Conjugation of Antibodies and Nanobodies to Nanoparticles for Targeted Cancer Immunotherapy

Valentina Lykhopiy 1, Juan C. Mareque-Rivas 1,2,3 1Theranostic Nanomedicine Laboratory, CIC biomaGUNE, 20014 Donostia/San Sebastián, Gipuzkoa, Spain 2Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Bizkaia, Spain... [ view full abstract ]

Valentina Lykhopiy ¹, Juan C. Mareque-Rivas ^1,2,3

¹Theranostic Nanomedicine Laboratory, CIC biomaGUNE, 20014 Donostia/San Sebastián, Gipuzkoa, Spain

²Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Bizkaia, Spain

³Department of Chemistry, Swansea University, Singleton Park, Swansea, SA2 8PP, UK

Antibody-conjugated iron oxide nanoparticles (Ab-IONPs) have shown great potential in theranostic applications due to their ability to target specific cells and tissue more accurately and with high specificity. An exciting new application of these constructs could be in cancer immunotherapy –an area currently generating tremendous interest and promising results. In one of our approaches, hybrid NPs are coated with two different antibodies to simultaneously target receptors on the T cells and cancer cells. In this study, we describe the first step in developing one such dual-targeting hybrid NP system, to be constructed with an anti-CD3 antibody and an anti-HER2 nanobody.

We engineered IONPs conjugated with a defined number of antibodies or nanobodies. Anti-CD3 antibody that induces the activation of T cells or a tumour-targeting anti-HER2 nanobody were conjugated to the surface of IONPs using thiol-maleimide chemistry. IONPs coated with DSPE-PEG(2000)-amino were reacted with sulfo-SMCC, while the antibodies/nanobodies were activated with Traut’s reagent through lysines. The resulting constructs were purified and characterised by different techniques, including gel filtration, size-exclusion chromatography, DLS and TEM. We assayed in vitro the functionality of the IONP-attached antibodies and nanobodies by flow cytometry and ELISA.

Thiol-maleimide “click” reactions provided successful bioconjugation of an anti-CD3 antibody and an anti-HER2 nanobody to the IONP-filled micelles. The number of conjugated antibodies per nanoparticle can be adjusted. In addition, we show that the covalently attached proteins on the surface of the IONPs are functional and recognise their target receptors. The chemistry and constructs offer a highly flexible and customizable system which may be tailored for cancer immunotherapy and other applications.

We gratefully acknowledge funding from the European Union through the PET3D project (H2020-MSCA-ITN-2015, Grant No. 675417). We also thank Catarina Xavier (In vivo Cellular and Molecular Imaging Lab, Vrije Universiteit Brussel) for the anti-HER2 nanobody supply.