Journal: Journal for Immunotherapy of Cancer

Article Title: Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells

doi: 10.1136/jitc-2021-003752

Figure Lengend Snippet: Generation and characterization of Fabrack-CAR for universal CAR T cell platform. (A) Schematic representation of protein structure of Fabrack-CAR on a cell. Amino acid sequence of meditope and linker: CQFDLSTRRLQC-SAPASSASAPSAASAPA. (B) Schematic representation of the lentiviral expression cassette of conventional HER2 scFv CAR and candidates of Fabrack-CAR, which incorporated an extracellular domain with or without CH3 spacer, a CD28 transmembrane (TM) domain, and a cytoplasmic region comprizing either CD28/CD3ζ, or 41BB/CD3ζ signaling domains for T cell signaling. A truncated nonsignaling CD19 (CD19t) following a T2A ribosomal skip sequence was expressed separately from CAR construct for tracking successfully transduced cells. (C) The ability of Fabrack-CAR binding to memAb or meFab. CHO-S cells transfected with HER2 scFv CAR or candidates of Fabrack-CAR were examined for their binding to αHER2 memAb and meFab conjugated with Alexa-647 by flow cytometry, with gating on CD19t+cells. Ipilimumab, an αCTLA-4 non-memAb, was used as a negative control (mean±SEM, ***p<0.001). (D) HER2-binding ability of HER2 scFv CAR, αHER2 memAb, or meFab-coupled meditope-CAR. The binding of HER2 extracellular domain (ECD)-Pacific Blue to cells were examined by flow cytometry. Experiments were done in technical triplicates (mean±SEM, ***p≤0.001). (E) Validation of Fabrack expression on Jurkat cells. Fabrack positive cells were confirmed by staining cells with αFc-PE or αCD19-PECy7 antibody followed by flow cytometry. Two independent experiments were done. (F) Validation of multiple memAbs binding to Fabrack Jurkat cells by flow cytometry. The binding of memAbs to Fabrack Jurkat cells was analyzed by flow cytometry after cells were stained with secondary anti-human kappa-Alexa-647. MemAbs only bound to Fabrack Jurkat cells, whereas Jurkat cells without Fabrack expression did not have memAb binding. All data are representative of at least two independent experiments. CAR, chimeric antigen receptor; memAbs, meditope-enabled monoclonal antibodies.

Article Snippet: Jurkat-NFAT-Luc cells (Invivogen #jktl-nfat) were cultured in RPMI.

Techniques: Sequencing, Expressing, Construct, Binding Assay, Transfection, Flow Cytometry, Negative Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells

doi: 10.1136/jitc-2021-003752

Figure Lengend Snippet: Activation of Fabrack Jurkat cells in the presence of target cells and a memAb. Fabrack with CH3-CD28 construct was examined in this figure. (A) NFAT regulated luciferase in Fabrack Jurkat cells was used as an index to measure T cell activity. NFAT-RE: NFAT-response element. (B) MemAb-mediated concentration-dependent activation of Fabrack Jurkat cells. Four-fold serial dilution of αHER2 memAb (upper left), αEGFR/HER3 memAb (upper right), αCDH6 memAb (lower left), and αCD33 memAb (lower right) was prepared to redirect Fabrack Jurkat cells to target cells. After 6 hour incubation, luciferase substrate was added in each well and luminescence was measured immediately. The data are representative of two independent experiments (mean±SEM). (C) Illustration showing that antibody without a meditope-enabled site blocked memAb-mediated Fabrack Jurkat cell activation. (D) Clinical trastuzumab dose-dependently blocked αHER2 memAb-mediated Fabrack Jurkat cell activation. Trastuzumab at 40 nM (red bar) or 400 nM (green bar) was used to block 4 nM αHER2 memAb-mediated activation of Fabrack Jurkat cells. During incubation, HER2 memAb and clinical trastuzumab were present simultaneously. After 6 hours incubation, luciferase substrate was added in each well and luminescence was measured immediately. Experiments were done in technical duplicates. Significance versus blue bar is indicated (mean±SEM, **p< 0.01, ***p< 0.001). (E) Clinical trastuzumab slightly blocked HER2 memAb-mediated Fabrack Jurkat cell activation when target cells were premixed with 100 nM HER2 memAb and washed out. Experiments were done in technical duplicates. Significance versus red bar is indicated (mean±SEM, *p<0.05, ***p<0.001). (F) Illustration shows that meFab binding to both Fabrack Jurkat cells and target cells. (G) Activation of Fabrack Jurkat cells was mediated by αHER2 memAb or meFab in the presence of HER2 expressing breast (MCF7 and SKBR3) or ovarian (OVCAR3 and SKOV3) cancer cell lines. Non-meditope-enabled anti-HER2 pertuzumab IgG or Fab were used as negative controls. All data are representative of at least two independent experiments (mean±SEM). NFAT-RE: NFAT-response element. memAb, meditope-enabled monoclonal antibody.

Article Snippet: Jurkat-NFAT-Luc cells (Invivogen #jktl-nfat) were cultured in RPMI.

Techniques: Activation Assay, Construct, Luciferase, Activity Assay, Concentration Assay, Serial Dilution, Incubation, Blocking Assay, Binding Assay, Expressing

Journal: Neural Development

Article Title: Ptf1a is expressed transiently in all types of amacrine cells in the embryonic zebrafish retina

doi: 10.1186/1749-8104-4-34

Figure Lengend Snippet: Primary antibody information

Article Snippet: rb anti-PKCβ1 , 1:150 , Santa-Cruz Biotechnology, (C-16) sc-209 , Carboxyl terminus of PKC β1 of human origin , Western blot in NIH/3t3, 3611-RF, A-431, HeLA and Jurkat whole cell lysate (manufacturer).

Techniques: Recombinant, Western Blot, Purification, Immunoprecipitation, Binding Assay, Clone Assay, Staining