Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice

Abstract

Development of neutralizing IntroductionOver the past decades protein therapeutics such as hormones, enzymes, blood coagulation factors, or Abs have provided effective treatment for numerous diseases. 1 Treatment commonly requires frequent high-dose administration of protein therapeutics and, although generally considered safe, they often induce immune responses. 2 The factors that underlie immunogenicity of biomedical products can be related to the structure of protein, such as the presence of promiscuous T-cell epitopes 3 or posttranslational modifications, 4 but also to the formulation of the biomolecule. 5 Treatment-related parameters such as dosage, frequency, route of administration, and concomitant infections may also contribute to the induction of antidrug immune responses. 2 In patients with protein deficiencies administered therapeutics may be recognized by the immune system as non-self, thereby greatly increasing the risk of Ab development. 2 Hemophilia A is an X-linked bleeding disorder that is caused by a deficiency in blood coagulation factor VIII (FVIII). Conventional treatment comprising frequent administration of FVIII often results in formation of neutralizing Abs, which inhibit FVIII activity. 6,7 Both treatment-related factors, such as intensive treatment episodes, 8 and genetic risk factors can contribute to the development of inhibitors. Polymorphic sites in genes involved in the adaptive immune response have been associated with anti-FVIII Ab formation. 9-11 Development of high-affinity IgG Abs directed against FVIII is a CD4 ϩ T cell-dependent process. 12,13 Endocytosis of FVIII by professional APCs comprises the initial step leading to activation of helper T cells. Uptake and transfer of Ags through the lyso-endosomal pathway results in intracellular processing and presentation of FVIII-derived peptides on MHC II molecules to CD4 ϩ helper T cells. 14 Here, we hypothesized that prevention of FVIII uptake by APCs will lead to diminished T-and B-cell responses. Previously, we have shown that endocytosis of FVIII by APCs is mediated via its C1 domain, because administration of a mAb directed toward an antigenic surface in the C1 domain reduced inhibitor titers in FVIII-deficient mice. 15 With the use of an Ab-guided mutagenesis strategy we designed a C1 domain variant of FVIII which displayed a strongly reduced internalization by APCs. In vivo studies revealed that this C1 domain variant showed decreased immunogenicity in a murine model for inhibitor development in hemophilia A. Our findings provide a novel paradigm for the reduction of the intrinsic immunogenicity of FVIII by modulating its uptake by APCs. Methods MaterialsFicoll-Paque Plus (GE Healthcare), CD14 microbeads (Miltenyi Biotech), and human recombinant GM-CSF and IL-4 (both Cellgenix Technology Transfer) were used for generation of human monocyte-derived dendritic cells (MDDCs); M-CSF (PeproTech) was used to generate human monocytederived macrophages (MDM⌽s). For culturing murine BM-derived DCs (BMDCs), mouse recombinant GM-CSF ...

Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

“…Moreover, the same spike contributes to FVIII immunogenicity in a murine model of hemophilia A (17). The same membrane-binding spike also contributes to the interaction of FVIII with the antibody KM33 (14,17).…”

“…Besides lipid binding, the C1 domain residues 2092-2093 also contribute to the uptake of FVIII by cells that express lowdensity lipoprotein receptor-related protein (LRP) and by monocyte-derived dendritic cells (16,17). Moreover, the same spike contributes to FVIII immunogenicity in a murine model of hemophilia A (17).…”

“…The same membrane-binding spike also contributes to the interaction of FVIII with the antibody KM33 (14,17). This anti-C1 domain antibody has been derived from the immune repertoire of a patient with severe hemophilia A (18) and is of particular interest because it also inhibits, besides FVIII activity, the interaction with von Willebrand factor (VWF) and LRP, cellular uptake, and immunogenicity (16,17,19). Apparently, the epitope of this antibody provides a "hot spot" for multiple macromolecular interactions and biological processes involving FVIII.…”

“…Cells were permeabilized employing 0.25% saponin in TBS containing 1% (w/v) BSA and incubated with labeled EL14 antibody. Cells were washed twice with TBS/0.5% (w/v) BSA and analyzed by flow cytometry using a BD Biosciences LSR II flow cytometer as described (16,17).…”

“…However, the tandem orientation of the two C domains in the crystal structures suggests that the C1 domain could also mediate membrane binding. Indeed, mutagenesis studies provided direct evidence that the C1 domain contributes to phospholipid binding as well and that this particularly involves the spike containing residues Lys-2092 to Phe-2093 (14, 15).Besides lipid binding, the C1 domain residues 2092-2093 also contribute to the uptake of FVIII by cells that express lowdensity lipoprotein receptor-related protein (LRP) and by monocyte-derived dendritic cells (16,17). Moreover, the same spike contributes to FVIII immunogenicity in a murine model of hemophilia A (17).…”