GloBody Technology: Detecting Anti-Drug Antibody against VH/VL domains

Abstract

The occurrence of anti-drug antibodies following administration of therapeutic monoclonal antibody to patients is a growing problem that is attracting attention from frontline clinicians. Ideally, an initial indicative point of care test would provide guidance to seek testing approved by the regulatory authorities. Here we describe a platform for the detection of IgG anti-drug antibodies that may provide an initial screen for all therapeutic monoclonal antibodies. Synthetic genes encoding Nanoluciferase polypeptides were inserted between the variable heavy and light domain encoding region of known antibody drugs (alemtuzumab and adalimumab) to generate recombinant single chain GloBodies, which retain the drug antibody paratopes and Nanoluciferase activity. In the presence of anti-drug antibodies, the GloBody is bound by specific IgG in the sample. These complexes are captured on immobilised Protein G and the luciferase activity determined. The amount of light generated being indicative of the anti-drug IgG antibody levels in serum. It should be possible to assemble GloBody reagents for all therapeutic monoclonal antibodies and adapt the capture phase to include additional specific isotypes. The assay has the potential to be developed for use with a drop of blood allowing initial pre-screening in a point of care setting.

Figure 1

(a) A schematic for the assembly of a GloBody. (i) The Alemtuzumab scFv was designed with NcoI/NotI directional cloning sites and a 5 amino acid linker between the VH and VL with a unique in frame BamHI site. (ii) A dual Nanoluc was designed flanked by in-frame BamHI sites and inserted in to the scFv to generate (iii) GloBody expression cassette. (b) A 3D model of the Alemtuzumab/ tandem dual Nanoluc luciferase fusion antibody. Molecular model (ribbon representation) of the CAMPATH-1H antigen-binding fragment (Fv) (PDB 1BEY) fused with the two Nanoluc luciferase (PDB 5IBO) separated by a short linker sequence. The Fv variable region heavy chain (VH) is coloured green whilst the Fv variable region light chain (VL) is coloured purple. First Nanoluc (orange) and the second nanoluc (olive) is fused in between these domains and is separated by two short amino acid linker sequences (blue).

Figure 2

(a) A schematic of the assay format. Anti-alemtuzumab antibodies present in serum bind to the Alem Globody. The total IgG is captured on Protein G allowing detection of the retained luciferase. In panel (b) in the absence of ADA the Globody is not retained. To determine the specificity of the assay, (c) Alem GloBody was incubated with ADA against alemtuzumab, adalimumab, ustekinumab and trastuzumab. Luciferase signal was only detected with ADA against alemtuzumab. (d) Conversely, Adali GloBody was incubated with ADA against alemtuzumab, adalimumab, ustekinumab and trastuzumab. Luciferase signal was detected with ADA against adalimumab, but no luciferase signal was detected with ADA against alemtuzumab, ustekinumab or trastuzumab.

Figure 3

Detection of ADA in serum samples from patients treated with alemtuzumab. (a) In patient 1, initially dosed with alemtuzumab at month 0 and a 1^st serum sample taken at month 5, then dosed at month 12 and a 2^nd serum sample taken at month 17. The control blank and the 50 µg/mL ADA positive included as reference points. Luciferase activity above the limit of detection were observed at both time points. In patient 2, (b) dosed with alemtuzumab at month 0 and then at month 12 prior to the 1^st serum sample at month 14 and a 2nd serum sample taken at month 26. The control blank and the 50 µg/mL ADA positive included as reference points. Luciferase activity above the limit of detection was observed at 2 months after the 2^nd infusion which then decreased to below the LoD at month 26.