In Ho Song

Cancer-targeted Nucleic Acid Delivery and Quantum Dot Imaging Using EGF Receptor Aptamer-conjugated Lipid Nanoparticles

Co-application of fluorescent quantum dot nanocrystals and therapeutics has recently become a promising theranostic methodology for cancer treatment. We developed a tumor-targeted lipid nanocarrier that demonstrates notable efficacy in gene delivery as well as tumor bio-imaging. Coupling of aptamer molecules against the EGF receptor (EGFR) to the distal termini of lipid nanoparticles provided the carrier with tumor-specific recognition capability. The cationic lipid component, referred to as O,O’-dimyristyl-N-lysyl glutamate (DMKE), was able to effectively complex with anionic small-interfering RNA (siRNA). The hydrophobic quantum dots (Q-dots) were effectively incorporated in hydrophobic lipid bilayers at an appropriate Q-dot to lipid ratio. In this study, we optimized the liposomal formula of aptamer-conjugated liposomes containing Q-dots and siRNA molecules (Apt-QLs). The anti-EGFR Apt-QLs exhibited remarkable EGFR-dependent siRNA delivery as well as fluorescence imaging, which were analyzed in cultured cancer cells and tumor xenografts in mice. These results imply that the formulation of Apt-QLs could be widely utilized as a carrier for tumor-directed gene delivery and bio-imaging.

Immuno-PET Imaging and Radioimmunotherapy of 64Cu-/177Lu-Labeled Anti-EGFR Antibody in Esophageal Squamous Cell Carcinoma Model

Immuno-PET provides valuable information about tumor location, phenotype, susceptibility to therapy, and treatment response, especially to targeted radioimmunotherapy. In this study, we prepared antiepidermal growth factor receptor (EGFR) antibody via identical chelator, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA), labeled with 64Cu or 177Lu to evaluate the EGFR expression levels using immuno-PET and the feasibility of radioimmunotherapy in an esophageal squamous cell carcinoma (ESCC) model. Methods: Cetuximab was conjugated with p-SCN-Bn-PCTA and radiolabeled with 64Cu or 177Lu. In vitro EGFR expression levels were determined and compared using flow cytometry and cell binding assay. In vivo EGFR expression levels were evaluated via immuno-PET imaging of 64Cu-cetuximab and biodistribution analysis. Micro-SPECT/CT imaging, biodistribution, and radioimmunotherapy studies of 177Lu-cetuximab were performed in the ESCC model. Therapeutic responses were monitored using 18F-FDG PET and immunohistochemical staining. Results: 64Cu- or 177Lu-labeled antibodies showed high radiolabeling yield (>98%), stability (>90%), and favorable immunoreactivity. In vitro EGFR status measured by cell binding assay was correlated with the flow cytometry data. Immuno-PET, micro-SPECT/CT, and biodistribution demonstrated specific uptake in ESCC tumors depending on the EGFR expression levels. Tumor accumulation of 64Cu- and 177Lu-cetuximab was peaked at 48 and 120 h, respectively. Radioimmunotherapy with 177Lu-cetuximab showed significant inhibition of tumor growth (P < 0.01) and marked reduction of 18F-FDG SUV compared with that of control (P < 0.05). Terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity and Ki-67 staining indices increased and decreased, respectively, in the radioimmunotherapy group compared with other groups (P < 0.01). Conclusion: 64Cu-cetuximab immuno-PET represented EGFR expression levels in ESCC tumors, and 177Lu-cetuximab radioimmunotherapy effectively inhibited the tumor growth. The diagnostic and therapeutic convergence radiopharmaceutical 64Cu-/177Lu-PCTA-cetuximab may be useful as a diagnostic tool in patient selection and a potent radioimmunotherapy agent in EGFR-positive ESCC tumors.

Immuno-PET imaging based radioimmunotherapy in head and neck squamous cell carcinoma model

The epidermal growth factor receptor (EGFR) is one of the most comprehensively studied molecular targets in head and neck squamous cell carcinoma (HNSCC). However, inherent and acquired resistance are serious problems and are responsible for limited clinical efficacy and tumor recurrence. In this study, we evaluated the feasibility of immuno-positron emission tomography (PET) imaging and radioimmunotherapy (RIT) with 64Cu-/177Lu-PCTA-cetuximab in cetuximab-resistant SNU-1066 HNSCC xenografted model. The cellular uptake of 64Cu/177Lu-3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA)-cetuximab showed good correlation with western blot and flow cytometry analysis in EGFR expression level of various HNSCC cells. 177Lu-PCTA-cetuximab selectively killed cetuximab-resistant SNU-1066 cells in vitro. 64Cu-/177Lu-PCTA-cetuximab specifically accumulated in SNU-1066 tumor and those uptakes were peaked at 48 h and 7 day, respectively in biodistribution, PET and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. RIT with single dose of 177Lu-PCTA-cetuximab exhibited significant tumor regression and markedly reduced 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) uptake, compared to other groups. Proliferation index were dramatically decreased and apoptotic index increased in RIT group. These results suggest that a diagnostic and therapeutic convergence radiopharmaceutical, 64Cu-/177Lu-PCTA-cetuximab has the potential of target selection using immuno-PET imaging and targeted therapy by RIT in EGFR expressing cetuximab-resistant HNSCC tumors.

Development of 64Cu-NOTA-Trastuzumab for HER2 targeting: radiopharmaceutical with improved pharmacokinetics for human study

The purpose of this study was to develop 64Cu-labeled trastuzumab with improved pharmacokinetics for human epidermal growth factor receptor 2 (HER2). Methods: Trastuzumab was conjugated with SCN-Bn-NOTA and radiolabeled with 64Cu. Serum stability and immunoreactivity of 64Cu-NOTA-trastuzumab were tested. Small-animal PET imaging and biodistribution studies were performed in a HER2-positive breast cancer xenograft model (BT-474). The internal dosimetry for experimental animals was determined using the image-based approach with the Monte Carlo N-particle code. Results: 64Cu-NOTA-trastuzumab was prepared with high radiolabeling yield and radiochemical purity (>98%) and showed high stability in serum and good immunoreactivity. Uptake of 64Cu-NOTA-trastuzumab was highest at 48 h after injection as determined by PET imaging and biodistribution results in BT-474 tumors. The blood radioactivity concentrations of 64Cu-NOTA-trastuzumab decreased biexponentially with time in both mice with and mice without BT-474 tumor xenografts. The calculated absorbed dose of 64Cu-NOTA-trastuzumab was 0.048 mGy/MBq for the heart, 0.079 mGy/MBq for the liver, and 0.047 mGy/MBq for the spleen. Conclusion: 64Cu-NOTA-trastuzumab was effectively targeted to the HER2-expressing tumor in vitro and in vivo, and it exhibited a relatively low absorbed dose due to a short residence time. Therefore, 64Cu-NOTA-trastuzumab could be applied to select the right patients and right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread.

556. Another Hepatic Control Region for Sustained Expression of Human Factor IX in the Mouse Liver

For long-term gene expression mediated by plasmid vectors, the plasmids should remain stable in the episomal state with transcriptional and post-transcriptional activities inside nucleus. There was an important report that a hepatic control region (referred to HCR I) maintained plasmids in the stable episomal state in nucleus, preventing loss of plasmids. Furthermore, the HCR has an enhancer activity, thus leading to high expression of transgenes in hepatic cells. There is another hepatic control region, called the second hepatic control region (referred to HCR II). In this research, we have tried to compare the functional activities of HCR I and HCR II in hepatic transgene expression. Their sequences in the functional region have 85% homology. We constructed the plasmid vector containing HCR II and encoding human factor IX (pBS-HCRII-IX), that was compared with the plasmid containing HCR I (pBSHCRHPI-IXA, provided by Dr. Miao, U. Wa). Several other plasmids containing HCR I and HCR II (pBS-HCRI-HCRII-IX and pBS-HCRII-HCRI-IX) were also constructed. These four different plasmids were administered to mice by the hydrodynamic method. There was no significant differences among the expression levels of human factor IX by the four different vectors for 3 months; about 1.2 mg/ml (24% of the normal level) on day 1 and about 0.2 mg/ml (4% of the normal level) on day 85. Also, blood clotting activities of human factor IX produced by the four vectors were similar among each other for the same period of time. Meanwhile, the factor IX level induced by a control plasmid (pAAVCMV-hFIX) without HCR was drastically diminished in 72 hours. Interestingly, the significant amount of anti-human factor IX antibody was detected in the mice treated with the control plasmid, but not in the mice treated with the plasmids containing HCR. At this moment, we have not seen any functional differences among the four different plasmids containing HCR. Further analysis of human factor IX expression and blood coagulation in hemophilia B mice treated with the HCR-containing plasmids for a longer period of time will give us a better understanding about HCR functions in hepatic transgene expression.