Serum Albumin as a Carrier of Immunostimulatory Oligodeoxynucleotides for Cancer Therapy Public Deposited

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  • March 21, 2019
Creator
  • Guley, Patrick C.
    • Affiliation: Eshelman School of Pharmacy
Abstract
  • Toll-like receptor 9 (TLR9) is an endosomal receptor expressed on immune cells. The receptor recognizes microbial DNA, which contains a higher frequency of unmethylated CpG sequences than host DNA. Oligodeoxynucleotides containing such CpG motifs (CpG) are potent activators of TLR9, causing release of inflammatory cytokines and initiating both the innate and adaptive immune response. CpG has been successfully used to treat solid tumors, but its use is limited by its unfavorable pharmacokinetics. In preclinical studies, CpG was effective only when injected peritumorally; i.e., ineffective when administered systemically. In an attempt to overcome this limitation, CpG was derivatized with a maleimide moiety to allow a chemical reaction with free thiols. The derivative is referred to as CpG-mal throughout this dissertation. Albumin, the most abundant serum protein, contains one free thiol, Cys34, which comprises greater than 80% of the serum thiol content. Consequently, upon intravenous injection, the CpG-mal will react with circulating albumin to form a 1:1 conjugate in a predictable manner. Serum pharmacokinetics and biodistribution of phosphodiester CpG-mal was investigated in tumor-bearing mice using a PET/CT imaging procedure. For this series of studies, a novel tyrosine-containing crosslinker was synthesized to facilitate radioiodination of the CpG with 124I in high yields. Imaging studies revealed the reaction between the CpG-mal and albumin was fast enough to outcompete the high renal clearance of CpG, with the reaction complete within minutes. The new CpG-albumin conjugate displayed a similar distribution pattern and plasma half-life as albumin; half-life was increased 70-fold and tumor accumulation was increased 30-fold over control CpG. In vitro plasma stability studies showed that albumin conjugation lead to a 1.5-fold reduction in the rate of enzymatic degradation of phosphodiester CpG. An in vitro macrophage activation assay indicated that phosphodiester CpG-albumin conjugates were weak agonists of TLR9. However, phosphorothioate CpG-albumin adducts were able to interact with TLR9 to initiate cytokine release from J774 macrophage cells, although this activity was reduced compared to control phosphorothioate CpG. The activation was independent of crosslinker length, and introducing a reducible disulfide crosslinkage did not enhance the activity. Pharmacokinetics and biodistribution of phosphorothioate CpG were measured using [3H]-labeled CpG. The [3H]-CpG-mal had a longer plasma half-life than control CpG, however, the liver accumulation was significantly increased. This liver uptake led to a less striking increase in tumor accumulation of CpG-mal than control CpG. In vivo tumor growth inhibition studies using a CT26 colon carcinoma model showed both the CpG-mal and control CpG were equally efficacious and caused complete tumor regression in 6/10 mice. On the other hand, in the 4T1 model no tumor regression was observed presumably due to lack of tumor-associated antigens from the 4T1 cells.
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  • In Copyright
Advisor
  • Cho, Moo J.
Degree
  • Doctor of Philosophy
Graduation year
  • 2013
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