Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.Cataloged from PDF version of thesis. Page 175 blank.Includes bibliographical references (p. 161-174).Immunostimulatory therapies that activate immune response pathways are of great interest for overcoming the immunosuppression present in advanced tumors. Agonistic antibodies against the co-stimulatory receptors CD40 and CD137, Toll-Like Receptor (TLR) ligands such as CpG oligonucleotides, and immunostimulatory cytokines such as IL-2 have all previously demonstrated potent, synergistic anti-tumor effects. However, the clinical use of such therapies is significantly hampered by the severe, dose-limiting inflammatory toxicities provoked upon systemic exposure. We hypothesized that by anchoring immunomodulatory agents to lipid nanoparticles we could retain the bio-activity of therapeutics in the local tumor tissue and tumordraining lymph node, but limit systemic exposure to these potent molecules. We first prepared liposomes bearing surface-conjugated anti-CD40 and CpG and assessed their therapeutic efficacy and systemic toxicity compared to soluble versions of the same immuno-agonists, injected intratumorally in established solid tumors in mice. Anti-CD40/CpG-coupled liposomes significantly inhibited primary tumor growth and induced a survival benefit similar to locally injected soluble anti-CD40+CpG. Biodistribution analyses following local delivery showed that the liposomal carriers successfully sequestered anti-CD40 and CpG in vivo, reducing leakage into systemic circulation while allowing draining to the tumor-proximal lymph node. Contrary to locally administered soluble immunotherapy, anti-CD40/CpG liposomes did not elicit significant increases in serum levels of ALT enzyme, systemic inflammatory cytokines, or overall weight loss, confirming that off-target inflammatory effects had been minimized. Thus, these results confirmed the development of a delivery strategy capable of inducing robust antitumor responses concurrent with minimal systemic side effects. We next assessed the dissemination of the tumor-specific immune response that had been primed by locally administered, liposome-conjugated therapy. Since anti-CD40/CpG-coupled liposomes were unable to consistently induce the rejection of a secondary distal tumor challenge, we adapted the strategy of liposome-coupled delivery for the administration of anti-CD 137 and IL-2, two potent T cell-stimulatory agents. Local intra-tumoral therapy using anti-CD137-liposomes + IL-2-liposomes induced the highly potent inhibition of primary treated tumors and achieved a majority of complete cures, while successfully minimizing systemic exposure and eliminating symptoms of inflammatory toxicity, including lethality. In addition, 100% of anti-CD 137 + IL-2 liposome-treated mice were protected against a secondary distal tumor challenge, and demonstrated a significant delay in the progression of simultaneously inoculated, distal untreated tumors. Subsequent analyses confirmed that anti-CD137-liposomes and IL-2-liposomes bound specifically to cytotoxic T cells (CTLs) within the treated tumor, and that the depletion of CTLs abrogated the therapeutic anti-tumor response. Overall, these results indicated the effective local priming of an adaptive tumor-specific response, capable of mediating local, systemic, and memory anti-tumor immunity. The versatility of this liposome conjugation strategy suggests that we have developed a generalizable tool enabling the local delivery of highly potent immunomodulatory agonists in the absence of systemic toxicity, which could substantially improve the clinical applicability of such agents in cancer therapy.by Brandon Kwong.Ph.D
