Single-domain antibodies targeting the von Willebrand factor A3 domain

Abstract

We isolated a single-domain antibody (designated KB-VWF-D3.1) targeting the A3 domain, whose epitope overlaps with the collagen-binding site. The binding of KB-VWF-D3.1 was demonstrated to be independent of VWF multimer size. However, its interaction with VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis at the A2 domain regulates the exposure of its epitope at the A3 domain. Therefore, we used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments demonstrated that this single-domain antibody could detect a loss of 10% intact VWF. By comparing plasma from volunteers with that from congenital VWD patients, the levels of intact VWF were significantly reduced in all VWD types, with the most severe reduction occurring in VWD type 2A (IIA), in which mutations promote ADAMTS13-mediated proteolysis. Surprisingly, mild proteolysis was also observed in VWD types 1 and 2M. Thus, this single-domain antibody proved to be highly sensitive for detecting mild proteolysis in plasma from AVWS patients and congenital VWD patients, including types 1 and 2M. The present invention relates to an isolated single-domain antibody that targets at least one region of the A3 domain of VWF, wherein the region comprises the following sequence: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4.

Description

[Technical Field]

[0001] Field of the invention: The present invention is in the field of coagulation, more specifically the present invention relates to nanobodies targeting the A3 domain of Factor Von Willebrand, and their use in diagnostic methods. [Background technology]

[0002] Background of the invention: Von Willebrand factor is a multimeric protein, the degree of which regulates its interaction with platelets. Multimerization of VWF occurs during its synthesis in megakaryocytes or endothelial cells. 1,2 In this process, two VWF subunits (with the domain structure: D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK) first covalently link via a disulfide bridge between the two C-terminal CK domains. These prodimers are then processed into multimers via N-terminal coupling of the D'-D3 regions, and the D1-D2 portion (also known as the VWF propeptide) is removed during this process. The size of the multimers in endothelial cells is highly variable, ranging from dimers to very large multimers with more than 40 subunits. Upon secretion, VWF multimers are subject to regulated proteolysis by ADAMTS13, a metalloprotease that cleaves VWF at the Tyr1605-Met1606 peptide bond within its A2 domain. 3 Importantly, proteolysis occurs only when the cleavage site is decoded, which is usually buried within the A2 domain. 4 Several events can expose the ADAMTS13 cleavage site. First, upon stimulation of secretion, multiple VWF multimers assemble on the endothelial surface and form elongated fibrillar bodies that are proteolyzed by ADAMTS13. 5~7 Second, VWF unfolds during circulation under conditions of high shear stress or impaired blood flow. 8Increased blood flow obstruction, as seen in patients with severe aortic stenosis or those requiring mechanical circulatory support, can lead to excessive VWF degradation, which later became known as acquired von Willebrand syndrome (AVWS). 9~11 Third, mutations within VWF can lead to exposure of the ADAMTS13 proteolytic site; such mutations are most frequently seen in von Willebrand disease (VWD) type 2A (IIA) (also known as VWD-2A group 2) and VWD-2B. 12,13 Excessive proteolysis of VWF is accompanied by increased loss of high molecular weight (HMW) multimers, resulting in decreased platelet binding and decreased collagen binding, thereby increasing the risk of bleeding. 9 The classical approach to visualize the extent of VWF degradation is to analyze the multimer pattern using SDS-agarose electrophoresis. 14 This approach is laborious, not standardized, and requires 24–72 hours depending on the method used. Alternatively, collagen or platelet binding assays can be used. 15 However, this assay has low specificity in that it does not distinguish between defective multimerization and excessive degradation. Finally, Kato and coworkers described a monoclonal antibody that binds to the newly formed C-terminus within the A2 domain. Using this antibody, they successfully measured ADAMTS13 activity in patients with thrombotic thrombocytopenic purpura. 16 In addition, the present inventors and others have used a similar antibody (MAB27642) to monitor VWF proteolysis in patients. 17,18 However, using this antibody, the inventors found that it was unable to detect mild degradation, for example, in samples from patients with non-severe aortic stenosis or congenital VWD-1 or 2M. 5 .

[0003] Therefore, there is a need to develop new tools to detect mild degradation of VWF in patients.

[0004] Summary of the Invention: The present invention relates to an isolated single domain antibody directed against at least one region of the von Willebrand factor A3 domain, said region comprising the following sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4. In particular, the invention is defined by the claims.

[0005] Detailed description of the invention: We isolated a single-domain antibody (designated KB-VWF-D3.1) targeting the A3 domain, whose epitope overlaps with the collagen-binding site. The binding of KB-VWF-D3.1 was demonstrated to be independent of VWF multimer size. However, its interaction with VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis at the A2 domain regulates the exposure of its epitope at the A3 domain. Therefore, we used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments demonstrated that this single-domain antibody could detect a loss of 10% intact VWF. By comparing plasma samples from volunteers with those from congenital VWD patients, we found that intact VWF levels were significantly reduced in all VWD types, with the most severe reduction observed in VWD type 2A (IIA), in which mutations promote ADAMTS13-mediated proteolysis. Surprisingly, mild proteolysis was also observed in VWD types 1 and 2M. Thus, this single-domain antibody proved to be highly sensitive in detecting mild proteolysis in plasma from patients with AVWS and congenital VWD, including types 1 and 2M.

[0006] Single-domain antibodies targeting at least one region of the von Willebrand factor A3 domain Thus, in a first aspect, the present invention relates to an isolated single domain antibody that targets at least one region of the von Willebrand factor A3 domain, wherein said region comprises the following sequence: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4.

[0007] "Isolated," when referring to a single domain antibody according to the present invention, means that the designated molecule is present in the substantial absence of other biological macromolecules of the same type.

[0008] As used herein, the term "single domain antibody" (sdAb) has its general meaning in the art and refers to a single heavy chain variable domain antibody of the type that can be found in camelid mammals that naturally lack light chains. Such single domain antibodies are also called VHHs or "nanobodies®." For a general description of (single) domain antibodies, see the prior art cited above, as well as EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol, 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388. The amino acid sequence and structure of a single domain antibody can be considered to consist of four framework regions or "FRs," referred to in the art and herein 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 separated by three complementarity-determining regions or "CDRs," 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. A single domain antibody can therefore be defined as an amino acid sequence having the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 through FR4 refer to framework regions 1 through 4, respectively, and CDR1 through CDR3 refer to complementarity-determining regions 1 through 3. In the context of the present invention, the amino acid residues of single domain antibodies are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http: / / imgt.cines.fr / ).

[0009] The term "VWF" has its common meaning in the art and refers to human von Willebrand factor (VWF), a blood glycoprotein involved in blood coagulation. VWF is a monomer composed of several homologous domains: D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK, each of which serves a distinct function. Naturally occurring human VWF protein has the amino acid sequence shown in GeneBank Accession number NP_000543.2. The monomers are then arranged into dimers or multimers by cross-linking cysteine ​​residues via disulfide bonds. Thus, VWF multimers can be quite large, consisting of more than 40 monomers, and are also called high-molecular-weight (HMW) VWF multimers. Upon secretion, VWF multimers are subject to regulated proteolysis by ADAMTS13, a metalloprotease that cleaves VWF at the Tyr1605-Met1606 peptide bond within its A2 domain. 3 Importantly, proteolysis occurs only when the cleavage site is decoded, which is usually buried within the A2 domain. 4 Several events can expose the ADAMTS13 cleavage site. First, upon stimulation of secretion, multiple VWF multimers assemble on the endothelial surface and form elongated fibrillar bodies that are proteolyzed by ADAMTS13. 5~7 Second, VWF unfolds during circulation under conditions of high shear stress or impaired blood flow. 8 Increased blood flow obstruction, as seen in patients with severe aortic stenosis or those requiring mechanical circulatory support, can lead to excessive VWF degradation, which later became known as acquired von Willebrand syndrome (AVWS). 9~11 Third, mutations within VWF can lead to exposure of the ADAMTS13 proteolytic site; such mutations are most frequently seen in von Willebrand disease (VWD) type 2A (IIA) (also known as VWD-2A group 2) and VWD-2B. 12,13Excessive proteolysis of VWF is accompanied by increased loss of high molecular weight (HMW) multimers, resulting in decreased platelet binding and decreased collagen binding, thereby increasing the risk of bleeding. 9 .

[0010] In a particular embodiment, the single domain antibody according to the invention targets at least one region of the collagen binding site of the A3 domain of VWF.

[0011] In a particular embodiment, the single domain antibody of the invention binds to an epitope comprising residues Val1732-Val1747 (region 1), Leu1755-Gln1769 (region 2), Asp1771-His1786 (region 3) and Val1805-Asn1818 (region 4) of VWF.

[0012] In certain embodiments, the single domain antibodies according to the invention target: A region comprising or consisting of the following amino acid sequence: VLQYGSITTIDVPWNV (SEQ ID NO:1); A region comprising or consisting of the following amino acid sequence: LSLVDVMQREGGPSQ (SEQ ID NO:2); A region comprising or consisting of the following amino acid sequence: DALGFAVRYLTSEMH (SEQ ID NO:3); and / or A region comprising or consisting of the following amino acid sequence: VDSVDAAADAARSN (SEQ ID NO:4);

[0013] In a particular embodiment, the single domain antibodies of the invention detect alterations in the exposure of their epitope within the collagen binding site of the A3 domain.

[0014] Given their unique properties, the single domain antibodies according to the invention are suitable for use as diagnostic tools to investigate whether the loss of larger multimers is due to ADAMTS13-mediated proteolysis.

[0015] In a particular embodiment, the single domain antibodies of the present invention detect intact VWF that has not been degraded by ADAMTS13.

[0016] In a particular embodiment, the single domain antibodies according to the invention specifically bind to intact VWF (i.e. VWF that has not been proteolyzed / degraded by ADAMTS13).

[0017] As used herein, the term "specifically binds" means that the single domain antibody binds only to an antigen of interest, e.g., non-proteolyzed VWF, and does not exhibit cross-reactivity with VWF proteolyzed by ADAMTS13. In other words, in certain embodiments, the single domain antibody of the present invention does not detect intact VWF cleaved by ADAMTS13.

[0018] In a particular embodiment, the single domain antibodies according to the invention are capable of determining VWF degradation in a biological sample, such as a plasma sample.

[0019] The inventors have isolated nanobodies that distinguish between proteolytic and non-proteolytic VWF, leading to the identification of a single domain antibody (designated KB-VWF-D3.1) that targets the A3 domain, the epitope of which overlaps with the collagen binding site. In a particular embodiment, the single domain antibody KB-VWF-D3.1 is characterized by the complementarity determining regions (CDRs) as described below (Table A):

[0020] [Table 1]

[0021] In a particular embodiment, the present invention provides an isolated single domain antibody targeting the A3 domain of VWF, said sdAb comprising a CDR1 having at least 70% identity to the sequence shown as SEQ ID NO:5, a CDR2 having at least 70% identity to the sequence shown as SEQ ID NO:6, and a CDR3 having at least 70% identity to the sequence shown as SEQ ID NO:7.

[0022] In particular, the present invention relates to an isolated single domain antibody (sdAb) comprising a CDR1 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the sequence shown as SEQ ID NO:5, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the sequence shown as SEQ ID NO:6, and a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the sequence shown as SEQ ID NO:7.

[0023] Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87(6):2264-2268 (1990)).

[0024] In some embodiments, an isolated single domain antibody of the present invention comprises a CDR1 having the sequence set forth as SEQ ID NO:5, a CDR2 having the sequence set forth as SEQ ID NO:6, and a CDR3 having the sequence set forth as SEQ ID NO:7.

[0025] An isolated single domain antibody directed against albumin according to the present invention, wherein the sdAb is KB-VWF-D3.1 (SEQ ID NO:8).

[0026] In some embodiments, the isolated single domain antibody of the present invention has the sequence shown as SEQ ID NO:8.

[0027] In a particular embodiment, the isolated single domain antibody according to the invention has the sequence shown as SEQ ID NO:9: [ka]

[0028] In a particular embodiment, an isolated single domain antibody targeting the A3 domain of VWF according to the present invention, said single domain antibody having the sequence of the variable heavy chain (VHH) shown as SEQ ID NO:8.

[0029] It is also worth noting that sdAb KB-VWF-D3.1 cross-reacts with murine VWF, which is of interest for preclinical evaluation and toxicology studies.

[0030] In some embodiments, the single domain antibody is a "humanized" single domain antibody. As used herein, the term "humanized" refers to a single domain antibody of the present invention in which the amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (particularly in the framework sequence) with one or more amino acid residues present at the corresponding positions in a VH domain from a conventional chain antibody of human origin. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be selected such that the resulting humanized single domain antibody still retains the advantageous properties of the single domain antibody of the present invention. Those skilled in the art are able to determine and select a suitable humanizing substitution or a suitable combination of humanizing substitutions.

[0031] In accordance with the present invention, the single domain antibodies of the present invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and producing proteins are well known to those skilled in the art.

[0032] The single domain antibodies and polypeptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols, such as those described in Stewart and Young; Tam et al., 1983; Merrifield, 1986; and Barany and Merrifield, Gross and Meienhofer, 1979. The single domain antibodies and polypeptides of the present invention can also be synthesized by solid-phase techniques using a representative peptide synthesizer, such as the Model 433A manufactured by Applied Biosystems Inc. The purity of any given protein produced through automated peptide synthesis or through recombinant methods can be determined using reverse-phase HPLC analysis. The chemical authenticity of each peptide can be established by any method known to those skilled in the art.

[0033] As an alternative to automated peptide synthesis, recombinant DNA techniques may be used, in which a nucleotide sequence encoding a selected polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression, as described herein below. Recombinant methods are particularly preferred for producing longer polypeptides.

[0034] A variety of expression vector / host systems can be used to contain and express peptide or protein coding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see Babe et al., 2000); or animal cell systems. Those skilled in the art are aware of various techniques for optimizing mammalian expression of proteins, see, for example, Kaufman, 2000; Colosimo et al., 2000. Mammalian cells useful for producing recombinant proteins include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), 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 known to those skilled in the art and are briefly described herein below. Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art. Host cell lines can be selected for their particular ability to process expressed proteins or to produce certain post-translational modifications that may be useful in providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves a "prepro" form of the protein, may also be important for correct insertion, folding and / or function.Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be selected to ensure the correct modification and processing of the introduced foreign protein.

[0035] Recombinant production of the single domain antibodies and polypeptides of the present invention will require the use of vectors containing polynucleotide molecules encoding the single domain antibodies and polypeptides of the present invention. Methods for preparing such vectors and producing host cells transformed with such vectors are well known to those skilled in the art. Polynucleotide molecules used in such endeavors will generally be ligated to vectors containing selectable markers and origins of replication for propagation in a host. These elements of expression constructs are well known to those skilled in the art. Generally, expression vectors contain DNA encoding a given protein operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators or enhancers, mRNA ribosomal binding sites, and appropriate sequences for controlling transcription and translation.

[0036] The terms "expression vector," "expression construct," or "expression cassette" are used interchangeably throughout this specification and are meant to include any type of genetic construct containing a nucleic acid encoding a gene product in which part or all of the nucleic acid coding sequence can be transcribed.

[0037] The selection of a suitable expression vector for expressing a peptide or polypeptide of the invention will, of course, depend on the particular host cell used and is within the skill of one of ordinary skill in the art.

[0038] For expression, appropriate signals, such as enhancers / promoters from both viral and mammalian sources, that can be used to drive the expression of a nucleic acid of interest in a host cell must be provided within the vector. Usually, the nucleic acid to be expressed is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate the specific transcription of a gene. A nucleotide sequence is operably linked when the regulatory sequence is functionally related to the DNA encoding a protein of interest (e.g., a single-domain antibody). Thus, if the promoter nucleotide sequence directs the transcription of a given DNA sequence, the promoter nucleotide sequence is operably linked to that sequence.

[0039] In a particular embodiment, the present invention relates to a nucleic acid sequence encoding an isolated single domain antibody of the present invention.

[0040] In a particular embodiment, the present invention relates to a nucleic acid sequence encoding the heavy chain of an isolated single domain antibody of the present invention.

[0041] In a particular embodiment, the present invention relates to a vector comprising a nucleic acid of the present invention.

[0042] In a particular embodiment, the present invention relates to host cells engineered to express the isolated single domain antibodies of the present invention.

[0043] Diagnostics: We used KB-VWF-D3.1 to monitor VWF degradation in plasma samples, and spiking experiments showed that this single-domain antibody could detect a loss of 10% intact VWF.

[0044] Therefore, in a second aspect, the present invention relates to the use of a single domain antibody according to the invention for determining the level of VWF degradation in a biological sample.

[0045] In a particular embodiment, the present invention relates to a method for determining the level of VWF degradation in a subject in need thereof, comprising the steps of: i) contacting an isolated single domain antibody of the present invention with a biological sample; ii) determining the level of intact VWF by the isolated single domain antibody (KB-VWF-D3.1); iii) comparing the level determined in step ii) with its corresponding predetermined reference value; and iv) concluding that the level of VWF degradation is elevated when the level of intact VWF is lower than a predetermined reference value, or concluding that the level of VWF degradation is not elevated when the level of intact VWF determined in step ii) is higher than a predetermined reference value.

[0046] In a further embodiment, a method according to the invention, wherein a loss of 10% of intact VWF is detected by using a single domain antibody according to the invention.

[0047] In a further embodiment, a method according to the invention, wherein a loss of at least 10% of intact VWF is detected by using a single domain antibody according to the invention. As used herein, the term "at least 10%" means 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99 or 100%.

[0048] In a particular embodiment, the present invention relates to a method for determining the level of VWF degradation in a subject in need thereof, comprising the steps of: i) contacting an isolated single domain antibody of the present invention with a biological sample; ii) determining the level of intact VWF by the isolated single domain antibody (KB-VWF-D3.1); iii) determining the level of total VWF by antibody (polyclonal or monoclonal); iv) calculating the ratio of the level determined in step ii) to the level of total VWF antigen determined in step iii); v) comparing the ratio determined in step iv) with a predetermined corresponding reference value; and vi) concluding that the level of VWF degradation is elevated when the ratio determined in step iv) is lower than a predetermined reference value, or concluding that the level of VWF degradation is not elevated when the ratio determined in step iv) is higher than a predetermined reference value.

[0049] As used herein, the term "biological sample" refers to any biological sample obtained from a subject for the purpose of in vitro evaluation. In some embodiments, the biological sample is a bodily fluid sample. Examples of bodily fluids are blood, serum, plasma, amniotic fluid, brain / spinal cord fluid, liquor, cerebrospinal fluid, sputum, throat and pharyngeal secretions, and other mucosal secretions, synovial fluid, peritoneal fluid, tears, lymph, and urine.

[0050] In certain embodiments, the biological sample is a blood sample.

[0051] As used herein, the term "blood sample" means a whole blood, serum, or plasma sample obtained from a subject.

[0052] In certain embodiments, the blood sample is a plasma sample. Plasma samples may be obtained using methods well known in the art. For example, blood may be collected from a subject according to standard venipuncture procedures using trisodium citrate buffer. Plasma may then be obtained from the blood sample according to standard procedures, including, but not limited to, centrifuging the blood sample at about 1,500 × g for about 15-20 minutes (room temperature), followed by pipetting the plasma layer.

[0053] In certain embodiments, the sample has been previously obtained from the subject.

[0054] As used herein, the term "intact VWF" refers to VWF that has not been proteolytically degraded by the enzyme ADAMTS13.

[0055] As used herein, the term "total VWF" refers to the entire VWF, including any form of proteolyzed VWF (including VWF proteolyzed by ADAMTS13) and non-proteolyzed VWF.

[0056] As used herein, the term "ratio" refers to the relationship between two groups or quantities that describes how much greater one is than the other, in this case allowing a comparison of the levels of intact VWF detected by the single domain antibodies of the invention and total VWF detected by polyclonal or monoclonal antibodies.

[0057] The level of intact VWF as defined above may be determined, for example, by an immunoassay such as capillary electrophoresis-mass spectrometry (CE-MS), flow cytometry, mass cytometry or enzyme-linked immunosorbent assay (ELISA) performed on the sample.

[0058] In a specific embodiment, intact VWF is considered to be VWF recognized by KB-VWF-D3.1. Briefly, samples containing undigested VWF, ADAMTS13-digested VWF, or a mixture of both were incubated in wells coated with KB-VWF-D3.1. Alternatively, plasma samples were used. Bound VWF was probed with a polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine.

[0059] VWF-deficient plasma was spiked with various amounts of purified rVWF and degraded VWF and incubated in a microtiter plate coated with KB-VWF-D3.1. Bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. The solid line illustrates the best linear fit, with the 95% confidence interval indicated by dotted lines. The vertical line indicates 90% intact rVWF supplemented with 10% degraded VWF (Figure 7A).

[0060] In some embodiments, the level of intact VWF peptide is determined by immunoassay.

[0061] As used herein, the term "immunoassay" encompasses any assay in which a capture reagent (i.e., KB-VWF-D3.1) is immobilized on a support and the detection of an analyte of interest (i.e., rVWF or cleaved VWF) is carried out through the use of an antibody directed against the analyte of interest (i.e., intact VWF). Such assays include, but are not limited to, agglutination tests; enzyme-labeled and mediated immunoassays, such as enzyme-linked immunosorbent assays (ELISAs); biotin / avidin-type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis-mass spectrometry (CE-MS), etc. The reaction generally involves revealing a label, such as a fluorescent, chemiluminescent, radioactive, enzyme-labeled, or dye molecule, or other method for detecting the formation of a complex between an antigen and one or more antibodies that react with it. Immunoassays include competitive, direct reaction, or sandwich-type assays.

[0062] Typically, bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. Single domain antibodies are labeled with a detectable molecule or substrate, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art. Labels are known in the art and generally provide a signal (either directly or indirectly). As used herein, the term "labeled," with respect to antibodies or aptamers, is intended to encompass direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substrate, such as a radioactive agent or fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)), to the antibody or aptamer, as well as indirect labeling of the probe or antibody by reactivity with a detectable substrate. Antibodies or aptamers may be labeled with radioactive molecules by any method known in the art. For example, radioactive molecules include I <123> , I <124> , In <111> ,Re <186> ,Re <188> Examples of suitable antibodies include, but are not limited to, radioactive atoms for scintigraphy studies such as FITC conjugate and / or PE conjugate. Preferably, the antibody against VWF is already conjugated to a fluorophore (e.g., FITC conjugate and / or PE conjugate).

[0063] In a particular embodiment, a single domain antibody according to the invention conjugated to a detectable label.

[0064] In a particular embodiment, the single domain antibody according to the invention, wherein the detectable label is a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label or a bioluminescent label.

[0065] In a particular embodiment, the antibody of the invention, wherein the label is selected from the group consisting of β-galactosidase, glucose oxidase, peroxidase (eg, horseradish peroxidase) and alkaline phosphatase.

[0066] In some embodiments, the level of intact VWF is determined by enzyme-labeled and mediated immunoassay (ELISA).

[0067] In some embodiments, the level of intact VWF is determined by direct ELISA: single domain antibodies according to the invention are immobilized directly on the surface of a multi-well plate and detected with a biotin-conjugated detection antibody specific for VWF, which is directly conjugated to a detection system (horseradish peroxidase (HRP)-conjugated streptavidin or other detection molecule).

[0068] In some embodiments, the level of intact VWF is determined by indirect ELISA. Single domain antibodies are immobilized directly on the surface of a multiwell plate and detected with an unconjugated primary detection antibody specific for VWF. A conjugated secondary antibody directed against the host species of the primary antibody is then added. A substrate then generates a signal proportional to the amount of degraded VWF bound in the well.

[0069] In some embodiments, the level of intact VWF is determined by sandwich ELISA.

[0070] According to the present invention, a "sandwich" ELISA refers to an immunoassay in which free VWF can be sandwiched between two antibodies that specifically bind to free VWF. Typically, the single domain antibodies according to the present invention are conjugated to a detection system (such as horseradish peroxidase (HRP)-conjugated streptavidin or other detection molecules).

[0071] In another embodiment, the level of intact VWF is determined by immunohistochemical staining. Typically, immunohistochemical staining of biological material obtained from a subject is performed using a single domain antibody according to the invention. In a particular embodiment, the antibody is a polyclonal antibody against whole VWF.

[0072] By comparing plasma samples from volunteers with those from congenital VWD patients, we found that intact VWF levels were significantly reduced in all VWD types, with the most severe reduction observed in VWD type 2A (IIA), in which mutations promote ADAMTS13-mediated proteolysis. Surprisingly, mild proteolysis was also observed in VWD types 1 and 2M. Thus, this single-domain antibody proved to be highly sensitive in detecting mild proteolysis in plasma from patients with AVWS and congenital VWD, including types 1 and 2M.

[0073] Thus, in a third aspect, the present invention relates to an in vitro method for diagnosing a bleeding episode in a subject in need thereof, comprising the steps of: i) contacting an isolated single domain antibody of the present invention with a biological sample; ii) determining the level of intact VWF by the isolated single domain antibody (KB-VWF-D3.1); iii) comparing the level determined in step ii) with its corresponding predetermined reference value; and iv) concluding that the subject is prone to or at risk of having a bleeding episode when the level of intact VWF determined in step ii) is lower than a predetermined reference value, or concluding that the subject is unlikely to or not at risk of having a bleeding episode when the level of intact VWF determined in step ii) is the same as the predetermined reference value.

[0074] In a further embodiment, a method according to the invention, wherein a loss of 10% of intact VWF is detected by using a single domain antibody according to the invention.

[0075] In a further embodiment, a method according to the invention, wherein a loss of at least 10% of intact VWF is detected by using a single domain antibody according to the invention.

[0076] In a particular embodiment, the present invention relates to an in vitro method for diagnosing a bleeding episode in a subject in need thereof, comprising the steps of: i) contacting a biological sample with a single domain antibody according to the invention; ii) determining the level of intact VWF by the isolated single domain antibody (KB-VWF-D3.1); iii) determining the level of total VWF by antibody (polyclonal or monoclonal); iv) calculating the ratio of the level determined in step ii) to the level of total VWF antigen determined in step iii); v) comparing the ratio determined in step iv) with a predetermined corresponding reference value; and vi) concluding that the subject is prone to or at risk of having a bleeding episode when the ratio determined in step iv) is lower than a predetermined reference value, or that the subject is unlikely to or not at risk of having a bleeding episode when the ratio determined in step iv) is identical to the predetermined reference value.

[0077] As used herein, the term "diagnosing" refers to classifying a disease or condition, determining the severity of a disease, monitoring disease progression, predicting the outcome of a disease and / or the likelihood of recovery. In the context of the present invention, the method of the present invention is capable of diagnosing a bleeding episode.

[0078] As used herein, the term "subject" refers to any mammal, such as rodents, felines, canines, and primates. Specifically, in the present invention, the subject is a human. In certain embodiments, the subject is a human susceptible to a disease that induces bleeding episodes.

[0079] As used herein, the term "bleeding" refers to the extravasation of blood from any component of the circulatory system. Thus, a "bleeding episode" encompasses unwanted, uncontrolled, and often excessive bleeding associated with surgery, trauma, or other forms of tissue injury, as well as unwanted bleeding in patients with bleeding disorders. In particular, unexplained bleeding episodes are associated with ventricular assist devices (VADs) and may occur in part due to acquired von Willebrand syndrome (aVWS). AVWS is characterized by the loss of high-molecular-weight (HMW) multimers of VWF. This loss of multimers can occur when VWF is subjected to high shear stress, as occurs with VADs.

[0080] In certain embodiments, the bleeding episode occurs in a disease state selected from the group consisting of acquired von Willebrand syndrome VWD-type 1, type 2A (IIA) (also referred to as type 2A-group 2), type 2A (IIE) (also referred to as type 2A-group 1), types 2B and 2M, severe aortic stenosis, and patients receiving ECMO.

[0081] In certain embodiments, a bleeding episode occurs when increased VWF degradation by ADAMTS13 is observed.

[0082] In a specific embodiment, multimers were analyzed via SDS-agarose electrophoresis. The relative amount of multimers with more than 10 bands was determined by comparison with normal pooled plasma. Plasma samples from subjects suffering from bleeding episodes were analyzed for total antigens using a polyclonal antibody and for intact VWF using KB-VWF-D3.1. Normal pooled plasma was used as a calibrator. The ratio of intact VWF / total VWF antigen is presented. Each individual sample is represented by a filled symbol. Statistical analysis was performed via one-way ANOVA with Dunnett's correction for multiple comparisons. The ratio of intact VWF / total VWF antigen is plotted against the relative amount of large multimers for samples from subjects with severe aortic stenosis and ECMO subjects.

[0083] In a further embodiment, the present invention relates to an in vitro method for diagnosing a disease associated with decreased ADAMTS13 activity in a subject in need thereof, comprising the steps of: i) contacting a biological sample with a single domain antibody according to the invention; ii) determining the level of intact VWF by the isolated single domain antibody (KB-VWF-D3.1); iii) determining the level of total VWF by antibody (polyclonal or monoclonal); iv) calculating the ratio of the level determined in step ii) to the level of total VWF antigen determined in step iii); v) comparing the ratio determined in step iv) with a predetermined corresponding reference value; and vi) concluding that the subject is susceptible to or at risk of developing a disease associated with decreased ADAMTS13 activity when the ratio determined in step iv) is higher than a predetermined reference value, or that the subject is unlikely to be susceptible to or not at risk of developing a disease associated with decreased ADAMTS13 activity when the ratio determined in step iv) is similar to a predetermined reference value.

[0084] As used herein, the term "disease associated with decreased ADAMTS13 activity" refers to a disease in which the activity of ADAMTS13 is decreased. In this case, VWF degradation is decreased. Therefore, the level of intact VWF increases with the administration of the single domain antibody of the present invention.

[0085] In certain embodiments, decreased ADAMTS13 activity results in less VWF degradation, which induces the formation of thrombi.

[0086] Therefore, the disease associated with decreased ADAMTS13 activity refers to any disease in which thrombus formation occurs. In a specific embodiment, the disease associated with decreased ADAMTS13 activity is selected from the group consisting of, but not limited to, immune thrombotic thrombocytopenic purpura, hereditary thrombotic thrombocytopenic purpura, hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP) syndrome, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) such as Covid-19, HIV, dengue, chikungunya, and malaria.

[0087] As used herein, the term "predetermined reference value" refers to a threshold or cutoff value. A "threshold," "reference value," or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold can also be arbitrarily selected based on existing experimental and / or clinical conditions, as recognized by those of ordinary skill in the art. For example, retrospective measurement of the concentration of a marker of the present invention (e.g., intact VWF) in appropriately stored historical subject samples can be used to establish a predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median value measured in a population of subjects for a marker of the present invention (e.g., intact VWF). In some embodiments, the threshold should be determined to obtain optimal sensitivity and specificity according to the test's function and benefit / risk balance (clinical consequences of false positives and false negatives). Typically, optimal sensitivity and specificity (and therefore, threshold) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the concentration of the marker of the present invention (e.g., intact VWF) in a reference group, algorithm analysis can be used to statistically process the expression level determined in the sample to be tested, thereby obtaining a classification criterion with significance for sample classification. The full name of ROC curve is receiver operating characteristic curve, and it is also known as receiver operating characteristic curve. It is mainly used in clinical biochemistry diagnostic tests. ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It uses image synthesis method to reveal the relationship between sensitivity and specificity. A series of different cutoff values ​​(threshold or critical value, i.e., the boundary value between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity values ​​and specificity values. Then, sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw the curve. The larger the area under the curve (AUC), the higher the diagnostic accuracy. On an ROC curve, the point closest to the top left corner of the coordinate system is the critical point, which has both high sensitivity and high specificity. The AUC value of an ROC curve is between 1.0 and 0.5.When AUC>0.5, the closer the AUC is to 1, the better the diagnostic result. When AUC is 0.5-0.7, the accuracy is low. When AUC is 0.7-0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably performed using a computer. For example, existing software or systems in the art, such as MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), may be used to plot the ROC curve.

[0088] In some embodiments, the predetermined corresponding reference value is typically determined by carrying out a method comprising the following steps: a) providing a collection of samples from a subject; b) for each sample provided in step a) providing information on the actual clinical profile of the subject (healthy or suffering from a bleeding episode); c) providing a series of arbitrary quantitative values; d) determining the concentration of a marker of the invention (e.g. intact VWF) for each sample contained in the collection provided in step a); e) for one particular arbitrary quantitative value provided in step c), classifying the blood samples into two groups: (i) a first group including samples exhibiting a quantitative value at a level lower than the arbitrary quantitative value contained in the series of quantitative values; (ii) a second group including samples exhibiting a quantitative value at a level higher than the arbitrary quantitative value contained in the series of quantitative values; thereby obtaining two groups of samples for the particular quantitative value, and the samples in each group are listed separately; f) calculating the statistical significance between (i) the quantitative values ​​obtained in step e) and (ii) the actual clinical profiles of the subjects from which the samples contained in the first and second groups defined in step f) are derived; g) repeating steps f) and g) until all of the optional quantitative values ​​provided in step d) have been tested; h) setting said predetermined corresponding reference value as consisting of any quantitative value calculated in step g) with the highest statistical significance (most significant).

[0089] Therefore, in some embodiments, the predetermined corresponding reference value thus allows for the discrimination between healthy subjects and subjects suffering from an inflammatory disease. In fact, a high statistically significant value (e.g., a low P-value) is generally obtained not only for a single arbitrary quantitative value, but also for a range of continuous arbitrary quantitative values. Therefore, in an alternative embodiment of the present invention, instead of using a clear predetermined corresponding reference value, a range of values ​​is provided. Thus, a minimum statistically significant value (a minimum significance threshold, e.g., a maximum threshold P-value) is arbitrarily set, and a range of multiple arbitrary quantitative values ​​having a higher statistically significant value (more significant, e.g., lower P-value) calculated in step g) is retained, thereby providing a range of quantitative values. This range of quantitative values ​​includes the "cut-off" value described above. For example, according to this particular embodiment of the "cut-off" value, a diagnosis can be determined by comparing the concentration of a marker of the present invention (e.g., intact VWF) with the identified range of values. In certain embodiments, the cutoff value therefore consists of a range of quantitative values, e.g., a quantitative value centered around the quantitative value at which the highest statistically significant value is found (e.g., generally, the smallest p-value is found).

[0090] In some embodiments, the methods of the present invention are performed in vitro or ex vivo.

[0091] kit In a fourth aspect, the present invention relates to a kit suitable for use in a method for diagnosing a bleeding episode in a subject as described above, comprising a single domain antibody as described above that specifically reacts with intact VWF and instructions for use.

[0092] The kit of the present invention can contain a single domain antibody coupled to a solid support, such as a well plate or beads (e.g., Sepharose beads). A kit can be provided containing an antibody for detecting and quantifying intact VWF protein in vitro, for example, by ELISA or Western blot. Such single domain antibodies useful for detection can be provided with a label, such as a fluorescent or radiolabel.

[0093] In a particular embodiment, the present invention relates to a kit for carrying out the method of the present invention, said kit comprising means for measuring the expression level of a biomarker of the present invention.

[0094] In certain embodiments, kits of the present invention may include instructional materials containing directions (eg, protocols) for practicing the diagnostic methods.

[0095] The kits may include probes, primers, macroarrays, or microarrays as described above. The kits may further include hybridization reagents or other appropriately packaged reagents and materials required for a particular hybridization protocol (including solid phase matrices, if applicable, and standards). Alternatively, the kits of the present invention may include amplification primers, which may be pre-labeled or contain affinity purification or attachment moieties. The kits may further include amplification reagents and other appropriately packaged reagents and materials required for a particular amplification protocol.

[0096] In certain embodiments, the present invention provides diagnostic kits containing anti-total VWF antibodies (monoclonal or polyclonal), including single domain antibodies, antibody conjugates, of the present invention. The diagnostic kit is a package containing at least one single domain antibody of the present disclosure (e.g., either in lyophilized form or as an aqueous solution) and one or more reagents useful for performing a diagnostic assay (e.g., a diluent, a labeled antibody that binds to the anti-total VWF antibody, a suitable substrate for the labeled antibody, positive controls and reference standards, VWF in a form suitable for use as a negative control).

[0097] Alternatively, the kit can include a labeled antibody that binds to the anti-VWF monoclonal / polyclonal antibody and is conjugated to an enzyme. When the anti-total VWF monoclonal or other antibody is conjugated to an enzyme for detection, the kit can include substrates and cofactors required for the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., block buffers or lysis buffers), can also be included. The anti-total VWF antibody included in the diagnostic kit can be immobilized on a solid surface, or alternatively, a solid surface (e.g., a slide) on which the antibody can be immobilized is included in the kit. The relative amounts of the various reagents can be varied widely to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. The antibody and other reagents, including excipients that, upon dissolution, provide a reagent solution having the appropriate concentration, can be provided (individually or in combination) as dry powders (usually lyophilized).

[0098] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0099] drawing: [Figure 1]Generation of anti-VWF nanobodies. A: Flow diagram of the screening approach using recombinant VWF (rVWF) and cleaved VWF to isolate anti-VWF nanobodies that discriminate between intact and cleaved VWF. B–D: Dose response of rVWF (black circles) and cleaved VWF (gray circles) to immobilized single-domain antibodies KB-VWF-D3.1 (5 μg / ml; panel B) or KB-VWF-F1.1 (5 μg / ml; panel D). Panel C compares rVWF and plasma-derived VWF (pdVWF), both added at a concentration of 5 μg / ml. Bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent the mean ± SD of 4–8 experiments. E: Binding of various concentrations of KB-VWF-D3.1 (0–5 μg / ml) to immobilized rVWF or degraded VWF (both 5 μg / ml). Bound KB-VWF-D3.1 was probed with a peroxidase-labeled polyclonal rabbit anti-cMyc antibody and detected by hydrolysis of 3,3',5,5'-tetramethylbenzidine. [Figure 2] Determination of the binding epitope for KB-VWF-F1.1. A: ADAMTS13 activity was measured using its fluorescent substrate, FRETS-VWF73. The substrate (2 μM) was preincubated with the indicated concentrations of single-domain antibodies. Residual ADAMTS13 activity was calculated and plotted against the concentration of single-domain antibodies. B: Immobilized cleaved VWF (5 μg / ml) was incubated with KB-VWF-F1.1 (1 μg / ml) in the absence or presence of various concentrations of an anti-A2 domain antibody (MAB27642; R&D Systems) that recognizes the N-terminal portion (Asp1596-Tyr1605) of the ADAMTS13 cleavage site. The residual binding of KB-VWF-F1.1 (set to 1.0 in the absence of competitor) is presented as a function of the molar ratio of antibody MAB27642 to KB-VWF-F1.1. For both panels, data represent the mean ± SD of three experiments. [Figure 3]KB-VWF-D3.1 binds to the VWF A3 domain. A: Binding of KB-VWF-D3.1 (1 μg / ml) to various concentrations of VWF domain-Fc fusion protein (0–10 nM) captured on an anti-human Fc antibody. Bound KB-VWF-D3.1 was probed using a peroxidase-labeled polyclonal rabbit anti-cMyc antibody and detected by hydrolysis of 3,3',5,5'-tetramethylbenzidine. A1-Fc: gray squares; A2-Fc: black triangles; A3-Fc: black circles; D4-Fc: white circles. Data represent the mean ± SD of three experiments. B: Amino acid sequence of the VWF A3 domain. Residues bearing the epitope for KB-VWF-D3.1 are in bold. Residue 22, previously reported to be involved in collagen binding, is boxed. C: Inhibition of pd-VWF binding to collagen type III by KB-VWF-D3.1 (filled circles), monoclonal antibody Mab505 (gray squares), and single-domain antibody C37h (open circles). Residual pd-VWF binding is presented versus single-domain antibody / antibody concentration. Data represent the mean ± SD of three experiments. D: Binding of pd-VWF (filled symbols) or degraded VWF (open symbols) to immobilized Mab505 (5 μg / ml; circles) or C37h (5 μg / ml; squares). Bound pd-VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent the mean ± SD of three experiments. [Figure 4]Determination of the binding epitope for KB-VWF-D3.1. A. Binding of VWF domain-Fc fusion proteins to immobilized KB-VWF-D3.1 (5 μg / ml). Bound fragments were probed using a peroxidase-labeled monoclonal anti-human Fc antibody and detected according to 3,3',5,5'-tetramethylbenzidine hydrolysis. A1-Fc: gray triangles; A2-Fc: white circles; A3-Fc: black circles; D4-Fc: white squares. B. Immobilized KB-VWF-D3.1 (5 μg / ml) was incubated with recombinant VWF deletion variants (1 μg / ml). Bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected according to 3,3',5,5'-tetramethylbenzidine hydrolysis. Blood was perfused through a collagen-coated PL chip at a shear rate of 2000 s-1 using a CT-TAS Plus device. Pressure (a marker of clot formation) was measured in real time during perfusion. Perfusion performed in the presence of KB-VWF-D3.1 (20 μg / ml) resulted in a smaller area under the curve compared to the control (48.5 (95% CI: 48.1-48.9) vs. 70.3 (95% CI: 67.4-73.3)). For panels A & B, data represent the mean ± SD of triplicate experiments. For panel C, the solid line represents the mean of triplicate experiments, and the gray area (delineated by the dotted line) represents the standard error. [Figure 5] Binding of VWF with various multimer sizes to KB-VWF-D3.1. A: Binding of HMW-VWF (filled circles) and MMW-VWF (open circles) to immobilized KB-VWF-D3.1 (5 μg / ml). B: Binding of multimeric rVWF (filled circles) and dimeric VWF / delta-pro variant (gray squares) to immobilized KB-VWF-D3.1 (5 μg / ml). In both panels, bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent the mean ± SD of three to four independent measurements. [Figure 6]ADAMTS13-mediated proteolysis regulates VWF binding to KB-VWF-D3.1 and KB-VWF-F1.1. Samples were analyzed for total VWF antigen using a polyclonal antibody, for the presence of intact VWF using KB-VWF-D3.1, and for the presence of degraded VWF using KB-VWF-F1.1. The ratios of intact VWF / total VWF antigen (filled circles; left y-axis) and degraded VWF / total VWF antigen (gray squares; right y-axis) are presented versus exposure time to ADAMTS13. Normal pooled plasma was used as a calibrator for KB-VWF-D3.1, while a degraded VWF preparation was used as a calibrator for KB-VWF-F1.1. Data represent the mean ± SD of three independent experiments. [Figure 7] Detection of intact VWF in congenital VWD. A: VWF-deficient plasma was spiked with various amounts of purified rVWF and degraded VWF and incubated in a microtiter plate coated with KB-VWF-D3.1. Bound VWF was probed using a peroxidase-conjugated polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent the mean ± SD of three to four independent measurements. The solid line illustrates the best linear fit, and the 95% confidence interval is indicated by a dotted line. The vertical line indicates 90% intact rVWF complemented with 10% degraded VWF. B-C: Patient plasma samples were analyzed for total antigen using a polyclonal antibody and for intact VWF using KB-VWF-D3.1. Normal pooled plasma was used as a calibrator. The ratio of intact VWF / total VWF antigen is presented. Each individual sample is represented by a filled symbol. Statistical analysis was performed via one-way ANOVA with Dunnett's correction for multiple comparisons (Panel B) or Mann-Whitney (Panel C). D: Multimers were analyzed via SDS-agarose electrophoresis. The relative amount of multimers with more than 10 bands was determined via comparison with normal pooled plasma (NPP). E: The ratio of intact VWF / total VWF antigen is plotted against the relative amount of large multimers. Correlations were determined using Graphpad Prism software. [Figure 8] Detection of intact VWF in AVWS. A: Multimers were analyzed via SDS-agarose electrophoresis. The relative amount of multimers with more than 10 bands was determined via comparison with normal pooled plasma (NPP). B: Patient plasma samples were analyzed for total antigen using a polyclonal antibody and for intact VWF using KB-VWF-D3.1. Normal pooled plasma was used as a calibrator. The ratio of intact VWF / total VWF antigen is presented. Each individual sample is represented by a filled symbol. Statistical analysis was performed via one-way ANOVA with Dunnett's correction for multiple comparisons. Control samples were identical to those presented in Figure 5. C-D: The ratio of intact VWF / total VWF antigen is plotted against the relative amount of large multimers for samples from a patient with severe aortic stenosis (AS; panel C) and an ECMO patient (panel D). [Example]

[0100] Materials & Methods Code of ethics: All volunteers and patients provided written informed consent in accordance with the Declaration of Helsinki. Patients with VWD were included in the French VWD Cohort Multicenter Database (Centre Reference Maladie Willebrand). 19 The cohort's database and biobank were declared and approved by the French Data Protection Authority (CNIL-1245379 / DEC-19252, CODECOH-DC-2008-642). Patients with severe aortic stenosis (WITAVI trial, NCT02628509) and those receiving extracorporeal mechanical oxidation (ECMO; WITECMO-H trial, NCT03070912) were included in the study. All protocols were approved by the local audit and ethics committee.

[0101] Isolation of anti-VWF nanobodies Phage libraries encoding synthetic single-domain antibodies 20 Anti-VWF nanobodies were isolated using the library (3 × 10 9 The phage (10 clones) were incubated with streptavidin-coated beads loaded with biotinylated rVWF. Unbound phage were then incubated with beads loaded with biotinylated degraded VWF. Three rounds of phage display were performed, and the depletion step was repeated for each round. 12 unique sequences were obtained through this procedure (Figure 1A).

[0102] Analysis of VWF binding to nanobodies Purified rVWF (0–0.5 μg / ml) was incubated in wells coated with the single-domain antibodies KB-VWF-D3.1 or KB-VWF-1.1 (both at 5 μg / ml). Bound VWF was probed with a polyclonal anti-VWF antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine.

[0103] Detection of intact VWF Intact VWF was considered to be VWF recognized by KB-VWF-D3.1. Briefly, samples containing undigested VWF, ADAMTS13-digested VWF, or a mixture of both were incubated in wells coated with KB-VWF-D3.1 (5 μg / ml). Alternatively, plasma samples were used. Bound VWF was probed with a polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine.

[0104] Total VWF antigen Total VWF antigen was measured by ELISA using polyclonal rabbit anti-VWF antibody as described 21 .

[0105] result Selection of anti-VWF nanobodies To isolate nanobodies that could discriminate between intact and proteolytically digested VWF, we applied a selection strategy using rVWF and digested VWF, generating 12 unique sequences (Figure 1A, data not shown). The purified nanobodies were tested for their interaction with rVWF and digested VWF. Ten of the 12 nanobodies exhibited similar binding to both VWF preparations, whereas two were characterized by differential binding. Initially, rVWF associated with the immobilized single-domain antibody KB-VWF-D3.1 in a dose-dependent manner, whereas binding of digested VWF to this single-domain antibody was strongly reduced (Figure 1B). In these assays, pd-VWF consistently produced lower responses than rVWF (93 ± 4% compared with 100 ± 2%; p = 0.015; Figure 1C), likely due to the presence of digested VWF in normal plasma. In a supplementary assay, KB-VWF-D3.1 bound to immobilized cleaved VWF 8-fold less efficiently than immobilized rVWF (Figure 1E). For KB-VWF-F1.1, efficient binding of cleaved VWF was detected, whereas binding of rVWF approached background levels (Figure 1D). Therefore, these two nanobodies were selected for further analysis.

[0106] Determination of the binding epitope for KB-VWF-F1.1 Since KB-VWF-F1.1 bound to cleaved but not intact VWF, we concluded that this single domain antibody binds to Tyr 1605 -Met 1606 The VWF A2 domain sequence Asp 1596 -Arg 1668 VWF-KB-F1.1 was tested in an ADAMTS13 activity assay using its substrate FRETS-VWF73, which contains the domain VWF / D4-CK (Figure 2A). KB-VWF-D3.1 and the control single-domain antibody KB-VWF-004 (against the domain VWF / D4-CK) did not affect the proteolysis of the substrate, whereas KB-VWF-F1.1 efficiently blocked the substrate conversion by ADAMTS13. This indicates that the epitope of VWF-KB-F1.1 is located in the domain Asp 1596 -Arg 1668Notably, KB-VWF-F1.1 and residue Arg 1596 -Tyr 1605 MAB27642, which targets VWF 1, did not compete for binding to cleaved VWF (Fig. 2B), suggesting that they recognize different epitopes within this region.

[0107] Determination of the binding epitope for KB-VWF-D3.1 To determine the epitope of KB-VWF-D3.1, we first analyzed the binding of this single-domain antibody to a series of rVWF fragments, namely, A1-Fc, A2-Fc, A3-Fc, and D4-Fc. Surprisingly, KB-VWF-D3.1 bound most efficiently to the A3-Fc fragment rather than the A2-Fc fragment (Fig. 3A). Binding was similar when the binding of the fragments to immobilized KB-VWF-D3.1 was assessed (Fig. 4A). Furthermore, binding of rVWF lacking the A3 domain to KB-VWF-D3.1 was undetectable, whereas deletion of other domains did not affect binding (Fig. 4B).

[0108] To narrow down its binding site within the A3 domain, molecular modeling was performed (data not shown). This approach demonstrated that all of the top 30 ranked conformations of the complex clustered similarly, suggesting that the single-domain antibody binds to the VWF A3 domain region Val. 1732 -Asn 1818 We found that KB-VWF-D3.1 docked onto four distinct stretches of amino acids within the epitope (data not shown). Interestingly, eight of the amino acids in the epitope of KB-VWF-D3.1 have previously been implicated in collagen binding (Figure 3B). 22 This indicates that the epitope of KB-VWF-D3.1 overlaps with the collagen-binding site. Based on this, we investigated the effect of KB-VWF-D3.1 on VWF binding to type III collagen by comparing it with two known A3 domain-binding antibodies (C37h single domain antibody) and the effect of KB-VWF-D3.1 on VWF binding to type III collagen. 23 and Mab505 monoclonal antibody 24) compared with those of the positive controls C37h and Mab505, which efficiently blocked pd-VWF-collagen interaction (Fig. 2F). KB-VWF-D3.1 also dose-dependently reduced pd-VWF binding to collagen, but less efficiently than the antibodies C37h and Mab505 (Fig. 3C). In addition, KB-VWF-D3.1 delayed VWF-dependent platelet adhesion to collagen under flow conditions (Fig. 4C). The overlapping epitopes of their epitopes with the collagen-binding site raise the question of whether C37h and Mab505 can discriminate between intact and cleaved VWF in the same way as KB-VWF-D3.1. However, both C37h and Mab505 exhibited similar binding to both intact rVWF and cleaved VWF (Fig. 3D). Thus, the single domain antibody KB-VWF-D3.1 is unique in binding to an epitope within the A3 domain whose exposure is regulated by proteolysis within the A2 domain.

[0109] Effect of multimer size on VWF binding to KB-VWF-D3.1 ADAMTS13-mediated proteolysis of VWF involves the Tyr 1605 -Met 1606This results in the loss of peptide bonds, thereby reducing multimer size. Therefore, we examined how multimer size affects VWF binding to immobilized KB-VWF-D3.1. First, we analyzed two separate pd-VWF preparations obtained from pd-VWF concentrates via gel filtration chromatography. One contained high-molecular-weight (HMW) multimers, while the other was enriched in medium-molecular-weight (MMW) multimers (data not shown). Both fractions exhibited similar binding to KB-VWF-D3.1 (Figure 5A). Next, we compared the binding of dimeric rVWF / delta-pro with that of full-length rVWF (Figure 5B). Both dimeric rVWF / delta-pro and rVWF bound to KB-VWF-D3.1 with similar half-maximal binding (0.2±1 μg / ml vs. 0.2±0.1 μg / ml; p=0.62). Apparently, VWF binding to immobilized KB-VWF-D3.1 is independent of its multimer size. The reduced binding of cleaved VWF to KB-VWF-D3.1 is due to the Tyr binding rather than a reduction in multimer size. 1605 -Met 1606 It is thought to result from proteolysis of peptide bonds.

[0110] Sequential proteolysis of VWF Next, we investigated the effect of time-dependent ADAMTS13 proteolysis on VWF binding to KB-VWF-D3.1 and KB-VWF-F1.1. Briefly, pd-VWF was exposed to shear stress in the presence of recombinant ADAMTS13, and samples were collected at the indicated time points (0–3 h). The multimerization pattern and binding to both nanobodies were analyzed. Exposure to ADAMTS13 resulted in a time-dependent decrease in pd-VWF multimer size (data not shown). As expected, proteolysis was inhibited in the presence of EDTA (a metal ion chelator that inactivates ADAMTS13). Concurrent with increased pd-VWF proteolysis, increased binding to KB-VWF-F1.1 was observed (Figure 6). In contrast, pd-VWF binding to KB-VWF-D3.1 was abolished in a complementary manner (Figure 6). These data demonstrate that the binding of both nanobodies to VWF depends on the extent of proteolysis by ADAMTS13.

[0111] Measurement of cleaved VWF in mixtures of intact and cleaved VWF Given the specificity of both nanobodies for intact and degraded VWF, respectively, we anticipated their usefulness in determining the extent of VWF proteolysis in patient samples. In preliminary experiments, KB-VWF-F1.1 lacked sufficient sensitivity to detect minor VWF proteolysis in plasma, so we focused the remainder of our studies on KB-VWF-D3.1. We first analyzed the extent to which increased proteolysis could be detected. We prepared different mixtures of purified rVWF and degraded VWF and determined the ratio of intact VWF / total VWF antigen. A dose-dependent decrease in this ratio was observed as the proportion of degraded VWF in the sample increased (Figure 7A). These experiments suggest that an increase of approximately 10% degraded VWF can be detected (p=0.0009 compared to 100% intact).

[0112] Analysis of plasma from patients with congenital VWD We then compared the variability of VWD in controls (n = 31) and VWD patients (n = 101) included in the French reference center for VWD. 19 Plasma samples obtained from patients were analyzed. The patient cohort consisted of patients with VWD-1 (n = 20), VWD-2A (n = 43), VWD-2B (n = 24), and VWD-2M (n = 14).

[0113] To determine the amount of intact VWF, we used normal pooled plasma as a calibrator and calculated the amount of antigen (=intact VWF) obtained with KB-VWF-D3.1 relative to the amount of total VWF antigen. By doing so, we found that the intact VWF / total antigen ratio for the control was 1.0 ± 0.2 (Figure 7B). The intact VWF / total VWF ratio decreased with each of the VWD types analyzed. The ratios were 0.7 ± 0.3 for VWD-1 (p = 0.0004), 0.5 ± 0.2 for VWD-2A (p < 0.0001), 0.6 ± 0.2 for VWD-2B (p < 0.0001), and 0.7 ± 0.2 for VWD-2M (p = 0.0148) (Figure 7B). Because VWD-2A is divided into two subtypes, VWD-2A-1 and VWD-2A-2 (where loss of multimers is dominated by defective multimerization and increased proteolysis, respectively), we analyzed samples from patients with VWD-2A-1 (n = 14) and patients with VWD-2A-2 (n = 29) separately. The ratio of intact VWF / total VWF antigen was significantly lower in the VWD-2A-2 group (0.4 ± 0.2) compared with the VWD-2A-1 group (0.6 ± 0.2; p = 0.0007; Figure 7C).

[0114] Because we did not expect to see a decrease in the intact VWF / total VWF antigen ratio in all VWD types, we also examined whether this decreased ratio corresponded to a potential loss of HMW multimers. Multimer analysis was available for a subset of samples, and we indeed observed a general relative decrease in HMW multimers (multimer bands >10) compared with normal pooled plasma in all patient groups, including VWD types 1 and 2M (Fig. 7D). Interestingly, there was a significant correlation between the intact VWF / total VWF antigen ratio and multimer size (r = 0.51; p < 0.0001; Fig. 7E). Thus, it appears that a large proportion of VWD patients experience increased proteolysis compared with the normal population.

[0115] Analysis of plasma from AVWS patients Next, we examined plasma samples from patients receiving ECMO support (n = 27) and patients with severe aortic stenosis (n = 17). Both patient groups were characterized by a loss of VWF HMW multimers, potentially caused by increased ADAMTS13-mediated proteolysis (Fig. 8A). Compared with normal controls, the ratio of intact VWF to total VWF antigen (measured by binding to KB-VWF-D3.1) was significantly reduced in both patient groups: 0.85 ± 0.09 (mean ± SD; p = 0.0017) and 0.78 ± 0.13 (p < 0.0001) for patients with severe aortic stenosis and those receiving ECMO, respectively (Fig. 8B). Of note, there was a significant correlation between the intact / total antigen ratio and the presence of HMW multimers (>10) in both groups, with p = 0.0463 in the severe aortic stenosis samples and p = 0.0452 in the ECMO samples (Figure 8C-D), which may suggest that the loss of larger multimers is indeed primarily due to proteolysis rather than other mechanisms.

[0116] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. [Table 2] TIFF2025541243000004.tif234169 TIFF2025541243000005.tif228169

Claims

1. An isolated single domain antibody that targets at least one region of the A3 domain of VWF, wherein the region comprises the following sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO:

4.

2. 2. An isolated single domain antibody targeting the A3 domain of VWF according to claim 1, comprising a CDR1 having the sequence shown as SEQ ID NO: 5, a CDR2 having the sequence shown as SEQ ID NO: 6, and a CDR3 having the sequence shown as SEQ ID NO:

7.

3. 3. An isolated single domain antibody targeting the A3 domain of VWF according to claim 2, comprising a CDR1 having at least 70% identity with the sequence shown as SEQ ID NO: 5, a CDR2 having at least 70% identity with the sequence shown as SEQ ID NO: 6, and a CDR3 having at least 70% identity with the sequence shown as SEQ ID NO:

7.

4. 2. The isolated single domain antibody directed against von Willebrand factor (VWF) according to claim 1, which is KB-VWF-D3.1 (SEQ ID NO: 8).

5. 1. A method for determining a level of VWF degradation in a subject in need thereof, comprising the steps of: i) contacting an isolated single domain antibody according to claims 1 to 4 with a biological sample; ii) determining the level of intact VWF by isolated single domain antibody (KB-VWF-D3.1); iii) determining the level of total VWF by antibody (polyclonal or monoclonal); iv) calculating the ratio of the level determined in step ii) to the level of total VWF antigen determined in step iii); v) comparing the ratio determined in step iv) with a predetermined corresponding reference value; and vi) concluding that the level of VWF degradation is elevated when the ratio determined in step iv) is lower than a predetermined reference value, or concluding that the level of VWF degradation is not elevated when the ratio determined in step iv) is higher than a predetermined reference value.

6. 1. An in vitro method for diagnosing a bleeding episode in a subject in need thereof, comprising: i) contacting a biological sample with a single domain antibody according to the invention; ii) determining the level of intact VWF by isolated single domain antibody (KB-VWF-D3.1); iii) determining the level of total VWF by antibody (polyclonal or monoclonal); iv) calculating the ratio of the level determined in step ii) to the level of total VWF antigen determined in step iii); v) comparing the ratio determined in step iv) with a predetermined corresponding reference value; and vi) concluding that the subject is prone to or at risk of suffering from a bleeding episode when the ratio determined in step iv) is lower than a predetermined reference value, or that the subject is unlikely to or not at risk of suffering from a bleeding episode when the ratio determined in step iv) is identical to the predetermined reference value.

7. The method according to claims 5 to 6, wherein the biological sample is a plasma sample.

8. 8. The method of claims 6-7, wherein the bleeding episode occurs in a disease state selected from the group consisting of acquired von Willebrand syndrome VWD-type 1, type 2A(IIA) (also called type 2A-group 2), type 2A(IIE) (also called type 2A-group 1), type 2B and type 2M, severe aortic stenosis, and patients receiving ECMO.

9. A nucleic acid sequence encoding the isolated single domain antibody of claim 1.

10. A nucleic acid sequence encoding the heavy chain of the isolated single domain antibody of claim 1.

11. A vector comprising the nucleic acid according to claims 9 to 10.

12. A host cell engineered to express the isolated single domain antibody of claim 1.

13. A kit comprising at least one single domain antibody according to claims 1 to 4.

14. 14. The kit of claim 13, comprising a solid support, a label, such as a fluorescent or radioactive label, and instructions for use.

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