Abstract 3636: Enhancement of trastuzumab selectivity via cyclic complex formation

Abstract

Abstract Therapeutic antibodies rely on high affinity interactions between antibody and a cell surface receptor that is overexpressed on the cancer cell. These receptors are not highly expressed on healthy cells, and therefore antibody binding to healthy cells is less frequent than tumors. This provides antibody therapeutics a significant advantage over small molecule drugs. Nevertheless, non-selective targeting and non-specific toxicity still remains a serious drawback. This is well exemplified by trastuzumab, a monoclonal antibody that is used in the treatment of Her-2 overexpressing breast carcinomas. Trastuzumab has significantly improved the prognosis of breast cancer patients, however, it has been associated with clinically significant cardiac toxicity. Trastuzumab related cardiac toxicity has been associated with non-selective targeting of cardiomyocyte Her-2, which plays a protective role in adult heart. Here, we developed a targeting strategy where we enhanced selectivity of trastuzumab by delivering it as thermodynamically stable cyclic complexes formed via interactions with synthetic trivalent Her-2 mimotopes. Trastuzumab is in a dynamic equilibrium between complex-bound and cell-surface bound form, where only cells with a high receptor density is able to recruit the antibody to their surface by shifting the equilibrium towards dissociation of cyclic complexes. As a result, delivering trastuzumab as cyclic complexes provide enhanced selectivity for targeting tumors. To form cyclic complexes of trastuzumab, we first synthesized trivalent mimotopes from a small library of Her-2 mimotopes. Stoichiometric mixing of synthetic trivalent mimotopes and trastuzumab yielded stable cyclic complexes. Dynamic light scattering results confirmed the expected ∼ 20 nm hydrodynamic radius for the complex. Moreover, expected stoichiometry was confirmed via analytical ultracentrifugation, which yielded a calculated molecular weight of 420 KDa. We measured one order of magnitude increase in the Kd for the binding of trastuzumab to trivalent mimotope over that of monovalent mimotope by SPR, which was further supported with ITC results. To verify selectivity of complexed antibody for high receptor density surfaces, we generated high or low receptor density surfaces in an ELISA assay, and determined that cyclic complexes of trastuzumab showed only 16% of the activity of free trastuzumab towards low density surfaces while they showed equal activity towards high density surfaces. Taken altogether, here we describe design and application of cyclic antibody complexes that are engineered to improve selectivity of trastuzumab for Her-2 overexpressing tumor cells, with the long term goal of reducing trastuzumab associated cardiac toxicity. These studies form the proof of principal studies of a novel delivery method that can be applied to all antibody therapeutics to further improve their selectivity of the diseased target tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3636. doi:10.1158/1538-7445.AM2011-3636