Journal: Vaccines

Article Title: Advancing Pyrogen Testing for Vaccines with Inherent Pyrogenicity: Development of a Novel Reporter Cell-Based Monocyte Activation Test (MAT)

doi: 10.3390/vaccines13101009

Figure Lengend Snippet: Performance characteristics of the MAT method for OMPC. ( A ) Dose–response curves illustrating the evaluation of relative pyrogenicity at 25%, 50%, 100%, and 200% compared to a reference batch. ( B ) Linear regression analysis depicting the logarithmic linear relationship between observed and expected relative pyrogenicity across five levels: 25%, 50%, 100%, 200%, and 300%. Each level is represented by 6 to 10 data points. ( C ) Dose–response curves for the formulation buffer in the MAT, both with and without OMPC. The formulation buffer did not interfere with the MAT method. ( D ) Dose–response curves for OMPC in the MAT, with varying concentrations of spiked pyrogens: endotoxin (LPS, USP RSE). ( E ) Dose–response curves for OMPC in the MAT with spiked LTA-SA at different concentrations. The concentrations indicated in the figures represent the final concentrations in the wells.

Article Snippet: OMPC materials and the formulation buffer were provided by Merck & Co., Inc., West Point, PA, USA manufacturing site.

Techniques: Formulation

Journal: Cancer Research Communications

Article Title: IMGN853 Induces Autophagic Cell Death in Combination Therapy for Ovarian Cancer

doi: 10.1158/2767-9764.CRC-24-0215

Figure Lengend Snippet: Effects of IMGN853 on the survival and inhibition of tumor growth of mice bearing ovarian high-grade serous carcinoma (HGSC) PDX tumors. A, Main clinical and pathologic characteristics of PDX tumor tissues from patients with HGSC. Date of Implantation (DATE) B, Schematic representation of PDX experiments. PDX mouse models were established by surgical implantation of HGSC tumors via intraovarian injection. Treatment with either formulation buffer (control) or IMGN853 (5.2 mg/kg, once weekly) was initiated approximately 3 weeks following tumor implantation in the recipient NOD/SCID mice. Black dots represent 1, 2, and 3 months, respectively, after PDX tumor implantation. C, Kaplan–Meier survival curves for HGSC PDX 2414–bearing mice treated with formulation buffer (blue) or IMGN853 (red). P < 0.001, log-rank test. D, Left, Representative confocal IF images showing terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining (green) in the control formulation buffer and IMGN853 treatment groups. Right, The fluorescence intensity from the green channel over that of the blue channel was quantified using Imaris v. 9.1.2. The sum of fluorescence intensity is shown in relative fluorescence unit (RFU). E, The HGSC PDX model 2428 was surgically implanted in NOD/SCID mice. Twenty-one days after implantation, mice were randomized to receive buffer (control) or IMGN853 (5.2 mg/kg, once weekly) for 6-dose treatment. Tumor weights (left), numbers of nodules (middle), and body weights (right) of the mice were recorded at necropsy. P values were determined by a two-tailed, nonparametric t test. Error bars ± SDs. NACT, neoadjuvant chemotherapy; WT, wild-type.

Article Snippet: Formulation buffer (ImmunoGen, Inc.) served as a control for IMGN853.

Techniques: Inhibition, Injection, Formulation, Control, Tumor Implantation, End Labeling, TUNEL Assay, Staining, Fluorescence, Two Tailed Test

Journal: Cancer Research Communications

Article Title: IMGN853 Induces Autophagic Cell Death in Combination Therapy for Ovarian Cancer

doi: 10.1158/2767-9764.CRC-24-0215

Figure Lengend Snippet: IMGN853 treatment increases autophagic flux and cell death in FOLR1 + ovarian high-grade serous carcinoma (HGSC) models. A, Representative confocal IF images showing expression patterns of the autophagy markers LC3B (green) and beclin-1 (green) in the control- (formulation buffer, top row) and IMGN853-treated PDX tumors (bottom row). B, Fluorescence intensity in each IF image was quantified using Imaris v. 9.1.2. The sum of fluorescence intensity for each marker is shown in relative fluorescent units (RFU); tissues treated with IMGN853 showed higher expression of LC3 and beclin-1. C, Representative images from transmission electron microscopy showed autophagic vesicles formed in the orthotopic OVCAR-8 tumors treated with control agent (top) or IMGN853 (bottom). Substantial amounts of APs (green) and ALs (yellow) were identified in the IMGN853-treated OVCAR-8 tumor cells, whereas tight cell–cell junctions and condensed patterns of mitochondria were observed in control tumors, in which only the minimum amounts of AP present. Substructure was observed at 20,000× (left) and 50,000× (right) magnifications. Representative ultrathin sections in representative areas from n = 5 fields are shown. D, Quantification of autophagic vacuoles including APs and ALs per image at 20,000× magnification (approximately 44 μm 2 ). P values were determined by a two-tailed, nonparametric t test. Error bars ± SDs. E, ptfLC3–pEGFP-RFP–expressing OVCAR-8 cells were treated with control (top) or IMGN853 (bottom) for 48 hours and imaged using a Leica confocal microscope for another 24–48 hours. Time-lapse videos were recorded to assess autophagy flux (represented by GFP-LC3 and RFP-LC3 colocalization during cell division) under the control condition or the accumulation of late-stage ALs (represented by RFP-LC3) leading to autophagic cell death under the IMGN853 condition (available as Supplementary Movies S1 and S2). The time points are shown above each image, n = 3 for each treatment.

Article Snippet: Formulation buffer (ImmunoGen, Inc.) served as a control for IMGN853.

Techniques: Expressing, Control, Formulation, Fluorescence, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Microscopy

Journal: Cancer Research Communications

Article Title: IMGN853 Induces Autophagic Cell Death in Combination Therapy for Ovarian Cancer

doi: 10.1158/2767-9764.CRC-24-0215

Figure Lengend Snippet: IMGN853 induces beclin-1–dependent autophagic cell death in FOLR1 + HGSC cell lines. A, Percentages of autophagic vacuoles in FOLR1 + OVCAR-8 and OVCA432 cells and in FOLR1 low A2780 cells treated with control (formulation buffer), IMGN853 alone, HCQ alone, or IMGN853+HCQ for 48 hours and analyzed by acridine orange staining followed by FACS. n = 6; error bars represent SEMs. B, Percentages of SYTOX + dead cells among FOLR1 + OVCAR-8 and OVCA432 cells and FOLR1 low A2780 cells treated with control (formulation buffer), IMGN853 alone, HCQ alone, or IMGN853+HCQ for 48 hours and analyzed by FACS. n = 6; error bars represent SEMs. C, Stable knockdown of beclin-1( BECN1 ) human shRNAs (A, B, C, and D, OriGene) were performed in OVCAR-8 cells. β-Actin was applied as the loading control. shRNA-B is selected for the subsequent experiments. The blots are representative of two biological repeats. shRNA, short hairpin RNA. D, Levels of LC3B-II and cleaved caspase-3 in OVCAR-8 cells WT (left) and shBECN1 knockdown (right) treated with control, IMGN853 alone, HCQ alone, or IMGN853+HCQ for 48 hours. β-Actin was used as the loading control. Each western blot was representative of two biological repeats. CTL, control, WT, wild-type.

Article Snippet: Formulation buffer (ImmunoGen, Inc.) served as a control for IMGN853.

Techniques: Control, Formulation, Staining, Knockdown, shRNA, Western Blot