Anti-protein S single-domain antibody and polypeptide containing the same

Abstract

Vitamin K-dependent protein S (PS) is a natural anticoagulant that acts as a nonenzymatic cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). We identified an anti-PS nanobody that significantly enhances the APC-cofactor activity of PS through an unknown mechanism. Interestingly, this nanobody exerts antithrombotic effects in injured mesenteric microvessels of mice. Consequently, it constitutes a novel class of antithrombotic agent that may be used to treat acute microthrombosis in pathological conditions such as sepsis, COVID-19, distal microvascular thrombosis due to stroke, or sickle cell disease. Therefore, the present invention relates to isolated single-domain antibodies (sdAbs) against protein S (PS) and polypeptides containing them.

Description

[Technical Field]

[0001] The present invention relates to single-domain antibodies on the conformation of anti-protein S (PS), polypeptides containing the same, and their use in the therapeutic field in particular. [Background technology]

[0002] Vitamin K-dependent protein S (PS) is a natural anticoagulant that acts as a non-enzymatic cofactor for both activated protein C (APC) and tissue factor pathway inhibitors (TFPIs). Historically, PS has been described as enhancing the proteolytic activity of APC against activated factor V (FVa) and activated factor VIII (FVIIIa), thereby very effectively limiting thrombin production. PS has also been reported to enhance the inhibitory effect of TFPI-α on FVa (Hackeng et al. 2006), although the physiological relevance of such cofactor activity is not precisely known. Furthermore, PS has recently been described as a direct inhibitor of activated factor IX (FIXa) (Plautz et al. 2018). The physiological importance of PS is demonstrated by the clinical symptoms observed in patients with PS deficiency. Mild PS deficiency is associated with an increased risk of venous thrombosis, while severe PS deficiency results in a dramatic and life-threatening thrombotic phenotype (i.e., fulminant purpura with microvascular thrombosis and disseminated intravascular coagulation, particularly in cutaneous blood vessels). In mice, complete PS deficiency leads to embryonic lethality due to severe thrombocoagulation disorders and massive intracerebral hemorrhage (Saller et al. 2009; Burstyn-Cohen et al. 2009). In hemophilic mice, PS is highly expressed in the joints, which may partly explain the highly anticoagulant environment observed in hemophilic joints (Prince, Bologna et al. 2018). Interestingly, the absence of PS or its pharmacological inhibition by polyclonal antibodies results in a significant reduction of hemophilia.

[0003] Therefore, the inventors aimed to develop nanobodies that are targeted to PS as an original and powerful tool for regulating the anticoagulant activity of PS. Nanobodies, or single-domain antibodies (sdAbs), are the variable region (VHH) of heavy-chain-only antibodies (HcAbs) found in camelids. Despite their small size of 15 kDa, isolated nanobodies can perfectly recognize their own antigens. Furthermore, their small size and physicochemical properties offer various advantages compared to conventional immunoglobulins. For example, they maintain high stability even after being separated from the residue of the original HcAb. They are also highly soluble and are thought to have excellent in vivo tissue penetration. As a result, nanobodies have emerged as a novel and promising type of therapeutic antibody. Moreover, they can be expressed in E. coli, and by combining them with other nanobodies, polyvalent or polyspecific species can be easily generated. One advantage of nanobodies is that, compared to conventional monoclonal antibodies, they can recognize latent epitopes via a prominent complementarity-determining region 3 (CDR3). Therefore, the inventors hypothesized that by using anti-PS nanobodies, it might be possible to identify original antibodies that can unexpectedly modulate the anticoagulant activity of PS.

[0004] There is still a need to develop safer antithrombotic drugs. In fact, currently used antiplatelet drugs (e.g., aspirin and clopidogrel) or anticoagulants (e.g., heparin derivatives, warfarin) are known to increase the risk of bleeding because they inhibit the physiological hemostatic response. In contrast, enhancing the anticoagulant activity of APC with anti-PS nanobodies may have only a minor effect on hemostasis and may not increase the risk of bleeding.

[0005] Physiological agents such as high-density lipoprotein (Griffin et al. 1999; Fernandez et al. 2015), cardiolipin (Fernandez et al. 2000), or skeletal muscle myosin (Heeb et al. 2019) have been reported to enhance the anticoagulant activity of APC. However, no drug that enhances the anticoagulant activity of APC has yet been developed. Interestingly, a pharmacokinetic activator of protein C is currently being studied as a candidate antithrombotic agent. This activator is the thrombin variant W215A / E217A (WE thrombin or AB002), which has lost its coagulogenic properties but can activate protein C into APC (Cantwell et al. 2000). Systemic administration of exogenous APC or systemic activation of protein C by high levels of soluble thrombomodulin in patients (Dargaud et al. Blood 2015) is associated with an increased bleeding tendency, whereas administration of AB002 only slightly impairs hemostasis (Gruber et al. 2002). This is thought to be due to the local action of AB002 on the serum surface, where APC may be purified in situ by AB002, limiting its escape into circulation (Gruber et al. 2007). This may explain why AB002 has been described as potently inhibiting thrombus propagation without significant systemic anticoagulant effects in various animal models of thrombosis (Gruber et al. 2002; Tucker et al. Blood 2020).

[0006] The nanobody for PS developed by the present inventors can be proposed for the treatment of acute conditions in which microvascular thrombosis, such as sepsis or stroke, is the main pathogenesis. [Overview of the project]

[0007] The inventors utilized a platform developed at UMR_S1176, which allows screening of a large library of nanobodies generated from PS-immunized llamas, to identify anti-PS nanobodies. They identified a highly surprising anti-PS nanobody that enhances PS-APC cofactor activity through an unknown mechanism. Intriguingly, this nanobody exerts antithrombotic activity in the mesenteric microvessels of injured mice. As a result, it constitutes a novel class of antithrombotic agents for the treatment of acute microthrombosis in conditions such as sepsis, COVID-19, or stroke-induced distal microvascular thrombosis.

[0008] Accordingly, the present invention refers to isolated single-domain antibodies (sdAbs) against protein S (PS) and polypeptides comprising the same. In particular, the present invention is defined by the claims. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows schematic diagrams of the monovalent and divalent nanobodies used in this study. The oligonucleotide sequences of the monovalent PS003 and KB013 nanobodies were cloned into the pET28 plasmid between the restriction enzyme sites of PstI and BstEII to generate nanobodies flanked by an N-terminal His6 tag and a C-terminal HA tag sequence. Two oligonucleotide sequences of the monovalent nanobodies (PS003, KB004, PS004) were fused via a (GGGS)4 linker, synthesized, and cloned into the pET28 plasmid between the PstI and BstEII sites to similarly generate divalent nanobodies (PS003biv, KB004biv, PS004biv) flanked by an N-terminal His6 tag and a C-terminal HA tag sequence. [Figure 2]Figure 2 shows the binding of PS003 to various vitamin K-dependent proteins in ELISA. Recombinant human FIX (FIX), recombinant human FX (FX), plasma-derived protein Z (ProZ), recombinant human Gas6 (Gas6), and recombinant human PS (PS) were immobilized in ELISA wells, and the binding affinity of 20 nM PS003 was analyzed. [Figure 3] Figure 3 shows the binding of PS003 to PS and Gas6 in ELISA. PS and Gas6 have high homology (47% homology) and both contain SHBG-like domains, so the binding of PS003 to immobilized rhPS and rhGas6 was further analyzed directly by ELISA. The results showed that PS003 binds strongly to rhPS but not to rhGas6, confirming the specificity of PS003 to rhPS. [Figure 4] Figure 4 shows the epitope mapping of PS003. rhPS, a recombinant SHBG-like domain of PS (rhSHBG), and BSA were immobilized (60 μL at 10 μg / mL in TBS containing 5 mM CaCl2), and the binding of PS003 was analyzed directly by ELISA. These results suggest that PS003 can bind to rhSHBG, and that the epitope of PS003 is localized within the C-terminal SHBG-like domain of PS. On the other hand, PS004 did not bind to rhSHBG (data not shown). This suggests that the epitope of PS004 is localized in the N-terminal region of PS. [Figure 5] Figure 5 shows the binding of recombinant human and plasma-derived PS to immobilized PS003 in solution in ELISA. Purified PS003 (60 μL at 10 μg / mL) was immobilized in ELISA wells, and the binding of two different forms of PS was analyzed. These results indicate that PS003 binds to either recombinant or plasma-derived human PS, and that the binding of PS003 to PS is not limited to non-native immobilized forms of PS. [Figure 6]Figure 6 shows a comparison of the binding of PS003 and PS003biv to immobilized PS in direct ELISA. rhPS (60 μL at 2.5 μg / mL in TBS containing 5 mM CaCl2) was immobilized in ELISA wells, and the binding of PS003 and PS003biv (0-200 nM) was analyzed with a peroxidase-conjugated anti-His6 tagged polyclonal antibody. Three individual experiments were performed in a simple manner, and the results were expressed as a percentage of the maximum binding amount for each nanobody. The binding curves showed that both PS003 and PS003biv efficiently bound to immobilized rhPS. To further compare the binding ability of PS003 and PS003biv to PS, the affinity of PS003 and PS003biv to rhPS was evaluated as described (Beatty et al. J Immunol Methods 1987) by obtaining similar binding curves for rhPS immobilized at increasing concentrations (0.6, 1.25, and 5 mg / mL in TBS containing 5 mM CaCl2) in three simplified individual experiments. For each nanobody, the dissociation constant (KD) was determined using an equation based on the law of mass action. Based on this method, the KDs of PS003 and PS003biv were 26.8±2.7 nM and 13.8±5.7 nM, respectively, suggesting that PS003biv binds to rhPS with a slightly higher affinity (1.9 times). [Figure 7]Figure 7 shows the epitope mapping of PS003biv and the specificity of PS003biv to PS. Recombinant human PS (rhPS), recombinant PS containing only the SHBG-like region (rSHBG), recombinant human Gas6 (rhGas6), or BSA (60 μL at 10 mg / mL in TBS containing 5 mM CaCl2) were immobilized in ELISA wells, and the binding of PS003biv (0.5 nM in TBS-0.1% Tween-5 mM CaCl2) was analyzed using a peroxidase-conjugate anti-His6 tagged polyclonal antibody. The results are expressed as the percentage of Abs450 nm obtained on rhPS. Three individual experiments were simplified and performed. As a result, PS003biv efficiently bound to rSBHG, and therefore, the epitope of PS003biv was localized within the SHBG-like region of PS. Since this region is only found in Gas6, the fact that PS003biv does not bind to rhGas6 strongly suggests that PS003biv is specific to PS. [Figure 8A-D]Figures 8A-D show the enhancing effects of PS003 and PS003biv on APC cofactor activity in APTT-based plasma coagulation assays (STACLOT® PS, Stago). Figure 8A shows the results of measuring the ability of rhPS (final concentration 5 nM) to act as a cofactor for APC using a commercially available APTT-based plasma coagulation assay (STACLOT® PS, Stago). In this assay, APC prolonged the coagulation time of PS-deficient plasma, and 5 nM rhPS, when added together with APC, further prolonged the coagulation time. Figure 8B shows the dose-dependent effect of rhPS (final concentration 0-10 nM) in our APTT-based APC cofactor activity assay. Figure 8C shows the results of testing the effects of PS003 and PS003biv using the ability of rhPS (final concentration 6 nM) to enhance the anticoagulant activity of APC. PS003, KB013 (control monovalent nanobody), PS003biv, and KB004biv (control divalent nanobody) were pre-incubated with rhPS at room temperature for 15 minutes, and a mixture of rhPS ± nanobody was added in our assay. The final concentrations of rhPS and nanobody were 6 nM and 2 μM, respectively. The experiment was performed in triplicates. In Figure 8D, the results are expressed as the ratio of the coagulation time in the presence of rhPS (t+PS) to the coagulation time in the absence of rhPS (t-PS). An independent Student's t-test was used for statistical testing. The results showed that both PS003 and PS003biv enhanced the APC-cofactor activity of rhPS, and PS003biv had a greater enhancing effect on rhPS-APC-cofactor activity than PS003. [Figure 9A-B]Figures 9A-B show the effects of PS003 and PS003biv on the APC-cofactor activity of PS in an in vitro FVa inactivation assay. The ability of PS003 and PS003biv to enhance the APC-cofactor activity of rhPS was evaluated using purified protein in an in vitro assay that measured the specific proteolytic inactivation of FVa by APC in the presence of rhPS. In Figure 9A, the slope was determined for each rhPS concentration in the FVa inactivation mixture, and the FVa activity value was expressed as the ratio of the slope obtained in the presence of rhPS to the slope obtained in the absence of rhPS. Three simplified experiments were performed. In Figure 9B, residual FVa activity was measured for each condition using the prothrombinase assay as described above, and compared with the FVa activity obtained when rhPS was pre-incubated in the absence of nanobodies or antibodies (TBS). Three simplified experiments were conducted, and an independent Student's t-test was used for statistical analysis (***P<0.001). [Figure 10A-C] Figures 10A-C show the effects of PS003 and PS003biv on the TFPI-cofactor activity of rhPS. An in vitro assay was developed to evaluate the ability of rhPS to enhance the direct inhibition of FXa by IFPIα. In Figure 10A, recombinant human full-length TFPIα expressed in E. coli was used at a final concentration of 5 nM to inhibit the amide degradation activity of FXa. In Figure 10B, the ability of rhPS to enhance the inhibitory activity of TFPIα after pre-incubation for 15 minutes at room temperature with blocking rabbit polyclonal anti-PS antibody (α-PS) (DAKO, final concentration 0.5 μM) or rabbit IgG (DAKO, final concentration 0.5 μM) was investigated. Figure 10C evaluates the ability of rhPS to enhance TFPIα inhibitory activity after pre-incubation for 15 minutes at room temperature with PS003 and PS003biv, or their respective monovalent (KB013) and bivalent (KB004biv) control nanobodies (final concentration 10 μM). Results are expressed as a percentage of rhPS relative to TFPIα-cofactor activity in the absence of nanobodies (TBS). Three simplified experiments were performed, and independent Student's t-tests were used for statistical testing. [Figure 11]Figure 11 shows a comparison of the binding of PS003biv and PS004biv to immobilized mouse PS in direct ELISA. PS004biv is a proprietary anti-human PS nanobody generated from a monovalent nanobody (PS004) identified by selecting rhPS immobilized in an ELISA well. PS004biv binds strongly to rhPS in direct ELISA, but in contrast to PS003biv, its epitope is localized within the N-terminus of PS and not within the SHBG-like domain of PS (data not shown). The binding of PS003biv and PS004biv to immobilized rhPS was analyzed by ELISA. The results showed that PS003biv bound to rmPS, but PS004biv did not. This suggests that PS004biv, along with PS003biv, can be used as a control bivalent nanobody in our in vivo FeCl3-induced thrombosis model. [Figure 12A-B]Figures 12A-B show the in vivo antithrombotic effect of PS003biv in a mouse FeCl3-induced thrombosis model. FeCl3 injury was induced in 4-5 week old C57BL6 / JRccHsd male mice essentially as previously described (Ayme et al. 2017; Adam et al. 2010). To facilitate visualization of thrombus formation, platelets from anesthetized mice were fluorescently labeled in vivo by intravenous infusion of rhodamine 6G (3.3 mg / kg, i.e., 2.5 μL / g at 1 mg / mL in 0.9% NaCl) into the posterior orbital plexus. PS003biv (10 mg / kg), PS004biv (10 mg / kg), or a colleague's TBS buffer (Ctl) was administered simultaneously, diluted in 0.9% NaCl. Alternatively, low molecular weight heparin (LMWH, Robenox) 200 UI / Kg was subcutaneously injected after intravenous administration of rhodamine 6G. After circulating labeled platelets for 10 minutes, FeCl3 solution (10% in water) was locally administered into the mesenteric vessels, and thrombus growth was observed in real time using an inverted epifluorescence microscope (×10). One vein and one artery were analyzed for each mouse. Statistical analysis was evaluated by the Kruskal-Wallis and Dunn tests. In Figure 12A, the control bivalent anti-VWF (KB004biv) used in our APC-cofactor activity assay could not be used in the FeCl3-induced thrombosis model because treatment of mice with this nanobody resulted in a delay in occlusion time in the veins and arterioles of one mouse. Therefore, a control bivalent anti-PS nanobody (PS004biv) that cannot bind to recombinant mouse PS was used. Our thrombosis model was sensitive to anticoagulants, as treatment with LMWH (200 UI / kg, SC) delayed occlusion time in both veins and arterioles (n=6 mice). Treatment with PS004biv (n=6 mice) did not affect occlusion time, but treatment with PS003biv resulted in a significant delay in occlusion time in veins (n=10 mice). A similar trend was observed in arterioles of mice treated with PS003biv (n=9 mice), but the difference was not statistically significant.Figure 12B shows that in the mesenteric vessels of mice administered PS003biv, thrombus stability was lower and embolization occurred at a higher rate compared to thrombi formed in the mesenteric vessels of mice not administered nanobodies (not shown) or control mice administered PS004biv nanobodies. [Figure 13] Figure 13 shows the effect of PS003biv on physiological hemostasis in a mouse tail clip bleeding model. Anesthetized C57 / BL6 mice were either intravenously injected with PS003biv (10 mg / kg) or subcutaneously injected with low molecular weight heparin (LMWH) (Lovenox, 200 UI / kg). Bleeding time was defined as the time to the first cessation of bleeding. Blood samples were also collected over 20 minutes to quantify the total blood loss. Each bar represents the mean value obtained from multiple mice evaluated. Standard one-way ANOVA with Turkey's multiple comparison test was used for statistical analysis of variance. [Modes for carrying out the invention]

[0010] Protein S (PS) is a natural anticoagulant that acts as a cofactor for activated protein C (APC) and tissue factor pathway inhibitors (TFPIs). The inventors hypothesized that regulating PS activity would be an effective approach in treating coagulation disorders. Therefore, they created an immunotherapy library of single-domain antibodies (sdAbs) from llamas immunized with human PS and selected sdAbs that target PS using phage display.

[0011] The inventors identified an sdAb that strongly binds to PS, exhibits anticoagulant activity in vitro, and shows antithrombotic activity in vivo.

[0012] (definition) As used herein, the terms "protein S" or "PS" have their general meaning in the art and refer to a vitamin K-dependent plasma glycoprotein primarily synthesized in the liver. Protein S exists in the circulatory system in two forms: free and complex, bound to complement protein C4b-binding protein (C4BP). In humans, protein S is encoded by the PS1 gene. Protein S is a natural anticoagulant that acts as a non-enzymatic cofactor for both activated protein C (APC) and tissue factor pathway inhibitors (TFPI). In fact, PS has historically been reported to enhance the proteolytic activity of APC against activated factor V (FVa) and activated factor VIII (FVIIIa), and to very effectively limit thrombin production. PS has also been widely reported to enhance the inhibitory effect of TFPIα on FXa (Hackeng et al. 2006), although the physiological relevance of such cofactor activity is not precisely understood. Furthermore, PS has recently been described as a direct inhibitor of activating factor IX (FIXa) (Plautz et al. 2018).

[0013] As used herein, the term “single-domain antibody” has its general meaning in the art and refers to a single heavy-chain variable domain of an antibody of the type found in camelid mammals, naturally lacking a light chain. Such single-domain antibodies are also called VHH or “nanobody®”. For a general description of (single) domain antibodies, refer to the prior art cited above, as well as European Patent No. 0368684, Ward et al. (Nature 1989 Oct 12;341(6242):544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and International Publication Nos. 06 / 030220 and 06 / 003388. Nanobodies have a molecular weight about one-tenth that of a human IgG molecule, and the physical diameter of the protein is only a few nanometers. As a result of their small size, camel nanobodies have the ability to bind to antigen sites that are functionally invisible to larger antibody proteins. Camel nanobodies are useful as reagents for detecting antigens undetectable by conventional immunological methods, and also as therapeutic agents. Therefore, because nanobodies can exert biological effects by binding to specific sites in grooves or narrow fissures of target proteins, they can exhibit capabilities closer to those of classical low-molecular-weight drugs than classical antibodies. Furthermore, due to their low molecular weight and compact size, nanobodies are highly heat-resistant, stable against extreme pH and proteolysis, and exhibit low antigenicity. In addition, nanobodies readily migrate from the circulatory system to tissues, and some can cross the blood-brain barrier, allowing them to treat disorders affecting nerve tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. (See U.S. Patent Application No. 2004 / 0161738, published August 19, 2004.) These characteristics, coupled with low antigenicity to humans, indicate significant therapeutic potential.The amino acid sequence and structure of a single-domain antibody are thought to be composed of four framework regions or "FRs", which are referred to in the art and in this specification as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively. These framework regions are interrupted by three complementarity-determining regions or "CDRs", which are referred to in the art as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3", respectively. Thus, a single-domain antibody can be defined as an amino acid sequence having the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 represent framework regions 1 to 4, and CDR1 to CDR3 represent complementarity-determining regions 1 to 3. In the context of the present invention, the amino acid residues of the single-domain antibody are numbered according to the general numbering for the VH domain provided by the international ImMunoGeneTics information system amino acid numbering (http: / / imgt.cines.fr / ).

[0014] As used herein, the term "amino acid sequence" has its general meaning and is the sequence of amino acids that confers the primary structure on a protein. According to the present invention, an amino acid sequence can be modified with one, two or three conservative amino acid substitutions without a significant loss of interaction binding ability. "Conservative amino acid substitution" means that an amino acid can be substituted with another amino acid having a similar side chain. Families of amino acids having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0015] According to the present invention, a first amino acid sequence having at least 70% identity to a second amino acid sequence means that the first sequence has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to the second amino acid sequence. The identity of amino acid sequences is typically determined using a suitable sequence alignment algorithm such as BLAST P (Karlin and Altschul, 1990) and default parameters.

[0016] According to the meaning of this invention, "identity" is calculated by comparing two sequences aligned within a comparison window. Sequence alignment allows for the determination of the number of common positions (nucleotides or amino acids) between two sequences within the comparison window. The identity percentage is then obtained by dividing the number of common positions by the total number of positions within the comparison window and multiplying by 100. The determination of the sequence identity percentage can be done manually or by a well-known computer program.

[0017] As used herein, the terms “purification” and “isolation” refer to the sdAb of the present invention and mean the presence of the sdAb in the substantial absence of other biomolecules of the same type. As used herein, the term “purification” means the presence of at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and even more preferably at least 98% by weight of the antibody, relative to the total weight of the polymer present.

[0018] As used herein, the term “nucleic acid molecule” has its general meaning in the art and refers to a DNA or RNA molecule.

[0019] As used herein, the term “specifically binding” means that, when evaluated using recombinant proteins, their epitopes, or native proteins present on the surface of isolated target cells, the antibody binds only to the target antigen, such as protein S (PS), and does not cross-react to other antigens.

[0020] (Single-domain antibodies and polypeptides) The target sequence in this application is shown in Table 1 below.

[0021] [Table 1]

[0022] In a first embodiment, the present invention relates to an isolated single-domain antibody (sdAb) against protein S (PS).

[0023] In a first embodiment, the present invention relates to an isolated single-domain antibody (sdAb) that specifically binds to protein S (PS).

[0024] In some embodiments, the isolated single-domain antibody according to the present invention is a PS agonist antibody.

[0025] As used herein, the term "PS agonist" antibody means an antibody that exhibits PS activity. According to the present invention, a "PS agonist" antibody means an antibody that can enhance the APC cofactor activity of PS, that is, an antibody that can enhance the proteolytic activity of APC against activated factor V (FVa) and activated factor VIII (FVIIIa). According to the present invention, a "PS agonist" antibody means an antibody that can enhance the anticoagulant activity of protein S.

[0026] Therefore, in some embodiments, the isolated single-domain antibody according to the present invention enhances the APC-cofactor activity of PS.

[0027] In some embodiments, the isolated single-domain antibodies according to the present invention bound to either recombinant or plasma-derived human PS and did not significantly interfere with their TFPI-cofactor activity.

[0028] According to the present invention, a single-domain antibody against PS enhances the APC-cofactor activity of PS.

[0029] In some embodiments, the isolated single-domain antibody according to the present invention exhibits antithrombotic activity.

[0030] As used herein, the term "antithrombotic activity" has a general meaning in the art and refers to activity that inhibits the formation of thrombi. According to the present invention, isolated single-domain antibodies inhibit the formation of thrombi and / or dissolve thrombi.

[0031] Tests for determining the ability of antibodies exhibiting antithrombotic activity are well known to those skilled in the art. Tests for determining the ability of antibodies to specifically enhance the APC-cofactor activity of PS are well known to those skilled in the art and include thrombus-based assays such as prothrombin time (PT) assays, activated partial thromboplastin time (APTT) assays (see Figures 8C-8D), specific one-step coagulation assays, calibrated automated thrombinography (CAT) or other thrombin generation assays, FVa inactivation assays (see Figure 9) and FVIIIa inactivation assays, and TFPIα-cofactor activity assays (see Figure 10).

[0032] In particular, the present invention relates to an isolated single-domain antibody (sdAb) comprising CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3 ("PS003 derivative").

[0033] In some embodiments, the isolated single-domain antibody according to the present invention has at least 70% identity with respect to the sequence of SEQ ID NO: 4 ("PS003 derivative").

[0034] In some embodiments, the isolated single-domain antibody according to the present invention has at least 70% identity to the sequence of SEQ ID NO: 4 and includes CDR1, CDR2, and CDR3 of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0035] In some embodiments, the isolated single-domain antibody according to the present invention contains the sequence of SEQ ID NO: 4 ("PS003").

[0036] In some embodiments, the isolated single-domain antibody according to the present invention has the sequence of SEQ ID NO: 4.

[0037] Furthermore, it is worth noting that sdAb "PS003" also enhances the APC-cofactor activity of PS.

[0038] Furthermore, it is noteworthy that sdAb "PS003" did not significantly inhibit the TFPI-cofactor activity of recombinant or plasma-derived human PS when it bound to them.

[0039] In some embodiments, the isolated single-domain antibody is a "humanized" single-domain antibody.

[0040] As used herein, the term “humanized” means a single-domain antibody of the present invention whose amino acid sequence is “humanized” to correspond to the amino acid sequence of a naturally occurring VHH domain, that is, “humanized” by substituting one or more amino acid residues (particularly the framework sequence) in the amino acid sequence of a naturally occurring VHH sequence with one or more amino acid residues located at the corresponding position in the VH domain of a conventional chain antibody from humans. Methods for humanizing single-domain antibodies are well known in the art. Typically, it is desirable that the humanization substitutions be selected such that the resulting humanized single-domain antibody still retains the desirable properties of the single-domain antibody of the present invention. Those skilled in the art can determine and select an appropriate humanization substitution or a suitable combination of humanization substitutions.

[0041] In some embodiments, the single domain of the present invention is conjugated with further therapeutic agents used to treat anticoagulant disorders.

[0042] Further aspects of the present invention provide cross-competitive single-domain antibodies that cross-compete for binding of the single-domain antibody of the present invention to PS. In some embodiments, the cross-competitive single-domain antibody of the present invention cross-competes for binding of PS to a single-domain antibody comprising CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3.

[0043] In some embodiments, the cross-competitive single-domain antibody of the present invention cross-competes with the binding of PS to a single-domain antibody containing or consisting of the sequence of SEQ ID NO: 4.

[0044] As used herein, the term “cross-competition” means single-domain antibodies that share the ability to bind to a specific region of an antigen. In this disclosure, “cross-competing” single-domain antibodies have the ability to interfere with the binding of another single-domain antibody to an antigen in a standard competitive binding assay. Such single-domain antibodies may, according to a non-limiting theory, bind to the same, related, or nearby (e.g., structurally similar or spatially adjacent) epitopes as the competing single-domain antibody. Cross-competition exists if the binding of single-domain antibody A to single-domain antibody B is reduced by at least 60%, specifically at least 70%, and more specifically at least 80%, compared to a positive control lacking one of the single-domain antibodies, and vice versa. As those skilled in the art will understand, competition can be evaluated in different assay setups. One suitable assay involves the use of Biacore technology (e.g., by using a BiAcore3000 instrument (Biacore, Uppsala, Sweden)) which can measure the degree of interaction using surface plasmon resonance technology. Another assay for measuring cross-competition employs an ELISA-based approach. Furthermore, a high-throughput process for "binning" antibodies based on antibody cross-competition is described in International Patent Application No. 2003 / 48731.

[0045] According to the present invention, the cross-competitive antibody described above retains the activity of a single antibody comprising CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3.

[0046] According to the present invention, the cross-competitive antibody described above retains the activity of a single antibody containing or consisting of the sequence of SEQ ID NO: 4.

[0047] Therefore, in some embodiments, the cross-competitive single-domain antibody of the present invention is a PS agonist antibody.

[0048] In some embodiments, the cross-competitive single-domain antibodies of the present invention enhance the APC-cofactor activity of PS.

[0049] In some embodiments, the cross-competitive single-domain antibodies of the present invention bound to either recombinant or plasma-derived human PS and did not significantly inhibit their TFPI-cofactor activity.

[0050] A further aspect of the present invention relates to a polypeptide comprising at least one single-domain antibody of the present invention.

[0051] Typically, the polypeptide of the present invention comprises a single-domain antibody of the present invention, which is fused to at least one further amino acid sequence at its N-terminus, its C-terminus, or both its N-terminus and C-terminus, i.e., fused to provide a fusion protein. According to the present invention, a polypeptide comprising a single single-domain antibody is referred to herein as a “monovalent” polypeptide. A polypeptide comprising, or essentially consisting of, two or more single-domain antibodies according to the present invention is referred to herein as a “polyvalent” polypeptide. Typically, a polyvalent polypeptide may be a divalent, trivalent, or tetravalent antibody.

[0052] In some embodiments, the polypeptides of the present invention enhance the APC-cofactor activity of PS.

[0053] In some embodiments, the polypeptides of the present invention bound to either recombinant or plasma-derived human PS and did not significantly inhibit their TFPI-cofactor activity.

[0054] In some embodiments, the polypeptide comprises at least one single-domain antibody of the present invention and at least one other binding unit (i.e., directed to other epitopes, antigens, targets, proteins, or polypeptides), which is typically also a single-domain antibody. Such polypeptides are referred herein as "polyspecific" polypeptides, in contrast to polypeptides containing the same single-domain antibody ("monospecific" polypeptides). Accordingly, in some embodiments, the polypeptide of the present invention may provide at least one further binding site directed to any desired protein, polypeptide, antigen, antigenic determinant, or epitope. The binding site may be directed to the same protein, polypeptide, antigen, antigenic determinant, or epitope as directed in the single-domain antibody of the present invention, or it may be directed to a different protein, polypeptide, antigen, antigenic determinant, or epitope than that of the single-domain antibody of the present invention.

[0055] In some embodiments, the polypeptide of the present invention comprises at least one single-domain antibody having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3.

[0056] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies, CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3.

[0057] In some embodiments, the polypeptide of the present invention comprises two, three, four, or five single-domain antibodies, each containing a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3.

[0058] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having at least 70% identity with respect to the sequence of SEQ ID NO: 4.

[0059] In some embodiments, the polypeptide of the present invention has at least 70% identity with the sequence of SEQ ID NO: 4 and comprises at least two single-domain antibodies including CDR1, CDR2, and CDR3 of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0060] In some embodiments, the polypeptide of the present invention comprises at least two single-domain antibodies having the sequence of SEQ ID NO: 4.

[0061] In some embodiments, the polypeptide of the present invention comprises two, three, four, or five single-domain antibodies having the sequence of SEQ ID NO: 4.

[0062] In some embodiments, the polypeptide of the present invention comprises a sequence having at least 70% identity with the sequence of SEQ ID NO: 5 ("PS003biv derivative").

[0063] In some embodiments, the polypeptide of the present invention comprises the sequence of SEQ ID NO: 5 ("PS003biv").

[0064] In some embodiments, the polypeptide of the present invention has the sequence of SEQ ID NO: 5 ("PS003biv").

[0065] In some embodiments, single-domain antibodies of the polypeptide of the present invention may be bound to each other directly (i.e., without the use of a linker) or via a linker. The linker is typically a linker peptide and, according to the present invention, may be selected to allow binding of the two single-domain antibodies to each of at least two different epitopes in the PS. The appropriate linker depends particularly on the epitopes, specifically the distance between the epitopes in the PS to which the single-domain antibodies bind, and will be evident to those skilled in the art after some limited routine experiments as arbitrarily described herein. Alternatively, two single-domain antibodies that bind to the PS may be linked to each other via a third single-domain antibody (in this case, the two single-domain antibodies may be bound directly to the third single-domain antibody or via a suitable linker). Such a third single-domain antibody may be, for example, a single-domain antibody that results in an increased half-life. For example, the latter single-domain antibody may be a single-domain antibody that can bind to (human) serum proteins such as (human) serum albumin or (human) transferrin, as further described herein. In some embodiments, two or more single-domain antibodies conjugated to PS are linked in series (directly or via a suitable linker), and a third (single) single-domain antibody (which may provide an increased half-life as described above) is linked directly or via a linker to one of these two or more single-domain antibodies. A suitable linker consists of an amino acid sequence, which is described herein in relation to a particular polypeptide of the present invention and is not limited to, for example, nine or more amino acids, more preferably at least 17 amino acids, and may have a length of, for example, about 20 to 40 amino acids. However, the upper limit is not definitive and is selected for convenience reasons, for example, with regard to the production of biopharmaceuticals of such polypeptides. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to whom the anti-EGFR polypeptide of the present invention is administered.A useful group of linker sequences are those derived from the hinge region of heavy-chain antibodies, as described in International Publications 96 / 34103 and 94 / 04678. Other examples include polyalanine linker sequences such as Ala-Ala-Ala. Further preferred examples of linker sequences include Gly / Ser linkers of different lengths, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0066] The “bispecific” polypeptide of the present invention is a polypeptide comprising at least one single-domain antibody against a first antigen (i.e., protein S, PS) and at least one further binding site against a second antigen (i.e., different from PS), and the “triply specific” polypeptide of the present invention is a polypeptide comprising at least one single-domain antibody against a first antigen (i.e., PS), at least one further binding site against a second antigen (i.e., different from PS), and at least one further binding site against a third antigen (i.e., different from both the first and second antigens).

[0067] In some embodiments, the additional binding site is directional to a serum protein so as to increase the half-life of the single-domain antibody. Typically, the serum protein is albumin.

[0068] Typically, one or more additional binding sites may include one or more portions, fragments, or domains of conventional chain antibodies (and especially human antibodies) and / or heavy chain antibodies. For example, the single-domain antibodies of the present invention may optionally bind to conventional (typically human) VH or VL via a linker sequence.

[0069] In some embodiments, the polypeptide comprises a single-domain antibody of the present invention conjugated to an immunoglobulin domain. For example, the polypeptide comprises a single-domain antibody of the present invention conjugated to an Fc moiety (e.g., human Fc). The Fc moiety may be useful in increasing the half-life and production of the single-domain antibody of the present invention. For example, the Fc moiety can bind to serum proteins and thus increase the half-life of the single-domain antibody. In some embodiments, at least one single-domain antibody may optionally be conjugated to one or more (typically human) CH1 and / or CH2 and / or CH3 domains via a linker sequence. For example, a single-domain antibody conjugated to a suitable CH1 domain may be used, for example, with a suitable light chain to generate an antibody fragment / structure similar to a conventional Fab fragment or F(ab')2 fragment, but in which one or (in the case of an F(ab')2 fragment) one or both of the conventional VH domains are replaced with the single-domain antibody of the present invention. In some embodiments, one or more single-domain antibodies of the present invention may be conjugated (optionally via appropriate linker or hinge regions) to one or more constant domains (e.g., two or three constant domains that can be used as part of / to form an Fc moiety), an Fc moiety, and / or to one or more antibody moieties, fragments, or domains that confer one or more effector functions to the polypeptide of the present invention, and / or may be conferred the ability to bind to one or more Fc receptors. For example, for this purpose, and not limited thereto, one or more further amino acid sequences may include one or more CH2 and / or CH3 domains of an antibody, such as from a heavy chain antibody, more typically from a conventional human chain antibody, and / or may form an Fc region from, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), from IgE, or from other human Ig such as IgA, IgD, or IgM.For example, International Publication No. 94 / 04678 describes heavy chain antibodies containing a camelid VHH domain or its humanized derivative (i.e., a single-domain antibody), each providing a single-domain antibody and an immunoglobulin consisting of two heavy chains containing human CH2 and CH3 domains (but not a CH1 domain), wherein the camelid CH2 and / or CH3 domains are substituted with human CH2 and CH3 domains, and this immunoglobulin has effector function due to the CH2 and CH3 domains and can function without the presence of any light chains.

[0070] In some embodiments, the polypeptide is as described in International Publication No. 2006 / 064136. In particular, the polypeptide may consist of i) a first fusion protein in which the CL constant domain of an antibody is fused to the C-terminus of a single-domain antibody according to the present invention (i.e., a single antibody against PS), and ii) a second fusion protein in which the CH1 constant domain of an antibody is fused to the C-terminus of a single-domain antibody against an antigen different from PS, with its N-terminus being fused. In other specific embodiments, the polypeptide consists of a first fusion protein in which the CH1 constant domain of an antibody is fused to the C-terminus of a single-domain antibody against an activation trigger molecule on effector cells (e.g., CD16), with its N-terminus being fused to the C-terminus of a single-domain antibody according to the present invention (i.e., PS).

[0071] In some embodiments, the polypeptides of the present invention are conjugated into further therapeutic agents used for the treatment of thrombotic disorders.

[0072] In some embodiments, the single-domain antibody or polypeptide of the present invention used in the therapeutic method of the present invention may be modified to improve its therapeutic effect. Such modifications of the therapeutic compound may be used to reduce toxicity, increase circulation time, or alter biodistribution. For example, the toxicity of a potentially important therapeutic compound can be significantly reduced by combining it with various drug carrier vehicles that alter biodistribution.

[0073] One strategy for improving drug viability is the use of water-soluble polymers. Various water-soluble polymers have been shown to alter in vivo distribution, improve cellular uptake, change physiological barrier permeability, and alter clearance rates from the body. Water-soluble polymers have been synthesized to obtain targeted or sustained-release effects by incorporating the drug moiety as terminal groups, part of the backbone, or pendant groups in the polymer chain.

[0074] Polyethylene glycol (PEG) is widely used as a drug carrier due to its high biocompatibility and ease of modification. Its attachment to various drugs, proteins, and liposomes has been shown to lead to improved residence times and reduced toxicity. While PEG can bind to activators via hydroxyl groups at the ends of its chain and other chemical methods, PEG itself is limited to a maximum of two activators per molecule. In a different approach, copolymers of PEG and amino acids have been explored as novel biomaterials that maintain PEG's biocompatibility while offering the advantages of having numerous binding sites per molecule (allowing for more drug loading) and being synthetically designed to suit various applications. Those skilled in the art are familiar with PEGylation techniques for effective drug modification. For example, drug delivery polymers consisting of alternating polymers of PEG and trifunctional monomers such as lysine have been used by VectraMed (Plainsboro, NJ). PEG chains (typically less than 2000 Daltons) bind to the α- and α-amino groups of lysine via stable urethane bonds. Such copolymers maintain the desirable properties of PEG and provide reactive pendant groups (carboxylic acid groups of lysine) at predetermined intervals, tightly controlled along the polymer chain. These reactive pendant groups can be used for derivatization, crosslinking, or conjugation to other molecules. These polymers are useful for producing stable, long-circulating prodrugs by modifying the polymer molecular weight, the PEG segment molecular weight, and the cleavable bond between the drug and the polymer. The PEG segment molecular weight affects the drug / conjugate interval and the amount of drug per molecular weight of the conjugate (smaller PEG segments result in a higher drug load). Generally, increasing the overall molecular weight of a block copolymer conjugate increases the circulating half-life of the conjugate. Nevertheless, the conjugate must have a molecular weight below a threshold that is easily degraded or limits pharyngeal filtration (e.g., less than 45 kDa).Furthermore, in addition to the polymer backbone, which is important for maintaining circulating half-life and biodistribution, linkers may be used to maintain the therapeutic agent in the form of a prodrug until it is released from the backbone polymer by a specific trigger, typically enzymatic activity in the target tissue. For example, this type of tissue-activated drug delivery is particularly useful when delivery to a specific site of biodistribution is required, and the therapeutic agent is to be released at or near the lesion site. Libraries of binding groups for use in activated drug delivery are well known to those skilled in the art and can be obtained based on enzyme kinetics, the prevalence of the active enzyme, and the cleavage specificity of selected disease-specific enzymes (see, for example, the techniques established by VectraMed, Plainsboro, NJ). Such linkers may be used to modify the polypeptides of the present invention described herein for therapeutic delivery.

[0075] According to the present invention, the single-domain antibodies and polypeptides of the present invention can be produced by conventional automated peptide synthesis methods or recombinant expression. The general principles for designing and producing proteins are well known to those skilled in the art.

[0076] The single-domain antibodies and polypeptides of the present invention can be synthesized in solution or on a solid support according to the prior art. Various automated synthesizers are commercially available and can be used according to known protocols, as described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. Alternatively, the single-domain antibodies and polypeptides of the present invention can be synthesized by solid-phase technology employing exemplary peptide synthesizers such as the Model 433A from Applied Biosystems Inc. The purity of a given protein produced by automated peptide synthesis or recombinant methods can be determined by reverse-phase HPLC analysis. Chemical certification of each peptide can be established by methods well known to those skilled in the art.

[0077] In other embodiments, the single-domain antibodies and polypeptides of the present invention are modified to increase their biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced, as described in U.S. Patent No. 6,277,375 by Ward: T252L, T254S, T256F. Alternatively, to increase the biological half-life, the antibody may be modified within the CH1 or CL region to include a salvage receptor-binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and 6,121,022 by Presta et al. Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.immunol.24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions therein that improve binding to FcRn in the Fc region. Such Fc variants include those having one or more substitutions of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0078] Another modification of the single-domain antibody or polypeptide of the present invention as envisioned by the present invention is pegylation. Antibodies can be pegylated, for example, to increase their biological (e.g., serum) half-life. To pegylate an antibody, the antibody or a fragment thereof is reacted with PEG, such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more polyethylene glycol (PEG) groups are attached to the antibody or antibody fragment. Pegylation can be carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to include any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegyling proteins are well known in the art and can be applied to the antibodies of the present invention. For example, one can refer to European Patent No. 0154316 by Nishimura et al. and European Patent No. 0401384 by Ishikawa et al.

[0079] Other modifications of the single-domain antibody or polypeptide of the present invention envisioned by the present invention include a conjugate or fusion protein of at least the antigen-binding domain of the antibody of the present invention with a serum protein such as human serum albumin or a fragment thereof, and the modification is intended to increase the half-life of the resulting molecule. Such an approach is described, for example, by Balance et al. in European Patent No. 0322094. Another possibility is to fusion at least the antigen-binding domain of the antibody of the present invention with a protein capable of binding to serum proteins such as human serum albumin to increase the half-life of the resulting molecule. Such an approach is described by Nygren et al. in European Patent No. 0486525.

[0080] Polysialic acid (PSA) is a technology that uses the naturally occurring polymer polysialic acid (PSA) to extend the activity lifetime and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (sugar). When used in drug delivery of proteins and therapeutic peptides, polysialic acid provides a protective microenvironment during conjugation. This increases the activity lifetime of circulating therapeutic proteins and prevents them from being recognized by the immune system. PSA polymers are found naturally in the human body. They have been adopted by certain species of bacteria that have evolved over millions of years to coat their walls with them. These naturally polysialylated bacteria can cover the body's defense systems through branching mimicry. PSA is the ultimate natural stealth technology and can be easily produced in large quantities with specific physical properties from such bacteria. Bacterial PSA is chemically identical to PSA in the human body and is therefore completely non-immunogenic when bound to proteins.

[0081] Other techniques include the use of antibody-conjugated hydroxyethyl starch (HES) derivatives. HES is a modified natural polymer derived from waxy maize starch and can be metabolized by enzymes in the body. HES solutions are typically administered to supplement deficient blood volume and improve the rheological properties of the blood. Hesylation of antibodies can enhance their bioactivity by increasing molecular stability and extending their circulating half-life by reducing renal clearance. Various HES antibody conjugates can be customized by changing various parameters such as the molecular weight of HES.

[0082] (Nucleic acids, vectors, recombinant host cells, and their use) Recombinant DNA technology is used as an alternative to automated peptide synthesis. This involves inserting a nucleotide sequence encoding a selected protein into an expression vector, transforming or transfecting it into suitable host cells, and culturing them under conditions suitable for expression, as described below. Recombinant methods are particularly preferred for producing longer polypeptides.

[0083] Various expression vectors / host systems can be used to express sequences encoding peptides or proteins. These include, but are not limited to, recombinant bacteriophages, microorganisms such as bacteria transformed with plasmids or cosmid DNA expression vectors, yeast transfected with yeast expression vectors (Giga-Hama et al., 1999), insect cell lines infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002), plant cell lines transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmid, see Babe et al., 2000), or animal cell lines. Those skilled in the art are aware of various techniques for optimizing mammalian protein expression, see, for example, Kaufman, 2000 and Colosimo et al., 2000. Mammalian cells useful for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (COS-7, etc.), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are well known to those skilled in the art and are briefly described below. Mammalian host systems for recombinant protein expression are also well known to those skilled in the art. Host cell lines may be selected for their ability to process the expressed protein or to produce certain post-translational modifications useful for providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Also, post-translational processes that cleave the "prepro" form of the protein may be receptive to correct insertion, folding, and / or function. Various host cells, such as CHO, HeLa, MDCK, 293, and WI38, possess specific cellular and characteristic mechanisms for such post-translational activity and can be selected to ensure the correct modification and processing of introduced foreign proteins.

[0084] In the recombinant production of the single-domain antibody and polypeptide of the present invention, it may be necessary to use a vector containing a polynucleotide molecule for encoding the single-domain antibody and polypeptide of the present invention. Methods for preparing such vectors and for generating host cells transformed with such vectors are well known to those skilled in the art.

[0085] Therefore, a further subject of the present invention is nucleic acid molecules encoding single-domain antibodies and / or polypeptides according to the present invention.

[0086] Typically, the nucleic acids are DNA or RNA molecules, which may be contained in appropriate vectors such as plasmids, cosmids, episomes, artificial chromosomes, phages, or viral vectors. As used herein, the terms “vector,” “cloning vector,” and “expression vector” mean a vehicle capable of introducing a DNA or RNA sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. The terms “expression vector,” “expression construct,” or “expression cassette” are used interchangeably throughout this specification and mean any type of gene construct containing nucleic acids in which part or all of the nucleic acid coding sequence encodes a transcribed gene product.

[0087] Therefore, a further aspect of the present invention relates to a vector comprising the nucleic acid of the present invention. Such a vector may include regulatory elements such as promoters, enhancers, and terminators for causing or inducing the expression of the antibody upon administration to a subject. Examples of promoters and enhancers used in expression vectors in animal cells include the initial promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney's mouse leukemia virus (Kuwana Y et al. 1987), and the promoter and enhancer of immunoglobulin H chain (Mason JO et al. 1985) (Gillies SD et al. 1983). Any expression vector for animal cells can be used as long as it can insert and express the gene encoding the human antibody C region. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other examples of plasmids include replication plasmids containing origins of replication, or integrated plasmids such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenovirus, retrovirus, herpesvirus, and AAV vectors. Such recombinant viruses can be generated by techniques well known in the art, such as transfection of packaging cells or transient transfection of helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, in International Publication No. 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5882877, U.S. Patent No. 6013516, U.S. Patent No. 4861719, U.S. Patent No. 5278056 and International Publication No. 94 / 19478.

[0088] The selection of an expression vector suitable for the expression of the peptide or polypeptide of the present invention naturally depends on the specific host cell used and is within the scope of the art for those skilled in the art.

[0089] Expression requires that the vector be supplied with appropriate signals, such as enhancers / promoters from both viral and mammalian sources, which can be used to promote the expression of the target nucleic acid in the host cell. Typically, the nucleic acid to be expressed is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism necessary to initiate a specific transcription of a gene. A nucleotide sequence is operably ligated if the regulatory sequence is functionally relevant to the DNA encoding the target protein (e.g., a single-domain antibody). Thus, if a promoter nucleotide sequence directs the transcription of a sequence, the promoter nucleotide sequence is operably ligated to a given DNA sequence.

[0090] Further aspects of the present invention relate to host cells transfected, infected, or transformed with nucleic acids and / or vectors according to the present invention.

[0091] The term "transformation" refers to the process of introducing an "external" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell, causing the host cell to express the introduced gene or sequence and produce a desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is considered "transformed."

[0092] The nucleic acids of the present invention can be used to produce the antibodies of the present invention in a suitable expression system. The term “expression system” means a host cell and a suitable vector under suitable conditions for the expression of a protein encoded by foreign DNA, which is delivered, for example, by a vector and introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established cell cultures (e.g., those produced from lymphoblasts, fibroblasts, embryonic cells, nerve cells, adipocytes, etc.). Furthermore, the present invention includes, for example, mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. The present invention also relates to a method for generating recombinant host cells expressing the antibody according to the present invention, the method comprising the following steps: (i) introducing a recombinant nucleic acid or vector as described above into competent host cells in vitro or ex vivo; (ii) culturing the obtained recombinant host cells in vitro or ex vivo; and (iii) optionally selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used for the generation of the antibody of the present invention.

[0093] The antibodies of the present invention can be appropriately separated from the culture medium by conventional immunoglobulin production methods such as protein A-Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0094] (Treatment methods and use) The single-domain antibodies and polypeptides of the present invention enhance APC but have little to no effect on the TFPI-cofactor activity of PS. The single-domain antibodies and polypeptides of the present invention exert in vivo antithrombotic activity in a mouse thrombosis model.

[0095] Therefore, the single-domain antibodies and polypeptides of the present invention are particularly suitable for the prevention or treatment of thrombotic disorders in subjects requiring them.

[0096] In yet another embodiment, the present invention relates to a single-domain antibody and / or polypeptide of the present invention for use as a pharmaceutical agent.

[0097] In certain embodiments, the present invention relates to single-domain antibodies and / or polypeptides of the present invention for treating thrombotic disorders in subjects requiring such treatment.

[0098] In other words, the present invention relates to a method for preventing or treating thrombotic disorders in a subject requiring such treatment, comprising administering an effective amount of the single-domain antibody and / or polypeptide of the present invention to the subject.

[0099] As used herein, the term “subject” means a mammal. In a preferred embodiment of the present invention, the subject according to the present invention means any subject (preferably human) that is suffering from or susceptible to thrombotic disorders.

[0100] As used herein, the term “thrombotic disorder,” also known as coagulation disorder or thrombosis, has a general meaning in the art and refers to a hereditary or acquired condition that increases the risk of excessive thrombus formation. When a blood vessel is damaged, blood begins to leak out or into the surrounding tissue. Normal coagulation is important at the time of injury because it stops bleeding at the wound site and initiates the healing process. However, when the blood tends to clot too much, it is called hypercoagulation or thrombosis. In healthy individuals, there is a homeostatic balance between procoagulant, anticoagulant, and fibrinolytic forces. Many genetic, acquired, and environmental factors can disrupt this balance favorable to coagulation, leading to pathological conditions such as thrombus formation in the veins (e.g., deep vein thrombosis (DVT)), arteries (e.g., myocardial infarction, ischemic stroke), or ventricles. Thrombi can obstruct blood flow at the site of formation or break off and embolize, blocking distant blood vessels (e.g., pulmonary embolism, embolic stroke). Acquired disorders are usually the result of surgery, trauma, medication, or medical conditions that increase the risk of thrombotic disorders.

[0101] According to the present invention, thrombotic disorders include mutations in the prothrombin gene, deficiencies in innate proteins that prevent coagulation such as antithrombin, protein C and protein S, increased levels of coagulation factors such as factor VII, factor IX and factor XI, fibrinolytic abnormalities such as factor V Leiden defect, hypoplasminogenemia, plasminogen disorders and increased levels of plasminogen activator inhibitor (PAI-1), fibrinolytic dysfunction, central venous catheterization, restenosis due to stents, obesity, hypercoagulation during pregnancy, antiphospholipid syndrome, cancer, homocystinemia, sticky platelet syndrome, pulmonary embolism (PE), myeloproliferative disorders such as polycythemia vera or essential platelet syndrome, paroxysmal nocturnal hemoglobinuria (PNH), and heparin-induced disorders. This includes iatrogenic thromboembolism such as thromboembolism induced by thrombocytopenia (HIT) or hemophilia treatment (emiguzimab, phytosilan, etc.), inflammatory bowel syndrome such as ulcerative colitis and Crohn's disease, acquired immunodeficiency syndrome (AIDS), COVID-19, nephrotic syndrome, acute microthrombosis, distal microvascular thrombosis, deep vein thrombosis (DVT), Paget-Schlotter disease, Budd-Chiari syndrome, portal vein thrombosis, renal vein thrombosis, cerebral venous sinus thrombosis, jugular vein thrombosis and cavernous vein thrombosis, embolic diseases such as marginal ischemia, sepsis, anemia, sickle cell disease, cerebral malaria, pulmonary embolism and cerebral embolism, and cardiovascular diseases such as stroke, myocardial infarction (heart attack), atrial fibrillation, coronary artery disease, congestive heart failure and implantation of artificial heart valves.

[0102] In some embodiments, the thrombotic disorder is selected from the group consisting of, but is not limited to, sepsis, sickle cell anemia, embolism (pulmonary and cerebral), and cardiovascular disease.

[0103] In some embodiments, the thrombotic disorder is sepsis or stroke.

[0104] In some embodiments, the thrombotic disorder is sickle cell anemia.

[0105] As used herein, the term “sepsis” has a general meaning in the art and refers to a serious medical condition characterized by a systemic inflammatory state. In addition to symptoms associated with an induced infection, sepsis is characterized by the presence of acute inflammation throughout the body, and is therefore frequently accompanied by fever and elevated (leukocytosis) or decreased (leukocytosis) white blood cell count and below-average body temperature, as well as vomiting. In particular, sepsis is defined as an abnormal immune response to infection and a life-threatening organ dysfunction, defined by a sequential organ failure score of 2 or higher. Infection may be suspected or proven, or the clinical syndrome may be characterized by an infection. Septic shock is defined by infection and the need for intravascular pressure to maintain mean arterial pressure above 65 mmHg and arterial lactate above 2 mmol / L.

[0106] As used herein, the term “stroke” means any condition resulting from an interruption, reduction, or cessation of the flow of blood or oxygen to any part of the brain. In particular, the term “stroke” includes, but is not limited to, ischemic stroke, transient ischemic attack (TIA), and hemorrhagic stroke.

[0107] As used herein, the term “embolism” refers to the retention of an embolus, a substance that causes blockage within a blood vessel. An embolus may be a thrombus (thrombus), a fat globule (fat embolus), a bubble of air or other gas (gas embolus), or a foreign body. Embolisms come in various forms, and in the context of this invention, embolism is caused by a thrombus and is selected from the group consisting of, but not limited to, arterial embolism, venous embolism, or paradoxical embolism. Generally, arterial embolism can cause blockage in any part of the body. It is a major cause of infarction (tissue death due to blockage of blood supply). When an embolus forms in the brain from the heart or carotid artery, it is likely to cause ischemic stroke. Generally, venous embolism refers to embolus formed in the veins throughout the body that pass to the right side of the heart and always affect the lungs. This can form pulmonary embolism, which blocks the major arteries of the lungs and can be a complication of deep vein thrombosis. The most common site of pulmonary embolism is the femoral vein. The most common site of actual blood clot formation is the deep veins of the calf. Typically, venous embolism results in pulmonary embolism or cerebral embolism.

[0108] As used herein, the term “cardiovascular disease” is also known as “arterial vascular disease” and is a general term used to classify a number of conditions affecting the heart, heart valves, blood, and vascular system of the body. It includes any disease affecting the heart or blood vessels, and includes, but is not limited to, metabolic syndrome, syndrome X, atherosclerosis, atherothrombosis, stable and unstable angina, stroke, diseases of the aorta and its branches (aortic stenosis, thrombosis, or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and all transient and permanent ischemic cardiovascular events. As used herein, arterial vascular disease generally does not mean non-ischemic disease, but most commonly means ischemic disease or pre-ischemic disease. As used herein, "atherosclerosis" and "atherothrombosis" refer to a systemic inflammatory disease state associated with a complex inflammatory response to multifaceted vascular pathology, including inflammatory leukocytes as a source of endothelial inflammation activation, thrombus formation stimulation, smooth muscle cells as an amplifier of the inflammatory response between coagulation-promoting substances and thrombosis, and platelets as a mediator of inflammation and thrombosis. Arteries harden and narrow due to the accumulation of a substance called "plaque" on their inner walls. As plaque develops and grows, the inside of the artery narrows ("stenosis"), impairing blood flow. Stenosis or plaque rupture can occlude part or all of the blood vessels in the affected area. As a result, tissue supplied by the blood vessels may be deprived of oxygen (ischemia) and undergo cell death (necrosis). "CAD" or "coronary artery disease" is an arterial vascular disease that develops when the arteries that supply blood to the heart muscle (coronary arteries) become atherosclerotic, calcified, and / or narrowed. When blood flow to the heart muscle decreases, the heart muscle may not receive enough oxygen, leading to necrosis, as blood carries a large amount of essential oxygen. CAD includes acute coronary syndrome (ACS), myocardial infarction (heart attack), angina pectoris (stable and unstable types), and conditions such as arteriosclerosis and atherothrombosis that occur in the blood vessels that supply a large amount of oxygen to the heart. "CVD" or "cerebrovascular disease" refers to arterial vascular diseases such as arteriosclerosis and atherothrombosis that occur in the blood vessels that supply oxygen-rich blood to the face and brain. This term is often used to mean "hardening" of the carotid arteries that supply blood to the brain.It is known as a comorbidity with CAD and / or PAD (peripheral artery disease). It is also called ischemic disease, or disease that causes insufficient blood flow. CVD includes conditions such as cerebral ischemia, acute ischemic stroke, stroke, ischemic ischemic stroke, hemorrhagic ischemic stroke, aneurysm, mild cognitive impairment (MCI), and transient ischemic attack (TIA). Ischemic CVD is thought to be closely related to CAD and PAD, while non-ischemic CVD can have multiple pathophysiological aspects.

[0109] As used herein, “sickle cell disease” or “SCD” has the general meaning in the art and refers to a hereditary blood disorder in which red blood cells take on an abnormal, rigid, sickle-shaped form. Sickling of red blood cells reduces cellular flexibility and consequently increases the risk of various life-threatening complications. The term includes sickle cell anemia, hemoglobin SC disease, and hemoglobin sickle β-salestemia. This monogenic disorder is characterized by variant hemoglobin S (HbS) and chronic intravascular hemolysis. Patients with sickle cell disease often experience episodes of acute pain due to vascular occlusive crisis (VOC). VOC is the most common complication of sickle cell anemia and is a frequent reason for emergency room visits and hospitalization.

[0110] As used herein, the term "vascular occlusive crisis" (VOC) has its general meaning in the art and refers to a condition in which the supply of oxygen to tissues is impaired due to occlusion of microvessels, causing injury. VOCs can be extremely painful and should be considered a medical emergency.

[0111] Here, the present inventors demonstrate that the single-domain antibody and polypeptide of the present invention can suppress vascular occlusive crisis in a mouse model.

[0112] In certain embodiments, the present invention relates to a single-domain antibody and / or polypeptide of the present invention for use in suppressing vascular occlusive crises in subjects requiring such suppression.

[0113] In some embodiments, the subjects suffer from sickle cell anemia.

[0114] In other words, the present invention relates to a method for preventing or treating vascular occlusive crisis (VOC) in a subject requiring such treatment, comprising administering an effective amount of the single-domain antibody and / or polypeptide of the present invention to the subject.

[0115] Typically, the single-domain antibodies and polypeptides of the present invention, as well as the classic treatments for thrombotic disorders as described above, are administered to a subject in a therapeutically effective amount. As used herein, the terms “treatment” or “to treat” mean both prophylactic or preventive treatment and curative or disease-modifying treatment, and include treatment to subjects at risk of or suspected of having the disease, and subjects diagnosed with suffering from the disease or condition, and include suppression of clinical relapse. Treatment may be administered to subjects with a medical disability or those likely to eventually acquire a disability, to prevent, cure, delay the onset, reduce the severity or improve one or more symptoms of the disability or recurrent injury, or to extend the subject’s survival time beyond what would be expected in the absence of such treatment. “Treatment regimen” means a treatment pattern for the disease, e.g., a pattern of administration used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The terms “induction regimen” or “induction period” mean a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. The general purpose of an induction regimen is to provide the subject with a high level of medication during the initial period of a treatment regimen. An induction regimen may employ a “loading regimen” (partially or entirely), which may include administering a larger dose of medication than the physician employs during the maintenance regimen, administering medication more frequently than the physician employs during the maintenance regimen, or both. The terms “maintenance regimen” or “maintenance period” refer to a treatment regimen (or part of a treatment regimen) used to maintain a subject during treatment for a disease, for example, to maintain the subject in remission for an extended period (several months or several years). A maintenance regimen may employ continuous treatment (e.g., administering medication at regular intervals such as weekly, monthly, or yearly) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment on relapse, or treatment when certain predetermined criteria [e.g., pain, disease onset, etc.] are met).

[0116] As used herein, “therapeutically effective amount” refers to the minimum amount of activator necessary to provide a patient with therapeutic benefit. For example, “therapeutically effective amount of activator” for a patient is the amount of activator that induces, enhances, or causes improvement in pathological symptoms, disease progression, or physical condition related to the disease affecting the patient. It will be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutically effective dose level for a particular patient will be determined by a variety of factors, including the patient’s age, weight, general health, sex and diet, the timing of administration of the particular compound to be employed, the route of administration and excretion rate, the duration of treatment, the agents used in combination with or concurrently with the particular polypeptide to be employed, and similar factors well known in the medical art. For example, it is well known to those skilled in the art to start administration of a compound at a level lower than the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can vary over a wide range of 0.01 to 1000 mg per adult per day. Preferably, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient to adjust the dosage to the patient being treated according to their symptoms. The pharmaceutical product typically contains about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg of the active ingredient. The effective dose is usually supplied at a dose level of 0.0002 mg / kg (body weight) to about 100 mg / kg (body weight) per day.

[0117] As used herein, the terms “administer” or “give” mean the act of injecting or otherwise physically delivering a substance (e.g., a nanobody or polypeptide according to the present invention) to a substance present outside the body, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or other methods of physical delivery described herein or known in the art. When a disease or its symptoms are to be treated, the administration of the substance is typically performed after the onset of the disease or its symptoms. When a disease or its symptoms are to be prevented, the administration of the substance is typically performed before the onset of the disease or its symptoms.

[0118] In other embodiments, the single-domain antibody and / or polypeptide according to the present invention may be delivered in association with a vector. The single-domain antibody or drug conjugate of the present invention is contained in a suitable vector such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. Thus, a further object of the present invention relates to a vector comprising the single-domain antibody or drug conjugate of the present invention. Typically, the vector is a viral vector, which is an adeno-associated virus (AAV), retrovirus, bovine papillomavirus, adenovirus vector, lentiviral vector, vaccinia virus, polyomavirus, or infectious virus. In some embodiments, the vector is an AAV vector. As used herein, the term “AAV vector” means a vector obtained from adeno-associated virus serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and their variants. AAV vectors have one or more AAV wild-type genes deleted, preferably all or part of the rep and / or cap genes, but may retain a functional flanking ITR sequence. Retroviruses can be selected as gene delivery vectors due to their ability to integrate their genes into the host genome, transport large amounts of foreign genetic material, infect a wide range of species and cell types, and package in specific cell lines. To construct a retroviral vector, a nucleic acid encoding the gene of interest is inserted into the viral genome in place of a specific viral sequence to create a non-replicating virus. To produce virions, a packaging cell line is constructed containing the gag, pol, and env genes but without the LTR and / or packaging components. When a recombinant plasmid containing cDNA is introduced into this cell line along with the retroviral LTR and packaging sequence (e.g., by calcium phosphate precipitation), the RNA transcript of the recombinant plasmid is packaged into viral particles by the packaging sequence and secreted into the culture medium. The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene delivery. Retroviral vectors can infect a wide variety of cells.Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. This higher complexity allows the virus to modulate its life cycle, such as during latent infection. Examples of lentiviruses include human immunodeficiency viruses (HIV1, HIV2) and simian immunodeficiency viruses (SIV). Lentiviral vectors are constructed by multiple attenuation of HIV pathogenic genes, for example, by deleting the env, vif, vpr, vpu, and nef genes to create biologically safe vectors. Lentiviral vectors are well known in the art, for example, U.S. Patents 6,013,516 and 5,994,136, both of which are incorporated herein by reference. Generally, vectors are plasmid-based or virus-based and are configured to have essential sequences for incorporating foreign nucleic acids, for selection, and for the transfer of nucleic acids into host cells. The gag, pol, and env genes of the vector of interest are also well known in the art. Accordingly, the relevant genes are cloned into a selected vector and subsequently used to transform the target cells of interest. Recombinant lentiviruses that can infect non-dividing cells transfected with two or more vectors having packaging functions, i.e., gag, pol, and env, as well as rev and tat, are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. This describes generating packaging cells using a first vector capable of supplying nucleic acids encoding the viral gag and pol genes and another vector capable of supplying nucleic acids encoding the viral env. Introducing the vectors supplying the heterologous genes into these packaging cells yields producer cells that release infectious viral particles having the desired exogenous genes. Env is preferably an amphipathic envelope protein that enables the transformation of human and other species cells.Typically, the nucleic acid molecules or vectors of the present invention include “regulatory sequences,” which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRESs"), enhancers, etc., that provide for the replication, transcription, and translation of the coding sequence in the receptor cell. These regulatory sequences do not necessarily have to be present indefinitely, as long as the selected coding sequence can be replicated, transcribed, and translated in a suitable host cell. Other nucleic acid sequences are “promoter” sequences, which are used herein in the usual sense to mean a nucleotide region containing a DNA regulatory sequence, where the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating transcription of the downstream (3' direction) coding sequence. Transcription promoters may include “inducible promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressive promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.”

[0119] In certain embodiments, the single-domain antibodies and polypeptides according to the present invention may be used in combination with classic treatments for thrombotic disorders.

[0120] Accordingly, the present invention relates to a method for preventing or treating thrombotic disorders in subjects requiring such treatment, comprising applying to the subject i) an effective amount of the single-domain antibody and / or polypeptide of the present invention and ii) a classical treatment as a combined formulation for the treatment of thrombotic disorders.

[0121] As used herein, the term “classical treatment of thrombotic disorders” means any natural or synthetic compound and / or thrombectomy used in the treatment of thrombotic disorders.

[0122] According to the present invention, compounds used in the treatment of thrombosis include vitamin K antagonists such as coumarin, warfarin, asenocumarol, fenprocumon, atromentin, fluindione and phenindione; heparin and derivative substances such as enoxaparin, dalteparin, nadroparin and tinsaparin; factor Xa synthetic pentasaccharide inhibitors such as fondaparinux, hydrabarinux and hydrabiotapalinux; direct-acting oral anticoagulants such as dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban; direct thrombin inhibitors such as hirudin, repirudine, bivalirudine, argatroban and dabigatran; antithrombin proteins, batroxobin, hementin, tissue plasminogen activators (tPA), and The following can be selected from the group consisting of recombinant tissue plasminogen activators (rtPAs) such as teplase, reteplase, urokinase, and tenectoplase; streptokinase, anistreplase, clopidogrel, prasugrel, ticagrelor, aspirin, triflusal, cangerol, ticlopidine, cryostazol, borapaxer, absiximab, eptifivatide, tyrofiban, dipyridamole, thromboxane inhibitors, and tertroban; platelet GPVI inhibitors such as ACT017 and rebacept; P-selectin inhibitors such as chrysanlizumab; protein C activators such as AB002 (WE thrombin) and soluble thrombomodulin (BDCA-3); or recombinant activated protein C (APC).

[0123] As used herein, the term “thrombectomy” has its general meaning in this art and refers to an interventional treatment to remove a blood clot from a blood vessel. It is commonly performed in cerebral arteries (interventional neuroradiology). Stent-retriever thrombectomy can be performed in an angiography suite under general anesthesia or conscious sedation. Typically, a coaxial catheter system is pushed into the arterial circulation via percutaneous access in the right femoral artery. Finally, a microcatheter is positioned beyond the occlusion, a stent-retriever is deployed to capture the clot, and the stent is withdrawn from the artery, usually with continuous aspiration using a larger catheter. A different technique for cerebral thrombectomy is direct aspiration. This involves pushing a large, soft aspiration catheter into the occluded vessel and directly aspirating and retrieving the clot, achieving a higher recanalization rate when combined with the stent-retriever method.

[0124] As used herein, the terms “combination therapy,” “combination treatment,” or “combination therapy” refer to treatment using multiple drugs. Combination therapy may also be a two-drug therapy or a dual therapy.

[0125] The drugs used in the combination therapy according to the present invention are administered to the target simultaneously, separately, or sequentially.

[0126] As used herein, the term "simultaneous administration" means administering two active ingredients simultaneously or substantially simultaneously via the same route. The term "separate administration" means administering two active ingredients simultaneously or substantially simultaneously via different routes. The term "sequential administration" means administering two active ingredients at different times, with the same or different routes of administration.

[0127] (Pharmaceutical composition and kit of the present invention) Typically, the single-domain antibodies and polypeptides of the present invention (either alone or contained in a vector) can be combined with pharmaceutically acceptable excipients and optionally a sustained-release matrix such as a biodegradable polymer to form a pharmaceutical composition. Therefore, the single-domain antibodies and polypeptides of the present invention are administered to a target in the form of a pharmaceutical composition.

[0128] "Medicinal" or "medically acceptable" means, as appropriate, molecules and compositions that, when administered to mammals, particularly humans, do not produce harmful, allergic, or other unpleasant reactions. A medically acceptable carrier or excipient means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation aid.

[0129] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active principle can be administered to animals and humans alone or in combination with another active principle as a mixture with a conventional pharmaceutical support in unit dose forms. Suitable unit dose forms include oral administration forms such as tablets, gel capsules, powders, granules, and oral suspensions or solvents, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms, and rectal administration forms.

[0130] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle as an injectable formulation. These may be isotonic and sterile saline (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures thereof), or lyophilized compositions that, by adding drying, particularly sterile water or saline, optionally enable the formation of an injectable solution.

[0131] Forms of pharmaceuticals suitable for injection include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the rapid preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0132] Solutions containing the inhibitor of the present invention as a free base or a pharmaceutically acceptable salt can be prepared in water by appropriately mixing with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0133] The single-domain antibody and / or polypeptide of the present invention may be incorporated into a composition in neutral or salt form. pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0134] Furthermore, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained by, for example, the use of coating agents such as lecithin, maintaining the required particle size when dispersed, and the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0135] Sterile injectable solutions are prepared by incorporating the required amount of the active compound into a suitable solvent, along with some of the other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques to obtain any additional desired components from the active ingredient powder and its pre-sterilized filtered solution.

[0136] Once formulated, the solution is administered in a therapeutically effective amount using a method appropriate to the dosage form. While the formulation can be easily administered in various dosage forms, such as the injectable solutions described above, drug-releasing capsules may also be used.

[0137] For parenteral administration in aqueous solutions, the solution must be appropriately buffered as needed, and the liquid diluent must first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be well known to those skilled in the art in light of this disclosure. The dosage will inevitably vary somewhat depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dosage for each individual subject.

[0138] In addition to the inhibitors of the present invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration, liposomal formulations, time-release capsules, and any other forms currently in use.

[0139] The pharmaceutical composition of the present invention may comprise any further agents used for the treatment of thrombotic disorders.

[0140] In one embodiment, the additional activator may be included in the same composition or administered separately.

[0141] In other embodiments, the pharmaceutical compositions of the present invention relate to compound formulations for simultaneous, separate, or sequential use in the prevention and treatment of thrombotic disorders.

[0142] Finally, the present invention also provides a kit comprising at least one single-domain antibody or polypeptide of the present invention. The kit comprising the isolated single-domain antibody and / or polypeptide of the present invention finds use in therapeutic methods.

[0143] The present invention is further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed as limiting the scope of the present invention. [Examples]

[0144] [Example 1] <Materials and Methods> (Selection of PS003 nanobody using phage display) Anti-PS nanobodies were identified essentially as previously described for anti-VWF nanobodies (Ayme et al. 2017). Briefly, immunization of one llama (L. glama) with recombinant human PS (rhPS) was outsourced to the Centre de Recherche en Cancerologie (Universite Aix-Marseille, Marseille, France). Blood was collected for the isolation of peripheral blood lymphocytes, and the total mRNA of the lymphocytes was used to construct a single-domain antibody (sdAb) library. Briefly, the total mRNA was used for cDNA synthesis by reverse transcriptase with CH2' primers. sdAb coding DNA fragments were obtained by nested PCR, and then the fragments were cloned into a pHEN6 phagemide vector. Competent TG1E. coli cells (ThermoFischer Scientific) were transformed using the ligated vectors, and 10 7A library of more than 100 transformants was successfully constructed. Phages exposed to each sdAb were rescued by infecting the cultures in this library with M13KO7-helper phage. The phage particles were incubated with Dynabeads M-450 epoxy beads coated with purified rhPS (1 mg / mL) in 50 mM Tris containing 2% BSA and 5 mM CaCl2, and 150 mM NaCl pH 7.4 (TBS buffer) at room temperature for 1 hour. The magnetic beads were washed nine times with TBS containing 0.1% Tween20 and 5 mM CaCl2, and twice with TBS containing 5 mM CaCl2. The captured phages were eluted by incubation at room temperature for 30 minutes with 500 μL of TBS containing 1 mg / mL trypsin. 500 μL of eluted phage was diluted with 500 μL of TBS, and 5 μL of the eluted phage was serially diluted and used to infect TG1E.coli, which was then seeded on plates and PS-specific enrichment was evaluated. The remaining eluted phage solution was rescued using M13KO7-helper phage and amplified, and a new round of enrichment was performed. Enrichment was performed for two consecutive rounds. After the second round of enrichment, 5 μL of eluted phage was serially diluted and used to infect TG1E.coli, which was then seeded on plates and ampicillin-resistant single colonies were obtained. To isolate genuine PS-specific nanobodies, these TG1 clones were cultured overnight in 0.5 mL of 2YT medium in 96-well deep-well culture plates, and nanobody expression was induced with 1 mM IPTG. Periplasm extracts containing nanobodies were prepared as described, and binding to immobilized rhPS or BSA was tested by direct ELISA. This allowed us to identify a powerful and unique PS binder, which we named PS003.

[0145] (Construction of PS003 and PS003biv nanobodies) To enable bacterial expression in the cytoplasm, the cDNA sequence of PS003 was cloned into a pET28 plasmid between the 5'PstI and 3'BstEII restriction sites. In this pET28 format, the PS003 protein sequence is flanked by an N-terminal His6 tag and a C-terminal hemagglutinin (HA) tag to facilitate purification and detection (Figure 1). To potentially increase the affinity and activity of PS003, a bivalent form of PS003biv (named PS003biv) was constructed by fusing two cDNA sequences of PS003 via a flexible (GGGS)4 linker (Figure 1). The cDNA sequence of PS003biv was synthesized (ProteoGenix, France) and cloned into a pET28 plasmid between the PstI and BstEII restriction sites. Monovalent anti-VWF nanobodies (KB013) and bivalent anti-VWF nanobodies (KB004biv) were used as controls in in vitro functional assays. The cDNA sequences of these nanobodies were cloned into the pET28 plasmid, as described above for PS003 and PS003biv. In our in vivo FeCl3-induced thrombosis model, a bivalent anti-PS nanobody was used as a control. This anti-PS nanobody was named PS004biv and was constructed from a monovalent nanobody (PS004) identified by selecting phage particles on PS immobilized in ELISA wells. Through three rounds of enrichment, a monovalent nanobody (PS004) that strongly and specifically binds to ELISA-immobilized PS was identified. The cDNA sequence of PS004biv was synthesized and cloned into the pET28 plasmid. All nanobodies used in this study are sandwiched between an N-terminal His6 tag and a C-terminal HA tag, and all pairs of nanobodies are linked by a (GGGS)4 linker.

[0146] (Nanobody expression and purification) Competent T7 Shuffle E. coli cells (New England Biolabs) were transformed with plasmids encoding monovalent and bivalent nanobodies. Each nanobody was subjected to 0.4°C in LB medium containing 30 μg / mL kanamycin. <OD600mm Single colonies resistant to kanamycin to <0.6 were cultured. Subsequently, cytoplasmic expression of nanobodies was induced by the addition of 0.1 mM IPTG, and nanobodies were produced at 20°C for 16 hours. The bacterial pellet was resuspended in 50 mM NaH2PO4 containing 10 μg / mL lysozyme (Sigma) and 25 U / mL benzonase (Sigma), and 0.3 M NaCl pH 7.4, and SigmaFAST protease inhibitor (Sigma) was added. The suspension was sonicated and centrifuged at 12000 rpm at 4°C for 30 minutes. The eluate was frozen at -20°C.

[0147] Monovalent nanobodies were purified by immobilized metal ion chromatography (IMAC). In short, the eluate was thawed at 37°C and centrifuged at 4700 rpm at 20°C for 30 minutes. The supernatant was loaded at 1 mL / min onto a HiTrap TALON Crude column (GE Healthcare) pre-equilibrium with 50 mM NaH2PO4 and 0.3 M NaCl pH 7.4. The column was washed with more than 20 column volumes of 50 mM NaH2PO4 and 0.3 M NaCl pH 7.4, and then again with more than 20 column volumes of 50 mM NaH2PO4 and 0.3 M NaCl pH 7.4 containing 10 mM imidazole. The bound nanobodies were eluted with 50 mM NaH2PO4 and 0.3 M NaCl pH 7.4 containing 150 mM imidazole, and the fraction (1 mL) was collected. The protein content in each fraction was measured using OD. 280nm The fraction was determined by measurement, pooled, and dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS buffer). The dialysate was finally concentrated using an Amicon Ultra-15 centrifugal filter (3 kDa cutoff) (Merck Millipore).

[0148] Divalent nanobodies were purified by protein A affinity chromatography. In short, the eluate was thawed at 37°C and centrifuged at 4700 rpm at 20°C for 30 minutes. The supernatant was loaded at 0.5–1 mL / min onto a HiTrap Protein A Fast Flow column (GE Healthcare) pre-equilibrium with 50 mM NaH2PO4 and 0.3 M NaCl pH 7.4. The column was washed with 50 mM NaH2PO4 and 0.3 M NaCl pH 7.4 (more than 20 column volumes), and the bound nanobodies were eluted with 0.1 M glycine pH 2.7. Fractions (1 mL) were collected in tubes containing 100 μL of 1 M TrisHCl pH 8.5. The protein content in each fraction was measured using OD (Optical Spectroscopy). 280nm The fraction was determined by measurement, pooled, and dialyzed against TBS buffer. The precipitation was finally concentrated using an Amicon Ultra-15 centrifugal filter (3 kDa cutoff) (Merck Millipore).

[0149] (Epitope mapping of PS003) A recombinant form of PS containing only the SHBG-like domain (rSHBG) had been previously expressed and purified (Saposnik et al. 2003). BSA, purified rhPS, and purified rSHBG (60 μL at 8 μg / mL in TBS containing 5 mM CaCl2) were immobilized on a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of wash buffer (TBS containing 5 mM CaCl2 and 0.1% Tween20), and blocked at room temperature for 1 hour with TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of wash buffer, and the immobilized concentration of PS003 (2 nM in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at 37°C for 1 hour. The wells were washed with 3 × 200 μL of washing buffer, and peroxidase-labeled polyclonal anti-HA tag antibody (Abcam, 1 μg / mL in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer, and 50 μL of TMB was added. The reaction was stopped by adding 50 μL of 2 M H2SO4. The results showed that the PS003 epitope was located within the SHBG-like domain of PS (Figure 4). Furthermore, the PS003 epitope was not found within the SHBG-like domain of Gas6 (Figure 4). Non-conserved amino acid residues between the SHBG-like domains of PS and Gas6 are considered to be candidates for mediating the interaction between PS003 and PS.

[0150] <Result> (Specificity of PS003 over rhPS) To determine the specificity of PS003, the binding ability of purified PS003 to various vitamin K-dependent proteins, including PS and homologous domains (Gla and EGF-like domains), was tested. Recombinant human PS (rhPS), recombinant human FIX (BeneFIX, Pfizer), recombinant human FX (Haematologic technologies), plasma-derived protein Z (Hyphen BioMed), and recombinant human Gas6 (rhGas6) (Clauser et al. 2012) (60 μL at 10 μg / mL in TBS containing 5 mM CaCl2) were immobilized on a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of washing buffer (TBS containing 0.1% Tween20 and 5 mM CaCl2), and blocked at room temperature for 1 hour using TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of washing buffer, and a constant concentration of purified PS003 (20 nM in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer, and peroxidase-labeled polyclonal anti-HA tag antibody (Abcam, 1 g / mL in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer, and 50 μL of TMB buffer was added. The reaction was stopped by adding 50 μL of 2 M H2SO4. As a result, PS003 strongly bound only to rhPS (Figure 2), suggesting that PS003 specifically binds to PS.

[0151] Vitamin K-dependent Gas6 exhibits high homology to PS (47% of the total) and, in contrast to other vitamin K-dependent proteins, also contains an SHBG-like domain. To further confirm the specificity of PS003 to PS, rhPS and rhGas6 (60 μL at 10 μg / mL in TBS containing 5 mM CaCl2) were immobilized in a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of washing buffer (TBS containing 5 mM CaCl2 and 0.1% Tween20) and blocked at room temperature for 1 hour with TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of washing buffer and incubated with increasing concentrations of PS003 (0–200 nM, 50 μL / well in TBS containing 5 mM CaCl2 and 1% BSA) at room temperature for 1 hour. The wells were washed with 3 × 200 μL of wash buffer, and peroxidase-labeled polyclonal anti-HA tag antibody (Abcam, 1 μL / mL in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at room temperature for 1 hour. The wells were washed with 3 × 200 μL of wash buffer, and 50 μL of TMB buffer was added. The reaction was stopped by adding 50 μL of 2 M H2SO4. As a result, PS003 bound strongly to rhPS in a dose-dependent manner, but did not bind to rhGas6 (Figure 3), confirming the specificity of PS003 to rhPS.

[0152] (Conjugation of recombinant and plasma-derived PS to immobilized PS003 in sandwich ELISA) In our phage display strategy, PS003 was selected on immobilized rhPS covalently bound to magnetic beads. Furthermore, PS003 was found to bind strongly to rhPS immobilized in ELISA wells. To rule out the possibility that PS003 only recognizes non-native immobilized rhPS, the binding of rhPS in solution to immobilized PS003 was analyzed by sandwich ELISA. Also, since PS003 selected recombinant PS, the binding of plasma-derived PS in solution to PS003 was analyzed by the same sandwich ELISA. Briefly, rhPS and purified plasma-derived human PS (Haematologic Technologies) (60 μL at 10 μg / mL in TBS containing 5 mM CaCl2) were immobilized on a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of washing buffer (TBS containing 5 mM CaCl2 and 0.1% Tween20) and blocked at room temperature for 1 hour with TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of washing buffer and incubated with increasing concentration PS003 (0-200 nM in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer and incubated with peroxidase-labeled polyclonal anti-HA tag antibody (Abcam, 1 μL / mL in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer and 50 μL of TMB buffer was added. The reaction was stopped by adding 50 μL of 2 M H2SO4. As a result, PS003 bound to recombinant or plasma-derived human PS (Figure 5), demonstrating that the binding of PS003 to PS is not limited to non-native immobilization forms.

[0153] (Comparison of binding of PS003 and PS003biv to immobilized PS in ELISA) Recombinant human PS (rhPS) (60 μL at 2.5 μg / mL in TBS containing 5 mM CaCl2) was immobilized in a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of washing buffer (TBS containing 5 mM CaCl2 and 0.1% Tween20) and blocked at room temperature for 1 hour with TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of washing buffer and incubated at room temperature for 1 hour with increasing concentrations of PS003 (0–200 nM in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well). The wells were washed with 3 × 200 μL of washing buffer, and peroxidase-labeled polyclonal anti-His6 tag antibody (Abcam, 1 μg / mL in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at room temperature for 1 hour to detect nanobodies. The wells were washed with 3 × 200 μL of washing buffer, and 50 μL of TMB buffer was added. The reaction was stopped by adding 50 μL of 2 M H2SO4. Three simplified individual experiments were performed for each nanobody, and the results are expressed as the percentage of maximum binding for each nanobody. The binding curves showed that both PS003 and PS003biv efficiently bound to immobilized rhPS (Figure 6).

[0154] To further compare the binding ability of PS003 and PS003biv to PS, the affinity of PS003 and PS003biv to rhPS was evaluated as described (Beatty et al. J Immunol Methods 1987) by obtaining binding curves for rhPS immobilized at increasing concentrations (0.6, 1.25, 2.5, and 5 μg / mL in TBS containing 5 mM CaCl2) in three simplified individual experiments. For each nanobody, the dissociation constant (K) was evaluated. D The value was determined using an equation based on the law of mass action.

[0155] Based on this method, the K of PS003 and PS003biv DThe concentrations were 26.8±2.7 nM and 13.8±5.7 nM, respectively, suggesting that PS003biv binds to rhPS with a slightly higher affinity (1.9 times).

[0156] (Epitope mapping of PS003biv to PS and the specificity of PS003biv) rhPS, recombinant PS SHBG-like region alone (rSHBG) (Saposnik et al. 2003), recombinant human Gas6 (rhGas6), or BSA (60 μL at 10 μg / mL in 50 mM Tris containing 5 mM CaCl2, 150 mM NaCl, pH 7.4 (TBS)) were immobilized on a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of washing buffer (TBS containing 5 mM CaCl2 and 0.1% Tween20), and the cells were blocked at room temperature for 1 hour with TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of washing buffer, and 0.5 nM PS003biv (TBS containing 5 mM CaCl2, 0.1% Tween20, and 2% BSA, 50 μL / well) was incubated at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer, and peroxidase-labeled polyclonal anti-His6 tag antibody (Abcam, 1 μg / mL in TBS containing 5 mM CaCl2, 0.1% Tween20, and 2% BSA, 50 μL / well) was incubated at room temperature for 1 hour to detect bound nanobodies. The wells were washed with 3 × 200 μL of washing buffer, and 50 μL of TMB buffer was added. The reaction was stopped by adding 50 μL of 2 M H2SO4. Three simplified individual experiments were performed for each nanobodies, and the results were obtained on rhPS. 450nm It is expressed as a percentage. Three individual experiments were simplified and performed.

[0157] As a result, PS003biv efficiently bound to rhSBHG (Figure 7), indicating that the epitope of PS003biv is located within the SHBG-like region of PS. Since this region is only found in Gas6, the fact that PS003biv did not bind to rhGas6 (Figure 7) strongly suggested that PS003biv is specific to PS.

[0158] (Enhancing effects of PS003 and PS003biv on APC-cofactor activity of PS in APTT-based plasma coagulation assay) We measured the ability of rhPS to act as a cofactor for APC in the inactivation of FVa and FVIIIa using a commercially available APTT-based plasma coagulation assay (STACLOT® PS, Stago) on a KC4 Coagulometer (Stago). Briefly, 25 μL of rhPS diluted in TBS containing 0.1% BSA was added to 25 μL of PS-deficient plasma (R1 reagent) together with APC (R2 reagent, 25 μL) and bovine FVa (R3 reagent, 25 μL). After incubation at 37 °C for 2 minutes, coagulation was induced by adding 25 μL of 50 mM CaCl2. In this assay, APC prolonged the clotting time of PS-deficient plasma, and when rhPS (final concentration 5 nM) was added together with APC, the clotting time was further prolonged in a dose-dependent manner (Figure 8A). This prolongation reflects the APC-cofactor activity of rhPS. In this assay, rhPS did not prolong the clotting time in the absence of APC (Figure 8A). In this assay, the ability of rhPS to enhance the anticoagulant activity of APC was abolished by a polyclonal anti-PS antibody (DAKO, A0384) that has been well documented to strongly block the APC-cofactor activity of rhPS (data not shown), further indicating that this assay is highly dependent on the presence of rhPS.

[0159] The volume response curve showed that rhPS dose-dependently prolonged the clotting time in the presence of a certain concentration of APC (Figure 8B). To detect the inhibitory or stimulatory effects of the nanobody, an intermediate concentration of rhPS (6 nM) at which the ratio t +PS / t -PS is 2 or less was selected.

[0160] Next, we tested the effect of PS003 and PS003biv on the ability of rhPS (6 nM) to enhance the anticoagulant activity of APC. PS003, KB013, PS003biv, and KB004biv were pre-incubated at 10 μM with rhPS (30 nM) in TBS containing 0.1% BSA at room temperature for 15 minutes. A mixture of rhPS ± nanobodies (25 μL) was added to 25 μL of PS-deficient plasma (R1 reagent) along with APC (R2 reagent, 25 μL) and bovine FVa (R3 reagent, 25 μL). After incubation at 37°C for 2 minutes, coagulation was induced by adding 25 μL of 50 mM CaCl2. The final concentrations of rhPS and nanobodies were 6 nM and 2 μM, respectively. Experiments were performed in triplicates.

[0161] In the presence of rhPS and APC, the solidification time was approximately doubled in the absence of APC (TBS), and in the presence of monovalent (KB013) and divalent (KB004biv) nanobodies (final concentration 2 μM), reflecting the normal APC-cofactor activity of rhPS (Figure 8C). On the other hand, in the presence of PS003 and PS003biv (final concentration 2 μM), the solidification time was further extended by 2.8 and 3.6 times, respectively, thus demonstrating that PS003 and PS003biv have a remarkable effect in enhancing the APC-cofactor activity of rhPS compared to their respective control nanobodies (Figure 8C). Furthermore, the enhancing effect of PS003biv on the APC-cofactor activity of rhPS was greater than that of PS003 alone.

[0162] The previous results showed that the coagulation time (t) in the presence of rhPS +PS ) Coagulation time (t) in the absence of rhPS -PS This was expressed as a ratio to (Figure 8D). An independent Student's t-test was used as the statistical test.

[0163] (Effects of PS003 and PS003biv on PS APC-cofactor activity in an in vitro FVa inactivation assay) The ability of PS003 and PS003biv to enhance the APC-cofactor activity of rhPS was evaluated using purified protein in an in vitro assay measuring the specific proteolytic inactivation of FVa by APC in the presence of rhPS. Plasma-derived human FVa (Haematologic Technologies, 80 nM) was inactivated for 20 minutes with plasma-derived human APC (Haematologic Technologies, 0.5 nM) in the presence of 25 μM PC / PE / PS phospholipid vesicles and rhPS (0-100 nM) ("FVa inactivation mixture") in 50 mM Tris containing 5 mM CaCl2, 0.2% PEG, and 0.2% BSA, 150 mM NaCl, pH 7.4 (TBS). The reaction was stopped by diluting the FVa inactivation mixture with TBS containing 5 mM CaCl2, 0.2% PEG, and 0.2% BSA (1:10). Next, a prothrombinase assay using plasma-derived human prothrombin (Haematologic Technologies, 200 nM) and FXa (Enzyme Research Laboratories, 200 nM) was performed to measure residual FVa activity in a TBS containing 5 mM CaCl2, 0.2% PEG, 0.2% BSA, and 50 μM PC / PE / PS phospholipid vesicles. The amide degradation activity of thrombin was tracked using a chromogenic substrate (pNAPEP0238, 200 μM) in a TBS containing 10 mM EDTA, 0.2% PEG, and 0.2% BSA, and the slope of the progression curve was calculated. The slope was determined for each rhPS concentration in the FVa inactivation mixture, and the FVa activity value was expressed as the ratio between the slope obtained in the presence of rhPS and the slope obtained in the absence of rhPS. Three experiments were performed in a simple manner.

[0164] As a result, rhPS was shown to dose-dependently and very efficiently enhance the ability of APC to inactivate FVa (Figure 9A), and a rhPS concentration of 6 nM was selected to evaluate the effects of PS003 and PS003biv. To confirm that our assay was dependent on the presence of rhPS, we also used a polyclonal anti-PS antibody (DAKO, A0384), which has been primarily described as potently blocking the APC-cofactor activity of rhPS. Then, 80 nM FVa was inactivated for 20 minutes using 0.5 nM APC and 25 μM PC / PS / PE phospholipid vesicles, and 6 nM rhPS was pre-incubated for 15 minutes with or without nanobodies (PS003, PS003biv, control monovalent nanobodies KB013 and control bivalent nanobodies KB004biv, final concentration 10 μM), rabbit polyclonal anti-PS antibody (α-PS, DAKO, final concentration 0.5 μM), and rabbit IgG (DAKO, final concentration 0.5 μM) (TBS). The residual FVa activity was measured under each condition using the prothrombinase assay as described above, and compared with the FVa activity obtained when rhPS was pre-incubated in the absence of nanobodies or antibodies (TBS). The three experiments were simplified and an independent Student's t-test was used for statistical testing. *** P<0.001).

[0165] As a result, in this APC-cofactor activity assay, blocking anti-PS antibodies (α-PS, DAKO) effectively inhibited the APC-cofactor activity of rhPS (Figure 9B). In contrast to the APTT-based APC-cofactor activity observed in the assay, PS003 and PS003biv did not show any enhancing effect on the APC-cofactor activity of rhPS in this "reductionist" FVa inactivation assay (Figure 9B).

[0166] (Effects of PS003 and PS003biv in in vitro TFPIα-cofactor activity assay of PS) An in vitro assay was developed to evaluate the ability of rhPS to enhance the direct inhibition of FXa by TFPIα. The amidogenesis activity of plasma-derived human FXa (Enzyme Research Laboratories, final concentration 0.2 nM) against FXa-specific chromogenic substrates (pNAPEP, Cryopep, 400 μM) was monitored every 8 seconds for 60 minutes in 100 μL of TBS containing 10 mM CaCl2, 0.2% PEG, 0.2% BSA, and 25 μM PC / PS / PE phospholipid vesicles. Recombinant human full-length TFPIα expressed in E. coli (obtained from Tilman Hackeng, Maastricht, The Netherlands) was used at a final concentration of 5 nM to inhibit the amidogenesis activity of FXa. We selected experimental conditions in which TFPIα alone was weakly inhibited, but rhPS (final concentration 20 nM) effectively enhanced TFPIα-mediated inhibition of FXa (Figure 10A). In the absence of TFPIα, rhPS did not affect the amide degradation activity of FXa (data not shown).

[0167] The ability of rhPS to enhance TFPIα inhibitory activity was abolished by pre-incubating with rabbit polyclonal anti-PS antibody (α-PS) (DAKO, final concentration 0.5 μM) for blocking at room temperature for 15 minutes, but not with rabbit IgG (DAKO, final concentration 0.5 μM) (Figure 10B).

[0168] The ability of rhPS to enhance TFPIα inhibitory activity was evaluated when rhPS was pre-incubated for 15 minutes at room temperature with PS003 and PS003biv, or their respective monovalent (KB013) and divalent (KB004biv) control nanobodies (final concentration 10 μM). The dynamics constant (k) for inhibition of FXa by TFPIα under each condition was evaluated. obs The ) was calculated from the progress curve, as previously described (Ndonwi et al. 2010). The results were expressed as a percentage of rhPS relative to TFPIα-cofactor activity in the absence of nanobodies (TBS), and three simplified experiments were performed. Independent Student's t-tests were used for statistical testing.

[0169] As a result, in this in vitro functional assay, PS003 and PS003biv did not enhance the TFPIα-cofactor activity of rhPS, but rather slightly inhibited it (Figure 10C).

[0170] (Conjugation of PS003biv and PS004biv to immobilized recombinant mouse PS (rmPS)) For rhPS, recombinant mouse PS (rmPS) was expressed in HEK293 cells in the presence of 10 μg / mL vitamin K1 and purified by two-step anion exchange chromatography as previously described (Fernandez et al. 2009). rmPS (60 μL at 10 μg / mL in TBS containing 5 mM CaCl2) was immobilized in a 96-well NUNC Maxisorp plate at 4°C for 16 hours. The wells were washed with 3 × 200 μL of wash buffer (TBS containing 5 mM CaCl2 and 0.1% Tween20) and blocked at room temperature for 1 hour with TBS containing 5 mM CaCl2 and 5% BSA. The wells were washed with 3 × 200 μL of wash buffer and incubated with increasing concentrations of PS003biv and PS004biv (0–50 nM, 50 μL / well in TBS containing 5 mM CaCl2 and 1% BSA) at room temperature for 1 hour. The wells were washed with 3 × 200 μL of washing buffer, and peroxidase-labeled polyclonal anti-HA tag antibody (Abcam, 2 μg / mL in TBS containing 5 mM CaCl2 and 1% BSA, 50 μL / well) was incubated at room temperature for 1 hour to detect bound nanobodies. The wells were washed with 3 × 200 μL of washing buffer, and 50 μL of TMB buffer was added. The reaction was stopped by adding 50 μL of 2 M H2SO4.

[0171] As a result, PS003biv strongly bound to rmPS (Figure 11), indicating that PS003biv can be tested in mouse models of thrombosis and bleeding. PS004biv did not bind to rmPS in this assay (Figure 11), and therefore may be usable as a control nanobody for PS003biv in our mouse in vivo model.

[0172] Since mouse and human PS have 78% sequence homology between their SHBG-like regions, this result may also be useful in identifying the epitope of PS003biv. In fact, candidate amino acid residues are likely conserved in the SHBG-like regions of human and mouse PS, but not in the SHBG-like region of human Gas6.

[0173] (In vivo antithrombotic effect of PS003biv in a mouse FeCl3-induced thrombosis model) Ferric chloride (FeCl3) injury was induced in 4-5 week old C57BL6 / JRccHsd male mice, essentially as previously described (Ayme et al. 2017; Adam et al. 2010). To facilitate visualization of thrombus formation, platelets from pentobarbital-anesthetic mice were fluorescently labeled in vivo by intravenous injection of rhodamine 6G (3.3 mg / kg, i.e., 2.5 μL / g of rhodamine 6G at 1 mg / mL in 0.9% NaCl) into the posterior orbital plexus. PS003biv (10 mg / kg), PS004biv (10 mg / kg), or the same amount of TBS buffer (Ctl) diluted in 0.9% NaCl was administered simultaneously. Alternatively, to confirm that the thrombosis model was sensitive to pharmacological inhibition of coagulation, 200 UI / kg of low molecular weight heparin (LMWH, Lovenox) was subcutaneously injected after intravenous administration of rhodamine 6G alone. After circulating labeled platelets for 10 minutes, FeCl3 solution (10% in water) was locally administered into the mesenteric vessels, and thrombus formation was observed in real time using an inverted epifluorescence microscope (×10) (Nikon Eclipse TE2000-U). One vein and one artery were analyzed for each mouse. Statistical analysis was evaluated via the Kruskal-Wallis and Dunn tests. The results showed that PS003biv exerted antithrombotic effects in the mesenteric vessels of mice in an FeCl3-induced thrombosis model. Treatment of mice with PS003biv delayed venous occlusion in particular (Figure 12A). Similar trends were observed in the arterioles of mice administered PS003biv compared to control nanobodies, but the difference was not statistically significant (Figure 12A). Furthermore, PS003biv administration was associated with increased thrombus stability and a higher incidence of thromboembolism (Figure 12B). These clear antithrombotic effects of PS003biv may, at least in part, reflect the enhancing effect of PS003biv on the APC-cofactor activity of rhPS.

[0174] It should be noted that the control bivalent anti-VWF nanobody (KB004biv) used in our APC- and TFPI-cofactor activity assays was not used in the FeCl3-induced thrombosis model. In fact, treatment of mice with this nanobody resulted in delayed occlusion time in the veins and arteries of one mouse. Therefore, we decided to use our own proprietary bivalent anti-PS nanobody (PS004biv) that cannot bind to recombinant mouse PS (Figure 11).

[0175] (Effect of PS003biv on physiological hemostasis in a mouse tail clip bleeding model) Anesthetized C57 / BL6 mice were either intravenously injected with PS003biv (10 mg / kg) or subcutaneously injected with low molecular weight heparin (LMWH) (Lovenex, 200 UI / kg) as described in the FeCl3-induced thrombosis model. The tails were immersed in 0.9% NaCl at 37°C for 10 minutes, 3 mm from the tip of the tail was cut, and immediately immersed in a tube containing 10 mL of 0.9% NaCl at 37°C. Bleeding time was defined as the time of first cessation of bleeding. Blood samples were also collected over 20 minutes to quantify total blood loss. Each bar represents the mean value obtained from multiple mice evaluated. Standard one-way ANOVA with Turkey's multiplexed comparison test was used for statistical analysis of variance.

[0176] As a result, administration of 200 UI / kg of low molecular weight heparin (LMWH) significantly prolonged bleeding time and significantly increased bleeding volume, indicating that this mouse bleeding model is sensitive to pharmacological coagulation inhibition (Figure 13). This dose of LMWH also significantly prolonged occlusion time in our mouse FeCl3-induced thrombosis model (Figure 12A). In contrast to LMWH, PS003biv had no significant effect on either bleeding time or bleeding volume (Figure 13). These results support our hypothesis that the enhancement of PS's anticoagulant activity by PS003biv administration is not related to impaired physiological hemostasis. Therefore, this study suggests the therapeutic potential of PS003biv as a potent and safe antithrombotic agent.

[0177] <Consideration> We propose that PS003 / PS003biv nanobodies may be useful in the treatment of sickle cell disease (SCD). SCD is a genetic disorder caused by a point mutation in the HBB gene, which leads to polymerization of hemoglobin S (HbS) during deoxygenation, causing red blood cells to deform into a sickle shape. Such sickling impairs the permeability of red blood cells through microvessels, making them prone to hemolysis. Hemolysis of red blood cells activates vascular endothelial cells, releasing harmful mediators such as leukocytes and platelets that adhere to the activated endothelium. These pathological phenomena ultimately lead to microvessel occlusion, causing recurrent, painful vascular occlusive crises (VOCs), which are characteristic of SCD. These vascular occlusion phenomena ultimately lead to peripheral organ failure and, in many cases, premature death.

[0178] The pathophysiology of VOCs is complex, involving interactions between sickle cells, endothelial cells, platelets, and leukocytes. Furthermore, SCD patients are generally considered to have a chronic hypercoagulable state, as evidenced by elevated levels of thrombin-antithrombin complex (TAT), prothrombin fragment F1.2, and D-dimer in these patients (Ataga et al. Hematology Am Soc Hematol Educ Program 2007). This hypercoagulable state is associated with an increased risk of venous thromboembolism and stroke, which are commonly reported in SCD patients (Sparkenbaugh and Pawlinksi. JTH 2017; Brunson et al. Br J Haematol 2017; Shet et al. Blood 2018). However, chronic coagulation activation in SCD may also locally induce and / or enhance vascular inflammation, a key pathophysiological feature of SCD. In fact, it has long been recognized that coagulation and inflammation amplify each other in various thromboinflammatory diseases, and this crosstalk between coagulation and inflammation is considered to be central to the pathophysiology of vascular occlusion in SCD (Sparkenbaugh et al. Br J Haematol 2013).

[0179] Tissue factor (TF) expression has been shown to be increased in leukocytes in SCD patients and mouse models of SCD, suggesting that TF likely contributes to hypercoagulation in SCD. While leukocyte TF is considered the most likely source of TF contributing to coagulation activation in SCD (Sparkenbaugh and Pawlinski, JTH 2017), TF is also inductively expressed in vascular endothelial cells. Little is known about the role of the contact system in hypercoagulation in SCD. FXII can be activated in various cell types (sickle cells and endothelial cells, etc.), as well as at phosphatidylserine-exposed sites on microvesicles derived from endothelial cells, platelets, or monocytes. This can be inferred from studies (Yang et al., Front Immunol 2017) suggesting that FXII can bind to phosphatidylserine exposed by apoptotic cells, leading to its rapid cleavage and activation. Such phosphatidylserine-mediated activation of FXII may be a trigger for coagulation activation in SCD, separate from TF. Alternatively, FXII and the contact system may be activated by mast cell-derived products such as glycosaminoglycans and heparin, or by glycated hemoglobin released by hemolysis (Sparkenbaugh and Pawlinski. JTH 2017).

[0180] Exposure of phosphatidylserine on the surfaces of sickle cells, endothelial cells, and microvesicles is a key driver of increased coagulation in SCD. This phosphatidylserine exposure significantly accelerates the rate of coagulation and can also promote thrombin detachment and activation (Ansari et al. Thromb Haemost 2019). Interestingly, PS has a high affinity for anionic phospholipid membranes containing phosphatidylserine, suggesting that PS may accumulate at these sites and exert an important anticoagulant role in locally limiting thrombin production. However, widespread intravascular or extravascular exposure of phosphatidylserine can trap PS and deplete it from the plasma pool. This would be consistent with the apparent acquired deficiency of PS observed in SCD patients in various studies (Whelihan et al. JTH 2016). Since it has been found that hepatic expression of PS is downregulated by hypoxia via HIF-1α, acute or chronic hypoxia may also be involved in the reduction of plasma PS levels observed in SCD patients (Pilli et al. Blood 2018). It is unclear how such PS deficiency contributes to hypercoagulation and exacerbates thrombotic tendencies in SCD patients. Interestingly, protein C deficiency is also observed in SCD patients (Whelihan et al. JTH 2016), suggesting that the anticoagulant protein C pathway may be more extensively altered in SCD. Indeed, a combined deficiency of PS and protein C in SCD is expected to significantly affect the ability of activated protein C (APC) to exert anticoagulant activity. This is supported by APC resistance observed in the plasma of SCD patients, even though elevated FVIII levels in SCD patients may also be a contributing factor (Wright et al. 1997; Whilihan et al. 2016).

[0181] Importantly, local coagulation activation and thrombin generation may play a significant direct role in the pathophysiology of VOCs, independently of fibrin production during thrombus formation. In fact, thrombin not only cleaves fibrinogen to produce fibrin, but is also a potent activator of endothelial cells, platelets, and leukocytes, particularly through PAR1 activation. In endothelial cells, thrombin exerts PAR1-dependent pro-inflammatory, pro-apoptotic, and barrier-disrupting effects (Flaumenhaft and De Ceunynck. Trends Pharmacol Sci 2017). Furthermore, thrombin-mediated PAR1 activation in endothelial cells induces exocytosis of Viver-Parade bodies, including Willebrand factor (VWF) and P-selectin (Cleator et al. Blood 2014), promoting or enhancing the interaction between sickle cells and endothelial cells. Furthermore, such exocytosis of Weiber-Palades bodies may release soluble mediators of vascular thromboinflammation, such as angiopoietin-2. In addition, thrombin may directly or indirectly induce the exposure of phosphatidylserine in endothelial cells, promoting and continuing coagulation and thrombin production on their surface.

[0182] Therefore, even without thrombus formation and widespread coagulation activation, localized and low-level thrombin generation initiated by the TF-endothelial surface contact system can be an initial trigger for vascular inflammation and occlusion. Recently, direct oral anticoagulants targeting anti-TF antibodies, FXa (rivaroxaban), and thrombin (dabigatran), as well as PAR1 antagonists (borapaxer), significantly suppressed hemoglobin-induced microvasoconstriction in a mouse model of VOC (Sparkenbaugh et al. Blood 2020). Therefore, pharmacological targeting of thrombin-mediated endothelial PAR1 activation appears to be an attractive therapeutic strategy for preventing and / or mitigating VOC in SCD. Furthermore, this study suggests that appropriately controlling thrombin generation on the endothelial surface with natural anticoagulants such as PS, APC, and tissue factor pathway inhibitors (TFPIs) may be important in limiting VOC.

[0183] PS exhibits high affinity for phosphatidylserine exposed on the activated endothelial surface and possesses the unique ability to function as a cofactor for both APC and TFPIα. By stimulating the anticoagulant activity of both APC and TFPIα, PS may play a central role in limiting thrombin production on the endothelial cell surface in SCD. Consequently, PS may be an important negative regulator of thrombin-induced vascular occlusion, although this has not yet been confirmed by experimental studies.

[0184] We propose that enhancing the APC-cofactor activity of PS with PS003 / PS003biv nanobodies could be a novel therapeutic strategy for preventing or mitigating vascular occlusive events in SCD patients. In particular, since SCD patients exhibit PS deficiency and APC resistance, the antithrombotic properties of PS003 / PS003biv may contribute to reducing the risk of venous thromboembolism and stroke in patients undergoing treatment.

[0185] [References] Throughout this application, various references describe the state of the art to which the present invention pertains. The disclosures of these documents are incorporated into this disclosure by reference.

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Claims

1. An isolated single-domain antibody (sdAb) against protein S (PS) that enhances the APC-cofactor activity of PS, The sdAb is a single-domain antibody comprising CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO:

3.

2. An isolated single-domain antibody according to claim 1, comprising the sequence of SEQ ID NO:

4.

3. A polypeptide comprising at least one single-domain antibody as described in claim 1.

4. The polypeptide according to claim 3, comprising at least two single-domain antibodies according to claim 1.

5. The polypeptide according to claim 3, comprising two single-domain antibodies according to claim 1.

6. The polypeptide according to claim 4, comprising the sequence of sequence number 5.

7. A nucleic acid molecule encoding a single-domain antibody according to claim 1 and / or a polypeptide according to claim 3.

8. A vector comprising the nucleic acid described in claim 7.

9. Host cells transfected, infected, or transformed with the nucleic acid described in claim 7 and / or the vector described in claim 8.

10. Use of the single-domain antibody and / or polypeptide according to Claim 3 for producing a pharmaceutical composition for preventing or treating thrombotic disorders.

11. Use of the single-domain antibody and / or polypeptide according to Claim 3 for producing a pharmaceutical composition for preventing or treating vascular occlusive crisis.

12. The use according to claim 10, wherein the thrombotic disorder is sepsis, sickle cell anemia, embolism (lung and brain), stroke, or cardiovascular disease.

13. A pharmaceutical composition comprising the single-domain antibody described in claim 1 and / or the polypeptide described in claim 3.

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