WO2024126685A1

WO2024126685A1 - Single-domain antibody targeting von wilebrand factor a3-domain

Info

Publication number: WO2024126685A1
Application number: PCT/EP2023/085828
Authority: WO
Inventors: Olivier Christophe; Caterina CASARI; Sophie SUSEN; Cécile DENIS; Petrus Lenting
Original assignee: Institut National de la Santé et de la Recherche Médicale; Centre Hospitalier Regional Universitaire De Lille; Institut Pasteur De Lille; Université Paris-Saclay; Université de Lille
Priority date: 2022-12-15
Filing date: 2023-12-14
Publication date: 2024-06-20

Abstract

Inventors have isolated a single-domain antibody (designated KB-VWF-D3.1) targeting the A3-domain, the epitope of which overlaps the collagen-binding site. Binding of KB-VWF-D3.1 proved independent of VWF multimer size. However, its interaction with VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2-domain modulates exposure of its epitope in the A3-domain. Inventors therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact-VWF could be detected using this single-domain antibody. By comparing plasma from volunteers to that of congenital VWD-patients, intact-VWF levels were significantly reduced for all VWD-types, and most severely in VWD-type 2A(IIA) in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, low-grade proteolysis in VWD-type 1 and -type 2M was also observed. Thus, this single-domain antibody proved sensitive to detect low-grade degradation in plasma from patients with AVWS and congenital VWD, including types 1 and 2M. The present invention relates to an isolated single domain antibody targeting at least one region of A3-domain of VWF, wherein the region comprising the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4.

Description

SINGLE-DOMAIN ANTIBODY TARGETING VON WILEBRAND FACTOR A3- DOMAIN

FIELD OF THE INVENTION:

The invention is in the field of coagulation, more particularly, the invention relates to nanobodies targeting A3 domain of Factor Von Willebrand and their uses in diagnosis method.

BACKGROUND OF THE INVENTION:

Von Willebrand factor is a multimeric protein, the extent 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) are first covalently linked via disulfide bridging between two C-terminal CK-domains. These pro-dimers are then processed into multimers viaN-terminal coupling of the D’-D3 regions, with the D1-D2 portion (also known as VWF propeptide) being eliminated during this event. The multimer size in endothelial cells is highly variable and varies from dimers to ultra-large multimers with >40 subunits. Upon secretion, VWF multimers are susceptible to regulated proteolysis by ADAMTS13, a metalloprotease that cleaves VWF in its A2-domain at the Tyrl605-Metl606 peptide bond.³ Importantly, proteolysis occurs only upon decryption of the cleavage site, which normally lies buried within the A2-domain.⁴ There are several occurrences that allow for the exposure of the ADAMTS13-cleavage site. First, multiple VWF multimers assemble at the endothelial surface upon stimulated secretion, forming elongated fibers that are proteolyzed by ADAMTS13.⁵'⁷ Second, VWF unfolds during circulation under conditions of increased shear stress or disturbed blood flow.⁸ When disturbed blood flow is exaggerated, like in patients having severe aortic stenosis or those who require mechanical circulatory support, excessive VWF degradation may occur, which is then referred to as acquired von Willebrand syndrome (A VWS).^9-11 Third, mutations within VWF may provoke exposure of the ADAMTS13 proteolytic site, and such mutations are most frequently found in von Willebrand disease (VWD)-type 2A(IIA) (also referred to as VWD-type 2A-group 2) and VWD-type 2B.^12,13 Excessive proteolysis of VWF is associated with an increased loss of high molecular weight (HMW)-multimers, which results in reduced platelet binding and reduced collagen binding, thereby increasing the risk of bleeding.⁹ The classic approach to visualize the extent of VWF degradation is to analyze the multimeric pattern using SDS-agarose electrophoresis.¹⁴ This approach is laborious, non-standardized and requires 24-72h, dependent on the method that is used. Alternatively, collagen- or platelet-binding assays are used¹⁵, which are less specific in that they do not distinguish between impaired multimerization and excessive degradation. Finally, Kato and colleagues described a monoclonal antibody that binds to the newly formed C -terminal end in the A2-domain. This antibody was successfully used to measure ADAMTS13 activity in patients with thrombotic thrombocytopenic purpura.¹⁶ In addition, we and others used a similar antibody (MAB27642) to monitor VWF proteolysis in patients.^17,18 However, while using this antibody, we noticed that this antibody was unable to detect low-grade degradation, for instance in samples from patients with non-severe aortic stenosis or with congenital VWD-type 1 or 2M. ⁵

Thus, there is a need to develop new tools to detect low-grade degradation of VWF in patients.

SUMMARY OF THE INVENTION:

The invention relates to an isolated single domain antibody directed against to at least one region of Von Willebrand Factor A3-domain, wherein said region comprising the following sequences: 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.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have isolated a single-domain antibody (designated KB-VWF-D3.1) targeting the A3 -domain, the epitope of which overlaps the collagen-binding site. Binding of KB- VWF - D3.1 proved independent of VWF multimer size. However, its interaction with VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2-domain modulates exposure of its epitope in the A3-domain. Inventors therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact- VWF could be detected using this single-domain antibody. By comparing plasma from volunteers to that of congenital VWD-patients, intact- VWF levels were significantly reduced for all VWD-types, and most severely in VWD-type 2A(IIA) in which mutations promote ADAMTS13- mediated proteolysis. Unexpectedly, low-grade proteolysis in VWD-type 1 and -type 2M was also observed. Thus, this single-domain antibody proved sensitive to detect low- grade degradation in plasma from patients with AVWS and congenital VWD, including types 1 and 2M. Single-domain antibody targeting at least one region of Von Willebrand Factor A3- domain

Accordingly, in a first aspect, the invention relates to an isolated single domain antibody targeting at least one region of Von Willebrand Factor A3-domain, wherein the region comprising the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4.

By "isolated" it is meant, when referring to a single-domain antibody according to the invention, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.

As used herein the term "single-domain antibody" (sdAb) has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single-domain antibody are also called VHH or "nanobody®". For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends Biotechnol, 2003, 21(1 1):484- 490; and WO 06/030220, WO 06/003388. The amino acid sequence and structure of a singledomain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3 " or "FR3"; and as "Framework region 4" or "FR4" respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementary Determining Region 1” or “CDR1”; as "Complementarity Determining Region 2" or "CDR2" and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single-domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single-domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/).

The term "VWF" has its general meaning in the art and refers to the human von Willebrand factor (VWF) which is a blood glycoprotein involved in blood clotting. VWF is a monomer composed of several homologous domains each covering different functions: D1-D2- D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. The naturally occurring human VWF protein has an aminoacid sequence as shown in GeneBank Accession number NP 000543.2. Monomers are subsequently arranged into dimers or multimers by crosslinking of cysteine residues via disulfide bonds. Multimers of VWF can thus be extremely large and can consist of over 40 monomers also called high molecular weight (HMW)-multimers of VWF. Upon secretion, VWF multimers are susceptible to regulated proteolysis by ADAMTS13, a metalloprotease that cleaves VWF in its A2-domain at the Tyrl605-Metl606 peptide bond.³ Importantly, proteolysis occurs only upon decryption of the cleavage site, which normally lies buried within the A2-domain.⁴ There are several occurrences that allow for the exposure of the ADAMTS13-cleavage site. First, multiple VWF multimers assemble at the endothelial surface upon stimulated secretion, forming elongated fibers that are proteolyzed by AD AMTS 13.^5-7 Second, VWF unfolds during circulation under conditions of increased shear stress or disturbed blood flow.⁸ When disturbed blood flow is exaggerated, like in patients having severe aortic stenosis or those who require mechanical circulatory support, excessive VWF degradation may occur, which is then referred to as acquired von Willebrand syndrome (A VWS).^9-11 Third, mutations within VWF may provoke exposure of the ADAMTS13 proteolytic site, and such mutations are most frequently found in von Willebrand disease (VWD)-type 2A(IIA) (also referred to as VWD-type 2A-group 2) and VWD-type 2B.^12,13 Excessive proteolysis of VWF is associated with an increased loss of high molecular weight (HMW)-multimers, which results in reduced platelet binding and reduced collagen binding, thereby increasing the risk of bleeding.⁹

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.

In a particular embodiment, the single-domain antibody according to the invention binds to an epitope comprising residues Vall732-Vall747 (region 1), Leul755-Glnl769 (region 2), Aspl771-Hisl786 (region 3) and Vall805-Asnl818 (region 4) of VWF.

In a particular embodiment, the single-domain antibody according to the invention targets:

- the region comprising or consisting of the following amino acid sequences: V L Q Y G S I T T I D V P W N V (SEQ ID NO: 1);

- the region comprising or consisting of the following amino acid sequences: L S L V D V M Q R E G G P S Q (SEQ ID NO: 2);

- the region comprising or consisting of the following amino acid sequences: D A L G F A V R Y L T S E M H (SEQ ID NO: 3); and/or - the region comprising or consisting of the following amino acid sequences: V D S V D A A A D A A R S N (SEQ ID NO: 4);

In a particular embodiment, the single-domain antibody according to the invention detects changes in the exposure of its epitope within the collagen-binding site of the A3 -domain.

In view of its unique characteristics, the single-domain antibody according to the invention is suitable to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

In a particular embodiment, the single-domain antibody according to the invention detects the VWF intact which is not degraded by the AD AMTS 13.

In a particular embodiment, the single-domain antibody according to the invention specifically binds to intact VWF (i.e VWF not proteolyzed/degraded by ADAMTS13).

As used herein, the term "specifically binds to" means that the single-domain antibody only binds to the antigen of interest, e.g. non-proteolyzed VWF, and does not exhibit crossreactivity to proteolyzed VWF by AD AMTS 13. In other word, in a particular embodiment, the single-domain antibody according to the invention does not detect the VWF intact degraded by the AD AMTS 13.

In a particular embodiment, the single-domain antibody according to the invention allows to determine the VWF degradation in a biological sample such as plasma sample.

The inventors have isolated nanobodies distinguishing between proteolyzed and non- proteolyzed VWF, leading to the identification of a single-domain antibody (designated KB- VWF-D3.1) targeting the A3-domain, the epitope of which overlaps 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):

Table A: Sequences of KB-VWF-D3.1 domains.

In a particular embodiment, the isolated single-domain antibody targeting A3-domain of VWF according to the invention, wherein said sdAb comprises a CDR1 having at least 70% of identity with sequence set forth as SEQ ID NO: 5, a CDR2 having at least 70% of identity with sequence set forth as SEQ ID NO: 6 and a CDR3 having at least 70% of identity with sequence set forth as SEQ ID NO: 7.

In particular, the 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%, even more preferably at least 99% sequence identity with sequence set forth as SEQ ID NO: 5, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with sequence set forth as SEQ ID NO: 6 cand a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with sequence set forth as SEQ ID NO: 7.

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)).

In some embodiments, the isolated single-domain antibody according to the invention comprises a CDR1 having a sequence set forth as SEQ ID NO: 5, a CDR2 having a sequence set forth as SEQ ID NO: 6 and a CDR3 having a sequence set forth as SEQ ID NO: 7.

The isolated single-domain antibody directed against albumin according to the invention wherein said sdAb is KB-VWF-D3.1 (SEQ ID NO: 8).

In some embodiments, the isolated single-domain antibody according to the invention has the sequence set forth as SEQ ID NO: 8.

In a particular embodiment, the isolated single-domain antibody according to the invention has the sequence set forth as SEQ ID NO: 9:

MAEVQLQASGGGFVQPGGSLRLSCAASGTGSHFDSMGWFRQAPGKEREFVS AISREQTVEPYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAGWKYVTW MGHLKFKAKERYWGQGTQVTVSS. In a particular embodiment, the isolated single domain antibody targeting A3-domain of VWF according to the invention, wherein said single domain antibody has a sequence of variable heavy chain (VHH) set forth as SEQ ID NO: 8.

It should be further noted that the sdAb KB-VWF-D3.1 cross-react with murine VWF, which is of interest for preclinical evaluation and toxicological studies.

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 invention wherein an amino acid sequence that corresponds 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 (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional chain antibody from a human being. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be chosen such that the resulting humanized single domain antibodies still retain the favorable properties of single-domain antibodies of the invention. The one skilled in the art is able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions.

According to the invention, the single domain antibody of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art.

The single domain antibodies and polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as 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 invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art.

As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a polypeptide of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

A variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. 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 virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus 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 e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful in recombinant protein productions 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, PC 12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide 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, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

In the recombinant production of the single domain antibodies and polypeptides of the invention, it would be necessary to employ vectors comprising polynucleotide molecules for encoding the single domain antibodies and polypeptides of the invention. Methods of preparing such vectors as well as producing host cells transformed with such vectors are well known to those skilled in the art. The polynucleotide molecules used in such an endeavor may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. These elements of the expression constructs are well known to those of skill in the art. Generally, the expression vectors include DNA encoding the given protein being operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation.

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 coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.

The choice of a suitable expression vector for expression of the peptides or polypeptides of the invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan.

Expression requires that appropriate signals be provided in the vectors, such as enhancers/promoters from both viral and mammalian sources that may be used to drive expression of the nucleic acids of interest in host cells. Usually, the nucleic acid being expressed is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the protein of interest (e.g., a single domain antibody). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence.

In a particular embodiment, the invention relates to a nucleic acid sequence encoding the isolated single domain antibody of the invention.

In a particular embodiment, the invention relates to a nucleic acid sequence which encodes a heavy chain of the isolated single domain antibody of the invention.

In a particular embodiment, the invention relates to a vector comprising the nucleic acid of the invention.

In a particular embodiment, the invention relates to a host cell engineered to express the isolated single domain antibody of the invention.

Diagnosis method:

Inventors have used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact- VWF could be detected using this singledomain antibody. Accordingly, in a second aspect, the invention relates to use of the single domain antibody according to the invention for determining the level of VWF degradation in a biological sample.

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

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

In a further embodiment, the method according to the invention, a loss of at least 10% intact- VWF is detected by using the single domain antibody according to the invention. As used herein, the term “at least 10%” refers to 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 %.

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

As used herein, the term “biological sample” refers to any biological sample obtained from the subject for the purpose of evaluation in vitro. In some embodiments, the biological sample is a body fluid sample. Examples of body fluids are blood, serum, plasma, amniotic fluid, brain/spinal cord fluid, liquor, cerebrospinal fluid, sputum, throat and pharynx secretions and other mucous membrane secretions, synovial fluids, ascites, tear fluid, lymph fluid and urine.

In a particular embodiment, the biological sample is blood sample.

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

In a particular embodiment, the blood sample is a plasma sample. A plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the subject following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about l,500*g for about 15-20 minutes (room temperature), followed by pipeting of the plasma layer.

In a particular embodiment, the sample has been previously obtained from the subject.

As used herein, the term “intact- VWF” refers to VWF which is not proteolytically degraded by the enzyme AD AMTS 13.

As used herein, the term “total- VWF” refers to whole VWF which contains any form proteolyzed VWF (including VWF proteolyzed by ADAMTS13) and non-proteolyzed VWF.

As used herein, the term “ratio” refers to the relationship between two groups or amounts that expresses how much bigger one is than the other. In this case, the ratio allows to compare the level of intact- VWF detected by the single domain antibody according to the invention and the total VWF detected by a polyclonal or monoclonal antibody.

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

In a particular embodiment, the intact- VWF is referred as VWF being recognized by KB-VWF-D3.1. Briefly, wells coated with KB-VWF-D3.1 were incubated with samples containing non-proteolyzed VWF, ADAMTS13-proteolyzed VWF or a mixture of both. Alternatively, plasma samples were used. Bound VWF was probed using polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine.

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

In some embodiments, the level of intact- VWF peptide is determined by immunoassay.

As used herein, the term “Immunoassays” encompass any assay wherein a capture reagent (i.e KB-VWF-D3.1) is immobilised on a support and wherein detection of an analyte of interest (i.e rVWF or degraded- VWF ) is performed through the use of antibodies directed against the said 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 spectroscopy technique (CE-MS) etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Immunoassays includes competition, direct reaction, or sandwich type assays.

Typically, the bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine. The single domain antibody is labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer may be labelled with a radioactive molecule by any method known in the art. For example, radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as I<123>, I<124>, In<l 11>, Re<186>, Re<188>. Preferably, the antibodies against VWF are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE-conjugated).

In a particular embodiment, the single domain antibody according to the invention which is conjugated with a detectable label.

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 bio luminescent label.

In a particular embodiment, the antibody according to the invention wherein the label is selected from the group consisting of P-galactosidase, glucose oxidase, peroxidase (e.g. horseradish perodixase) and alkaline phosphatase.

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

In some embodiments, the level of intact- VWF is determined by direct ELISA. The single domain antibody according to the invention is directly immobilized to a surface of a multi-well plate and detected with a biotin-conjugated detection antibody specific for the VWF. This antibody is directly conjugated to a detection system (horseradish peroxidase (HRP)- conjugated Streptavidin or other detection molecules).

In some embodiments, the level of intact- VWF is determined by indirect ELISA. The single domain antibody is directly immobilized to a surface of a multi-well plate and detected with an unconjugated primary detection antibody specific for the VWF. A conjugated secondary antibody directed against the host species of the primary antibody is then added. Substrate then produces a signal proportional to the amount of VWF degraded bound in the well.

In some embodiments, the level of intact- VWF is determined by sandwich ELISA.

According to the invention, “sandwich” ELISA refers to an immunoassay wherein free VWF may be sandwiched between two antibodies that specifically bind to free VWF. Typically, the single domain antibody according to the invention is conjugated with a detection system (such as horseradish peroxidase (HRP)-conjugated Streptavidin or other detection molecules). In another embodiment, the level of intact- VWF is identified by immunohistochemistry. Typically, an immunohistochemistry of biological obtained from a subject is performed by using the single domain antibody according to the invention. In a particular embodiment, the antibody is a polyclonal antibody against VWF total.

By comparing plasma from volunteers to that of congenital VWD-patients, intact- VWF levels were significantly reduced for all VWD-types, and most severely in VWD-type 2A(IIA) in which mutations promote ADAMTS13- mediated proteolysis. Unexpectedly, low-grade proteolysis in VWD-type 1 and -type 2M was also observed. Thus, this single-domain antibody proved sensitive to detect low-grade degradation in plasma from patients with AVWS and congenital VWD, including types 1 and 2M.

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

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

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

As used herein term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. In the context of the invention, the method according to the invention allows to diagnose a bleeding episode.

As used herein term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a human who is susceptible to have a disease which triggers a bleeding episode.

A used herein, the term "bleeding" refers to extravasation of blood from any component of the circulatory system. A "bleeding episode" thus encompasses unwanted, uncontrolled and often excessive bleeding in connection with surgery, trauma, or other forms of tissue damage, as well as unwanted bleedings in patients having bleeding disorders. More particularly, unexplained bleeding episodes are associated with ventricular assist devices (VAD) and can occur in part due to acquired von Willebrand syndrome (a VWS). AVWS is characterised by loss of HMW-multimers of VWF. Loss of multimers can occur as VWF is subjected to increased shear stress, which occurs in presence of VADs.

In a particular embodiment, the bleeding episode occurs in the following disease condition which is selected from the group consisting of but not limited to: acquired von Willebrand syndrome, VWD-type 1, 2A(IIA) (also referred to as 2A-group 2), 2A(IIE) (also referred to as 2A-group 1), 2B and 2M, severe aortic stenosis, patients receiving ECMO.

In a particular embodiment, the bleeding episode occurs when an increase of VWF degradation by ADAMTS13 is observed. In a particular embodiment, multimers were analyzed via SDS-agarose electrophoresis. The relative amount of multimers exceeding 10 bands was determined via comparison to normal pooled plasma. Plasma samples from subjects suffering from a bleeding episode were analyzed for total antigen using polyclonal antibodies and for intact- VWF using KB-VWF- D3.1. Normal pooled plasma was used as calibrator. Presented is the ratio intact- VWF/total VWF-antigen. Each individual sample is represented by a closed symbol. Statistical analysis was performed via a one-way Anova with Dunnett’s correction for multiple comparisons. Plotted is the ratio intact- VWF/total VWF-antigen versus the relative amount of large multimers for samples from subjects with severe aortic stenosis and from ECMO subjects.

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

A used herein, the term "ADAMTS13 reduced activity related disease” relates to diseases in which the activity of ADAMTS13 is decreased. In this case, the degradation of VWF is decreased. Accordingly, the level of intact- VWF is increased with the single domain antibody of the invention. When the In a particular embodiment, a decreased ADAMTS13 activity occurs low VWF degradation which triggers to the formation of blood clots.

Accordingly, the ADAMTS13 reduced activity related disease refers to all disease where a blood clot is formed. In a particular embodiment, the ADAMTS13 reduced activity related disease is selected from the group consisting of but not limited to: immune Thrombotic Thrombocytopenic Purpura, hereditary Thrombotic Thrombocytopenic Purpura), Hemolysis Elevated Liver enzymes Low Platelet (HELLP)-syndrome, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) such as Covid-19, infectious diseases such as, HIV, Dengue, Chikungunya and malaria.

As used herein, the term “predetermined reference value” refers to a threshold value or a cut-off value. A "threshold value", “reference value” or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the concentration of the marker of the invention (e.g. intact- VWF) in properly banked historical subject samples may be used in establishing the predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median measured in the population of the subjects for the marker of in the invention (intact- VWF for example). In some embodiments, the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) 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 invention (intact- VWF for example) in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1- specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined corresponding reference value is typically determined by carrying out a method comprising the steps of a) providing a collection of samples from subjects; b) providing, for each sample provided at step a), information relating to the actual clinical profile of the subject (healthy or suffering from a bleeding episode); c) providing a serial of arbitrary quantification values; d) determining the concentration of the marker of the invention (intact- VWF for example) for each sample contained in the collection provided at step a); e) classifying said blood samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical profile of the subjects from which samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined corresponding reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g).

Thus in some embodiments, the predetermined corresponding reference value thus allows discrimination between healthy subject and subjects suffering from an inflammatory disese. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite predetermined corresponding reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the diagnosis can be determined by comparing the co centration of the marker of the invention (intact- VWF for example) with the range of values which are identified. In certain embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found).

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

Kit

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

Kits of the invention can contain a single domain antibody coupled to a solid support, e.g., well plate or beads (e.g., sepharose beads). Kits can be provided which contain antibodies for detection and quantification of intact- VWF protein in vitro, e.g. in an ELISA or a Western blot. Such single domain antibody useful for detection may be provided with a label such as a fluorescent or radiolabel. In a particular embodiment, the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of the biomarker of the invention.

In a particular embodiment, the kit according to the invention may include instructional materials containing instructions (e.g., protocols) for the practice of diagnostic methods.

The kits may include probes, primers macroarrays or microarrays as above described. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively, the kit of the invention may comprise amplification primers that may be pre labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

In a particular embodiment, the invention provides diagnostic kits containing the single domain antibody of the invention, anti- total-VWF antibodies (monoclonal or polyclonal) including antibody conjugates. The diagnostic kit is a package comprising at least one single domain antibody of the 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., diluents, a labeled antibody that binds to an anti- total-VWF antibody, an appropriate substrate for the labeled antibody, VWF in a form appropriate for use as a positive control and reference standard standard, a negative control).

Alternatively, the kit can include a labeled antibody which binds an anti-VWF monoclonal/polyclonal antibody and is conjugated to an enzyme. Where the anti-total-VWF monoclonal antibody or other antibody is conjugated to an enzyme for detection, the kit can include substrates and cofactors required by the enzyme (e.g. , a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives can be included, such as stabilizers, buffers (e.g. , a block buffer or lysis buffer), and the like. Antitotal VWF antibodies included in a 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 for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Antibodies and other reagents can be provided (individually or combined) as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Generation of anti-VWF nanobodies.

A: Flow-diagram of screening approach using recombinant VWF (rVWF) and degraded- VWF for the isolation of anti-VWF nanobodies that distinguish between intact and degraded- VWF. B-D: Dose-response of rVWF (black circles) and degraded- VWF (grey circles) to immobilized single-domain antibody KB-VWF-D3.1 (5 pg/ml; panel B) or KB-VWF-F1.1 (5 pg/ml; panel D). Panel C compares rVWF to plasma-derived VWF (pdVWF), both added at a concentration of 5 pg/ml. Bound VWF was probed using peroxidase-labeled polyclonal anti- VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine. Data represent mean±SD of 4-8 experiments. E: Binding of various concentrations of KB-VWF-D3.1 (0-5 pg/ml) to immobilized rVWF or degraded- VWF (both 5 pg/ml) Bound KB-VWF-D3.1 was probed using peroxidase-labeled polyclonal rabbit anti-cMyc antibodies and detected following 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 fluorogenic substrate FRETS-VWF73. Substrate (2 pM) was preincubated with indicated concentrations of single-domain antibody. Residual ADAMTS13 activity was calculated and plotted against single-domain antibody concentration. B: Immobilized degraded- VWF (5 pg/ml) was incubated with KB-VWF-F1.1 (1 p/ml) in the absence or presence of various concentrations an anti-A2 domain antibody that recognizes the N-terminal portion (Asp 1596-Tyr 1605) of the AD AMTS 13 -cleavage site (MAB27642; R&D systems). Presented is residual binding of KB-VWF-F1.1 (with no competitor being set as 1.0) versus molar ratio antibody MAB27642 over KB-VWF-F1.1. For both panels, data represent 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 pg/ml) to various concentrations of VWF domain-Fc fusion proteins (0-10 nM) that were captured onto anti-human Fc antibodies. Bound KB-VWF- D3.1 was probed using peroxidase-labeled polyclonal rabbit anti-cMyc antibodies and detected following hydrolysis of 3,3’,5,5’-tetramethylbenzidine. Al-Fc: grey squares; A2-Fc: black triangles; A3-Fc: black circles; D4-Fc: white circles. Data represent mean±SD of 3 experiments. B: Amino acid sequence of the VWF A3 -domain, with the residues harboring the epitope for KB-VWF-D3.1 in bold. Residues previously reported to be involved in collagen binding²² are boxed. C: Inhibition of pd-VWF binding to collagen-type III by KB-VWF-D3.1 (closed circles), monoclonal antibody Mab505 (grey squares) and single-domain antibody C37h (open circles). Presented is residual pd-VWF binding versus single-domain antib ody/antibody concentration. Data represent mean±SD of three experiments. D: Binding of pd-VWF (closed symbols) or degraded- VWF (open symbols) to immobilized Mab505 (5 pg/ml; circles) or C37h (5 pg/ml; squares). Bound pd-VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine. Data represent mean±SD of 3 experiments.

Figure 4: Determination of the binding epitope for KB-VWF-D3.1. A. Binding of VWF domains-Fc fusion proteins to immobilized KB-VWF-D3.1 (5 pg/ml). Bound fragments were probed using peroxidase-labeled monoclonal anti-human Fc antibodies, and detected following hydrolysis of 3,3’,5,5’-tetramethylbenzidine. Al-Fc: grey triangles; A2-Fc: white circles; A3-Fc: black circles; D4-Fc: white squares. B. Immobilized KB-VWF-D3.1 (5 pg/ml) was incubated with recombinant VWF-deletion variants (1 pg/ml). Bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected following hydrolysis of 3,3’,5,5’-tetramethylbenzidine. C. Blood was perfused over collagen-coated PL-chips using the T-TAS Plus equipment at a shear rate of 2000 s-1. During perfusion, pressure (a marker for thrombus formation) is measured in real-time. The Area Under the Curve was lower for perfusions performed in the presence of KB-VWF-D3.1 (20 pg/ml) compared to control (48.5 (95%CI: 48.1-48.9) versus 70.3 (95%CI: 67.4-73.3). For panels A & B, data represent mean±SD of 3 experiments. For panel C, solid lines represent the mean of three experiments and the grey area (delineated by the dotted lines) the standard error.

Figure 5: Binding of VWF with varying multimer size to KB-VWF-D3.1

A : Binding of HMW-VWF (closed circles) and MMW-VWF (open circles) to immobilized KB-VWF-D3.1 (5 pg/ml). B : Binding of multimeric rVWF (closed circles) and the dimeric VWF/delta-pro variant (grey squares) to immobilized KB-VWF-D3.1 (5 pg/ml). In both panels, bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine. Data represent mean±SD of 3-4 independent measurements.

Figure 6: ADAMTS13-mediated proteolysis modulates binding of VWF to KB- VWF-D3.1 and KB-VWF-F1.1. Samples were analyzed for total VWF-antigen using polyclonal antibodies, for the presence of intact- VWF using KB-VWF-D3.1 and for the presence of degraded- VWF using KB-VWF-F1.1. Presented is the ratio intact- VWF/total VWF-antigen (closed circles; left Y-axis) and the ratio degraded- VWF/total VWF-antigen (grey squares; right Y-axis) versus exposure time to ADAMTS13. Normal pooled plasma was used as calibrator for KB-VWF-D3.1, whereas a degraded- VWF preparation was used as calibrator for KB-VWF-F 1.1. Data represent mean±SD of 3 independent experiments.

Figure 7: Detection of intact VWF in congenital VWD

A: VWF-deficient plasma was spiked with different amounts of purified rVWF and degraded- VWF, and incubated in microtiter plates coated with KB-VWF-D3.1. Bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine. Data represent mean±SD of 3-4 independent measurements. The solid line illustrates the best linear fit, with 95% confidence interval indicated with the dotted lines. The vertical line indicates 90% intact rVWF supplemented with 10% degraded- VWF B-C: Patient plasma samples were analyzed for total antigen using polyclonal antibodies and for intact- VWF using KB-VWF-D3.1. Normal pooled plasma was used as calibrator. Presented is the ratio intact- VWF/total VWF-antigen. Each individual sample is represented by a closed symbol. Statistical analysis was performed via a 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 exceeding 10 bands was determined via comparison to normal pooled plasma (NPP). E: Plotted is the ratio intact- VWF/total VWF-antigen versus the relative amount of large multimers. Correlation was determined in 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 exceeding 10 bands was determined via comparison to normal pooled plasma (NPP). B: Patient plasma samples were analyzed for total antigen using polyclonal antibodies and for intact- VWF using KB-VWF-D3.1. Normal pooled plasma was used as calibrator. Presented is the ratio intact- VWF/total VWF-antigen. Each individual sample is represented by a closed symbol. Statistical analysis was performed via a one-way Anova with Dunnett’s correction for multiple comparisons. Control samples were identical to those presented in Figure 5. C-D: Plotted is the ratio intact- VWF/total VWF-antigen versus the relative amount of large multimers for samples from patients with severe aortic stenosis (AS; panel C) and from ECMO patients (panel D).

EXAMPLE:

Material & Methods Ethics statement:

All volunteers and patients provided informed written consent according to the Declaration of Helsinki. Patients with VWD were selected from the French cohort multicentric database of VWD (Centre Reference Maladie Willebrand).¹⁹ The database and biobank of this cohort are declared to and approved by the French data protection authority (CNIL- 1245379/DEC-19252, CODECOH-DC-2008-642). Patients with severe aortic stenosis (WIT AVI-trial, NCT02628509) and patients receiving extracorporal mechanical oxidation (ECMO; WITECMO-H-trial; NCT03070912) were included in the study. All protocols were approved by the local review and ethics committees.

Isolation of anti-VWF nanobodies

A synthetic single-domain antibody-encoding phage-library²⁰ was used to isolate anti- VWF nanobodies. The library (3xl0⁹ clones) was incubated with streptavidin-coated beads loaded with biotinylated rVWF. Unbound phages were then incubated with beads loaded with biotinylated degraded- VWF. Three rounds of phage-display were performed, with the depletion step being repeated every round. Twelve unique sequences were obtained via this procedure (Fig. 1A).

Analysis of VWF binding to nanobodies

Wells coated with single-domain antibody KB-VWF-D3.1 or KB-VWF-1.1 (both 5 pg/ml) were incubated with purified rVWF (0-0.5 pg/ml). Bound VWF was probed using polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine.

Detecting intact-VWF

Intact- VWF is referred as VWF being recognized by KB-VWF-D3.1. Briefly, wells coated with KB-VWF-D3.1 (5 pg/ml) were incubated with samples containing non-proteolyzed VWF, ADAMTS13-proteolyzed VWF or a mixture of both. Alternatively, plasma samples were used. Bound VWF was probed using polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine.

Total VWF-antigen

Total VWF-antigen was measured in an ELISA using polyclonal rabbit anti-VWF antibodies as described.²¹

Results

Selection of anti-VWF nanobodies

To isolate nanobodies that distinguish between intact or proteolyzed VWF, a selection method using rVWF and degraded- VWF was applied, generating 12 unique sequences (Figure 1A and data not shown). Purified nanobodies were tested for interaction with rVWF and degraded- VWF. Whereas 10 of 12 nanobodies displayed similar binding to both VWF preparations, two of them were characterized by differential binding. First, rVWF associated in a dose-dependent manner to immobilized single-domain antibody KB-VWF-D3.1, whereas binding of degraded- VWF to this single-domain antibody was strongly reduced (Figure IB). In these assays, pd-VWF yielded responses that were consistently lower than rVWF (93±4% compared to 100±2%; p=0.015; Figure 1C), probably due to degraded- VWF present in normal plasma. In complementary assays, KB-VWF-D3.1 bound 8-fold less efficiently to immobilized degraded- VWF compared to immobilized rVWF (Figure IE). As for KB -VWF -F 1.1, efficient binding of degraded- VWF was detected, whereas binding of rVWF approached background levels (Figure ID). Therefore, these two nanobodies were selected for further analysis.

Determination of the binding epitope for KB-VWF-F1.1

Since KB-VWF-F 1.1 bound to degraded- VWF but not intact- VWF, we anticipated that this single-domain antibody recognizes the region surrounding the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ cleavage site. This was tested in an ADAMTS 13 -activity test utilizing its substrate FRETS-VWF73, which contains the VWF A2-domain sequence Asp¹⁵⁹⁶-Arg¹⁶⁶⁸ (Figure 2A). Whereas KB- VWF-D3.1 and control single-domain antibody KB-VWF-004 (against the region VWF/D4- CK) left substrate proteolysis unaffected, KB-VWF-F 1.1 efficiently interfered with substrate conversion by ADAMTS13. This suggests that the epitope of VWF-KB-F1.1 is located within the region Asp¹⁵⁹⁶-Arg¹⁶⁶⁸. Of note, KB-VWF-F 1.1 and MAB27642, which targets residues Arg¹⁵⁹⁶-Tyr¹⁶⁰⁵, did not compete for binding to degraded- VWF (Figure 2B), suggesting they recognize different epitopes within this region.

Determination of the binding epitope for KB-VWF-D3.1

To determine the epitope of KB-VWF-D3.1, we first analyzed binding of this singledomain antibody to a series of rVWF fragments, ie. Al-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 (Figure 3 A). Binding was similar when binding of fragments to immobilized KB- VWF -D3.1 was assessed (Figure 4A). Moreover, no binding of rVWF lacking the A3 domain to KB-VWF- D3.1 could be detected, whereas deletion of other domains left binding unaffected (Figure 4B).

To refine its binding site within the A3 -domain, molecular modeling was performed(data not shown). This procedure revealed that the top 30-ranked conformations of the complex all clustered similarly, with the single-domain antibody docking onto 4 separate amino acid-stretches within the VWF A3-domain region Val¹⁷³²-Asn¹⁸¹⁸ (data not shown). Interestingly, 8 of the amino acids included in the epitope of KB-VWF-D3.1 have previously been recognized as being relevant for collagen binding (Figure 3B)²², indicating that the epitope of KB-VWF-D3.1 overlaps the collagen binding site. Consequently, we compared the effect of KB-VWF-D3.1 to that of two known A3-domain binding antibodies (the C37h single-domain antibody²³ and the Mab505 monoclonal antibody²⁴) on binding of VWF to collagen type III. The positive controls C37h and Mab505 efficiently blocked pd-VWF-collagen interactions (Figure 2F). KB-VWF-D3.1 also dose-dependently reduced binding of pd-VWF to collagen, but less efficiently than antibodies C37h and Mab505 (Figure3C). In addition, KB-VWF-D3.1 delayed VWF-dependent platelet-adhesion to collagen under flow-conditions (Figure 4C). Having their epitope overlapping the collagen binding site in common, it raises the question whether C37h and Mab505 can distinguish between intact and degraded- VWF akin to KB- VWF-D3.1. However, both C37h and Mab505 displayed similar binding to both intact rVWF and degraded- VWF (Figure 3D). Thus, single-domain antibody KB-VWF-D3.1 is unique in binding to an epitope within the A3-domain, the exposure of which is modulated upon proteolysis within the A2-domain.

Effect multimer size on VWF binding to KB-VWF-D3.1

Proteolysis of VWF by ADAMTS13 results in loss of the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond, thereby reducing multimer size. We therefore tested how multimer size affects binding of VWF to immobilized KB-VWF-D3.1. First, we analyzed two distinct pd-VWF preparations that were obtained from pd-VWF concentrates via gel-filtration chromatography. One with high molecular weight (HMW)-multimers and one enriched in medium-sized molecular weight (MMW)-multimers (data not shown). Both fractions displayed similar binding to KB-VWF- D3.1 (Figure 5 A). Next, we compared binding of dimeric rVWF/delta-pro to 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±l pg/ml vs 0.2±0.1 pg/ml; p=0.62). Apparently, binding of VWF to immobilized KB-VWF-D3.1 is independent of its multimer size. Reduced binding of degraded- VWF to KB-VWF-D3.1 is conceivably originating from proteolysis of the Tyr¹⁶⁰⁵- Met¹⁶⁰⁶ peptide bond rather than from a reduction in multimer size.

Proteolysis of VWF over time

We next investigated the effect of ADAMTS 13 -proteolysis on VWF binding to KB- VWF-D3.1 and KB-VWF-F1.1 in a time-dependent manner. Briefly, pd-VWF was exposed to shear in the presence of recombinant ADAMTS 13 and samples were taken at indicated timepoints (0-3h). Multimeric pattern and binding to both nanobodies were analyzed. Exposure to ADAMTS 13 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 which renders ADAMTS 13 inactive. Concurrent with increased pd-VWF proteolysis, increased binding to KB-VWF-F1.1 was observed (Figure 6). In contrast, binding of pd-VWF to KB- VWF-D3.1 disappeared in a complementary fashion (Figure 6). These data validate that the binding of both nanobodies to VWF is dependent on the extent of proteolysis by AD AMTS 13.

Measuring degraded-VWF in mixtures of intact and degraded-VWF

Given the specificity of both nanobodies for intact and degraded-VWF, respectively, we anticipated that they could be useful to determine the extent of VWF proteolysis in patient samples. In preliminary experiments, KB-VWF-F1.1 lacked sufficient sensitivity to detect minor proteolysis of VWF in plasma, and we therefore focused for the remainder of the study on KB-VWF-D3.1. We first analyzed to what extent increased proteolysis would be detectable. Different mixtures of purified rVWF and degraded-VWF were prepared, and the ratio intact VWF/total VWF-antigen was determined. A dose-dependent decrease of this ratio was observed, when the percentage of degraded-VWF in the samples increased (Figure 7A). From these experiments, it seems that an increase of approximately 10% degraded-VWF can be detected (p=0.0009 compared 100% intact).

Analysis of congenital VWD-patient plasma

We then analyzed plasma samples obtained from controls (n=31) and VWD-patients included in the French reference center for VWD (n=101).¹⁹ The patient-cohort consisted of VWD-type 1 (n=20), VWD-type 2A (n=43), VWD-type 2B (n=24) and VWD-type 2M (n=14) patients.

To determine the amount of intact VWF, we calculated the amount of antigen obtained using KB-VWF-D3.1 (= intact- VWF) over the amount of total VWF-antigen, using normal pooled plasma as calibrator. By doing so, it appeared that the intact- VWF/total antigen ratio for controls was found to be 1.0±0.2 (Figure 7B). The ratio of intact- VWF/total VWF was decreased for each of the VWD-types analyzed. The ratio was 0.7±0.3 (p=0.0004) for VWD- type 1, 0.5±0.2 (p<0.0001) for VWD-type 2A, 0.6±0.2 (p<0.0001) for VWD-type 2B and 0.7±0.2 (p=0.0148) for VWD-type 2M (Figure 7B). Since VWD-type 2A is divided in two subtypes, ie. VWD-type 2A-group 1 and VWD-type 2A-group 2, in which the loss of multimers is dominated by impaired multimerization and increased proteolysis, respectively, we separately analyzed samples from patients with VWD-type 2A-group 1 (n=14) and VWD-type 2A-group 2 (n=29). The ratio intact- VWF/total VWF-antigen was significantly lower in VWD- type 2A-group 2 (0.4±0.2) compared to VWD-type 2A-group 1 (0.6±0.2; p=0.0007; Figure 7C).

Since it was unexpected to find a decreased ratio intact- VWF/total VWF-antigen in all VWD-types, we also verified whether this decreased ratio would correspond to a potential loss of HMW-mul timers. Multimer analysis was available for a subset of samples, and we indeed observed that in all patient groups, including VWD-type 1 and type 2M, there was on average a relative decrease in the HMW-multimers (>10 multimer bands) compared to normal pooled plasma, (Figure 7D). Interestingly, there was a significant correlation between the ratio intact- VWF/total VWF-antigen versus multimer size (r=0.51; p<0.0001; Figure 7E). Thus, it seems that in the majority of VWD-patients there is increased proteolysis compared to the normal population.

Analysis of AVWS-patient plasma

We next examined plasma from patients receiving ECMO-support (n=27) and from patients with severe aortic stenosis (n=17). Both patient groups are characterized by a loss of VWF HMW-multimers (Figure 8A), potentially caused by increased ADAMTS13-mediated proteolysis. Compared to normal control, the ratio of intact- VWF (measured by binding to KB- VWF-D3.1) over total VWF-antigen was significantly reduced for both patient groups: 0.85±0.09 (mean±SD; p=0.0017) and 0.78±0.13 (p<0.0001) for severe aortic stenosis and ECMO patients, respectively (Figure 8B). Of note, for both groups, there was a significant correlation between the ratio intact/total antigen and the presence of HMW-multimers (>10), with p-values being p=0.0463 for severe aortic stenosis-samples and p=0.0452 for ECMO- samples (Figures 8CD). This may suggest that the loss of larger multimers indeed is predominantly due to proteolysis rather than other mechanisms.

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

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Claims

CLAIMS:

1. An isolated single domain antibody targeting at least one region of A3-domain of VWF, wherein the region comprising the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4.

2. The isolated single domain antibody targeting A3-domain of VWF according to claim 2, wherein said single domain antibody comprises a CDR1 having a sequence set forth as SEQ ID NO: 5, a CDR2 having a sequence set forth as SEQ ID NO:6 and a CDR3 having a sequence set forth as SEQ ID NO:7.

3. The isolated single-domain antibody targeting A3-domain of VWF according to claim 2, wherein said sdAb comprises a CDR1 having at least 70% of identity with sequence set forth as SEQ ID NO: 5, a CDR2 having at least 70% of identity with sequence set forth as SEQ ID NO: 6 and a CDR3 having at least 70% of identity with sequence set forth as SEQ ID NO: 7.

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

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

6. 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 the single domain antibody according to the invention; ii) determining the level of intact- VWF with the isolated single domain antibody (KB- VWF-D3.1); iii) determining the level of total-VWF with an antibody (polyclonal or monoclonal); iv) calculating the ratio of the level determined at step ii) with the level of total VWF- antigen determined at step iii); v) comparing the ratio determined at step iv) with a predetermined corresponding reference value and vi) concluding that the subject is susceptible to have or is at risk of having a bleeding episode when the ratio determined at step iv) is lower than the predetermined reference value or concluding that the subject is not susceptible to have or is not at risk of having a bleeding episode when the ratio determined at step iv) is identical to the predetermined reference value.

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

8. The method according to claims 6 to 7 wherein the bleeding episode occurs in the following disease condition which is selected from the group consisting of but not limited to: acquired von Willebrand syndrome, VWD-type 1, 2A(IIA) (also referred to as 2A-group 2), 2A(IIE) (also referred to as 2A-group 1), 2B and 2M, severe aortic stenosis, patients receiving ECMO.

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

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

11. A vector comprising the nucleic acid of 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 of claims 1 to 6.

14. The kit according to claim 13 comprising a solid support, a label such as a fluorescent or radiolabel and use instructions.