Optimization of pretargeted radioimmunotherapy

Traditional cancer treatments include surgery, radiation and chemotherapy. The majority of patients with disseminated disease eventually succumb to their disease. To improve survival rates new treatments are needed, one promising approach is the use of radiolabeled antibodies, or radioimmunotherapy, which targets radiation specifically to cancer cells. A more effective method of radioimmunotherapy called pretargeting is a multi-step strategy using antibody-streptavidin conjugates followed by radiolabeled biotin. Although successful in curing lymphoma bearing nude mice there are limitations to this therapy. First, the tumor penetration of radiolabeled biotin has not been determined and if incomplete could diminish efficacy. Second, endogenous biotin is capable of blocking the biotin-binding sites of wild-type streptavidin, and thus reducing therapeutic efficacy.; To improve the penetration of the anti body-streptavidin conjugate a single chain antibody fragment was constructed and characterized. Design of the single chain antibody included residues for purification and conjugation. Methods to create single chain antibody-streptavidin conjugates were investigated using an engineered cysteine residue. Single chain antibody-streptavidin conjugate was assembled but quantities were insufficient for complete testing. Lymphoma bearing athymic mice were injected with labeled antibody-streptavidin and labeled biotin in separate experiments to determine the tumor penetration of each component. Antibody-streptavidin conjugate achieved complete tumor penetration, yet for the parameters tested labeled biotin was mainly found at the tumor periphery. As a method of mitigating the effects of endogenous biotin, a biotin binding system employing a streptavidin affinity mutant and a radiolabeled bis-biotin derivative was investigated both in vitro and in vivo. The reduced affinity of the streptavidin affinity mutant allowed exchange of biotin, and a bivalent biotin was retained by the streptavidin affinity mutant in vitro. Subsequent in vivo characterization revealed that mice with high levels of endogenous biotin are capable of achieving high uptake of bis-biotin in tumors pre-targeted with antibody-streptavidin affinity mutant conjugates, in contrast to wild-type streptavidin conjugates.; Developing a more complete characterization of pretargeting has provided information about the penetration and molecular interactions involved in pretargeting. Improved penetration and mitigating the effect of endogenous biotin may ultimately lead to superior therapeutic efficacy for pretargeted radioimunotherapy.