Modifying the Single Domain Antibody Framework as Platform for Enhanced Radiotherapy - PROJECT SUMMARY Targeted radiotherapeutics are an emerging class of highly effective treatments that leverage the potent therapeutic properties of radiation in a biologically focused manner to provide systemic killing of cells of interest with reduced non-target toxicity. These therapies have been utilized in cancer, inflammatory, degenerative and hyperproliferative diseases with great clinical efficacy. There are currently, however, two main limitations of targeted radiotherapeutics. The first is that the choice of targeting molecules is limited to a select number of peptides and small molecules. This is driven in large part by the second limitation, which is that a targeting molecule must have specific pharmacokinetic and pharmacodynamic properties that are not provided by traditional antibody targeting moieties. A targeted radiotherapeutic must combine high affinity and specificity for its target with rapid binding and blood clearance in order to minimize the radiation delivered to the bone marrow and clearance organs. A new class of antibodies, termed nanobodies, offers a combination of high affinity binding in a low molecular weight vector that endows rapid blood clearance by renal filtration. Nanobodies are growing in clinical use, however to date most have been derived from the immunization of dromedaries. Immunization- generated nanobodies have been demonstrated to have high retention in the kidneys, which is thought to be driven by a large number of positively charged amino acids in their framework that lead to endocytosis in the proximal tubules of kidneys. The inherent dependency of the tertiary structure of a nanobody on its primary sequence poses significant challenges to modifying the positively charged amino acids in existing nanobodies. Given these limitations, we have developed a novel nanobody framework that has greater than 90% reduction in kidney retention. This framework will serve as the basis of our proposal, in which we will test the hypothesis that reduced kidney retention can improve the therapeutic index of targeted radiotherapy. Additionally, we will explore whether previously isolated nanobodies from immunizations can have their complementarity determining regions engrafted into this framework. Nanobodies will also be designed to incorporate a sortase tag that allows for rapid and facile site-specific conjugation to bifunctional chelators for both imaging and therapeutic radionuclide incorporation. The radiolabeled nanobodies will be assessed in vitro for affinity, specificity and stability prior to in vivo PET imaging and radiotherapy studies. Overall, this proposal has the potential to generate a novel method for generating nanobodies with ideal pharmacokinetics for radiotherapy against virtually any target.
