DOI

10.17077/etd.5ttr-wjy3

Document Type

Dissertation

Date of Degree

Summer 2019

Access Restrictions

Access restricted until 09/04/2021

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacy

First Advisor

Salem, Aliasger K.

First Committee Member

Donovan, Maureen D.

Second Committee Member

Doorn, Jonathan A.

Third Committee Member

Stevens, Lewis L.

Fourth Committee Member

Govindarajan, Ramprakash

Abstract

Cancer immunotherapy is an approach to cancer therapy that involves the enhancement of the cancer patient’s own innate and/or adaptive immune systems to attack their own cancer. Clinically available cancer immunotherapies rely on different strategies: infusion of ex vivo manipulated autologous dendritic cells (DCs), infusion of genetically engineered autologous cytotoxic CD8+ T lymphocytes, stimulation of T lymphocyte proliferation, or inhibition of immunosuppressive pathways to improve T lymphocyte effector function. Nonetheless, only a small percentage of cancer patients receive benefit from immunotherapies and thus further improvements in clinical outcomes are required. Among numerous other therapeutic immunotherapies strategies being developed and tested, adenovirus serotype 5-based vectors (Ad5) have been well studied in preclinical and clinical settings. Preclinical research has shown that vaccination of mice with Ad5-OVA (an Ad5 encoding a model tumor antigen, chicken ovalbumin (OVA)) results in activation and proliferation of OVA-specific CD8+ T lymphocytes capable of specific killing of tumor cells that express OVA. This dissertation evaluates the potential of polymeric nanoparticles (NP) loaded with all-trans retinoic acid (ATRA), a vitamin A derivative with potent immunostimulatory effects, to improve the immunostimulatory and therapeutic effects of Ad5-OVA in a murine E.G7-OVA tumor model, a well described model that can be used for studying the immune response to Ad5-based immunotherapies.

In the first part of this work, poly(lactide-co-glycolide) NP loaded with ATRA (ATRA-PLGA-NP) were prepared and characterized. Next, the antitumor effect and the magnitude of the OVA-specific immune response due to Ad5-OVA vaccination versus ATRA-PLGA-NP (or ATRA soluble) plus Ad5-OVA combination treatment were compared in vivo. The results showed that the combination treatment using ATRA-NP, but not ATRA soluble, resulted in enhanced survival and enhanced levels of OVA-specific CD8+ T lymphocytes in peripheral blood, spleen, and tumor.

Next, cRGD- and mannose-functionalized PLGA-PEG NP were developed in an attempt to actively target the tumor neovasculature and DC-rich organs, respectively. The functionalization efficacy was confirmed by ex vivo fluorescence imaging studies. In vivo studies using E.G7-OVA-challenged mice showed that treatment with ATRA-loaded cRGD-functionalized PLGA-PEG-NP + Ad5-OVA, despite not enhancing the levels of OVA-CD8+ T lymphocytes in peripheral blood, substantially enhanced survival compared to either the combination of Ad5-OVA + non-functionalized ATRA-PLGA-PEG-NP or Ad5-OVA + conventional ATRA-PLGA-NP. On the contrary, treatment with mannose-functionalized PLGA-PEG-NP + Ad5-OVA, despite optimally enhancing the levels of OVA-CD8+ T lymphocytes in peripheral blood (compared to all other treatment groups), did not lead to enhanced survival compared to either the combination of Ad5-OVA + non-functionalized ATRA-PLGA-PEG-NP, Ad5-OVA + conventional ATRA-PLGA-NP, and over Ad5-OVA treatment alone. Although not investigated further in this dissertation, it was speculated that the observed trend in survival benefit provided by ATRA-PLGA-PEG-cRGD-NP + Ad5-OVA over the other NP formulations may have been due to higher levels of ATRA within the TME due to actively targeting the tumor vasculature, corroborating previous studies which demonstrated that ATRA functions as a potent stimulator of anti-tumor cellular immune responses within the tumor. The paradoxical results obtained with mannose-functionalized PLGA-PEG-NP are less readily explained.

In conclusion, it was demonstrated in this work that the co-administration of Ad5-OVA and ATRA-loaded NP formulations enhanced the tumor specific cellular immune response and the survival of tumor challenged mice compared to vaccination with Ad5-OVA alone.

Keywords

Adenovirus, All-trans retinoic acid, cRGD, Mannose, Nanoparticles, PLGA

Pages

xiii, 144 pages

Bibliography

Includes bibliographical references (pages 123-141).

Copyright

Copyright © 2019 Cristina Maria de Barros

Available for download on Saturday, September 04, 2021

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