Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.

The concept of cancer stem cells suggests that there are malignant stem-like cells within a tumor that are responsible for tumor renewal and resistance to cytotoxic therapies. Studies have identified glioma stem-like cells that extrude Hoechst 33342 dye, representing a double-negative "side population" (SP) thought to be selectively resistant to drug therapy. A CD133+ stem cell-like subpopulation has been isolated from a human glioma that was enriched for tumor-initiating cells. It is unknown whether CD133+ cells with similar phenotype persist in established glioma cell lines, or if CD133 is a marker of glioma stem-like cells in rodents. We investigated whether CD133+ and SP cells existed in the GL261 cell line, a syngeneic mouse glioma model that is widely used for preclinical and translational research. Intracerebral injection of less than 100 CD133+ GL261 cells formed tumors, whereas it required 10,000 CD133(-) cells to initiate a tumor. CD133+ GL261 cells expressed nestin, formed tumor spheres with high frequency, and differentiated into glial and neuronal-like cells. Similar to GL261, seven human glioma cell lines analyzed also contained a rare CD133+ population. Surprisingly, we found that CD133+ GL261 cells did not reside in the SP, nor did the majority ( approximately 94%) of CD133+ human glioma cells. These results demonstrate that the expression of CD133 in murine glioma cells is associated with enhanced tumorigenicity and a stem-like phenotype. This study also reveals a previously unrecognized level of heterogeneity in glioma cell lines, exposing several populations of cells that have characteristics of cancer stem cells.

A novel anti-cancer agent was constructed by fusing a gene encoding the scFV that targets both glycosylated and unglycosylated forms of CD133 to a gene fragment encoding deimmunized PE38KDEL. The resulting fusion protein, dCD133KDEL, was studied to determine its ability to bind and kill tumor-initiating cells in vitro and in vivo. The anti-CD133 scFV selectively bound HEK293 cells transfected with the CD133 receptor gene. Time course viability studies showed that dCD133KDEL selectively inhibited NA-SCC and UMSCC-11B, two head and neck squamous cell carcinomas that contain a CD133 expressing subpopulation. Importantly, the drug did not inhibit the viability of hematopoietic lineages measured by long-term culture initiating cell and colony-forming assays from sorted human CD34+ progenitor cells. In addition to in vitro studies, in vivo tumor initiation experiments confirmed that CD133 sorted cells implanted into the flanks of nude mice grew faster and larger than unsorted cells. In contrast, cells that were pretreated with dCD133KDEL prior to implantation showed the slowest and lowest incidence of tumors. Furthermore, UMSCC-11B-luc tumors treated with multiple intratumoral injections of dCD133KDEL showed marked growth inhibition leading to complete degradation of the tumors, not observed with an irrelevant control targeted toxin. Experiments in immunocompetent mice showed that toxin deimmunization resulted in a 90% reduction in circulating anti-toxin levels. These studies show that dCD133KDEL is a novel anti-cancer agent effective at inhibiting cell proliferation, tumor initiation, and eliminating established tumors by targeting the CD133 subpopulation. This agent shows significant promise for potential development as a clinically useful therapy.

A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter Correspondence to: Daniel A. Vallera, valle001@umn.edu. NIH Public AccessAuthor Manuscript J Neurooncol. Author manuscript; available in PMC 2012 June 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100 days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study.

Objectives While most women with ovarian cancer will achieve complete remission after treatment, the majority will relapse within two years, highlighting the need for novel therapies. Cancer stem cells (CSC) have been identified in ovarian cancer and most other carcinomas as a small population of cells that can self-renew. CSC are more chemoresistant and radio-resistant than the bulk tumor cells; it is likely that CSC are responsible for relapse, the major problem in cancer treatment. CD133 has emerged as one of the most promising markers for CSC in ovarian cancer. The hypothesis driving this study is that despite their low numbers in ovarian cancer tumors, CSC can be eradicated using CD133 targeted therapy and tumor growth can be inhibited. Methods Ovarian cancer cell lines were evaluated using flow cytometry for expression of CD133. In vitro viability studies with an anti-CD133 targeted toxin were performed on one of the cell lines, NIH:OVCAR5. The drug was tested in vivo using a stably transfected luciferase-expressing NIH:OVCAR5 subline in nude mice, so that tumor growth could be monitored by digital imaging in real time. Results Ovarian cancer cell lines showed 5.6% to 16.0% CD133 expression. dCD133KDEL inhibited the in vitro growth of NIH:OVCAR5 cells. Despite low numbers of CD133-expressing cells in the tumor population, intraperitoneal drug therapy caused a selective decrease in tumor progression in intraperitoneal NIH: OVCAR5-luc tumors. Conclusions Directly targeting CSC that are a major cause of drug resistant tumor relapse with an anti-CD133 targeted toxin shows promise for ovarian cancer therapy.

Dendritic cell (DC) vaccines have shown antitumor activity in experimental glioma models and in human glioma patients. The typical approach has been to generate the vaccine ex vivo, by pulsing DCs with tumor lysate or peptides, then administering the DCs back into the patient. This process requires significant expertise and expenses in DC generation. Immature DCs which present antigens to T cells in the absence of appropriate costimulatory signals can lead to induction of immune tolerance. Recent studies have shown that coadministration of toll-like receptor 9 agonists, CpG oligodeoxynucleotides, can promote DC vaccines to break immune tolerance to tumor antigens. We investigated the therapeutic efficacy of in vivo DC activation, by directly administering glioma cell lysate with CpG oligodeoxynucleotides (CpG/lysate), in glioma-bearing mice. Subcutaneous vaccination with CpG/lysate induced a significant increase (P<0.05) in the number of total T cells and activated DCs in lymph nodes draining the vaccination site as compared to mice treated with CpG or tumor lysate alone. Mice vaccinated with CpG/lysate exhibited over 2 times greater median survival than mice in the control groups (P<0.05). Up to 55% of mice vaccinated with CpG/lysate were rendered tumor-free as assessed by survival and bioluminescent imaging. Splenocytes taken from mice vaccinated with CpG/lysate elaborated significantly more IFN-gamma production and displayed greater tumor cell lysis activity compared with the control groups (P<0.05). These results suggest direct vaccination with CpG/lysate provides an alternative and effective approach to induce host antitumor immunity and warrants clinical investigation in the immunotherapy of cancer.

A drug of high potency and reduced immunogenicity is needed to develop a targeted biological drug that when injected systemically can penetrate to malignant B cells. Therefore, a novel deimmunized bispecific ligand-directed toxin targeted by dual high-affinity single-chain Fvs (scFv) spliced to PE38 with a KDEL COOH-terminus was genetically engineered. The aims were to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit antitoxin antibody responses, and show that mutated drug was effective against systemic B-cell lymphoma in vivo. Both human anti-CD22 scFv and anti-CD19 scFv were cloned onto the same single-chain molecule with truncated pseudomonas exotoxin (PE38) to create the drug. Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISAs were done to determine whether antitoxin antibodies were reduced. Finally, a powerful genetically altered luciferase xenograft model was used that could be imaged in real time to determine the effect on systemic malignant human B-cell lymphoma, Raji-luc. Patient B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B lymphoma were high in CD22 and CD19 expression. 2219KDEL7mut was significantly effective against systemic Raji-luc in mice and prevented metastatic spread. Mutagenesis reduced neutralizing antitoxin antibodies by ∼80% with no apparent loss in in vitro or in vivo activity. Because 2219KDEL7mut immunogenicity was significantly reduced and the drug was highly effective in vivo, we can now give multiple drug treatments with targeted toxins in future clinical trials. Mol Cancer Ther; 9(6); 1872-83. ©2010 AACR.

A bispecific ligand-directed toxin (BLT) consisting of human interleukin-13, epithelial growth factor, and the first 389 amino acids of diphtheria toxin was assembled in order to target human glioblastoma. In vitro, DTEGF13 selectively killed the human glioblastoma cell line U87-luc as well as other human glioblastomas. DTEGF13 fulfilled the requirement of a successful BLT by having greater activity than either of its monospecific counterparts or their mixture proving it necessary to have both ligands on the same single chain molecule. Aggressive brain tumors established intracranially (IC) in nude rats with U87 glioma genetically marked with a firefly luciferase reporter gene were treated with two injections of DTEGF13 using convection enhanced delivery resulting in tumor eradication in 50% of the rats which survived with tumor free status at least 110 days post tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The bispecific DTEGF13 MTD dose was measured at 2 microg/injection or 0.5 microg/kg and toxicity studies indicated safety in this dose. Combination of monospecific DTEGF and DTIL13 did not inhibit tumor growth. ELISA assay indicated that anti-DT antibodies were not generated in normal immunocompetent rats given identical intracranial DTEGF13 therapy. Thus, DTEGF13 is safe and efficacious as an alternative drug for glioblastoma therapy and warrants further study.