Publication Date

2016-10-28

Availability

Open access

Embargo Period

2016-10-28

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2016-10-14

First Committee Member

Eli Gilboa

Second Committee Member

Robert Levy

Third Committee Member

Diana Lopez

Fourth Committee Member

Thomas Malek

Fifth Committee Member

Lina Shehadeh

Sixth Committee Member

Smita K Nair

Abstract

Cancer is among the leading causes of death worldwide. According to the National Cancer Institute, in 2012, there were 8.2 million cancer-related deaths worldwide, 14 million new cases and the number of new cancer cases is expected to rise to 22 million within the next two decades. Currently, Standard therapies for most cancers include surgical removal, radiotherapy using ionizing radiation or chemotherapy using drugs such as doxorubicin etc. Although these therapies help control the disease severity, they lack specificity and elicit significant side effects through their targeting of normal tissues. These off-target effects range from blood disorders, hair loss, diarrhea and sometimes long-term damage to organs including lung, liver etc., thus affecting the patients’ lifestyle severely. In addition, these therapies are not curative, where patients eventually have recurrence of cancer. Immunotherapy for cancer is an attractive alternative as it has the potential to circumvent these pitfalls. The immune system is capable of recognizing cancer cells specifically, thus minimizing the off-target effects on healthy tissue. Immune system can also prevent recurrence of the disease due to its ability to generate long-term memory responses, thus conferring life-long protection. This feature of the immune system has helped in the eradication of deadly diseases such as small pox and can be harnessed against cancer to control the disease. However, immune response elicited against cancer even in response to vaccine or radiation is often weak and not curative. One approach to potentiate antitumor immunity is to enhance the long-term persistence of vaccine or radiation-induced anti-tumor CD8+ T cells. A characteristic CD8+ T cell response involves parallel generation of short-lived effector cells (SLEC) and memory precursor effector cells (MPEC) from naïve responding cells. Upon activation, about 95-98% of the T cells differentiate into SLECs, giving rise to immediate effectors, while only 2-5% differentiate into MPECs, giving rise to persistent memory subsets. The ability of these CD8+ memory subsets to confer host protection has been attributed to their enhanced proliferative capacity upon antigen re-encounter and their ability to mediate systemic immunity by residing in lymph nodes, helping to control the metastatic load in cancers. To this end, generation of these CD8+ memory subsets during a vaccine induced response, suggests an approach to mount an optimal anti-tumor immune response. Over the last decade, profiling of the transcriptional factors expressed by different T cell subsets upon activation has provided important insights into memory T cell differentiation. High levels of transcription factors Tcf-7, Eomes, Bcl-6 have been shown to favor MPECs while high levels of T-bet, Blimp-1 and mTOR have been shown to favor SLECs. Increased IL-2 signaling in activated CD8+ T cells, via the binding of IL-2 cytokine to CD25 (IL-2Ra), promotes the development of SLEC via the coordinated expression of intracellular mediators such as Blimp-1, whereas reduced IL-2 signaling was shown to favor the development of memory CD8+ T cells. Although blocking anti-CD25 antibodies can be used to reduce IL-2 signaling, systemic administration of CD25 antibodies could exert off-target effects such as interfering with the function of CD25 expressing CD4+Foxp3+ regulatory cells and therefore increase the risk of autoimmune pathology. it’s also desirable to achieve a modest reduction in the strength of IL-2 signaling in activated CD8+ T cells rather than a complete shut-down. To achieve this, we took advantage of RNA interference. We used siRNAs designed against CD25 mRNA to downregulate the levels of CD25 to achieve a decrease in IL-2 signaling. These CD25 siRNA were conjugated to a 4-1BB aptamer which targets activated CD8+ T cells. Aptamers are single-stranded DNA, RNA or peptide molecules capable of binding their target protein with equal or greater affinities than antibodies. The 4-1BB RNA aptamer binds 4-1BB, a Tumor Necrosis Factor Receptor that gets upregulated on CD8+ T cells upon TCR activation. 4-1BB targeted delivery of CD25 siRNAs downregulated levels of CD25 at mRNA and protein level. Using 4-1BB aptamer as a targeting agent was crucial in delivering these siRNAs to activated CD8+ T cells as no effect was observed when these siRNAs were conjugated to a scrambled aptamer. Downregulation of IL-2 signaling in activated CD8+ T cells enabled their differentiation into MPECs. This was evident from the change in transcriptional profile where activated CD8+ T cells treated with 4-1BB-CD25 had reduced levels of Blimp-1 and increased levels of Bcl-6 and Tcf-7. A higher proportion of 4-1BB-CD25 treated cells re-expressed CD62L and also exhibited improved response to IL-7 cytokine. Systemic administration of the 4-1BB aptamer-CD25 siRNA conjugate via tail vein injection downregulated CD25 mRNA only in 4-1BB activated CD8+ T cells. Treatment of mice bearing 4T-1 mammary carcinoma with the 4-1BB aptamer-CD25 siRNA conjugates enhanced the antitumor response of a cellular vaccine as well as that of local radiation therapy. To validate the approach of using 4-1BB targeted delivery of siRNAs to activated CD8+ T cells to enhance their persistence, we generated siRNAs against Axin-1. Axin-1 is the rate-limiting component of the b-catenin destruction complex. Reduction in Axin-1 levels decreases the activity of the destruction complex, thus turning on the WNT pathway, leading to activation of b-catenin/Tcf-7 complex and transcription of genes responsible for CD8+ memory generation. We tested the ability of 4-1BB aptamer targeted delivery of an Axin-1 siRNA to CD8+ T cells to enhance CD8+ T cell memory development and antitumor activity. Indicative of the generality of the approach, 4-1BB aptamer targeted reduction of Axin-1 in CD8+ T cells, enhanced persistence of activated CD8+ T cells, potentiating vaccine mediated anti-tumor immunity. This study shows that aptamer-targeted siRNA therapeutics can be used to specifically modulate the function of circulating CD8+ T cells, skewing their development into long-lasting memory CD8+ T cells, and thereby potentiate antitumor immunity.

Keywords

Cancer Immunotherapy; Aptamer-siRNA therapeutics; Persistence of anti-tumor CD8+ T cells; 4-1BB aptamer; IL-2 signaling

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