Document Type

Dissertation

Date of Degree

Fall 2014

Degree Name

PhD (Doctor of Philosophy)

Degree In

Immunology

First Advisor

George J. Weiner

Abstract

Cancer immunotherapy has proven to be challenging as it depends on overcoming multiple mechanisms that mediate immune tolerance to self-antigens. In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. Based on recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (1) Inducing immunogenic tumor cell death with the chemotherapeutic drug Doxorubicin (Dox). Dox enhances the expression of "eat-me" signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DCs). Due to the vesicant activity of Dox, microparticles (MPs) made of PLGA (a biodegradable polymer) can safely deliver Dox intratumorally and are effective vaccine adjuvants; (2) Enhancing antigen presentation and T cell activation using anti-OX40; (3) Sustaining T cell responses by checkpoint blockade using anti-CTLA-4. In vitro, Dox MPs were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by the lymphoma cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared to soluble Dox. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T cell infiltration into distant lymphoma tumors leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation of three-step therapy for immunotherapy of lymphoma patients.

Furthermore, we designed another in situ immunization approach using PLGA MPs loaded with both Dox and CpG oligodeoxynucleotides (CpG). The addition of CpG was to further enhance the Dox MP design by including an agent that addresses Step Two in situ, by enhancing tumor antigen presentation by DCs. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs than soluble Dox. In vivo, Dox/CpG MPs combined with anti-CTLA-4 and anti-OX40 generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent a versatile, efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.

Public Abstract

Tumor immunotherapy aims to activate the patient’s immune system to recognize and attack tumor cells, thus eradicating tumor cells remaining after chemotherapy. This can be achieved by many approaches, one of which is in situ immunization, which generates antitumor immune responses through manipulating the tumor itself.

Microparticles (MPs) made of a biodegradable polymer and injected into the tumor degrade slowly over time. When loaded with a drug, they allow for controlled release of the loaded drug into the tumor. Based upon this attractive property, we designed a three-step approach to in situ immunization: (1) Enhancing tumor cell death by loading MPs with Doxorubicin (Dox), a chemotherapy drug that enhances the expression of “eat-me” signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DCs); (2) Enhancing tumor antigen presentation and T cell activation with CpG (a bacterial component) loaded into the MPs and/or anti-OX40 (an antibody that stimulates OX40 on T cells) injected into the blood; (3) Sustaining T cell responses with anti-CTLA-4 (an antibody that blocks the inhibitory action of CTLA-4 on T cells) injected into the blood.

We show that our drug-loaded MPs are more toxic to tumor cells and less toxic to DCs. Using different tumor models in mice, we show that our three-step approach can generate a potent, long-lasting antitumor response to B cell lymphoma and reduce tumor burdens of T cell lymphoma and of melanoma. With the versatility of our design, three-step therapy thus represents an attractive immunotherapy option for patients.

Keywords

publicabstract, anti-CTLA-4, anti-OX40, biodegradable microparticles, CpG, Doxorubicin, in situ immunization

Pages

xvi, 117 pages

Bibliography

Includes bibliographical references (pages 106-117).

Copyright

Copyright 2014 Amani Makkouk

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