Introduction
Soluble antigens complexed with immunoglobulin G (IgG) antibodies can induce robust adaptive immune responses in vitro and in animal models of disease. Factor VIII immune complexes (FVIII-ICs) have been detected in individuals with hemophilia A and severe von Willebrand disease following FVIII infusions. Yet, it is unclear if and how FVIII-ICs affect antibody development over time.
Methods
In this study, we analyzed internalization of FVIII complexed with epitope-mapped FVIII-specific IgG monoclonal antibodies (MAbs) by murine bone marrow-derived dendritic cells (BMDCs) in vitro and antibody development in hemophilia A (FVIII-/-) mice injected with FVIII-IC over time.
Results
FVIII complexed with 2-116 (A1 domain MAb), 2-113 (A3 domain MAb), and I55 (C2 domain MAb) significantly increased FVIII uptake by BMDC but only FVIII/2-116 enhanced antibody titers in FVIII-/- mice compared to FVIII alone. FVIII/4A4 (A2 domain MAb) showed similar FVIII uptake by BMDC to that of isolated FVIII yet significantly increased antibody titers when injected in FVIII-/- mice. Enhanced antibody responses observed with FVIII/2-116 and FVIII/4A4 complexes in vivo were abrogated in the absence of the FVIII carrier protein von Willebrand factor.
Conclusion
These findings suggest that a subset of FVIII-IC modulates the humoral response to FVIII in an epitope-dependent manner, which may provide insight into the antibody response observed in some patients with hemophilia A.
Anti-drug antibodies to coagulation factor VIII (fVIII), often termed inhibitors, present the greatest economical and treatment related obstacle in the management of hemophilia A. Although several genetic and environmental risk factors associated with inhibitor development have been identified, the precise mechanisms responsible for the immune response to exogenous fVIII therapies remain undefined. Clinical trials suggest there is an increased immunogenic potential of recombinant fVIII compared to plasma-derived products. Additional biochemical and immunological studies have demonstrated that changes in recombinant fVIII production and formulation can alter fVIII structure and immunogenicity. Recently, one study demonstrated increased immunogenicity of the recombinant fVIII product Helixate in hemophilia A mice following oxidation with hypochlorite (ClO−). It is widely reported that protein aggregates within drug products can induce adverse immune reactions in patients. Several studies have therefore investigated the prevalence of molecular aggregates in commercial recombinant products with and without use-relevant stress and agitation. To investigate the potential link between oxidation-induced immunogenicity and molecular aggregation, we analyzed the recombinant fVIII product, Helixate, via sedimentation velocity analytical ultracentrifugation following oxidation with ClO−. At 80 μM ClO−, a concentration that reduced the specific-activity by 67%, no detectable increase in large molecular aggregates (s > 12 S) was observed when compared to non-oxidized fVIII. This lack of aggregates was demonstrated both in commercial excipient as well as a HEPES buffered saline formulation. These data suggest that oxidation induced immunogenicity is independent of aggregate-mediated immune response. Therefore, our data support multiple, independent mechanisms underlying fVIII immunogenicity.
The most significant complication for patients with severe cases of congenital or acquired hemophiliaAis the development of inhibitor antibodies against coagulation factor VIII (fVIII). The C2 domain of fVIII is a significant antigenic target of antifVIII antibodies. Here, we have utilized small angle x-ray scattering (SAXS) and biochemical techniques to characterize interactions between two different classes of anti-C2 domain inhibitor antibodies and the isolated C2 domain. Multiple assays indicated that antibodies 3E6 and G99 bind independently to the fVIII C2 domain and can form a stable ternary complex. SAXS-derived numerical estimates of dimensional parameters for all studied complexes agree with the proportions of the constituent proteins. Ab initio modeling of the SAXS data results in a long kinked structure of the ternary complex, showing an angle centered at the C2 domain of ∼130°. Guided by biochemical data, rigid body modeling of subunits into the molecular envelope of the ternary complex suggests that antibody 3E6 recognizes a C2 domain epitope consisting of the Arg2209-Ser2216 and Leu 2178-Asp2187 loops. In contrast, antibody G99 recognizes the C2 domain primarily through the Pro2221-Trp2229 loop. These two epitopes are on opposing sides of the fVIII C2 domain, are consistent with the solvent accessibility in the context of the entire fVIII molecule, and provide further structural detail regarding the pathogenic immune response to fVIII.
Background: The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain.
Objectives: To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions.
Methods: ELISA, flow cytometry, a thermal stability assay and hydrogen–deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length.
Results: The longest A1 fragment (rVWF1238–1493) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459–1478 at the C-terminus of rVWF1238–1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479–1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238–1461, or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271–1493, showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain.
Conclusion: The AIM is probably composed of residues 1238–1271 (N-AIM) and 1459–1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.
Haemophilia Published by John Wiley & Sons Ltd. Factor VIII (FVIII) is a multidomain blood plasma glycoprotein. Activated FVIII acts as a cofactor to the serine protease factor IXa within the membrane-bound tenase complex assembled on the activated platelet surface. Defect or deficiency in FVIII causes haemophilia A, a severe hereditary bleeding disorder. Intravenous administration of plasma-derived FVIII or recombinant FVIII concentrates restores normal coagulation in haemophilia A patients and is used as an effective therapy. In this work, we studied the biophysical properties of clinically potent recombinant FVIII forms: human FVIII full-length (FVIII-FL), human FVIII B-domain deleted (FVIII-BDD) and porcine FVIII-BDD bound to negatively charged phospholipid vesicles at near-physiological conditions. We used cryo-electron microscopy (Cryo-EM) as a direct method to evaluate the homogeneity and micro-organization of the protein-vesicle suspensions, which are important for FVIII therapeutic properties. Applying concurrent Cryo-EM, circular dichroism and dynamic light scattering studies to the three recombinant FVIII forms when bound to phospholipid vesicles revealed novel properties for their functional, membrane-bound state. The three FVIII constructs have similar activity, secondary structure distribution and bind specifically to negatively charged phospholipid membranes. Human and porcine FVIII-BDD induce strong aggregation of the vesicles, but the human FVIII-FL form does not. The proposed methodology is effective in characterizing and identifying differences in therapeutic recombinant FVIII membrane-bound forms near physiological conditions, because protein-containing aggregates are considered to be a factor in increasing the immunogenicity of protein therapeutics. This will provide better characterization and development of safer and more effective FVIII products with implications for haemophilia A treatment.
Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of-recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines.
In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/-CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/-MGAT1-CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the Nlinked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/-MGAT1-CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.
Bacterial toxin–antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin–antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin–antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin–antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression.
Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorder-to-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin–antitoxin system.
Inhibitory antibodies to factor VIII (fVIII inhibitors) are the most significant complication in the management of hemophilia A. The immunogenicity of fVIII may be driven in part by structural determinants within the fVIII molecule itself. Regions of non-identity between human and porcine fVIII possibly could drive differential immune responses. The goal of this study was to compare the overall antibody response and levels of antibodies to the individual fVIII domains in naïve hemophilia A mice immunized with human or porcine fVIII. Hemophilia A mice were immunized with human or porcine fVIII using a protocol that mimics human clinical use. Inhibitor and total anti-fVIII antibody titers were measured and the domain-specificity of antibodies from 1759 anti-fVIII hybridomas was determined. The overall immunogenicity of human and porcine fVIII was similar but significant differences in domain recognition were discovered. Anti-A2 and anti-C2 antibodies constituted the majority of inhibitors in both the human and porcine fVIII groups, similar to inhibitors that develop in humans. The proportions of anti-A2 or anti-C2 antibodies were not significantly different between the two groups. However, the specific inhibitory activity of anti-A2 antibodies was higher in the human fVIII group. Additionally, proportion of anti-C1 antibodies was significantly higher in the human fVIII group. In contrast, anti-A3 antibodies were more common in the porcine fVIII group. The differential immune response to human and porcine fVIII suggests that it may be possible to reduce the immunogenicity of fVIII by mutagenesis of the A2, A3 and C1 domains.
Summary
Objective
The pathogenicity of anti-human fVIII monoclonal antibodies (MAbs) was tested in a murine bleeding model.
Methods
MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 nM, followed by injection of human B domain-deleted factor VIII (fVIII) at 180 U/kg, producing peak plasma concentrations of ∼2 nM. At 2 hours, blood loss following a 4 mm tail snip was measured. The following MAbs were tested: 1) 4A4, a type I anti-A2 fVIII inhibitor, 2) I54 and 1B5, classical type I anti-C2 inhibitors, 3) 2-77 and B45, non-classical type II anti-C2 inhibitors, and 4) 2-117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units/mg IgG.
Results
All MAbs except 2-117 produced similar amounts of blood loss that were significantly greater than control mice injected with fVIII alone. Increasing the dose of fVIII to 360 U/kg overcame the bleeding diathesis produced by the type II MAbs 2-77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U/ml and 3 U/ml fVIII, while there was 40% residual activity at saturating concentrations of 2-77 at either concentration of fVIII.
Conclusions
For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose fVIII rather than bypassing agents may be warranted.
by
Kenneth C Childers;
Nathan G Avery;
Kevin A. Estrada Alamo;
Omar Davulcu;
Rose Marie Haynes;
Pete Lollar;
Christopher B Doering;
Carmen H Coxon;
P. Clint Spiegel Jr
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.