Functional characteristics and binding pattern toward rADAMTS13 of the 16 human anti-ADAMTS13 Fabs generated by phage display technology
| mAbs . | Inhibitory potential assessd by residual ADAMTS13 activity (%) . | Binding pattern toward rADAMTS13 as assesed by . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Clone . | IgG subclass . | CDR3 motif . | 10 nM . | 100 nM . | KD Biacore (M) . | ELISA 405 nm* . | CLIPS conformational epitope . | Dot-blot reactivity with† . | |
| Full-length . | MDTCS . | ||||||||
| 1a | IgG4 | 1 | 8.1 | <1 | 4.6E-09 | 2.611 | Group 1 | +++ | − |
| 1d | IgG4 | 3 | 29.3 | 1.6 | 4.7E-08 | 2.759 | Group 1 | +++ | − |
| 1e | IgG4 | 1 | 71.5 | <1 | 2.5E-08 | 1.460 | Group 1 | +++ | +++ |
| 1f | IgG4 | 1 | 69.2 | <1 | 1.5E-09 | 1.848 | ‡ | +++ | ++ |
| 1g | IgG4 | 1 | 124 | 100 | 1.0E-08 | 2.105 | ‡ | +++ | + |
| 1h | IgG4 | 3 | 100 | <1 | 7.2E-09 | 1.424 | Group 3 | +++ | ++ |
| 2a | IgG4 | 2 | 56.7 | 1.1 | 2.6E-08 | 1.210 | Group 3 | +++ | + |
| 2c | IgG4 | 1 | 92.5 | 5.4 | 7.9E-08 | 2.623 | Group 1 | +++ | + |
| 2h | IgG4 | 3 | 91.9 | 2.6 | 3.5E-08 | 1.859 | Group 3 | +++ | + |
| 2i | IgG4 | 3 | 72.5 | 1.3 | 9.2E-09 | 1.741 | Group 2 | +++ | + |
| 2j | IgG4 | 2 | 100 | <1 | 9.6E-08 | 1.063 | Group 2 | +++ | ++ |
| 3b | IgG4 | 7 | 100 | 3.5 | 9.0E-09 | 1.549 | Group 2 | +++ | +++ |
| 3c | IgG4 | 3 | 100 | <1 | 6.6E-08 | 2.818 | ‡ | +++ | +++ |
| 3h | IgG4 | 3 | 100 | <1 | 9.2E-08 | 1.373 | Group 1 | +++ | − |
| 3j | IgG4 | 8 | 100 | 39.2 | 9.2E-09 | 0.792 | Group 2 | +++ | +++ |
| 4a | IgG4 | 2 | 100 | 13.6 | 5.8E-08 | 1.401 | Group 2 | +++ | +++ |
| mAbs . | Inhibitory potential assessd by residual ADAMTS13 activity (%) . | Binding pattern toward rADAMTS13 as assesed by . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Clone . | IgG subclass . | CDR3 motif . | 10 nM . | 100 nM . | KD Biacore (M) . | ELISA 405 nm* . | CLIPS conformational epitope . | Dot-blot reactivity with† . | |
| Full-length . | MDTCS . | ||||||||
| 1a | IgG4 | 1 | 8.1 | <1 | 4.6E-09 | 2.611 | Group 1 | +++ | − |
| 1d | IgG4 | 3 | 29.3 | 1.6 | 4.7E-08 | 2.759 | Group 1 | +++ | − |
| 1e | IgG4 | 1 | 71.5 | <1 | 2.5E-08 | 1.460 | Group 1 | +++ | +++ |
| 1f | IgG4 | 1 | 69.2 | <1 | 1.5E-09 | 1.848 | ‡ | +++ | ++ |
| 1g | IgG4 | 1 | 124 | 100 | 1.0E-08 | 2.105 | ‡ | +++ | + |
| 1h | IgG4 | 3 | 100 | <1 | 7.2E-09 | 1.424 | Group 3 | +++ | ++ |
| 2a | IgG4 | 2 | 56.7 | 1.1 | 2.6E-08 | 1.210 | Group 3 | +++ | + |
| 2c | IgG4 | 1 | 92.5 | 5.4 | 7.9E-08 | 2.623 | Group 1 | +++ | + |
| 2h | IgG4 | 3 | 91.9 | 2.6 | 3.5E-08 | 1.859 | Group 3 | +++ | + |
| 2i | IgG4 | 3 | 72.5 | 1.3 | 9.2E-09 | 1.741 | Group 2 | +++ | + |
| 2j | IgG4 | 2 | 100 | <1 | 9.6E-08 | 1.063 | Group 2 | +++ | ++ |
| 3b | IgG4 | 7 | 100 | 3.5 | 9.0E-09 | 1.549 | Group 2 | +++ | +++ |
| 3c | IgG4 | 3 | 100 | <1 | 6.6E-08 | 2.818 | ‡ | +++ | +++ |
| 3h | IgG4 | 3 | 100 | <1 | 9.2E-08 | 1.373 | Group 1 | +++ | − |
| 3j | IgG4 | 8 | 100 | 39.2 | 9.2E-09 | 0.792 | Group 2 | +++ | +++ |
| 4a | IgG4 | 2 | 100 | 13.6 | 5.8E-08 | 1.401 | Group 2 | +++ | +++ |
Functional tests: inhibitory capacity against plasma-derived ADAMTS13 in a Bethesda-like assay determining the residual ADAMTS13 activity assessed by FRETS-VWF73 (in %). Binding pattern assays included affinity analysis by SPR (Biacore), specificity tests by ELISA (Figure 3), and epitope mapping performed by either conformational CLIPS technology and dot-blot technique on either recombinant full-length or on ADAMTS13 stop mutant (MDTCS-13, consisting of ADAMTS13–metalloprotease–disintegrin–thrombospondin type I repeat number 1–cys-rich and spacer domain MDTCS) (supplemental Materials).
ELISA data in Figure 3.
Dot-blot technique described in supplemental Data.
No binding pattern found by CLIPS technology.