Antibodies that are capable of simultaneously engaging two different antigens are known as bispecific antibodies (BAbs), and their unique properties have spawned a rapidly growing field of research and therapeutic development. Given the capability for BAbs to crosslink two antigens, they can be employed to recruit components of the immune system to cancerous cells, can better antagonize signaling pathways by simultaneously inhibiting two compensatory pathways, or can lead to better cell type targeting specificity, among many other therapeutic applications. In addition to the research objectives, this proposal possesses significant broader impact. First, it provides a new conceptual framework and toolkit for the creation of bispecific antibodies. In addition, bispecific antibodies discovered by researchers using the BAb platform could yield new therapeutic approaches for a variety of diseases including cancer, brain disease, and viral infection. Next, the project is designed to train one full-time graduate student and one part-time graduate student along with several undergraduate students in the protein engineering/chemical biology field to prepare them for careers in industry and academia. Research results will be integrated into courses that the PI teaches as protein engineering modules. Finally, research results will also be disseminated to high school students through the Wisconsin High School State Science Olympiad in an 'Applied Protein Modeling for Medical Uses' symposium that the PI will organize and host.
Collectively, the state-of-the-art BAb design strategies possess many drawbacks, with the most robust systems requiring laborious molecular redesign, cloning, and recombinant protein expression. These challenges limit the current approaches to the evaluation of a handful of BAbs when antibody discovery technologies can easily generate 10's or 100's of possible BAb combinations that should be tested. To address these issues, a new BAb platform that will enable the facile combinatorial assembly of BAbs is proposed. The platform will be based on yeast displayed antibody-intein fusions that can be chemically cleaved and site-specifically chemically functionalized. Subsequently, pairs of released and functionalized antibodies will be covalently linked resulting in bispecific antibodies. Importantly, all of the requisite steps for CliBAb assembly would be performed directly using yeast displayed antibody clones without any subcloning, reformatting or purification, greatly facilitating BAb antibody assembly and evaluation. In order to develop such a BAb platform, intein engineering will be used to maximize yeast surface display of antibody-intein fusion proteins. In parallel, the chemical handles will be optimized to drive efficient BAb formation. Finally, the BAb platform will be validated by creating and testing a set of 36 BAbs resulting from the combinatorial pairing of 8 different antibodies previously identified for cell binding and internalization.
This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Instrument Development for Biological Research Program of the Division of Biological Infrastructure.