ALBERT

Description: Abstract 3172 Poster Board III-112 Hemophilia B is an X-linked bleeding disorder that requires regular substitution of FIX preparations to restore coagulation. A rare but severe complication is the development of FIX inhibitors affecting about 3% of hemophilia B patients. Possible allergic reactions and challenging but often unsuccessful immune tolerance protocols complicate the treatment of affected patients. In the present study, five samples from patients with inhibitors against FIX (including one acquired inhibitor) were screened against random peptide libraries displayed on phage. A biopanning protocol was established to isolate phagotopes specific for FIX inhibitors and thus small peptides that mimic the epitopes of the inhibitors. Binding was confirmed by ELISA and the peptide sequences were determined by translating the DNA sequence of the phage. In total 65 peptide sequences were determined. For all patients except for the patient suffering from acquired FIX inhibitors consensus motifs were identified. These motifs include the amino acids FTK(T), TSL(T) and LRQ. In order to compare the motifs to the surface of FIX for epitope identification a novel structural model of human FIX was generated. Using this model epitopes were identified on the surface of the protease domain of FIX. The residues comprising the conformational epitopes are found in the regions AA290 - 330 and 380 - 390. Small peptides have been identified that mimic the epitopes for FIX inhibitors in hemophilia B patients. These mimotopes mimic conformational epitopes on the surface of the protease domain on FIX. Identifying inhibitor epitopes and isolating molecules that can possibly interfere with inhibitor binding might help to understand and to develop novel approaches to treat this rare but life-threatening complication. Disclosures Königs: Octapharma AG: Research Funding; Baxter AG: Research Funding.

Description: Key Points The fVIII C1 domain contributes significantly to the immune response against fVIII in acquired and congenital hemophilia inhibitor patients. B-cell epitopes identified for monoclonal murine and human C1 inhibitors are recognized by antibodies present in patients with hemophilia.

Description: The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice. As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein. To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response. In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The development of neutralizing anti-FVIII antibodies (inhibitors) with reduced or absent activity of substituted factor VIII (FVIII) remains the most serious complication of hemophilia A therapy (Kempton, 2014). Frequent and high doses of FVIII with or without bypassing products can reestablish immune tolerance in 60-70% of patients. Polymorphism in immune response genes including IL-10 and TNFa were identified as risk factors for inhibitor development (Astermark, 2006). Cross-sectional studies showed different cytokine profiles in patients with hemophilia, especially in those with history of an inhibitor (Oliveira, 2013). In this study cytokine profiles were monitored longitudinally during immune tolerance induction (ITI). Methods 107 plasma samples from 18 patients were collected during the RES.I.S.T Experienced and Naive trial, which included patients with a poor prognosis for ITI success (Gringeri, 2007). We quantified 14 cytokines in each sample by using a Human High Sensitivity T-Cell Discovery Array 14-Plex (Eve Technologies Corp, Calgary, AB, Canada). ELISA based FVIII antibody assays were used for anti-FVIII IgG detection. FVIII inhibitor titers (Bethesda assay, BU) were measured and available for the analysis. The cut-off for a positive inhibitor was 〉0,6 BU mL-1. Bethesda titers (BUpos) between 0,6 -