(454a) Eradication of Primary and Cancer Stem Cells By Chemically Self-Assembled Nanoring Targeted T-Cells

Over the past decade, it has become increasingly clear that harnessing a cancer patient’s T-cells to destroy tumor cells will be an important weapon in an oncologist’s therapeutic arsenal. Nevertheless, although selective tumor cell killing and significant clinical success against B-cell malignancies has been demonstrated, the same level of success against solid tumors has remained elusive.

To address this unmet need, our group has envisioned the potential of chemical biology and protein engineering to non-genetically modify T-cell membranes. We have shown that two dihydrofolate reductase molecules (DHFR²) fused to a single chain antibody (scFv) or peptide can spontaneously self-assemble upon the addition of the chemical dimerizer, bis-methotrexate (BisMTX), into highly stable multivalent targeted chemically self-assembled nanorings (CSANs). Recently, we have prepared anti-EpCAM/anti-CD3 CSANs and anti-CD133/anti-CD3 CSANs. EpCAM has been shown to be a ubiquitous marker of a number of epithelial cancers, including breast, prostate, lung, pancreatic and colon cancer, while CD133 has been shown to be a marker for a number of types of cancer stem cells. The bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes, thus forming Prosthetic Antigen Receptors (PARs) T-cells. (Upon incubation of the PAR T-cells with EpCAM+ and/or CD133+ cancer cells, T-cell activation and selective cell killing was observed. Furthermore, we demonstrated with murine xenograft models that the anti-EpCAM and anti-CD133 PAR T-cells are able to permanently eradicate tumors in animals.A unique feature of our approach is the ability to remove the CSANs from the T-cells by incubation with the FDA-approved non-toxic antibiotic trimethoprim, thus 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). Thus, PAR T-cells may afford and alternative and complimentary immunotherapy approach.