Derek W. Bartlett

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging.

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5′ end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by ≈50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics.

Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma.

The development of effective, systemic therapies for metastatic cancer is highly desired. We show here that the systemic delivery of sequence-specific small interfering RNA (siRNA) against the EWS-FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of metastatic Ewings sarcoma. The nonviral delivery system uses a cyclodextrin-containing polycation to bind and protect siRNA and transferrin as a targeting ligand for delivery to transferrin receptor-expressing tumor cells. Removal of the targeting ligand or the use of a control siRNA sequence eliminates the antitumor effects. Additionally, no abnormalities in interleukin-12 and IFN-alpha, liver and kidney function tests, complete blood counts, or pathology of major organs are observed from long-term, low-pressure, low-volume tail-vein administrations. These data provide strong evidence for the safety and efficacy of this targeted, nonviral siRNA delivery system.

Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging

Small interfering RNA (siRNA) molecules are potent effectors of post-transcriptional gene silencing. Using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function, the effects of target-specific and treatment-specific parameters on siRNA-mediated gene silencing are monitored in cells stably expressing the firefly luciferase protein. In vitro, luciferase protein levels recover to pre-treatment values within <1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting ∼10 days in subcutaneous tumors in A/J mice and 3–4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. To demonstrate the practical use of the model in treatment design, model calculations are used to predict the dosing schedule required to maintain persistent silencing of target proteins with different half-lives in rapidly dividing or nondividing cells. The approach of bioluminescent imaging combined with mathematical modeling provides useful insights into siRNA function and may help expedite the translation of siRNA into clinically relevant therapeutics for disease treatment and management.

Preclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiple cancer models.

Preclinical efficacy of i.v. IT-101, a nanoparticulate conjugate of 20(S)-camptothecin and a cyclodextrin-based polymer, was investigated in several mouse xenografts. The effects of different multiple dosing schedules on tumor growth of LS174T colon carcinoma xenografts are elucidated. All multiple dosing schedules administered over 15 to 19 days resulted in enhanced efficacy compared with untreated or single-dose groups. Further improvements in antitumor efficacy were not observed when the dosing frequency was increased from three weekly doses to five doses at 4-day intervals or 5 days of daily dosing followed by 2 days without dosing repeated in three cycles using similar cumulative doses. This observation was attributed to the extended release characteristics of camptothecin from the polymer. Antitumor efficacy was further evaluated in mice bearing six different s.c. xenografts (LS174T and HT29 colorectal cancer, H1299 non–small-cell lung cancer, H69 small-cell lung cancer, Panc-1 pancreatic cancer, and MDA-MB-231 breast cancer) and one disseminated xenograft (TC71-luc Ewings sarcoma). In all cases, a single treatment cycle of three weekly doses of IT-101 resulted in a significant antitumor effect. Complete tumor regression was observed in all animals bearing H1299 tumors and in the majority of animals with disseminated Ewings sarcoma tumors. Importantly, IT-101 is effective in a number of tumors that are resistant to treatment with irinotecan (MDA-MB-231, Panc-1, and HT29), consistent with the hypothesis that polymeric drug conjugates may be able to overcome certain kinds of multidrug resistance. Taken together, these results indicate that IT-101 has good tolerability and antitumor activity against a wide range of tumors.

Potent siRNA Inhibitors of Ribonucleotide Reductase Subunit RRM2 Reduce Cell Proliferation In vitro and In vivo

Purpose: Ribonucleotide reductase (RR) is a therapeutic target for DNA replication–dependent diseases such as cancer. Here, a potent small interfering RNA (siRNA) duplex against the M2 subunit of RR (RRM2) is developed and shown to reduce the growth potential of cancer cells both in vitro and in vivo. Experimental Design: Three anti-RRM2 siRNAs were identified via computational methods, and the potency of these and additional “tiling” duplexes was analyzed in cultured cells via cotransfections using a RRM2-luciferase fusion construct. Knockdown of RRM2 by the best duplex candidates was confirmed directly by Western blotting. The effect of potent duplexes on cell growth was investigated by a real-time cell electronic sensing assay. Finally, duplex performance was tested in vivo in luciferase-expressing cells via whole animal bioluminescence imaging. Results: Moderate anti-RRM2 effects are observed from the three duplexes identified by computational methods. However, the tiling experiments yielded an extremely potent duplex (siR2B+5). This duplex achieves significant knockdown of RRM2 protein in cultured cells and has pronounced antiproliferative activity. S.c. tumors of cells that had been transfected with siR2B+5 preinjection grew slower than those of control cells. Conclusions: An anti-RRM2 siRNA duplex is identified that exhibits significant antiproliferative activity in cancer cells of varying human type and species (mouse, rat, monkey); these findings suggest that this duplex is a promising candidate for therapeutic development.

Abstract LB-351: Evaluation of two engineered antibody fragments derived from a human CD20 antibody as tracers for immunoPET .

Background: CD20 is a cell surface antigen that is expressed on the majority of B-cell lymphomas and B-cell leukemias. Several monoclonal antibodies (mAbs), including the fully human IgG1, Ofatumumab, have been used successfully to target human CD20. Ofatumumab is FDA-approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and under ongoing clinical development for other hematological malignancies and autoimmune diseases. In this study Ofatumumab was engineered into smaller fragments including a minibody (scFv-C H 3; 80kDa) and a cys-diabody (scFv dimer; 50kDa) to enhance pharmacokinetic parameters and optimize possible application for immunoPET imaging. Methods: The variable (V) genes of Ofatumumab were reformatted into two minibodies (Mb) and four cys-diabodies (Cys-Db), differing in orientation of the V genes and linker lengths. Following in vitro characterization of each variant, a lead candidate from each format was produced at larger scale for in vivo evaluation. Ofatumumab, Mb and Cys-Db were radioiodinated with I-131 (t 1/2 8d) for biodistribution and I-124 (t 1/2 4.2d) for PET imaging studies using the Iodogen method. Female SCID mice bearing Ramos (CD20 + ) and BC-1 (CD20 − ) tumors were used. Blocking studies were performed by pre-injecting 0.2-1mg of unlabeled Ofatumumab 24h prior to imaging. The percent injected dose per gram (%ID/g) was determined in harvested tumor, blood and organs. Results: The lead Cys-Db and Mb were shown to have similar binding affinity (0.8 nM) to that of the parental Ofatumumab. Receptor specific binding was confirmed by competitive cell binding assays using both fluorophore-conjugated and radiolabeled antibody fragments. Blood clearances resulted in MRTs of 3.8h, 8.3h and 24.9h for Cys-Db, Mb and Ofatumumab, respectively. Due to its slower initial distribution, the Mb had a higher blood AUC (306%ID/g*h) than that of Ofatumumab (234% ID/g*h) and Cys-Db (102% ID/g*h). Biodistribution studies of Ofatumumab revealed significant uptake in spleen, bone and liver, which was attributed to Fc-mediated clearance as this was not observed with the antibody fragments. Imaging studies with Mb and Cys-Db showed clear delineation of Ramos xenografts; 1.3% ID/g Cys-Db at 8h and 2.7% ID/g Mb at 24h. This resulted in a tumor to blood ratio of 1.7 for the Cys-Db at 8h and 1.1 for the Mb at 24h. Prior administration of excess unlabeled Ofatumumab reduced tumor uptake of the Cys-Db by 50% at 8h and decreased uptake of the Mb by 17% at 24h. Conclusion: Our preliminary results show that antibody fragments derived from Ofatumumab can be used to target CD20 positive cells in vitro and in vivo . The favorable pharmacokinetics of the Cys-Db resulted in higher tumor to blood ratios at 8h and 24h compared to the Mb or Ofatumumab and support the further development of this fragment as a CD20-targeted immunoPET agent. Citation Format: Jean Gudas, Tove Olafsen, David Ho, Derek Bartlett, Arye Lipman, Jean Gudas, Katy Sorenson, Jennifer Keppler, Christian Behrenbruch, Anna Wu, Sharon Ashworth, Jan Passchier, Roger Gunn, Paul Matthews. Evaluation of two engineered antibody fragments derived from a human CD20 antibody as tracers for immunoPET . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-351. doi:10.1158/1538-7445.AM2013-LB-351

208. Physicochemical and Biological Characterization of Cyclodextrin-Based Polycation (CDP)/siRNA Composites Designed for Systemic Delivery

The feasibility of systemic nucleic acid delivery hinges on the ability to simultaneously incorporate numerous properties into the overall delivery system. For example, the delivery vehicle must have the following characteristics: (i) be small enough to travel through blood capillaries and enter tissues, but not be too small such that it is excreted rapidly through the kidneys; (ii) provide stability against degradation, yet willingly release its cargo once it arrives at the proper site; and (iii) avoid nonspecific interactions (i.e., avoid opsonization by immune system components) while still providing specific targeting to a given cell or molecule. We are developing a cyclodextrin-based polycation (CDP) that achieves a favorable balance in all of these design criteria. Here, we show the physicochemical characterization of the colloidal, composite particles formed with CDP and siRNAs through dynamic light scattering (DLS), multi-angle laser light scattering (MALLS), and gel mobility shift assays to quantitate the size and composition. Upon providing steric stabilization of the particles by surface PEGylation, we determine the average number of polyethylene glycol (PEG) molecules on each particle by isothermal titration calorimetry (ITC) and the average number of targeting ligands per particle using fluorescently labeled transferrin (Tf). Biological characterization of the particles reveals that they provide nuclease resistance to nucleic acids, do not aggregate in the presence of serum, and do not cause any observable hemolysis or aggregation of erythrocytes. Complement activation by polycations is often cited as a major concern for their in vivo application. We show that, at the concentrations required for in vivo delivery of siRNA, the complement activation is negligible for the PEGylated particles. This extensive physicochemical and biological characterization of the composite particles demonstrates their suitability for in vivo delivery of siRNA and provides a baseline for rational optimization of these particles as required for specific therapeutic applications.

70. Systemic Administration of siRNA Against EWS-FLI1 Using a Targeted, Non-Viral Formulation Inhibits Growth in a Disseminated Murine Model of Ewing's Sarcoma

Despite aggressive therapy, 40% of patients with localized Ewings family of tumors (EFT) and 80% of patients with detectable metastasis die due to tumor progression. More specific and effective treatments are needed, especially for high-risk cases. EWS-FLI1, the chimeric gene product of a chromosome translocation found in more than 90% of EFT, is crucial for maintaining EFT tumor growth. Endogenous expression of siRNA targeting the breakpoint of the EWS-FLI1 fusion gene (siEFBP) from a pol III promoter has previously been shown to reduce EWS-FLI1 transcript levels up to 80% and resulted in growth inhibition of the cultured EFT cell line TC135. Since EWS-FLI1 expression is tumor-specific, it serves as an excellent candidate for targeted therapy of EFT, especially metastatic disease.