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Introduction Hereditary thrombotic thrombocytopenic purpura (hTTP) is an ultra-rare thrombotic microangiopathy caused by autosomal recessively inherited severe ADAMTS13 deficiency. In respect to organ damage, we recently reported a heterogeneous clinical course with some patients having various degrees of organ damage while others are almost asymptomatic*. The long-term consequences of hTTP are still not fully known. Methods We analyzed the prevalence and development of renal disease in confirmed hTTP patients in the International Hereditary TTP Registry until July 2021. We studied the onset of kidney disease, the presence of additional co-morbidities and the possibility of a specific genotype-phenotype correlation. We classified different renal diseases of our hTTP patients, according to the ICD-10 code (version 2016) as described in Table 1. Results Thirty-five (21%) of 163 confirmed hTTP patients of the International TTP Registry with a mean age at enrolment of 38.2 years had kidney disease. 80% were Caucasian, 8.5% Asian and 11.5% indicated other ethnicities. According to the ICD-10 code classification, two patients had acute kidney disease (AKI, ICD code N17) not specified during acute TTP episodes. Among 18 patients with general CKD, we could identified three patients with chronic kidney disease (CKD, ICD code N18) stages 1-2; four with CKD stage 3, and four patient with CKD stage 5. In two patients, kidney disease was unspecified (ICD code N19). Five patients had diagnoses of certain specific renal diseases, such as acute glomerulonephritis (ICD code N00), unspecified nephritis syndrome (ICD code N05), recurrent and persistent hematuria (ICD code N02), and calculus of the kidney (ICD code N20) (Table 1). Seven patients developed end-stage renal disease requiring renal replacement therapy (ICD code Z99.2), which was temporarily needed in four additional patients (Z49.2). Two patients underwent kidney transplantation (ICD code Z94) (Table 1). The 163 hTTP patients experienced 625 acute disease episodes, of which 108 (17%) were associated with acute renal failure. 21/163 (13%) acute episodes were treated with hemodialysis. After experiencing several hTTP episodes with acute renal failure, 15 patients developed CKD at different stages. 26/35 (74%) hTTP patients with kidney disease presented also other co-morbidities: hypertension in 14 patients, 10 and 18 patients had suffered from transient ischemic attacks and/or strokes, and six patients had suffered from a myocardial infarction. We observed no apparent correlation between onset of CKD and presence of co-morbidities. Regular plasma prophylaxis was introduced in 26/35 hTTP patients with kidney disease; in the majority of them (64%) plasma treatment was started due to CKD or after CKD diagnosis. Of the 35 patients with renal disease, 15 were homozygous and 20 compound heterozygous ADAMTS13 mutation carriers. The most known ADAMTS13 mutation c.4143_4144dupA counts 33% of the whole hTTP patients in our database (54/163 patients). It was the most represented mutations in our cohort of hTTP patients with renal disease (60%, 21/35): 11 homozygous and 10 compound heterozygous carriers. Nevertheless, we did not observe a specific genotype-phenotype correlation. Conclusions Our analysis of the International Hereditary TTP Registry cohort (at present 163 patients) showed that the majority of patients had a diagnosis of hTTP before a diagnosis of kidney disease, especially CKD. We could not identify a correlation with the onset of additional co-morbidities or with the genotype; nevertheless we cannot exclude that it may be present. Considering that 43% (15/35) patients developed CKD after several acute episodes with acute renal failure, a closer and attentive care for those patients not yet developing CKD to avoid or delay the onset is highly recommended. Hence, to evaluate the contribution of acute hTTP episodes and of other co-morbidities to the development of kidney disease requires further in depth analyses. More frequent and long-term follow-up visits are needed to better understand the long-term outcome and development of kidney disease in hTTP patients. * Tarasco et al, Blood 2021 Figure 1 Figure 1. Disclosures Matsumoto: Sanofi: Consultancy; Takeda: Consultancy; Alexion Pharma: Consultancy; Asahi Kasei Pharma: Research Funding; Chugai Pharmaceutical: Research Funding; Alfesa Pharma: Patents & Royalties: ELISA for measuring ADAMTS13 activity. Lämmle: Takeda: Membership on an entity's Board of Directors or advisory committees; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Baxter: Other: Travel Support, Speakers Bureau; Alexion: Other: Travel Support, Speakers Bureau; Siemens: Other: Travel Support, Speakers Bureau; Bayer: Other: Travel Support, Speakers Bureau; Roche: Other: Travel Support, Speakers Bureau; Sanofi: Other: Travel Support, Speakers Bureau. Kremer Hovinga Strebel: Baxalta US Inc: Other: grant; Shire: Consultancy, Speakers Bureau; Ablynx: Consultancy, Speakers Bureau; Federal Office of Public Health: Consultancy; Insel Gruppe AG: Current Employment.

Introduction: Congenital thrombotic thrombocytopenic purpura (cTTP) is an autosomal recessive disease characterised by acute episodes of thrombotic microangiopathy. cTTP has a clinically heterogeneous course. Currently there are more than 150 disease-causing ADAMTS13 gene mutations reported, though a genotype-phenotype correlation of this ultra-rare disease remains incompletely understood. Characterization of mutations of the ADAMTS13 gene will be useful for carrier detection and genetic counselling. In 2006, the Hereditary TTP Registry (ClinicalTrials.gov NCT01257269; www.ttpregistry.net) was initiated in order to achieve this objective by identifying possible triggers of acute episodes of TTP and to document individual clinical courses and treatment requirements. Methods: The Hereditary TTP Registry is an international cohort study. Individual data were analysed from 121 cTTP patients, who were enrolled between 2006 and the end of 2017. Diagnosis of cTTP was confirmed by a severely deficient ADAMTS13 activity Results: The cTTP cohort consists of 54 and 52 patients from Europe and Asia (90% from Japan), respectively, 13 patients from the Americas and 2 patients from Africa. For 69% of the patients regular treatment was reported, which consisted predominantly of fresh frozen plasma infusion (84%). More compound heterozygous (n=76) than homozygous mutation carriers (n=45) were included. The female to male ratio was 1:1, and the median age at first diagnosis was 25.8 years (range from 0 to 81.3 years). Prior arterial thromboembolic events (mainly myocardial infarction, stroke, TIA) were present in 27% of patients at enrolment and at the age of 50 years more than half of the patients had experienced at least one such event. In the 121 patients, we identified 98 different ADAMTS13 mutations (missense 57; nonsense 12; frameshift due to deletions or insertions 21; splice site 8). The most frequent observed mutations were c.4143_4144dupA (exon 29; p.Glu1382Argfs*6; n=58 alleles), c.3178C>T (exon 24; p.Arg1060Trp; n=13), and c.577C>T (exon 6; p.Arg193Trp; n=11). Nineteen mutations have not been previously reported (8 missense, 3 splice site, 2 nonsense, and 6 frameshift mutations). Mutations are located across the whole ADAMTS13 gene and affect all domains (there was no "hotspot"). We did not find a correlation of the mutations with clinical or laboratory features, except for ADAMTS13 c.4143_4144dupA. For this mutation, the age at initial diagnosis was younger (median age 4.1 years, range 1.8 - 52.1) in compound heterozygous compared to homozygous patients (median 23.3 years, range 0.7 - 47.5,) (p=0.040). Patient numbers were equally distributed (p = 0.51); 16 patients were compound heterozygotes and 21 were homozygotes. All patients were of Scandinavian or Central European ancestry. For the other assessed clinical parameters, no significant difference between compound heterozygotes and homozygotes was observed. Conclusion: At present, the Hereditary TTP Registry is the largest cohort on cTTP, to our knowledge. The large number of different mutations as well as confounding factors, including multi-ethnic and geographical factors in our internationally compiled patient cohort make unravelling the genotype-phenotype correlation in cTTP challenging. Yet, for c.4143_4144dupA, we can conclude that homozygous patients with ADAMTS13 c.4143_4144dupA are clinically less severely affected than compound heterozygotes due to the later onset of the disease in homozygotes, whereas the clinical characteristics are similar between compound heterozygotes and homozygotes. Why homozygous carriers of c.4143_4144dupA, despite having no ADAMTS13 activity in plasma, may have a rather late onset of acute TTP remains to be further elucidated. Disclosures Friedman: Shire: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Knöbl:Ablynx: Consultancy, Other: Member of Advisory Board. Schneppenheim:CSL Behring: Consultancy; SHIRE: Consultancy. Kremer Hovinga Strebel:Ablynx: Other: Advisory Board; SHIRE: Other: Advisory Board, Research Funding.

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 2215 Introduction: Among survivors of TTP associated with severe ADAMTS13 deficiency, long-term outcomes other than relapse are not well documented. The recent report of persistent minor neurocognitive abnormalities attributed to subcortical microvascular thrombosis (Transfusion 2009;49:1092) suggested that other organs may also be affected by subclinical abnormalities. Therefore we documented renal function in patients in the Oklahoma TTP-HUS Registry who survived acute episodes of TTP associated with severe ADAMTS13 deficiency (activity Methods: ADAMTS13 activity has been measured in 299 of 321 (93%) patients, 1995–2010. Glomerular filtration rate (GFR) was calculated by the Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation (Ann Int Med 2009;150:604). Urine protein was measured by dipstick. Patient GFRs were compared to GFRs for the US population using data from the National Health and Nutrition Examination Survey (NHANES), 1998–2006, that had been analyzed in the CKD-EPI study. Results: 68 of 299 (23%) patients from the Oklahoma TTP-HUS Registry had severe ADAMTS13 deficiency. Fifty-five of the 68 (81%) patients with severe ADAMTS13 deficiency survived their initial episode of TTP. Among the 55 survivors, 48 (87%) have had ≥1 year of follow-up (median, 6.4 years; range, 1.0 – 14.9 years). The 7 patients whose follow-up was less than 1 year included 3 patients whose initial episode was within the past year; 2 with systemic lupus erythematosus who died within one year; 1 who relapsed and died at one year; 1 lost to follow-up. Sixteen (33%) of the 48 patients had 1–4 relapses. None of the 48 patients developed end stage renal disease and none developed severe chronic kidney disease (GFR Conclusion: Renal function of TTP patients with severe ADAMTS13 deficiency who had been followed for a median of 6.4 years following recovery from their initial episode was not different from the US population. 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 2519 TTP associated with acquired, ADAMTS13-deficiency and SLE are both autoimmune disorders that occur preferentially in young, black women and they have many similar clinical features. TTP may occur in patients previously diagnosed with SLE, or patients may develop SLE following recovery from TTP. In addition, TTP may be quite difficult to distinguish from SLE patients with severe hematologic manifestations. We compared the prevalence of SLE-associated autoantibodies in TTP patients to published population data using 95% confidence intervals (CI). The Oklahoma TTP Registry enrolled 292 consecutive patients with their first episode of clinically diagnosed TTP from 11–13-1995 (date of our initial ADAMTS13 measurement) to 7–31-2009; ADAMTS13 activity was measured in 271 (93%) patients; 64 (24%) patients had ADAMTS13 activity Autoantibody TTP (95% CI) Population % ANA ≥1:40 89% (78%–95%) 0–27% ≥1:120 56% (42%–68%) 0% Anti-dsDNA ≥1:30 43% (30%–56%) 3% Anti-Ro OD>0.350 17% (8%–29%) 3% aPL IgM ≥20 PL units 15% (7%–26%) 2% Because of the increased prevalence of SLE-associated autoantibodies, we evaluated our TTP patients for the America College of Rheumatology (ACR) criteria for SLE (presence of ≥4 of 11 criteria suggests the diagnosis of lupus); abnormalities associated with any TTP episode were not counted in this evaluation of clinical criteria for SLE. By definition ACR criteria can be fulfilled serially or simultaneously over a lifetime. Evaluations were completed between 6-1-2007 and 5-1-2009 on 38/42 (90%) eligible patients (alive, non-institutionalized, no previous SLE diagnosis) consisting of physical examination, review of available lifetime medical records, and serial laboratory evaluations. Patients have been followed for a median of 8.3 years (range, 1–14 years). During this time, 3 (8%) developed clinically evident SLE requiring treatment 1, 5, and 70 months after their initial TTP episodes. Among the other 35 patients, 3 (8%) have ≥4 SLE classification criteria by medical record review (1 had pre-existing Sjögren's syndrome and receives treatment; 2 have minimal clinical features and are not actively treated for SLE); 9 (24%) have 3 criteria; 16 (42%) have 2 criteria; 6 (16%) have 1 criterion; and 1 (2%) patient has no ACR criteria for SLE. All patients continue to be followed and clinically evaluated for potential intervention. SLE diagnosis is a clinical designation and because of the lack of disease modifying drugs, routine follow-up is standard of care unless the patient is symptomatic. Conclusions: [1] A high prevalence of SLE-associated autoantibodies was present in a cohort of consecutive patients with TTP associated with acquired severe ADAMTS13 deficiency. [2] The presence of anti-dsDNA, anti-Ro, aPL and high titers of ANA suggest that patients with ADAMTS13-deficient TTP may be at risk for developing SLE. [3] During long-term follow-up, 6 (16%) of 38 patients have developed overt SLE or ACR criteria without an established diagnosis of SLE. Careful continuing evaluation following recovery from TTP is important. Disclosures: No relevant conflicts of interest to declare.

Abstract 4654 Background: Hereditary TTP, also known as Upshaw-Schulman syndrome (USS), is a rare disorder and the result of recessively inherited ADAMTS13 gene (located on chromosome 9q34) mutations. The clinical presentation is variable and may vary from mild isolated thrombocytopenia to recurrent severe TTP episodes leading to organ damage or even death. The first acute TTP episode may occur during the neonatal period up to older age. The same variety applies to the treatment requirements as some patients need regular plasma infusion every two to three weeks to prevent recurrent episodes while others only need plasma therapy in situations of increased risk, such as pregnancy or during infections. Due to the rareness of USS evidence based guidelines are lacking as are knowledge of long-term outcome which emphasizes the need of a multicenter cooperation. Aim: We have established a long-term observational study with an electronic database system for hereditary TTP patients (www.ttpregistry.net, ClinicalTrials.gov NCT01257269) to gather as much information as possible about the clinical courses and laboratory investigations performed. We aim at the identification of triggers that set about acute TTP bouts and of factors influencing the clinical course with the goal to possibly elucidated the underlying causes of the variable clinical presentation of this rare monogenic disorder eventually leading to optimization of therapy and evidence based recommendations. The study is open to any patient diagnosed with hereditary TTP and his/her interested family members. Eligibility criteria are as follows: • ADAMTS13 activity ≤10% on two separate occasions at least 1 month apart; and • Absence of a functional ADAMTS13 inhibitor and • ≥2 ADAMTS13 gene mutations and/or full recovery and normal half-life of infused plasma ADAMTS13); or • Being a family members of a confirmed patient Clinical information and laboratory investigations are collected retrospectively up to enrollment as well as prospectively every 12 months following enrollment. Analysis of ADAMTS13 related parameters including molecular analysis of ADAMTS13 gene are offered free of charge to patients and all family members. Conclusion: Our long-term goal is to establish an international network and knowledge platform to exchange information and experience on USS, that helps to improve diagnosis, treatment and prevention of acute episodes with risk of permanent organ damage for affected patients. Physicians treating USS patients are invited to contact us for diagnostics of suspected patients or enroll their confirmed patients. Disclosures: Fujimura: Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. George:Baxter, Inc.: Consultancy; Alexion, Inc.: Consultancy; Amgen, Inc.: Consultancy, PI for clinical trial involving romiplostim, PI for clinical trial involving romiplostim Other, Research Funding. Kremer Hovinga Strebel:Baxter: Consultancy, Research Funding.

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.