CD19 CAR-targeted T cell dependence on conditioning chemotherapy and T cell dose.

<p>T cells were transduced with m1928z and infused at increasing doses (noted on axes) into congenic mice (n = 40) that had been conditioned with increasing doses of cyclophosphamide (noted above columns). Peripheral blood B and congenic T cells were evaluated by flow cytometry for B and T cell markers 4 weeks after adoptive transfer. (b and c) A repeat experiment (n = 44) was done while holding cyclophosphamide conditioning constant (300 mg/kg), but with increasing T cell doses. Mice were sacrificed 1 and 5 weeks after adoptive transfer with CD19 CAR-targeted congenic T cells and flow cytometry for B cell (B220) and congenic T cells (Thy1.1/Thy1.2) was performed on single-cell suspensions of the BM and spleen. All counts and percentages were done with Countbright beads (Invitrogen). Statistical significance (p <0.05) was calculated by one-way ANOVA and is denoted by asterisks. For all sections of this figure error bars represent the SEM.</p

Antigen-dependence of CD19 CAR-targeted T cells in the BM.

<p>B and congenic T cell counts from the BM of mice treated with cyclophosphamide (300 mg/kg) and increasing doses (labeled in bold) of congenic m1928z T cells. Mice were sacrificed 1 and 5 weeks after adoptive transfer with congenic T cells and flow cytometry for B cell (B220) and congenic T cells (Thy1.1/Thy1.2) was performed on single-cell suspensions of the BM. Counts were facilitated with Countbright beads (Invitrogen). E∶T ratio is the ratio of congenic donor T cells to B cells in the BM.% Dose is the m1928z T cell dose divided by the total number of BM congenic T cells, estimated by multiplying the femoral congenic T cell count by 15.8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061338#pone.0061338-Brentjens1" target="_blank">[5]</a>.</p

CD19 CAR-Targeted T Cells Induce Long-Term Remission and B Cell Aplasia in an Immunocompetent Mouse Model of B Cell Acute Lymphoblastic Leukemia

<div><p>Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.</p> </div

Adoptive transfer of CD19 CAR-targeted T cells into mice with leukemia.

<p>(a) Flow cytometry of bulk mouse T splenocytes double-transduced with GL-2A-m1928z. (b) Survival curve of mice injected with Eμ-ALL01 and not treated (No Rx), treated only with cyclophosphamide (CTX), or treated with cyclophosphamide (100-200 mg/kg IP) and CD19 CAR-targeted T cells (CTX+m1928z), displayed in (a). This data (n = 28) is pooled from 2 independent experiments. Another study (Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061338#pone.0061338.s002" target="_blank">Figure S2</a>) confirms that cyclophosphamide and control T cells mediate no survival advantage. (c) Flow cytometry of cells from the BM, spleen (SP), and lymph node (LN) of a single mouse treated with cyclophosphamide but sacrificed due to clinical deterioration. Splenocytes were also stained with isotype antibodies (ISO) as a control.</p

Adoptive transfer of CD8+ CD19 CAR-targeted T cells alone is sufficient for long-term persistence and B cell eradication.

<p>(a) OTI Thy1.2+ T cells were transduced with the m1928z CAR or the m19z CAR, which has a CD3ζ signal transduction domain but lacks the CD28 signal transduction domain. Untransduced T cells (UNT) are mock-transduced cells and are included as a negative control. Donor T cells (5×10⁶) were then adoptively transferred into congenic Thy1.1 mice (n = 19) 1 day after treatment with cyclophosphamide (300 mg/kg). (b) Peripheral blood B and OTI T cell counts were evaluated by flow cytometry 4 weeks after adoptive transfer. One-way ANOVA was done for the treatment groups' (UNT, m19z, m1928z) B cell counts (p = 0.0001) and also OTI T cell counts (p = 0.03). Error bars are the SEM. (c) A few of the mice in each group were sacrificed 7 weeks after adoptive T cell transfer. BM and spleen cells were analyzed by flow cytometry for B and congenic T cell markers. A representative plot is included from each group. Displayed cells were gated on Live and DUMP-negative cells, which were characterized with Mac1, Gr1, NK1.1, and Ter119 antibodies. (d) In another study (n = 17), peripheral blood counts of B and congenic T cells were performed from 3–22 weeks after adoptive transfer. This study included groups of control mice that were untreated wild-type (WT), or treated with cyclophosphamide alone (CTX), or treated with T cells modified with the m19Δz CAR, which lacks any signaling element. Statistical analyses (t tests) of the treatment groups (m19Δz and m1928z) were significant (p<0.05) when done on the B cell counts but not the OTI T cell counts (p = 0.47). Error bars are the SEM. * is Not Done.</p

Progressive disease in mice after injection with Eμ-ALL01 tumor cells.

<p>(a) Retro-orbital blood was collected from 5 mice on the days indicated after Eμ-ALL01 injection and analyzed serially over time with an A^CT diff cell counter (Beckman Coulter, Brea, CA). White cell count (WBC), Hemoglobin (Hgb), and Platelets were measured and statistically analyzed with t-tests, which were non-significant for Day 0 and Day 14 means. Comparison of the Day 0 and Day 32 means by t-tests were all significant (p values are noted on graph). Error bars are the standard error of the mean (SEM). The mice were sacrificed 4 weeks after injection with Eμ-ALL01 because of clinical deterioration and retro-orbital blood, bone marrow, and spleen were harvested for anatomical (b) and cellular (c) analyses, with representative images displayed. (b) Blood and tissue were used to prepare peripheral smears (left-panel) and H&E stained slides of sections of the bone marrow (middle-panel) and spleen (right-panel). (c) Single-cell suspensions were prepared from bone marrow (BM) and spleen (SP) and then incubated with antibodies specific for B220 and IgM. The panels on the top are cells isolated from disease-free, wild-type C57BL/6 mice (B6) and the panels on the bottom were injected with the Eμ-ALL01 cells. Cells displayed have been gated on live lymphoid cells.</p

Generation and <i>in vitro</i> function of the m1928z CAR.

<p>(a) Schematic of the genetic construct GL-2A-m1928z for the reporter gene (GFP) and CAR (m1928z). Depicted are the packaging signal ψ, splice donor (SD), splice acceptor (SA), the V_H and V_L regions of the scFV, and the extracellular (EC), transmembrane (TM), and cytosolic (C) regions. (b) GFP-expression in mouse T cells after transduction with GL-2A-m1928z retroviral supernatant (right-panel). The left-panel displays untransduced (UNT) T cells as a control. The gene-transfer efficiency, estimated as the GFP+ population, is derived from a single experiment with a double-transduction of a bulk population of mouse T splenocytes as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061338#pone.0061338-Lee1" target="_blank">[22]</a>. (c) Cytotoxic T lymphocyte antigen-specific killing was evaluated with a Chromium release assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061338#pone.0061338-Gong1" target="_blank">[39]</a>. Target cells (EL4-mCD19) are EL4 cells retrovirally transduced with mouse CD19. Effector cells are T cells transduced with GL-2A-m1928z. Control effector cells were transduced with GL-2A-h1928z since it is identical to GL-2A-m1928z except for the scFv, which is derived from an anti-human CD19 antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061338#pone.0061338-Brentjens1" target="_blank">[5]</a>. Effector to target ratio (x-axis) are based on the number of GFP⁺ T cells to EL4-mCD19 cells and were performed in triplicate. Killing efficiency (y-axis) was calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061338#pone.0061338-Gong1" target="_blank">[39]</a>. (d) Cytokine secretion by m1928z and h1928z T cells was evaluated after stimulation with 3T3-mCD19 cells. Stimulation with 3T3-mCD19 cells was performed in triplicate and supernatants were obtained 1 day after stimulation. Each supernatant was evaluated for cytokines also in triplicate (no dilution, 3×dilution, and 9×dilution). Error bars represent the SEM.</p

<i>In vivo</i> anti-leukemia abilities of 19-28z⁺, 19z1⁺ and 19z1-CD80⁺ T cells.

<p>(A) Representative ventral bioluminescence images of GFP-FFLuc NALM/6 bearing NSG mice non treated or injected with 2x10^<b>5</b> CAR^<b>+</b> 19z1^<b>+</b>, 19z1-CD80^<b>+</b> or 19-28z^<b>+</b> human T cells. Signal intensities are shown as photons/second/square centimeter/steradian. Mice treated with 19z1-CD80^<b>+</b> or 19-28z^<b>+</b> T cells showed relapsed disease at distinct anatomic sites including the periodontal region, CNS/calvarium and abdomen. (B) Bioluminescent tumor signal quantified per animal every week over a 120-day period. Quantification is the average photon count of the ventral and dorsal acquisition per animal at all given time points. One line represents one mouse. N = 7–8 mice per group or N = 15 for 19-28z^<b>+</b> T-cell group resulting from 2 pooled experiment (C) Survival is illustrated in the Kaplan-Meier curves. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. (D) Bone marrow hCD3^<b>+</b> T-cell absolute numbers evaluated 8 and 30 days after T-cell injection, as indicated. Each symbol indicates an individual mouse (n = 4). (E) Detailed bone marrow CD4^<b>+</b> and CD8^<b>+</b> T-cell absolute numbers gated on CD3^<b>+</b> cells showed in D.</p

shRNA-mediated CTLA-4 down-regulation.

<p>(A) CTLA-4 knock–down transient assay. Briefly, 293 T cells were costransfected with CTLA-4/GFP and shRNAs/reporter plasmids at a 1:9 ratio. 48h later, MFI of GFP was evaluated by FACS analysis in the shRNA reporter gene gate. (B) Real-time PCR relative quantification of CTLA-4 transcript in 19z1-CD80^<b>+</b> T cells transduced to express a control shRNA or the anti-CTLA-4 shRNA #3 and <i>in vitro</i> stimulated with aAPCs expressing CD19, as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130518#pone.0130518.g001" target="_blank">Fig 1</a>. One representative out of 3 experiments is shown. (C) Flow cytometry analysis of intracellular CTLA-4 in 19-28z^<b>+</b> T cells transduced to express a control shRNA or the anti-CTLA-4 shRNA #3 and activated twice on aAPC. Plots from one representative donor are shown. Bold numbers indicate the MFI of the CTLA-4 staining in the shRNA negative (left) or positive (right) populations. (D) Histograms show the average of the measured CTLA-4 MFI gated in the dsRed-shRNA positive population in each T-cell group, from 3 different donors, <i>P</i> = 0.029. (E) Cytokines measured in supernatants 24h after coculture of 19z1-CD80^<b>+</b> T cells expressing the indicated shRNA on aAPCs expressing CD19. One representative out of 2 experiments is shown. <i>P</i> = 0.0002.</p

CTLA-4 down-regulation increases 19z1-CD80⁺ T-cell efficiency but not that of 19-28z⁺ T cells.

<p>(A-B) Facs plots showing expression of the CAR and shRNAs traced by dsRed in 19z1-CD80^<b>+</b> T cells (A) or in 19-28z^<b>+</b> T cells (B), generated from the same PBMC, before <i>in vivo</i> infusion. (C) Treatment of NALM/6 bearing NSG mice with 2x10^<b>5</b> 19z1-CD80^<b>+</b> T cells expressing an anti-CTLA-4 shRNA statistically enhanced survival when compared with similar treatment with 19z1-CD80^<b>+</b> T cells expressing a control shRNA, <i>P</i> = 0.0002. Kaplan-Meier curves include the results of two pooled experiments. N = 11 or 12 mice per group. (D-E) No statistical difference in survivals of NALM/6 bearing NSG mice treated with 19-28z^<b>+</b> T cells expressing an anti-CTLA-4 shRNA or a control shRNA at two different doses: 2x10^<b>5</b> (D) and 1x10^<b>5</b> T cells (E). (F-G) Tumor burden weekly quantified by bioluminescence imaging of NALM/6 bearing mice treated with 2x10^<b>5</b> 19z1-CD80^<b>+</b> T cells (F) or 19-28z^<b>+</b> T cells (G) expressing an anti-CTLA-4 shRNA or control shRNA. One line represents one mouse.</p