Facile immunotargeting of nanoparticles against tumor antigens using site-specific biotinylated antibody fragments

Nanotechnology is actively being developed for preclinical and clinical oncology applications. Nanoparticle-based immunotargeting against tumor antigens with antibodies or antibody fragments is designed to increase the nanoparticle concentration at the tumor site. However, chemical-based strategies for bioconjugating antibody fragments to nanoparticles typically result in a functionally heterogeneous population of conjugates due to alteration of amino acids within the antigen binding site. The loss of function can be prevented by expressing recombinant antibodies that contain a unique bioconjugation site, which is isolated from the antigen binding site. Biobodies are antibody fragments biosynthetically biotinylated by yeast at a specific biotin acceptor site and secreted into the culture supernatant. The high specificity and affinity between streptavidin-labeled nanoparticles and soluble biobodies allow self-assembly of immunotargeted nanoparticles directly in the yeast culture supernatant. Here, we demonstrate the versatility of biobodies for nanoparticle immunotargeting using streptavidin-labeled superparamagnetic iron oxide nanoparticles as a general, modular scaffold. Biobody-mediated targeting was performed against two antigens (mesothelin and TEM1) that are upregulated in solid tumors. The technology for biosynthetic biotinylation can be extended to proteins other than antibody fragments and adopted by fields outside of oncology for directed modification of any streptavidin-functionalized surface.