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Thrombotic thrombocytopenic purpura: from patients literacy to immunoprofiling

Book - Dissertation

Thrombotic thrombocytopenic purpura (TTP) is a form of thrombotic microangiopathy characterized by a severe deficiency of ADAMTS13 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, member 13). This deficiency can be due to mutations in the gene encoding ADAMTS13 (Congenital TTP; cTTP) or to the presence of autoantibodies targeting ADAMTS13 (immune-mediated TTP; iTTP). The main function of the enzyme ADAMTS13 is the cleavage of ultra-large von Willebrand factor (UL-VWF) multimers into small multimers that are less prothrombotic. This process prevents the spontaneous interaction between UL-VWF and platelets. When ADAMTS13 is absent, UL-VWF multimers accumulate forming disseminated platelet-rich microthrombi in the circulation that block arterioles and capillaries. This systemic microvascular thrombosis can result in organ failure and can be life-threatening with a 90% mortality rate if left untreated. Prompt diagnosis and subsequent treatment decrease the mortality rate to 20%. However, patients who survive an acute episode are at risk of recurrent acute episodes. Therefore, clinicians and researchers are trying to gain more insight into the pathophysiology of the disease, so that these findings could lead to the development of novel strategies to prevent, diagnose and treat TTP. TTP is a rare disease in which the clinical suspicion of the disease is confirmed by an ADAMTS13 activity lower than 10%. The rarity of TTP, together with the inherent delays in obtaining the ADAMTS13 activity test results, leads to an initial misdiagnosis in 20% of the TTP patients. This misdiagnosis of TTP by health care providers suggests that disease awareness needs to be improved, not only for healthcare professionals but also for TTP patients. Well-informed patients get more out of their health care experience and as a result, have less disease complications, morbidities and mortality. For instance, in TTP, the education material provided by patient associations has shown to have beneficial effects on the management of the patients. However, currently it is not clear how much patients understand about TTP and which factors can affect their comprehension. Therefore, we analyzed the TTP literacy in TTP patients and investigated which factors are associated with a reduced disease understanding (AIM I). Through a survey, it became evident that patients already have substantial knowledge about the disease, but there were still significant knowledge gaps. These gaps were related to the origin of the disease, the importance of treatment, predisposing factors for TTP development and actions to take in an emergency setting. In addition, age and education level were found to influence disease comprehension. The continuous development of methodologies and the availability of biological reagents are used to expand the understanding of ADAMTS13 and TTP. For instance, studies using ADAMTS13 fragments or variants in immunoassays such as immunoprecipitation, ELISA and Western blot, showed that patients with iTTP exhibit a polyclonal immune response with antibodies mainly targeting the spacer domain. Small cohort studies in acute iTTP have shown that specifically residues Arg568, Phe592, Arg660, Tyr661, and Tyr665 (R568F592R660Y661Y665) are the main binding sites of anti-spacer autoantibodies. Therefore, active ADAMTS13 variants with reduced binding to anti-ADAMTS13 autoantibodies have been developed. However, other amino acid regions in the spacer domain are also targeted by anti-spacer autoantibodies. Hence, large cohort studies are required to gain more insight into immunogenic regions of the spacer domain, contributing to the identification of patients that could benefit from therapies using ADAMTS13 spacer variants. These studies would also allow to investigate the possible link between anti-spacer autoantibodies profiles and disease severity. Consequently, we developed and validated an ELISA that uses ADAMTS13/ADAMTS1 hybrids, in which regions from the spacer domain of ADAMTS13 were exchanged for the corresponding region in ADAMTS1, to epitope map anti-spacer autoantibodies in a large cohort of TTP patients in the acute and remission phase (AIM II). We found that amino acid regions 588-592, 602-610 and 657-666 are hotspots for anti-spacer autoantibody binding. However, dependent on the patient, also other regions in and outside the spacer domain were involved in anti-ADAMTS13 autoantibody binding. Additionally, we did not find major changes in the anti-spacer autoantibody profile during disease course. Finally, disease severity was not linked with certain anti-spacer autoantibody profiles. Antibodies have been essential tools to investigate the pathophysiology of TTP in vitro and to develop animal models for TTP. Our Laboratory is specialized in developing monoclonal anti-human ADAMTS13 antibodies that have led to novel insights in ADAMTS13 structure, the ADAMTS13 mode-of-action and the ADAMTS13 antigen levels in patients. Nevertheless, the high specificity of monoclonal antibodies for a particular antigen limits its use in immunoassays, because they are susceptible to changes in the epitope(s) in the antigen. In contrast, polyclonal antibodies can recognize different epitopes on the same antigen; hence, polyclonal antibodies are less susceptible to antigen changes. Thus, in the current study, we developed a polyclonal anti-human ADAMTS13 antibody with high specificity and sensitivity and validated its use in immunoassays such as ELISA, Western blot and immunocytochemistry (AIM III). Finally, animal models in TTP are used to unravel the pathophysiologic mechanism underlying TTP and to test novel therapeutics. Therefore, reagents cross-reacting, in particular with murine ADAMTS13, have been developed. In the current study, an antigen-based purification of a previously developed polyclonal anti-murine ADAMTS13 antibody was performed (AIM III). The process increased the purity of the antibody and enriched the specific anti-murine ADAMTS13 antibodies. This PhD investigated the TTP literacy in patients and revealed that age and education level are associated with low disease comprehension. Apparently, not all patients are aware about the origin of the disease, the importance of treatment, the predisposing factors evoking an acute TTP bout and which actions have to be taken in an emergency setting. Additionally, an ELISA to epitope map anti-spacer autoantibodies in TTP patients was created showing that the spacer domain contains three hotspot regions for autoantibody binding. The findings provided in this study might help in designing novel therapies aiming to provide active ADAMTS13 that escapes the immune response. Additionally, we showed that the immune response against the spacer domain did not change in the course of the disease. Finally, a polyclonal anti-human ADAMTS13 antibody was developed, while an antigen-based affinity purification of the polyclonal anti-murine ADAMTS13 antibody enriched the specific anti-murine ADAMTS13 antibodies. Thrombotic thrombocytopenic purpura (TTP) is a form of thrombotic microangiopathy characterized by a severe deficiency of ADAMTS13 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, member 13). This deficiency can be due to mutations in the gene encoding ADAMTS13 (Congenital TTP; cTTP) or to the presence of autoantibodies targeting ADAMTS13 (immune-mediated TTP; iTTP). The main function of the enzyme ADAMTS13 is the cleavage of ultra-large von Willebrand factor (UL-VWF) multimers into small multimers that are less prothrombotic. This process prevents the spontaneous interaction between UL-VWF and platelets. When ADAMTS13 is absent, UL-VWF multimers accumulate forming disseminated platelet-rich microthrombi in the circulation that block arterioles and capillaries. This systemic microvascular thrombosis can result in organ failure and can be life-threatening with a 90% mortality rate if left untreated. Prompt diagnosis and subsequent treatment decrease the mortality rate to 20%. However, patients who survive an acute episode are at risk of recurrent acute episodes. Therefore, clinicians and researchers are trying to gain more insight into the pathophysiology of the disease, so that these findings could lead to the development of novel strategies to prevent, diagnose and treat TTP. TTP is a rare disease in which the clinical suspicion of the disease is confirmed by an ADAMTS13 activity lower than 10%. The rarity of TTP, together with the inherent delays in obtaining the ADAMTS13 activity test results, leads to an initial misdiagnosis in 20% of the TTP patients. This misdiagnosis of TTP by health care providers suggests that disease awareness needs to be improved, not only for healthcare professionals but also for TTP patients. Well-informed patients get more out of their health care experience and as a result, have less disease complications, morbidities and mortality. For instance, in TTP, the education material provided by patient associations has shown to have beneficial effects on the management of the patients. However, currently it is not clear how much patients understand about TTP and which factors can affect their comprehension. Therefore, we analyzed the TTP literacy in TTP patients and investigated which factors are associated with a reduced disease understanding (AIM I). Through a survey, it became evident that patients already have substantial knowledge about the disease, but there were still significant knowledge gaps. These gaps were related to the origin of the disease, the importance of treatment, predisposing factors for TTP development and actions to take in an emergency setting. In addition, age and education level were found to influence disease comprehension. The continuous development of methodologies and the availability of biological reagents are used to expand the understanding of ADAMTS13 and TTP. For instance, studies using ADAMTS13 fragments or variants in immunoassays such as immunoprecipitation, ELISA and Western blot, showed that patients with iTTP exhibit a polyclonal immune response with antibodies mainly targeting the spacer domain. Small cohort studies in acute iTTP have shown that specifically residues Arg568, Phe592, Arg660, Tyr661, and Tyr665 (R568F592R660Y661Y665) are the main binding sites of anti-spacer autoantibodies. Therefore, active ADAMTS13 variants with reduced binding to anti-ADAMTS13 autoantibodies have been developed. However, other amino acid regions in the spacer domain are also targeted by anti-spacer autoantibodies. Hence, large cohort studies are required to gain more insight into immunogenic regions of the spacer domain, contributing to the identification of patients that could benefit from therapies using ADAMTS13 spacer variants. These studies would also allow to investigate the possible link between anti-spacer autoantibodies profiles and disease severity. Consequently, we developed and validated an ELISA that uses ADAMTS13/ADAMTS1 hybrids, in which regions from the spacer domain of ADAMTS13 were exchanged for the corresponding region in ADAMTS1, to epitope map anti-spacer autoantibodies in a large cohort of TTP patients in the acute and remission phase (AIM II). We found that amino acid regions 588-592, 602-610 and 657-666 are hotspots for anti-spacer autoantibody binding. However, dependent on the patient, also other regions in and outside the spacer domain were involved in anti-ADAMTS13 autoantibody binding. Additionally, we did not find major changes in the anti-spacer autoantibody profile during disease course. Finally, disease severity was not linked with certain anti-spacer autoantibody profiles. Antibodies have been essential tools to investigate the pathophysiology of TTP in vitro and to develop animal models for TTP. Our Laboratory is specialized in developing monoclonal anti-human ADAMTS13 antibodies that have led to novel insights in ADAMTS13 structure, the ADAMTS13 mode-of-action and the ADAMTS13 antigen levels in patients. Nevertheless, the high specificity of monoclonal antibodies for a particular antigen limits its use in immunoassays, because they are susceptible to changes in the epitope(s) in the antigen. In contrast, polyclonal antibodies can recognize different epitopes on the same antigen; hence, polyclonal antibodies are less susceptible to antigen changes. Thus, in the current study, we developed a polyclonal anti-human ADAMTS13 antibody with high specificity and sensitivity and validated its use in immunoassays such as ELISA, Western blot and immunocytochemistry (AIM III). Finally, animal models in TTP are used to unravel the pathophysiologic mechanism underlying TTP and to test novel therapeutics. Therefore, reagents cross-reacting, in particular with murine ADAMTS13, have been developed. In the current study, an antigen-based purification of a previously developed polyclonal anti-murine ADAMTS13 antibody was performed (AIM III). The process increased the purity of the antibody and enriched the specific anti-murine ADAMTS13 antibodies. This PhD investigated the TTP literacy in patients and revealed that age and education level are associated with low disease comprehension. Apparently, not all patients are aware about the origin of the disease, the importance of treatment, the predisposing factors evoking an acute TTP bout and which actions have to be taken in an emergency setting. Additionally, an ELISA to epitope map anti-spacer autoantibodies in TTP patients was created showing that the spacer domain contains three hotspot regions for autoantibody binding. The findings provided in this study might help in designing novel therapies aiming to provide active ADAMTS13 that escapes the immune response. Additionally, we showed that the immune response against the spacer domain did not change in the course of the disease. Finally, a polyclonal anti-human ADAMTS13 antibody was developed, while an antigen-based affinity purification of the polyclonal anti-murine ADAMTS13 antibody enriched the specific anti-murine ADAMTS13 antibodies. Establishing immunoprofiling as a new type of biomarker for stratification of acquired TTP patients: by developing a new bioassay for immunoprofiling of acquired TTP patients, which must be validate on plasmas of acquired TTP patients. To achieve this it is needed to develop monoclonal anti-idiotypic antibodies in mice. Mice will be separately immunized with the cloned and purified anti-ADAMTS13 autoantibodies from Sanquin and the autoantibodies obtained previously by another PhD student. Six days after the last protein injection, it is needed to fuse the mouse spleen cells with SP2/0 myeloma cells according to the method of Köhler and Milstein. For large-scale productions, hybridoma cells will be cultured in cell line flasks. In addition, the HybrifreeÒ technology (ICOSAGEN) for direct cloning of VH and VL regions from B-cells followed by recombinant antibody generation and production in CHO cells, will be used. To select paratope specific anti-idiotypic antibodies at KU Leuven and Icosagen, ELISA plates will be coated with purified recombinant human ADAMTS13. Constant amounts of anti-ADAMTS13 autoantibodies will be preincubated with a serial dilution of the anti-idiotypic antibodies and subsequently added to the ADAMTS13 coated plate. Bound anti-ADAMTS13 autoantibodies will be detected with goat anti-human IgG1 antibodies labelled with HRP. We will develop the new autoantibody screening assay at KU Leuven and Biokit by coating the paratope specific anti-idiotypic antibodies and adding dilution series of purified anti-ADAMTS13 autoantibodies. Human anti-ADAMTS13 autoantibodies will be detected with goat antihuman IgG antibodies labelled with HRP. The efficiency of the ELISA to detect specific anti-ADAMTS13 autoantibodies in the serum of acquired TTP patients will be done at Biokit by investigating whether the anti-idiotypic antibodies produced can indeed capture the autoantibodies from the serum of the patients from whom the anti-ADAMTS13 scFv autoantibodies were cloned. The ELISA will be further analytically validated using spiked sera and will be validated for sensitivity, specificity, precision, accuracy, range, recovery and inter- and intra-assay coefficient of variation. We will perform a small-scale screening of 20 sera of acquired TTP patients, with 10 patients known to have anti-ADAMTS13 autoantibodies with the epitope and 10 patients not having these types of antibodies in their sera. This will allow the development of a new tool (ELISA) to classify TTP patients according to their immune profile and identification of new biomarkers for TTP prognosis. In addition, besides an excellent research and thematic based training at KULeuven, Icosagen and Biokit, I am looking forward to receive a clinical training at AP-HP in Paris, translational training, training in management, business, ethical and legal skills and training in career development and personal skills; during eight network-wide training events with beneficiaries, partner organizations and non-academic beneficiaries involve in PROFILE.
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