Studies of a Murine Monoclonal Antibody Directed against DARC: Reappraisal of Its Specificity

Abstract

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

Global analysis of VHHs framework regions with a structural alphabet

“…Comparative molecular modelling had shown the difficulty to propose pertinent structural model of VHHs [26,[35][36], even with very sophisticated approaches as RosettaAntibody [71]. In fact, the results can be appreciated as less efficient than for classical antibodies as tested in AMA I and AMA II [88][89][90][91].…”

“…In addition the VHHs are interesting alternative to the use of monoclonal antibodies for therapy as shown by recent studies of their use against the Dengue virus [19], H5N1 influenza [20], viral infection [21], aflatoxins in agro-products [22], head and neck cancers [23], vascular endothelial growth factor implicated in cancers [24], venom therapy [25] and Plasmodium knowlesi malaria vector [26]. Phase II clinical trials are currently underway to evaluate the efficacy and toxicity of a therapeutic Nanobody TM in patients with thrombotic thrombocytopenic purpura [27] showing recent promising results [28].…”

MiR-29 Induces K562 Cell Apoptosis by Down-Regulating FoxM1

“…Flow cytometry: Cells were incubated at Annexin-V-FITC combining buffer at 1×10 5 cells. Flow cytometry was used to detect apoptosis of the transfected K562cells at 488 nm by determining the relative amount of Annexin V-FITC-positive cells [ 14 ].…”

VHH Structural Modelling Approaches: A Critical Review

“…Indeed, this high similarity is explained by two reasons: the extra disulphide bridge constraint and an entirely equivalent fold of the frameworks [ 73 ]. This model was compared to other (and distinct) V H H clones obtained from the same camel and helped to search for a peptide able to bind DARC; an analysis of the electrostatic surface determinants was particularly important [ 112 ].…”