Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

PLoS One. 2016 Aug 1;11(8):e0160388. doi: 10.1371/journal.pone.0160388. eCollection 2016.

Abstract

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy linked to a deficiency in the metalloprotease ADAMTS13. In the current study, a novel mouse model for acquired TTP was generated to facilitate development and validation of new therapies for this disease. Therefore, a large panel (n = 19) of novel anti-mouse ADAMTS13 (mADAMTS13) monoclonal antibodies (mAbs) of mouse origin was generated. Inhibitory anti-mADAMTS13 mAbs were identified using the FRETS-VWF73 assay. Four mAbs strongly inhibited mADAMTS13 activity in vitro (∼68-90% inhibition). Injecting a combination of 2 inhibitory mAbs (13B4 and 14H7, 1.25 mg/kg each) in Adamts13+/+ mice resulted in full inhibition of plasma ADAMTS13 activity (96 ± 4% inhibition, day 1 post injection), leading to the appearance of ultra-large von Willebrand factor (UL-VWF) multimers. Interestingly, the inhibitory anti-mADAMTS13 mAbs 13B4 and 14H7 were ideally suited to induce long-term ADAMTS13 deficiency in Adamts13+/+ mice. A single bolus injection resulted in full ex vivo inhibition for more than 7 days. As expected, the mice with the acquired ADAMTS13 deficiency did not spontaneously develop TTP, despite the accumulation of UL-VWF multimers. In line with the Adamts13-/- mice, TTP-like symptoms could only be induced when an additional trigger (rVWF) was administered. On the other hand, the availability of our panel of anti-mADAMTS13 mAbs allowed us to further develop a sensitive ELISA to detect ADAMTS13 in mouse plasma. In conclusion, a novel acquired TTP mouse model was generated through the development of inhibitory anti-mADAMTS13 mAbs. Consequently, this model provides new opportunities for the development and validation of novel treatments for patients with TTP. In addition, these newly developed inhibitory anti-mADAMTS13 mAbs are of great value to specifically study the role of ADAMTS13 in mouse models of thrombo-inflammatory disease.

MeSH terms

  • ADAMTS13 Protein / blood
  • ADAMTS13 Protein / genetics
  • ADAMTS13 Protein / immunology*
  • ADAMTS13 Protein / metabolism
  • Animals
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal / therapeutic use*
  • Antigen-Antibody Reactions
  • Disease Models, Animal
  • Enzyme-Linked Immunosorbent Assay
  • Epitope Mapping
  • Female
  • Humans
  • Male
  • Mice
  • Mice, Knockout
  • Platelet Count
  • Protein Multimerization
  • Purpura, Thrombotic Thrombocytopenic / drug therapy*
  • Purpura, Thrombotic Thrombocytopenic / metabolism
  • Purpura, Thrombotic Thrombocytopenic / pathology
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / therapeutic use
  • Reproducibility of Results
  • von Willebrand Factor / chemistry
  • von Willebrand Factor / genetics
  • von Willebrand Factor / metabolism

Substances

  • Antibodies, Monoclonal
  • Recombinant Proteins
  • von Willebrand Factor
  • ADAMTS13 Protein

Supplementary concepts

  • Thrombotic thrombocytopenic purpura, acquired

Grants and funding

Louis Deforche is supported by a PhD grant from the Agency for Innovation by Science and Technology (IWT, www.iwt.be), Flanders, Belgium (111507). Claudia Tersteeg is a Postdoctoral Fellow supported by the Research Foundation—Flanders (Fonds voor Wetenschappelijk Onderzoek, FWO, www.fwo.be), Belgium (12N0715N). This work was supported by research grants ‘Programmafinanciering KU Leuven’ (PF10/014), the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ of the Flemish government (FWO grant G.0584.11) and the ‘Onderzoekstoelage’ of the KU Leuven (OT grant OT/14/071). Hanspeter Rottensteiner is an employee of Baxalta Innovations and only provided recombinant VWF for our study. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The funders provided support in the form of salaries for authors [LD, CT] and research materials, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.