Chemically Controlled Assembly of Therapuetic Protein Nanostrctures

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DESCRIPTION (provided by applicant): The design and development of nanodevices capable of functioning both as biosensors and therapeutic agents would greatly advance the treatment of a number of human diseases, including cancer. Because they have already been used to deliver a variety of toxins, drugs and radionuclides to cancer tissues, monoclonal and recombinant antibodies could be harnessed as recognition ligands for anticancer nanostructures. The targeting of radionuclides to tumors by conjugation to antibodies has been shown to be a successful imaging and antitumor approach. Despite their preclinical and clinical success, the development of monoclonal antibody radionuclide conjugates suffers from a number of concerns, such as, poor imaging capability due to low renal clearance, toxic radionuclide bone deposition, conjugation chemistry that is incompatible with immunoreactivity and molecular heterogeneity. We propose to develop a protocol for the pharmacologically controlled assembly and disassembly of multivalent radioimmunotherapeutic nanostructures that have the potential for bispecificity. We will take advantage of our recent discovery of how to construct discrete chemically induced protein nanorings (8-30 nm dia.) from E. coli dihydrofolate reductase (DHFR2) fusion proteins. We will prepare DHFR2 molecules fused to a single chain antibody (scFv) that was developed by Dr. Daniel Vallera (co-Investigator) and binds to the B-cell lymphoma and leukemia antigen CD22. We will prepare DHFR2-anti-CD22 scFv's fusion proteins that are able to self-assemble into bivalent, tetravalent or octavalent species in the presence of a methotrexate dimerizer coupled to a fluorophore or chelated radionuclides. We will demonstrate that the antibody-nanorings are able to selectively bind and undergo intracellular B-leukemia cell uptake and trafficking in vitro. In addition, we will determine the in vivo biodistribution of the antibody nanorings, as well as the ability of timethoprim, a non-toxic E. coli DHFR inhibitor, to promote oligomer disassembly in vivo. We will also investigate the anti-tumor and tumor imaging properties of the DHFR2-anti-CD22 scFv nanorings with a mouse xenograft tumor model. Using molecular modeling and protein engineering principles, we will design DHFR molecules capable of heterodimerization. These mutant DHFRs will be used in future studies to prepare self-assembling bispecific antibody nanorings. The principles elucidated by this study will be applicable to the design of antibody-radionuclide nanorings that are capable of detecting and treating a wide range of cancers in the future.The development of nanoparticles that can home in on a tumor, report back on where the tumor is and destroy the tumor is the goal of our research. In our first attempt, we will develop a method to prepare radiolabeled antibody protein nanorings that can target B- cell leukemias. We will use these antibody-nanorings for both tumor imaging and antitumor therapy and demonstrate that we can remove the nanoparticles when needed.