Publication Date

2013-07-19

Availability

Embargoed

Embargo Period

2015-07-19

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2013-07-11

First Committee Member

Eli Gilboa

Second Committee Member

Diana Lopez

Third Committee Member

Robert Levy

Fourth Committee Member

Alberto Montero

Fifth Committee Member

Geoffrey Stone

Abstract

The host immune system plays an important role in the development and control of cancer, yet cancer cells often elaborate mechanisms to evade this regulation. Transforming growth factor beta (TGFβ) is a pleiotropic cytokine often overexpressed by cancers due to TGFβ’s growth-promoting effect on tumor cells and suppressive function on multiple cell types of the innate and adaptive immune systems. Genetic ablation of TGFβ signaling induces significant and toxic immune responses against host tissues, but when signaling is selectively ablated in cells of the adaptive immune system, such as CD8+ T cells, they become capable of mediating protection against various kinds of tumor challenge upon adoptive transfer. Similar results occur upon systemic blockade of TGFβ using monoclonal antibodies or small molecule inhibitors, yet dose-limiting toxicities exists and have been observed in human patients. The challenge addressed in this thesis work is to selectively render CD8+ T cells activated by therapeutic vaccination against tumor antigens transiently resistant to TGFβ-mediated immune suppression for the purpose of enhancing their proliferation and function against tumor cells in a clinically feasible manner. The approach consists of an RNA aptamer that binds murine 4-1BB, a costimulatory molecule up-regulated on CD8+ and CD4+ cells shortly after activation fused to small interfering RNAs (siRNAs) directed against message for various components of the TGFβ signaling cascade, including Smad3 and Arkadia/RNF111. A method for rapidly identifying and characterizing siRNAs amenable to 4-1BB aptamer incorporation is described in which the most active candidates were identified in multiple selection algorithms by choosing ones with the lowest melting temperatures. Not all siRNAs screened showed equivalent function between conjugated and unconjugated siRNA, yet this approach successfully identified candidates suitable for in vitro and in vivo studies. Conjugates targeting Smad3 and RNF111 reduced target mRNA expression in in vitro assays using activated OT-1 cells and in vivo studies where conjugates potentiated the effects of irradiated whole-cell vaccines in the B16-F10 and 4T-1 model systems relative to control conjugates. Additionally, using a chemical carcinogenesis model system, dimeric 4-1BB-Smad3 conjugates exhibited a trend towards increasing progression-free survival when administered shortly after chemical insult, yet a similar treatment with TGFβ monoclonal antibody worsened survival, demonstrating toxicity of systemic TGFβ blockade. In the final part of this dissertation, a human 4-1BB RNA aptamer selection is described. By combining traditional selection methods with emulsion polymerase chain reaction (ePCR) to reduce PCR enzymatic bias and high-throughput sequencing (HTS) of early selection rounds to identify highly enriching sequences many (>5) RNA aptamer candidates were identified, including one cross-reactive with both human and murine 4-1BB homologs. These binding RNA aptamer candidates remain to be tested for function (costimulation of human T cells and/or aptamer-mediated siRNA delivery). Thus, this dissertation work describes a specific application of RNA aptamer-siRNA conjugates in antagonizing tumor-induced immune suppression as well as describing a new approach to rapidly identify high-affinity RNA aptamer ligands.

Keywords

tumor immunology; small interfering RNA; RNA aptamer; SELEX; transforming growth factor beta; 4-1BB

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