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Introduction: Antibodies are the primary effector molecules in the humoral immune system. To create a diversity of receptors-antibodies B-cells undergo V(D)J gene recombination and somatic hypermutation. This allows the recognition of most of pathogens but also sometimes of self-antigen, which can lead to autoimmune diseases. Autoantibodies (Abs) neutralizing and/or accelerating the clearance of ADAMTS13 are present in nearly all acquired thrombotic thrombocytopenic purpura (aTTP) patients with severe ADAMTS13 activity ( Methods: Specimens were analyzed from 8 aTTP patients who were splenectomized due to a refractory course of the autoimmune disease. Splenic mononuclear cells were sorted by flow-cytometry into naïve and transitional (CD27-, IgD+), unswitched memory (CD27+, IgD+) and switched memory (CD27+, IgD-) as well as (CD27+, CD20-/CD19-, CD38+) plasma cells. The frequencies of highly positive anti-ADAMTS13 B-cells in each B-cell population were calculated. Library consisting of antibody cDNA from each B-cell population was deep sequenced on MiSeq Ilumina. Prime analysis were performed in CLC Genomics Workbench. High quality sequences were submitted to IMGT/high V-QUEST web-based analysis tool to determine for V(D)J genes alignments, and V(D)J junction (antigen binding region) and mutations analysis. The IMGT output was parsed into MySQL database for further analysis. In addition, from 4 patients anti-ADAMTS13 specific IgG bearing cells were individually sorted. Gene transcripts of single cells were reverse-transcribed followed by nested PCR of IgG heavy/light chains. V(D)J pairings were visualized with Circos software. In MySQL we compared single cell antigen specific sequence with global repertoire. Results: Anti-ADAMTS13 specific B-cells were detected in the spleen of all patients (average 0.01% of total B-cell population). Deep sequencing of the total B-cell repertoire revealed ~3 million productive sequences, from which ~2 million were unique. Splenic anti-ADAMTS13 specific B-cells of four aTTP patients revealed 80 antibodies with unique V(D)J junction. Among those most frequently used V-genes were IGHV1-69 and IGHV3-30 (15% and 12% respectively) in global repertoire 1.5% of antibodies were encoded with IGHV1-69 gene within respective population. The average identity to germline was lower in ADMTST13 specific B-cells (92% compare to 96%). Four V(D)J junctions were convergent in at least 2 patients (identical amino acid sequence). Conclusion: Anti-ADAMTS13 specific B-cells were found in all aTTP patients in different B-cell populations. We observed enrichment of some variable gene segments when comparing the specific anti-ADAMTS13 to the total splenic repertoire (mainly IGHV1-69). Finding convergent V(D)J junction is very promising and also confirms previous finding of our group where similar V(D)J junction were found among two patients. Currently we clone selected single sorted monoclonal Abs (Schaller et al, Blood 2014;124(23):3469-79). Functional testing will allow the selection of the inhibitory Abs to be used as tools to develop anti-idiotypic specific therapies for aTTP patients. Disclosures No relevant conflicts of interest to declare.

Background. Autoantibodies (Abs) neutralizing and/or accelerating clearance of the von Willebrand factor-cleaving protease-ADAMTS13 are the major culprits in acquired Thrombotic Thrombocytopenic Purpura (aTTP). Despite the first-line treatment with daily plasma exchange (PEX), acute aTTP is still today associated with a mortality rate of ~20% leaving survivors at high risk of relapse. To improve patient care and/or treatment of aTTP we explored the capacity of anti-idiotypic small molecules to specifically bind and potentially neutralize pathogenic anti-ADAMTS13 autoantibodies thereby restoring plasma-derived ADAMTS13 activity in acquired TTP patients. Method: As selecting tools we used previously generated spleen-derived inhibitory human monoclonal anti-ADAMTS13 antibodies consisting of the 4 antigen-binding CDR3 motifs shared by the two aTTP patients (Schaller et al, Blood 2014;124(23):3469-79). Either 3 mAbs representative for CDR3 Motif-1 or 3 mAbs for CDR3 Motif-3 or 2 mAbs representing CDR3 Motif-4 were pooled at equimolar concentrations by ribosomal display to screen for binders in two combinatorial small protein libraries (DARPins from Molecular Partners, Schlieren, Switzerland) containing either two (N2C) or three (N3C) randomized ankyrin repeat modules, respectively From each of the 6 libraries single anti-idiotypic DARPins were cloned into a pMPAG6 expression vector and purified by His-tag affinity chromatography. In total 22 unique anti-idiotypic DARPins, 4 N2C-DARPins and 2 N3C-DARPins selected by Motif-1, 9 N2C-DARPins by Motif-3 and 7 N2C-DARPins by Motif-4 mAbs were identified. Inhibitor targeting Strategy: The neutralization potential towards plasma-derived anti-ADAMTS13 Abs, was tested by pre-incubating the DARPins prior measuring the residual activity by functional FRETS assay, in a cohort of 50 acute aTTP patients with pathological inhibitor titers >0.4 BU/ml (diagnosed at our Center 2006-2014). First, equimolar pools (1000 nM) of anti-idiotypic DARPins (Motif-1, Motif-3 or Motif-4) were tested in 11/50 patients, secondly the contribution of the single DARPins from the responsive pools was assessed in 18/50 patients to finally test the entire cohort with a combination of all neutralizing anti-idiotypic DARPins. Results: The pool of all Motif-1 anti-idiotypic DARPins (n=6) neutralized inhibitor titers below the pathological threshold in 9/11 (82 %) aTTP patients tested, whereas only a slight reduction of the inhibitor titers (in average 15%) was observed for Motif-3 and Motif-4 DARPin pools, revealing the Motif-1 DARPins as the most universal neutralizers. To test the contribution of each Motif-1 anti-idiotypic DARPin to the observed inhibitor neutralization, single DARPins were tested in 18/50 aTTP revealing a reduction of the inhibitor of 20-50% (N2C-19) or 20% (N3C-74) in 5/18 patients tested. In contrast 90% (45/50) aTTP patients incubated with the pool of all 6 Motif-1 DARPins caused inhibitor titers to drop either completely or below the pathological threshold. The inhibitor titer was reduced below 1BU/ml in 3/50, but was irresponsive in 2/50 patients. Conclusions: Our data clearly show that anti-idiotypic DARPins can potently and universally neutralize inhibitory anti-ADAMTS13 Abs of aTTP patients. However an effective neutralization, restoring ADAMTS13 activity above 10-15% in most patients was only achieved when pooling all Motif-1 DARPins. If a combination of Motif-3 and/or Motif-4 DARPins with Motif-1 DARPins can additionally increase neutralization capacity also in relapsing patients is currently under investigation. Disclosures No relevant conflicts of interest to declare.

Abstract 3299 Background. Thrombotic thrombocytopenic purpura (TTP) is a rare disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia with schistocytes on the peripheral blood smear and a variable degree of ischemic organ dysfunction due to microvascular thrombosis. In the case of acquired TTP, circulating autoantibodies inhibit ADAMTS13 activity and/or increase ADAMTS13 clearance. The first-line treatment of an acute TTP bout is plasma exchange (PEX) therapy with the replacement of fresh frozen plasma. Up to 30% of TTP patients show an exacerbation under PEX. After initial improvement, platelet counts drop again which is typically associated with a strong increase of ADAMTS13 inhibitor titers, and therefore the phenomenon is also referred to as inhibitor boosting. Interestingly it is apparently not seen in other autoimmune diseases treated with PEX, like acquired hemophilia A or myasthenia gravis. The aim of this study was therefore to investigate if inhibitor boosting in acquired TTP is a general or ADAMTS13 specific phenomenon. Methods. Plasma samples, obtained day-to-day during PEX therapy supplemented with steroids until remission, of 9 patients suffering from acute TTP due to severe acquired ADAMTS13 deficiency, were analyzed for the following laboratory parameters: (a) C-reactive protein (CRP) and total IgG, (b) ADAMTS13 activity, (c) anit-ADAMTS13 antibody titers by ELISA, (d) functional inhibitor titers by FRETS assay, and (e) anti-TG and anti-TPO antibody titers. Results. At initial presentation inhibitor titers ranged from 0.7 - >10 BU/ml (a positive functional inhibitor titer is defined as >0.4 BU/ml by FRETS). All patients initially improved when PEX was started, however, in 7/9 cases a drop in platelet count was noted on day 7 (n=5), 8 or 9 (each n=1), which was associated with a substantial increase in anti-ADAMTS13 antibody and functional inhibitor titer in all seven patients. CRP was increased (>30mg/L) in one patient only, during the three days before the raise in inhibitor titer and a drop in platelet count was noted, while total IgG remained stable in all 9 patients throughout PEX. At presentation none of the patients had a positive result for the thyroid antibodies, however, all 9 patients developed positive anti-TPO antibody titers during treatment and three patients tested positive for anti-TG antibodies at the same time. Anti-TPO antibody peaks occurred after the anti-ADAMTS13 antibody peak in all patients (range days 9–17), except the two patients where no raise in anti-ADAMTS13 antibody titers was observed (anti-TPO peak on day 3 in both). The two patients without inhibitor boosting required only five and six PEX sessions, respectively to get into remission, of the other seven patients two didn't overcome the inhibitor boosting and became refractory and were subsequently successfully treated by splenectomy or Rituximab, and one patient died of sepsis on day 11. Conclusions. Inhibitor boosting in acquired TTP due to antibody-mediated severe ADAMTS1313 deficiency treated with PEX is common (78%) and cannot be overcome by standard first line treatment in half of the patients. The fact that significant titers of other autoantibodies than anti-ADAMTS13 antibodies occur during the acute disease episode is striking and points to a more general dysregulation of the immune response in acquired TTP. Investigation of further autoantibodies, cytokines, etc. to explore the role of B-cells and T-cells in this phenomenon in more detail are underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 1431 Background. Thrombotic Thrombocytopenic Purpura (TTP) is a severe, life-threatening disease and results from an impaired regulation of the von Willebrand factor multimer size due to a deficiency of its cleaving protease ADAMTS13. In case of acquired TTP, inhibitory autoantibodies neutralize and/or accelerate ADAMTS13 clearance. To better understand the role of these autoantibodies in the pathogenesis of TTP and to develop more specific therapies, molecular analysis of the pathological relevant isotype IgG4 is necessary. Rituximab, a B-cell depleting anti-CD20 mAb is used to treat various autoimmune diseases with variable success. Increasing evidence points at the spleen, rather than the bone marrow as the major reservoir of memory B cells, which are thus accessible to Rituximab. The analysis of the humoral immune response of splenic B-cells towards ADAMTS13 in TTP patients treated with/without Rituximab offers a unique opportunity to address resistance to Rituximab. Methods. Two patients suffering from acute TTP with high titers of anti-ADAMTS13 antibodies were splenectomized because of frequent relapses, which occurred in one case even after several courses of Rituximab (patient B). Splenic mononuclear cells were isolated and used to clone the patient's entire IgG4 repertoire using phage display technology. Two IgGk-IgGλFab libraries of ∼2 × 1010 members each were constructed and selected against recombinant ADAMTS13 coated on ELISA plates. Additionally an existing naïve IgM Fab library constructed from cord blood RNA was screened identically. To analyze the memory repertoire of Rituximab-resistant and non-resistant anti-ADAMTS13 IgG, the subset of switched memory B cells (CD19, CD27 IgG) was isolated by magnetic beads (Miltenyi Biotec) from the splenic mononuclear cells of both patients prior to immortalization by Epstein-Barr virus (EBV) transformation to generate monoclonal antibodies. Results. Phage display. After 5 rounds of selection a 105-fold enrichment in eluted phages was observed. An ELISA screening after the fourth panning round showed 16/34 clones displayed a strong binding to ADAMTS13. Sequencing of DNA encoding the heavy chain variable region (IgVH) of strong binding clones revealed that the Fabs of patient A were most homologous to germline genes IgVH1-3*01 (7/11) and IgVH4-28*01 (4/11). The usage of these germline genes was also observed in patient B (1/5 and 2/5 clones IgVH1 and IgVH4, respectively), in addition 2/5 clones used IgVH1-69*01 germline gene, which had been previously observed in other acquired TTP patients. Six out of 14 analyzed anti-ADAMTS13 Fabs of the naïve IgM Fab library showed the same restricted gene usage (VH1-2, VH1-69 and VH4-28), whereas 4/14 clones were found to comprise heavy chains encoded by IgVH6-1*02, 2/14 were encoded by IgVH3-48*02 and 2/14 by IgVH3-24*04. The homology to the closest germline gene for all VH genes was in the range of 81.6–99%, with the lowest mutation rate found in cord blood anti-ADAMTS13 IgM Fabs (range 1–7.7%) followed by the anti-ADAMTS13 Fabs of patient B (after Rituximab; range 2.9–13.6%, average 10%) and finally patient A (range 8.3–18.6%, average 15%), the mutation rate being in the range of an antigen-driven immune response. EBV transformation. Screening of the supernatant of single clones of EBV transformed memory B-cells for the presence of anti-ADAMTS13 IgG antibodies was positive in 5/125 (4%) of clones of patient A and in 8/109 (7.3%) of patient B. Sequencing of IgVH encoding DNA of 3 single clones to date revealed gene usage of IgVH1-3*01 in 2 clones (one patient A and B) with a mutation rate of 15.4% and one (patient A) being homolog to IgVH3-30*022 with a lower mutation rate of 8.2%, in the upper limit of the naïve repertoire. Conclusions. VH gene usage of specific anti-ADAMTS13 B-cells, including Rituximab-resistant specific anti-ADAMTS13 B-cells is genetically restricted to VH1, VH3 and VH4. Rituximab may deplete circulating specific anti-ADAMTS13 B cells. However, newly differentiated specific anti-ADAMTS13 B cells characterized by a lower degree of somatic mutation may evolve from the memory cell pool giving rise to pathological anti-ADAMTS13 antibody. Functional characterization (inhibitory capacity, affinity and epitope mapping) of these anti-ADAMTS13 Fabs are underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 194 Background. Acquired Thrombotic thromobcytopenic purpura (TTP) is the result of autoantibodies neutralizing and-or accelerating clearance of ADAMTS13. We have previously isolated mononuclear cells of two spleens donated to our laboratory by patients (A and B) splenectomized for frequently relapsing TTP to generate human monoclonal anti-ADAMTS13 antibodies. Preliminary characterization of the patient's entire IgG4 repertoire using phage display technology and Epstein Barr virus (EBV) transformation of switched memory B cells (CD19+, CD27+, IgG+) showed that the memory repertoire of Rituximab-resistant (B) and non-resistant (A) anti-ADAMTS13 IgG used the same restricted VH germline genes, namely VH1-3 (55%), VH1-69 (17%), VH3-30 (7%) and 4–28 (21%). In contrast, peripheral blood-derived IgG1 anti-ADAMTS13 antibody repertoire from 2 acquired TTP patients, inhibiting ADAMTS13 activity by maximal 15–40% used VH1-69 germline gene segment only (Luken et al, JTH 2006 and Pos et al, JTH 2009). To evaluate if the generated spleen-derived monoclonal anti-ADAMTS13 antibodies are of pathological relevance we started with their functional characterization. Methods. Both patients (A and B) were suffering from acute TTP with high titers of anti-ADAMTS13-Ab and therefore needed to be splenectomized because of frequent relapses, which occurred even after several courses of Rituximab (B). Both patients have remained in remission since splenectomy. Previous screening of the IgGk-IgGλ IgG4-Fab phage-display libraries and EBV-transformed clones on recombinant ADAMTS13 by ELISA revealed high ADAMTS13 specificity for 16/34 and 13/234 antibodies, respectively. Functional characterization of the phage-display clones included (a) determination of their inhbitiory capacity by functional FRETS inhibitor assay, (b) assessment of their affinity performed by either fluid-phase competition ELISA and/or plasma surface resonance (Biacore), and (c) epitope mapping by dot blot analysis testing their recognition of recombinant full-length wildtype ADAMTS13 and a truncated ADAMTS13 variant. Results. Detailed sequence analysis of all human monoclonal anti-ADAMTS13 analyzed showed seven unique CDR3 signatures in the variable heavy region of all clones generated using both techniques (phage display and EBV-transformation), three of which were seen in both patients. These CDR3 signatures are different from signatures found in peripheral blood-derived antibodies (Luken et al, JTH 2006 and Pos et al, Blood 2010). Strikingly, the anti-ADAMTS13 antibodies of EBV-transformed memory B cells used λ light chain exclusively, whereas the phage-display-derived clones showed usage of either κ- (A: 6/11 and B: 3/5) or λ-light chain (A: 5/11 and B: 2/5). Anti-ADAMTS13 IgG in supernatants of EBV-immortalized switched memory B-cell clones were mainly of subclass IgG4 (A: 60%; B: 25%) and IgG1 (A: 40%; B: 75%) thus IgG1 being the prevailing subclass in patient B, who relapsed despite several courses of Rituximab. Functional characterization of the anti-ADAMTS13 IgG4 antibodies revealed that 15/16 phage display clones are strongly inhibitory, showing 0–100% (A) and 60–100% (B) inhibition at 200 nM and even 0–92% (A) and 0–16% (B) inhibition at 20 nM. This represents a 5–6 times increase of inhibitory capacity when compared to previouse findingy by others, in the range of 2×10−7-7×10−10 M (A) and 1×10−9-6×10−10M (B) as measured by Biacore or/and competition ELISA. Epitope mapping revealed that 3/16 clones recognized a novel epitope at the C-terminus of ADAMTS13, in contrast to the previously reported N-terminally located spacer domain. Conclusions. The identification of several spleen-derived anti-ADAMTS13 clones displaying highly similar CDR3 variable heavy chains, in 2 unrelated acquired TTP patients is striking and novel. Our data points at the discovery of unique CDR3 signatures that characterize the pathological relevant, inhibitory antibodies paving thus the road to define their triggering ADAMTS13 epitope(s), while providing insight into the mode of success of splenectomy in relapsing TTP patients. Further functional characterization of the anti-ADAMTS13 EBV-derived clones and confirmation of our data by analysis of the immunological memory from two additional spleens are underway. Disclosures: No relevant conflicts of interest to declare.