ALBERT

Description: Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. Patients are commonly treated by substitution of recombinant (rFVIII) or plasma derived (pdFVIII) FVIII preparations. However, a significant number of patients treated develop an antibody response (inhibitors) against substituted FVIII. Various Immune Tolerance Therapies (ITTs) are applied to overcome the FVIII specific immune response. We recently reported on a novel approach to eliminate the FVIII specific immune response by several rounds of rituximab (Mabthera®) infusions after several failed ITT approaches. In this study, phage displayed random peptide libraries were screened with sera from a patient before, under and after rituximab treatment. After several rounds of biopanning more than 100 phage clones specific for FVIII-inhibitors were identified and sequenced. One amino acid motif - EVPN - was most common in isolated clones. Computer based analysis identified a conformational epitope in the A2 domain of FVIII. The clones specifically inhibit binding of inhibitor to FVIII. Also sera from other inhibitor patients cross reacts with isolated phage clones. Inhibitor sera also bind to synthetic peptides, which sequences correspond to the peptide insert on isolated phage. Functional studies to target inhibitor specific B cells with isolated peptides are currently being performed. Specific ligands for FVIII inhibitors were identified. The epitope was mapped to the A2 domain. When inhibitor relapsed after Rituximab treatment, the same epitope was recognized. Small peptides representing the epitope on the phage (mimotopes) bound to inhibitor positive sera and even cross-reactivity with heterologous sera was observed. The inhibitor specific peptide ligands may serve as useful tools to develop novel approaches for inhibitor elimination therapies.

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: Hemophilia B is an X-linked bleeding disorder. Substitution of recombinant (rFIX) or plasma derived (pdFIX) FIX preparations are required to restore coagulation. A rare but severe complication is the development of FIX inhibitors that are often accompanied by allergic reactions. Immune tolerance therapies (ITTs) for inhibitor elimination are difficult and can induce further allergic reactions. ITTs for FIX inhibitors are less established and less successful than for FVIII inhibitors. In this study, three phage displayed random peptide libraries were screened by biopanning to isolate small peptides that mimic the epitope of FIX inhibitors. In the patient, the inhibitor was eliminated by ITT but reappeared again. Plasma samples before ITT and at reappearance of inhibitor were used to isolate inhibitor specific phage clones. Phage clones showing specific reactivity with inhibitor positive plasma compared to control plasma by ELISA were amplified and DNA was sequenced to determine the peptide sequence displayed on the phage. Before ITT, 31 isolated phage clones specific for FIX inhibitors have been sequenced so far. 10/31 contain a common motif. After ITT, 24 clones specific for patient plasma have been sequenced. 10/24 contain a different common motif. Synthetic peptides have been generated according to the phage displayed sequence to demonstrate binding of inhibitors and test for cross reactivity. The surface of the model of FIX was screened for these motifs with a novel software tool. Different conformational epitopes could be identified on the surface of the molecule before and after ITT. Small peptides (mimotopes) have been identified that mimic epitopes for FIX inhibitors in a hemophilia B patient. The sequences can be aligned to different conformational epitopes on FIX. It appears that the epitope has changed in the course of the ITT. The mimotopes can also be used to target inhibitor specific B cells for a novel approach in inhibitor elimination in hemophilia B.

Description: In acquired hemophilia polyclonal autoantibodies (inhibitors) to coagulation factor VIII are generated. Inhibitors functionally interfere with interaction of molecules involved in the coagulation cascade for example the interaction of FVIII and the von Willebrand factor. Inhibitors in acquired hemophilia have been reported to bind either to the A2 or to C2 domain of FVIII. Random peptide phage display libraries were screened with patient plasma to identify small peptides mimicking inhibitor epitopes. Peptide sequences were used to map the inhibitor epitope to the surface of FVIII. The phage libraries included libraries with linear or cyclic 7mer or 12mer inserts. Biopanning was performed including positive selections and also negative selections to deplete irrelevant binders. Phages bound to inhibitors were eluted by pH shift or by competition with FVIII. Single phage clones were tested for their specificity for inhibitor positive plasma by ELISA. For specific binders, the sequence of the inserts was identified by DNA sequencing. For one patient 33 phage clones were analysed from three libraries and 28 peptide sequences mimicking the inhibitor epitopemimotopes - were determined. The peptide sequences map to two conformational epitopes on the A2 and A1 domain of FVIII. Clusters of six and four amino acids are the proposed binding regions on the A2 and A1 domain respectively. The matching conformational motifs contain PPLRQ and PPLS. Synthetic peptides corresponding to the sequence displayed on phage were generated and binding of inhibitors to peptides was confirmed by different ELISA formats. Currently, functional studies with synthetic peptides are being performed to further analyse the epitopes. Additionally, more patient samples are being screened to analyse further epitopes and understand immuno dominant regions of FVIII in acquired haemophilia and compare them to the immune response in hemophilia A. The mimotopes isolated could be a basis for the development of ligand effector conjugates to target inhibitor specific B cells.

Description: Introduction: Acquired haemophilia (AcH) is associated with the development of polyclonal autoantibodies against FVIII, which affect directly the A2 or C2 domain of the FVIII molecule. Immunomodulatory therapy regimes to normalize FVIII levels and to eliminate the inhibitor are essential options in the treatment of patients (pts) with AcH. The aims of the present study were to investigate the response to Rituximab® and to localize the inhibitor epitopes on the FVIII domains. Patients and Methods: In 5 pts with AcH (2 females,3 males; age: 64–81 yrs) the inhibitor titers ranged from 9 to 156 BU and the FVIII activities from

Description: Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. In hemophilia A recombinant (rFVIII) or plasma derived (pdFVIII) preparations are substituted to restore coagulation activity. However, a major problem of current treatment in hemophilia A is the development of an antibody response (inhibitors) to FVIIII. Various Immune Tolerance Therapies (ITTs) are being applied to overcome the FVIII specific immune response. In this study, phage displayed random peptide libraries were screened with various patients’ plasma samples. We recently reported about the identification of a conformational epitope in the A2 subunit of FVIII. Phages as well as corresponding synthetic peptides specifically block inhibitor binding and crossreact with heterologous plasma. Peptide ligands specifically stained primary B cells (CD19+/CD27+ double positive cells) of the inhibitor patient, but not of a healthy control by FACS analysis. To increase the potential of small synthetic peptide ligands as tools for the development of novel inhibitor elimination strategies, the peptide was expressed in a multimeric form in an eukaryotic expression system. In multimeric form the peptide ligand is highly stable and reveals better inhibitor blocking capacities than the synthetic peptide we sowed before. The multimer now serves as basis for the construction of LECs (Ligand Effector Conjugates) for the specific targeting and destruction of inhibitor specific B cells.

Description: Hemophilia A is an X-chromosome linked bleeding disorder resulting from the absence or nonfunctional expression of coagulation factor VIII (FVIII). About 30% of severe hemophiliacs develop neutralizing antibodies (inhibitors) to FVIII upon treatment with exogenous factor preparations. Specificity of these antibodies is often restricted to functional determinants in the A2 and C2 domain. Phage displayed random peptide libraries were screened with various plasma samples of high titer inhibitor patients and inhibitor specific peptide ligands were selected. In silco mapping of consensus amino acid motifs among peptides revealed conformational epitopes in A2 and C2. Selected ligands partially restored FVIII activity. Equimolar combination of these ligands enhanced blocking of inhibitors in autologous and heterologous patients’ plasma. Peptide ligands were fused to the C-terminal multimerization domain of the C4bp alpha-chain and expressed as multimers in 293T cells. Peptide multimers revealed improved binding of anti-FVIII IgG and prolonged in vitro half-life in comparison to single synthetic peptides. Selected peptide ligands were combined in heteromultimers by co-transfection of respective vectors, resulting in molecules binding both A2- and C2- specific IgG and blocking up to 100% of antibody binding to FVIII. Those molecules could provide a basis for the generation of novel peptide-based therapeutic approaches.

Description: Crohn's disease (CD) and ulcerative colitis (UC) have a chronic-remittent course. Optimal management of inflammatory bowel diseases (IBD) relies on early intervention, treat-to-target strategies and a tight disease control. However, it is challenging to assess the risk of relapses in individual patients. We investigated blood-based biomarkers for the confirmation of disease remission in patients with IBD. We retrospectively analyzed samples of 40 IBD patients (30 UC, 10 CD) enrolled in a tight-control follow-up study. Half of the patients had a flare during follow up. Serum was analyzed for S100A12 as well as S100A8/A9 and for 50 further biomarkers in a bead-based multiplex assay. The concentrations of 9 cytokines/chemokines and S100A8/A9 significantly differed in IBD patients with unstable remission (before flares) when compared to IBD patients with stable remission. Although the number of patients was small, ROC curve analyses revealed a number of biomarkers (IL-1β, IL-1RA, IL-8, IL13, IL-15, IL-21, IL-25, IFN-β, CXCL9, CXCL10, CXCL11, Galectin-1, G-CSF and S100A8/A9) that were elevated in patients with later occurring relapses. While earlier studies on peripheral biomarkers in IBD are limited to only few analytes, our study using a broad screening approach identified serum biomarkers with the potential to indicate unstable disease control in IBD, which may help to steer individual therapies to maintain remission.