Date on Master's Thesis/Doctoral Dissertation

5-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Committee Chair

Shirwan, Haval

Author's Keywords

SA-4-1BBL; Adjuvant; Cancer; SA-OX40L; Therapeutic vaccine; MPLA

Subject

Cancer vaccines; Cancer--Immunotherapy

Abstract

Cancer accounts for nearly one-quarter of deaths in the United States, exceeded only by heart diseases. Despite the development of various strategies to treat cancer, it remains one of the most deadly diseases worldwide due to the limited effects of treatments available. The limited efficacy of these current treatment modalities, such as surgery, radiotherapy, and chemotherapy, are often due to their association with adverse side effects arising from lack of specificity for tumors, and most importantly their failure of eliminating residual and micro-metastatic tumors, which can lead to recurrences. Therefore, there is a dire need to develop tumor-specific therapies that not only eliminate primary tumors, but also micro-metastasis and prevent recurrences. In this regard, therapeutic cancer vaccines based on tumor-associated antigens (TAAs) has evolved as a promising approach due to their safety profile, ease of production, storage, transportation, administration to a broad patient population and most importantly establishment and/or maintenance of long-term immunological memory critical for the control of recurrences, a major cause of cancer death. However, despite theoretical promise, development of therapeutic cancer vaccines has been facing numerous set-backs mostly due to the weak immunogenicity of T AAs, tolerance to self-T AAs and various immune evasion mechanisms employed by progressing tumors. Therefore, we hypothesized that use of natural costimulatory ligands of TNF family as adjuvant may overcome these limitations due to their effect on cells of innate, adaptive, and regulatory immunity without any sign of toxicity. The tumor necrosis factor receptor (TNFR)/TNF superfamily represents a crucial group of costimulatory receptor/ligands as most of the receptors of this family are inducibly expressed on various immune cells. Costimulatory receptors that are inducibly expressed or upregulated on activated T cells may serve as preferred targets for immunomodulation due to their potential to selectively target antigen-experienced T cells for expansion, survival, and establishment of long-term immunological memory. Among these family members, 4-1BB/4-1BBL signaling has recently been much appreciated as its signaling provides the essential survival signals, particularly in CD8+ T cells. 4-1BB signaling into T cells allows CD8+ T cell expansion, cytokine production, development of CTL effector function, and prevention of apoptotic cell death by up-regulating anti-apoptotic Bcl-xL and Bcl-2 molecules. As the aim of tumor immunotherapy is to generate long-lasting immune response, particularly CD8+ T cell specific response, for the destruction of tumor cells, in this project, we focused on the utilization of 4-1 BBL either alone or in combination with other immunomodulators, as a component of TAA-based subunit vaccines and tested its efficacy in preclinical mice tumor models. First, we report that a single immunization with a therapeutic vaccine formulation containing novel form of soluble SA-4-1BBL, and survivin (SVN), a bona fide self antigen, resulted into the eradication of SVN-expressing 3LL tumors in 75% of mice in the absence of autoimmunity. The efficacy of vaccine was further improved to complete tumor eradication with an additional vaccination 6 days after the first vaccination. CD8+ T cells and NK cells effector function was found to be critical for the efficacy of vaccine, but not the CD4 + T cells. Next, we tested the vaccine formulation containing combination of SA-4-IBBL and toll-like receptor 4 agonist monophosphoryl lipid A (MPL) with distinct mechanisms of action as a novel adjuvant system. A single immunization with both adjuvants and HPV E7 protein resulted in eradication of 100% of E7 expressing TC-1 tumors. Combined adjuvants had better therapeutic efficacy over the individual adjuvants, while SA-4-1BBL monotherapy outperformed MPL, 80% vs. 50%. Similarly, a single vaccination with SVN resulted in control/eradication of established 3LL pulmonary metastases that was further improved by a booster injection. The therapeutic efficacy of combined adjuvants as well as SA-4-1BBL as monotherapy was achieved in the absence of detectable toxicity and correlated with enhanced CD8+ T cell function and increased intratumoral CD8+ T effector/CD4+FoxP3+ T regulatory cell ratio. In marked contrast, vaccination with MPL as monotherapy resulted in an unfavorable intratumoral CD8+ T effector/CD4+FoxP3+ T regulatory cell ratio that played a definitive role in vaccine efficacy. Depletion of T regulatory cells improved MPL efficacy to 100%, whereas elimination of CD8+ T cells totally abrogated the efficacy of combined adjuvants. In last, we report that combination of SA-4-1BBL and SA-OX40L, another member of TNF ligand family, was also able to eradicate TC-I tumors in 100% of mice. This efficacy was mainly dependent on CD8+ T cells as depletion of these cells completely abrogated the efficacy. Importantly, combination of these two ligands was also able to eradicate a 3-4 mm established tumors in 50% of mice. Taken together, these data provide important mechanistic insight into the mode of action of SA-4-1BBL alone or in combination either MPL or SA-OX40L adjuvants and demonstrate its utility as a novel adjuvant system for the development of therapeutic TAA-based subunit cancer vaccines with significant clinical implications. These data also shed lights into the mode of action of MPL and SA-OX40L as a part of vaccine adjuvant systems and set the stage for their utilization in the development of new vaccine strategies.

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