Journal: Molecular Cancer Therapeutics

Article Title: Development of a Novel Bifunctional Anti-CD47 Fusion Protein with Improved Efficacy and a Favorable Safety Profile

doi: 10.1158/1535-7163.MCT-24-0917

Figure Lengend Snippet: CO-001 induces cancer cell phagocytosis via blocking the CD47 interaction with SIRPα and mediates direct PCCD. A, Jurkat cells (5 × 10 5 cells/mL) were incubated with 100 μg/mL human IgG4 isotype control, CO-001, B6H12, or 2D3, followed by incubation with 10 μg/mL FITC-conjugated SIRPα. B, CellTrace-labeled Jurkat cells co-cultured with DiO-labeled RAW264.7 macrophages and treated with CO-001, anti-CD47 sequence analogues AO-176 or magrolimab, benchmark anti-CD47 B6H12, or human IgG4 isotype control for 2 hours. C, Time-lapse imaging of Jurkat cells treated with the human IgG4 isotype control or CO-001 (1 μg/mL) at the indicated time points (H = hours) using the IncuCyte Live Cell Imaging System. Scale bar, 200 μm. D, Representative FACS plots showing Jurkat cells treated with 1 μg/mL the human IgG4 isotype control or CO-001 for 3 hours and then stained with annexin V and 7-AAD. The gated population, indicated by a red rectangle, indicates the population of the cells that is undergoing PCCD with 11% undergoing PCCD for IgG4-treated cells and 58% for CO-001 treated. E, Jurkat cells (5 × 10 5 cells/mL) were treated with CO-001 at the indicated concentrations in μg/mL or human IgG4 isotype control (1 μg/mL) for 0.5, 1, or 3 hours, followed by annexin V and 7-AAD staining. F, Reh cells (5 × 10 5 cells/mL) were treated with CO-001 or AO-176 at the indicated concentrations in μg/mL or human IgG4 isotype control (10 μg/mL) for 3 hours, followed by annexin V and 7-AAD staining. A , B , and E, Data presented as the mean ± SD. Significance was determined by ANOVA analysis *, P < 0.01 relative to isotype control, §, P < 0.01, relative to the sample treated with CO-001 at the same concentration, n = 3 to 5. G, Binding of CO-001 to various cell lines derived from hematologic malignancies (Jurkat, MOLT-4, CCRF-CEM, and KG-1a) and RBCs relative to the human IgG4 isotype control. The EC 50 was determined using four-parameter curve fitting analysis in GraphPad Prism. H, Hemagglutination assay of freshly isolated RBCs (2% v/v in PBS) incubated with increasing concentrations of the CO-001, sequence analogues of AO-176 and magrolimab, or IgG4 isotype control. A small punctate circle indicates no hemagglutination, whereas a diffuse hazy pattern indicates hemagglutination. MFI, mean fluorescence intensity.

Article Snippet: Target cancer cells were collected by centrifugation and stained with 1 μL/mL CellTrace Violet Cell Proliferation Dye according to the manufacturer’s protocol (Thermo Fisher Scientific, cat # C34557 ).

Techniques: Blocking Assay, Incubation, Control, Labeling, Cell Culture, Sequencing, Analogues, Imaging, Live Cell Imaging, Staining, Concentration Assay, Binding Assay, Derivative Assay, Hemagglutination Assay, Isolation, Fluorescence

Journal: Molecular Cancer Therapeutics

Article Title: Development of a Novel Bifunctional Anti-CD47 Fusion Protein with Improved Efficacy and a Favorable Safety Profile

doi: 10.1158/1535-7163.MCT-24-0917

Figure Lengend Snippet: Development of the CO-005 drug candidate with an improved RBC safety profile. A, Cartoon representation of the structure of the CO candidates CO-001, CO-004, and CO-005. B, Incubation of freshly isolated RBCs (2% v/v in PBS) with increasing concentrations of CO-001, CO-004, CO-005, or human IgG4 isotype control. A small punctate circle indicates no hemagglutination, whereas a diffuse hazy pattern indicates hemagglutination. C, Binding of CO-001, CO-005, CO-004, and human IgG4 isotype control to RBCs derived from a healthy human donor. D, Jurkat cells (5 × 10 5 cells/mL) were treated with CO-001, CO-004, or CO-005 at the indicated concentrations in μg/mL or human IgG4 isotype control (1 μg/mL) for 3 hours, followed by annexin V and 7-AAD staining. E, Phagocytosis activity of CO-001 fusion proteins in Jurkat cells using DiO-labeled RAW264.7 macrophages at the indicated concentrations for 2 hours. Phagocytosed target cells were analyzed by flow cytometry as %CellTrace + DiO + cells. D and E, Data are presented as the mean ± SD. Significance was determined by ANOVA analysis *, P < 0.01 relative to isotype control, §, P < 0.01, relative to the sample treated with CO-001 at the same concentration, n = 3 to 5. MFI, mean fluorescence intensity.

Article Snippet: Target cancer cells were collected by centrifugation and stained with 1 μL/mL CellTrace Violet Cell Proliferation Dye according to the manufacturer’s protocol (Thermo Fisher Scientific, cat # C34557 ).

Techniques: Incubation, Isolation, Control, Binding Assay, Derivative Assay, Staining, Activity Assay, Labeling, Flow Cytometry, Concentration Assay, Fluorescence

Journal: PLOS Pathogens

Article Title: Fatty acid desaturases link cell metabolism pathways to promote proliferation of Epstein-Barr virus-infected B cells

doi: 10.1371/journal.ppat.1012685

Figure Lengend Snippet: (A-B) LCL growth in the presence of indicated doses of A939572 (SCD1i) (n = 4, LCLs from 4 donors). Growth was measured using Cell Titer Glo. (C-D) LCL growth in the presence of SC-26196 (FADS2i) (n = 4, LCLs from 4 donors). Growth was measured as in A-B. (E) LCL growth in the presence of 10 μM SCD1i, 20 μM SC-26196 FADS2i, or both inhibitors (n = 4, LCLs from 4 donors). Growth was measured as in A-B. (F) Representative dot plots, gated on live and dead single cells, showing anti-BrdU (FITC) versus total DNA (7-AAD) to measure the number of cells in each stage of the cell cycle at 48 hours post treatment. (G) DMSO-normalized relative percentage of single cells in S phase after 48 hours of drug treatment (n = 3, LCLs from 3 donors). (H) DMSO-normalized relative live cell percentage and number after 72 hours of treatment with indicated drugs (n = 4, from 4 donors). Cell number measured using Trypan dye. (I) Bulk PBMCs (n = 6, PBMCs from 2 donors) were treated with 10 µM SCD1i and/or 20 µM FADS2i immediately following EBV infection, and CD19 + B cell proliferation was based on CellTrace Violet dilution over time. (J) PBMCs (n = 4, PBMCs from 2 donors) were stimulated with 2.5 μg/mL CpG and treated with 10 µM SCD1i and/or 20 µM FADS2i. CD19 + B cell proliferation was assessed based on Cell Trace Violet dilution after five days. (K) PBMCs (n = 4, PBMCs from 2 donors) were stimulated with 5 ng/mL CD40L+20 ng/mL IL-4 and treated with 10 µM SCD1i and/or 20 µM FADS2i. CD19 + B cell proliferation was assessed based on Cell Trace Violet dilution after five days. Statistical significance for pairwise comparisons determined using a Tukey’s post-hoc test or Dunnett’s post-hoc test (panels B & D) (* p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005).

Article Snippet: To track proliferation in early EBV-infected B cells, cells were stained with the fluorescent proliferation tracking dye CellTrace Violet (ThermoFisher, Cat# C34557 ).

Techniques: Infection

Journal: The Journal of Infectious Diseases

Article Title: Antibody-Based Antigen Delivery to Dendritic Cells as a Vaccination Strategy Against Ebola Virus Disease

doi: 10.1093/infdis/jiae613

Figure Lengend Snippet: T-cell response after DEC-205 targeting. A , Antimouse DEC205 antibody conjugated to allophycocyanin was used to determine the proportion of cross-presenting dendritic cells (DCs). The overlay plots show the percentage of DCs from total DCs (black) expressing CD24, CD11c, XCR1, CD8α, and DEC205 (turquoise) in the spleen (left) and intra-abdominal lymph nodes (right). B , CellTrace Violet–stained CD8 + T cells from mice immunized with Ebola virus (EBOV) nucleoprotein (NP) peptide (YQVNNLEEI) and complete Freund adjuvant and boosted with the peptide and incomplete Freund adjuvant were incubated with bone marrow–derived DCs in the presence of 5 µg of the isotype control (left) or NP-DEC205 (middle). OT-I T cells incubated with bone marrow–derived DCs in the presence of 5 µg of ovalbumin DEC205 served as an internal positive control (right). Proliferation was assessed by flow cytometry. C , WT → IFNAR −/− mice (n = 3) were immunized with the isotype control or NP-DEC205. Fourteen days ( C ) or 28 days ( D ) later, splenocytes were collected and stimulated with EBOV NP AGQFLSFASLFLPKLVVGEK (CD4 T-cell) or RVIPVYQVNNLEEICQLIIQ (CD8 T-cell) peptides. Interferon-γ–secreting cells were enumerated; results are represented as spot-forming units per 5 × 10 5 cells. Bars represent mean values ± standard error of the mean. Statistical significance was determined using unpaired Student t test: ** P ≤ .01, *** P ≤ .001. Data are representative of 2 independent experiments. Abbreviations: DCs, dendritic cells; DEC-205, lymphocyte antigen 75; LNs, lymph nodes; NP, nucleoprotein; OVA, ovalbumin; SFU, spot-forming units.

Article Snippet: Splenocytes were labeled with the CellTrace Violet proliferation dye (Thermofisher Scientific) according to the manufacturer's instructions.

Techniques: Expressing, Staining, Virus, Adjuvant, Incubation, Derivative Assay, Control, Positive Control, Flow Cytometry