Publication Date

2012-08-25

Availability

Open access

Embargo Period

2012-08-25

Degree Name

Master of Science (MS)

Department

Cancer Biology (Medicine)

Date of Defense

2012-05-02

First Committee Member

Eli Gilboa

Second Committee Member

Diana Lopez

Third Committee Member

Marta Torroella-Kouri

Fourth Committee Member

Joseph Rosenblatt

Fifth Committee Member

Mark Pegram

Abstract

Tumors that develop in patients with fully functional immune systems evolve different mechanisms of immune suppression that must be counteracted for tumor immunotherapy to be effective. The proposed therapeutic aims to co-stimulate T-cells via OX40, a co-stimulatory receptor expressed on activated T-cells. Signaling via OX40 leads to prolonged survival of activated T-cells, enhanced memory formation, and protection against immunosuppression. In addition to boosting co-stimulation, we aim to enhance T-cell activation by downregulating Cbl-b, an E3 ubiquitin ligase that suppresses T-cell activation. An RNA aptamer that binds OX40 was conjugated to anti-Cbl-b siRNA and was shown by a luciferase reporter assay to have approximately 80% knockdown efficiency. Its co-stimulatory function as a monomer was tested using proliferation assays, but showed no effect on polyclonal activated CD4+ T-cells. When the OX40 aptamer component was dimerized, the OX40-Cbl-b siRNA conjugate was shown to enhance activation of ova-specific CD4+ T-cells when compared to the control OX40-GFP siRNA conjugate, or monoclonal OX40 antibody. After testing for in vivo costimulatory effects, the conjugates were not found to be effective. Development of a trimeric OX40 aptamer and conjugate may yield better results in vivo as it would emulate the trimeric nature of OX40L.

Keywords

Aptamer; siRNA; OX40; Cbl-b; T-cell activation; Immunosuppression

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