Our group previously developed a platform of bioluminescent sensor proteins (LUMABS) that allow antibody detection directly in blood plasma using the camera of a smart phone as the sole piece of equipment (Arts et al (2016) Anal. Chem. 88, 4525). Antibody binding to LUMABS results in change in colour of the emitted light from green-blue to blue that can be detected directly in blood plasma, even at pM antibody concentrations. To establish LUMABS as a broadly applicable platform technology, we targeted five clinically important therapeutic antibodies, the Her2-receptor targeting trastuzumab, the anti-CD20 antibodies rituximab and obinutuzumab, the TNFα-binding Infliximab, and the EGFR-blocking cetuximab (Rosmalen et al (2018) Anal. Chem. 90, 3592-3599). The development of LUMABS sensors for the anti-CD20 antibodies was unsuccessful, because of a lack of epitope sequences with sufficient affinity. LUMABS sensors with physiologically relevant affinities and decent sensor responses were obtained for trastuzumab and cetuximab with affinities in the 10−7 M range. For both of these antibodies, LUMABS-based detection directly in plasma compared well to the analytical performance of commercial ELISA kits, which is the current gold-standard in the detection of these therapeutic antibodies. We also developed a new LUMABS sensor format that allows us to use the red fluorescent dye Cy3 as the BRET acceptor. This new class of ratiometric bioluminescent sensor proteins does not rely on direct modulation of BRET efficiency, but is based on competitive intramolecular complementation of split Nano-Luc luciferase (Ni et al (2019) ACS Sensors 4, 25-29). The new sensor design improved the dynamic response of a sensor for the therapeutic antibody cetuximab 4- fold, allowing the direct quantification of this anti-EGFR antibody in undiluted blood plasma.
An important step for translation into a point-of-care test is the development of low-cost, simple to use, but still reliable analytical devices. Together with the group of Prof. Daniel Citterio (Keio University, Yokohama, Japan) we developed a prototype of a paper-based device for use with BRET sensors such as LUMABS, allowing the detection of 3 different antibodies in whole blood (Tenda et al (2018) Angew. Chem. Int. Ed. 57, 15369). The devices use LUMABS sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume independent and fully reagent-free operation, including on device blood plasma separation. User operation is limited to the application of a single drop (20–30 µL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. This work was published as a Very Important Paper (VIP) in Angewandte Chemie and a press release on this work received a lot of media attention, including articles in national and international newspapers (Eindhovens Dagblad, De Volkskrant, De Morgen, National Geographic) and several radio stations. Our work was also chosen to feature as an example of “Drivers of Change” research at the Dutch Design Week (DDW) exhibition, attracting over 80,000 visitors.
During the project we have had discussions with a variety of stakeholders, including small and large diagnostic companies, clinical pharmacologists and fellow academic colleagues. Based on these discussions, we concluded that the best way to ensure translation of the LUMABS technology to the market, is to start a spin-of company, which could then act as a partner for other companies. We developed a strategy to start a spin-of company in the 2nd half of 2019. This strategy involves the application of specific grants available from the national and regional governments and a further discussion with clinicians and other stakeholders to further define the best application area. We also further strengthened our IP portfolio during the granting period. Together with our Japanese collaborators we filed a joint patent application on the paper-based devices for bioluminescent detection of biomarkers using luminescent sensing proteins and we applied for a provisional protection on the new platform of red-blue ratiometric bioluminescent sensor proteins.
