Eradication of Heterogeneous Tumors by T Cells Targeted with Combination Bispecific Chemically Self-assembled Nanorings

Cancer stem-like cells (CSCs) are often the root cause of refractive relapse due to their inherent resistance to most therapies and ability to rapidly self-propagate. Recently, the antigen CD133 has been identified as a CSC marker on several cancer types and aCD133 therapies have shown selective targeting against CSCs with minimal off-target toxicity. Theoretically, by selectively eliminating CSCs, the sensitivity to bulk tumor-targeting therapies should be enhanced. Previously, our laboratory has developed bispecific chemically self-assembled nanorings (CSANs) that successfully induced T-cell eradication of EpCAM-positive (EpCAM^þ) tumors. We reasoned that targeting both CSCs [CD133-positive (CD133^þ)] and the bulk tumor (EpCAM^þ) simultaneously using our CSAN platform should produce a synergistic effect. We evaluated aCD133/aCD3 CSANs as both a single agent and in combination with aEpCAM/aCD3 CSANs to treat triple-negative breast cancer (TNBC) cells, which express a subpopulation of CD133^þ cancer stem cells and EpCAM^þ bulk tumor cells. Furthermore, an orthotopic breast cancer model validated the ability of aCD133 and aEpCAM targeting to combine synergistically in the elimination of TNBC MDA-MB-231 cells. Complete tumor eradication only occurred when EpCAM and CD133 were targeted simultaneously and lead to full remission in 80% of the test mice. Importantly, the depletion and enrichment of CD133 TNBCs highlighted the role of CD133^þ cancer cells in regulating tumor growth and progression. Collectively, our results demonstrate that dual targeting with bispecific CSANs can be effective against heterogenous tumor cell populations and that elimination of primary and CD133^þ CSCs may be necessary for eradication of at least a subset of TNBC.