Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• 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.
• 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.
• D1-D2 portion also known as VWF propeptide
• 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.
• VWF von Willebrand syndrome
• VWF von Willebrand disease
• HMW high molecular weight
• collagen- or platelet-binding assays are used 15 , which are less specific in that they do not distinguish between impaired multimerization and excessive degradation.
• 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
• MAB27642 a similar antibody to monitor VWF proteolysis in patients. 17,18
• 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
• 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.
• said region comprising the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4.
• 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.
• 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.
• 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.
• single-domain antibody 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®".
• VHH single-domain antibody
• Single-domain antibody 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.
• 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.
• 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/).
• 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.
• HMW high molecular weight
• VWF multimers 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.
• 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).
• a VWS acquired von Willebrand syndrome
• VWF von Willebrand disease
• HMW high molecular weight
• the single-domain antibody according to the invention targets at least one region of the collagen-binding site of the A3-domain of VWF.
• 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.
• the single-domain antibody according to the invention targets:
• 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.
• 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.
• the single-domain antibody according to the invention detects the VWF intact which is not degraded by the AD AMTS 13.
• the single-domain antibody according to the invention specifically binds to intact VWF (i.e VWF not proteolyzed/degraded by ADAMTS13).
• 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.
• 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.
• a single domain antibody designated KB-VWF-D3.1
• 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.
• 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.
• 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.
• sdAb isolated single-domain antibody
• 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)).
• 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).
• the isolated single-domain antibody according to the invention has the sequence set forth as SEQ ID NO: 8.
• the isolated single-domain antibody according to the invention has the sequence set forth as SEQ ID NO: 9:
• 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.
• VHH variable heavy chain
• 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.
• 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.
• microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama e
• 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.
• 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.
• 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.
• 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.
• suitable transcriptional or translational regulatory sequences such as those derived from a mammalian, microbial, viral, or insect genes.
• regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation.
• expression vector expression construct
• expression cassette 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.
• 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.
• promoters/promoters from both viral and mammalian sources that may be used to drive expression of the nucleic acids of interest in host cells.
• 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.
• the invention relates to a nucleic acid sequence encoding the isolated single domain antibody of the invention.
• the invention relates to a nucleic acid sequence which encodes a heavy chain of the isolated single domain antibody of the invention.
• the invention relates to a vector comprising the nucleic acid of the invention.
• the invention relates to a host cell engineered to express the isolated single domain antibody of the invention.
• 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.
• 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.
• a loss of 10% intact- VWF is detected by using the single domain antibody according to the invention.
• a loss of at least 10% intact- VWF is detected by using the single domain antibody according to the invention.
• the term “at least 10%” refers to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
• 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.
• biological sample refers to any biological sample obtained from the subject for the purpose of evaluation in vitro.
• the biological sample is a body fluid sample.
• 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.
• the biological sample is blood sample.
• blood sample means a whole blood, serum, or plasma sample obtained from the subject.
• 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.
• the sample has been previously obtained from the subject.
• intact- VWF refers to VWF which is not proteolytically degraded by the enzyme AD AMTS 13.
• 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.
• CE-MS capillary electrophoresis-mass spectroscopy technique
• flow cytometry flow cytometry
• mass cytometry mass cytometry
• immunoassay such as an enzyme-linked immunosorbent assay (ELISA)
• the intact- VWF is referred as VWF being recognized by KB-VWF-D3.1.
• VWF being recognized by KB-VWF-D3.1.
• wells coated with KB-VWF-D3.1 were incubated with samples containing non-proteolyzed VWF, ADAMTS13-proteolyzed VWF or a mixture of both.
• 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).
• the level of intact- VWF peptide is determined by immunoassay.
• 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).
• a capture reagent i.e KB-VWF-D3.1
• detection of an analyte of interest i.e rVWF or degraded- 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.
• 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.
• 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.
• FITC fluorescein isothiocyanate
• PE phycoerythrin
• Cy5 Indocyanine
• An antibody or aptamer may be labelled with a radioactive molecule by any method known in the art.
• 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>.
• the antibodies against VWF are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE-conjugated).
• the single domain antibody according to the invention which is conjugated with a detectable label.
• 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.
• 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.
• the level of intact- VWF is determined by enzyme-labeled and mediated immunoassays (ELISA).
• ELISA enzyme-labeled and mediated immunoassays
• 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).
• HRP horseradish peroxidase
• 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.
• the level of intact- VWF is determined by sandwich ELISA.
• “sandwich” ELISA refers to an immunoassay wherein free VWF may be sandwiched between two antibodies that specifically bind to free VWF.
• 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).
• HRP horseradish peroxidase
• the level of intact- VWF is identified by immunohistochemistry.
• an immunohistochemistry of biological obtained from a subject is performed by using the single domain antibody according to the invention.
• the antibody is a polyclonal antibody against VWF total.
• 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.
• a loss of 10% intact- VWF is detected by using the single domain antibody according to the invention.
• a loss of at least 10% intact- VWF is detected by using the single domain antibody according to the invention.
• 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
• diagnosis 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.
• the method according to the invention allows to diagnose a bleeding episode.
• 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.
• 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).
• VAD ventricular assist devices
• VWS von Willebrand syndrome
• 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.
• 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.
• acquired von Willebrand syndrome VWD-type 1
• 2A(IIA) also referred to as 2A-group 2A-group 2A(IIE) (also referred to as 2A-group 1)
• 2B and 2M severe aortic stenosis
• the bleeding episode occurs when an increase of VWF degradation by ADAMTS13 is observed.
• 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.
• 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 ADAMTS
• ADAMTS13 reduced activity related disease relates to diseases in which the activity of ADAMTS13 is decreased.
• the degradation of VWF is decreased.
• the level of intact- VWF is increased with the single domain antibody of the invention.
• a decreased ADAMTS13 activity occurs low VWF degradation which triggers to the formation of blood clots.
• the ADAMTS13 reduced activity related disease refers to all disease where a blood clot is formed.
• 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.
• immune Thrombotic Thrombocytopenic Purpura Hereditary Thrombotic Thrombocytopenic Purpura
• HELLP Hemolysis Elevated Liver enzymes Low Platelet
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• infectious diseases such as, HIV, Dengue, Chikungunya and malaria.
• 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.
• 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).
• 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).
• the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
• ROC Receiver Operating Characteristic
• concentration of the marker of the invention intact- VWF for example
• 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 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.
• AUC area under the 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.
• AUC>0.5 the diagnostic result gets better and better as AUC approaches 1.
• AUC is between 0.5 and 0.7, the accuracy is low.
• AUC is between 0.7 and 0.9, the accuracy is moderate.
• 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.
• 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
• the predetermined corresponding reference value thus allows discrimination between healthy subject and subjects suffering from an inflammatory disese.
• high statistical significance values e.g. low P values
• a range of values is provided instead of using a definite predetermined corresponding reference value. 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.
• a range of quantification values includes a "cut-off value as described above.
• 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.
• 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).
• the methods of the present invention are performed in vitro or ex vivo.
• 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.
• 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.
• the kit according to the invention may include instructional materials containing instructions (e.g., protocols) for the practice of diagnostic methods.
• 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.
• 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.
• 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).
• 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.
• the kit can include a labeled antibody which binds an anti-VWF monoclonal/polyclonal antibody and is conjugated to an enzyme.
• the kit can include substrates and cofactors required by the enzyme (e.g. , a substrate precursor which provides the detectable chromophore or fluorophore).
• substrates and cofactors required by the enzyme e.g. , a substrate precursor which provides the detectable chromophore or fluorophore.
• 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 are a diagrammatic representation of 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.
• Figure 3 KB-VWF-D3.1 binds to the VWF A3-domain.
• 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 binding 22 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.
• 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 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
• 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.
• 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).
• 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.
• 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.
• 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.
• 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).
• a synthetic single-domain antibody-encoding phage-library 20 was used to isolate anti- VWF nanobodies.
• the library (3xl0 9 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).
• 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.
• 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.
• Veyradier A Boisseau P, Fressinaud E, et al. A Laboratory Phenotype/Genotype Correlation of 1167 French Patients From 670 Families With von Willebrand Disease: A New Epidemiologic Picture. Medicine (Baltimore). 2016;95(l l):e3038.

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