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Induction and decay kinetics of CD38-CAR expression.

<p>(A) schematic overview of CAR induction and decay assay. Black bars indicate the DOX incubation times, gray bars indicate the period of decay after the removal of DOX. (B and C) Representative results of five independent experiments of mean fluorescent intensity (MFI) of the CAR measured by staining with soluble his-tagged CD38 after 6, 24 or 48 hours incubation with (B) 10 or 1000 ng/ml DOX or 6, 24, 48 or 120 hours after washing of DOX (C) (an MFI of 600, observed by Mock cells was considered background expression). (D) A MM patient bone marrow sample with 20% MM cells was co-incubated with inducible, high affinity (028), CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule depicted in Fig 3A. are the CAR-dependent % lysis of CD138+/CD38+ MM cells (% lysed by CAR—% lysed by Mock). Presented is the representative data of n = 5. (E) Significant Pearson correlation of MFI of CAR expression as detected with soluble CD38 (sCD38) with % lysis of MM cells. High dose DOX R² = 0.60 and p = 0.012, low dose DOX R² = 0.61 and p = 0.015.</p

Off-tumor effect of inducible CD38-CAR T cells.

<p>(A) Representative flow cytometry density plots of MM-BM with CD38⁺/CD138⁺ cells (MM) after treatment with inducible mock (+/- 1000 ng/ml DOX for 48 hours) and inducible high affinity (028) CD38-CAR T cells (- DOX or + 1000 ng/ml DOX for 48 hours and 0, 24 or 120 hours after DOX removal). (B) Pooled data obtained from the analysis of five MM patient bone marrow samples (patient 1–5, see for their phenotype data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>) with ~20% MM cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>) were co-incubated with inducible high affinity (028) CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g003" target="_blank">Fig 3A</a>. Shown are the mean CAR-dependent % lysis of MM (CD138⁺/CD38⁺ ; open squares) and % lysis of healthy non-MM cells (CD138^-/CD56^-/CD38^+/-; grey diamonds) by inducible CD38-CAR T cells. Incubated with DOX for 24 hours 1000 ng/ml (upper left), 48 hours 1000 ng/ml (upper right), 24 hours 10 ng/ml (lower left), 48 hours 10 ng/ml (lower right). Presented is the pooled data from 5 independent experiments. Error bars indicate the mean +/- SEM. (Pt 1–5, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>).</p

Off-tumor effect of inducible low affinity CD38-CAR T cells.

<p>MM patient bone marrow samples (n = 4) with ~20% MM cells were co-incubated with inducible low affinity (B1) CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g003" target="_blank">Fig 3A</a>. Depicted are the average CAR-dependent lysis of MM cells (CD138⁺/CD38⁺ ; open squares) and lysis of healthy non-MM cells (CD138^-/CD56^-/CD38^+/- ; grey diamonds) by inducible CD38-CAR T cells. Incubated with DOX for 24 hours 1000 ng/ml (upper left), 48 hours 1000 ng/ml (upper right), 24 hours 10 ng/ml (lower left), 48 hours 10 ng/ml (lower right). Presented is the pooled data from 4 independent experiments. Error bars indicate the mean +/- SEM. (Pt 2–5, same pts as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g004" target="_blank">Fig 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>).</p

DOX dose-dependent induction of CD38-CAR expression and anti-MM cytotoxicity.

<p>(A) Lysis of luciferase-transduced CD38⁺ MM cell lines UM9 and RPMI8226 after co-incubation with Mock and inducible, high affinity (028) CD38-CAR, after treatment with no or 1000 ng/ml DOX for 48 hours. Grey lines indicate the lysis by constitutively expressed high affinity (028) CD38-CAR T cells The BLI signal from surviving MM cells was measured after 16 hours using a luminometer and the percentage lysis was calculated as indicated in the material & methods. Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD (B) Mean fluorescent intensity (MFI) of the CAR measured by staining with soluble CD38-his after 48 hours incubation with 0, 1, 10, 100 or 1000 ng/ml DOX, Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD. (C) The cytotoxic activity of untreated or DOX treated inducible CD38-CAR T cells against luc+ MM cell line UM9 after 16 hours. Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD. In all panels * indicates p value <0.05 and ** <0.01 using one-way analysis of variance and subsequent multiple comparison.</p

DOX induced CD38-CAR expression.

<p>(A) Schematic overview of constructs. The pRetroX-TRE3G vector with the P_TRE3GV inducible promoter controlling the transcription of Mock containing the marker LNGFR or the CD38-CAR (shown is high affinity scFv 028; the same vector design is also used for low affinity CARs A4 and B1), consisting of the single chain variable fragments, 4-1BB and CD3ζ and a LNGFR separated by a P2A sequence. These vectors were co-transduced with the pRetroX-TET-On 3G containing the transcription site for the transactivator protein rTta. (B) Representative flow cytometry density plots and histograms to determine CAR expression of the inducible CAR T cells, after 48 hours incubation with 0 or 1000 ng/ml DOX. The expression of the marker LNGFR was measured with an APC-conjugated antibody. CAR expression was measured by binding of his-tagged (HHHHHH) soluble CD38 (sCD38) protein to the ScFv domain, stained with PE-conjugated anti-His tag antibody.</p