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ADAMTS13 variants to escape autoantibodies in iTTP
tive substitutions inserted in a full-length context confer resistance to autoantibodies while generating a gain-of- function (GoF) ADAMTS13 with more activity than the wild-type form.26 These mutations were introduced cumu- latively. At present, it is not clear how the degree of con- servation of the amino-acid side-chains in the spacer exosite-3 RFRYY epitope affects both the autoantibody binding and the activity of ADAMTS13 variants.
In this study we present a refinement of the contribu- tion of the residues in the exosite-3 RFRYY epitope to overall response of patients’ autoantibodies. We included an extensive set of full-length spacer domain epitope vari- ants with different degrees of residue conservation (Figure 1). The mutations inserted included single mutations and multiple progressively cumulative mutations, to scrutinize in more detail how they affect the binding of patients’ autoantibodies. A total of 42 ADAMTS13 variants were screened for binding of autoantibodies from patients’ sam- ples. As expected, autoantibodies directed towards spacer domain exosite-3 constituted the most important subset of autoantibodies within the patients’ response. We also demonstrated that within our panels of full-length ADAMTS13 variants, several displayed significantly reduced binding of autoantibodies while retaining residual proteolytic enzyme activity. Our study provides candidate molecules that could be used as a template for the design
of novel therapeutic approaches that limit the binding of pathogenic autoantibodies to the spacer domain of ADAMTS13.
Methods
Patients
Patients’ sera were obtained from the French Reference Center for Thrombotic Microangiopathies (CNR-MAT, Paris, France), from patients in the acute phase of onset of immune TTP, before treatment. All patients had ADAMTS13 activity <10% (FRETS-VWF73) and detectable autoantibodies against ADAMTS13 in varying titers (>15 IU/mL, Technozyme ADAMTS13-INH ELISA kit; Technoclone, Vienna, Austria). All patients included were described in previous studies27,28 (Table 1). Informed consent was obtained from patients according to the Declaration of Helsinki. The study was approved by the Ethics Committee of Hôpital Pitié-Salpêtrière and Hôpital Saint- Antoine (Assistance Publique-Hôpitaux de Paris, France).
Assessment of reactivity of ADAMTS13 variants with patients’ samples
The design, construction, production, and computation- al modeling of human ADAMTS13 variants, as well as
Table 1. Patients’ clinical data.
Patient
TTP-007
TTP-008 TTP-012 TTP-017 TTP-030
TTP-041 TTP-042
TTP-049
TTP-052
TTP-057
TTP-058 TTP-075 TTP-076 TTP-079 TTP-080 TTP-081
TTP-085
TTP-093
Sex Age
M 55
M 74 M 55 F 48 F 62
F 68 F 45
F 35
F 24
M 52
F 76 M 27 F 45 F 37 M 61 M 42
F 85
ADAMTS13 (IU/mL) (mg/mL)*
IgG anti-
ADAMTS13 antigen
Treatment regimen Detected
in addition anti-spacer §§
ADAMTS13 activity (FRETS- VWF73, %)
<5
<5 <5 <5 <5
<5 <5
<5
<5
<5
<5 100 0.03
<5 >100 <0.02
Cerebral involvement
No
Yes (confusion)
No
Yes (headaches)
Yes (aphasia) idiotopes
Yes (seizure)
Yes (transient focal defect)
Yes (headaches,
visual disorders) Yes (aphasia)
No
Yes (stroke) Yes (headaches) No
No
No
Yes (stroke)
Yes (transient focal defect)
Yes (headaches,
§
100 0.05
120 0.05 260 <0.02 85 <0.02 59 0.35
69 0.74 69 0.16
52 0.14
175 0.10
87 0.11
>100 0.21 89 0.44 65 0.09
180 <0.02 <10 210 <0.02
+ steroids + rituximab + cyclophosphamide
+ steroids + rituximab No treatment †
+ steroids + rituximab PEX only
+ steroids + rituximab + steroids + rituximab
+ steroids
+ steroids + rituximab
+ steroids + rituximab
+ vincristine + splenectomy (anti-VWF antibodies)
+ rituximab + steroids + steroids + steroids PEX only
+ steroids + rituximab
+ steroids
+ rituximab
Others
Others Others Others
I-9 + II-1 + TTP73
II-1 + TTP73 idiotopes I-9 idiotope
I-9 + TTP73 idiotopes
I-9 + II-1 + TTP73 idiotopes
Others
Others
I-9 idiotope
I-9 + II-1 idiotopes
I-9 + TTP73 idiotopes
I-9 idiotope
I-9 + II-1 + TTP73
idiotopes Others
Others
to PEX
idiotopes
<5 <5 <5 <5
F 21 <5 160 0.02
transient focal defect)
PEX:plasmaexchange;M:male;F:female;VWF:vonWillebrandfactor;*DatafromRooseetal.2018.27 §DatafromSchelpeetal.2019.28
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