Hep3B Tumors Research Articles

Abstract 5667: CYT-303 FLEX-NKTM engager dose response efficacy mechanisms in HCC tumor model and safety in cynomolgus monkey toxicology studies support clinical evaluation in hepato-cellular carcinoma

Abstract BACKGROUND: CYT-303 is a multifunctional bispecific NK engager (NKE) targeting NK cell activating receptor NKp46 and tumor antigen Glypican-3 (GPC3) expressed in HCC (hepatocellular carcinoma). Cytovia’s proprietary FLEX-NKTM platform utilizes a novel FLEX-linker and human IgG1 back bone to allow for simultaneous binding to targeted cancer cells and NK cells. We evaluated CYT-303 dose response pharmacology efficacy mechanisms in HCC tumor models and conducted safety assessment studies in cynomolgus monkeys to support first in human clinical studies in HCC patients. METHODS: CYT-303 dose response pharmacology mechanistic studies were conducted in NSG-hIL-15 mice bearing Hep3B tumors and flow cytometry was used to assess bold and tumor NK cells. A 4-week CYT-303 repeat dose GLP toxicology study in cynomolgus monkeys at 6, 20 and 60 mg/kg dose was conducted following weekly intravenous infusions followed by a 6-week recovery period. CYT-303 toxicokinetic and anti-drug antibody (ADA) assessments were conducted using validated immunoassays. RESULTS: CYT-303 dose response in HCC tumor models showed increased tumor growth inhibition at the lower 2-3 mg/kg doses compared to higher 5-10 mg/kg doses consistent with the dose response previously reported in NK cell redirected cytotoxicity assays against HCC tumors. The increased anti- tumor efficacy observed at the lower doses were associated increased infiltration of PBNK’s to the tumor and corresponding reductions in PBNK’s in blood suggesting CYT-303 facilitates PBNK entry and retention in HCC tumors expressing GPC3 tumor antigen. CYT-303 safety assessment in the 4-week toxicology study in cynomolgous monkeys showed no treatment related toxicities or cytokine release and the NOAEL was determined to 60 mg/kg/week the highest dose administered in the study. No treatment related clinical signs, clinical pathology (hematology and clinical chemistry) and anatomic pathology (macroscopic, microscopic and organ weights) changes were observed. CYT-303 toxicokinetic analysis showed dose dependent increases in Cmax and AUC’s and no evidence for CYT-303 accumulation. CYT-303 anti-drug antibodies in monkeys were minimal and only observed in one low dose animal (1 out of 22) which also showed reduced CYT-303 exposures. Based on these pharmacology and toxicology results a pharmacologically active dose (PAD) based approach together with pharmacokinetic modeling would be utilized to estimate first in human dosing in HCC patients. CONCLUSIONS: CYT-303 efficacious doses identified in the HCC model together with safety in cynomolgus monkeys and planned human pharmacokinetic modeling studies support clinical evaluation of CYT-303 in first in human HCC clinical trials. Citation Format: Vishal Khairnar, Christine Waters, Solgalim Diaz, David De Franco, Jean Kadouche, Daniel Teper, Wei Li, Antonio Arulanandam. CYT-303 FLEX-NKTM engager dose response efficacy mechanisms in HCC tumor model and safety in cynomolgus monkey toxicology studies support clinical evaluation in hepato-cellular carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5667.

Abstract PO001: Preclinical activity of glypican-3 (GPC3) and NKp46 directed FLEX-NK™ engager antibody (CYT-303) in combination with iPSC derived natural killer cells (iNKs) or peripheral blood (PB) NK cells in hepatocellular carcinoma (HCC)

Abstract BACKGROUND: GPC3 is highly expressed in HCC while it is hardly expressed in adult normal tissues except placenta. Given the presence of dysfunctional NK cells in the HCC tumor microenvironment that contribute to poor anti-tumor immune responses, we sought to develop an iNK cell therapy in combination with GPC3 targeted NK cell engagers (NKE). FLEX-NK™ is a proprietary tetravalent IgG1-like multifunctional antibody NKE platform with a novel FLEX-linker to allow for simultaneous binding to different antigens on different cells. CYT-303 was constructed to engage NK cells via the activation receptor NKp46 and to target cancer cells via GPC3. We hypothesized that CYT-303 may be active in the tumor microenvironment where there are sufficient number and function of endogenous NK cells, but that this activity may be augmented by fresh functional NK cells. iNK cells derived from iPSC, a uniform starting material with unlimited self-renewal capabilities, can be expanded to produce a universal off-the-shelf allogeneic therapy. We investigated the combination of CYT-303 to enhance tumor specificity and potency of iNKs. METHODS: CYT-303 was expressed in CHOZEN cells and purified by step column chromatography. iNK’s were differentiated from iPSC’s and expanded using irradiated feeder cells. PB-NK cells were purified from healthy donor blood. iNK and PB-NK cytotoxicity against Hep3B tumors was evaluated in the presence of CYT-303 or isotype control antibodies at a fixed E/T ratio. CYT-303 pharmacokinetics in mice was evaluated following a single intravenous injection. Anti-tumor efficacy of the iNK combination with CYT-303 was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors following intra-tumoral injection of iNKs and intravenous dosing with CYT-303. Blood alpha-fetoprotein (AFP) levels were assessed by immunoassay. Cytokine release assessments for CYT-303 was evaluated in an in-vitro PBMC activation assay. RESULTS: iNK cells express high levels of multiple activation receptors including NKp46, but low levels of CD16. Despite low expression of CD16, natural cytotoxicity of iNK’s against Hep3B tumors was higher than with fresh PB-NK and CYT-303 redirected killing of Hep3B tumors was comparable to PB-NK’s. The CYT-303 pharmacokinetics study in mice showed a terminal half-life of 17 hrs. In a Hep3B tumor model in NSG-hIL-15 mice, the combination of CYT-303 and iNK’s showed significantly greater tumor growth inhibition compared to iNK’s alone plus an IgG1 isotype control. Blood AFP levels decreased in the CYT-303 plus iNK combination compared to iNK’s alone. Cytokine release assessment of CYT-303 in the human PBMC assay showed no evidence of cytokine release while high levels of cytokine release was observed with anti-CD28 (TGN1412) and CD3 antibody controls. CONCLUSIONS: The combination of NK cell engager CYT-303 and iNK cells is active in preclinical models of HCC with low levels of cytokine release. These data support further development of CYT-303 and iNKs to engage and empower NK cells as an off the shelf therapeutic approach in HCC. Citation Format: Antonio Arulanandam, Liang Lin, Hao-Ming Chang, Harish Potu, Vishal Khairnar, David Zou, Melissa Triggiano, Nejmi Dilmac, Yinan Yang, Shira Kahlon, Stanley Frankel, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Yoav Hershkovitz, Yaron Ilan, Wei Li. Preclinical activity of glypican-3 (GPC3) and NKp46 directed FLEX-NK™ engager antibody (CYT-303) in combination with iPSC derived natural killer cells (iNKs) or peripheral blood (PB) NK cells in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO001.

756P Glypican-3 (GPC3) and NKp46 directed FLEX-NK engager antibody (CYT-303) recruits natural killer (NK) cells to tumors in a preclinical hepatocellular carcinoma (HCC) mouse model

GPC3 is an oncofetal antigen that is highly expressed in HCC while minimally expressed in adult normal tissues except placenta. CYT-303 is a multifunctional bispecific NK engager targeting NK cell activating receptor NKp46 and GPC3 expressed on tumor cells constructed with Cytovia’s Flex-NK scaffold with a fully functional Fc. Previously, we reported CYT-303 in vitro treatment of Hep3B tumor cells demonstrated cytolysis and cytokine production as well as in vivo tumor growth inhibition when combined with both PBNK and iNK cells. Here we further characterized the CYT-303 dose-response for tumor growth inhibition in vivo and its influence on NK cell distribution. CYT -303 pharmacokinetics was evaluated in tumor-bearing mice. Anti-tumor efficacy and dose response of CYT-303 in the presence of human PBNKs or iNKs was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors. Biodistribution of the NK cells were analyzed by flow cytometry. CYT-303 pharmacokinetics in tumor bearing mice was similar to normal mice suggesting no target mediated clearance. In the Hep3B tumor model in PBNK or iNK injected NSG-hIL-15 mice, CYT-303 showed dose dependent tumor growth inhibition compared to control hIgG1 treated mice. Consistent with CYT-303 tumor growth inhibition, increased NK cells were detected in the tumor compared to control animals. Blood NK cells in CYT-303 treated animals were significantly lower compared to IgG1 isotype control treated mice suggesting CYT-303 could facilitate NK cell trafficking from blood into the tumor. Blood AFP (alpha fetoprotein), a biomarker in HCC, decreased with CYT-303 tumor growth inhibition. Pharmacologically active CYT-303 doses were identified. CYT-303 can help recruit NK cells to the tumor site to achieve tumor growth inhibition.

Abstract 576: Shark VNAR single domain-based CAR T cells targeting PD-L1 for cancer therapy

Abstract Background: Chimeric antigen receptor (CAR)-T therapy shows great potency against hematological malignancies, whereas it is difficult to transfer CAR T into solid tumors due to lack of appropriate antigenic targets and immunosuppressive tumor microenvironment. Checkpoint molecule PD-L1 is widely overexpressed on multiple tumor types, and PD-1/PD-L1 interaction is a primary mediator of immunosuppression in the TME. Here, we generated anti-PD-L1-specific shark single domain VNAR-based CAR T cell strategy and explored its anti-tumor efficacy in xenograft mouse models. Methods: We isolated anti-PD-L1 single domain antibodies from semi-synthetic shark VNAR phage display libraries. The binding of isolated VNARs was validated by ELISA, flow cytometry, and Octet. A peptide library based on human PD-L1 was synthesized to predict the epitope of select VNARs. Anti-tumor efficacy of CAR T cells was determined via cell luciferase-based cell assay as well as xenograft mouse models. Two tumor models, MDA-MB-231 (triple-negative breast cancer) and Hep3B (hepatocellular carcinoma or HCC) were used in the present study. Glypican-3 (GPC3) is a tumor-specific target in the Hep3B model. Results: We identified three anti-PD-L1 phage binders, B2, A11, and F5 from our shark phage libraries. All three binders showed cross-activity to human, mouse, and canine antigens, whereas only B2 functionally blocked the interaction of human PD-1 to PD-L1. Moreover, CAR (B2) T cells specifically lysed both MDA-MB-231 and Hep3B tumor cells by targeting constitutive and inducible expression of PD-L1. Importantly, the combination of anti-GPC3 CAR (hYP7) T cells with CAR (B2) T cells regress Hep3B tumors synergistically in mice. Conclusions: PD-L1-targeted shark VNAR single domain-based CAR T cell therapy is a novel strategy to treat breast cancer and liver cancer. This work provides a rationale for a potential use of anti-PD-L1 CAR T cells as a monotherapy or combination with a tumor-specific therapy in clinical studies for solid tumors. Citation Format: Dan Li, Hejiao English, Jessica Hong, Tianyuzhou Liang, Glenn Merlino, Chi-Ping Day, Mitchell Ho. Shark VNAR single domain-based CAR T cells targeting PD-L1 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 576.

Two Zn(II) coordination polymers with anticancer drug norcantharidin as ligands for cancer chemotherapy.

Here two Zn(II) coordination polymers [Zn20(DMCA)12]O12 (DMCA = demethylcantharic acid, DMCA-Zn1) and [Zn(DMCA)](H2O)2 (DMCA-Zn2) are synthesized from a broad-spectrum anticancer drug norcantharidin (NCTD) and Zn(NO3)2·6H2O under solvothermal conditions. By mechanical grinding with a biocompatible polymeric surfactant F127, ultrasonic treatment and filtration, DMCA-Zn1 and DMCA-Zn2 can be transformed into stable nanoparticles (DMCA-Zn1 NPs and DMCA-Zn2 NPs) suspended in water with average diameters of around 190 nm and 162 nm for drug delivery. The in vitro evaluation indicates that DMCA-Zn1 NPs and DMCA-Zn2 NPs can enter into HepG2 and Hep3B cancer cells via endocytosis and inhibit their proliferation. Meanwhile they exhibit relatively low toxicity to L927 normal cells. The in vivo evaluation confirms that DMCA-Zn1 NPs and DMCA-Zn2 NPs can more effectively inhibit the growth of Hep3B tumors with relatively few side effects compared with free NCTD. This approach can be extended to other anticancer drugs to construct nanodrug delivery systems for cancer treatment.

PET Imaging of Hepatocellular Carcinomas: 18F-Fluoropropionic Acid as a Complementary Radiotracer for 18F-Fluorodeoxyglucose.

Objective:To evaluate the preclinical value of 18F-fluoropropionic acid (18F-FPA) and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) for imaging HCCs.Methods:The 18F-FPA and 18F-FDG uptake patterns in 3 HCC cell lines (Hep3B, HepG2, and SK-Hep1) were assessed in vitro and in vivo. The 18F-FPA uptake mechanism was investigated using inhibition experiments with orlistat and 5-tetradecyloxy-2-furoic acid. The 18F-FPA PET imaging was performed in different tumor animal models and compared with 18F-FDG. We also evaluated the expressions of glucose transporter-1 (GLUT1), fatty acid synthase (FASN), and matrix metalloproteinase-2 (MMP2) in these cell lines.Results:In vitro experiments showed that the radiotracer uptake patterns were complementary in the HCC cell lines. Orlistat and 5-tetradecyloxy-2-furoic acid decreased the uptake of 18F-FPA. The tumor-to-liver ratio of 18F-FPA was superior to that of 18F-FDG in the SK-Hep1 and HepG2 tumors (P < .05). However, in the Hep3B tumors, the tumor-to-liver normalized uptake of 18F-FDG was higher than 18F-FPA (P < .01). FASN was highly expressed in cell lines with high 18F-FPA uptake, whereas GLUT1 was highly expressed in cell lines with high 18F-FDG uptake. The 18F-FPA uptake correlated with FASN (r = 0.89, P = .014) and MMP2 (r = 0.77, P = .002) expressions.Conclusions:PET imaging with 18F-FPA combined with 18F-FDG can be an alternative for detecting HCC.

Abstract 2549: Development of CAR T-cell therapy targeting glypican-3 in liver cancer

Abstract Background: Glypican-3 (GPC3) is a candidate therapeutic target in hepatocellular carcinoma (HCC). We have generated the HN3 and hYP7 antibodies that recognize the N-terminus and C-terminus of GPC3, respectively. Here, we engineered human T cells that express GPC3-specific chimeric antigen receptors (CARs) and evaluated their potential for the treatment of HCC. Methods: The GPC3-specific CARs (CAR.HN3 and CAR.hYP7) contain a CD3ζ chain and the 4-1BB co-stimulatory endodomain along with a truncated human EGFR polypeptide (huEGFRt) that retains the cetuximab epitope. A set of GPC3 positive-liver cancer cell lines including Hep3B, HepG2 and Huh7 were engineered to express the firefly luciferase gene. These cells were co-cultured with GPC3 CAR T cells at various ratios for 24 hours. A bioluminescent luciferase reporter assay was then used to evaluate the cytolytic activity of effector CAR T cells. We also evaluated the anti-tumor activity of CAR T cells in xenograft models with intraperitoneal injection of luciferase-expressing HCC cells into NOD/SCID/IL2gnull (NSG) mice. The tumor growth was measured via bioluminescence tumor imaging. Results: We found that the CAR.hYP7 cells targeting the C-terminus of GPC3 showed the highest cytolytic activity against GPC3-positive HCC cells. In two HCC intraperitoneal xenograft models (Hep3B and HepG2), luciferase-expressing HCC cells preferentially grew on the mouse liver. More importantly, we found that a single intraperitoneal treatment with a high dose of CAR.hYP7 cells (40 million per mouse) exhibited sustainable antitumor efficacy in the Hep3B model and all of the mice survived after 9 weeks post CAR T-cell treatment without recurrence. The serum levels of alpha fetoprotein (AFP) in mice treated with a high dose of CAR.hYP7 cells were less than 50 ng/ml, the threshold value for HCC diagnosis. Additionally, a single intraperitoneal treatment with a low dose of CAR.hYP7 cells (10 million per mouse) caused regression of liver tumor xenografts. In the low dose CAR.hYP7 group, Hep3B tumors in the mice grew locally and restricted to the fat tissues far from the mouse liver, indicating that CAR T cells prevented tumors from seeding and growing in the liver. Although CAR.HN3 cells targeting the N- terminus of GPC3 also exhibited high cytotoxicity to HCC cells in vitro, no significant Hep3B tumor growth inhibition was seen in the mice treated with CAR.HN3 cells. Conclusion: Our work suggests that GPC3-targeting CAR T cells, in particular CAR.hYP7, is a promising therapeutic intervention for liver cancer that can be translated to human use. Citation Format: Dan Li, Nan Li, Yifan Zhang, Haiying Fu, Madeline B. Torres, Qun Wang, Tim F. Greten, Mitchell Ho. Development of CAR T-cell therapy targeting glypican-3 in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2549.

Histotripsy for Non-Invasive Ablation of Hepatocellular Carcinoma (HCC) Tumor in a Subcutaneous Xenograft Murine Model.

Histotripsy fractionates tissue through a mechanical, non-invasive ultrasonic ablation process that precisely controls acoustic cavitation while utilizing real-time ultrasound (US) imaging guidance. This study investigates the potential, feasibility and tumor volume reduction effects of histotripsy for liver cancer ablation in a subcutaneous in vivo murine Hepatocellular Carcinoma (HCC) model. Hep3B tumors were generated in the right flanks of 14 NSG and 7 NOD-SCID mice. The mice were grouped as follows: A (acute, NSG with n=9 treatment and n=1 control), B (chronic, NSG with n=2 treatment and n=2 control) and C (chronic NODSCID, with n=6 treatment and n=1 control). Treatment was performed when the tumor diameters reached >5 mm. 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa) were delivered using a custom built 1 MHz therapy transducer attached to a motorized positioner, which scanned the transducer focus to traverse the targeted tumor volume, guided by real-time US imaging. Tumor ablation effectiveness was assessed by obtaining T1, T2 and T2* weighted MR images. Post euthanasia, treated tumor, brain, and lung tissue samples were harvested for histology. Histology of acute group A showed fractionation of targeted region with a sharp boundary separating it from untreated tissue. Groups B and C demonstrated effective tumor volume reduction post treatment on MRI as the homogenate and edema were resorbed within 23 weeks. However, as the tumor was subcutaneous, it was not possible to set adequate treatment margin and since the mice were immune-compromised, residual viable tumor cells eventually developed into tumor regrowth at 3-9 weeks after histotripsy. Groups B and C showed no signs of metastasis in the lung and brain. Our study successfully demonstrated the potential of histotripsy for non-invasive HCC ablation in a subcutaneous murine model. Additional work is ongoing to study the response of histotripsy in immune-competent orthotopic liver tumor models.

Abstract 1234: The novel FGFR4-selective inhibitor INCB062079 is efficacious in models of hepatocellular carcinoma harboring FGF19 amplification

Abstract Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with limited treatment options for advanced stage disease. Thus, there is a critical medical need for improved therapies. In approximately 10% of HCC, a focal amplicon at 11q13 harboring FGF19 has been reported. High levels of FGF19 have been shown to drive HCC tumor development and progression in preclinical models, suggesting that selective targeting of FGFR4, a high affinity receptor for FGF19, may be efficacious in these HCC tumors. INCB062079 a potent and selective irreversible inhibitor of FGFR4 (&amp;gt;250-fold vs FGFR1/2/3) suppresses the growth of HCC cell lines driven by amplification and overexpression of FGF19. In subcutaneous xenograft models of HCC, oral dosing of INCB062079 at tolerated doses resulted in dose-dependent inhibition of tumor growth with regressions observed at higher doses consistent with inhibition of FGFR4 signaling in the tumors. In combination with sorafenib, the only approved targeted therapy for HCC, FGFR4 inhibition exhibited additive tumor growth inhibition in the Huh7 model. To assess exposure of INCB062079 in orthotopic tumors after oral dosing, Hep3B tumors were implanted surgically into the liver and their development monitored by analysis of plasma alpha-fetoprotein (AFP). At efficacious doses, INCB062079 strongly suppressed the levels of AFP and FGF19 secreted by the tumors, and their levels correlated well with the reduction in terminal liver tumor mass, suggesting that these may be surrogate markers for response of HCC tumors to INCB062079. In two PDX models of HCC with amplification of FGF19 (4-6 CNV), INCB062079 administration reduced tumor growth by 50-66% at doses that were well tolerated. Additional surrogate markers for FGFR4 inhibition were explored including several parameters related to FGF19 regulation of bile acid metabolism. The mRNA levels of CYP7A1, encoding cholesterol 7a-hydroxlyase, the rate limiting enzyme in bile acid synthesis, were induced in the livers of cynomolgus monkeys upon dosing with INCB062079. Correspondingly there was a dose-dependent increase in fecal bile acids. In summary these data demonstrate that INCB062079 is highly and selectively efficacious in models of HCC with FGF19-FGFR4 oncogene addiction and elicits pharmacodynamic responses in primates providing support for the clinical evaluation of INCB062079 in genetically selected liver cancer patients. Citation Format: Bruce Ruggeri, Matthew Stubbs, Yan-ou Yang, Ashish Juvekar, Liang Lu, Sindy Condon, Darlise DiMatteo, Xiaoming Wen, Paul Collier, Timothy Burn, Liangxing Wu, Daniel Wilson, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Phillip CC Liu. The novel FGFR4-selective inhibitor INCB062079 is efficacious in models of hepatocellular carcinoma harboring FGF19 amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2017-1234

Abstract 2888: Lipid nanoparticle-mediated delivery of siRNA to extrahepatic tumors

Abstract While lipid nanoparticles (LNPs) proved to be effective in delivering siRNA to the liver and hepatocellular carcinoma (HCC) via systemic administration, they showed inferior or no efficacy in the extrahepatic tumor models. To understand the underlying mechanism for LNP activity in delivering siRNA to extrahepatic tumors, we determined siRNA biodistribution and gene-silencing activity of the LNP-siRNA nanoparticles containing different percentages of a polyethylene glycol (PEG) lipid, distinct PEG lipids with a short (C-14) or long (C-18) alkyl chain, or an identical composition with different particle sizes in mouse subcutaneous tumor xenograft models derived from either human HCC (Hep3B) or colon cancer (HT29 or HCT116) cells. We show that the LNPs containing C-18 DSA PEG exhibited a higher retention of siRNA in both Hep3B and HT29 tumors than those with C-14 DMA PEG following an intravenous administration. Intriguingly, LNPs containing 2% of DSA or DMA PEG resulted in similar levels (∼50%) of target knockdown in Hep3B tumors while those containing 6% of DSA or DMA PEG caused no target silencing. Also, none of these LNPs reduced target expression in HT29 tumors despite the retention of siRNA in tumor tissues. When LNPs with an identical composition (2% DMG PEG) but different particle sizes (80 nm vs. 120 nm) were tested, we observed a comparable and moderate suppression of target expression in Hep3B tumors, but no target knockdown in HCT116 tumors. This is despite the fact that the expression of low density lipid receptor, which is shown to be involved in mediating LNP efficacy, is detected in HT29 and HCT116 tumors. Finally, intratumor injection of LNPs only induced minimal target silencing in subcutaneous tumors while provoking significant target knockdown in the liver. Together, enhanced tumor retention of siRNA might not be readily translated to target silencing activity in tumors. Other factors such as the efficiency of transfecting tumor cells and the passive targeting effect through opsonization could impact on LNP efficacy in extrahepatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2888. doi:1538-7445.AM2012-2888

Critical roles of cellular glutathione homeostasis and jnk activation in andrographolide‐mediated apoptotic cell death in human hepatoma cells

Andrographolide (ANDRO), isolated from the traditional herbal medicine Andrographis paniculata, is reported to have the potential therapeutic effects for hepatocellular carcinoma (HCC) in our previous reports. Here, we investigated the mechanism of ANDRO-mediated apoptotic cell death, focusing on the involvement of cellular reduced glutathione (GSH) homeostasis and c-Jun NH(2) -Terminal kinase (JNK). Buthionine sulfoximine (BSO), an inhibitor of cellular GSH biosynthesis, significantly augmented ANDRO-induced cytotoxicity in hepatoma Hep3B and HepG2 cells. BSO depleted cellular GSH, and augmented ANDRO-induced apoptosis, inhibition of colony formation and JNK activation in Hep3B cells. All these effects could be reversed by GSH monoethyl ester (GSH.EE), whose deacetylation replenishes cellular GSH. BSO also augmented ANDRO-induced activation of apoptosis signal-regulating kinase 1 (ASK1), mitogen-activated protein kinase kinase-4 (MKK4) and c-Jun, which are all up-stream or down-stream signals of JNK. Further results showed that JNK inhibitor SP600125 and 420116 both reversed ANDRO-induced cytotoxicity, and SP600125 also decreased ANDRO-increased intracellular GSH and GCL activity. Finally, we showed that in nude mice bearing xenografted Hep3B tumors, BSO improved the inhibition of tumor growth by ANDRO. Taken together, our results suggest that there is a crosstalk between JNK activation and cellular GSH homeostasis, and ANDRO targets this to induce cytotoxicity in hepatoma cells.

A fully replication-competent adenovirus vector with enhanced oncolytic properties

We have studied the oncolytic efficacy of two adenovirus vectors named KD3 and INGN 007, which differ from each other only in that whereas KD3 has two small deletions in its e1a gene that restrict its replication to rapidly cycling cells, INGN 007 has wild-type e1a gene. Both vectors overexpress the adenovirus death protein (ADP). Both KD3 and INGN 007 effectively suppressed the growth of subcutaneous human A549 and Hep3B tumors in nude mice upon intratumoral injection, and contained the growth of subcutaneous LNCaP tumors after intravenous injection, making some tumors shrink or disappear. However, in a more demanding model, intravenous injections of neither KD3 nor wild-type Ad5 were effective against subcutaneous A549 tumors, whereas INGN 007 increased the mean survival time by 35%. INGN 007 was also effective in suppressing tumor growth in a challenging A549 orthotopic lung cancer model. INGN 007 was superior to dl1520 (ONYX-015) in repressing subcutaneous A549 tumors. Our results suggest that vectors such as INGN 007 might provide better antitumor efficacy in the clinic as well.

Treatment of hepatocellular carcinoma by AdAFPep/rep, AdAFPep/p53, and 5-fluorouracil in mice

Although conditionally replicable adenovirus (CRAd) has been used in the clinical treatment of hepatocellular carcinoma (HCC), it suffers from the inherent drawback of having relatively low antitumor activity. Here, we have sought to overcome this drawback. First, we combined CRAd (AdAFPep/Rep) driven by alpha-fetoprotein enhancer/promoter (AFPep) with a replication-incompetent adenovirus carrying a p53 transgene that is also driven by AFPep. The synergism of this combination produced a significantly improved tumoricidal effect on the human HCC cell line Hep3B, which has a relatively short doubling time in comparison with other human HCC cell lines, through the transactivation of p53 by early region 1A transcribed by AdAFPep/Rep. This synergistic interaction was augmented by the addition of a subtumoricidal dose (0.5 microg/mL) of 5-fluorouracil (5-FU), which enhanced p53 expression and facilitated the release of virions from tumor cells. When relatively large (10-mm-diameter) Hep3B tumors grown in nude mice were injected with the two viruses in combination, they showed significantly impaired growth in comparison with those treated with each virus separately. The growth suppression effect of the virus combination was enhanced by a low dose (600 microg) of 5-FU. Survival of the tumor-bearing mice treated with these three agents was significantly longer than that of control mice. Moreover, the tumor completely disappeared with the repeated injection of these agents. This combination strategy holds promise for the treatment of relatively large and rapidly growing HCCs that may be encountered clinically.

Adeno‐associated virus‐mediated expression of kallistatin suppresses local and remote hepatocellular carcinomas

The current treatments for hepatocellular carcinoma (HCC) are poor, particularly for metastatic HCC. Intraportal transfusion of adeno-associated virus (AAV) leads to long-term and persistent transgenic expression in livers. Kallistatin, a novel angiogenesis inhibitor, exhibits anti-tumor activity. The aim of the study was to investigate whether intraportal injection of AAV-kallistatin could suppress local and metastatic HCC in mice. An AAV vector encoding kallistatin was constructed, and its transduction efficiency by intraportal transfusion in livers was examined by RT-PCR, immunohistochemical and Western blotting analysis. The anti-tumor activity was tested in three HCC models including hepatic and subcutaneous human Hep3B HCC tumors in BALB/c athymic (nu/nu) mice, and subcutaneous mouse BNL HCC tumors in BALB/c mice. Tumor cell proliferation in situ was examined by anti-Ki-67 staining, and apoptosis by TUNEL. Gene transfection by rAAV-kallistatin inhibited proliferation of human umbilical vein endothelial cells and HCC cells in vitro. Intraportal injection of rAAV-kallistatin resulted in persistent and specific expression of kallistatin in livers detected by RT-PCR and immunohistochemical analysis, and kallistatin protein in circulation detected by Western blotting analysis. Intraportal injection of rAAV-kallistatin significantly suppressed angiogenesis and growth of hepatic Hep3B tumors. The kallistatin released by hepatocytes into the circulation suppressed remote Hep3B and BNL tumors established subcutaneously. The rAAV-kallistatin gene therapy significantly inhibited tumor cell proliferation and induced apoptosis. Intraportal injection of rAAV-kallistatin suppressed hepatic and subcutaneous HCC tumors, relying on its anti-angiogenic and anti-proliferative activities.

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein.

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G(0) to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1(-) E3(-) replication-defective vector expressing beta-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10(-4) PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.