Factor VIII: Perspectives on Immunogenicity and Tolerogenic Strategies.
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ABSTRACT:
SUBMITTER: Scott DW
PROVIDER: S-EPMC6978909 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
Publications
Factor VIII: Perspectives on Immunogenicity and Tolerogenic Strategies.
Scott David W DW Pratt Kathleen P KP
Frontiers in immunology 20200117
Therapeutic treatment of bleeds with FVIII can lead to an antibody response that effectively inhibits its function. Herein, we review the factors that contribute to this immunogenicity and possible ways to overcome it. ...[more]
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Project description: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.
| S-EPMC4149818 | biostudies-literature
Project description:Replacement therapy with exogenous recombinant factor VIII (rFVIII) to control bleeding episodes results in the development of inhibitory antibodies in 15% to 30% of hemophilia A patients. The inhibitory antibodies are mainly directed against specific and universal immunodominant epitopes located in the C2 domain. Previously we have shown that complexation of O-phospho-L-serine (phosphatidylserine head group) with the phospholipid binding region of the C2 domain can lead to an overall reduction in the immunogenicity of rFVIII. Here, we have investigated the hypothesis that dicaproyl phosphatidylserine, a short-chain water-soluble phospholipid, can reduce the immunogenicity of rFVIII. Circular dichroism and fluorescence spectroscopy studies suggest that dicaproyl phosphatidylserine interacts with rFVIII, causing subtle changes in the tertiary and secondary structure of the protein. Sandwich enzyme-linked immunosorbent assay studies indicate that dicaproyl phosphatidylserine probably interacts with the phospholipid binding region of the C2 domain. The immunogenicity of FVIII-dicaproyl phosphatidylserine complexes prepared at concentrations above and below the critical micellar concentrations of the lipid were evaluated in hemophilia A mice. Our results suggest that micellar dicaproyl phosphatidylserine may be useful to reduce the immunogenicity of rFVIII preparations.
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Project description:Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evidence has highlighted that recombinant factor VIII produced in baby hamster kidney cells is more immunogenic than factor VIII produced in Chinese hamster ovary cells. Glycosylation differences have been hypothesized to influence the immunogenicity of these synthetic concentrates. In two hemophilia A mouse models, baby hamster kidney cell-derived factor VIII elicited a stronger immune response compared to Chinese hamster ovary cell-derived factor VIII. Furthermore, factor VIII produced in baby hamster kidney cells exhibited accelerated clearance from circulation independent of von Willebrand factor. Lectin and mass spectrometry analysis of total N-linked glycans revealed differences in high-mannose glycans, sialylation, and the occupancy of glycan sites. Factor VIII desialylation did not influence binding to murine splenocytes or dendritic cells, nor surface co-stimulatory molecule expression. We did, however, observe increased levels of immunoglobulin M specific to baby hamster kidney-derived factor VIII in naïve hemophilia A mice. De-N-glycosylation enhanced immunoglobulin M binding, suggesting that N-glycan occupancy masks epitopes. Elevated levels of immunoglobulin M and immunoglobulin G specific to baby hamster kidney-derived factor VIII were also observed in healthy individuals, and de-N-glycosylation increased immunoglobulin G binding. Collectively, our data suggest that factor VIII produced in baby hamster kidney cells is more immunogenic than that produced in Chinese hamster ovary cells, and that incomplete occupancy of N-linked glycosylation sites leads to the formation of immunoglobulin M- and immunoglobulin G-factor VIII immune complexes that contribute to the enhanced clearance and immunogenicity in these mouse models of hemophilia A.
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Project description:Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein-lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.
| S-EPMC2758112 | biostudies-literature
Project description:Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance.
| S-EPMC4936482 | biostudies-literature
Project description:A main complication of treatment of patients with hemophilia A is the development of anti-factor VIII (fVIII) antibodies. The immunogenicity of fVIII potentially is a function of its procoagulant activity, which may result in danger signals that drive the immune response. Alternatively, intrinsic structural elements in fVIII may be particularly immunogenic. Finally, VWF, the carrier protein for fVIII in plasma, may play a role in immune recognition. We compared the immunogenicity of wild-type (wt) B domain-deleted fVIII and 2 inactive fVIII molecules, R372A/R1689A fVIII and V634M fVIII in fVIII(-/-) and fVIII(-/-)/VWF(-/-) mice. R372A/R1689A fVIII lacks proteolytic recognition sites and is not released from VWF. In contrast, V634M fVIII undergoes proteolytic cleavage and dissociation from VWF. No significant difference was observed in the immunogenicity of wt fVIII and V634M fVIII. R372A/R1689A fVIII was slightly less immunogenic in a subset of immunization regimens tested. High doses of wt fVIII were required to produce an immune response in fVIII(-/-)/VWF(-/-) mice. Our results indicate that a main component of the immune response to fVIII is independent of its procoagulant function, is both positively and negatively affected by its association with VWF, and may involve intrinsic elements of fVIII structure.
| S-EPMC3448263 | biostudies-literature
Project description:Development of neutralizing antibodies against biotherapeutic agents administered to prevent or treat various clinical conditions is a longstanding and growing problem faced by patients, medical providers and pharmaceutical companies. The hemophilia A community has deep experience with attempting to manage such deleterious immune responses, as the lifesaving protein drug factor VIII (FVIII) has been in use for decades. Hemophilia A is a bleeding disorder caused by genetic mutations that result in absent or dysfunctional FVIII. Prophylactic treatment consists of regular intravenous FVIII infusions. Unfortunately, 1/4 to 1/3 of patients develop neutralizing anti-FVIII antibodies, referred to clinically as "inhibitors," which result in a serious bleeding diathesis. Until recently, the only therapeutic option for these patients was "Immune Tolerance Induction," consisting of intensive FVIII administration, which is extraordinarily expensive and fails in ~30% of cases. There has been tremendous recent progress in developing novel potential clinical alternatives for the treatment of hemophilia A, ranging from encouraging results of gene therapy trials, to use of other hemostatic agents (either promoting coagulation or slowing down anti-coagulant or fibrinolytic pathways) to "bypass" the need for FVIII or supplement FVIII replacement therapy. Although these approaches are promising, there is widespread agreement that preventing or reversing inhibitors remains a high priority. Risk profiles of novel therapies are still unknown or incomplete, and FVIII will likely continue to be considered the optimal hemostatic agent to support surgery and manage trauma, or to combine with other therapies. We describe here recent exciting studies, most still pre-clinical, that address FVIII immunogenicity and suggest novel interventions to prevent or reverse inhibitor development. Studies of FVIII uptake, processing and presentation on antigen-presenting cells, epitope mapping, and the roles of complement, heme, von Willebrand factor, glycans, and the microbiome in FVIII immunogenicity are elucidating mechanisms of primary and secondary immune responses and suggesting additional novel targets. Promising tolerogenic therapies include development of FVIII-Fc fusion proteins, nanoparticle-based therapies, oral tolerance, and engineering of regulatory or cytotoxic T cells to render them FVIII-specific. Importantly, these studies are highly applicable to other scenarios where establishing immune tolerance to a defined antigen is a clinical priority.
| S-EPMC6965068 | biostudies-literature
Project description:Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.
| S-EPMC5789939 | biostudies-literature
Project description:BackgroundUp to 30% of hemophilia A patients develop inhibitory antibodies against the infused factor VIII (FVIII). The development of a deimmunized FVIII is an unmet high medical need. Although improved recombinant FVIII (rFVIII) products evolved within the last years, the immunogenicity has not been solved. A deimmunized FVIII could reduce the probability of inhibitor development, providing safer therapy.ObjectiveTo develop a deimmunized FVIII molecule by modifying major histocompatibility complex (MHC) class II presentation, leading to a functional but less immunogenic molecule.MethodsWe performed (1) in silico prediction of potentially immunogenic T cell epitopes and their modification by amino acid substitutions in the FVIII sequence, (2) evaluation of functional and structural similarity of the modified rFVIII to unmodified FVIII and registered products, and (3) confirmation of the reduced immunogenicity by in vitro testing.ResultsA partially deimmunized fully functional FVIII molecule incorporating 19 amino acid substitutions was generated. The substitutions led to a reduction of the immunogenicity score, indicating a reduced immunogenicity based on in silico calculations. This was confirmed in an in vitro dendritic cell (DC)--T cell assay. Using this assay, cells from healthy donors proved the significantly reduced immunogenicity of the modified FVIII variant by revealing less proliferation of T helper cells to this variant than to the unmodified FVIII.ConclusionIn silico predictions resulted in a partially deimmunized FVIII. This FVIII is fully functional and was demonstrated to be less immunogenic in in vitro testing. This approach may result in a reduction of the inhibitor risk for patients with hemophilia A.
| S-EPMC8456792 | biostudies-literature
Project description:Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma ("CRM-negative"), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ?50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ?20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.
| S-EPMC4123441 | biostudies-literature