The online version of this article has a Supplementary Appendix. BackgroundThe majority of patients diagnosed with thrombotic thrombocytopenic purpura have autoantibodies directed towards the spacer domain of ADAMTS13. Design and MethodsIn this study we explored the epitope specificity and immunoglobulin class and immunoglobulin G subclass distribution of anti-ADAMTS13 antibodies. The epitope specificity of antispacer domain antibodies was examined using plasma from 48 patients with acute acquired thrombotic thrombocytopenic purpura by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Using similar methods, we also determined the presence of anti-TSP2-8 and CUB1-2 domain antibodies in this cohort of patients.
SummaryThe eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so-called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti-FVIII antibodies during ITI. FVIII-specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti-FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti-FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low-titre inhibitor patients, anti-FVIII antibodies consisted primarily of subclass IgG1 whereas, anti-FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti-FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti-FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.
To cite this article: Luken BM, Kaijen PHP, Turenhout EAM, Kremer Hovinga JA, van Mourik JA, Fijnheer R, Voorberg J. Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura. J Thromb Haemost 2006; 4: 2355-64.See also Knö bl P. Unraveling the immunologic response in thrombotic thrombocytopenic purpura. This issue, pp 2352-4.Summary. Background: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). Objective: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. Methods: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. Results: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. Conclusion: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.
We have cloned a human Hevin cDNA from omental adipose tissue of different patients by reverse transcription polymerase chain reaction and shown a sequence variation due to a possible polymorphism at amino acid position 161 (E/G). Hevin protein expressed in vitro showed molecular weights of approximately 75 kDa and 150 kDa, suggesting that Hevin may form a homodimer in vitro. Using Northern blots and a human expressed sequence tAg database analysis, Hevin was shown to be widely expressed in human normal or non-neoplastic diseased tissues with various levels. In contrast to this, its expression was strongly down-regulated in most neoplastic cells or tissues tested. However, neither the mechanism nor the physiological meaning of this down-regulation is known. As an initial step towards investigating the functional role of Hevin in cell growth and differentiation, we transiently or stably expressed this gene in cancer cells (HeLa 3S) that are devoid of endogenous Hevin and measured DNA synthesis (cell proliferation) by 5-bromo-2′-deoxyuridine incorporation. Hevin was shown to be a negative regulator of cell proliferation. Furthermore, we have shown that Hevin can inhibit progression of cells from G1 to S phase or prolong G1 phase. This is the first report which describes the function of Hevin in cell growth and proliferation. Through database analysis, Hevin was found to be located on chromosome 4 which contains loss of heterozygosity of many tumour suppressor genes. Taken together, these results suggest that Hevin may be a candidate for a tumour suppressor gene and a potential target for cancer diagnosis/therapy. © 2000 Cancer Research Campaign
Development of neutralizing IntroductionOver the past decades protein therapeutics such as hormones, enzymes, blood coagulation factors, or Abs have provided effective treatment for numerous diseases. 1 Treatment commonly requires frequent high-dose administration of protein therapeutics and, although generally considered safe, they often induce immune responses. 2 The factors that underlie immunogenicity of biomedical products can be related to the structure of protein, such as the presence of promiscuous T-cell epitopes 3 or posttranslational modifications, 4 but also to the formulation of the biomolecule. 5 Treatment-related parameters such as dosage, frequency, route of administration, and concomitant infections may also contribute to the induction of antidrug immune responses. 2 In patients with protein deficiencies administered therapeutics may be recognized by the immune system as non-self, thereby greatly increasing the risk of Ab development. 2 Hemophilia A is an X-linked bleeding disorder that is caused by a deficiency in blood coagulation factor VIII (FVIII). Conventional treatment comprising frequent administration of FVIII often results in formation of neutralizing Abs, which inhibit FVIII activity. 6,7 Both treatment-related factors, such as intensive treatment episodes, 8 and genetic risk factors can contribute to the development of inhibitors. Polymorphic sites in genes involved in the adaptive immune response have been associated with anti-FVIII Ab formation. 9-11 Development of high-affinity IgG Abs directed against FVIII is a CD4 ϩ T cell-dependent process. 12,13 Endocytosis of FVIII by professional APCs comprises the initial step leading to activation of helper T cells. Uptake and transfer of Ags through the lyso-endosomal pathway results in intracellular processing and presentation of FVIII-derived peptides on MHC II molecules to CD4 ϩ helper T cells. 14 Here, we hypothesized that prevention of FVIII uptake by APCs will lead to diminished T-and B-cell responses. Previously, we have shown that endocytosis of FVIII by APCs is mediated via its C1 domain, because administration of a mAb directed toward an antigenic surface in the C1 domain reduced inhibitor titers in FVIII-deficient mice. 15 With the use of an Ab-guided mutagenesis strategy we designed a C1 domain variant of FVIII which displayed a strongly reduced internalization by APCs. In vivo studies revealed that this C1 domain variant showed decreased immunogenicity in a murine model for inhibitor development in hemophilia A. Our findings provide a novel paradigm for the reduction of the intrinsic immunogenicity of FVIII by modulating its uptake by APCs. Methods MaterialsFicoll-Paque Plus (GE Healthcare), CD14 microbeads (Miltenyi Biotech), and human recombinant GM-CSF and IL-4 (both Cellgenix Technology Transfer) were used for generation of human monocyte-derived dendritic cells (MDDCs); M-CSF (PeproTech) was used to generate human monocytederived macrophages (MDM⌽s). For culturing murine BM-derived DCs (BMDCs), mouse recombinant GM-CSF ...
Key Points• ADAMTS13 derived peptides presented on HLA-DR; implications for acquired TTP.• CUB2 domain peptide binds to risk-allele HLA-DRB1*11.Autoantibodies directed against ADAMTS13 prohibit the processing of von Willebrand factor multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura (TTP). Recently, HLA-DRB1*11 has been identified as a risk factor for the development of acquired TTP. Here, we identified ADAMTS13-derived peptides presented on MHC class II alleles from 17 healthy donors. Dendritic cells from a panel of both HLA-DRB1*11-positive and -negative donors were pulsed with ADAMTS13, and the HLA-DR-presented peptide repertoire was analyzed by mass spectrometry. Interestingly, at low antigen concentrations, HLA-DRB1*11-or DRB1*03-positive donors presented a limited number of CUB2-derived peptides. Pulsing of dendritic cells using higher concentrations of ADAMTS13 resulted in the presentation of larger numbers of ADAMTS13-derived peptides by both HLA-DRB1*11-positive and -negative donors. Although the presented peptides were derived from several ADAMTS13 domains, inspection of the peptide profiles revealed that CUB2 domainderived peptides were presented with a higher efficiency when compared with other peptides. Remarkably, dendritic cells from DRB1*11 donors pulsed with higher concentrations of ADAMTS13-present derivatives of a single CUB2-derived peptide. We hypothesize that functional presentation of CUB2-derived peptides on HLA-DRB1*11 contributes to the onset of acquired TTP by stimulating low-affinity, self-reactive CD41 T cells. (Blood. 2013;121(17):3502-3510) Introduction Acquired thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening disorder characterized by the production of autoantibodies directed toward ADAMTS13, a plasma metalloprotease responsible for the cleavage of ultra-large von Willebrand factor (UL-VWF) multimers.1-3 Deficiency of ADAMTS13 causes the accumulation of UL-VWF strings on the surface of endothelial cells of the vessel wall and in the circulation. 4 Prolonged exposure of UL-VWF strings on the surface of endothelial cells promotes platelet adhesion, leading to low platelet counts and microvascular thrombosis. 5,6 The majority of the inhibitory anti-ADAMTS13 antibodies that develop in patients affected by acquired TTP targets a single epitope composed of Arg568, Phe592, Arg660, Tyr661, and Tyr665 on the outer surface of the spacer domain. This exposed region is crucial for binding of ADAMTS13 to unfolded VWF A2 domain.7-9 Antibodies targeting the CUB 1-2 and TSP2-8 regions of ADAMTS13 have also been detected in the plasma of several patients with acquired TTP. 10,11 The pathogenic role of anti-TSP2-8 and anti-CUB1-2 is still unclear. The carboxy-terminal ADAMTS13 TSP2-8 and CUB 1-2 regions not only modulate processing of VWF under flow 12-14 but are also necessary for the binding of ADAMTS13 to endothelial cells. 15A number of studies have shown that anti-ADAMTS13 antibodies detected in the plasma ...
Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domain.
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