Journal: Advanced Science

Article Title: BiTE‐Secreting CAR‐ γδ T as a Dual Targeting Strategy for the Treatment of Solid Tumors

doi: 10.1002/advs.202206856

Figure Lengend Snippet: Characterization of mRNA‐driven Nb‐CAR.BiTE‐ γδ T cells. A) Nb‐CAR expression was assessed after lentiviral transduction or mRNA electroporation. γδ T cells (1 × 10 6 ) were electroporated with 2 µg Nb‐CAR/CAR.BiTE IVT mRNA or transduced with lentiviral Nb‐CAR/CAR.BiTE particles (MOI = 3). The Nb‐CAR expression on the indicated days was determined through flow cytometry using a specific antibody against VHH. B,C) Superior cell growth and Nb‐BiTE secretion using the mRNA delivery strategy. B) Cell number and viability were recorded during 7 d after transfection of the mRNA or lentiviral Nb‐CAR.BiTE transgene. C) Contents of PD‐L1‐targeted Nb‐BiTE in the supernatants from mRNA‐ or lentiviral‐driven Nb‐CAR.BiTE‐ γδ T cell cultures were detected by an ELISA‐based coating with recombinant full‐length PD‐L1. D) Phenotype and purity of mRNA‐driven Nb‐CAR.BiTE‐ γδ T cells. T effector and central memory phenotype, activating marker, and purity of Nb‐CAR.BiTE mRNA‐electroporated γδ T cells were analyzed through flow cytometry using specific antibodies against CD27, CD45RA, CD3, NKG2D, V δ 2, V γ 9, αβ TCR, γδ TCR, CD4, CD8, CD19, CD14, CD66b, or CD56. Isotype staining was used as a gating control for positive staining. E) Increased secretion of granzyme B and IFN‐ γ , but not IL‐17A, from Nb‐CAR.BiTE‐ γδ T cells after coculture with A549 or MDA‐MB‐231 cells. The contents of granzyme B (upper panel), IFN‐ γ (middle panel), and IL‐17A (bottom panel) in culture supernatants were measured using ELISA. These results showed that mRNA‐driven Nb‐CAR.BiTE‐ γδ T cells would be an effective strategy for the manufacture of Nb‐CAR.BiTE‐ γδ T cells. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3–4, * p < 0.05; ** p < 0.01; and *** p < 0.001 based on paired Student's t‐tests.

Article Snippet: Antibodies specific for CD3 ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals.

Techniques: Expressing, Transduction, Electroporation, Flow Cytometry, Transfection, Enzyme-linked Immunosorbent Assay, Recombinant, Marker, Staining, Control

Journal: Advanced Science

Article Title: BiTE‐Secreting CAR‐ γδ T as a Dual Targeting Strategy for the Treatment of Solid Tumors

doi: 10.1002/advs.202206856

Figure Lengend Snippet: Evaluation of Nb‐BiTE secreted from Nb‐CAR‐ γδ T cells. A–D) Functional binding of Nb‐BiTE released from Nb‐CAR‐ γδ T cells. A) Diagram shows the cell non‐contact coculture system for Nb‐CAR.BiTE‐ γδ T cells and target cells (left panel). Secreted Nb‐BiTE from Nb‐CAR‐ γδ T cells was detectable on target cells. We seeded 1 × 10 5 isolated primary CD3‐positive cells from PBMCs, A549, H1975, or MDA‐MB‐231 cells on the bottom, with or without exposure to 5 × 10 5 Nb‐CAR.BiTE‐ γδ T cells on the top, in impenetrable wells for 24 h. Subsequently, the bottom cells were harvested and Nb‐BiTE signals were detected by flow cytometry using specific antibody against VHH (right panel). B) Secreted Nb‐BiTE from Nb‐CAR‐ γδ T cells promoted CD3‐positive cell proliferation. We cocultured 1 × 10 6 isolated primary CD3 + cells, parental, or Nb‐CAR‐ γδ T cells with or without 1 × 10 6 parental, Nb‐CAR, or Nb‐CAR.BiTE‐ γδ T cells on the top well for 6 days. Subsequently, the CD3 + cell numbers were normalized to the non‐coculture group. C) Nb‐BiTE released from Nb‐CAR‐ γδ T cells strengthened effector cells against PD‐L1‐expressing tumor cells. Schematic illustration of the non‐contact coculture system for Nb‐CAR.BiTE‐ γδ T cells, which release Nb‐BiTE to engage CD3 + effectors and PD‐L1‐overexpressing tumor cells (left panel). The PD‐L1 level on PD‐L1‐overexpressing A549 and PD‐L1 knockdown MDA‐MB‐231 stable clones were examined by flow cytometry analysis (right panel). D) Purity of the isolated CD4‐ and CD8‐positive and CD3‐/CD56 + cells from PBMCs were checked by flow cytometry. E) We cocultured 1 × 10 5 PD‐L1‐overexpressing A549 cells with an individual healthy donor‐derived CD4 + , CD8 + , NK, parental γδ T, or Nb‐CAR‐ γδ T cells on the bottom well and with or without 5 × 10 5 Nb‐CAR.BiTE‐ γδ T cells on the top well for 72 h. F) PD‐L1 determined the cell‐killing ability of effector cells when exposed to released Nb‐BiTE. PD‐L1‐stable knockdown or scramble‐control MDA‐MB‐231 cells (1 × 10 5 ) were cocultured with an individual healthy donor‐derived CD4 + , CD8 + , NK, parental γδ T, or Nb‐CAR‐ γδ T cells on the bottom well and with 5 × 10 5 Nb‐CAR.BiTE‐ γδ T cells on the top well for 72 h. G,H) PD‐L1‐targeted Nb‐BiTE enhanced anti‐tumor responses of T cells. MDA‐MB‐231 or PD‐L1‐overexpressing A549 cells were cocultured with or without the isolated CD4 + or CD8 + cells at an E:T ratio of 5:1; or γδ T cells at an E:T ratio of 3:1 in the presence/absence of recombinant Nb‐BiTE (1 ng mL ‐1 ). G) After 24, 48, or 72 h of coculture, we determined the cytotoxicity induced by these effector cells. H) After 48 h of coculture, the supernatants were harvested for detecting the contents of granzyme B and IFN γ by ELISA. Specific lysis was determined using LIVE/DEAD cell‐mediated cytotoxicity assay. These results suggested that the secreted PD‐L1 targeting Nb‐BiTE from Nb‐CAR.BiTE‐ γδ T could trigger bystander effector cells active against PD‐L1‐expressing tumor cells. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 4, *, # , X p < 0.05; **, ## , XX p < 0.01; and ***, ### , XX X p < 0.001 based on paired Student's t‐test. For sub‐Figure G, * represents significant differences between Nb‐BiTE and effector cells (Nb‐BiTE vs effector cells); # represents significant differences between Nb‐BiTE and effector cells+Nb‐BiTE; X represents significant differences between effector cells and effector cells+ Nb‐BiTE.

Article Snippet: Antibodies specific for CD3 ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals.

Techniques: Functional Assay, Binding Assay, Isolation, Flow Cytometry, Expressing, Knockdown, Clone Assay, Derivative Assay, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Lysis, Cytotoxicity Assay

Journal: Cell

Article Title: BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection

doi: 10.1016/j.cell.2021.03.026

Figure Lengend Snippet:

Article Snippet: For ACE2 assays the following was used for control, 1:200 Goat IgG Alexa Fluor 647-conjugated antibody, and assay 1:200 anti-human ACE2 AlexFluor 647 conjugated antibody and 1:200 anti-human CD90 (all RnD Systems) and were incubated for 60 min at 4°C in Binding Buffer.

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Infection, Software

Journal: International Journal of Molecular Sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant

doi: 10.3390/ijms241814140

Figure Lengend Snippet: ACE2, HS, and SDC expression profiles of 293T and 293T-ACE2 cells. ACE2, HS, and SDC expression in 293T and 293T-ACE2 cells were assessed using fluorescently labeled specific antibodies with imaging flow cytometry. ( A , B ) ACE2 and HS expression profile of 293T and 293T-ACE2 cells. The representative flow cytometry histograms and cellular images show the ACE2 and HS expression of 293T and 293T-ACE2 cells treated with the fluorescent ACE2 and HS antibodies. Scale bar = 20 μm. ( C ) SDC expression profile of 293T and 293T-ACE2 cells. The representative flow cytometry histograms and fluorescent cellular images show the SDC expression of 293T cells and 293T-ACE2 cells treated with the APC-labeled respective SDC antibodies. Scale bar = 20 μm. ( D – F ) Detected ACE2, HS, and SDC expression values were normalized to those of 293T cells as standards. The bars represent the mean ± SEM of three independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: ACE2 expression was measured with human ACE2 AF 647-conjugated antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB9332R) and respective isotype control (mouse IgG2A AF 647-conjugated isotype control, RnD Systems, cat. no. IC003R), according to the manufacturer’s protocol.

Techniques: Expressing, Labeling, Imaging, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Cellular uptake of WT SCV2, Delta, and Omicron variants into 293T- and ACE2-overexpressing 293T-ACE2 cells. The cells were exposed to 1 MOI of heat-inactivated WT SCV2, Delta, and Omicron variants for 4 h at 37 °C. After incubation, the cells were washed, trypsinized, fixed, permeabilized, and treated with a primary SARS-CoV-2 spike (1000–1200 aa), followed by a fluorescently labeled (AF 633) secondary antibody. Cellular uptake was then analyzed with imaging flow cytometry. ( A – D ) Representative flow cytometry histograms and brightfield (BF) and fluorescent cellular images showing the intracellular fluorescence of the virus-exposed 293T and 293T-ACE2 cells. Scale bar = 20 μm. ( E ) Detected fluorescence intensities were normalized to WT SCV2-treated 293T cells as standards. The bars represent the mean + SEM of five independent experiments. Experimental data are presented as dots. Statistical significance was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: ACE2 expression was measured with human ACE2 AF 647-conjugated antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB9332R) and respective isotype control (mouse IgG2A AF 647-conjugated isotype control, RnD Systems, cat. no. IC003R), according to the manufacturer’s protocol.

Techniques: Incubation, Labeling, Imaging, Flow Cytometry, Fluorescence, Virus

Journal: Scientific Reports

Article Title: Specific detection of interferon regulatory factor 5 (IRF5): A case of antibody inequality

doi: 10.1038/srep31002

Figure Lengend Snippet: IRF5 antibody details.

Article Snippet: We also purchased two recently available pre-conjugated antibodies, PE-conjugated IC8447P (R&D; RDIC8447P-PE) and AF647-conjugated FAB8447R (Novus Biologicals; FAB8447R-AF647), for testing.

Techniques: Immunohistochemistry-IF

Journal: Cells

Article Title: Generation of Chimeric Antigen Receptors against Tetraspanin 7

doi: 10.3390/cells12111453

Figure Lengend Snippet: Protein panning yields TSPAN7-specific binders. ( A ) Screening ELISA of 96 scFv binders (hg010 A1-H12) after the 3rd round of protein-based panning. scFv were stained using secondary staining of the appending His tag by anti-His tag antibody and HRP conjugate. ( B ) Screening ELISA of 92 scFv binders (hg015 A1-H11) obtained by the same procedure but after the 5th round. ( C ) Example of scFv binder screening on antigen-transfected HEK293T cells in FACS and score calculation. Cells were stained with soluble scFv hg015-C7 and detected using secondary staining of the appending His tag by PE anti-His tag antibody. TSPAN7 antigen expression was measured using GFP coexpression. Specific binding score was calculated according to the formula below. ( D ) FACS Screening of TSPAN7-transfected HEK293T cells for the binders obtained in ( A ). ( E ) FACS Screening of TSPAN7-transfected HEK293T cells for the binders obtained in B. Positive control of an scFv detecting a transfected control antigen on cells in blue. Medium-only control in orange. ( F ) Representative plots of specific scFv binders hg015-C7, hg015-D12, hg015-G9, and hg015-H5 showing binder TSPAN7-transfected HEK293T cells. Medium control (−) and unrelated specific binder as positive control (+) shown left. Binders are also indicated with asterisks in ( E ).

Article Snippet: TSPAN7 expression was verified with flow cytometry staining with an anti-TSPAN7 Alexa FluorTM 647-conjugated antibody (FAB9179R, R&D Systems, Wiesbaden, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Transfection, Expressing, Binding Assay, Positive Control, Control

Journal: Cells

Article Title: Generation of Chimeric Antigen Receptors against Tetraspanin 7

doi: 10.3390/cells12111453

Figure Lengend Snippet: Peptide panning generated a single binder detecting a TSPAN7 peptide. ( A ) Screening ELISA of scFv binders generated by peptide panning. Change fold of A450-620 absorption signals of ELISA on TSPAN7 peptide and streptavidin control antigen. ScFv binder Kro68-D9 indicated with asterisk. ( B ) Flow cytometric staining of Kro68-D9 on TSPAN7-transfected HEK293T cells. ScFv staining with anti-His-PE antibody. TSPAN7 expression reported by GFP. Medium-control left, Kro68-D9 scFv staining on human TSPAN7 and murine TSPAN7-transfected HEK293T cells middle and right, respectively. ( C ) Staining of peptide binder Kro68-D9 on murine beta cell line MIN6. Binding of Kro68-D9 on MIN6 shown in blue. Binding of an unspecific control scFv in red. ( D ) Immunohistochemistry staining on paraffin pancreas sections. Murine and human sections left and right, respectively. First row shows isotype control antibody. Second row shows the positive control with insulin staining, respectively. Below, staining of islets with Kro68-D9 is shown. All pictures 20× magnification.

Article Snippet: TSPAN7 expression was verified with flow cytometry staining with an anti-TSPAN7 Alexa FluorTM 647-conjugated antibody (FAB9179R, R&D Systems, Wiesbaden, Germany).

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Control, Staining, Transfection, Expressing, Binding Assay, Immunohistochemistry, Positive Control

Journal: Cells

Article Title: Generation of Chimeric Antigen Receptors against Tetraspanin 7

doi: 10.3390/cells12111453

Figure Lengend Snippet: Kro68-D9 CAR T cells are activated by TSPAN7. ( A ) Murine T cells were transduced with 2nd generation short-hinge (CD8-based) CAR containing TSPAN7-specific scFv binder Kro68-D9 and Thy1.1 as reporter gene under control of IRES. ( B ) Schematic overview of CAR T cell activation assay. ( C ) BalbC CD4+ T cells can be efficiently transduced by TSPAN7-specific CAR Kro68-D9. Thy1.1 and CD69 expression is shown for CD4+ pre-gated T cells after stimulation with TSPAN7. Thy1.1+ cells (red gate) show strong proliferation indicated by CFSE dilution compared to untransduced cells (blue gate). Cross indicates MFI within gates. ( D ) Kro68-D9 CAR T cells specifically upregulate CD69 and increased proliferation after stimulation with TSPAN7. Stimulation on CD3/CD28 beads and control antigen is shown. A control CAR specific to the control antigen is shown as a further control. Data are presented as mean ± SD ( n = 3). p values determined by two-way ANOVA and multiple comparison testing (Benjamini, Krieger and Yekutieli post hoc test). * p < 0.033, ** p < 0.002, *** p < 0.001, ns = not significant.

Article Snippet: TSPAN7 expression was verified with flow cytometry staining with an anti-TSPAN7 Alexa FluorTM 647-conjugated antibody (FAB9179R, R&D Systems, Wiesbaden, Germany).

Techniques: Transduction, Control, Activation Assay, Expressing, Comparison

Journal: Cells

Article Title: Generation of Chimeric Antigen Receptors against Tetraspanin 7

doi: 10.3390/cells12111453

Figure Lengend Snippet: Kro68-D9 CAR can detect TSPAN7 protein and peptide but not surface-expressed TSPAN7 on cells. ( A ) 2nd generation CAR constructs used for cloning of scFvs. Short-hinge CAR shown left and long-hinge CAR right, respectively. ( B ) Schematic overview of used CAR construct—short- and long-hinge CAR, respectively. ( C ) TSPAN7 can be stained on the surface of transfected HEK293T cells. Anti-TSPAN7 antibody staining on untransfected (blue) and transfected cells (red). ( D ) CAR activation in murine hybridoma cells following stimulation by target. NFAT activation is reported by GFP and anti-Fab antibody was used for CAR staining. Activation patterns are shown for short-hinge (above) and long-hinge (below) CARs. CARs were tested with TSPAN7 peptide used for panning, TSPAN7 protein, medium (control), HEK293T transfected with human TSPAN7 and untransfected HEK293T (control).

Article Snippet: TSPAN7 expression was verified with flow cytometry staining with an anti-TSPAN7 Alexa FluorTM 647-conjugated antibody (FAB9179R, R&D Systems, Wiesbaden, Germany).

Techniques: Construct, Cloning, Staining, Transfection, Activation Assay, Control