Pooled screening for CAR function in glioblastoma
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ABSTRACT:
PROVIDER: PRJNA1081276 | ENA |
REPOSITORIES: ENA
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Project description:Human primary CD3+ T cells expressing IL13Ra2-targeted chimeric antigen receptors (CARs) that had been identified through high throughput pooled screening were rechallenged with an IL13Ra2+ human glioblastoma cell line , then stained with CITESEQ antibodies indicative of T cell memory and exhaustion state and prepared for single cell sequencing in order to determine their phenotypic responses to chronic tumor challenge relative to that of a clinical standard CAR.
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Project description:Glioblastoma (GBM) is the most prevalent primary malignant brain tumor, containing self-renewing stem-like GBM stem cells (GSCs) that have been a focus of immunotherapies. Chimeric antigen receptor (CAR) T cell therapy has shown evidence of clinical activity, but overall limited responses in patients with GBMs. Here, we interrogated molecular determinants of CAR T cell-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. CRISPR screening of CAR T cells identified dependencies for their effector functions, including TLE4 and IKZF2. Targeted knockout of these genes in CAR T cells robustly enhanced antitumor efficacy against GBM patient-derived xenografts (PDXs). Bulk and single cell-RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for their susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered the tumor-immune signaling axis and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs are promising strategies to discover avenues and inform potential combinatorial approaches for enhancing CAR T cell therapeutic efficacy against GBM, and can be extended to reveal key mediators of immunotherapy responses across solid tumors.
2021-05-19 | GSE163401 | GEO
Project description:Glioblastoma (GBM) is the most prevalent primary malignant brain tumor, containing self-renewing stem-like GBM stem cells (GSCs) that have been a focus of immunotherapies. Chimeric antigen receptor (CAR) T cell therapy has shown evidence of clinical activity, but overall limited responses in patients with GBMs. Here, we interrogated molecular determinants of CAR T cell-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. CRISPR screening of CAR T cells identified dependencies for their effector functions, including TLE4 and IKZF2. Targeted knockout of these genes in CAR T cells robustly enhanced antitumor efficacy against GBM patient-derived xenografts (PDXs). Bulk and single cell-RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for their susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered the tumor-immune signaling axis and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs are promising strategies to discover avenues and inform potential combinatorial approaches for enhancing CAR T cell therapeutic efficacy against GBM, and can be extended to reveal key mediators of immunotherapy responses across solid tumors.
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Project description:Glioblastoma (GBM) is the most prevalent primary malignant brain tumor, containing self-renewing stem-like GBM stem cells (GSCs) that have been a focus of immunotherapies. Chimeric antigen receptor (CAR) T cell therapy has shown evidence of clinical activity, but overall limited responses in patients with GBMs. Here, we interrogated molecular determinants of CAR T cell-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. CRISPR screening of CAR T cells identified dependencies for their effector functions, including TLE4 and IKZF2. Targeted knockout of these genes in CAR T cells robustly enhanced antitumor efficacy against GBM patient-derived xenografts (PDXs). Bulk and single cell-RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for their susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered the tumor-immune signaling axis and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs are promising strategies to discover avenues and inform potential combinatorial approaches for enhancing CAR T cell therapeutic efficacy against GBM, and can be extended to reveal key mediators of immunotherapy responses across solid tumors.
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Project description:This is an in vitro genome-wide CRISPR/cas9 screen in human glioblastoma stem cells, screening for genes essential for survival of these cells. These cells express cas9 and have been transfected with a guide RNA library causing gene knockouts. We will analyse the sequencing data for depletion of guide RNAs. In this particular study, we will do RNA sequencing to correlate CRISPR with expression levels in specific cancer cell subpopulations. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2016-03-22 | E-ERAD-448 | biostudies-arrayexpress
Project description:Pooled CRISPR screening with single-cell transcriptome read-out
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Project description:Scalable, compressed phenotypic screening using pooled perturbations (PDAC screens)
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Project description:A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive tumor microenvironment (TME), which is densely populated and supported by protumoral glioma-associated microglia and macrophages (GAMs). Targeting CD47, a don't-eat-me signal overexpressed by tumor cells, disrupts the CD47-SIRPalpha axis and induces GAM phagocytic function. However, antibody-mediated CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. To overcome these limitations, we combined local CAR T cell therapy with paracrine GAM modulation to effectively eliminate GBM. To this end, we engineered a new CAR T cell against epidermal growth factor receptor variant III (EGFRvIII) that constitutively secretes a signal regulatory protein gamma (SIRPgamma)-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in a dose-dependent manner in vitro and eradicated orthotopically xenografted EGFRvIII-mosaic GBM by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon tumor re-challenge. Combining anti-CD47 antibodies with anti-EGFRvIII CAR T cells failed to achieve a similar therapeutic effect, underscoring the importance of sustained paracrine GAM modulation. Multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry on GBM-xenografted brains showed that anti-EGFRvIII-SGRP CAR T cells accelerated GBM clearance, increased CD68+ cell trafficking to tumor scar sites and promoted GAM-mediated tumor cell uptake. In a peripheral lymphoma mouse xenograft model, anti-CD19-SGRP CAR T cells had superior efficacy to conventional anti-CD19 CAR T cells. Validation on human GBM explants revealed that anti-EGFRvIII-SGRP CAR T cells had a similar tumor-killing capacity to anti-EGFRvIII CAR monotherapy but showed a slight improvement in the maintenance of tumor-infiltrated CD14+ cells. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate immune TME. This results in the additive elimination of bystander EGFRvIII- tumor cells in a manner that overcomes the main mechanisms of CAR T cell therapy resistance, including tumor innate immune suppression and antigen escape.
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