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ADAMTS13 variants to escape autoantibodies in iTTP
It is possible that the differences observed may be due to post-translational modifications, such as different glycosy- lation patterns that may result from the CHO-based tran- sient expression system that we used in our study.48,49 To address this we also expressed the GoF variant in HEK293 cells, and under these conditions the GoF variant displayed increased activity, compared to the respective HEK293 wild-type ADAMTS13, in both FRETS-VWF73 and in the VWF multimer assay (Online Supplementary Figure S7). In the context of our ELISA, the GoF variant expressed in both CHO and HEK293 cells behaved in a similar way against the patients’ samples (Online Supplementary Figure S7), and importantly our CHO-produced GoF variant was also inhibited by patients’ autoantibodies (Figure 5), as anticipat- ed by our ELISA results (Figure 2).
In conclusion, we have shown that less conservative mutations make it possible to obtain ADAMTS13 variants that resist the majority of patients’ autoantibodies and retain residual levels of activity in vitro. Our data suggest a
trade-off between the resistance towards patients’ autoan- tibody binding and the loss of proteolytic activity in spac- er exosite-3 ADAMTS13 variants. The study also reveals the importance of the side-chains of the RFRYY epitope in the spacer domain for autoantibody binding and ADAMTS13 activity, and it provides a basis for the devel- opment of novel therapeutic interventions that prevent the binding of pathogenic autoantibodies to the spacer domain of ADAMTS13.
Acknowledgments
This project received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement number 675746 (PRO- FILE). The authors would like to thank Tiiu Männik, Kristiina Karro, Urve Toots (Icosagen AS), Elien Roose (KU Leuven) and Ellie Karampini (Sanquin) for their technical advice, and to all the patients from CNR-MAT who consented to the use of their materials.
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