Thermo Fisher Scientific,
Keywords: antibodies, immunoglobulin gamma
Summary:Antibodies, specifically Immunoglobulin gamma (IgG), comprise an indispensable part of the biochemist's toolbox. Though small-molecule chemical probes provide great utility in cellular analysis, their development is idiosyncratic, and only a handful of targets have specific probes available for purchase. In contrast to small organic molecules, antibodies are produced in vivo in animals, and can be generated against nearly any target. Because of these attractive properties, five out of the ten best-selling pharmaceuticals in 2015 were monoclonal antibodies (and another of the top ten, Etanercept, is an antibody fusion protein). It is the specific binding to a target that makes antibodies so valuable, but binding to a target is often only one step in the mechanism of action. In some cases it is desirable to destroy the antibody-targeted cells, particularly when those cells are cancerous or otherwise harmful. The immune system itself can be used to promote cell destruction, through recognition of the Fc receptor by immune cells, in the process of antibody-mediated phagocytosis or antibody-mediated cytotoxicity. But designing an antibody that will elicit an immune response is not trivial, and cancerous cells often have ways of evading detection by the immune system, so antibody-dependent cell cytotoxicity will not work in all cases. Antibody-drug conjugates (ADCs) were invented to achieve the targeting benefits of the antibody, as well as a custom-tailored payload to be delivered to the target. The key problem to ADCs, and the reason there are so few, is that the process of attaching the payload to the antibody has typically been ill-defined. Amine-reactive or thiol-reactive chemistries are not specific enough to guarantee the attached payload does not interfere with the antibody's action—both the targeting and the Fc recognition can be disrupted by conjugation. SiteClick™ technology allows for site-specific attachment of cargo to the branched glycan structure attached to amino acid 297 of all IgG antibodies, so that the antigen binding site is not perturbed. The ability to modify natural antibodies distinguishes SiteClick technology from other defined conjugation technologies, which require engineered proteins, synthetic tRNAs, mutated enzymes, and unnatural amino acids. Several fluorescent dyes were conjugated to the anti-cancer monoclonal antibody drugs Herceptin (Trastuzumab) and Gazyva (obinutuzumab), and the conjugates were shown to have similar in vitro functionality as the unconjugated antibodies. Internalization of these research model antibodies into breast cancer and leukemia cells was visualized using pH-sensitive fluorescent dyes, which become brighter in acidic lysosomal compartments. Both model antibodies were efficiently internalized into target cells, suggesting that they are strong candidates for further development to become antibody drug conjugates.