Seunguk Oh

In vivo vaccination with tumor cell lysate plus CpG oligodeoxynucleotides eradicates murine glioblastoma

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-γ 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.

Targeting Tumor-Initiating Cancer Cells with dCD133KDEL Shows Impressive Tumor Reductions in a Xenotransplant Model of Human Head and Neck Cancer

A novel anticancer 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, 2 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 before 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 that was not observed with an irrelevant control-targeted toxin. Experiments in immunocompetent mice showed that toxin deimmunization resulted in a 90% reduction in circulating antitoxin levels. These studies show that dCD133KDEL is a novel anticancer 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. Mol Cancer Ther; 10(10); 1829–38. ©2011 AACR.

A Novel Reduced Immunogenicity Bispecific Targeted Toxin Simultaneously Recognizing Human Epidermal Growth Factor and Interleukin-4 Receptors in a Mouse Model of Metastatic Breast Carcinoma

Purpose: To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand–directed toxin (BLT) targeted by dual high-affinity cytokines with a PE38KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo. Experimental Design: A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE38). 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 ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231. Results: EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by ∼90% with no apparent loss in in vitro or in vivo activity. Conclusions: Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials. (Clin Cancer Res 2009;15(19):6137–47)

Targeting CD133 in an in vivo ovarian cancer model reduces ovarian cancer progression

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.

Transposon-based interferon gamma gene transfer overcomes limitations of episomal plasmid for immunogene therapy of glioblastoma.

Despite improvements in gene delivery technology, transient expression of plasmid DNA has limited the efficacy of nonviral vectors applied to cancer gene therapy. We previously developed plasmid DNA vectors capable of transgene integration and long-term expression in human glioblastoma cells by utilizing the Sleeping Beauty (SB) transposable element. In this study, we compared the efficacy of interferon gamma (IFN-γ) immunogene therapy using episomal or SB vectors in a syngeneic GL261 glioma model. Gene delivery was achieved by intratumoral convection-enhanced delivery of DNA/polyethylenimine complexes. Only mice treated with SB transposase-encoding DNA to facilitate chromosomal integration exhibited a significant increase in survival (P<0.05). SB-mediated intratumoral gene transfer caused sustained IFN-γ expression assessed by reverse transcription-polymerase chain reaction, of both vector-derived and endogenous IFN-γ, whereas expression following episomal plasmid gene transfer was undetectable within 2 weeks. Median survival was enhanced further when SB-mediated IFN-γ gene transfer was combined with CpG oligodeoxynucleotides as adjuvant therapy. Prolonged survival positively correlated with tumor regression measured by in vivo bioluminescent imaging, and enhanced T-cell activation revealed by the ELISPOT assay. SB appears to improve the efficacy of cytokine gene therapy using nonviral vectors by enhancing the duration of transgene expression.

Heterodimeric Bispecific Single-Chain Variable-Fragment Antibodies Against EpCAM and CD16 Induce Effective Antibody-Dependent Cellular Cytotoxicity Against Human Carcinoma Cells

A heterodimeric bispecific biological recombinant drug was synthesized by splicing DNA fragments from two fully humanized single-chain variable-fragment (scFV) antibody fragments forming a novel drug simultaneously recognizing the CD16 natural killer (NK) cell marker and the cancer marker epithelial cell adhesion molecule (EpCAM). The drug precipitously enhanced the killing of human carcinomas of the prostate, breast, colon, head, and neck even at very low effector:target ratios. The drug EpCAM16 rendered even nonactivated NK cell-proficient killers and activated them to kill via degranulation and cytokine production. Studies show that bispecific antibodies can be used to induce proficient killing of the carcinoma targets that ordinarily are resistant to NK-mediated killing. Apparently, the innate immune system can be effectively recruited to kill cancer cells using the bispecific antibody platform and EpCAM targeting.

Bioengineering a Unique Deimmunized Bispecific Targeted Toxin That Simultaneously Recognizes Human CD22 and CD19 Receptors in a Mouse Model of B-Cell Metastases

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.

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

Background:Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy.Methods:To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut.Results:In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14–16 days post tumour innoculation.Conclusion:These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.

Efficacy of nonviral gene transfer in the canine brain

OBJECT The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain. METHODS Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression. RESULTS Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS). CONCLUSIONS Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.

Structural elucidation of new antibiotic peptides, atroviridins A, B and C from Trichoderma atroviride

Three peptaibols, atroviridins A–C, were isolated from the culture broth of Trichoderma atroviride. The amino acid sequences were determined by mass spectrometry and two-dimensional NMR experiments. They are composed of 20 residues with a high ratio of α-aminoisobutyric acid, and these all are determined as new peptaibols.