Continued development of our Chemically Self-Assembled Antibody Nanorings (CSANs) has led to novel structural alterations to enhance overall functionality and broaden therapeutic application through the use of adoptive T cell therapy. Through spontaneous nanoring formation with a dimerization agent (bismethotrexate, bisMTX) and monomeric antibody fusion proteins (DHFR-DHFR-scFv) we are able to form highly stable bivalent or octavalent nanorings. Previous studies utilizing DHFR-DHFR-antiCD3 fusion proteins have demonstrated the promise of CSANs as targeted, drug delivery agents towards the treatment of T cell leukemia. In this study, improvement of overall CSAN bioreactivity was evaluated through the incorporation of a PEG-3000 or PEG-10,000 onto the C-terminus of an antiDR5-DHFR-DHFR protein through enzymatic and bioorthogonal farnesylation. Results of both FACS and confocal studies showed that uptake by RAW macrophage 264.7 cells of the PEGylated CSANs was significantly reduced compared to an unpegylated control, thus indicating that in future in vivo studies that the modified CSANS may potentially exhibit reduced non-specific uptake by the liver and spleen and increased tumor accumulation. Additionally, two novel DHFR-DHFR-antiEpCAM proteins were constructed and evaluated for their use in T cell directed therapy towards breast carcinoma. Two methods were employed in the redirection of T cells, the first through formation of a bispecific antiCD3-antiEpCAM CSAN, and the second through the incorporation of a phospholipid moiety into the bisMTX dimerizer leading to the creation of antiEpCAM, lipid-based CSANs. T cell redirection studies carried with both the bispecific CSANs and lipid-CSANs demonstrated that upon incubation with the CSANs, T cells were stably modified for several days and were successfully directed towards EpCAM expressing carcinoma cells. Further studies revealed that the T cells redirected towards target cancer cells with either bispecific CSANs and lipid-CSANs significantly increased target cell lysis when compared to untreated or non-treated controls, with up to 95% target cell lysis achieved with lipid-CSAN treated T cells. Overall, these results demonstrate that through modification of the CSANs a reduction in macrophage uptake is achievable through the use of pegylated CSANs. In addition, bispecific and lipid-CSANs can be used to non-genetically prepare T-cells capable of specific tumor cell lysis and thus a potentially new adoptive T cell therapy approach.
University of Minnesota Ph.D. dissertation. August 2013. Major: Medicinal Chemistry. Advisor: Dr. Carston R. Wagner. 1 computer file (PDF); xiii, 197 pages.
Gabrielse, Kari A..
Chemically self-assembled antibody nanorings: a multifaceted approach to anti-cancer therapeutics using techniques including drug delivery, bispecific targeting, and T cell surface modification..
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