Publication Date

2017-09-29

Availability

Open access

Embargo Period

2017-09-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2017-08-11

First Committee Member

Eli Gilboa

Second Committee Member

Thomas Malek

Third Committee Member

Diana Lopez

Fourth Committee Member

Robert Levy

Fifth Committee Member

Duane Mitchell

Abstract

Cancer is one of the leading causes of death in the world. Current treatments for cancer include chemotherapy, radiation therapy, surgery, immunotherapy and transplantation. While these treatments seem successful in some cases, they fail to provide long-term protection. Chemotherapy and radiation therapy lack specificity whereas immunotherapy, although it has the specificity is not very effective due to the ability of cancer cells to invade the host immune system. Over the years, many tumors have developed ways to prevent or escape their immune-based elimination. Therefore, counteracting tumor-associated (derived) immune suppression represents a potential platform for therapeutic intervention. A key mediator of immune suppression is the cytokine, transforming growth factor beta (TGFß) secreted by most tumor cells. The TGFß signaling pathway plays a vital and dual role in the progression of cancer. In the early stages of cancer, TGFß signaling exhibits tumor suppressive effects such as cell cycle arrest, apoptosis and inhibition of cell-cell adhesion. During later stages, TGFß promotes metastasis via evasion of immune surveillance, pro-metastatic cytokine secretion and production of autocrine mitogens. Due to the profound tumor promoting effects of TGFß, many clinical studies in cancer patients have targeted the TGFß pathway using antisense oligonucleotide, antibodies, soluble TGFßRII-Fc fusion decoys, or small molecule TGFßRI and Smad3 inhibitors. Owing to the broad range of roles TGFß plays in tissue homeostasis, these non-specific treatments elicit minimal clinical response and significant side effects. A likely reason, a common limitation of many cancer drugs, is that the physiological roles of TGFß in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFß may be sufficient to inhibit tumor growth. One approach to avoid the broad effects of TGFß therapy is to limit TGFß inhibition to tumor specific CD8+ T cells. Here we propose to develop a clinically feasible and broadly applicable method to render vaccine-activated CD8+ T cells resistant to TGFß inhibition using RNAi to inhibit key steps in the canonical TGFß signaling pathway. The siRNA is targeted to vaccine activated CD8+ T cells in the mouse by conjugation to a 4-1BB binding oligonucleotide (ODN) aptamer ligand. In this work, siRNAs targeting different mediators of the TGFß pathway, namely Smad4, TGFßRII and Foxp1, were conjugated to a murine 4-1BB aptamer. In vitro, all tested candidates knocked down their targets in a 4-1BB dependent delivery manner. When tested for function in vitro, only 4-1BB-Smad4 conjugate rendered T cells partially resistant to TGFß inhibition, and treatment of tumor bearing mice with systemically administered 4-1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFß to tumor-specific T cells will not interfere with its multiple physiological roles and hence reduce the risk of toxicity. TGFß mediated inhibition of tumor immunity is a major impediment in developing effective immune-based treatment for cancer. This approach serves as an adjunct therapy in combination with other forms of immune-based therapy applicable to patients. This study described 4-1BB aptamer-mediated delivery of siRNAs that knock-down key mediators of the canonical TGFß signaling pathway, namely Smad4, TGFßRII and Foxp1, in activated CD8+ T cells to render them insensitive to TGFß signaling, thereby enhancing vaccine-induced antitumor immune response.

Keywords

TGFbeta; cancer immunotherapy; preclinical mouse models; tumor targeting; aptamers; siRNA

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