David Filpula

A RNA antagonist of hypoxia-inducible factor-1α, EZN-2968, inhibits tumor cell growth

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the α-subunit of HIF-1 (HIF-1α), which occurs in response to hypoxia or activation of growth factor pathways, is associated with poor prognosis in many types of cancer. Therefore, down-regulation of HIF-1α protein by RNA antagonists may control cancer growth. EZN-2968 is a RNA antagonist composed of third-generation oligonucleotide, locked nucleic acid, technology that specifically binds and inhibits the expression of HIF-1α mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC50, 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor cell growth. Additionally, down-regulation of HIF-1α protein by EZN-2968 led to reduction of its transcriptional targets and of human umbilical vein endothelial cell tube formation. In vivo, administration of EZN-2968 to normal mice led to specific, dose-dependent, and highly potent down-regulation of endogenous HIF-1α and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted with DU145 cells treated with EZN-2968. Ongoing phase I studies of EZN-2968 in patients with advanced malignancies will determine optimal dose and schedule for the phase II program. [Mol Cancer Ther 2008;7(11):3598–608]

Single-chain Fv designs for protein, cell and gene therapeutics

One decade after the invention of single-chain Fv (sFv) proteins and methods for their facile engineering in microbial systems, there have been several recent engineering advances which combine the avidity, effector and pharmacological properties of intact monoclonal antibodies (mAbs) with the simplicity of the sFv formats. These developments may serve as the prelude for a new wave of immunomolecule therapeutics which can surpass mAbs in efficacy, biodistribution and abundance of applications. Additionally, sFv formats are now used extensively in the fields of gene and cell therapy as intracellular target-binding drugs; targeting agents for vectors with redesigned tropism; and engineered chimeric receptors for targeted immune cells.

Abstract 602: Silencing of the androgen receptor (AR) with a novel mRNA antisense oligonucleotide causes antitumor effects in xenograft models of prostate cancer

Background: The AR is a ligand-activated transcriptional factor that plays an important role in prostate cancer. While androgen-deprivation therapies typically inhibit tumor growth, tumor recurrence due to the castration resistant form of the disease frequently occurs and leads to mortality. Nevertheless, castration-resistant tumors are still dependent on a functional (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)-based antisense oligonucleotide (ON), EZN-4176, which down-modulated AR and inhibited tumor growth. Methods: The target mRNA down-modulation, growth inhibition, luciferase activity, protein inhibition, and reduction of prostate specific antigen (PSA; an AR downstream target and biomarker of prostate cancer) were evaluated by qRT-PCR, MTS, SteadyGlo, western blot analysis, and ELISA assay, respectively, in LNCaP cancer cell lines. A mismatched LNA antisense oligo (EZN-4176MM) and an AR negative cell line (PC-3 prostate cancer) served as controls. Additionally, the effect of EZN-4176 on DHT-induced AR transcriptional activity was evaluated in LNCaP cells containing an AR-responsive luciferase reporter. In vivo, therapeutic efficacy of EZN-4176 was evaluated in the AR-positive CWR22 (androgen dependent). Results: In vitro, in the absence of any transfection reagent, EZN-4176 but not the mismatched control (EZN-4176MM) inhibited the growth of DHT-induced LNCaP cells. The growth reduction correlated with AR down-modulation and its down-stream target gene expression and AR-responsive luciferase activity. In vivo, EZN-4176, prepared in the absence of a delivery vehicle, demonstrated dose-dependent inhibition of mRNA of AR, PSA, and TMPRSS2 as well as protein level of AR in androgen-sensitive CWR22 tumor xenografts, resulting in significant tumor growth inhibition (TGI) (∼55%) (similar to high-dose Casodex ® [bicalutamide] treatment). Cy5.5-labeled EZN-4176 was shown to localize in subcutaneous CWR22 or LNCaP tumor xenografts using xenogen imaging system. In castration-resistant tumor cells, EZN-4176 potently and specifically inhibited the growth of C4-2b cells as well as down-regulated AR-luciferase reporter systems. More importantly, AR and PSA mRNA were inhibited in both castration-resistant C4-2b and 22RV1 tumor xenografts. Conclusions: Our data suggest that AR mRNA down modulation by EZN-4176 inhibits prostate tumor growth and provides a novel strategy to treat advanced prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 602.

Abstract 600: Targeting the hedgehog pathway by LNA (locked nucleic acid) oligonucleotide-based GLI2 RNA antagonists, EZN-4482 and EZN-4496, in vitro and in vivo

Background: Aberrent activation of hedgehog (Hh) signaling has been implicated in the initiation and growth of a number of human malignancies, including those derived from skin, lung, liver, prostate and breast. The GLI family proteins are zinc finger transcription factors and the downstream effectors of Hh pathway, controling expression of the target genes such as GLI1, Ptch1, Cyclin D, and BCL2. Since amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway are found in cancer and cancer stem cells, specific down regulation of GLIs with antisense molecules may offer a novel and effective therapeutic approach for cancer treatment. We report here the biological activities of LNA-based GLI2 mRNA antagonist, EZN-4482 and EZN-4496. Methods: In vitro, the ability of the antagonists to down modulate their direct target, GLI2 mRNA, and to inhibit cell growth was evaluated by qRT-PCR and MTS, respectively, in multiple cell lines of various origins. To identify and investigate the molecular events associated with targeted downmodulation of GLI2, expression of Hh pathway components and downstream effectors were analyzed by gene expression profiling and immnomoblotting analysis. In vivo, GLI2 mRNA down-modulation in PC3 xenograft tumor was evaluated after intravenous administration of the antagonists. The effect of antagonists on tumor growth and survival of mice were evaluated in PC3 prostate tumor grown on the flank and in A549 lung carcinoma cells that had metastasized to the liver respectively. Results: Both antagonists are specific and potent down-modulators of GLI2 mRNA (by transfection, IC 50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 600.

Abstract C141: In vitro and in vivo characterization of two novel β‐catenin RNA antagonists, EZN‐3889 and 3892

Background: β‐Catenin is an important signaling molecule in the Wnt pathway that plays a key role in tumorgenesis. In the absence of Wnt signaling, the cytoplasmic level of β‐catenin is kept low due to rapid proteasomal‐mediated degradation of GSK3β phosphorylated β‐catenin. Activation of Wnt signaling leads to the inactivation of GSK3β, resulting in stabilization and accumulation of β‐catenin in the cytoplasm. Consequently, β‐catenin translocates into the nucleus, where it binds with members of the T‐cell factor (Tcf)/lymphocyte enhancer‐binding factor family of transcription factors and activates the expression of many target genes important for cancer development. Most colon cancers have activating mutations in the APC tumor suppressor or in β‐catenin itself. Furthermore, activating β‐catenin mutations have been found in a variety of other tumors such as melanomas, hepatocellular carcinomas, skin, breast, and prostate cancer, whereas β‐catenin is not activated in most normal tissues. Therefore, inhibition of β‐catenin is likely to have therapeutic effects in many cancers. We report here the identification of two β‐catenin LNA‐based mRNA‐antagonists, EZN‐3889 and EZN‐3892. Material and Methods:In vitro, the ability of the compounds to inhibit mRNA, cell growth, and reporter gene were evaluated by qRT‐PCR, MTS, and luciferase assays respectively, in multiple cell lines. In vivo, β‐catenin mRNA down‐modulation in liver and human tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the compounds. Results: These molecules were potent down‐modulators of β‐catenin (IC50 = 0.1 to 3 nM) as well as protein (> 80% at 20 nM) of multiple cell lines, when transfected into tumor cells. Growth inhibition appeared to correlate with the status of either APC mutation or activating β‐catenin mutations, suggesting that β‐catenin is the driver of cancer cells. Interestingly, we found that these two antagonists specifically down modulated β‐catenin mRNA and protein effectively in multiple cell lines with a long‐lasting effect in the absence of any tranfection reagent. In mice, both molecules significantly down modulated β‐catenin mRNA in the liver. Conclusions: β‐catenin antagonists potently and specifically inhibited β‐catenin mRNA expression both in vitro and in vivo after intravenous injection. Further studies will examine the antitumor efficacy of the compounds. hese novel agents specifically inhibit a transcription factor that has been difficult to target with conventional agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C141.

Abstract C146: LNA (locked nucleic acid) oligonucleotide based GLI2 RNA antagonist EZN‐4482 effectively inhibited tumor growth and increased survival of animals with liver metastasis

Background: The Hedgehog (Hh) pathway has been implicated in the growth of a number of human malignancies and cancer stem cells. In the presence of Hh ligands, PTCH1 inhibition of a membrane protein, Smoothened (SMO), is relieved. SMO activation triggers a cascade of downstream events that ultimately activate the zinc finger transcription factors GLI2 and GLI3 and induce transcription of their target genes such as GLI1, PTCH1, Cyclin D, and BCL2. Emerging evidence suggests that GLI1 and GLI2 represent the main activators of Hh pathway due to amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway. Therefore, specific down regulation of GLIs with RNA antagonists such as siRNA and antisense molecules may offer an effective therapeutic approach for cancer treatment. We reveal here the biological activities of LNA‐based GLI2 mRNA antagonist, EZN‐4482 and 4496. Material and Methods:In vitro, the ability of the antagonists to down modulate mRNA and cell growth was evaluated by qRT‐PCR and MTS respectively. In vivo, GLI2 mRNA down‐modulation in tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the antagonists. The effect of antagonists on tumor growth and survival of mice were evaluated in PC3 prostate tumor grown on the flank and in A549 lung carcinoma cells that had metastasized to the liver respectively. Results: Both antagonists, when transfected into tumor cells, were potent down‐modulators of GLI2 mRNA (IC50 ≤ 2 nM), resulting caspase ativation and growth inhibition. Interestingly, we found that without any transfection reagent, 1–10 micromolar concentrations of these antagonists were able to down modulate GLI2 mRNA and inhibit the growth of multiple cell lines while their scrambled control oligonucleotides showed no effect, suggesting that the effect was specific. In mice, EZN‐4482 (3 mg/kg given q3d x4) down modulated GLI2 mRNA in PC3 prostate tumors. Moreover, using murine specific probes, we found EZN‐4482 also inhibited GLI1/2 and PTCH1 in the same tumors. Moderate tumor growth inhibition was found with EZN‐4482 in the PC3 xenograft model. Additionally, we show that EZN‐4482 prolonged the survival of animals with liver metastasis derived from A549 cells. Conclusions: GLI2 antagonists potently and specifically inhibited GLI2 mRNA expression and tumor growth in two tumor models. The possible mechanisms of efficacy include inhibition of mRNA levels of GLI1/2 and PTCH1 in mouse stromal cells. While small molecule inhibitors of SMO are being evaluated clinically, this approach is limited to tumors where pathway activation is upstream of GLIs or tumors that are resistant to such inhibitors. Therefore, a GLI2 RNA antagonist may be an effective therapy to treat a broad spectrum of cancers including ones that fail treatment with SMO inhibitor therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C146.

Production of engineered IgM-binding single-chain antibodies inEscherichia coli

SummaryTwo single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human IgM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed inEscherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small IgM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.