises the chimeric molecule of the invention and an anticoagulant agent, preferably selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin, Argatroban, Heparin, UFH, LMWH, Fondaparinux, Danaparoid, Direct oral anticoagulants (DOACs) such as Dabigatran and Rivaroxaban, and Vitamin K antagonists such as warfarin, and any combination thereof.

[0361] In some aspects, the pharmaceutical composition or the combined therapy comprises a chimeric molecule comprising an anti-fibrin antibody covalently linked to a thrombolytic agent, preferably alteplase or a fragment thereof, and an additional therapeutic agent that is an anticoagulant molecule, preferably selected from the group consisting of Bivalirudin, hirudin, bivalirudin, lepirudin, desirudin, Argatroban, Heparin, UFH, LMWH, Fondaparinux, Danaparoid, Direct oral anticoagulants (DOACs) such as Dabigatran and Rivaroxaban, and Vitamin K antagonists such as warfarin, and any combination thereof.

[0362] In some aspects, the pharmaceutical composition or the combined therapy comprises a chimeric molecule comprising an anti-fibrin antibody covalently linked to anticoagulant agent, preferably hirudin and an additional therapeutic agent that is an anticoagulant molecule, preferably selected from the group consisting of Bivalirudin, hirudin, bivalirudin, lepirudin, desirudin, Argatroban, Heparin, UFH, LMWH, Fondaparinux, Danaparoid, Direct oral anticoagulants (DOACs) such as Dabigatran and Rivaroxaban, and Vitamin K antagonists such as warfarin, and any combination thereof.

[0363] In some aspects, the pharmaceutical composition or the combined therapy comprises a chimeric molecule comprising or consisting of an anti-fibrin antibody covalently linked to a thrombolytic agent, preferably a recombinant tissue-type plasminogen activator's fragment comprising or consisting of a protease, especially a serine protease and an anti-fibrin antibody covalently linked to an anticoagulant agent, preferably hirudin. Uses

[0364] The chimeric molecules, nucleic acids, vectors, host cells, compositions and methods of the present invention have numerous in vitro and in vivo utilities and applications. Particularly, any one of the chimeric molecules, nucleic acid molecules, group of nucleic acid molecules, vectors, host cells, pharmaceutical composition or combined therapy provided herein may be used in therapeutic methods and / or for therapeutic purposes, in particular as a medicament and / or for the prevention and / or treatment of conditions, disorders and diseases. Examples of disorders and diseases to be treated are more particularly described hereafter. Such disorders and diseases are preferably caused by, related to or associated with thrombosis and / or blood clots.

[0365] In some aspects, the disease or disorder is selected from the group consisting of ischaemic stroke, myocardial infarction, venous thromboembolism (including pulmonary embolism and / or deep vein thrombosis), arterial thrombosis (including peripheral arterial occlusion), venous thrombosis (e.g. intracerebral).

[0366] Preferably, the disease is a thromboembolic venous disease or venous thrombosis. Typically, the disease associated with venous thrombosis is deep vein thrombosis or pulmonary embolism.

[0367] Alternatively, the disease or disorder is an arterial thrombosis or is associated with arterial thrombosis. Typically, the disease associated with arterial thrombosis is selected from the group consisting of ischemic stroke, peripheral arterial disease, myocardial infarction and acute limb ischemia.

[0368] The present disclosure is also related to the uses of the present chimeric molecule, pharmaceutical composition or combined therapy in methods for preventing or treating thrombosis in a subject, to the chimeric molecule, pharmaceutical composition or combined therapy for use in the prevention or treatment of thrombosis or for the manufacture of a medicament for the prevention or treatment of thrombosis.

[0369] As used herein, the term "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and / or a prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and / or eliminating, reducing and / or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly. A prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and / or delaying the progression and / or the incidence of a disease or the risk of its occurrence. In certain aspects, such a term refers to the improvement or eradication of a disease, disorder, condition or symptoms associated with it. In other aspects, this term refers to minimizing the spread or the worsening of the disease, disorder, condition or symptoms. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a chimeric molecule of the invention, a pharmaceutical composition or a combined therapy to a subject in need thereof, typically who has or is susceptible to have a disorder / disease.

[0370] The present invention also relates to a method for treating a disease in a subject comprising administering to said subject a therapeutically effective amount of the chimeric molecule or the pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent such as described herein.

[0371] The present invention particularly relates to a chimeric molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition or a combined therapy comprising such for use as a medicament. As used herein, the term "medicament" refers to any substance or composition with curative or preventive properties against disorders or diseases.

[0372] It also relates to the use of a chimeric molecule as described herein; a nucleic acid or a vector encoding such, or a pharmaceutical composition or a combined therapy comprising such for use in the treatment of a disease and / or disorder or for treating a disease and / or disorder in a subject. It also concerns the use of a chimeric molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, a pharmaceutical composition or a combined therapy comprising such as disclosed herein in the manufacture of a medicament for treating a disease and / or disorder in a subject in need thereof.

[0373] It also concerns the use of a chimeric molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, a pharmaceutical composition or a combined therapy comprising such as disclosed herein in the manufacture of a medicament for treating thrombosis or a disease or disorder associated with thrombosis.

[0374] Finally, it relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition, combined therapy or a chimeric molecule to the subject.

[0375] The present invention particularly relates to a method for treating thrombosis or a disease or disorder associated with thrombosis in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the chimeric molecule, nucleic acid, group of nucleic acid molecules or vector encoding such, pharmaceutical composition or combined therapy such as disclosed herein. In one aspect, the treatment method comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a chimeric molecule, nucleic acid, vector, pharmaceutical composition or a combined therapy as described herein.

[0376] "An effective amount" or a "therapeutic effective amount" as used herein refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The "effective amount" will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0377] In some aspects, the therapeutic effective amount of the chimeric molecule, pharmaceutical composition or combined therapy is a concentration within a range of 0.05 mg / ml to about 50 mg / ml.

[0378] As used herein, the terms "subject", "individual" or "patient" are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human. However, the term "subject" can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others.

[0379] Preferably, the subject to treat is a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old or of at least 70 years old.

[0380] A subject in need of a treatment may be a human having, at risk for, or suspected of having a disease. Such a patient can be identified by routine medical examination.

[0381] The chimeric molecule, pharmaceutical composition or combined therapy may be administered as a single dose or in multiple doses.

[0382] The form, the route of administration and the dose of administration can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, size, sex, and / or general physical condition. Kits

[0383] Any of the chimeric molecules or compositions described herein may be included in a kit provided by the present invention.

[0384] In the context of the present invention, the term "kit" means two or more components (one of which corresponding to the chimeric molecule, the nucleic acid molecule, the vector or the cell of the invention) packaged in a container, recipient or otherwise. A kit can hence be described as a set of products and / or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit. The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.

[0385] Particularly, a kit according to the invention may comprise:

[0386] - a chimeric molecule as described herein,

[0387] - a pharmaceutical composition or combined therapy as described herein,

[0388] - a nucleic acid molecule or a group of nucleic acid molecules encoding said chimeric molecule,

[0389] - a vector comprising said nucleic acid molecule or group of nucleic acid molecules, and / or

[0390] - a host cell comprising said vector or nucleic acid molecule or group of nucleic acid molecules.

[0391] The kit may thus include, in suitable container means, the pharmaceutical composition, combined therapy chimeric molecules, and / or host cells of the present invention, and / or vectors encoding the nucleic acid molecules of the present invention, and / or nucleic acid molecules or related reagents of the present invention. In some embodiments, means of taking a sample from an individual and / or of assaying the sample may be provided. The compositions comprised in the kit according to the invention may particularly be formulated into a syringe compatible composition.

[0392] The instructions related to the use of the chimeric molecule or pharmaceutical composition described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, means for reconstituting the chimeric molecule and / or means for diluting the chimeric molecule of the invention. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual).

[0393] Finally, the invention concerns the kit disclosed herein for use in the treatment of thrombosis or of a disease or disorder associated with thrombosis in a subject, such as described herein.

[0394] Further aspects and advantages of the present invention are described in the following Figures and Examples, which should be regarded as illustrative and not limiting. BRIEF DESCRIPTION OF THE FIGURES

[0395] Figure 1 : Examples of chimeric molecules according to the invention

[0396] Figure 2 : Characterization of ADC-HirlO's anticoagulant effect. A. Enzymatic activity of ADC-HirlO in comparison to free Hirudin. Fluorogenic assay to determine the inhibitory activity of ADC-HirlO compared to hirudin's. By using the two VO curves obtained at different hirudin concentrations, the linear regression's slopes for each molecule between 0 and 12 ng / ml of hirudin, were -5.82 x 10-2for hirudin and -3.8 x 10-2for ADC-HirlO. With those slopes, it was determined that ADC-HirlO inhibits 65% of all thrombin molecules inhibited by hirudin alone. N= 12 for each condition (Hirudin and ADC-HirlO). [Hir]: hirudin concentration. B. Anticoagulant effect of ADCs compared to hirudin in an activated partial thromboplastin time (aPTT). Anticoagulant effect of ADC-HirlO in comparison to free Hirudin and to other ADCs wherein hirudin is fused in C-terminus of the heavy chain by a flexible or rigid peptide linker (ADC-Hir8 and ADC-Hir9, respectively). aPTT was performed on human PFP to test the anticoagulant effect of ADCs-Hir in vitro. Results were analysed using Kruskal-Wallis test. For each curve, every point was compared to the point corresponding to 0 pg / mL of hirudin. Stars were added on points that are significantly increased compared to their respective control at 0 pg / mL. For all ADCs- Hir points at 0 pg / mL of hirudin concentration, r59D8 was added at 116 pg / mL. It corresponds to the antibody concentration when 10 pg / mL of hirudin was added. N=3 for each ADC, and hirudin.

[0397] Figure 3: Stability of ADC-HirlO's anticoagulant effect in comparison to uncoupled hirudin and uncoupled anti-fibrin antibody. Results of aPTT performed from the blood samples collected. ADC- HirlO ([Hir]: 0,1 mg / kg), r59D8 ([Hir]: 0 mg / kg) or Hirudin (10 mg / kg) were injected IV. TO, Tl, T6, T24 and T30 correspond to the collection of blood samples (BS) respectively before injection, 1, 6, 24 and 30 hours after injection. r59D8 was injected at an antibody dose of 2,9 mg / kg, corresponding to the antibody concentration used to inject ADCs at 0,25 mg / kg of hirudin. ADC-HirlO and hirudin at 1 and 6 hours post-injection were compared to r59D8 by a Kruskal-Wallis and an ANOVA test, respectively. At 24 and 30 hours post injection, ADC-HirlO was compared to control by an unpaired T-test.

[0398] Figure 4. ADC-HirlO's efficacy in preventing thrombosis in a pulmonary embolism model. A. A dose dependent effect of ADC-HirlO at different hirudin concentrations. Platelet consumption was determined using a ratio between the first and the second blood sample. ADC-HirlO and r59D8 were statistically compared using an ANOVA test. B. Ability of ADC-HirlO to inhibit thrombosis compared to an equivalent dose of hirudin. ADC-HirlO's efficiency to prevent platelet consumption at a hirudin dose of 0,015 mg / kg was compared to an equivalent dose of hirudin: 0,01 mg / kg. Platelet consumption is directly correlated with thrombi formation. The more platelets are consumed, the more thrombi are formed. All three conditions were statistically compared to a one-way ANOVA. Before performing the ANOVA test, Grubb's test was ran and one mouse was excluded in the ADC-HirlO's condition. « No TF » means no tissue factor (procoagulant) injected. NaCI was injected instead.

[0399] Figure 5. Effect of ADC-HirlO in a mouse model for venous thrombosis based on the transient inhibition of natural anticoagulants. A. Survival curve indicating the affected mice in time, according to their treatment. B. Number of mice that were affected when the experiment was ended. ADC-HirlO- treated groups were compared to the control group using a Fisher-exact test. C. Body weight of mice monitored during the experiment. All groups were analysed and compared to control group by using a two-way ANOVA. D. Platelet count of mice 22 hours after being injected with siRNA. The platelet count was determined by a ratio with a normal platelet count (mice injected only with PBS). Blood was collected moments before mice were sacrificed.

[0400] Figure 6 : Impact ADC-HirlO on haemostasis in comparison to uncoupled hirudin and uncoupled antifibrin antibody (r59D8). r59D8, ADC-HirlO at hirudin doses between 0.01 and 0.05 mg / kg) and hirudin (10 mg / kg) were injected IV in mice. Then, the tail was cut (3 mm) and bleedings were observed for 30 min. Bleeding time corresponds to the time it takes for the mouse to stop bleeding for the first time. Total bleeding time is the sum of all bleeding times. All conditions were compared to r59D8 conditions using a Kruskal-Wallis test. "Bleeding time" corresponds to the first blood arrest.

[0401] Figure 7: Regression curves of rtPAs' enzymatic activity. rtPA is such as described in SEQ ID NO: 18, the ADCs tested in this figure comprise the sequence of rtpa serine protease domain, such as described in SEQ ID NO: 19.

[0402] Figure 8: Comparison of rtPAs enzymatic activity in absence of fibrin. A. Comparison of rtPAs' affinity for the substrate (km). B. Comparison of rtPAs' catalytic activity (kcat). It represents the amount of substrate catalysed by rtPA per second. C. Comparison of rtPAs' catalytic efficiency. It represents the global capacity of rtPA to catalyse the enzyme. One-way ANOVAs were used for the three graphs. rtPA is such as described in SEQ ID NO: 18, the ADCs tested in this figure comprise the sequence of rtPA serine protease domain, such as described in SEQ ID NO: 19.

[0403] Figure 9: Tinzaparin's impact on haemostasis and its efficacy in preventing thrombosis in a pulmonary embolism model. A. Impact of Tinzaparin on haemostasis. Mice were injected subcutaneously with NaCI or tinzaparin at 300, 175 or 75 Ul / kg. One hour later, the tail was cut (3 mm) and bleedings were observed for 30 min. Bleeding Time (i.e., first blood arrest) corresponds to the time it takes for the mouse to stop bleeding for the first time. Total bleeding time is the sum of all bleeding times. 175 Ul / kg corresponds to the dose that is used in humans. All conditions were compared to NaCI using a Kruskal- Wallis test. B. A dose dependent effect of tinzaparin at two different concentrations (75 to 300 Ul / kg) to identify the efficient dose in the pulmonary model: 300 Ul / kg. Tinzaparin doses or NaCI were injected 1 hour before the experiment to be at tinzaparin's maximal antithrombotic effect. Its efficiency to prevent platelet consumption was determined by using a ratio between the first and the second blood sample. Both doses were statistically compared to NaCI by using an ANOVA test. C. Ability of ADC-HirlO to inhibit thrombosis compared to tinzaparin. ADC-HirlO's efficiency to prevent platelet consumption at a hirudin dose of 0,05 mg / kg was compared to tinzaparin at 300 Ul / kg. Tinzaparin's maximal dose (300 Ul / kg), injected one hour subcutaneously before the experiment, was chosen as the only dose that does not impact haemostasis (75 Ul / kg) does not have an effect in this model. ADC-HirlO's dose, injected right before injecting tissue factor (TF) injection was chosen at 0.05 mg / kg as it is the maximal dose that does not have an impact on haemostasis. All conditions were compared using a one-way ANOVA.

[0404] Figure 10: Capacity of ADC-HirlO to inhibit thrombus formation in a FeCI3injury model of the carotid artery, compared to an equivalent dose of hirudin. Hirudin was injected at 0.065 mg / kg, and ADC- HirlO was injected at 0.1 mg / kg. They were both injected before inducing the thrombus. Platelets were marked using DIOC6. A. Area under curve (AUC) of platelet area during thrombus formation (20 min). B. Maximal platelet area during the same lapse of time. Both graphs were statistically analysed using a one-way ANOVA.

[0405] Figure 11: Bleeding time of ADC-HirlO compared to hirudin at the same equivalent doses used in the FeCh injury model (0.1 and 0.065 mg / kg respectively). Mice were injected intravenously with r59D8, ADC-HirlO or hirudin. The tail was cut (3 mm) and bleedings were observed for 30 min. "Bleeding time" corresponds to the First blood arrest, i.e., the time it takes for the mouse to stop bleeding for the first time. Total bleeding time is the sum of all bleeding times. All conditions were compared using an ANOVA test.

[0406] EXAMPLES

[0407] MATERIALS AND METHODS

[0408] Materials

[0409] Activated cephalin, calcium chloride (CaCI2) and FlucaKit® were purchased from Diagnostica Stago (Asniere-sur-scene, France). Human Serum Albumin (HSA), HEPES and thrombin were from Sigma Aldrich (Lyon, France). Tissue Factor (TF) used was thromborel® S from Siemens (Nanterre, France). Alexa 647 labeling kit, DAPI (ref: D3571) and Dulbecco's phosphate-buffered saline (PBS) were from ThermoFisher Scientific (lllkirch-Graffenstaden, France), and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) from Molecular Probes (Paisley, United Kingdom). Recombinant hirudin was purchased from Transgene (lllkirch-Graffenstaden, France). Bovin serum albumin (BSA) and glycine were from Euromedex (Souffelweyersheim, France). Cryomatrix was purchased from Epredia (Netherlands). Anti- fibrinogen (ref: 4F7) antibody was from Bioss (Nanterre, France), the mouse isotypical MOPC-21 was purchased from Sigma Aldrich (Lyon, France). The mouse antifibrin (59D8), and the rat anti-GPIba (RAM-1 IgG) antibodies were produced and coupled to a fluorophore on site.

[0410] Mice

[0411] C57BL / 6J male and female mice (Charles River Laboratories) between 6 and 12 weeks old were used in the experiments.

[0412] Preparation of human Platelet-Free Plasma (PFP)

[0413] Whole blood (15 mL) was collected into a tube containing 3.8% buffered sodium citrate (ratio blood / sodium citrate 1: 9). Whole blood was centrifuged at 2,200 g for 10 min at room temperature. The supernatant was collected to be centrifuged again at 13,000 g for 5 min to obtain PFP.

[0414] Human PFP in activated partial thromboplastin time (aPTT)

[0415] Hirudin, ADCs-Hir or r59D8 were added into 50 pL of human PFP to obtain hirudin concentrations between 0 and 10 pg / mL. 50 pL of activated cephalin was added and incubated for 180 s. Then, 50 pL of CaCL (0.025 M) was added, and time for PFP coagulation was recorded. For all ADCs-Hir points at 0 pg / mL, r59D8 was added at 116 pg / mL. It corresponds to the antibody concentration when 10 pg / mL of hirudin was added.

[0416] Microfluidic model for ADC-HirlO

[0417] Microfluidic flow chambers were prepared as previously described in Ahmed et al., Res Pract Thromb Haemost. 2021;5(5):el2551. Channels (dimensions: 1 mm length, 0.1 mm height, 40 mm long) were coated with 12 nM TF for 1 hours at room temperature. Then, they were blocked with 1% Human Serum Albumin (HSA) for 30 min at room temperature. Citrated PFP was recalcified with 20 mM CaCI2 and perfused at 500 s1through the channel. As soon as the experimenter sees in differential interference contrast (DIC) the first fibrin fibres form in the channel, non-recalcified citrated PFP with the tested antibody (ADC-HirlO, r59D8, 59D8 or MOPC-21) coupled to dylight 647 (10 pg / ml) was perfused through the microfluidic channel. The fluorescence was pictured after 10 min of perfusion.

[0418] Microfluidic model for ADC-rtPA

[0419] Microfluidic flow chambers were prepared as previously described. Briefly, recalcified human citrated platelet free plasma (PFP) (CaCL 20 mmol / L) was perfused at 750 s1through a microfluidic chamber coated with 12 nM of tissue factor (TF) (Thromborel S). All perfusion experiments were carried out for 10 min at 37°C. Once the first fibrin fibers formed, one of the ADCs-rtPA, r59D8 or rtPA at 1 pg / ml of rtPA was perfused in PFP. In DIC, we looked if it cleaved fibrin. It was determined by eye that there was no fibrinolysis if the fibrin remained intact. If there was less fibrin fibers, but a lot of fibrin remained in the middle after the end of the flow, the fibrinolysis was moderate. If the majority of the fibrin was cleaved in the middle and if there was only a small amount of remaining fibrin fiber on the border of the channel, this significates that there had been a good fibrinolysis.

[0420] Fluorogenic assay (hirudin)

[0421] Recombinant 59D8 (r59D8), ADC-HirlO or hirudin were diluted into HEPES 0.35 M and BSA 2 mg / mL to obtain hirudin concentrations between 0 and 45 ng / mL. The mixture was incubated with thrombin at 0.9 UI / mL for 10 min. FluCa Kit® peptide was added as recommended by manufacturer and the fluorescence was recorded for 20 minutes by the TECAN plate reader (Infinite 200 (Lyon, France)) (Excitation: 460 nm). The final hirudin concentration obtained for ADC-HirlO or hirudin alone was between 0 and 15 ng / mL. Each measure was performed in duplicate and the slope (V0) was determined for each curve. V0 for every kinetic activity was calculated for each hirudin concentration, giving two curves. Then, the coefficient of each linear regression was determined with VOs from 0 to 12 ng / ml. A ratio of these to slopes was done to determine the difference of two agents' inhibitory activity. The well at 0 ng / ml hirudin concentration for ADC-HirlO was diluted with r59D8 at the highest concentration of ADC-HirlO used in this experiment.

[0422] Fluorogenic assay (rt-pa)

[0423] 7-amino-4-methylcoumarin (A191) is the fluorescent product of pefafluor. It was used for a calibration curve. Pefafluor was added in wells for a final concentration between 12.5 and 250 pM. ADCs-rtPA or rtPA alone were added for a final concentration at 0.5 pg / mL of rtPA. The kinetic was followed for 20 min at 37 °C and the fluorescence was measured every 30 seconds.

[0424] ADC-HirlO's pharmacokinetic in mice using aPTT

[0425] Mice were anesthetized with isoflurane 4% to inject in retro-orbital (RO) the r59D8, ADC-HirlO ([hir] : 0.1 mg / kg) or hirudin (hir) (10 mg / kg). The mouse was anesthetized with xylazine (20 mg / kg) and ketamine (100 mg / kg) before any injection, 1, 6, 24 or 30 hours post-injection, to collect 3.15% citrated blood through the abdominal aorta (ratio blood / sodium citrate 9:1). Blood was centrifuged at 3,500 g for 5 min at 4°C. Plasma was kept and centrifuged at 12,000 g for 5 min at 4°C. The final PFP was frozen at -80°C to later be tested through an aPTT.

[0426] In vivo thrombosis model - Ferric chloride (FeCh)-induced arterial thrombosis

[0427] Mice were anesthetized with xylazine (10 mg / kg) and ketamine (100 mg / kg) and platelets were labelled by administering DlOCg IV (200 pM, 2.5 pL / g). r59D8, the anti-fibrin antibody 59D8 or an isotypical antibody (MOPC-21), coupled to dylight 647, were injected RO (3.8 pg / kg). FeCI3-mediated thrombosis was then induced by applying a 1x3 mm long Whatman filter paper saturated with 7.5% FeCI3laterally to the exposed carotid artery for 2.5 min. Thrombus formation was recorded by intravital microscopy for 20 min (fluorescence macroscope : Leica Z16 APO with plan Apol / x0.57, Leica Microsystems (Nanterre, France)).

[0428] In vivo thrombosis model - Thromboembolism (TE) model (ADC used in prevention)

[0429] A cervicotomy was performed on anesthetized mice (xylazine (10 mg / kg) and ketamine (100 mg / kg)) and a first 3.15% citrated blood sample was collected through the jugular vein (ratio citrate / blood 9:1, 100 pL). r59D8, ADC-HirlO ([hir]: 0.015, 0.03 mg / kg) or hir (0.01 mg / kg) were injected in RO. Then, tissue-factor (TF) was injected in the jugular vein. After 2 min, a new blood sample of 100 pL was collected through the second jugular vein. The platelet count was measured with an haematology analyser (Element HT5), and a percentage of consumed platelets was determined using the ratio between the second blood sample and the first one. The dose of TF (125 to 150 pL / kg) was determined every day on control mice to obtain more than 35% of platelet consumption.

[0430] Venous thrombosis model via siRNA injection

[0431] Mice develop a severe thrombotic coagulopathy after the transient inhibition of natural anticoagulants using small interfering (si)RNAs. This mouse model for venous thrombosis was introduced and characterized previously in Heestermanset al., . Blood 2019, 133, 2090-2099 and Safdar et al., Blood 2013, 121, 4413-4416. SiRNAs targeting protein C and antithrombin were injected in the tail vein (TV) at 15 pM / siRNA / kg, after complexation with invivofectamine 3.0. Pilot experiments showed that in our current setup mice developed venous thrombosis 24 hours after injection, characterized by the formation of large thrombi leading to vessel occlusion in large veins of the head. The unique phenotype is macroscopically scored by the formation of large haemorrhages and swellings around the mandibular area. Before being affected, 22 hours after siRNA injection, r59D8 or ADC-HirlO ([Hir]:0.45or 0.05 mg / kg) were injected in the TV. Subsequently mice were closely monitored and weighed regularly, until their sacrifice 12 hours later. Mice were scored as 'affected' when they displayed with the typical phenotype as described above (Fig 5.B). 300 pL of citrated blood was collected RO from the side of the mouse head that was not affected. Blood counts were determined using a haematological blood counter (MS4, MELET Schloesing Laboratories).

[0432] In vivo thrombosis model - Thromboembolism (TE) model (ADC used in prevention)

[0433] A cervicotomy was performed on anesthetized mice (xylazine (10 mg / kg) and ketamine (100 mg / kg)) and a first 3.15% citrated blood sample was collected through the jugular vein (ratio citrate / blood 9:1, 100 pL). r59D8, ADC-HirlO ([hir]: 0.015, 0.03 mg / kg) or hirudin (0.01 mg / kg) were injected in RO. Then, tissue-factor (TF) was injected in the jugular vein. After 2 min, a new blood sample of 100 pL was collected through the second jugular vein. The platelet count was measured with an haematology analyser (Element HT5), and a percentage of consumed platelets was determined using the ratio between the second blood sample and the first one. The dose of TF (125 to 150 pL / kg) was determined every day on control mice to obtain more than 35% of platelet consumption.

[0434] Tail bleeding time (ADC-Hir dose effect) r59D8, ADC-HirlO ([hir]: 0.01, 0.015, 0.03, and 0.05 mg / kg) and hirudin (10 mg / kg) were injected RO on anesthetized mice (Isoflurane 4%). A 3 mm segment from the distal tail of mice was cut, as described in Janus Bell et al., Thromb Haemost. 2022 May;122(5):767-776, to determine the bleeding time and the volume of blood loss. The amputated tail was immersed in a 0.9% saline at 37°C and observed until the bleeding stops, this timing corresponding to the bleeding time (maximum 30 min). The tube containing saline and blood was homogenized and centrifuged. A lysis buffer (NH4CI 150 mM, KHCO3 1 mM, EDTA 0.1 mM, pH 7.2) was added to the pellet. After further homogenization, the optical density was read by the TECAN plate reader at 540 nm and compared to a calibration curve to determine the volume of blood loss. The calibration curve was prepared by adding 50 pL of blood sample in 450 pL of lysis buffer diluted at 20 - 10 - 5 - 2 and lpL / mL.

[0435] Tail bleeding time (Tinzaparin dose effect)

[0436] NaCI and tinzaparin doses (75, 175 or 300 Ul / kg) were injected subcutaneously one hour before the experiment on anesthetized mice (Isoflurane 4%). One hour post-injection, when the effect of tinzaparin is supposed to be at its maximum, 3 mm segment from the distal tail of mice was cut, as described in Janus Bell et al., Thromb Haemost. 2022 May;122(5):767-776, to determine the bleeding time and the volume of blood loss. The amputated tail was immersed in a 0.9% saline at 37°C and observed until the bleeding stops, this timing corresponding to the bleeding time (maximum 30 min). The tube containing saline and blood was homogenized and centrifuged. A lysis buffer (NH4CI 150 mM, KHCO3 1 mM, EDTA 0.1 mM, pH 7.2) was added to the pellet. After further homogenization, the optical density was read by the TECAN plate reader at 540 nm and compared to a calibration curve to determine the volume of blood loss. The calibration curve was prepared by adding 50 pL of blood sample in 450 pL of lysis buffer diluted at 20 - 10 - 5 - 2 and lpL / mL.

[0437] In vivo thrombosis model - Thromboembolism (TE) model (Tinzaparin dose effect)

[0438] One hour before the experiment, mice were injected subcutaneously with NaCI or Tinzaparine (75 or 300 Ul / kg). One hour later, a cervicotomy was performed on anesthetized mice (xylazine (10 mg / kg) and ketamine (100 mg / kg)) and a first 3.15% citrated blood sample was collected through the jugular vein (ratio citrate / blood 9:1, 100 pL). Then, tissue-factor (TF) was injected in the jugular vein. After 2 min, a new blood sample of 100 pL was collected through the second jugular vein. The platelet count was measured with an haematology analyser (Element HT5), and a percentage of consumed platelets was determined using the ratio between the second blood sample and the first one. The dose of TF (125 to 150 p.L / kg) was determined every day on control mice to obtain more than 34% of platelet consumption.

[0439] In vivo thrombosis model - Thromboembolism (TE) model (Tinzaparin vs ADC-HirlO)

[0440] One hour before the experiment, mice were injected subcutaneously with NaCI (r59D8 or ADC-HirlO groups) or Tinzaparine (300 Ul / kg). One hour later, a cervicotomy was performed on anesthetized mice (xylazine (10 mg / kg) and ketamine (100 mg / kg)) and a first 3.15% citrated blood sample was collected through the jugular vein (ratio citrate / blood 9:1, 100 pL). r59D8, ADC-HirlO ( [hir] : 0.05 mg / kg) or NaCI for tinzaparin group, were injected in RO. Then, tissue-factor (TF) was injected in the jugular vein. After 2 min, a new blood sample of 100 pL was collected through the second jugular vein. The platelet count was measured with an haematology analyser (Element HT5), and a percentage of consumed platelets was determined using the ratio between the second blood sample and the first one. The dose of TF (125 to 150 pL / kg) was determined every day on control mice to obtain more than 34% of platelet consumption.

[0441] In vivo thrombosis model - Ferric chloride (FeCh)-induced arterial thrombosis

[0442] Mice were anesthetized with xylazine (10 mg / kg) and ketamine (100 mg / kg) and platelets were labelled by administering DlOCg IV (200 pM, 2.5 pL / g). r59D8, ADC-HirlO or Hirudin were injected RO at respectively 0, 0.1 and 0.065 mg / kg of hirudin. r59D8 was added at 440 pg / mL. It corresponds to the antibody concentration when 0.2 mg / kg of hirudin is injected. FeCI3-mediated thrombosis was then induced by applying a 1x3 mm long Whatman filter paper saturated with 7.5% FeCI3laterally to the exposed carotid artery for 2.5 min. Thrombus formation was recorded by intravital microscopy for 20 min (fluorescence macroscope : Leica Z16 APO with plan Apol / x0.57, Leica Microsystems (Nanterre, France)).

[0443] Tail bleeding time (ADC-Hir vs Hirudin) r59D8 ([Hir]: 0 mg / kg), ADC-HirlO ([hir]: 0.1 mg / kg) and hirudin (0.065 mg / kg) were injected RO on anesthetized mice (Isoflurane 4%). A 3 mm segment from the distal tail of mice was cut, as described in Janus Bell et al., Thromb Haemost. 2022 May;122(5):767-776, to determine the bleeding time and the volume of blood loss. The amputated tail was immersed in a 0.9% saline at 37°C and observed until the bleeding stops, this timing corresponding to the bleeding time (maximum 30 min). The tube containing saline and blood was homogenized and centrifuged. A lysis buffer (NH4CI 150 mM, KHCO31 mM, EDTA 0.1 mM, pH 7.2) was added to the pellet. After further homogenization, the optical density was read by the TECAN plate reader at 540 nm and compared to a calibration curve to determine the volume of blood loss. The calibration curve was prepared by adding 50 pL of blood sample in 450 pL of lysis buffer diluted at 20 - 10 - 5 - 2 and lpL / mL. Statistical analysis

[0444] Statistical analyses were performed with GraphPad Prism software (see figure legends).

[0445] Chimeric molecules

[0446] When reference is made to "ADC" in the Example section, this term means a chimeric molecule of the invention. The following chimeric molecules have been used in the Examples:

[0447] ADC-HirlO comprises a molecule of hirudin as described in SEQ ID NO: 15 covalently linked by its C- terminal end to the N-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a flexible peptide linker as described in SEQ ID NO: 30.

[0448] ADC-Hir9 comprises a molecule of hirudin as described in SEQ ID NO: 15 covalently linked by its N- terminal end to the C-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a rigid peptide linker as described in SEQ ID NO: 37.

[0449] ADC-Hir8 comprises a molecule of hirudin as described in SEQ ID NO: 15 covalently linked by its N- terminal end to the C-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a flexible peptide linker as described in SEQ ID NO: 30.

[0450] ADC-rtPA 1 comprises a molecule of alteplase's serine protease as described in SEQ ID NO: 19 covalently linked by its N-terminal end to the C-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a flexible peptide linker as described in SEQ ID NO: 30.

[0451] ADC-rtPA 2 comprises a molecule of alteplase's serine protease as described in SEQ ID NO: 19 covalently linked by its N-terminal end to the C-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a rigid peptide linker as described in SEQ ID NO: 37.

[0452] ADC-rtPA-3 comprises a molecule of alteplase's serine protease as described in SEQ ID NO: 19 covalently linked by its C-terminal end to the N-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a flexible peptide linker as described in SEQ ID NO: 30.

[0453] ADC-rtpa-4 comprises a molecule of alteplase's serine protease as described in SEQ ID NO: 19 covalently linked by its C-terminal end to the N-terminal end of the heavy chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a rigid peptide linker as described in SEQ ID NO: 37.

[0454] ADC-rtPA-5 comprises a molecule of alteplase's serine protease as described in SEQ ID NO: 19 covalently linked by its N-terminal end to the C-terminal end of the light chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a flexible peptide linker as described in SEQ ID NO: 30.

[0455] ADC-rtPA-6 comprises a molecule of alteplase's serine protease as described in SEQ ID NO: 19 covalently linked by its C-terminal end to the N-terminal end of the light chain of an anti-fibrin IgGl antibody comprising a VH sequence as set forth in SEQ ID NO: 7 and a VL sequence as set forth in SEQ ID NO: 8, by a flexible peptide linker as described in SEQ ID NO: 30.

[0456] The chimeric molecules comprise a peptide signal in N-terminus of each polypeptide chain, as described in SEQ ID NO: 9 and 10, respectively.

[0457] RESULTS

[0458] Example 1. Development of antibody-drug conjugates targeting fibrin to provide a localized anticoagulation.

[0459] The inventors generated recombinant antibody-drug conjugates (ADCs) comprising a mouse anti-fibrin antibody (59D8), humanized in its Fc region. The antibody was coupled or not to two molecules of recombinant hirudin (Hir), a potent direct thrombin inhibitor originally isolated from leech, Hirudo medicinalis or to a recombinant tissue-type plasminogen activator (rtPA) (Figure 1). The positioning of the hirudin and rtPA molecules and the linkers used to couple the ADCs, and the amino acid sequence of the anti-fibrin antibody, are detailed in the Material and Method section. The ADCs coupled to Hir or rtPA were produced in Chinese hamster ovary (CHO) cells with a yield ranging from 21 to 52 mg / L, while r59D8 was expressed at a yield of 120 mg / L (data not shown). The ADCs showed a high level of purity exceeding 95%, as evidence by SDS-PAGE analysis (data not shown), confirming that nearly all antibody chains were properly coupled to a Hir or rtPA molecule.

[0460] Example 2. Characterization of the anticoagulant effect and targeting potential of the ADCs.

[0461] To quantify precisely the inhibitory activity of hirudin linked to ADC-HirlO, the inventors used a fluorogenic assay measuring thrombin activity and compared it to hirudin alone (Figure 2A). With the two curves obtained with hirudin concentrations, the linear regression's slopes for each molecule between 0 and 12 ng / ml of hirudin, were -5.82 x 10-2for hirudin and -3.8 x 10-2for ADC-HirlO, indicating that at a similar hirudin concentration ADC-HirlO inhibits 65% of all thrombin molecules inhibited by hirudin alone. From there, for all in vivo models comparing hirudin to ADC-HirlO, hirudin doses were adjusted to have the same enzymatic activity for both molecules. Using an aPTT-based assay, the inventors provide evidence that ADC-HirlO significantly delays the coagulation time at 5 pg / ml (ADC- HirlO ([hir]: 0 pg / ml): 32.9 ± 1.3 s; ADC-HirlO ([hir]: 5 pg / ml): 119 ± 1.8 s; p= 0.0078) (Figure 2B).

[0462] The inventors next determined the potential of ADC-HirlO to target fibrin. Perfusing the fluorophore- coupled ADC-HirlO, r59D8 or 59D8 through a microfluidic chamber coated with fibrin showed a similar time-dependent increase in fluorescence, with a signal that was limited to the fibrin fibres (data not shown). This binding to fibrin was not observed when the isotypical antibody MOPC-21 was perfused.

[0463] Together, these results indicate that the ADC-HirlO presents an anticoagulant effect, and appears to be particularly efficient in targeting fibrin and a fibrin-rich thrombus both in vitro and in vivo.

[0464] Example 3. Pharmacokinetic of ADC-HirlO in mice.

[0465] The pharmacokinetic of ADC-HirlO in mice was determined by using a plasma aPTT assay, which evaluated the potential to prolong plasma clotting 1, 6, 24 and 30 hours post IV injection. The inventors observed that the ADC-HirlO significantly delayed coagulation time up to 30 hours post-injection, compared to r59D8 (r59D8 ([hir]: 0 mg / kg): 27.9 ± 2.5 s; 36.1 ± 4.4 s; p=0,0253). Concerning the administration of hirudin alone, while a very high dose (10 mg / kg) delayed more significantly coagulation time 1 hour post-injection compared to coagulation time before injection, it returned to basal levels 6 hours after injection (Hirudin (TO): 28.02 ± 2.8 s; Hirudin (Tl): 120 ± 0 s; Hirudin (T6): 32.9 ± 5.2 s; p= 0.0139 (TO vs Tl); p= 0.6468 (TO vs T6)) (Figure 3). This experiment highlights the increased stability of the ADC-HirlO compared to hirudin, even when administered at a hundred times higher dose.

[0466] Example 4. Efficacy of ADC-HirlO in preventing thrombosis in pulmonary embolism model.

[0467] To evaluate a dose dependant anti-thrombotic effect of ADC-HirlO, it was evaluated in a pulmonary embolism model in which ADC-HirlO was injected IV before the highly procoagulant tissue factor. Blood samples were collected before any injection, and 2 min after TF IV injection to identify platelet consumption (Figure 4A). The inventors identified the smallest hirudin dose (0.015 mg / kg) for ADC- HirlO, able to significantly decrease the percentage of platelets consumed compared to r59D8 (r59D8: 73.1± 19.2 %; ADC-HirlO ([Hir]: 0.015 mg / kg): 26.9 ± 9.6%; p= 0.0002 (r59D8 vs ADC-HirlO ([Hir]: 0.015 mg / kg);; (Fig. 4A). An equivalent dose of hirudin (0.01 mg / kg) significantly decreased platelet consumption as well. Nevertheless, platelet consumption was significantly higher than mice injected with ADC-HirlO (r59D8 ([hir]: 0 mg / kg): 73.1 ± 19.2 %; ADC-HirlO ([hir]:0.015 mg / kg): 26.9 ± 9.6 %; hirudin (0.01 mg / kg): 49.8 ± 26%; p= <0.0005 (r59D8 vs ADC-HirlO); p=0.0413 (ADC-HirlO vs hirudin and r59D8 vs hirudin)) (Fig. 4B).

[0468] Example 5. Impact of tinzaparin in mice and its antithrombotic effect compared to ADC-HirlO. Tinzaparin is a low molecular weight heparin used in clinic. Tinzaparin is commonly used to prevent and treat deep vein thrombosis (DVT) or pulmonary embolism (PE). The inventors thus characterized the impact on haemostasis and the antithrombotic effect of this molecule to be able to compare it properly to ADC-HirlO.

[0469] To evaluate the impact that tinzaparin has on haemostasis, tail bleeding time was performed on mice by a cut to the tail. One hour before cutting the tail, mice were injected with tinzaparin doses ranging from 75 to 300 Ul / kg. It appeared that tinzaparin at 75 Ul / kg did not significantly increase tail bleeding time, blood loss and total bleeding time compared to NaCI, while tinzaparin at 175 and 300 Ul / kg increased significantly the total bleeding time and the blood loss compared to control (Fig. 9A).

[0470] To evaluate a dose dependant anti-thrombotic effect of tinzaparin, 75 or 300 Ul / kg of tinzaparin were injected subcutaneously one hour before injecting the highly procoagulant tissue factor (TF). Blood samples were collected before TF injection, and 2 min after TF IV injection to identify platelet consumption. Tinzaparin at 75 Ul / kg did not decrease platelet consumption (Fig. 9B). Nevertheless, platelet consumption was significantly lower in mice injected with 300 Ul / kg compared to control (Fig. 9B). Through this experiment, 300 Ul / kg was the dose chosen to compare tinzaparin with ADC-HirlO in the pulmonary embolism model.

[0471] The pulmonary embolism model was used to compare ADC-HirlO and tinzaparin's antithrombotic effect. Mice were injected subcutaneously with NaCI for ADC-HirlO and r59D8 conditions, or tinzaparin 300 Ul / kg one hour before starting the experiment. One hour later, a first blood sample was collected. Then mice were injected RO with r59D8 ([hir]: 0 mg / kg), ADC-HirlO ([hir]: 0.05 mg / kg) or NaCI for tinzaparin condition. Tissue factor (TF) was injected intravenously and 2 minutes later, the second blood sample was collected. Both doses decreased platelet consumption significantly compared to control. ADC-HirlO decreases more efficiently platelet consumption compared to tinzaparin at their respective doses (Figure 9C).

[0472] Example 6. Effect of ADC-HirlO in a mouse model for venous thrombosis based on the transient inhibition of natural anticoagulants.

[0473] Wild type mice were injected with siRNA targeting natural anticoagulants protein C and antithrombin in the TV, which without further intervention results in venous thrombosis, as showed by Heestermans et al. Blood 2019; 133: 2090-2099. Importantly, development of venous thrombosis (VT) depended on thrombin, as showed by Safdar et al. Blood 2013; 121: 4413-4416 . This VT phenotype is scored macroscopically by haemorrhages and swellings around the mandibular area. 22 hours after the first injection, so before the mice develop VT, r59D8 (control group, concentration) or ADC-HirlO at 0.45, and 0.05 mg / kg of hirudin concentration were injected in the tail vein. In the following hours the mice were weighted and checked regularly to score for development of venous thrombosis (VT). Mice in the control group developed venous thrombosis at an expected rate, based on the results from pilot experiments. Remarkably, mice treated with 0.45 mg / kg ADC-HirlO appeared to be completely protected from VT (r59D8: 7 / 8 affected vs [Hir] 0.45 mg / kg: 0 / 8 affected, p=0.001). Mice injected with ADC-HirlO at 0.05 mg / kg of hirudin, displayed a lower frequency of thrombosis as compared to the control group (2 / 8 mice affected, p= 0.041,) (Fig 5. A and B). These results seemingly correspond with a condition in which mice are partly protected from venous thrombosis. The only group of mice that had a significant weight loss between the moment siRNAs were injected (0 hours) up to their sacrifice (31 hours) were the control group (r59D8) (P=0.047) (Fig 5.C). In the control group, the number of circulating platelets was also significantly lower compared to mice treated with ADC-HirlO (P= <0.0001and 0.006, compared to 0.45and 0.05 mg / kg of hirudin, respectively) (Fig. 5.D).

[0474] The same blood sample was then centrifuged to collect plasma. Serum amyloid A (SAA) protein, that increases in an inflammation context, was measured in plasma. Usually, inflammation and thrombosis are closely related. Inflammation parameters tend to increase when a thrombus forms. All groups treated with ADC-HirlO had a decrease in SAA concentration compared to r59D8 mice (data not shown). This indicates that ADC-HirlO has an effect on inflammation and reduces thrombosis.

[0475] Remarkably, the individual mice that developed venous thrombosis in the 0.05 mg / kg of hirudin group showed a number of circulating platelets comparable to the control group. In a parallel experiment, siRNA-injected mice were injected with un-complexed Hir just before onset of the thrombotic phenotype. Most likely because of the rapid turnover of Hirudin in the mouse circulation, the inventors did not see any differences in onset of venous thrombosis between the control group and the hirudin- treated group (6 / 8 vs. 7 / 8 mice affected, respectively, p=1.00, data not shown). In conclusion, in a severe thrombin-dependent mouse model for venous thrombosis ADC-HirlO prevents thrombosis in a dose-dependent manner.

[0476] Example 7. Impact of ADC-HirlO on haemostasis in mice.

[0477] To evaluate the impact ADC-HirlO has on haemostasis, tail bleeding time was performed on mice by a cut to the tail. Shortly before cutting the tail, mice were injected with hirudin doses on ADC-HirlO ranging from 0.01 to 0.05 mg / kg. It appeared that ADC-HirlO did not significantly increase tail bleeding time, blood loss and total bleeding time compared to r59D8 (Figure 6).

[0478] Example 8. Effect of ADC-HirlO in the arterial mouse model of FeCI3injury model on the carotid artery.

[0479] To evaluate the antithrombotic effect of ADC-HirlO compared to hirudin at an equivalent enzymatic hirudin dose, the FeCU injury model on the carotid artery was used. A dose effect of hirudin was made (data not shown) to determine a dose of hirudin that had a decreased antithrombotic effect in this model. This dose was used to compare hirudin and ADC-HirlO at an equivalent enzymatic dose of hirudin. After having injected DlOCg, r59D8 ([hir]: 0 mg / kg), ADC-HirlO ([hir]: 0.1 mg / kg) or hirudin (0.065 mg / kg), were injected before inducing thrombosis. Then, for 20 min, thrombus formation was observed through platelet fluorescence thanks to DIOC6. ADC-HirlO mice formed thrombi of lower size, with an area under curve and a maximal platelet area that are significantly lower compared to hirudin and control (Fig. 10A and B)

[0480] The impact of ADC-HirlO on hemostasis at the dose used in FeCU injury model was evaluated in a tail bleeding time model. Shortly before cutting the tail, r59D8 ([Hir]: 0 mg / kg), ADC-HirlO ([hir]: 0.1 mg / kg) or hirudin (0.065 mg / kg) injected RO in mice. At this dose, ADC-HirlO increased the total bleeding time compared to control and hirudin. However, it did not increase the blood loss. It means that the mice bled for a longer time but not abundantly. Therefore, ADC-HirlO had a moderate impact on hemostasis at this dose (Figure 11).

[0481] Example 9. Chimeric molecules comprising rtPA have an enzymatic effect

[0482] As demonstrated in Figure 7, three ADCs-rtPA induced fibrinolysis: ADC-rtPA3, 4 and 6. These constructions have a rtPA molecule fused in N-terminus of the light chain or heavy chain of the antifibrin antibody. r59D8 and ADC-rtPA 1, did not induce any fibrinolysis. ADC-2 and 5 induced a weak fibrinolysis, under the capacity of free rtpa. This constructions have a rtPA molecule fused in C-terminus of the light chain or heavy chain of the anti-fibrin antibody and are therefore different from the claimed constructions.

[0483] Similar results were obtained in another experiment (Figure 8). ADC-rtPA3, 4 and 6 show an enzymatic activity. The three molecules did not have a different affinity compared to rtPA alone.

[0484] ADC-rtPA3 had an increased catalytic activity and ADC-rtPA3 and 4 had an increased catalytic efficiency compared to rtPA in absence of fibrin. ADC-rtPA3 and 4 have both a rtPA molecule fused in N-terminus of the heavy chain of the anti-fibrin antibody.

Claims

Claims1. A chimeric molecule comprising:(i) an anti-fibrin antibody or an antigen binding fragment thereof; and(ii) one or more antithrombotic molecule(s), said antithrombotic molecule being a protein, a polypeptide or a peptide; wherein the C-terminal end of the antithrombotic molecule is covalently linked to the N-terminal end of the heavy and / or light chain of the anti-fibrin antibody or antigen binding fragment thereof.

2. The chimeric molecule of claim 1, wherein the anti-fibrin antibody or an antigen binding fragment thereof comprises:- a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and- a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion.

3. The chimeric molecule of claim 1 or 2, wherein the anti-fibrin antibody or an antigen binding fragment thereof comprises or consists of (a) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 85% sequence identity thereto and (b) a light chain variable domain (VL) comprising or consisting of anamino acid sequence of SEQ ID NO: 8 or a mutant thereof having at least 85% sequence identity thereto, .wherein the amino acid variations are outside of the CDRs.

4. The chimeric molecule of any one of claims 1-3, wherein the antithrombotic molecule is an anticoagulant molecule.

5. The chimeric molecule of any one of claims 1-4, wherein the antithrombotic molecule is selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any variant or fragment thereof.

6. The chimeric molecule of any one of claims 1-5, wherein the antithrombotic molecule is hirudin, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having at least 85 % sequence identity thereto.

7. The chimeric molecule of any one of claims 1-3, wherein the antithrombotic molecule is a thrombolytic molecule.

8. The chimeric molecule of claim 7, wherein the thrombolytic molecule is a plasminogen activator selected from the group consisting of alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof.

9. The chimeric molecule of claim 7, wherein the thrombolytic molecule is alteplase's serine protease comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof having at least 85 % sequence identity thereto.

10. The chimeric molecule of any one of claims 1-9, wherein the chimeric molecule comprises one or more antithrombotic molecule(s) that are anticoagulant molecules and / or thrombolytic molecules, and wherein:- the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules, preferably hirudin, and the N-terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several thrombolytic molecules, preferably alteplase or a fragment thereof, or vice-versa; or- the N-terminal end of the heavy chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules, preferably hirudin, and to one or several thrombolytic molecules, preferably alteplase or a fragment thereof; or- the N-terminal end of the light chain of the anti-fibrin antibody or an antigen binding fragment thereof is covalently linked to one or several anticoagulant molecules, preferably hirudin, and to one or several thrombolytic molecule(s), preferably alteplase or a fragment thereof.

11. The chimeric molecule of any one of claims 1-10, wherein:- the antithrombotic molecule is covalently linked to the anti-fibrin antibody or antigen binding fragment thereof by a non-cleavable peptide linker, preferably comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 27-44; and / or- the chimeric molecule comprises one or more peptide signal, said peptide signal being in N- terminus of the antithrombotic molecule and comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 9 if the antithrombotic molecule is covalently linked to the N-terminus of the heavy chain variable domain or in SEQ ID NQ:10 if the antithrombotic molecule is covalently linker to the N-terminus of the light chain variable domain, or any variant of SEQ ID NO 9 or 10 comprising one, two or three amino acid modifications selected from substitution, addition or deletion or any combination thereof.

12. The chimeric molecule of any one of claims 1-6 and 11, wherein the molecule comprises or consists of : a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting :- a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and- a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an aminoacid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more hirudin molecule, preferably comprising or consisting of SEQ ID NO: 15; wherein the C-terminal end of the hirudin molecule is covalently linked to the N-terminal end of the heavy chain of the anti-fi brin antibody or antigen binding fragment thereof, preferably by a peptide linker, said peptide linker preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 27-44, more preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30.

13. The chimeric molecule of any one of claims 1-3 and 7-11, wherein the molecule comprises or consists of: a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting :- a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and- a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more molecule of alteplase's serine protease, preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 19; wherein the C-terminal end of the hirudin molecule is covalently linked to the N-terminal end of the light chain of the anti-fibrin antibody or antigen binding fragment thereof, preferably by a peptidelinker, said peptide linker preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 27-44, more preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30.

14. The chimeric molecule of any one of claims of any one of claims 1-3 and 7-11, wherein the molecule comprises or consists of: a) an anti-fibrin antibody or an antigen binding fragment thereof comprising or consisting :- a heavy-chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a heavy chain complementary determining region (HCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 2 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a heavy chain complementary determining region (HCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 3 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and- a light-chain variable domain (VL) comprising a light chain complementary determining region (LCDR1) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 4, optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, a light chain complementary determining region (LCDR2) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 5 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion, and a light chain complementary determining region (LCDR3) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 optionally with one, two or three modifications selected from the group consisting of amino acid substitution, addition, and deletion; b) one or more o molecule of alteplase's serine protease, preferably comprising or consisting of an amino acid sequence as described in SEQ ID NO: 19; wherein the C-terminal end of the hirudin molecule is covalently linked to the N-terminal end of the heavy chain of the anti-fibrin antibody or antigen binding fragment thereof, preferably by a peptide linker, said peptide linker preferably comprising or consisting of an amino acid sequence as set forth in SEQ ID NO : 27-44, more preferably in SEQ ID NO: 27-35, even more preferably of SEQ ID NO: 30.

15. An isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding the chimeric molecule according to any one of claims 1-14.

16. A vector, comprising the nucleic acid or group of nucleic acid molecules according to claim 15.

17. A host cell, comprising the vector according to claim 16 or the nucleic acid or group of nucleic acid molecules of claim 15.

18. A method for producing the chimeric molecule according to any one of claims 1-14, comprising a step of culturing a host cell according to claim 17 and optionally a step of isolating the chimeric molecule.

19. A pharmaceutical composition comprising the chimeric molecule according to any one of claims 1-14, the nucleic acid or group of nucleic acid molecules according to claim 15, the vector of claim 16, or the host cell of claim 17.

20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition comprises: a) at least two chimeric molecules wherein a first chimeric molecule comprises one or more anticoagulant molecules, preferably hirudin, fused to the anti-fibrin antibody or antigen binding fragment thereof, and a second chimeric molecule comprises a thrombolytic agent, preferably alteplase or a fragment thereof fused to the anti-fibrin antibody or antigen binding fragment thereof; and / or b) one or more additional therapeutic agent, preferably an antithrombotic agent selected from (i) a thrombolytic agent selected from the group consisting of tissue-type plasminogen activator (t-PA), alteplase (rt-PA), reteplase (r-PA), Tenecteplase (TNK-tPA), anistreplase, desmoteplase, streptokinase, urokinase (u-Pa), staphylokinase and any variant or fragment thereof; (ii) an anticoagulant agent selected from the group consisting of hirudin, bivalirudin, lepirudin, desirudin and any variant or fragment thereof, Argatroban, Heparin, UFH, LMWH, Fondaparinux, Danaparoid, Direct oral anticoagulants (DOACs) such as Dabigatran and Rivaroxaban, and Vitamin K antagonists such as warfarin and (iii) any combination thereof.

21. The pharmaceutical composition according to claim 19 or 20, for use as a medicament.

22. The pharmaceutical composition for use according to claim 21, for use in the prevention or treatment of thrombosis or a disease or disorder associated with thrombosis.

23. Use of the chimeric molecule of any one of claims 1-14 or of the pharmaceutical composition according to claim 19 or 20 in the manufacture of a medicament for treating thrombosis or a disease or disorder associated with thrombosis in a subject in need thereof.

24. A method for treating thrombosis or a disease or disorder associated with thrombosis in a subject in need thereof, comprising administering a therapeutic effective amount of the chimericmolecule of any one of claims 1-15 or of the pharmaceutical composition according to any one of claim 19 or 20.

25. The use of claim 21 or 22 or the method of claim 23, wherein the disease or disorder associated with thrombosis is associated with venous thrombosis or arterial thrombosis.

26. The use of claim 25 or the method of claim 25, wherein the disease or disorder associated with thrombosis is selected from the group consisting of ischemic stroke, myocardial infarction, venous thromboembolism, deep vein thrombosis, pulmonary embolism and peripheral arterial occlusion.

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