Liposomal irinotecan + 5-FU/LV is approved for treating patients with mPDAC following progression with gemcitabine-based therapy. Liposomal irinotecan + 5-FU/LV + OX (NALIRIFOX) is being investigated 1L in a phase I/II study of patients with locally advanced/mPDAC. Serum CA 19-9 levels are typically elevated in such patients and post-treatment decreases are associated with prolonged survival. We report exploratory survival analyses from the phase I/II study for subgroups defined by post-treatment changes in CA 19-9 levels. Following dose exploration, the regimen of liposomal irinotecan 50 mg/m2 (free base), OX 60 mg/m2, 5-FU 2400 mg/m2 and LV 400 mg/m2, on days 1 and 15 of 28-day cycles, was selected for dose expansion and 25 more patients were enrolled. In total, 32 patients received the selected (maximum tolerable) dose. Tumors (RECIST v1.1) and serum CA 19-9 were assessed at screening, every 8 weeks and at end of treatment (EoT); tumor assessments continued after EoT. Progression-free and overall survival (PFS and OS) were compared across subgroups defined by best change within the first 16 weeks (data cut-off 26 Feb 2020). In total, 30/32 patients had a baseline CA 19-9 measurement (median 315.5 U/mL, range 2–127115) of whom 22 had measurements by week 16 (analysis set) with median best change –49.4% (range –100%, +376%). Survival data (Table) were similar for patients with above-normal (≥ 37 U/mL) baseline CA 19-9 levels (n = 17; median best change –35.9% [range –100%, +278%]).Table: 1529PPFSPP 50/60 N = 32All n = 22Analysis set, best CA 19-9 change≥ 20% decrease≥ 50% decreaseYes n = 14No n = 8Yes n = 11No n = 11Progressed/died,a n (%)17 (53.1)12 (54.5)6 (42.9)6 (75.0)4 (36.4)8 (72.7)Median, mo (95% CI)9.2 (7.69, 11.96)9.6 (7.59, 32.30)32.3 (7.95, 32.30)7.6 (1.48, 9.56)11.2 (7.95, NE)7.6 (1.48, 32.30)HR (95% CI)--0.13 (0.04, 0.50)0.33 (0.09, 1.12)OSDied, n (%)20 (62.5)14 (63.6)7 (50.0)7 (87.5)6 (54.6)8 (72.7)Median, mo (95% CI)12.6 (8.74, 18.69)12.7 (8.74, 22.54)22.5 (12.39, NE)8.2 (2.50, 18.69)22.5 (11.60, 22.54)9.2 (4.83, NE)HR (95% CI)-0.24 (0.08, 0.75)0.55 (0.19, 1.60)a Patients who progressed/died after new therapy or >16 wks after last non-PD assessment were censored. Open table in a new tab a Patients who progressed/died after new therapy or >16 wks after last non-PD assessment were censored. 1L NALIRIFOX reduced CA 19-9 levels in patients with locally advanced/mPDAC. Median OS and PFS were numerically higher in patients with a ≥20% decrease within the first 16 weeks. CA 19-9 is a potential biomarker of post-NALIRIFOX outcomes.

Liposomal irinotecan + 5-fluorouracil/leucovorin (5-FU/LV) is approved for adults with metastatic pancreatic ductal adenocarcinoma following progression with gemcitabine-based therapy. We report results from an open-label phase I/II study (NCT02551991) of adults with untreated, unresectable, locally advanced/metastatic PDAC receiving liposomal irinotecan + 5-FU/LV + oxaliplatin (NALIRIFOX). Eligible patients were adults with ECOG performance status (PS) ≤ 1 and adequate organ function who received NALIRIFOX (liposomal irinotecan 50 mg/m2 (free base), 5-FU 2400 mg/m2, LV 400 mg/m2, oxaliplatin 60 mg/m2) on days 1 and 15 of each 28-day cycle. The primary endpoint was safety; secondary endpoints included progression-free survival (PFS), overall survival (OS), best overall response, overall response rate (ORR), disease control rate at 16 weeks (DCR16) and duration of response (DoR). RECIST v1.1 was assessed at screening, every 8 weeks and at treatment end. Overall, 32 patients were included (median [range] age 58.0 [39–76] years; 43.8% men; 87.5% metastatic disease; 56.3% ECOG PS 1). In total, 22 patients experienced grade ≥ 3 treatment-emergent adverse events (TEAEs): neutropenia (31.3%), febrile neutropenia (12.5%), hypokalemia (12.5%), diarrhea (9.4%), nausea (9.4%) and decreased neutrophil count (9.4%). Serious TEAEs were reported in 17 patients: nausea (9.4%) and febrile neutropenia (9.4%). TEAEs led to 3 deaths (none treatment-related), to dose adjustment in 26 and discontinuation in 8 patients. Median (95% CI) PFS and OS were 9.2 (7.69–11.96) months and 12.6 (8.74–18.69) months, respectively. One patient, with locally advanced disease, had complete response, 10 partial response, and 15 stable disease. ORR (95% CI) was 34.4 (18.6–53.2) %, DCR16 was 71.9 (53.3–86.3) % and median (95% CI) DoR was 9.4 (3.52, NE) months. First-line NALIRIFOX raised no new safety signals in patients with locally advanced/metastatic PDAC; anti-tumour activity was promising. The randomized phase III NAPOLI-3 study (NCT04083235) will compare NALIRIFOX with gemcitabine + nab-paclitaxel.

Liposomal irinotecan + 5‑fluorouracil/leucovorin (5-FU/LV) is approved for adults with metastatic pancreatic ductal adenocarcinoma (PDAC) following progression with gemcitabine-based therapy. We report long-term follow-up results (data cut-off 26 Feb 2020) from an open-label phase 1/2 study (NCT02551991; EudraCT 2015-003086-28) of adults with previously untreated, unresectable, locally advanced/metastatic PDAC receiving liposomal irinotecan + 5-FU/LV + oxaliplatin (NALIRIFOX). Following dose exploration (Part 1A), the dose selected for expansion (Part 1B), based on dose-limiting toxicities and cumulative safety data, was liposomal irinotecan 50 mg/m2 (free base), 5-FU 2400 mg/m2, LV 400 mg/m2, oxaliplatin 60 mg/m2 on days 1 and 15 of each 28-day cycle. The analyses included patients receiving the selected dose (pooled population 50/60): 7 patients from Part 1A and 25 from Part 1B. Patients were aged ≥ 18 years with ECOG performance status score ≤ 1 and adequate organ function. The primary endpoint was safety and tolerability; secondary efficacy endpoints were progression-free survival (PFS; primary efficacy endpoint), overall survival (OS), best overall response, overall response rate (ORR), disease control rate at 16 weeks (DCR16) and duration of response (DoR); exploratory endpoints included tumour subtype. Disease was assessed (RECIST v1.1) at screening, end of treatment and every 8 weeks. Archival tumour samples were subtyped (Moffitt schema) using the PurISTSM RNAseq assay (GeneCentric Therapeutics, Inc). The PP 50/60 comprised 32 patients (median age 58.0 years [range 39-76]; 14 [43.8%] men; 28 [87.5%] with metastatic disease at diagnosis; 18 [56.3%] with ECOG performance status score 1; 1 receiving study treatment at data cut-off). In total, 22 of these patients had grade ≥ 3 treatment-related treatment-emergent adverse events (TEAEs); the most common were neutropaenia (31.3%), febrile neutropaenia (12.5%), hypokalaemia (12.5%), diarrhoea (9.4%), nausea (9.4%) and decreased neutrophil count (9.4%); vomiting occurred in 6.3% of patients, while fatigue and peripheral neuropathy were not reported. Serious TEAEs (SAEs) were reported in 17 patients; 10 of these patients had SAEs considered related to treatment, most commonly nausea (9.4%) and febrile neutropaenia (9.4%). TEAEs leading to death occurred in 3 patients (malignant gastrointestinal obstruction, upper gastrointestinal haemorrhage, disease progression); none were considered related to treatment. TEAEs led to dose adjustment in 26 patients and discontinuation (of oxaliplatin or all four study drugs) in 8. Median PFS (95% CI) was 9.2 months (7.69, 11.96) and median OS was 12.6 months (8.74, 18.69). Complete response was observed in 1 patient (with locally-advanced disease), partial response in 10, and stable disease in 15. ORR (95% CI) was 34.4% (18.6, 53.2), DCR16 was 71.9% (53.3, 86.3) and median DoR was 9.4 months (3.52, NE). Tumour subtype and response data were available for 9 patients in the PP 50/60 (classical, n=8, PFS range 7.7-17.8 months; basal-like, n=1, PFS 9.6 months). No new safety signals were observed with first-line NALIRIFOX in patients with locally advanced/metastatic PDAC, and anti-tumour activity was promising. The ongoing randomized phase 3 NAPOLI-3 study (NCT04083235; EudraCT 2018-003585-14) will compare NALIRIFOX with gemcitabine + nab-paclitaxel.

Liposomal irinotecan + 5‑fluorouracil/leucovorin (5-FU/LV) is approved for adults with metastatic pancreatic ductal adenocarcinoma (PDAC) following progression with gemcitabine-based therapy. We report long-term follow-up results (data cut-off 26 Feb 2020) from an open-label phase 1/2 study (NCT02551991; EudraCT 2015-003086-28) of adults with previously untreated, unresectable, locally advanced/metastatic PDAC receiving liposomal irinotecan + 5-FU/LV + oxaliplatin (NALIRIFOX). Following dose exploration (Part 1A), the dose selected for expansion (Part 1B), based on dose-limiting toxicities and cumulative safety data, was liposomal irinotecan 50 mg/m2 (free base), 5-FU 2400 mg/m2, LV 400 mg/m2, oxaliplatin 60 mg/m2 on days 1 and 15 of each 28-day cycle. The analyses included patients receiving the selected dose (pooled population 50/60): 7 patients from Part 1A and 25 from Part 1B. Patients were aged ≥ 18 years with ECOG performance status score ≤ 1 and adequate organ function. The primary endpoint was safety and tolerability; secondary efficacy endpoints were progression-free survival (PFS; primary efficacy endpoint), overall survival (OS), best overall response, overall response rate (ORR), disease control rate at 16 weeks (DCR16) and duration of response (DoR); exploratory endpoints included tumour subtype. Disease was assessed (RECIST v1.1) at screening, end of treatment and every 8 weeks. Archival tumour samples were subtyped (Moffitt schema) using the PurISTSM RNAseq assay (GeneCentric Therapeutics, Inc). The PP 50/60 comprised 32 patients (median age 58.0 years [range 39-76]; 14 [43.8%] men; 28 [87.5%] with metastatic disease at diagnosis; 18 [56.3%] with ECOG performance status score 1; 1 receiving study treatment at data cut-off). In total, 22 of these patients had grade ≥ 3 treatment-related treatment-emergent adverse events (TEAEs); the most common were neutropaenia (31.3%), febrile neutropaenia (12.5%), hypokalaemia (12.5%), diarrhoea (9.4%), nausea (9.4%) and decreased neutrophil count (9.4%); vomiting occurred in 6.3% of patients, while fatigue and peripheral neuropathy were not reported. Serious TEAEs (SAEs) were reported in 17 patients; 10 of these patients had SAEs considered related to treatment, most commonly nausea (9.4%) and febrile neutropaenia (9.4%). TEAEs leading to death occurred in 3 patients (malignant gastrointestinal obstruction, upper gastrointestinal haemorrhage, disease progression); none were considered related to treatment. TEAEs led to dose adjustment in 26 patients and discontinuation (of oxaliplatin or all four study drugs) in 8. Median PFS (95% CI) was 9.2 months (7.69, 11.96) and median OS was 12.6 months (8.74, 18.69). Complete response was observed in 1 patient (with locally-advanced disease), partial response in 10, and stable disease in 15. ORR (95% CI) was 34.4% (18.6, 53.2), DCR16 was 71.9% (53.3, 86.3) and median DoR was 9.4 months (3.52, NE). Tumour subtype and response data were available for 9 patients in the PP 50/60 (classical, n=8, PFS range 7.7-17.8 months; basal-like, n=1, PFS 9.6 months). No new safety signals were observed with first-line NALIRIFOX in patients with locally advanced/metastatic PDAC, and anti-tumour activity was promising. The ongoing randomized phase 3 NAPOLI-3 study (NCT04083235; EudraCT 2018-003585-14) will compare NALIRIFOX with gemcitabine + nab-paclitaxel.

Liposomal irinotecan + 5-fluorouracil/leucovorin (5-FU/LV) is approved for adults with metastatic pancreatic ductal adenocarcinoma (mPDAC) following progression with gemcitabine-based therapy. First-line (1L) liposomal irinotecan + 5-FU/LV + oxaliplatin (NALIRIFOX) is being investigated in a phase I/II trial of patients with locally advanced/mPDAC (NCT02551991). Serum CA 19-9 levels are typically elevated in patients with mPDAC and post-treatment reductions in CA 19-9 levels have been associated with prolonged survival. We report the results of an exploratory survival analysis from the phase I/II trial of 1L NALIRIFOX in mPDAC for subgroups defined by post-treatment changes in CA 19-9 level. Eligible patients were adults with ECOG performance status (PS) ≤ 1 and adequate organ function who received 1L NALIRIFOX (liposomal irinotecan 50 mg/m2 (free base), 5-FU 2400 mg/m2, LV 400 mg/m2, oxaliplatin 60 mg/m2) on days 1 and 15 of each 28-day cycle. Tumors (RECIST v1.1) and serum CA 19-9 were assessed at screening, every 8 weeks and at end of treatment. Progression-free (PFS) and overall survival (OS) were compared for subgroups defined by best change in CA 19-9 level over the first 16 weeks of treatment (≥ 20% and ≥ 50% decrease; data cut-off 26 Feb 2020). In total, 32 patients were eligible for the trial, of whom 30 had a baseline CA 19-9 measurement (median [range] 315.5 [2–127115] U/mL) and 22 had a repeat CA 19-9 measurement by Week 16 (analysis set). Overall, NALIRIFOX reduced CA 19-9 level: median (range) best change of –49.4 (–100, +376)%. Median OS and PFS were numerically higher in patients with a CA 19-9 decrease ≥ 20% by Week 16 (Table)Table: 193PAll N = 32Analysis set n = 22Analysis set, by best change in CA 19-9 by Week 16≥ 20% decrease≥ 50% decreaseYes n = 14No n = 8Yes n = 11No n = 11PFSProgressed/died,a n (%)17 (53.1)12 (54.5)6 (42.9)6 (75.0)4 (36.4)8 (72.7)Median (95% CI) months9.2 (7.69, 11.96)9.6 (7.59, 32.30)32.3 (7.95, 32.30)7.6 (1.48, 9.56)11.2 (7.95, NE)7.6 (1.48, 32.30)HR (95% CI)–0.13 (0.04, 0.50)0.33 (0.09, 1.12)OSDied, n (%)20 (62.5)14 (63.6)7 (50.0)7 (87.5)6 (54.6)8 (72.7)Median (95% CI) months12.6 (8.74, 18.69)12.7 (8.74, 22.54)22.5 (12.39, NE)8.2 (2.50, 18.69)22.5 (11.60, 22.54)9.2 (4.83, NE)HR (95% CI)–0.24 (0.08, 0.75)0.55 (0.19, 1.60)a Patients who progressed/died after new therapy or >16 weeks after last non-progressive disease assessment were censored. Open table in a new tab . a Patients who progressed/died after new therapy or >16 weeks after last non-progressive disease assessment were censored. 1L NALIRIFOX reduced CA 19-9 levels in patients with locally advanced/mPDAC. CA 19-9 is a potential biomarker of post-NALIRIFOX outcomes in these patients.

Background Liposomal irinotecan (nal-IRI) is a liposomal formulation of irinotecan which prolongs circulation of irinotecan and its active metabolite SN-38. This analysis describes the population pharmacokinetics (PK) of nal-IRI in patients with various tumour types, including untreated mPC. Methods Plasma concentration data for total irinotecan (tIRI) and SN-38 from an open-label, phase 2 study of nal-IRI plus 5-fluorouracil/leucovorin and oxaliplatin in patients with untreated mPC (NCT02551991; N = 48) were pooled with data from six other nal-IRI studies (five phase 1/2, one phase 3) in various tumour types. Data from overall 440 patients were used in the population PK model for tIRI and SN-38 after nal-IRI administration. PK parameters were estimated with non-linear mixed effects modelling. The adequacy of the model was assessed based on the uncertainty of parameter estimates, and on advanced evaluation methods such as visual predictive check. Potential covariates such as patient demographics and genotype were investigated to examine inter-individual variability. Results tIRI is described by a two-compartment model with first-order elimination. SN-38 is formed directly by a first-order constant from the central compartment of nal-IRI or after using a transit compartment. In the pooled population (N = 440), clearance was 0.1 L/h and 150 L/h for tIRI and SN-38, respectively. Central and peripheral volumes of distributions for tIRI were 4 L and 0.4 L, respectively. Consistent with previous data, tIRI clearance was 80% higher in patients of Asian ethnicity (n = 154/440) than other populations. Increasing bilirubin levels were associated with lower SN-38 clearance, and tIRI and SN-38 clearances were 20% lower in females than males. The UGT1A128 7/7 homozygous genotype (6% of the study population) had no statistically significant impact on SN-38 clearance. Model evaluation was satisfactory for both tIRI and SN-38. Conclusions The PK of nal-IRI and SN-38 in patients with mPC is well described by the population model. The results suggest that UGT status has no impact on the PK of nal-IRI. Editorial acknowledgement Oxford PharmaGenesis, Oxford, UK for providing editorial support, which was sponsored by Ipsen, Abingdon, UK. Legal entity responsible for the study Ipsen. Funding Ipsen. Disclosure T. Macarulla: Honoraria (institution): Shire Pharmaceuticals; Honoraria (institution): Roche; Honoraria (institution): Tesaro; Honoraria (institution): Baxter; Honoraria (institution), Travel / Accommodation / Expenses: Sanofi; Honoraria (institution): Celgene; Honoraria (institution): QED Therapeutics; Honoraria (institution): Genzyme Europe; Honoraria (institution): Baxalta; Honoraria (institution), Travel / Accommodation / Expenses: Bayer; Honoraria (institution): Incyte; Honoraria (institution): Genzyme; Travel / Accommodation / Expenses: Merck; Travel / Accommodation / Expenses: H3 Biomedicine. K. Brendel: Full / Part-time employment: Ipsen. Z.A. Wainberg: Advisory / Consultancy: Lilly; Advisory / Consultancy: Merck; Advisory / Consultancy: BMS; Advisory / Consultancy: Bayer; Advisory / Consultancy: Novartis; Advisory / Consultancy: Ipsen. F. Dayyani: Advisory / Consultancy: Array; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eisai; Advisory / Consultancy, Speaker Bureau / Expert testimony: Genentech; Speaker Bureau / Expert testimony: Ipsen; Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Sirtex. B. Zhang: Full / Part-time employment: Ipsen. B. Belanger: Full / Parttime employment: Ipsen. Y. Moore: Full / Part-time employment: Ipsen. A. Pedret-Dunn: Full / Parttime employment: Ipsen. F. Maxwell: Full / Part-time employment: Ipsen. A. Dean: Advisory / Consultancy, Non-paid: Shire; Advisory / Consultancy, Non-paid: Specialised Therapeutics Australia; Travel / Accommodation / Expenses, Grant: Amgen. All other authors have declared no conflicts of interest.

Introduction: nal-IRI+5-FU/LV is approved for patients with mPAC after disease progression following gemcitabine-based therapy. The current study (NCT02551991) is a phase 1/2, open-label trial to assess the safety, tolerability, and dose-limiting toxicities (DLTs) of nal-IRI+5-FU/LV+OX (NAPOX) for the first-line treatment of patients with mPAC and to determine phase 3 dosing. Methods: Following 4 dose exploration cohorts (Part 1A), a recommended dose for dose expansion (Part 1B) was selected based on DLTs and cumulative safety (nal-IRI 50 mg/m2 [free-base equivalent; FBE], OX 60 mg/m2, LV 400 mg/m2, 5-FU 2400 mg/m2 on days 1 & 15 of each 28-day cycle). The expansion phase enrolled 25 patients at the selected dose level, with 32 subjects treated at the selected dose level (pooled population; PP 50/60). Patients were age ≥18 yrs with previously untreated locally advanced or mPAC, ECOG performance status ≤1, and adequate organ function. The primary endpoint was safety and tolerability, with secondary assessments based on 19/Feb/2019 data cut-off when all patients had completed their second scheduled tumor assessment after 16 weeks of treatment. Results: 56 patients were enrolled and treated, with 32 patients from (n = 7) and; Dose Expansion Cohort (n = 25) included in the PP 50/60 analysis (n = 29 mPDAC; n=3 locally advanced PDAC). 9 DLTs were reported by 5 patients across the 4 dose exploration cohorts (diarrhea, n = 2; vomiting, anal fissure, anal inflammation, proctalgia, neutropenic infection, neutropenic sepsis, and febrile neutropenia, all n = 1), including 1 patient in (febrile neutropenia). Treatment-related TEAEs Grade 3 or higher were reported by 39 of 56 patients (50/60 PP, n = 20/32: neutropenia, n = 9; febrile neutropenia, hypokalemia, both n = 4; diarrhea, nausea, both n=3; anemia, vomiting, both n = 2), with no reported Grade 3 or higher fatigue or peripheral neuropathy. Serious adverse events (SAEs) were reported by 31 of 56 patients (50/60 PP, n = 14/32), with n = 23 patients reporting treatment-related SAEs (50/60 PP, n = 10/32 patients: nausea, febrile neutropenia, both n = 3; diarrhea, vomiting, both n = 2; colitis, enterocolitis, stomatitis, anemia, pneumonia, and pyrexia, all n = 1). 15 patients reported TEAEs leading to discontinuation (50/60 PP, n = 4/32), with 36 patients requiring dose adjustment due to AEs (50/60 PP, n = 23/32). 23 of 32 patients (71.9%) in the 50/60 PP achieved disease control at 16 weeks (DCR16wk). Best overall response in the 50/60 PP was complete response (CR) in 1 patient (diagnosed with locally advanced Stage III disease), partial response (PR) in 10 patients, and stable disease (SD) in 15/32 patients (sum of CR+PR+SD: 81.3%), with an overall response rate (ORR) of 34%. At data cut-off, 15/32 patients in the PP 50/60 remain on treatment. Preliminary analysis of median progression-free survival and median overall survival are not yet mature for evaluation. Conclusion: In the first-line treatment of patients with mPAC, NAPOX (nal-IRI 50 mg/m2 (FBE), OX 60 mg/m2, LV 400 mg/m2, and 5-FU 2400 mg/m2) appears manageable, with promising anti-tumor activity (DCR16wk of 71.9%, sum of CR+PR+SD: 81.3%, and ORR of 34%) warranting further clinical assessment. This study is ongoing, with additional analyses planned.

The treatment of advanced non-small cell lung cancer (NSCLC) may be changing, but the cisplatin-based doublet remains the foundation of treatment for the majority of patients with advanced NSCLC. In this respect, changes in practice to various aspects of cisplatin use, such as administration schedules and the choice of methods and frequency of monitoring for toxicities, have contributed to an incremental improvement in patient management and experience. Chemoresistance, however, limits the clinical utility of this drug in patients with advanced NSCLC. Better understanding of the molecular mechanisms of cisplatin resistance, identification of predictive markers and the development of newer, more effective and less toxic platinum agents is required. In addition to maximising potential benefits from advances in molecular biology and associated therapeutics, modification of existing cisplatin-based treatments can still lead to improvements in patient outcomes and experiences.

Human cell lines are permissive for LuIII, a member of the rodent group of autonomous parvoviruses. However, LuIII vectors pseudotyped with feline panleukopaenia virus (FPV) capsid proteins can transduce feline cells but not human cells. Feline transferrin receptor (FelTfR) functions as a receptor for FPV. Transfection of Rh18A, a human rhabdomyosarcoma cell line, with FelTfR enabled transduction by vector with FPV capsid. This was not true of other human lines, suggesting restriction at some additional, post-entry, level(s) in human cells other than Rh18A. It seemed a reasonable hypothesis that a second blockage might be in nuclear delivery mediated by the N-terminal region of the minor capsid protein, VP1. We therefore generated virions containing an LuIII-luciferase genome, packaged using chimaeric VP1 molecules (N-terminal region of LuIII VP1, fused with body of FPV, and vice versa) together with the major capsid protein, VP2, of FPV or LuIII. The virions were tested for ability to transduce feline and human cells. Our hypothesis predicted that the N-terminal region of LuIII VP1 should allow transduction of human cells expressing FelTfR, while the FPV N-terminal region should not allow transduction of human cells (except for Rh18A). The experimental results did not bear out either of these predictions. Therefore, the VP1 N-terminal region appears not to be a major determinant of permissiveness for LuIII, versus FPV, capsid in human cells.

Targeting the transcription of a toxin gene to activated endothelial cells might be used for inhibiting angiogenesis in solid tumors. As a model, we transiently transfected human endothelial cells (HUVEC) in culture with expression plasmids for the toxic A-chain of diphtheria toxin (DT-A), using electroporation (achieving ≈70% transfection efficiency). Protein synthesis in HUVEC was highly sensitive to DT-A expression from constitutive viral promoters. E-selectin is strongly expressed on HUVEC activated by TNF α or TPA. We therefore tested a human E-selectin promoter (−547 to +33) for targeting transcription of DT-A or reporter genes to HUVEC. Luciferase reporters were efficiently expressed in HUVEC from this promoter, with or without an enhancer responsive to Ets-1. Expression was increased by TNF α or TPA. DT-A showed highly preferential expression (increased by TNF α or TPA) in HUVEC, compared with WI38 human fibroblasts. HUVEC expressing DT-A were killed via apoptosis. Overall expression levels were influenced by alternative ‘backbone’ sequences used in the expression plasmids. We propose that delivery of transcriptionally regulated expression plasmids for DT-A in vivo , using cationic lipids that show preferential accumulation in activated or proliferating endothelium, may offer a novel means of inhibiting undesired angiogenesis.

Parvoviruses are small, icosahedral viruses (approximately 25 nm) containing a single-strand DNA genome (approximately 5 kb) with hairpin termini. Autonomous parvoviruses (APVs) are found in many species; they do not require a helper virus for replication but they do require proliferating cells (S-phase functions) and, in some cases, tissue-specific factors. APVs can protect animals from spontaneous or experimental tumors, leading to consideration of these viruses, and vectors derived from them, as anticancer agents. Vector development has focused on three rodent APVs that can infect human cells, namely, LuIII, MVM, and H1. LuIII-based vectors with complete replacement of the viral coding sequences can direct transient or persistent expression of transgenes in cell culture. MVM-based and H1-based vectors with substitution of transgenes for the viral capsid sequences retain viral nonstructural (NS) coding sequences and express the NS1 protein. The latter serves to amplify the vector genome in target cells, potentially contributing to antitumor activity. APV vectors have packaging capacity for foreign DNA of approximately 4.8 kb, a limit that probably cannot be exceeded by more than a few percent. LuIII vectors can be pseudotyped with capsid proteins from related APVs, a promising strategy for controlling tissue tropism and circumventing immune responses to repeated administration. Initial success has been achieved in targeting such a pseudotyped vector by genetic modification of the capsid. Subject to advances in production and purification methods, APV vectors have potential as gene transfer agents for experimental and therapeutic use, particularly for cancer therapy.

BioTechniquesVol. 33, No. 1 BenchmarksOpen AccessStorage of Various Cell Lines at -70°C or -80°C in Multi-Well Plates While Attached to the SubstratumJoe Corsini, Francoise Maxwell & Ian H. MaxwellJoe CorsiniChadron State College, Chadron, NE, USA, Francoise MaxwellChadron State College, Chadron, NE, USA & Ian H. MaxwellChadron State College, Chadron, NE, USAPublished Online:17 Sep 2018https://doi.org/10.2144/02331bm05AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit FiguresReferencesRelatedDetailsCited ByPollen derived macromolecules serve as a new class of ice-nucleating cryoprotectants19 July 2022 | Scientific Reports, Vol. 12, No. 1In-Plate Cryopreservation of 2D and 3D Cell Models: Innovative Tools for Biomedical Research and Preclinical Drug DiscoverySLAS Discovery, Vol. 26, No. 1Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleationCryobiology, Vol. 93A Simple and Rapid Cryopreservation Technique for Ciliates: A Long‐Term Storage Procedure Used for Marine Scuticociliates12 April 2019 | Journal of Eukaryotic Microbiology, Vol. 66, No. 5Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation21 August 2013 | Journal of Tissue Engineering and Regenerative Medicine, Vol. 10, No. 10The AMERE project: Enabling real-time detection of radiation effects in individual cells in deep spacePlanetary and Space Science, Vol. 74, No. 1Biobanking in microbiology: From sample collection to epidemiology, diagnosis and research1 November 2005 | FEMS Microbiology Reviews, Vol. 29, No. 5Rapid cryopreservation of five mammalian and one mosquito cell line at −80°C while attached to flasks in a serum free cryopreservativeBiological Procedures Online, Vol. 7, No. 1Serum-free cryopreservation of five mammalian cell lines in either a pelleted or suspended stateBiological Procedures Online, Vol. 6, No. 1 Vol. 33, No. 1 Follow us on social media for the latest updates Metrics Downloaded 2,839 times History Published online 17 September 2018 Published in print July 2002 Information© 2018 Author(s)PDF download

The autonomous parvoviruses are small, non-enveloped, single strand DNA viruses. They occur in many species and they have oncolytic properties. We are modifying the capsid of feline panleukopenia virus (FPV), a parvovirus which normally infects feline cells, with the goal of targeting human tumor cells for potential cancer therapy. Using recombinant viruses transducing a luciferase reporter, we show that insertion of a cyclically constrained, integrin-binding peptide at an exposed position on the FPV capsid enables transduction of an alpha(v) integrin-expressing human rhabdomyosarcoma cell line (Rh18A). These cells were not transduced by virus with the unmodified FPV capsid. Transduction of Rh18A was specifically inhibited by an alpha(v) integrin blocking antibody. However, other human tumor lines expressing alpha(v) integrins were not transduced by virus with either the modified or unmodified capsid. We conclude that modification of the FPV capsid to bind alpha(v) integrins can contribute to, but is not generally sufficient for, redirecting infection to human tumor cells. The permissiveness of Rh18A cells presumably involves additional factors unique to this line among various human cell lines tested.

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control ('TET ON' system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

Autonomous parvoviruses are small, single strand DNA viruses which preferentially replicate in transformed and tumor cells, causing cell death by expression of the cytotoxic nonstructural protein, NS1. Several parvoviruses of the rodent group, including LuIII, efficiently infect human transformed cell lines. The potential for systemic use of these viruses in targeting metastases might be enhanced if NS1 expression and viral replication could be controlled by an innocuous drug such as tetracycline. We therefore substituted prokaryotic tetracycline operator sequences for part of P4 of LuIII, the promoter responsible for transcription of the mRNAs for nonstructural proteins. The resulting construct unexpectedly showed constitutive expression in transiently transfected cells, as indicated by efficient excision and amplification of viral replicative form (RF) DNA. This was apparently due to self-stimulatory transcriptional transactivation by NS1. This problem was overcome by cotransfection with a plasmid expressing a chimera of the repressor of the tetracycline operon with a KRAB transrepression domain. These conditions allowed efficient control of transcription and RF amplification by the tetracycline derivative, doxycycline. These observations form a basis for developing a therapeutic agent based on a drug- controlled parvovirus.