Antibody mediated targeting for the treatment and diagnosis of colorectal cancer
Urva, Shweta Ramesh
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An estimated 1 million new cases of colorectal cancer are diagnosed worldwide each year, leading to almost 500,000 deaths annually. Traditional chemotherapeutic options currently available for treatment are associated with poor response rates; wide spread systemic toxicity and development of drug resistance. Monoclonal antibody based approaches have the potential to specifically target antigens identified to be overexpressed on cancerous cells compared to normal organs thereby increasing treatment selectivity, however this approach is plagued with numerous problems such as impaired tumor blood supply, poor lymphatic drainage, inadequate and slow antibody infiltration across the tumor. Therefore, the exists a significant need to develop new treatment approaches with the ability to selectively target necrotic tissue as well as maintain sustained concentrations within the tumor to have a curative effect. The overall goal of this research is to investigate a new multicomponent drug delivery strategy comprising of an "antibody targeting fraction", and an "effector fraction" comprising of an active cytotoxic drug conjugated with a peptide acting as a cell permeation enhancer. The major hypothesis of this dissertation is that the proposed targeting approach would increase the tumor selectivity of the model drug, doxorubicin while minimizing drug concentrations at distant normal organs. This in turn would enable cancer treatment at lower doses compared to administration of the drug alone. A LS174T adenocarcinoma xenograft model expressing tumor associated antigen (TAA), carcinoembryonic antigen (CEA) was developed in athymic nude mice. Plasma pharmacokinetics of T84.66 antibody, directed against carcinoembryonic antigen (CEA) was studied over a dose range (1, 10 and 25 mg/kg) in the presence and absence of CEA expressing xenografts. T84.66 plasma disposition was linear across the dose range studied in the absence of xenografts. Compared to control mice, T84.66 clearance was significantly increased in the presence of xenografts suggesting the presence of a "target mediated elimination" pathway. A physiologically based pharmacokinetic (PBPK) model was developed to characterize the plasma disposition data and the model was further used to predict antibody concentrations within the tumor tissue. Experimentally determined tumor concentrations compared well with the model predictions. Further investigation of T84.66 pharmacokinetics demonstrated that antibody clearance increased with increasing tumor volumes. Plasma concentrations in xenograft bearing mice on day 7 following T84.66 administration was significantly less than corresponding values in control mice (p < 0.05). Based on these results we hypothesized that plasma T84.66 levels can be used as a marker for screening of CEA positive tumors. Based on predetermined threshold values of T84.66 plasma concentrations, we were able to screen for CEA bearing intraperitoneal tumors with a sensitivity of 93.3 % and specificity of 100%. A high performance liquid chromatographic (HPLC) assay was developed to quantify doxorubicin concentrations in murine biological samples. Doxorubicin plasma, tissue and tumor pharmacokinetics were studied across a dose range following intravenous administration (2, 5 and 10 mg/kg). A physiologically based pharmacokinetic (PBPK) model capable of characterizing and predicting doxorubicin disposition in plasma and various tissues was developed. Administration of doxorubicin in conjugation with the transduction domain-low molecular weight protamine (LMWP) known to facilitate cell penetration failed to demonstrate any significant difference in drug disposition when compared to administration of doxorubicin alone. Additionally electrostatic binding of the doxorubicin transduction domain conjugate (DOX-LMWP) to T84.66 (anti-CEA IgG) also failed to enhance tumor concentrations of doxorubicin. This dissertation thoroughly examines various aspects of the proposed drug delivery strategy and proposes a new screening method for colorectal cancer, based on the target mediated elimination of an antibody directed against the selectively overexpressed tumor associated carcinoembryonic antigen.