title: Coagulation factor VII variants resistant to inhibitory antibodies.
abstract: Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.
authors.forename: Alessio
authors.initials: A
authors.lastname: Branchini
authors.forename: Marcello
authors.initials: M
authors.lastname: Baroni
authors.forename: Caroline
authors.initials: C
authors.lastname: Pfeiffer
authors.forename: Angelika
authors.initials: A
authors.lastname: Batorova
authors.forename: Muriel
authors.initials: M
authors.lastname: Giansily-Blaizot
authors.forename: Jean F
authors.initials: JF
authors.lastname: Schved
authors.forename: Guglielmo
authors.initials: G
authors.lastname: Mariani
authors.affiliation: Prof. Francesco Bernardi, Department of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy, Tel.: +39 0532 974425, Fax: +39 0532 974484, E-mail: ber@unife.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
mesh_terms: D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D001777:Blood Coagulation; D005167:Factor VII / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005168:Factor VII Deficiency / Q000188:drug therapy; D015942:Factor VIIa / Q000737:chemistry / Q000276:immunology* / Q000627:therapeutic use; D015951:Factor Xa / Q000096:biosynthesis; D016368:Frameshift Mutation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000737:chemistry / Q000276:immunology; D007518:Isoantibodies / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000627:therapeutic use; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000096:biosynthesis
chemical_list: D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007518:Isoantibodies; D011994:Recombinant Proteins; D005167:Factor VII; C103587:recombinant FVIIa; D015942:Factor VIIa; D013917:Thrombin; D015951:Factor Xa
keywords: Inhibitory antibodies; carboxyl-terminus; factor VII deficiency; recombinant proteins; replacement therapy