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N.A.G. Graça et al.
expression and purification of anti-ADAMTS13 mono- clonal antibodies I-9 and II-1 are given in the Online Supplementary Methods (see Online Supplementary Figures S1 and S2, and Online Supplementary Table S2). Figure 1 pro- vides an overview of the variants that were created and included in this study. Several panels of conservative (Y↔F), semi-conservative (Y/F→L), and non-conservative (Y/F→N) mutations and alanine (Y/F/R→A) substitutions, as well as a panel of alanine and lysine hybrids were employed in this study. For the non-conservative asparagine mutations no putative N-glycosylation sites were introduced. The rationale for the choice of each type of mutation is presented in the Online Supplementary Methods. For ease, from here on throughout the text the variants will be referred to by the final mutated epitope with the residue replacement underlined (e.g., RFRYY is the wild-type and RFRLL is a double leucine mutant carry- ing Y661L/Y665L mutations). Murine antibodies 3H9 (anti-metalloprotease), and biotinylated 19H4 (anti-TSP8) and 17G2 (anti-CUB1) have been previously described.29,30 Anti-V5 conjugated with horseradish peroxidase was obtained from Invitrogen® (Catalog # R961-25).
To determine the binding of autoantibodies in patients’ samples to the ADAMTS13 variants described, a sand- wich enzyme-linked immunosorbent assay (ELISA) was developed in-house. Full details are provided in the Online Supplementary Methods. Briefly, antibody 3H9 was used to coat 96-well plates to capture ADAMTS13 from cell cul- ture medium (coating done at 1 mg/mL). Each patient’s sample was tested for binding of autoantibodies against the variants described above. The samples were assessed against 200 ng/well (1.05 nmol/well) of each ADAMTS13 full-length variant or against equal molar quantities of the MDTCS variants (78.75 ng/well), in duplicate. A pool of monoclonal antibodies against human-IgG1, IgG2, IgG3 and IgG4, each conjugated with horseradish peroxidase (Sanquin, the Netherlands) was used for detection. In each plate, a monoclonal antibody II-1 dilution curve against the full-length wild-type ADAMTS13 was fitted with a four-parameter fit model (GraphPad Prism 5.0). Serum reactivity (i.e., binding) against each ADAMTS13 variant was converted to II-1 equivalent units (ng/mL) through interpolation, and a ratio of the interpolated signals was expressed as a percentage of wild-type binding. A heat map with the average values obtained was used for data comparison (Figure 2). The median reactivity for each ADAMTS13 variant was calculated from its average reac- tivities against each patient’s sample. An example of how the calculations were performed is given in Online Supplementary Figure S3.
Assessment of the activity of ADAMTS13 variants
All variants were assessed for activity through FRETS- VWF73 assays, as previously described,31 using a Fluoroskan Ascent plate reader (Thermo Electron Corporation) with modifications. All FRETS assays were done using the supernatants of cell cultures, and a modi- fied buffer was added to maintain the pH at 6.032 (compo- sition: CaCl2 25 mM, Bis-Tris 20 mM, Tris-HCl 20 mM, HEPES 20 mM, Tween20 0.005%). The recombinant full- length wild-type ADAMTS13 was used as the calibrator for the assay (concentration range: 0.025-0.4 mg/mL). Supernatant from Chinese hamster ovary (CHO) cells not producing ADAMTS13 was included as a control. All vari- ants were tested at the same molar concentration of 1.05
nM (0.2 mg/mL for full-length variants and 0.07875 mg/mL for MDTCS variants). An example of a calibration curve is shown in Online Supplementary Figure S4.
Selected variants were additionally tested for activity in a static VWF multimer assay as described in detail in the Online Supplementary Methods.
Results
The majority of autoantibodies in patients with immune thrombotic thrombocytopenic purpura target the spacer exosite-3 RFRYY epitope
All patients’ samples were assessed for binding to each of the ADAMTS13 variants described with an in-house developed sandwich ELISA. Figure 2 shows the reactivity of all the different ADAMTS13 variants towards each patient’s sample in a heat map format. To assess the potential presence of anti-TSP2-8 and CUB1-2 domain antibodies, we first compared the reactivity of the patients’ samples with full-length wild-type ADAMTS13 and the MDTCS variants. Two patients (TTP-057 and 085) showed a loss of signal with wild-type MDTCS of more than 70%. All other patients showed either similar signals to the full-length wild-type enzyme or, in a limited number of cases, losses up to 48%. The MDTCS-AAAAA variant caused a major reduction in signal compared to wild-type MDTCS in all patients (with the exception of patient TTP-057). The same 5x ala mutations in a full- length context (AAAAA) caused large increases in signal for six patients compared to MDTCS 5x ala (6/18; 33%). These findings show that the autoantibodies targeting the spacer exosite-3 RFRYY epitope comprise the majority of the antibody mixture in most patients’ autoantibody repertoire (16/18 cases, 89%). In rare cases, the repertoire was composed exclusively of autoantibodies targeting the TSP2-8 and/or CUB1-2 domains of ADAMTS13 (1/18 cases, 5%). However, in 12/18 cases (67%) the repertoire was composed almost exclusively of autoantibodies tar- geting the RFRYY epitope in the spacer domain.
Autoantibody resistance is obtained with non-conservative or alanine mutations
Next we assessed the efficacy of each type of mutation at inducing autoantibody resistance. We designed variants that retained R568 and R660 intact, as well as variants in which these residues were mutated. Within the variants in which R568 and R660 were kept intact, variants contain- ing conservative mutations of aromatic residues were still recognized by autoantibodies present in the patients’ sera (Figure 2). Semi-conservative mutations in the spacer domain epitope showed reduced binding to patients’ anti- bodies with signals in the range of 30%-75% when com- pared to the wild-type form in 15 patients, and below 30% in one patient. The non-conservative asparagine mutations were the most successful of the aromatic residue mutations at escaping binding by patients’ autoan- tibodies, presenting signals within the range of 0%-75% for 16 patients. RFRNY and RFRYN mutants were the least successful of this panel at escaping the autoantibody response, followed by RFRNN. The RNRNN mutant had the lowest median reactivity of all aromatic residue triple mutants (14%). Alanine mutations of aromatic residues (i.e., RARYY, RFRAY, RFRYA, RARAY, RARYA, RFRAA and RARAA) followed the same trend as the asparagine
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haematologica | 2020; 105(11)