Prosthetic Antigen Receptors As A Platform For Solid Tumor Immunotherapy

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Prosthetic Antigen Receptors As A Platform For Solid Tumor Immunotherapy

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2017-12

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The ability to engineer and reprogram cell surfaces has significant potential for enabling the use of cell based therapies in cancer treatment. Unfortunately, a majority of current strategies utilize either genetic engineering or chemical modifications which have a number of significant drawbacks. To address these concerns our lab developed Prosthetic Antigen Receptors (PARs), a non-genetic system, to direct selective cell-cell interactions. PARs are formed by engineering fusion proteins that contain a scFv fused to two E. Coli dihydrofolate reductase (DHFR2) molecules which spontaneously assemble into octomeric chemically self-assembled nanorings (CSANs) upon the addition of a chemical dimerizer, bis-methotrexate (Bis-MTX). This thesis provides the field with foundational work addressing the functional effects of PARs in a solid tumor model, both in vitro and in vivo. Chapter 2 initially addresses the in vivo stability, circulation and tissue distribution of CSANs using a radiolabeled construct affording the direct visualization of in vivo tissue localization and ex vivo organ biodistribution by microPET/CT imaging and tissue-based gamma counting, respectively. As anticipated, CSANs displayed an in vivo profile between that of rapidly clearing small molecules and slow clearing antibodies. In Chapter 3 we discuss both the in vitro and in vivo development of anti-EpCAM PARs which are then applied to an in vivo orthotopic Breast Cancer Model. Our results demonstrated that anti-EpCAM/anti-CD3 PARs were found to stably bind T-cells for >4 days, and treating EpCAM+ MCF-7 breast cancer cells with anti-EpCAM/anti-CD3 PAR-functionalized T-cells resulted in the induction of IL-2, IFN-γ and MCF-7 cytotoxicity. Furthermore, an orthotopic breast cancer model validated the ability of anti-EpCAM/anti-CD3 PAR therapy to direct T-cell lytic activity towards EpCAM+ breast cancer cells in vivo leading to tumor eradication. Following the in vivo success of anti-EpCAM PAR therapy we chose to explore, Chapter 4, the use of both anti-EpCAM/anti-CD3 and anti-CD133/anti-CD3 CSANs in conjunction. Notably, when applied to a triple negative breast cancer model we found a synergistic effect from targeting EpCAM and CD133; in fact, full tumor eradication was only elicited when both antigens were simultaneously targeted. Due to the growing need of a more modifiable CSAN platform we developed monovalent streptavidin (mSA)-DHFR2 fusion proteins. When incorporated into bispecific CSANs, Chapter 5, we were able to rapidly analyze the activation and directed cell lysis of several targeting constructs simultaneously. Additionally, in Chapter 6 we further adapted mSA CSANs into a universal cell membrane labeling technique. This was accomplished by hydrophobically inserting phospholipids conjugated to biotin into the cell membrane. Heterobifunctional CSANs containing mSA are then stably bound to the biotin moieties. PAR therapy has several unique innovations, such as the capability of quickly reprogramming T cell membranes in hours rather than in days which is typically seen with standard CAR therapy. Additionally, our approach has the capability to remove the PARs from T-cells by incubation with the FDA approved antibiotic trimethoprim, at clinically relevant concentrations, allowing the pharmacological deactivation of T cells. Collectively, our results demonstrate PAR modified T-cells have the potential to be a viable cancer immunotherapy targeting solid tumors.

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University of Minnesota Ph.D. dissertation. December 2017. Major: Medicinal Chemistry. Advisor: Carston Wagner. 1 computer file (PDF); xx, 250 pages.

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Petersburg, Jacob. (2017). Prosthetic Antigen Receptors As A Platform For Solid Tumor Immunotherapy. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/202157.

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