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A.S. Schelpe et al.
treatment initiation) and a score <3 has a negative predic- tive value.39 To check whether the disease outcome param- eter could be linked with specific anti-spacer idiotope pro- files, we performed χ2-based analysis. However, none of the anti-spacer idiotope profiles could be linked with a score of ≥3 (χ2, not significant) (Figure 6A). In line with this, there was no link between the anti-spacer idiotope profiles and the individual factors related to the score by Benhamou et al.39 (age: ANOVA, not significant; cerebral involvement and high LDH levels: χ2, not significant) (Online Supplementary Figure S1).
We next used the same approach to investigate whether anti-spacer idiotope profiles could be linked with the applied treatment strategy. We, therefore, compared the anti-spacer idiotope profiles in patients treated with PEX with/without rituximab and patients treated with PEX with/without rituximab and additional treatment(s) (either steroids or other immunosuppressive drugs, e.g. cyclophosphamide, bortezomib; or/and caplacizumab or/and splenectomy) (Online Supplementary Table S2). However, also treatment could not be linked with anti- spacer idiotope profiles (χ2, not significant) (Figure 6B).
Discussion
In this study, we successfully generated three anti-idio- typic antibodies that specifically recognized the idiotopes of anti-spacer autoantibodies II-1, TTP73, and I-9. With this anti-idiotypic assay, we could for the first time identi- fy the presence or absence of anti-spacer II-1, TTP73, and I-9 idiotopes in iTTP patients. In addition, grouping the patients according to the absence or presence of one, two or three of the anti-spacer idiotopes revealed an until now unknown insight into the anti-spacer II-1, TTP73 and I-9 idiotopes in these patients. Although the resulting idiotope profiles could not be linked with disease severity, our data show that anti-idiotypic antibodies are interest- ing tools to determine an antibody profile in patients with any autoimmune disease.
Many studies have used groups of ADAMTS13 domains to identify which ADAMTS13 domains (e.g. MDTCS, MDT, CS, T2-C2, T2-T8, C1-C2) are targeted by anti-ADAMTS13 autoantibodies in individual iTTP patients. All these studies concluded that the immune response in iTTP patients is polyclonal with an immuno- dominant epitope in the cysteine-rich/spacer domain.8,10- 12,29,43-45 Antibody profiling based on these data stratifies patients according to either the presence or absence of anti-ADAMTS13 antibodies against certain domain(s). Only two studies investigated the link between domain specificity of anti-ADAMTS13 antibodies and disease severity or platelet counts. Thomas et al.29 stratified iTTP patients according to having either anti-MDTCS or anti- T2-C2 autoantibodies but could not identify a link with disease severity. On the other hand, Zheng et al.10 report- ed an inverse correlation between the presence of IgG antibodies against the T2-T8 and/or C1-C2 domains and platelet counts on admission. In our study, we used anti- idiotypic antibodies to stratify iTTP patients according to the presence or absence of anti-ADAMTS13 antibodies with specific idiotopes. By using an anti-idiotypic anti- body, we can, therefore, investigate whether a specific anti-ADAMTS13 idiotope is present or absent in an indi- vidual iTTP patient. Indeed, with our three anti-idiotypic
antibodies, we determined the previously unknown anti- spacer II-1, TTP73, and I-9 idiotope profiles in 151 iTTP patients in acute phase. Eighteen of the 151 iTTP patients had all three anti-spacer idiotopes in their plasma, 63 patients had none of the anti-spacer idiotopes, and 70 patients had either one or a combination of two of the anti-spacer idiotopes in their plasma, showing that the presence of these three anti-spacer idiotopes is not a com- mon feature in iTTP patients. In addition, the anti-spacer autoantibody II-140 used in this study is a well character- ized iTTP patient autoantibody that targets the R568- F592-R660-Y661-Y665 epitope in the ADAMTS13 spacer domain43 and is a strong inhibitor of ADAMTS13 activi- ty.40 Although approximately 50% of the iTTP patients have inhibitory anti-ADAMTS13 autoantibodies,29,46 it is still not known if all these patients have a II-1 idiotope in their plasma. Using our anti-idiotypic antibody against the anti-spacer II-1 idiotope, we now provide a novel insight into the incidence of this anti-spacer II-1 idiotope in iTTP patients. Indeed, our study showed that only 34% of the patients had this anti-spacer idiotope in their plasma. A wider understanding of the diversity of inhibitory anti- ADAMTS13 autoantibodies that target the R568-F592- R660-Y661-Y665 epitope is important in view of the development of a targeted antibody therapy. In addition, anti-idiotypic antibodies allow the study of epitope spreading observed in iTTP patients by following the pres- ence of specific idiotopes over time. An additional advan- tage of using anti-idiotypic antibodies for antibody profil- ing is that the antigen itself is not needed for the profiling assay.21,47 Production of recombinant ADAMTS13 and its fragments in the case of iTTP is more expensive and com- plex than producing and purifying murine anti-idiotypic antibodies.
Finally, we investigated whether we could establish a link between these anti-spacer idiotope profiles and dis- ease severity (disease outcome and applied treatment strategy). However, the current idiotope profiles did not allow specific profiles that are linked with disease severity to be identified. On the one hand, this could be due to the
Figure 5. Anti-spacer idiotope profiles in acute immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients. Acute iTTP patients (n=151) were stratified according to the presence (+) or absence (-) of II-1, TTP73 and I-9 idiotopes, as determined in Figure 4. n: number.
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haematologica | 2019; 104(6)