Journal: PLoS ONE

Article Title: Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of V L and V H Domains Targeting CD123 + Tumors

doi: 10.1371/journal.pone.0159477

Figure Lengend Snippet: (A) Schematic diagrams of conventional and chimeric scFv specific for CD123. CARs 1 to 4: CD123-specific CARs generated by fusing V L and V H chains of mAbs specific to CD123. CARs 5–9: Chimeric scFvs created by mix-and-matching V L and V H chains. The scFvs of CARs 1–9 were fused to the signaling domains of CD28 and CD3ζ via CD8α hinge and TM domains. CAR-10 was derived by fusing the chimeric scFv from CAR 6 to the CD3ζ and CD28 endo-domains via the IgG4 hinge and CD28 TM domains. (B) Expansion kinetics of CARs 1–4 (left) and CARs 5–10 (right) over a period of 28 days from day 1 following electroporation of SB CAR plasmids. Data are pooled from 3 donors; graph displays mean ± SEM (C) CAR expression on Day 21 after electroporation. CAR expression was detected by CD123 recombinant protein fused to Fc followed by serial staining with fluorescence-labeled anti-Fc and anti-CD3 antibodies. (D) in vitro lysis of CD123 + target cells Nalm 6, TF1, 293T-parental cells, CD123-transfected 293T cells, and 123 neg by CAR + T cells. Histograms represent the mean ± SEM, n = 3. (E) CAR + T cell killing of BM-derived target cells. Mononuclear cells were isolated from normal human bone marrow samples and sorted for expression of lineage markers into lineage-positive (Lin + ) and lineage-negative (Lin neg ) groups. The latter presumable reflects the HSC pool. The BM-derived cells were then labeled with PKH-26 and incubated with CAR + T cells for 2 days before vitality was assessed by flow cytometry. The percent lysis compared to controls is shown. Histograms represent the mean ± SEM of 3 replicates. (F) Interferon-γ release by CAR + T cells after exposure to CD123. Day 28 CD123-specific CAR + T cells were incubated for 24 hours with Nalm-6 cells (CD123 + ), 293T cells (CD123 neg ), or alone, then the supernatant tested for cytokine expression using Biolegend plex Th1 cytokine capture beads, measured by flow cytometry. Results for IFN-γ are shown; other cytokines were not detectible over background. Histograms represent mean ± SEM for 2 replicates from 2 different experiments.

Article Snippet: The TF1 cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Generated, Derivative Assay, Electroporation, Expressing, Recombinant, Staining, Fluorescence, Labeling, In Vitro, Lysis, Transfection, Isolation, Incubation, Flow Cytometry

Journal: PLoS ONE

Article Title: Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of V L and V H Domains Targeting CD123 + Tumors

doi: 10.1371/journal.pone.0159477

Figure Lengend Snippet: (A) Overlay histograms display the flow cytometric analysis of CD123 expression on AML cell lines MV4-11, Molm-13, TF1, OCI-AML3, EL4-Parental and EL4-CD123. Isotype control is shown in grey, and specific staining by the unfilled black line. (B) Specific lysis of CD123-CD28 and CD123-CD137 CAR + T cells against AML cell lines EL4, CD123 neg OCI-Ly19, MV4-11, TF1, EL4-CD123, Molm-13, and OCI-AML3 assessed with a 4 hour chromium release assay. Histograms represent mean ± SEM, n = 3 (C) Flow cytometric analysis of CD123 expression on primary AML samples used in the co-culture assay depicted in (D) . Lysis of PKH-26 labeled primary AML cells by CD123-CD28 or CD123-CD137 CAR T cells at 1:1 ratio for 72 hours. CD19-specific CAR + T cells were used as a negative control.

Article Snippet: The TF1 cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Expressing, Control, Staining, Lysis, Release Assay, Co-culture Assay, Labeling, Negative Control

Journal: PLoS ONE

Article Title: Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of V L and V H Domains Targeting CD123 + Tumors

doi: 10.1371/journal.pone.0159477

Figure Lengend Snippet: (A) Schematic of the TF1 xenograft model. 2.5 × 10 6 TF1- effLuc -mKate cells were injected intravenously into NSG mice on day 0. On Day 5, tumor engraftment was quantified using non-invasive bioluminescence imaging (BLI), and mice were randomly divided into 3 groups: untreated (control), CD123-CD28-treated, or CD123-CD137-treated. CAR-treated mice were given infusions of T cells followed by IL-2 treatment and BLI on day 5, 11 and 20. Untreated mice received no T cells. (B) BLI images of mice display an overlay of luciferase activity, using the color scale shown on the right, displayed over the white-light image of the mice. (C) Histograms represent the luciferase activity measured by BLI for each group (** p < 0.01). (D) Kaplan-Meier curves display the survival analysis of xenograft mice treated with CD123-specific CAR T cells (** p < 0.01).

Article Snippet: The TF1 cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Injection, Imaging, Control, Luciferase, Activity Assay