Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Generation of clinical grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of B-cell malignancies

ABSTRACT: Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical grade tumor-redirected TSCM cells starting from naÃ¯ve precursors. CD8+CD62L+CD45RA+ naÃ¯ve T cells enriched by streptamer-based serial positive selection were activated by CD3/CD28 engagement in the presence of IL-7, IL-21 and the glycogen synthase-3Î² inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions allowed for the generation of CD19-CAR modified TSCM cells that were phenotypically, functionally and transcriptomically equivalent to their naturally occurring counterpart. Compared with T cell products currently under clinical investigation, CD19-CAR modified TSCM cells exhibit enhanced metabolic fitness, persistence and anti-tumor activity against systemic acute lymphoblastic leukemia xenografts. Based on these findings, we have initiated a phase 1 clinical study to evaluate the activity of CD19-CAR modified TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation. Three healthy human blood donors provided lymphocyte-enriched apheresis blood for this study after informed consent. From all samples, total RNA was isolated using an miRNeasy Mini Kit (Qiagen), processed by Ambionâ??s WT expression kit, fragmented and labeled with a WT Terminal Labeling Kit (Affymetrix), hybridized to WT Human Gene 1.0 ST arrays (Affymetrix) and stained on a Genechip Fluidics Station 450 (Affymetrix), all according to the respective manufacturer's instructions. Samples represent exon-level and gene-level analyses.

ORGANISM(S): Homo sapiens

SUBMITTER: Luca Gattinoni

PROVIDER: E-GEOD-68003 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies.

Sabatino Marianna M Hu Jinhui J Sommariva Michele M Gautam Sanjivan S Fellowes Vicki V Hocker James D JD Dougherty Sean S Qin Haiying H Klebanoff Christopher A CA Fry Terry J TJ Gress Ronald E RE Kochenderfer James N JN Stroncek David F DF Ji Yun Y Gattinoni Luca L

Blood 20160525 4

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8(+)CD62L(+)CD45RA(+) naive T cells enriched by streptamer-based seri ...[more]

PMID: 27226436

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Project description:Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of AML expressing CDX2, and observed that PPARγ agonists derepress KLF4 and are preferentially toxic to CDX2-positive leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells; provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer; and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a new therapeutic modality in a large proportion of AML patients. Experiment 1 (Samples 1-6): The transcriptional changes induced by Cdx2 were assessed ex vivo in primary murine hematopoietic stem and progenitor cells. Bone marrow cells were harvested from 3x15 C57BL/6 mice, and differentiated cells were removed by incubation with rat antibodies against lineage antigens (CD3, CD4, CD8, Gr-1, B220, CD19, IL-7R, Ter119) followed by depletion with magnetic beads (Dynabeads, Invitrogen). Lineage-depleted cells were stained with APC-conjugated anti-c-Kit and PE-Cy5-conjugated goat anti-rat antibodies, and c-Kit+ Lin– cells were sorted using a BD FACSAria cell sorter (BD Biosciences). Cells were plated in RetroNectin-coated tissue culture dishes (Takara Bio) and transduced twice with pMSCV-Cdx2-IRES-GFP or pMSCV-IRES-GFP retroviral constructs. After 48 hours, GFP-positive cells were sorted using a BD FACSAria cell sorter. Experiment 2 (Samples 7-10): The transcriptional changes induced by Cdx2 were assessed in vivo in Cdx2-initiated murine leukemias and compared with those of leukemias initiated by 5 different MLL fusion oncogenes (previously published data, available at GSE13690). Bone marrow cells isolated from C57BL/6 mice were transduced with pMSCV-Cdx2-IRES-GFP, and 8x10e5 GFP-positive cells were injected into lethally irradiated syngeneic recipient mice after 48 hours, followed by injection of 1x10e6 spleen cells from primary leukemic animals into sublethally irradiated secondary recipients. Spleen cells from secondary leukemic animals were harvested.

Dataset Information

Generation of clinical grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of B-cell malignancies

Publications

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies.

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