Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Abstract

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Figure 1. Screening of DARPin clones after third selection round.

A) Crude extracts of 96 individual DARPin clones were tested for binding to the target antibody, Bo2C11. Extracts were diluted 1∶10 and binding of DARPins was revealed using a murine anti-His₆ antibody and a peroxidase-labeled anti-mouse IgG antibody. Clones 1–12 are shown in row A, clones 13–24 in row B and so on. Clones chosen for retesting are highlighted in dark grey. B) Re-testing of positive clones. The highlighted clones depicted in Fig.1A were again tested on Bo2C11 (black bars), an isotype control antibody (grey bars) and the blocking agent (white bars) diluted 1∶10. DARPin binding was revealed with a murine anti-His₆ antibody and a horseradish-peroxidase labeled goat anti-mouse antibody.

Figure 2. Sequence analysis of Bo2C11–specific DARPin clones.

Plasmid DNA of positive clones was sequenced and aligned with the N2C DARPin consensus sequence. In the consensus module position X allows any amino acid except Cys, Pro or Gly and Z can be either His, Asp or Tyr. Based on the sequence homologies, DARPins were grouped to group 1(†) or group 2 (#), respectively. Identical residues are displayed as dots.

Figure 3. Specificity analysis of selected DARPin clones by ELISA.

Purified DARPin proteins were tested for binding to the human monoclonal anti-FVIII antibody Bo2C11 (black bars), human IgG subclass antibodies of non-relevant specificity (IgG1 dark grey bars, IgG2 grey bars, IgG3 light grey bars, IgG4 white bars) and the coating control (PBS containing 0.15% Casein) (striped bars) in ELISA. DARPin binding was revealed using a monoclonal mouse antibody directed against the N-terminal RGS-His₆-tag of DARPins and a horseradish-peroxidase-labeled goat anti-mouse antibody. A) shows the binding of 150 nM of monomeric DARPins eBo01 and eBo38. B) shows the binding of 4 nM of dimeric DARPins eBo01-38 and eBo38-38. As control in A) and B), PBS-C was incubated without DARPins on the human antibodies.

Figure 4. Bo2C11 binding to FVIII in presence of dimeric anti-FVIII antibody-specific DARPins.

Inhibition of anti-FVIII antibody (Bo2C11) binding was performed at the EC₅₀ value of Bo2C11 on FVIII. 800 ng/ml Bo2C11 was mixed 1∶1 with increasing concentrations of eBo01-38 (black circles), eBo38-01 (black triangles), eBo38-38 (black squares) or a dimeric DARPin of non-relevant specificity (open circles). The mixtures were pre-incubated at room temperature for 30 min and then added to FVIII-coated wells. The amount of Bo2C11 binding to FVIII was revealed with a horseradish-peroxidase-labeled anti-human IgG antibody. The results were normalized to 800 ng/ml Bo2C11 mixed 1∶1 with buffer on FVIII. Data represent mean and standard deviation of duplicates. Shown is one representative of 3 individual experiments.

Figure 5. Anti-Bo2C11 DARPins neutralize inhibitory activity of Bo2C11.

Different concentrations of three dimeric DARPins recognizing the monoclonal anti-FVIII antibody Bo2C11 (eBo01-38: closed circles; eBo38-01: closed triangles; eBo38-38: closed squares) or a dimeric control DARPin (open circles) were mixed 1∶1 with 6 nM of Bo2C1, added to human standard plasma and analyzed in the modified Bethesda assay. 3 nM of the monoclonal anti-FVIII antibody Bo2C11 corresponded to 1 Bethesda Unit (BU) (dotted line). Values were normalized to standard plasma and expressed as residual FVIII activity.

Figure 6. Dimeric DARPins recognize Bo2C11 spiked in healthy plasma.

A) Detection of Bo2C11 by DARPins in ELISA. DARPin eBo01-38 (closed circles) or a dimeric control DARPin (open circles) were coated at 69 nM in PBS on a microtiter plate. 3-fold serial dilutions (60 nM – 9.3 pM) of Bo2C11 in a plasma-pool from healthy donors (1∶100 in PBS containing 0.15% casein and 0.1% tween-20) were incubated on immobilized DARPins. Bo2C11-binding to DARPins was detected using a horseradish-peroxidase-labeled sheep anti-human IgG antibody. The dotted line represents the detection limit of Bo2C11 in plasma on DARPin eBo01-38 (mean of diluted plasma + 2 SD). B) Catching of Bo2C11 from human plasma with DARPins. An anti-His₆ antibody was dotted at 1 µg (a), 0.1 µg (b) or 0.01 µg (c) to a membrane, respectively. Human standard plasma diluted 1∶50 alone or spiked with either 2 µg/ml Bo2C11 (Bo2C11) or a control human IgG (X) was mixed with eBo38-38 (eBo), a DARPin specific for the control IgG (DX) or nothing (-). Complexes of human IgG and DARPins were detected using a peroxidase-labeled murine anti-human IgG antibody. As controls the detection antibody was incubated on the membrane alone and the presence of the anti-His₆ antibody on the membrane was confirmed with a peroxidase-labeled sheep anti-murine IgG antibody (amIgG).