Factor VIII Protein Research Articles

The 3.2 Å structure of a bioengineered variant of blood coagulation factor VIII indicates two conformations of the C2 domain

BackgroundCoagulation factor VIII represents one of the oldest protein‐based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. ObjectivesTo structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain‐deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. MethodsThe structure of ET3i was characterized with X‐ray crystallography and tandem mass spectrometry‐based glycoproteomics. ResultsHere, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N‐linked glycans with LC‐MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. ConclusionsThese improved structures yield a more confident model for next‐generation engineering efforts to develop FVIII therapeutics with longer half‐lives, higher expression levels, and lower immunogenicity.

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII

Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.

Neutralizing and Non-Neutralizing Anti-FVIII Antibodies in Black and White Hemophilia A Subjects: A Natural History Profile

The most common complication in hemophilia A (HA) treatment, affecting 25-30% of severe HA patients, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 gene haplotypes H1-H5 are defined by combinations of nonsynonymous SNPs encoding FVIII sequence variants D1241E, M2238V and R484H. F8 haplotypes H2-H5 are more prevalent in individuals with black African ancestry, while >90% of the white population has the H1 haplotype. This study used a validated Luminex-based assay to determine total anti-FVIII antibody titers in plasma from 395 HA (189 black, 206 white) and 23 non-HA control subjects, measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3 and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic Bethesda assay with the Nijmegen modification. Linear B-cell epitopes recognized by antibodies in human plasma samples were characterized using commercial peptide microarrays with imprinted 15-mer peptides spanning the FVIII A1, A2, C1 and C2 domains, with binding interactions detected using fluorescent-labeled anti-human IgG antibodies. Neither total nor inhibitory antibody titers correlated with F8 haplotype. FVIII peptides with the D1241E and M3348V polymorphisms showed low antibody reactivity, indicating they do not comprise linear B-cell epitopes. Similarly, antibodies from subjects with H3 and H5 haplotypes, who were necessarily infused with FVIII products having a different haplotype than that of their endogenous, (dysfunctional) F8 sequence, did not show haplotype-correlated differential binding to the three BDD-FVIII or full-length FVIII proteins, indicating the polymorphic M2238V or D1241E sites do not correspond to immunodominant, conformational B-cell epitopes. Interestingly, the BDD-FVIII proteins were significantly more reactive with antibodies in plasma than were two commercial full-length recombinant FVIII products. Overall, results of this study indicated that low-titer FVIII-reactive antibodies are readily detected in most HA subjects and in a majority of healthy non-HA controls. The observed stronger immunoreactivity of BDD-FVIII suggests that B-domain removal exposes novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains. Disclosures Pratt: Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding. Peters:Sanofi: Employment. Mann:Haematologic Technologies: Other: Owner; Stago: Consultancy; Novo Nordisk: Consultancy; Takeda: Consultancy; Shire: Consultancy; Baxalta: Consultancy.

Elispots and Ex Vivo Expansion of FVIII-Specific T-Cell Clones Confirm Specificity of CD4+ T-Cell Responses to Self-FVIII in Healthy Non-Hemophilic Blood Donors

The existence of natural antibodies recognizing endogenous factor VIII (FVIII) and of FVIII-specific CD4+ T-cell responses in some healthy, non-hemophilic blood donors has been appreciated for >20 years. The Conti-Fine group measured CD4+ T-cell proliferation following in vitro stimulation with FVIII protein or synthetic FVIII peptides. More recently, FVIII-specific CD4+ T-cell lines were expanded from PBMCs isolated from large blood volumes donated by healthy individuals, and estimates of specific precursor frequency (~2/million CD4+ T cells) were calculated on the basis of interferon (IFN)-gamma ELISPOT assays of FVIII-stimulated cells (Meuniere et al., Blood Advances 1(21): 1842-7). Escape of these self-reactive precursor cells from thymic editing via deletion or anergy and their subsequent persistence in the periphery may contribute to the rare but potentially severe autoimmune reactions to FVIII ("acquired hemophilia A") and to the unusual immunogenicity of therapeutic FVIII administered i.v. to hemophilia A patients. The present study sought to further characterize CD4+ T-cell responses to endogenous FVIII and to map epitopes recognized by these self-reactive cells. We were particularly interested to learn if these cells recognize multiple epitopes in FVIII or if they respond to only several immunodominant epitopes. Accordingly, IFN-gamma ELISPOT assays were carried out by stimulating CD4+ T cells with 15-mer FVIII peptides having 12-residue overlaps and spanning the FVIII A1, A2, A3, C1 and C2 domains. For efficient mapping, initial assays utilized large pools of peptides, and positive responses were then "decoded" by ELISPOTs using smaller peptide pools or individual peptides. Blood samples were obtained from healthy controls under approved IRB protocols. The ELISPOT assays utilized CD4+ T cells isolated by negative selection, with irradiated autologous PBMCs as antigen presenting cells. Anti-CD49d/CD28 monoclonal antibodies were added for co-stimulation to increase the sensitivity of the assay and cells were cultured with IL-7 to improve cell viability. As a result, this assay required smaller blood volumes, but it should be noted that lower-avidity T-cell responses were likely detected that might be missed in ELISPOT assays without these modifications. Relevance of such low-avidity self-reactive cells is provided by the clinical observation, consistent with basic immunological principles, that risk factors for autoimmune responses to FVIII include old age (pro-inflammatory), trauma, surgery and postpartum status, all of which up-regulate T-cell co-stimulatory factors. The first subject had HLA-DRB1*01:01 and HLA-DRB1*08:04 alleles. Stimulation with large peptide pools and rFVIII protein indicated recognition of epitopes in at least 3 FVIII domains. Additional ELISPOTs tested the immunogenicity of 15 peptides corresponding to FVIII peptides previously demonstrated to be presented on dendritic cells from 2 individuals with an HLA-DRB1*01:01 allele (van Haren et al., Mol Cell Proteomics. 2011;10(6)), ensuring that our assays included tests of naturally processed FVIII peptides. Two of these peptides, both from the FVIII A1 domain, produced ELISPOT readings above background levels. T cells were then stimulated with these peptides for 19 days, stained with peptide-loaded MHC Class II (HLA-DRB1*01:01) tetramers, sorted and expanded for another 14 days. Tetramer staining then confirmed isolation of CD4+ T-cell clones recognizing one of these peptides. T cells that recognize their cognate antigen with high avidity are significant drivers of allo- and autoimmune responses. Lower-avidity T cells, however, can play significant roles in pro-inflammatory settings. Tetramer staining validated our ELISPOT-based identification of specific epitopes in FVIII. We are now carrying out ELISPOT assays using pooled peptides followed by individual FVIII peptides as stimulants, to estimate the repertoire of FVIII-specific CD4+ T cells in healthy non-hemophilic individuals. Mapping of HLA-restricted T-cell epitopes will also enable future tetramer-based isolation and phenotypic characterization of these rare T cells without expanding them in culture. This will allow us to investigate the interesting question of what peripheral tolerance mechanisms prevent expansion of these self-reactive cells in vivo, except in rare cases of FVIII autoimmunity. . Disclosures Pratt: Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

Thrombin: A Driver or Attenuator of Bone Disease on Hemophilia?

Introduction: Hemophilic bone disease is a pathology inherent to factor VIII (FVIII) and factor IX (FIX) deficiency present in both adult and pediatric persons with hemophilia (PWH). This pathology is also present in mouse models. We have previously demonstrated that FVIII replacement restores lost mineralization activity in osteoblast-like cells from FVIII-deficient mice. We hypothesized that bone disease in PWH is secondary to decreased thrombin generation in PWH. Methods: Murine bone marrow stromal cells isolated from FVIII knockout (FVIII-/-) and wild-type (FVIII+/+) C57BL mice and human osteosarcoma (HOS) TE85 cells were cultured in α-MEM with 0-10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL Amphotericin B. Ten millimolar ß-glycerophosphate and 50 µg/mL ascorbic acid were added to induce differentiation and mineralization. Mineralized deposits were stained with alizarin red. Alkaline phosphatase (ALP) activity and culture protein content were measured using colorimetric assays. Results: In , the addition of thrombin in serum-free conditions increases cell proliferation ~25% (p=5x10-4, ANCOVA) and decreases mineralization up to 84% (p=1x10-6) (Figure 1). Thrombin's effect on ALP activity in HOS TE85 cells is negative at low cell numbers and positive at high cell numbers (Figure 1). Osteoblast-like cells from FVIII-/- mice mineralize 70% less than those from FVIII+/+ cells (p=3x10-6). Administering FVIII to cells from FVIII-/- mice ameliorates the mineralization deficiency (Figure 1), while mineralization in cells from FVIII+/+ mice does not change (p=0.66). FVIII-/- cells express 30% lower levels of ALP activity than FVIII+/+ (p=3x10-4). FVIII+/+ cells proliferate 26% more rapidly than FVIII-/- cells (p=4x10-7, ANCOVA), but this is not affected by the addition of FVIII in vitro (p=0.45). The addition of FVIII causes a ~33% decrease in ALP activity in osteoblast-like cells from both FVIII-/- (p=2x10-4) and FVIII+/+ (p=5x10-5) mice. Although thrombin impacts osteoblast differentiation and function, it has paradoxical effects on their mineralization, suggesting thrombin deficiency is not the sole driver of reduced bone health in PWH. Deficient mineralization in cells from FVIII-/- mice compared to those from FVIII+/+ mice is corrected with FVIII replacement in vivo or in vitro, suggesting that circulating FVIII has a positive role in these cells' mineralization. More rapid proliferation in cells from FVIII+/+ mice likely contributes to the increase in mineralization, yet, as proliferation is unchanged by FVIII replacement, it appears unlikely that this effect occurs via the same mechanism. Thrombin causes increased proliferation of HOS TE85 cells, yet also causes a significant decline in mineralization activity (Figure 1). Whether this is a paradoxical effect present only in this transformed cell line is yet undetermined. ALP activity increases when HOS TE85 cells are given thrombin in vivo during the log phase of growth (Figure 1), indicating a positive effect on the osteoblast-like function of the cells. However, this notion is undermined by the decrease in mineralization. Identifying the role of FVIII, FIX, thrombin, and active protein C in bone mineralization in PWH is proving to be a challenging undertaking, yet it remains critical, especially as new drugs with the potential to supplant FVIII prophylaxis hit the market. Future experiments will include thrombin and a thrombin inhibitor, a PAR1 agonist and antagonist, a PAR2 antagonist, and an APC neutralizing antibody. Upcoming findings will be discussed. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership.

Bleeding in Mild Hemophilia A Due to a Splice-Site F8 Mutation May be Fully Abrogated By Prothrombotic Gene Variants

Introduction: While Factor VIII (FVIII) activity correlates with phenotype in most Hemophilia A (HA) males, the study of rare individuals with discrepant bleeding phenotypes can provide insight into F8 genotype-bleeding phenotype relationships, refine HA classification and improve clinical management. We present an adult male subject who carries a F8 missense mutation, c.5999G>C (p.Gly2000Ala), but remarkably has had no bleeding despite numerous challenges, including multiple surgeries. To resolve genotype-phenotype discrepancy, we evaluated F8 transcripts and screened for additional gene variants by exome sequencing. Methods: We performed clinical laboratory assays including one-stage FVIII:C and chromogenic FVIII assays, FVIII antigen ELISA, VWF antigen and activity assays, and von Willebrand Disease (VWD) Type 2 Normandy binding activity assay. The F8 c.5999G>C mutation is proposed to affect F8 splicing, and result in a transcript that deletes exon 19. To evaluate F8 splice variants, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using primers that distinguish FVIII isoforms. Samples were normalized relative to a housekeeping gene, GAPDH. Normalized relative fold expression for each primer pair was calculated by the 2−ΔΔCT Livak method. Whole exome sequencing was performed at 80X coverage. Sequencing reads were mapped to the GRCh37/hg19 reference genome with BWA-MEM, and high-quality variant calls were made using GATK4.0 with annotation based on refSeq 1.9, employing best practice recommendations. Results: Clinical assays confirmed reduced FVIII activity levels consistent with mild HA. By qRT-PCR, the F8 mutation results in a partial splicing defect. Exon 19 deleted transcripts predominate, but are likely not secreted. Indeed, the reduced levels of full-length F8 isoform closely mirror measured FVIII protein and activity levels. These findings explain FVIII levels, but to do not explain the absent phenotype. Exome sequencing analysis first confirmed the F8 mutation and excluded other genetic causes of FVIII deficiency. Additionally, we excluded thrombophilic mutations proposed to attenuate bleeding severity in HA, including Factor V Leiden (c.1601G>A, p. R534Q) and the c.*97G>A (p.G20210A) mutation in the F2 gene encoding prothrombin. To identify additional variants that may modify hemostasis, we then screened for non-pathogenic variants in 96 genes that are known to be mutated in bleeding disorders or that alter FVIII clearance or half-life. Of 352 single nucleotide variants (SNVs) in these genes, we prioritized 10 non-synonymous variants with annotated effects on hemostasis. Of these, four are associated with functional effects that would potentiate bleeding phenotype in coagulation factor deficiency. In contrast, five variants are associated with prothrombotic effects and are strong candidates for ameliorating bleeding. Of these, two non-pathogenic VWF variants, rs1063856 and rs7962217, have been shown to increase FVIII. Three other variants in F5 reside within the R2 haplotype. R2 has effects in the absence of Factor V Leiden and can increase thrombin generation and FVIII levels. Conclusion: The identification of multiple variants that are expected to attenuate bleeding suggests that a single gene variant is unlikely to ameliorate bleeding phenotype in this subject, particularly in the presence of additional variants that promote bleeding. These results suggest evaluation of non-pathogenic variants in non-F8 genes may further explain other cases of discrepant HA not resolved by clinical assays. While these new modulators of HA phenotype require functional confirmation, they provide new avenues for therapeutic development. Disclosures Eyster: Bayer: Other: research support; Baxalta: Other: research support; Shire: Other: research support; NovoNordisk: Other: research support; SPARK: Other: research support.

The Dendritic Cell HLA-Class-II/Therapeutic Factor VIII (FVIII) Peptidome Is Influenced in Unanticipated Ways By the B-Domain of FVIII and the FVIII Chaperon Protein, Von Willebrand Factor: The Outrigger and Glycosylation-Umbrella (GUMB) Hypotheses

Background: Patients with the X-linked bleeding disorder hemophilia A have impaired blood clotting due to deficient or absent factor VIII (FVIII) coagulant activity. Bleeding can be managed by infusions of any of several plasma-derived (pd) or recombinant (r) therapeutic FVIII proteins (tFVIIIs). The efficacy of tFVIIIs can be eliminated, however, if neutralizing anti-tFVIII-antibodies called "inhibitors" develop. Since the development of inhibitors is T-helper-cell dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules comprise an important early determinant. Accumulating evidence suggests that the presence of the FVIII chaperon protein, von Willebrand factor (VWF), in either pdFVIII or rFVIII concentrates, decreases the immunogenicity of these tFVIIIs by reducing their uptake by antigen presenting cells, especially dendritic cells (DCs). Objectives: Use a native (i.e., non-engineered) full-length (FL) tFVIII without ((−)) or with ((+)) pdVWF in DC-protein processing and presentation assays (PPPAs) followed by mass-spectrometric and peptide-proteomic analyses to identify and quantify the DP-, DQ-, and DR-bound/tFVIII-derived-peptides in individual HLAcII repertoires. Compare the number of peptides in the subset from any of the five globular domains (A1, A2, A3, C1 and C2) or three acidic-residue-rich connecting segments (a1, a2 and a3), which we collectively refer to as the non-B-domain (NBD) portion of a tFVIII, with the number of peptides from its non-globular "outrigger like" B-domain (BD) that contains ~80% of the N-linked glycans of a FL-FVIII molecule despite containing only 908 amino acid residues, i.e. slightly less than 40% of its 2,332 total residues. Methods: DC-PPPAs were performed using monocyte-derived (Mo)DCs obtained from 12 healthy blood donors. The tFVIII tested was a FL-rFVIII (Advate®) used (−) or (+) pdVWF (i.e., FL-rFVIII − pdVWF and FL-rFVIII + pdVWF) and the resulting data consists of counts of tFVIII-derived peptides presented on and extracted from HLAcII molecules. Difference of proportion tests were used to compare the effect of pdVWF as well as the NBD- and BD-regions of the FL tFVIII on the peptide counts. Results: FL-rFVIII − pdVWF yielded significantly more peptides (p<0.05) than FL-rFVIII + pdVWF from the NBD portions but not the BD. Interestingly, for the FL-rFVIII − pdVWF preparation, the NBD portions yielded significantly more peptides (p<0.05) than the BD, but this pattern was reversed for the FL-rFVIII + pdVWF preparation in that the NBD portions yielded significantly less peptides (p<0.05) than the BD. Conclusions: The Outrigger Hypothesis posits that in the presence of VWF the heavily glycosylated BD acts as an "outrigger" and renders this portion of a FL tFVIII relatively more likely to be internalized, proteolytically processed and HLAcII-presented. However, in the absence of VWF, the N-linked glycans individually act to protect the amide bonds in the underlying peptide bond backbone of a tFVIII from proteolytic processing, as posited by the GUMB Hypothesis. Our results support both hypotheses as important determinants in the pathogenesis of inhibitor development. Disclosures Howard: Haplogenics Corporation: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Luu:Haplogenics Corporation: Employment. Hofmann:CSL Behring: Employment. Dinh:Haplogenics Corporation: Employment. Mead:CSL Behring: Employment. Powell:Haplogenics: Membership on an entity's Board of Directors or advisory committees. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees. Eugene:CSL Behring: Employment.

Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts

Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA.Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold.Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.

In silico and in vitro evaluation of the impact of mutations in non-severe haemophilia A patients on assay discrepancies.

Haemophilia A (HA) is caused by a lack or reduced amount of factor VIII protein (FVIII). About one-third of patients with non-severe HA carrying specific missense mutations show discrepant results between FVIII activity (FVIII:C), measured by one-stage or chromogenic two-stage assays. The aim of this study was to elucidate the mechanism underlying the assay discrepancy in vitro and in silico. Thirteen missense mutations in the Factor 8-gene associated with discrepant results in patients were transiently expressed. FVIII:C of the mutations was determined using two one-stage assays (FVIII:C1st, FVIII:CBonn) and a two-stage chromogenic assay (FVIII:Cchr). Furthermore, thrombin generation test (TGT) and in silico analysis were performed to investigate the haemostatic potential as well as the structural impact of the variants, respectively. For the majority (9/13) of the analysed mutations, the discrepancy was confirmed. Moreover, we established a modified TGT protocol for in vitro characterization of FVIII. Hence, TGT parameters were significantly impaired in the group of variants associated with higher chromogenic values. Additionally, in silico analysis revealed the impact of the mutations on FVIII protein structure leading to assay discrepancy. Moreover, the data shows that also among one-stage clotting assays, assay discrepancy is observed. Our results show that for the majority of mutations, application of a global assay like TGT method could help to improve diagnosis or correct assessment of the severity of HA.

Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs

Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIII gene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.

Single-molecule measurement and bioinformatics analysis suggest a preferred orientation of human coagulation factor VIII on hydrophobic interfaces

Investigating the adsorption behavior of coagulation proteins on interfaces will contribute to better understating blood clotting and to the development of biocompatible materials. In this work, atomic force microscopy (AFM)-based peakforce quantitative nanomechanical mapping (PF-QNM) was combined with bioinformatics tool to study the adsorption and orientation of coagulation factor VIII (FVIII) on both hydrophilic and hydrophobic interfaces by the height and mechanical measurement of single protein molecules. We found that interfacial hydrophilicity/hydrophobicity greatly influence the heights and Young's modulus of individual proteins. Compared to on the hydrophilic mica surface, FVIII proteins appear bigger vertical sizes while similar lateral sizes on the HOPG surface. The water accessible surface area analysis indicate stronger apolar properties C1 and C2 domains than others, suggesting a preferred orientation through the strong hydrophobic interactions between HOPG and the hydrophobic residues interface of the protein domains. These results provide novel insights on the adsorption and binding mechanism of the FVIII on cell membrane and will be helpful for the design of anticoagulant materials.

Reprogrammed CD4+ T Cells That Express FoxP3+ Control Inhibitory Antibody Formation in Hemophilia A Mice.

Coagulation Factor VIII (FVIII) replacement therapy in hemophilia A patients is complicated by the development of inhibitory antibodies, which often render the treatment ineffective. Previous studies demonstrated a strong correlation between induction of regulatory T cells (Treg) and tolerance to the therapeutic protein. We, therefore, set out to evaluate whether the adoptive transfer of FVIII-specific CD4+ Treg cells prevents inhibitor response to FVIII protein therapy. To this end, we first retrovirally transduced FoxP3+ into FVIII-specific CD4+ cells, which resulted in cells that stably express FoxP3, are phenotypically similar to peripherally induced Tregs and are antigen specific suppressors, as judged by in vitro assays. Upon transfer of the FVIII-specific CD4+ FoxP3+ cells into hemophilia A mice, development of inhibitory antibodies in response to administering FVIII protein was completely suppressed. Suppression was extended for 2 months, even after transferred cells were no longer detectable in the secondary lymphoid organs of recipient animals. Upon co-transfer of FoxP3+-transduced cells with the B cell depleting anti-CD20 into mice with pre-existing inhibitory antibodies to FVIII, the escalation of inhibitory antibody titers in response to subsequent FVIII protein therapy was dramatically reduced. We conclude that reprogramed FoxP3 expressing cells are capable of inducing the in vivo conversion of endogenous FVIII peripheral Tregs, which results in sustained suppression of FVIII inhibitors caused by replacement therapy in recipient hemophilia A animals.

Enhancing FVIII Expression By Modifying B-Domain Sequence for Hemophilia A Gene Therapy

Background: We have previously shown that conserving six N-linked glycosylation sites of the human factor VIII (FVIII) B-domain sequences in a synthetic linker, V3 (31aa/N6), increased FVIII expression by 3-fold over B-domain deleted FVIII (BDD-FVIII) following AAV mediated gene transfer in mice (McIntosh et. al. Blood 2013). These N-linked glycosylation consensus sequences (N-X-T/S) are highly conserved across different species but their role in the context of transgenic FVIII expression following gene transfer remains unknown. We, therefore, undertook systemic B-domain linker engineering studies to elucidate the mechanism by which the V3-linker improves FVIII expression following AAV mediated gene transfer.Methods: A series of FVIII variants were made by modifying the B-domain linker size and amino acid sequence and then cloned into AAV or lentiviral vectors. The latter were used for making stable FVIII producer cell lines to facilitate biochemical characterization including glycome profile using MALDI-TOF mass spectrometry. The AAV vectors, in which expression of the FVIII gene was directed by a small synthetic liver specific promoter (HLP) were used to assess transgene expression in-vitro and in murine models.Results: Purified recombinant FVIII-V3 had the same biological activity as that of full-length and BDD FVIII protein concentrates when assessed by thrombin generation and vWF/FVIII binding affinity assays (Dissociation constant (KD) <1nM) suggesting that V3 linker did not interfere with the potency of FVIII. Replacement of the asparagine residues with alanine (FVIII-V5) within the six N-linked glycosylation motifs resulted in a >20 reduction in glycan structures but the expression profile and biological properties of FVIII-V5 were comparable to those of FVIII-V3. In contrast, FVIII-V1 (44aa), in which the N-linked glycosylation triplets in FVIII-V3 were flanked by two additional amino acids that are normally adjacent to these moieties in the native FVIII B-domain, was expressed at 3-fold low levels when compared to FVIII-V3 and V5 in mice. Similarly, randomly scrambling the amino acid sequence of the V3-linker (FVIII-V2) without modifying size resulted in a 7-fold decrease in FVIII expression levels in mice. Notably, FVIII-V3 and V5 mRNA levels were 4-fold higher than the levels observed with BDD-FVIII and FVIII-V1 variants. Deletion analysis of the V3 B-domain linker identified a shorter form (FVIII-N2 19aa/N2) that exhibited similar levels of FVIII secretion to those observed with FVIII-V3.Conclusion: We have critically evaluated the role of the FVIII B domain V3 linker on human FVIII expression in human cell lines and mice following AAV mediated gene transfer. In all cases, the FVIII-V3 variant performed better than the BDD-FVIII, in large part due to an increase in FVIII-V3 mRNA levels. Although the FVIII-V3 molecule is fully glycosylated, our studies show that glycan content did not significantly influence FVIII expression but increasing size and modifying amino-acid content of the linker, markedly reduced FVIII expression following AAV mediated gene transfer in mice. Therefore, the results presented here support the use of FVIII-V3 for gene therapy for hemophilia A in our on-going investigator lead Phase 1 trial. DisclosuresMcIntosh:Freeline: Consultancy, Equity Ownership; BioMarin: Patents & Royalties. Dane:Freeeline: Employment, Equity Ownership. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties.

Optimized FVIII-Domain-Based Chimeric Antigen Receptors to Specifically Target FVIII Inhibitor-Producing B Cells in Hemophilia a

Hemophilia A (HA) is a X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). Optimal clinical management centers on FVIII protein concentrate replacement. However, up to 30% of patients with severe HA develop neutralizing antibodies to FVIII (inhibitors) upon exposure to therapeutic FVIII. Inhibitors neutralize the infused FVIII and, thus, pose a significant challenge in the management of these patients. Immune tolerance induction (ITI) using high-dose FVIII infusions can eliminate inhibitors but is not effective at generating long-term eradication in all patients. Thus, we developed an immunotherapy to more effectively eliminate FVIII-specific B cells to induce lasting eradication of inhibitors.Adoptive T cell immunotherapies using chimeric antigen receptor (CAR)-modified T cells (CARTs) are showing encouraging results in the treatment of cancer. We previously reported a proof-of-concept CART approach to eliminate autoantibodies to desmoglein-3, implicated in pemphigus vulgaris, by targeting non-malignant B cells. Here, using a similar strategy, we constructed two FVIII-based CARs using isolated FVIII A2 and C2 domains, which are commonly targeted by inhibitorsBoth FVIII fragments were used as the CAR extracellular domain followed by CD8a-derived hinge and transmembrane domains and intracellular signaling domains derived from 4-1BB and CD3z (BBz), as has been used in a recently FDA-approved CD19-targeted CAR (CART19) that have shown efficacy in patients. A2- and C2-CARs were introduced into primary human T cells using lentivirus vectors and were found to express on the cell surface. Primary expansion following anti-CD3/CD28 activation and lentivirus transduction yielded comparable population doublings of CART19 and A2-CARTs over 9-11 days (mean, 4.3 vs. 3.7; p=0.16). However, C2-CARTs consistently achieved fewer doublings compared to CART19 (mean 4.3 vs. 2.6; p=0.01). Additionally, C2-CARTs maintained larger cell volumes following initial activation compared to A2 and CART19, reminiscent of other CARs that demonstrate a high-level of ligand-independent basal activation. Consistent with hyper-activation and activation-induced cell death, flow cytometric analysis of C2-CART cultures showed prolonged expression of CD69, increased levels of cell death, and gradual loss of CAR+ cells compared to A2 and CART19 cultures. The FVIII C2 domain contains hydrophobic surfaces involved in binding to phospholipid membranes and von Willebrand Factor. We hypothesized that these hydrophobic regions may cause unfavorable interactions that result in CAR clustering and, in-turn, ligand-independent signaling. However, since these regions are also targeted by FVIII inhibitors, mutation of hydrophobic residues to improve CAR function would likely result in loss of binding to intended targets. Thus, we tested two independent strategies to improve function of C2-CARTs, both of which maintain native FVIII domain sequences. First, to block unfavorable interactions with C2 hydrophobic surfaces, we tested whether addition of C2 domain specific antibodies to the CART culture would improve expansion. Indeed, addition of anti-C2 antibodies improved C2-CART expansion by approximately one population doubling and reduced both cell volume and CD69 expression in C2-CARTs. As an alternate and perhaps more easily translatable approach, we replaced the BBz CAR signaling domain with a killer immunoglobulin receptor (KIR)-based multi-chain signaling system, which we previously utilized to improve function of a mesothelin-targeted CAR. KIR-based signaling effectively “rescued” the C2 CAR, and showed an expansion and activation profile comparable to CART19. Lastly, KIR-based FVIII CARs, demonstrated specific lysis of and cytokine response to target cells engineered to express surface anti-FVIII, even in the presence of soluble inhibitors.These data indicate that unfavorable interactions may be ameliorated directly by blocking agents or, indirectly, by altering intracellular signal transduction domains. These strategies have allowed us to construct optimized FVIII-based CARTs to target FVIII-specific B cells that are responsible for producing FVIII inhibitors in patients with HA. DisclosuresDoshi:Bayer Hemophilia Awards Program: Research Funding. Milone:Novartis: Patents & Royalties.

Elimination of factor VIII‐specific B cells by immunotoxins composed of a single factor VIII domain fused to Pseudomonas exotoxin A

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA exvivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested exvivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors.

P059 Analysis of HLAcII peptidomes presented by dendritic cells (DCs) from healthy donors and hemophilia-A (HA) patients with or without factor VIII (FVIII) inhibitors after ex vivo administration of different therapeutic FVIII proteins (tFVIIIs)

Aim T cells specific for tFVIIIs in HA patients can induce neutralizing FVIII antibodies called inhibitors. DCs that present tFVIII derived peptides in their HLAcII repertoire may activate T cells. We employed DC protein processing and presentation assays (DCAs) to quantify tFVIII peptides in HLAcII peptidomes and used these as measures of the immunogenic potential (IP) of tFVIIIs as a function of their associated HLAcII/tFVIII peptide density. Methods We used DCs from 28 healthy donors and 5 HA patients [2 inhibitor (Inh) positive and 3 Inh negative] in 3 DCAs. In DCA 1, DCs from healthy donors 1–12 were given an equimolar mix of 5 recombinant (r) FVIIIs. In DCA 2, DCs from healthy donors 13–24 were given a plasma derived (pd) FVIII + vWF or 1 of 2 rFVIIIs (BDT or FL) ± vWF. In DCA 3, DCs from healthy donors 25–28 and HA patients 1–5 were incubated with pdFVIII or FL, BDD, or BDT rFVIII, all + vWF (Fig. 1A). After DC lysis and DR, DQ and DP isolation, we identified tFVIII peptides by LC-MS/MS. We performed a log linear analysis of the DR bound tFVIII peptides to identify determinants of Inh risk (Fig. 1B-E) with random effects to control for potential donor- and DCA-clustering. Space limits prevent description of the DQ and DP peptides. Results The IP of tFVIIIs was greater in HA patients than in healthy donors and in Inh positive than in Inh negative patients (Fig. 1B-E). We found that BDT + vWF and FL ± vWF had lesser and greater IP, respectively, than pdFVIII + vWF; the IP of BDD + vWF was comparable to that of pdFVIII + vWF. The rFVIII mix had the greatest IP. The higher IP in HA patients versus healthy donors and in Inh positive vs Inh negative patients reflect significant correlations of 0.91 and 0.52 with the presence/absence of the FVIII Inh risk allele DRB1*15:01. Download : Download high-res image (400KB) Download : Download full-size image Conclusions DC protein processing and presentation is affected by HLAcII repertoires but not by HA patient or FVIII Inh status after accounting for correlation with DRB1*15:01. Relative to pdFVIII, rFVIIIs have different IP with BDT and FL being significantly less and greater, respectively. T.E. Howard: 4. Scientific/Medical Advisor; Company/Organization; Haplomics Biotechnology. M. Hofmann: 5. Employee; Company/Organization; CSL Behring. L.V. Dinh: 5. Employee; Company/Organization; Haplomics Biotechnology. J. Powell: 5. Employee; Company/Organization; CSL Behring. E. Maraskovsky: 5. Employee; Company/Organization; CSL Limited.

Neoangiogenesis of gastric submucosa-invasive adenocarcinoma.

Early gastric cancer may be defined as mucosal or submucosal invasive carcinoma, and exhibits a good prognosis: 90% of patients survive >10 years. Early gastric cancer infrequently exhibits lymph node metastasis, although submucosal invasion, the presence of vascular invasion and/or lymphatic permeation are independent risk factors for lymph node metastasis in early gastric cancer. The analysis of tumor lymphangiogenesis and angiogenesis are important to determine the extent of invasive progression and metastasis in patients. Previously, the presence of vessels expressing the D2-40 antibody and the factor-VIII protein has been identified immunohistochemically. The vessels that are immunoreactive for D2-40 and factor-VIII are morphologically similar to lymphatic vessels or small-size veins, also termed venules. In the present study, the association between tumor invasion and neoangiogenesis in early gastric cancer was examined. The D2-40/factor-VIII double-stained vessel (DSV) density was analyzed, in addition to lymphatic and blood vessel (vein and artery) density, using 46 submucosa-invasive and 50 mucosal carcinomas, and 20 non-neoplastic gastric tissues. The lymphatic density and DSV density of submucosa beneath the carcinoma and submucosa of the surrounding region in submucosa-invasive carcinoma were significantly increased (P<0.001) in comparison with those in mucosal carcinoma or non-neoplastic gastric tissue. No significant difference was observed in blood vessel density between non-neoplastic gastric, mucosal carcinoma and submucosa-invasive carcinoma tissues other than that of mucosa. The present study suggests the potential for the presence of D2-40/factor-VIII DSV and the importance of this vessel for neoangiogenesis in early gastric cancer.

Human Dendritic Cells Synergistically Activated by FVIII Plus LPS Induce Activation of Autologous CD4+ T Cells.

The most severe side effect in haemophilia A (HA) treatment is the development of anti-factor VIII antibodies, also called inhibitors. Why inhibitors develop in a proportion of treated HA patients and how this can be prevented remains largely unanswered. Among numerous theories, the presence of immunological danger signals, associated with events such as surgery or infection, has been proposed to play a role. In this study, we demonstrate that human dendritic cells (DC) synergistically activated by a combination of factor VIII (FVIII) concentrate plus the bacterial danger signal lipopolysaccharide (LPS) induce a significantly stronger activation of autologous CD4+ T cells than DC pretreated with FVIII or LPS alone. The observed T cell activation is dependent on antigen processing, presentation on MHC class II molecules and costimulation via CD86. Of note, FVIII plus LPS pretreated DC predominantly induce the activation of memory T cells and a minor proportion of naive T cells. Collectively, our data support a model in which immunological danger signals plus FVIII concentrates synergistically increase human CD4+ T cell responses to FVIII protein.

Molecular Characterization of Iranian Patients with Inherited Coagulation Factor VII Deficiency.

Coagulation factor VII (FVII) is a key enzyme of the extrinsic coagulation cascade that is predominantly produced by hepatocytes. The F7 gene mutations cause FVII deficiency with considerable molecular and phenotypic heterogeneity. We characterized the molecular alterations of the F7 gene and their corresponding mRNA transcripts in Iranian patients from eight unrelated families. The mutations were detected by polymerase chain reaction (PCR)-sequencing of all F7 gene exons, their flanking intronic sequences, as well as their corresponding cDNA fragments. Homozygous P303T, C91S and R304Q mutations were detected in patient 2, patient 5, and patient 6, respectively. Patient 7 was a compound heterozygote for S282R and H348R and patient 8 was a compound heterozygote for R304Q and IVS7+7A>G mutations. Furthermore, our investigation revealed three heterozygous individuals, patient 1 and patient 3 with the A244V mutation who were symptomatic and patient 4 with V(–39)I mutation who was also asymptomatic. The F7 mRNA expression analysis revealed that, except the transcript of V(–39)I, other mutation-harboring transcripts were expressed at detectable levels. In conclusion, this report reinforces the genetic and phenotypic heterogeneity of FVII deficiency. The findings of the mRNA study implied that decreased FVII protein activity subsequent to missense mutations does not completely reflect the degradation of mutation-harboring mRNA.