Abstract 2917: Tumor eradication with T-cells targeted with multivalent chemically self-assembled nanorings
Authors: Wagner Carston R. 
Published: June 2022

Abstract
Due to the heterogeneity of cellular dependences on signaling pathways and the potential development of drug resistance, there is an emerging recognition that future cancer therapy will require individualized drug combinations to produce long term remission. In addition, to truly address metastasis and relapse, methods of targeting quiescent tumor initiating cancer stem cells (CSC), which are resistant to chemotherapy and radiation, is of critical importance. Consequently, new methods that can target both primary tumor cells and CSC are needed. Previously, our group has shown that two dihydrofolate reductase molecules (DHFR2) fused to distinct targeting ligands can spontaneously self-assemble upon the addition of a chemical dimerizer (bis-methotrexate (BisMTX)) into highly stable, multivalent, chemically self-assembled nanorings (CSANs). When an anti-CD3 scFv is fused, the bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes. Consequently, we refer to the modified T-cells as Prosthetic Antigen Receptor (PAR) T-cells. A unique feature of our approach is the ability to remove the CSANs from the T-cells by incubation with the FDA-approved antibiotic trimethoprim, allowing us to deactivate the modified cells pharmacologically. In addition, we have shown that the CSANs have negligible immunogenicity in mice and do not induce naïve human T-cell anergy (i.e, unresponsiveness).Recently, we have prepared anti-EpCAM/anti-CD3 CSANs, which target triple negative cancer (TNBC) primary tumor cells, and anti-CD133/anti-CD3-CSANs, which target TNBC cancer stem cells. The bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes, thus either anti-EpCAM or anti-CD133 PAR T-cells. Upon incubation of CD133+ or EpCAM+ TNBC cells with the CSANs rapid and selective cell killing was observed. We have also demonstrated with an orthotopic murine cancer model that anti-EpCAM CSANs significantly reduce tumor size, while anti-CD133 CSANs block tumor proliferation. When dosed with a combination of anti-EpCAM CSANs and anti-CD133 CSANs tumor T-cell induced eradication of the tumors is observed. Further analysis revealed that initial tumor growth kinetics is dependent on the percentage of CD133+ tumor cells and that regardless of the starting point, the number of CD133+ tumor cells reaches a homeostatic percentage in the tumor. Whether CD133+ tumor cells arise from self-renewal or de-differentiation is yet to be determined. Regardless, dosing with either CSANs was found to be non-toxic. Taken together, our results demonstrate that CSAN modified T-cells targeting both primary tumor and CSC are necessary for TNBC tumor eradication and that CD133+ TNBC tumor cells maybe be necessary and sufficient for TNBC tumor initiation and proliferation.
Citation Format: Carston R. Wagner. Tumor eradication with T-cells targeted with multivalent chemically self-assembled nanorings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2917.