reproducibility-harmonization-research-using-biological-standards

Reproducibility and Harmonization in Research Using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide

Metrology - the science of measure - is a subject few biological scientists are taught about in their training to their detriment; the application of ...

National Institute for Biological Standards and Control

Medicines and Healthcare products Regulatory Agency

Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter-donor variability limit the application of such assays, especially with many patients on prophylaxis.

Microfluidic hemophilia models using blood from healthy donors.

The coagulation factors (F)V and VIII are homologous proteins that support hemostasis through their regulation of FX activity. Hemophilia A (HA) patients have reduced FVIII activity and a prolonged bleeding time that is corrected through the administration of exogenous FVIII. Around one-third of severe HA patients develop FVIII neutralizing antibodies, known as "inhibitors," which neutralize FVIII activity and preclude them from further FVIII therapy.

FVIII inhibitors display FV-neutralizing activity in the prothrombin time assay.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1-mediated clearance.

Article	Year
Microfluidic hemophilia models using blood from healthy donors. Research and practice in thrombosis and haemostasis\| PubMed ID: 31989085	2020
FVIII inhibitors display FV-neutralizing activity in the prothrombin time assay. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 33914406	2021
Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients. British journal of haematology\| PubMed ID: 33973229	2021
Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor. Blood\| PubMed ID: 37192299	2023

Carmen H. Coxon

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