Inhibitory Spleen-Derived Anti-ADAMTS13 Antibodies Are Characterized by a Limited Number of Variable Heavy Chain CDR3 Signatures in Patients with Relapsing Acquired Thrombotic Thromobocytopenic Purpura

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.

Restricted Immunglobulin Gene Usage of Spleen-Derived Specific Anti-ADAMTS13 B-Cells In Relapsing TTP Treated with or without Rituximab.

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.