Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)–associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor’s MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although .12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)–recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain–deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/ present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII

The national blueprint for future basic and translational research to understand factor VIII immunogenicity: NHLBI State of the Science Workshop on factor VIII inhibitors

Introduction Inhibitor formation against coagulation factor VIII (FVIII) is an unresolved serious problem in replacement therapy for the X‐linked bleeding disorder haemophilia A. Although FVIII inhibitors have been extensively studied, much of the basic mechanism of this immune response remains to be uncovered. Aim Within the NHLBI State of the Science Workshop on Factor VIII Inhibitors, Working Group 3 identified three scientific priorities for basic and translational research on FVIII inhibitor formation. Methods A larger list of potential areas of research was initially developed as a basis for subsequent prioritization. Each scientific goal was further evaluated based on required effort, potential impact, approach, methods, technologies and models. Results The three priorities include the following: activation signals and immune regulation that shape the response to FVIII (including innate immunity, microbiome, adaptive immunity and regulatory T cell studies in humans); utility of animal models and non‐animal approaches (in silico, genetic, single‐cell/sorted population “omics,” in vitro) to help predict inhibitor formation and identify novel therapeutics; and impact of the source of FVIII, its structure and von Willebrand factor on immunogenicity and tolerance. Conclusions Early interactions between FVIII and the immune system, biomarker development and studies in different patient groups (previously treated or untreated, with or without inhibitor formation, patients undergoing immune tolerance induction or gene therapy) deserve particular emphasis. Finally, linking basic to clinical studies, development of a repository for biospecimens and opportunities for interdisciplinary research training are important components to solving the urgent problem of inhibitor formation

Effect of HLA DR epitope de-immunization of Factor VIII \u3ci\u3ein vitro\u3c/i\u3e and \u3ci\u3ein vivo\u3c/i\u3e

T cell-dependent development of anti-Factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatic tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3 × E16 mice. Consistent with immunoinformatic predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use

In silico-in vitro screening of protein-protein interactions: towards the next generation of therapeutics.

International audienceProtein-protein interactions (PPIs) have a pivotal role in many biological processes suggesting that targeting macromolecular complexes will open new avenues for the design of the next generation of therapeutics. A wide range of "in silico methods" can be used to facilitate the design of protein-protein modulators. Among these methods, virtual ligand screening, protein-protein docking, structural predictions and druggable pocket predictions have become established techniques for hit discovery and optimization. In this review, we first summarize some key data about protein-protein interfaces and introduce some recently reported computer methods pertaining to the field. URLs for several recent free packages or servers are also provided. Then, we discuss four studies aiming at developing PPI modulators through the combination of in silico and in vitro screening experiments

Molecular Characterization and Inhibition of Antibodies Elicited Against Galactosyltransferase Knockout Pig Xenografts

On average, 21 people in the United States die every day while waiting for a transplantable organ. Moreover, at the end of 2014, nearly 130,000 additional individuals were still on the transplant waiting list. Xenotransplantation, the transplantation of cells and/or organs from a member of another species, is both a viable supplement for, and bridge to, allotransplantation. Pigs are the favored organ donor because they: 1) breed and grow rapidly, 2) can be genetically modified, and 3) present few ethical concerns. However, due in part to their phylogenetic distance, several barriers must be successfully overcome before clinical xenotransplantation can be fully realized. Antibody deposition is presently a barrier to successful cardiac, renal, and non-encapsulated islet xenotransplantation; although all xenografts are likely susceptible to antibody-mediated damage. The purpose of this dissertation is twofold: 1) to provide further molecular characterization of the elicited antibodies which mediate rejection of porcine xenografts, and 2) to identify clinically applicable small molecules capable of selectively inhibiting these antibodies. We have found that vascular pig xenografts, and isolated porcine pancreatic islets, both elicit antibody responses encoded by structurally related antibody gene progenitors. These structurally related antibody gene progenitors are both known to encode human antibodies capable of inhibiting clotting factor VIII (FVIII). Therefore, we subsequently identified FVIII as a novel xenoantigen, and provided a preliminary molecular analysis of the interaction between FVIII and the antibodies elicited after xenotransplantation. Additionally, our analyses strongly suggested that it was feasible for a single reagent to inhibit the majority of the antibodies elicited against transplanted xenografts. Consequently, we screened for, and identified, a single clinically relevant small molecule drug which, when tested in vitro, inhibited elicited antibody from multiple xenotransplant settings. However, it was only when utilized in combination with a previously identified experimental reagent that it was possible to reduce the post-transplant antibody to levels below, or comparable to, those present before transplantation. These results challenge long-standing presumptions with regard to the nature of xenoantigens and their role in antibody-mediated xenograft rejection. Furthermore, identification of a clinically relevant selectively inhibitory small molecule should expedite transition into large animal work

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity

A New Mechanism-Driven Bypassing Strategy for Direct Factor Xa Inhibitors Reveals An Unexpected Property of Target Specific Anticoagulants

Hemostasis is a crucial component of vascular homeostasis that prevents blood loss while also maintaining vascular patency. Hemostasis is achieved, in part, through a cascade of serine proteases that are sequentially activated, culminating in formation of the effector protease, thrombin. Normally, this process is tightly regulated, but loss of regulation can lead to bleeding or excessive clot formation (thrombosis). In the event of thrombosis, anticoagulation is the mainstay of care. Numerous pharmacokinetic problems with the oral anticoagulant warfarin prompted the development of new oral agents that directly inhibit the serine proteases of coagulation. In particular, several active site inhibitors of coagulation factor Xa (FXa) have recently been approved and are at least as effective as warfarin for the prevention of thrombosis. However, they, like warfarin, increase the risk of bleeding, and there are no approved countermeasures to treat or prevent bleeding with these direct FXa inhibitors. We evaluated whether a variant of FXa (FXaI[16]L) could reverse the effects of the direct FXa inhibitor rivaroxaban. FXaI[16]L has poor active site function and a long plasma half-life but has high catalytic activity at the site of vascular injury, making it an effective pro-hemostatic agent. FXaI[16]L potently reversed the effects of rivaroxaban in in vitro studies and animal models of coagulation. Kinetic studies revealed that both FXaI[16]L and wt-FXa are highly inhibited by rivaroxaban at therapeutic concentrations when bound to the cofactor FVa. Despite this high level of inhibition with rivaroxaban, both FXaI[16]L and wt-FXa support thrombin generation. To explain this discrepancy, we measured the kinetics of FXa inhibition by antithrombin III (ATIII), a key regulator of FXa activity in plasma. Rivaroxaban impaired ATIII-dependent FXa inhibition by creating a pool of reversibly-inhibited FXa, and kinetic simulations indicated that, under these conditions, a steady-state of free, uninhibited FXa is established. Thus, there is a paradoxical increase in the level of free FXa which explains how FXaI[16]L can generate thrombin in the presence of rivaroxaban. These results reveal a previously unreported, unintended consequence of direct FXa inhibitors that may have important implications. Further, FXaI[16]L may be able to fill the unmet clinical need for a rapid, hemostatic reversal agent for these new anticoagulants