Significant Bystander Effect Research Articles

Preclinical studies of BB-1701, a HER2-targeting eribulin-containing ADC with potent bystander effect and ICD activity.

Several HER2-targeting antibody-drug conjugates (ADC) have gained market approval for the treatment of HER2-expressing metastasis. Promising responses have been reported with the new generation of ADCs in patients who do not respond well to other HER2-targeting therapeutics. However, these ADCs still face challenges of resistance and/or severe adverse effects associated with their particular payload toxins. Eribulin, a therapeutic agent for the treatment of metastatic breast cancer and liposarcoma, is a new choice of ADC payload with a distinct mechanism of action and safety profile. We've generated a novel HER2-tageting eribulin-containing ADC, BB-1701. The potency of BB-1701 was tested in vitro and in vivo against cancer cells where HER2-expressing levels vary in a large range. Bystander killing effect and toxin-induced immunogenic cell death (ICD) of BB-1701 were also tested. In comparison with HER2-targeting ADCs with DM1 and Dxd payload, eribulin-containing ADC demonstrated higher in vitro cytotoxicity in HER2-low cancer cell lines. BB-1701 also effectively suppressed tumors in models resistant to DM1 or Dxd containing ADCs. Mode of action studies showed that BB-1701 had a significant bystander effect on HER2-null cells adjacent to HER2-high cells. In addition, BB-1701 treatment induced ICD. Repeated doses of BB-1701 in nonhuman primates showed favorable pharmacokinetics and safety profiles at the intended clinical dosage, route of administration, and schedule. The preclinical data support the test of BB-1701 in patients with various HER2-expressing cancers, including those resistant to other HER2-targeting ADCs. A phase I clinical trial of BB-1701 (NCT04257110) in patients is currently underway.

Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

CBMS-3 Potent bystander effect in suicide gene therapy using genetically engineered stem cells from human exfoliated deciduous teeth

HSV thymidine kinase (TK)/ganciclovir (GCV) has a long history of application in malignant glioma and we have previously demonstrated its bystander effect on gliomas using several stem cell types as a vehicle. The main reason for applying stem cells is that they have a unique tumor-trophic activity that allows them to deliver TK genes efficiently to nearby the tumor. Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells easily harvested from dental pulp and no studies have reported suicide gene therapy using SHED as a carrier for malignant gliomas. For transduction of SHED with the HSVTK gene (SHEDTK), we used HSVTK retrovirus-producing cells.In vitro experiments showed a significant migration ability of SHEDTK toward tumor-conditioned medium and representative tumor growth factors. We also detected a significant bystander effect of SHEDTK on gliomas in the presence of GCV. In vitro time-lapse imaging showed that both SHEDTK and glioma cells underwent gradual morphological apoptosis and activation of caspase 3/7 was observed in both cell types. In intracranial tumor models using nude mice, SHEDTK migrated around the U87 cell mass implanted in the contralateral hemisphere. Additionally, coculture suspensions of SHEDTK and U87-luciferase cells were xeno-transplanted followed by intraperitoneal administration of GCV for 10 days. All mice of treatment group survived for more than 100 days, whereas those treated without GCV died of tumor growth with median survival of 42 days after tumor implantation. Furthermore, pre-existing intracranial U87 model mice were injected intratumorally with SHEDTK followed by GCV administration as described above. The tumor volume was significantly reduced during the treatment period, and over-all surivial in treatment group is prolonged significantly to that of control groups. These results indicate that SHEDTK-based suicide gene therapy might offer a new promising therapeutic modality for human malignant gliomas.

Spotlight on Trastuzumab Deruxtecan (DS-8201,T-DXd) for HER2 Mutation Positive Non-Small Cell Lung Cancer.

Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene that, when mutated or overexpressed, plays an important role in oncogenesis. The landscape of HER2-positive breast cancer has changed dramatically over the past 2 decades with the FDA approval of a growing number of agents (antibodies, tyrosine kinase inhibitors, and antibody-drug conjugates) targeting the HER2 receptor. HER2 inhibition has also been approved for HER2-positive gastric cancer. HER2 is amplified in 9% and mutated in 3% of lung cancer. Historically, HER2-targeted therapy for lung cancer with trastuzumab, pertuzumab, and trastuzumab emtansine has failed to demonstrate a survival benefit. Trastuzumab deruxtecan (T-DXd) is a novel antibody–drug conjugate with a tetrapeptide linker, which delivers a topoisomerase I inhibitor with a drug-to-antibody ratio of 7~8. The potency of the active payload, as well as its significant bystander effect, resulted in significant anti-tumor activity. The DESTINY-Lung01 trial evaluated T-DXd in HER2-positive non-squamous non-small cell lung cancer (NSCLC) and reported a progression-free survival of 14 months in HER2-mutated NSCLC, earning its breakthrough designation by the FDA. In this review, we will discuss the structural characteristics, pharmacodynamics, and pharmacokinetics of T-DXd. We will also shed light on the preclinical and ongoing clinical trials of T-DXd along with future directions in the management of HER2 positive lung cancer.

Spatially-resolved pharmacokinetic/pharmacodynamic modelling of bystander effects of a nitrochloromethylbenzindoline hypoxia-activated prodrug.

Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates forany diffusion limitations of the prodrug in tumour tissue. Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively. NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug. The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.

Abstract 955: Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998

Abstract Stage-specific embryonic antigen-4 (SSEA-4) ceramide, a globo-series hexasaccharide glycosphingolipid, has been reported to be a tumor-associated antigen. Here, we present a novel antibody-drug conjugate (ADC) targeting SSEA-4 to evaluate its potential as a therapeutic agent for cancer treatment. OBI-998 is an ADC comprising the humanized anti-SSEA-4 antibody (OBI-898) that is conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE) through maleimide and PEGylated cleavable linkers. We demonstrated the specificity of the naked antibody to SSEA-4 by screening against a panel of 21 related carbohydrate antigens using ELISA. OBI-998 displayed potent cytotoxic activity against cells (SKOV3) with a high level of SSEA-4 expression at sub-nanomolar potency but no effect on the viability of cells (SKBR3) with negligible SSEA-4 expression. The bystander killing effect of OBI-998 was shown by the transferring of conditioned medium from SKOV3 cells treated with 1 or 5 nM of OBI-998 to SKBR3 cells. Furthermore, significant bystander effects were observed by co-culturing high (NCI-N87) and low (PANC-1/GFP) SSEA-4-expressing cell lines at different ratios in the presence of 5 or 10 nM of OBI-998. OBI-998 was found to be rapidly internalized into cancer cells within 5 minutes upon binding to its target on the cell surface by confocal microscopy. OBI-998 showed significant anti-tumor efficacy in multiple cancer cell–derived xenograft models at doses of 1, 3, and 10 mg/kg in a dose-dependent manner. More importantly, OBI-998 at a dose of 10 mg/kg showed complete tumor regression in an EGFR-triple mutation non–small cell lung cancer xenograft model, which is resistant to the current last-line tyrosine kinase inhibitor, osimertinib. Pharmacokinetic analysis of OBI-998 revealed that total antibody and the conjugated antibody exhibited similar pharmacokinetic profiles, suggesting OBI-998 is highly stable in vivo. The biodistribution study in HCC1428 tumor-bearing mice indicated that MMAE accumulated in the tumor site at a higher level compared with other blood-rich organs. In addition, the MMAE levels in tumors reached peak level at 24 hours post-treatment, which was much higher than the maximum levels in other organs, and the level was sustained for 168 hours with tumor-to-muscle ratio of 329. OBI-998 is a novel ADC targeting SSEA-4 that possesses desired properties such as high target specificity, rapid internalization, potent cytotoxicity, and significant bystander effects. Furthermore, OBI-998 showed a high level of deposition and a persistent presence of MMAE in tumors and significant anti-tumor efficacy in a variety of animal models. Taken together, these results support the further development of OBI-998 as a therapeutic agent for SSEA-4-targeting cancer therapy. Citation Format: I-Ju Chen, Chun-Chung Wang, Chi-Sheng Shia, Chung-Chen Su, Chi-Huan Lu, Hui-Wen Chang, Ping-Tzu Chiu, Yueh-Chin Wu, Ming-Tain Lai, Wei-Chien Tang, Hsin-Yi Tung, Ren-Yu Hsu. Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 955.

Letter: COVID-19 Pandemic-The Bystander Effect on Stroke Care in Michigan.

Letter: COVID-19 Pandemic-The Bystander Effect on Stroke Care in Michigan.

Sacituzumab govitecan (SG) in patients (pts) with previously treated metastatic endometrial cancer (mEC): results from a phase I/II study.

6081 Background: Unselected pts with EC who progressed on prior chemotherapy have a poor prognosis with limited treatment options. SG is a novel antibody-drug conjugate that targets Trop-2, a cell surface glycoprotein highly expressed in many epithelial tumors. It is conjugated to deliver SN-38, the active metabolite of irinotecan, via a proprietary hydrolyzable linker. Preclinical studies show SG has activity against chemotherapy-resistant EC and significant bystander effect against EC with heterogenous Trop-2 expression (Perrone E. Mol Oncol. 2019). Methods: The phase I/II basket study (NCT01631552) evaluated pts unselected for Trop-2 with advanced solid tumors who received intravenous SG (days 1 and 8 of 21-day cycles), until progression or unacceptable toxicity. CT/MRI scans were obtained at 8-week intervals for response assessment by RECIST 1.1. We report results for mEC pts who progressed after ≥1 prior systemic therapy and were treated with SG 10 mg/kg. Endpoints include safety, objective response rate (ORR), clinical benefit rate (CBR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results: 18 mEC pts (all women; 17 white and 1 black; median age 69 years [range, 41–76]) had a median 3.5 (range 2–6) prior lines of therapy. All pts received prior treatment with platinum therapies. At a median follow-up of 12.7 months, the ORR (95% CI) was 22.2% (6.4–47.6), with 4 partial responses. CBR (95% CI) was 44.4% (21.5–69.2), with 8 of 18 pts having either an objective response or stable disease ≥6 months. The DOR of responders ranged from 9.1 to 26.6 months, with 2 of 4 responders having a duration of ≥18 months. Median PFS (95% CI) was 3.2 months (1.9–9.4), and median OS (95% CI) was 11.9 months (4.7–not calculable). Key grade ≥3 TRAEs in the overall basket study safety population (n=495) included neutropenia (28%), neutrophil count decrease (14%), anemia (10%), diarrhea (8%), fatigue (6%), and febrile neutropenia (5%). A similar safety profile was seen in the mEC cohort. Conclusions: Median OS in unselected pts with mEC who progressed on prior platinum therapy is ~10 months with an ORR of ~10%. SG monotherapy showed clinical activity in pts with relapsed/refractory mEC, consistent with previous preclinical findings, and support further clinical investigation (NCT04251416). The phase II TROPiCS-03 (NCT03964727) study in pts with metastatic solid tumors selected based on elevated Trop-2 expression by a validated IHC assay will also provide further insights. Clinical trial information: NCT01631552.

Preclinical Activity of Sacituzumab Govitecan, an Antibody-Drug Conjugate Targeting Trophoblast Cell-Surface Antigen 2 (Trop-2) Linked to the Active Metabolite of Irinotecan (SN-38), in Ovarian Cancer.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Sacituzumab govitecan (SG) is a novel antibody-drug-conjugate (ADC) targeting trophoblast-antigen-2 (Trop-2), a cell surface glycoprotein highly expressed in many epithelial tumors, to deliver SN-38, the active metabolite of irinotecan. This study aimed to evaluate Trop-2 expression in EOC tissues and the preclinical activity of SG against primary EOC cell lines and xenografts.Methods: Trop-2 expression was assessed in 90 formalin-fixed-paraffin-embedded tumors and nine primary tumor cell lines by immunohistochemistry (IHC) and flow cytometry, respectively. Trop-2 expression and cell viability after exposure to SG in primary tumor cell lines, non-targeting control ADC, and SG-parental antibody hRS7 were evaluated using flow-cytometry-based-assays. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2– EOC cell lines was tested in vitro using 4 h Chromium-release-assays. In vivo activity of SG was evaluated against Trop-2+ EOC xenografts.Results: Moderate-to-strong staining was seen in 47% (42/90) of ovarian tumors by IHC while 89% (8/9) of the primary EOC cell lines overexpressed Trop-2 by flow cytometry. EOC Trop-2+ were significantly more sensitive to SG compared to control ADC (p < 0.05). Both SG and hRS7 mediated high ADCC activity against Trop-2+ cell lines. SG also induced significant bystander killing of Trop-2– tumor cells admixed with Trop-2+ EOC cells. In in vivo experiments SG treatment demonstrated impressive anti-tumor activity against chemotherapy-resistant EOC xenografts.Conclusion: SG demonstrates remarkable preclinical activity against biologically aggressive and chemotherapy-resistant EOC cell lines and a significant bystander effect against EOC cell lines with heterogenous Trop-2 expression. Clinical trials are warranted.

Lentivirus-mediated CDglyTK gene-modified free flaps by intra-artery perfusion show targeted therapeutic efficacy in rat model of breast cancer

BackgroundFree flap-mediated gene therapy in the tumor bed following surgical resection is a promising approach in cancer targeted treatment of residual disease. We investigated the selective killing efficacy of a lentivirus-mediated cytosine deaminase-thymidine kinase (CDglyTK) gene in transplanted breast cancer delivered into a free flap by intra-artery perfusion.MethodsProliferation, apoptosis, and cell cycle of rat SHZ-88 breast cancer cells transfected with a lentivirus-mediated CD/TK gene were measured following treatment with ganciclovir and 5-flucytosine in vitro. A model of residual disease of breast cancer in a rat superficial inferior epigastric artery (SIEA) flap model was used to study the therapeutic potential of a double suicide CD/TK and prodrug system in vivo.ResultsKilling efficacy of the double suicide CD/TK and prodrug system on SHZ-88 cells was mediated by increased apoptosis and cell cycle arrest at the G1 phase with significant bystander effect. Following recombinant lentivirus transfection of rat SIEA flap by intra-artery perfusion, CD/TK gene expression was limited to the flap, and the volume and weight of transplanted tumors were significantly reduced without observable toxicity.ConclusionsSIEA flaps transfected with a lentivirus-mediated CDglyTK gene by intra-artery perfusion effectively suppress transplanted breast tumor growth without obvious systemic toxic effects in rats.

Abstract 4481: BT5528, an EphA2-targeting Bicycle Toxin Conjugate (BTC): Profound efficacy without bleeding and coagulation abnormalities in animal models

Abstract Ephrin receptor A2 (EphA2) is part of the Ephrin receptor family of cell-cell junction proteins highly overexpressed in several solid tumours, and is associated with poor prognosis. EphA2 has long been a high-value target for cancer therapeutics, but development of an Antibody Drug Conjugate targeting EphA2 (MEDI-547) was stopped in early clinical development after severe adverse events were seen (Annunziata et al, 2013). We have developed a series of Bicycle Toxin Conjugates (BTC®) comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to EphA2, which is covalently linked to a toxin payload via a cleavable linker. The small size of BTCs offers a significant advantage over other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation, renal clearance and improved tumour penetration. Bicycle binders for EphA2 were identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of Bicycles, conjugated to cleavable linkers &amp; toxins to form Bicycle Toxin Conjugates (BTCs). We selected the candidate BTC BT5528 from a panel of &amp;gt;75 BTCs, based on in vivo efficacy, tolerability and drug-like properties. BT5528 is effective in EphA2-expressing xenograft models, with complete tumour regression seen from 0.5mg/kg weekly. Expression of EphA2 is high across a range of cancers of high unmet medical need. A good correlation with efficacy was seen across cell- and patient- derived xenograft models including lung, breast, oesophageal, ovarian, prostate, gastric and sarcoma tumours. No significant efficacy is seen in tumours without EphA2 expression. Rapid tumour penetration, renal elimination and significant bystander effect produce efficacy through a “hit &amp; run” approach, with low systemic exposure to conjugate or free toxin. Efficacy is seen with dosing intervals of 1 or 2 weeks, despite a terminal half-life of ~30min for BTCs across species. In simple xenografts (CDX, ~200mm3), efficacy of BT5528 &amp; anti-EphA2 ADC are comparable. In large &amp; more complex models (heterogeneous PDX, &amp;gt;1000mm3), the ADC shows reduced efficacy, while the efficacy of BT5528 is maintained. Clinical development of MEDI-547 was terminated when patients showed profound bleeding &amp; coagulation toxicity, consistent with the nature of toxicology seen in preclinical studies. In contrast, BT5528 shows no sign of bleeding in toxicology studies in rat and NHP and no changes in coagulation parameters or liver enzymes. The EphA2 targeting BTC BT5528 shows potent anti-tumour activity in a range of solid tumour xenograft models without the limiting toxicity observed with previous Antibody Drug Conjugate approaches. IND-enabling studies for BT5528 are currently underway. Citation Format: Gavin S. Bennett, Amy Brown, Gemma Mudd, Johanna Lahdenranta, Nicholas Keen. BT5528, an EphA2-targeting Bicycle Toxin Conjugate (BTC): Profound efficacy without bleeding and coagulation abnormalities in animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4481.

Antitumor effects of a dual-specific lentiviral vector carrying the Escherichia coli purine nucleoside phosphorylase gene

The Escherichia coli purine nucleoside phosphorylase/Fludarabine phosphate (ePNP/Fludara) suicide system has several drawbacks, such as side-effects and the low efficiency of ePNP expression. In this study, we evaluated the antitumor effects of the dual-specific 8HSEs-hTERTp-ePNP/Fludara suicide system under hyperthermia in vitro and in vivo. Luciferase activities from the 8HSEs-hTERT and CMV promoters were compared using the dual luciferase assay in SW480 (high hTERT expression) and MKN74 cells (hTERT-negative) in the presence and absence of hyperthermia. Then, we investigated the effects of overexpressing the suicide gene ePNP using 8HSEs-hTERT-driven lentiviral vectors with Fludara on in vitro cell viability, side-effects, apoptosis, cycle distribution, colony formation and in vivo xenograft tumor growth. At 43°C, luciferase activity from the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN74 cells. Importantly, luciferase activities from the 8HSEs-hTERT promoter were much higher than from the CMV promoter in hTERT-expressing SW480 cells under heated conditions. The in vitro quantitative analysis showed a 4-fold higher ePNP protein expression from the 8HSEs-hTERT promoter at 43°C than at 37°C in SW480 cells and the ePNP mRNA expression in SW480 cells at 43°C was also higher than at 37°C. Conversely, ePNP mRNA and protein expression were low, almost absent, in hTERT-negative MKN74 cells with or without hyperthermia. After Fludara addition, cell cytotoxicity assays showed that the significant inhibitory effect of the 8HSEs-hTERTp-ePNP on SW480 cells was dose- and time-dependent with hyperthermia. The 8HSEs-hTERTp-ePNP/Fludara suicide system significantly inhibited SW480 cell viability, colony formation, cell cycle progression and induced apoptosis in vitro, but also induced significant bystander effects, especially under the heated conditions. At the protein level, the suicide system significantly promoted Bax, caspase-3 and p53 expression and suppressed Bcl-2 expression. In sections from mouse xenografts, TUNEL assays showed that the suicide system reduced xenograft growth and induced SW480 apoptosis. These results indicated that the combinatorial cancer- and heat-specific promoter system has great potential for improving the efficacy of cancer treatment with hyperthermia. The 8HSEs-hTERTp-ePNP/Fludara system may serve as a powerful strategy for cancer gene therapy combined with hyperthermia.

Metabolic Rewiring Drives Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells.Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b).Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Central Nervous System Injury - A Newly Observed Bystander Effect of Radiation.

The unintended side effects of cancer treatment are increasing recognized. Among these is a syndrome of long-term neurocognitive dysfunction called cancer/chemotherapy related cognitive impairment. To date, all studies examining the cognitive impact of cancer treatment have emphasized chemotherapy. Radiation-induced bystander effects have been described in cell culture and, to a limited extent, in rodent model systems. The purpose of this study was to examine, for the first time, the impact of non-brain directed radiation therapy on the brain in order to elucidate its potential relationship with cancer/chemotherapy related cognitive impairment. To address this objective, female BALB/c mice received either a single 16 gray fraction of ionizing radiation to the right hind limb or three doses of methotrexate, once per week for three consecutive weeks. Mice were sacrificed either 3 or 30 days post-treatment and brain injury was determined via quantification of activated astrocytes and microglia. To characterize the effects of non-brain directed radiation on brain glucose metabolism, mice were evaluated by fluorodeoxygluocose positron emission tomography. A single fraction of 16 gray radiation resulted in global decreases in brain glucose metabolism, a significant increase in the number of activated astrocytes and microglia, and increased TNF-α expression, all of which lasted up to 30 days post-treatment. This inflammatory response following radiation therapy was statistically indistinguishable from the neuroinflammation observed following methotrexate administration. In conclusion, non-brain directed radiation was sufficient to cause significant brain bystander injury as reflected by multifocal hypometabolism and persistent neuroinflammation. These findings suggest that radiation induces significant brain bystander effects distant from the irradiated cells and tissues. These effects may contribute to the development of cognitive dysfunction in treated human cancer patients and warrant further study.

Effect of VP22-CD genetically engineered vectors on killing glioma cells in vitro

Objective s To construct CD and VP22-CD genetically engineered vectors and to investigate their killing effects on glioma cells in vitro. Methods CD and VP22 gene segments were obtain using polymerase chain reaction(PCR). The obtained gene fragments were inserted into pEGFP-N1 vectors using in-fusion gene cloning method. CD and VP22-CD gene engineering vectors were constructed. The constructed gene engineering vectors were transfected into the glioma cells C6 and U87 cells using the liposome transfection method, and the transfection efficiency was observed through in vitro light microscope and fluorescence microscope. The kill rate of glioma cells after transfection was assessed after adding the prodrug 5-fluorocytosine using MTT colorimetric method. Results (1)CD gene or VP22-CD fusion gene was inserted into the pEGFP-CD vectors, constructing and obtaining pEGFP-CD and pEGFP-VP22-CD vectors.(2)The pEGFP-CD and pEGFP-VP22-CD vectors were transfected into C6 or U87 glioma cells. The transfection rates were 2.5% and 3.7%, 2% and 4.1%, respectively.(3)Transfection of pEGFP-CD or pEGFP-VP22-CD vectors in the C6 or U87 cells and after adding prodrug 5-fluorocytosine, the survival rates were 30.36±0.63% and 11.75±1.01%, 28.78±3.62% and 18.26±2.27%, respectively. Compared with the transfection of pEGFP-N1 group, there was significant difference(all P<0.05).(4)After adding prodrug 5-fluorocytosine in C6 or U87 cells, There was significant difference between the transfection of pEGFP-VP22-CD group and the transfection of pEGFP-CD group(all P<0.05). Conclusions The CD/5-FC system has significant killing effect and bystander effect on the malignant glioma cells in vitro, and shuttle protein VP22 can further promote the killing effect of the system on cells. Key words: Glioma; CD/5-FC system; Bystander effect; Shuttle protein VP22

SC-07 * CYCLIC INTRANASAL APPLICATION OF NEURAL STEM CELL-MEDIATED ENZYM/PRODRUG THERAPY USING A NOVEL HSV-THYMIDINE KINASE VARIANT INHIBITS INTRACEREBRAL GLIOMA GROWTH AND IMPROVES SURVIVAL

We have recently introduced the concept of intranasal application of NSCs as a noninvasive and direct alternative delivery method for glioma-targeting NSCs. Here, we demonstrate that the repeated non-invasive intranasal administration of tumor-targeting NSC is able to deliver a suicide gene (TK.007) to intracerebrally growing human glioblastoma xenografts. Murine NSC were genetically modified to stably express a novel, codon-optimized HSVtk(A168H) mutant (TK.007). The biological activity of the NSC-mediated TK.007/ganciclovir (GCV) system was assessed in cell survival and bystander assays using various human glioma cell lines. Therapeutic effects of intratumoral and intranasal NSC-TK007 applications alone and the sequential combinations of both was tested using an intracranial U87 human glioblastoma model in nude mice. Therapeutic efficiency was determined by assessment of tumor size by 7T MR-imaging and by establishment of Kaplan-Meier survival curves. Tumor targeted migration was demonstrated by intravital imaging of luciferase-expressing NSC. Cell survival and bystander assays confirmed the GCV catalytic activity of NSC-expressed TK007 in a GCV dose dependent manner and a significant bystander effect at 12.5% of cells. Tumor targeted migration was mediated by intracerebral gradients of SDF-1. In glioma-bearing mice a single intratumoral application of NSC-TK007 followed by systemic prodrug application of GCV lead to a significant tumor growth inhibition of 72% versus the control groups at day 22 and translated in a prolonged survival time. Mice recieving a cyclic intranasal application of NSC-TK.007 alone displayed a signifcant longer survival than the controls. This effect was significantly enhanced when cyclic intranasal NSC-TK.007 therapy was preceded by a single intratumoral application of NSC-TK007 and led to a significant tumor growth inhibition of >95% and significantly improved survival time. Our findings establish that the intranasal application of therapeutically-modified NSC is a safe and non-invasive application method which allows a chronic treatment during the disease course.

REPEATED INTRANASAL APPLICATION OF NEURAL STEM CELL-MEDIATED ENZYM/PRODRUG THERAPY USING A NOVEL HSV-THYMIDINE KINASE VARIANT IMPROVES THERAPEUTIC EFFICIENCY IN AN INTRACRANIAL GLIOBLASTOMA MODEL

BACKGROUND: Neural stem cells (NSCs) have an inherent brain tumor tropism that can be exploited for targeted delivery of therapeutic genes to invasive gliomas. We have recently introduced the concept of intranasal application of NSCs as a noninvasive and direct alternative delivery method for glioma-targeting NSCs. Here, we demonstrate that the repeated non-invasive intranasal administration of tumor-targeting NSC is able to deliver a novel suicide gene (TK.007) to intracerebrally growing human glioblastoma xenografts. METHODS: Murine NSC were genetically modified to stably express a novel, codon-optimized HSVtk(A168H) mutant (TK.007). The biological activity of the NSC-mediated TK.007/ganciclovir (GCV) system was assessed in cell survival and bystander assays using various human glioma cell lines. Therapeutic effects of intratumoral and intranasal NSC-TK007 applications alone and the sequential combinations of both was tested using an intracranial U87 human glioblastoma model in nude mice. All animals received 50 mg/kg GCV i.p. for five consecutive days. In all experiments two control groups received either NaCl instead of GCV or NSC containing the control vector instead of NSC-TK.007. Therapeutic efficiency was determined by assessment of tumor size by 7T MR-imaging and by establishment of Kaplan-Meier survival curves. RESULTS: Cell survival and bystander assays confirmed the GCV catalytic activity of NSC-expressed TK007 in a GCV dose dependent manner and a significant bystander effect at 12.5% of cells. Tumor targeted migration was demonstrated by intravital imaging of luciferase-expressing NSC. In glioma-bearing mice a single intratumoral application of NSC-TK007 followed by systemic prodrug application of GCV lead to a significant tumor growth inhibition of 72% versus the control groups at day 22 and translated in a prolonged survival time. Mice recieving a cyclic intranasal application of NSC-TK.007 alone displayed a signifcant longer survival than the controls. This effect was significantly enhanced when cyclic intranasal NSC-TK.007 therapy was preceded by a single intratumoral application of NSC-TK007 and led to a significant tumor growth inhibition of >95% and improved survival time. CONCLUSIONS: Our data demonstrates that the NSC-mediated TK.007/GCV therapy, which is based on the clinically most widely used suicide system to date, is safe, nontoxic, and effective in glioblastoma-bearing mice. Most importantly, our findings establish that the intranasal application of therapeutically-modified NSC is a safe and non-invasive application method which allows a chronic treatment during the disease course. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

Antibody-directed Double Suicide Gene Therapy Targeting of MUC1- Positive Leukemia Cells In Vitro and In Vivo

Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

Induction of lethal bystander effects in human breast cancer cell cultures by DNA-incorporated Iodine-125 depends on phenotype

Purpose: This study uses a three-dimensional cell culture model to investigate lethal bystander effects in human breast cancer cell cultures (MCF-7, MDA-MB-231) treated with 125I-labeled 5-iodo-2 -deoxyuridine (125IdU). These breast cancer cell lines respectively form metastatic xenografts in nude mice in an estrogen-dependent and independent manner.Materials and methods: In the present study, these cells were cultured in loosely-packed three-dimensional architecture in a Cytomatrix™ carbon scaffold. Cultures were pulse-labeled for 3 h with 125IdU to selectively irradiate a minor fraction of cells, and simultaneously co-pulse-labeled with 0.04 mM 5-ethynyl-2′-deoxyuridine (EdU) to identify the radiolabeled cells using Click-iT® EdU and flow cytometry. The cultures were then washed and incubated for 48 h. The cells were then harvested, serially diluted, and seeded for colony formation. Aliquots of cells were subjected to flow cytometry to determine the percentage of cells labeled with 125IdU/EdU. Additional aliquots were used to determine the mean 125I activity per labeled cell. The percentage of labeled cells was about 15% and 10% for MCF-7 and MDA cells, respectively. This created irradiation conditions wherein the cross-dose to unlabeled cells was small relative to the self-dose to labeled cells. The surviving fraction relative to EdU-treated controls was measured.Results: Survival curves indicated significant lethal bystander effect in MCF-7 cells, however, no significant lethal bystander effect was observed in MDA-MB-231 cells.Conclusions: These studies demonstrate the capacity of 125IdU to induce lethal bystander effects in human breast cancer cells and suggest that the response depends on phenotype.