GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura

Abstract

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and life-threatening blood disorder characterized by the formation of blood clots in small blood vessels. It is caused by antibodies targeting the A disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13), which plays a role in cleaving von Willebrand factor. Most patients with iTTP have autoantibodies against specific domains of the ADAMTS13 protein, particularly the cysteine-rich and spacer domains. This study aimed to identify ADAMTS13 muteins that are resistant to autoantibodies and maintain their enzymatic activity. A panel of muteins was generated using rational and random mutagenesis methods and screened for autoantibody binding and ADAMTS13 activity. The selected muteins were assessed for pharmacodynamic biomarkers and pharmacokinetic profiles in the iTTP-mimic and wild-type mice, respectively. GC1126A was the most effective variant for escaping autoantibodies and had a longer half-life than the wild-type ADAMTS13 fragment (MDTCS). In the iTTP-mimic mouse model, GC1126A treatment significantly improved platelet counts, lactate dehydrogenase levels, and ADAMTS13 residual activity. In addition, GC1126A outperformed recombinant human wild-type ADAMTS13 (rh WT-ADAMTS13) and caplacizumab in terms of platelet recovery and sustained effectiveness. Results from the ex vivo study using plasma from patients with iTTP showed that GC1126A exhibited higher residual activity than rh WT-ADAMTS13, particularly in patients with high autoantibody titers. These findings suggest that GC1126A could be a promising new treatment option for patients with iTTP.

Fig. 1

The process and outcomes of candidate screening and the pharmacokinetic profile of candidates. (a) The diagram shows the binding domains of anti-ADAMTS13 neutralizing antibodies (Nabs). (b) The strategy and process of candidate screening. (c) The specific activity of selected candidates in the media following transient expression (n = 1 or 2). (d) The relative residual activity of selected candidates in the presence of 9 Nabs (n = 1 or 2). (e) The plasma concentration profiles after the intravenous administration of the selected candidates or controls (MDTCS or MDTCS-Fc) at the dose level of 160 IU/kg. Each point represents the mean and standard error of the mean (n = 4 mice per time point).

Fig. 2

In vivo screening results of the four selected candidates in mice. (a) The study design for candidate screening. (b) The residual activity in the plasma of the mice that received the respective treatments. Values below the limit of quantification were regarded as zero. Outliers were identified using the ROUT method (Q = 1%) and excluded from each group. Each bar represents the mean and standard error of the mean (n = 5–12 mice per group).

Fig. 3

Efficacy of GC1126A in the iTTP-mimic mouse model. (a) Study design for GC1126A prophylactic efficacy, (b)–(e) results of platelet count (b), number of red blood cells (RBC) (c), hematocrit percent (d), and lactate dehydrogenase (LDH) activity levels (e). (f) Study design for GC1126A therapeutic efficacy, (g)–(j) results of platelet count (g), number of RBC (h), hematocrit percent (i), and LDH activity levels (j). The dosage for the nine neutralizing antibodies (Nabs) was 0.54 mg/kg for prophylactic studies and 0.23 mg/kg for therapeutic efficacy studies. The dosage for recombinant human von Willebrand factor (rhVWF) was 2000 IU/kg. For GC1126A, the dosage was 7000 IU/kg (0.293 mg/kg) for prophylactic studies and 4000 IU/kg (0.084 mg/kg) for therapeutic efficacy studies. Outliers were identified using Grubbs’ test (α = 0.05) and excluded from each data group. Statistical significance was determined using the one-way analysis of variance followed by Tukey’s test (α = 0.05). Each bar represents the mean and standard error of the mean (n = 4–15 mice per group).

Fig. 4

Autoantibody escaping ability of GC1126A using the iTTP patient’s plasma. (a) Relative residual activity of ADAMTS13 and GC1126A in the presence of autoantibodies from iTTP patient’s plasma (n = 2). Each patient’s plasma was diluted to inhibitory antibody concentrations of 0.5, 1, 2, and 3 BU/ml. Each diluted plasma was mixed with the same molar concentration (3 nM) of ADAMTS13 or GC1126A. Relative activity is the ratio of activity remaining after neutralizing antibodies in the patient’s plasma to activity when each substance is free of neutralizing antibodies. (b) Binding level of autoantibodies from patients with iTTP to ADAMTS13 and GC1126A. Statistical significance was determined using the one-way analysis of variance followed by Tukey’s test (α = 0.05). Each bar represents the mean and standard error of the mean (n = 3).

Fig. 5

Relationship between the anti-ADAMTS13 inhibitor titer and GC1126A EC50. The distribution of the amount of GC1126A required to achieve 0.5 IU/ml activity (EC50) for different patient samples at inhibitor titers of 0.6–0.9 BU/ml (n = 4), 1 BU/ml (n = 23), 3 BU/ml (n = 15), 6 BU/ml (n = 9), and 9 BU/ml (n = 5) (the observed data are shown with closed circles). The estimated regression line and the 95% confidence interval are depicted in solid and dotted lines, respectively. The linear regression analysis yielded the following relationship: EC50 (µg/mL) = 0.0347 + 0.0396 × anti-ADAMTS13 inhibitor titer (BU/mL) (R² = 0.5842).

Fig. 6

Comparison of therapeutic efficacy of GC1126A, rh WT-ADAMTS13, and caplacizumab in mitigating platelet count reduction in the iTTP-mimic mouse model. (a) Study design for therapeutic efficacy comparison. (b) Platelet count results. (c) Residual activity of ADAMTS13 in the plasma of an iTTP-mimic mouse model. Values below the limit (0.03 IU/mL) are also not present on the graph. (d) Inhibitory antibody levels (BU levels). Values below the limit (0.42 BU/mL) are also not present on the graph. (b)–(d) Outliers were identified using the Grubbs’ test (α = 0.05) and excluded from each group. Each bar represents the mean and standard error of the mean (n = 4 mice per group).

Fig. 7

Comparison of therapeutic efficacy of GC1126A, rh WT-ADAMTS13, and caplacizumab in restoring severely reduced platelet counts in the iTTP-mimic mouse model. (a) Study design for therapeutic efficacy comparison. (b) Platelet count results. Outliers were identified using the Grubbs’ test (α = 0.05) and excluded from each group. Each bar represents the mean and standard error of the mean (n = 3–10 mice per group).