Document Type

Dissertation

Date of Degree

Summer 2014

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacy

First Advisor

Aliasger K Salem

Abstract

Immunotherapy has been widely investigated in cancer, infectious diseases and allergies for prevention or amelioration of disease progression. In the case of vaccines, the key cellular target in stimulating an effective and appropriate immune response is the professional antigen presenting cell or dendritic cell (DC). Cancer vaccines are primarily aimed at the activation of a tumor-specific cytotoxic T lymphocyte (CTL) response whilst vaccines to allergies are aimed at reducing IgE responses. Such vaccines normally involve the administration tumor-associated antigens (TAAs) for cancer, or antigens (Ags) derived from infectious microbes and allergens in the case of allergies. Ags, whether derived from tumor or allergen, can be combined with adjuvants, that include immunostimulatory molecules recognized by the pathogen associated receptors expressed by DCs and can trigger the activation/maturation of DCs. Co-delivery of an appropriate adjuvant with an Ag can stimulate DCs to subsequently promote a robust Ag-specific CTL response which may favor anti-tumor immunity.

Cancer vaccines have been widely investigated in the clinics as a complementary therapy to surgery, radiation and chemotherapy. Activation of CTLs against tumor cells that express TAAs could lead to the complete eradication of a cancer and prevent its reoccurrence. In this study I developed microparticles using a polyanhydride polymer prepared from 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) that has shown inherent adjuvant properties. I prepared 50:50 CPTEG:CPH microparticles encapsulating a model tumor Ag, ovalbumin (OVA), and synthetic oligonucleotide containing an unmethylated CpG motif, CpG, as an adjuvant. CpG has shown significant potential as an adjuvant for TAA-based vaccines leading to significant anti-tumor immune activity. I have shown that mice vaccinated with OVA-encapsulated 50:50 CPTEG:CPH microparticles developed OVA-specific CTL responses. These mice showed enhanced survival compared to the control treatment groups when challenged with OVA expressing tumor cells .

In a more novel in-situ cancer vaccine, TAAs from dying tumor cells (caused by certain chemotherapeutic drugs) can be used as the source of Ags delivered to DCs. The presence of an adjuvant with dying cancer cells can assist in appropriate maturation of DCs so as to promote the generation of an effective tumor/TAA-specific CTL response against released TAAs. In this work I developed a therapeutic in situ tumor vaccine encapsulating a chemotherapeutic drug and CpG. Doxorubicin (Dox) is a widely used chemotherapeutic drug that induces tumor cells to undergo an immunogenic form of apoptosis. Sustained release of Dox in solid tumors of mice can cause the release of a variety of TAAs which can be presented by DCs and, in the presence of CpG, stimulate a strong anti-tumor CTL response. I prepared formulations of poly(lactic-co-glycolic acid) (PLGA) particles loaded with Dox and CpG which demonstrated sustained release of their cargo. I show that among various formulations of Dox and CpG, co-delivery of Dox and CpG in the same PLGA particles in-vivo showed the highest reduction in tumor growth and longest survival when compared to treatment groups of PLGA particles delivering Dox and CpG either alone or in combination.

PLGA particles have also been investigated as a prophylactic vaccine delivery system that generates a robust Ag-specific CTL response. This system has been employed for the development of vaccines against various infectious diseases and allergies. However, there has been conflicting opinions regarding the optimum size of PLGA particles required to stimulate an active CTL response. Thus, I developed different sizes of PLGA particles encapsulating OVA and CpG to study the relationship of particle size with the magnitude of OVA-specific CTL responses. I showed that the degree of particle uptake and activation of DCs increased with decreasing size of PLGA particles. I also showed that immunization of mice with 300 nm sized particles demonstrated a higher proportion of OVA-specific CTLs and increased the secretion of IgG2a antibody responses. I also evaluated the efficacy of these particles with a clinically relevant Ag, Dermatophagoides pteronyssinus-2 (Der p2). Mice vaccinated with different sizes of PLGA particles loaded with CpG and coated with Der p2 displayed different magnitudes and types of immune activation against Der p2. The small sized particles decreased the airway hyperresponsiveness associated with allergy-induced asthma. The presence of CpG in the PLGA particle vaccines also reduced the airway hyperresponsiveness.

This thesis research has contributed to the identification and development of a delivery system for Dox in combination with CpG which gives sustained release of these molecules within tumors and show the longest survival in tumor bearing mice. This study also optimized the size of PLGA particles for the delivery of vaccine to produce a robust Ag specific immune response for development of vaccination against intracellular diseases, cancer and allergy.

Keywords

Allergy, Cancer, drug delivery, injectable, PLGA, Vaccine

Pages

xix, 167 pages

Bibliography

Includes bibliographical references (pages 148-167).

Copyright

Copyright 2014 Vijaya B. Joshi

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