scholarly journals Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma

2020 ◽  
Vol 12 (533) ◽  
pp. eaaw2672 ◽  
Author(s):  
Dongrui Wang ◽  
Renate Starr ◽  
Wen-Chung Chang ◽  
Brenda Aguilar ◽  
Darya Alizadeh ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Regression ◽  
Surface Expression ◽  
Matrix Metalloproteinase 2 ◽  
Cell Surface Expression ◽  
Binding Potential ◽  
Car T Cells ◽  
Car T ◽  
Peptide Toxin ◽  
Effector Activity

Although chimeric antigen receptor (CAR) T cells have demonstrated signs of antitumor activity against glioblastoma (GBM), tumor heterogeneity remains a critical challenge. To achieve broader and more effective GBM targeting, we developed a peptide-bearing CAR exploiting the GBM-binding potential of chlorotoxin (CLTX). We find that CLTX peptide binds a great proportion of tumors and constituent tumor cells. CAR T cells using CLTX as the targeting domain (CLTX-CAR T cells) mediate potent anti-GBM activity and efficiently target tumors lacking expression of other GBM-associated antigens. Treatment with CLTX-CAR T cells resulted in tumor regression in orthotopic xenograft GBM tumor models. CLTX-CAR T cells do not exhibit observable off-target effector activity against normal cells or after adoptive transfer into mice. Effective targeting by CLTX-CAR T cells requires cell surface expression of matrix metalloproteinase–2. Our results pioneer a peptide toxin in CAR design, expanding the repertoire of tumor-selective CAR T cells with the potential to reduce antigen escape.

scholarly journals Chlorotoxin Redirects Chimeric Antigen Receptor T Cells for Specific and Effective Targeting of Glioblastoma

2020 ◽  
Author(s):  
Dongrui Wang ◽  
Renate Starr ◽  
Wen-Chung Chang ◽  
Brenda Aguilar ◽  
Darya Alizadeh ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Matrix Metalloproteinase 2 ◽  
Cell Surface Expression ◽  
Binding Potential ◽  
Car T Cells ◽  
Car T

AbstractWhile chimeric antigen receptor (CAR) T cells have demonstrated antitumor activity against glioblastoma (GBM), tumor heterogeneity remains a critical challenge. To more effectively target heterogeneous GBMs, we report the development of a novel peptide-based CAR exploiting the GBM-binding potential of chlorotoxin (CLTX). CLTX bound a greater proportion of tumor cells than GBM-associated antigens EGFR, HER2 and IL13Rα2. CAR T cells bearing CLTX as the targeting domain (CLTX-CAR), mediated potent in vitro and in vivo anti-GBM activity, and efficiently targeted tumors lacking expression of other GBM-associated antigens. Importantly, CLTX-CAR T cells exhibited no observable off-target effector activity against normal cells, or when adoptively transferred into mice. Effective targeting by CLTX-CAR T cells required cell surface expression of matrix metalloproteinase-2 (MMP-2). Our results are the first demonstration of a peptide toxin utilized as a CAR targeting domain, expanding the repertoire of tumor-selective CAR T cells with the potential to reduce antigen escape.One Sentence SummaryChimeric antigen receptors incorporating chlorotoxin as the tumor targeting domain recognize and kill glioblastoma with high specificity and potency.

scholarly journals Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2789-2789
Author(s):  
Lindsey F Call ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Cynthia Nourigat-Mckay ◽  
LaKeisha Perkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

scholarly journals Identifying Factors in Multiple Myeloma Controlling Response to B-Cell Maturation Antigen (BCMA)-Targeted Immunotherapy Using CRISPR-Based Functional Genomics

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1926-1926
Author(s):  
Poornima Ramkumar ◽  
Jaime Leong ◽  
Meghan Seyler ◽  
Stratton J Georgoulis ◽  
Axel Hyrenius Wittsten ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Novel Genes ◽  
Car T Cells ◽  
B Cell Maturation ◽  
Genome Wide ◽  
Car T ◽  
Druggable Targets

Abstract Introduction: Multiple myeloma (MM) is the second most common blood cancer in the United States. Recent breakthroughs in immunotherapy have the potential to transform MM treatment. An immunotherapy target that shows considerable promise in myeloma is the B-cell maturation antigen (BCMA). BCMA is specifically expressed in myeloma cells and plasma cells, making it an ideal target in myeloma. Results from two early-phase clinical trials using anti-BCMA therapy, showed remarkable response in most patients. Although immunotherapy has been promising, recent findings suggest that patients can develop resistance to such therapies by lowering the levels of the target. Here we employ our innovative CRISPR-interference/activation (CRISPRi/a)-based functional genomics platform to identify mechanisms that regulate BCMA expression, which would enable us to design strategies to improve the efficacy of available BCMA immunotherapy agents. Methods: The CRISPRi/a platform utilizes a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor domain to silence genes (CRISPRi) or to activator domains to activate transcription (CRISPRa). The CRISPR machinery is targeted to specific genes using single guide-RNAs (sgRNA). We have engineered a panel of myeloma cell lines to express components of the CRISPRi system. Here we transduced CRISPRi-AMO1 cell line with sgRNAs targeting the human genome. The sgRNA expressing cells were stained with a fluorescent-tagged BCMA antibody and FACS sorted into cells expressing high and low levels of BCMA. The cells were processed for next generation sequencing to determine the frequency of sgRNA in each of these populations. To develop BCMA chimeric antigen receptor-T (CAR-T) cells, CD8+ T cells were transduced with BCMA CAR construct specifically recognizing BCMA. To examine the anti-myeloma activity of the BCMA CAR-T cells, CAR-T cells were co-cultured with MM cell lines at a ratio of 1:2 (Effector:Target) for 24hrs. The cells were analyzed by flow cytometry for expression of CD69, an activation marker on T cells and for apoptosis of cancer cells using propidium iodide. Results: Our FACS-based genome-wide CRISPRi screen identified several genes and pathways regulating BCMA cell surface expression. In addition to previously reported gamma-secretase complex and transcription factor POU2AF1 we identified genes involved in peroxisome biogenesis, subunits of the proteasome, transcription factors and a few druggable targets regulating cell surface expression of BCMA. We are currently validating the novel genes identified from our primary genome-wide screen in a panel of MM cell lines and developing MM cell lines expressing CRISPRa machinery to perform gain-of-function screens that will complement our CRISPRi screen. Furthermore, we have developed active CAR-T cells targeting BCMA and demonstrated its efficacy in MM cell lines expressing different levels of BCMA. We are currently testing the novel genes identified from our CRISPRi screen in combination with BCMA CAR-T cells to identify genes that alter sensitivity to BCMA immunotherapy. Conclusions: Our studies have identified several novel genes and pathways regulating BCMA expression including some druggable targets. Through these studies, we expect to uncover mechanisms regulating expression of BCMA and its impact on sensitivity to BCMA immunotherapy, and pinpoint potential combination therapy targets that pre-empt resistance to BCMA immunotherapy. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

scholarly journals Preclinical Development of a T-Cell ALL CAR Demonstrates That Differences in CAR Membrane Distribution May Impact Efficacy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4019-4019
Author(s):  
Haneen Shalabi ◽  
Haiying Qin ◽  
Kelsey Wanhainen ◽  
Jillian Smith ◽  
Rimas Orentas ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression. Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone. Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface. Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells. Disclosures Orentas: Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.

TCRab Deficient CAR T-Cells Targeting CD123: An Allogeneic Approach of Adoptive Immunotherapy for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2555-2555 ◽  
Author(s):  
Roman Galetto ◽  
Céline Lebuhotel ◽  
Agnès Gouble ◽  
Nuria Mencia-Trinchant ◽  
Cruz M Nicole ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Surface Expression ◽  
Car T Cells ◽  
Healthy Donors ◽  
Car T ◽  
Acute Myeloid

Abstract The remissions achieved using autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas have encouraged the use of CAR technology to treat different types of cancers by targeting distinct tumor-specific antigens. Since the current autologous approach utilizes CAR T-cells manufactured on a "per patient" basis, we propose an alternative approach based on the use of a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" CAR T-cell-based frozen products. In the present work we have adapted this allogeneic platform to the production of T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). Multiple antigen recognition domains were screened in the context of different CAR architectures to identify candidates displaying activity against cells expressing variable levels of the CD123 antigen. The three lead candidates were tested in an orthotopic human AML cell line xenograft mouse model. From the three candidates that displayed comparable activity in vitro, we found two candidates capable of eradicating tumor cells in vivo with high efficiency. Subsequently, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). Editing of the TRAC gene can be achieved at high frequencies, and allows efficient amplification of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition, we show that TCR-deficient T-cells display equivalent in vitro and in vivo activity to non-edited T-cells expressing the same CAR. We have performed an initial evaluation of the expression of CD123 in AML patients and found an average cell surface expression of CD123 was of 67% in leukemic blasts (95% CI 48-82), 71% in CD34+CD38+ cells (95% CI 56-86), and 64% in CD34+CD38- (95% CI 41-87). Importantly, we have found that CD123 surface expression persists in CD34+CD38-CD90- cells after therapy in at least 20% of patients in remission (n=25), thus emphasizing the relevance of the target. Currently, the sensitivity of primary AML cells to CAR T-cells is being tested. Finally, we will also present our large scale manufacturing process of allogeneic CD123 specific T-cells from healthy donors, showing the feasibility for this off-the-shelf T-cell product that could be available for administration to a large number of AML patients. Disclosures Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

scholarly journals CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

2021 ◽  
Author(s):  
Katherine Mueller ◽  
Nicole Piscopo ◽  
Matthew Forsberg ◽  
Louise Saraspe ◽  
Amritava Das ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tumor Regression ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cells traditionally harbor viral vectors that encode the CAR transgene in the genome. However, viral vector manufacturing typically is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. Here, CAR T cells were generated within nine days using recombinant SpCas9 protein and nucleic acids, without any viral vectors or animal components. In comparison to traditional retroviral CAR T cells, nonviral CRISPR CAR T cells exhibit TRAC-targeted genomic integration of the CAR transgene, higher frequency of gene expression signatures associated with a memory phenotype, low receptor signaling prior to infusion, and potent cytotoxicity against GD2+ neuroblastoma in vitro and in vivo. This proof-of-principle study eliminating viral vectors and animal components during CAR gene transfer could enable more flexible and scalable manufacturing of clinically-relevant, high-quality CAR T cells to treat cancers, including solid tumors.

scholarly journals DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

Cancers ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 54
Author(s):  
Sripathi M. Sureban ◽  
Robert Berahovich ◽  
Hua Zhou ◽  
Shirley Xu ◽  
Lijun Wu ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Strategy ◽  
Cell Surface Expression ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Car T Cells ◽  
Hematological Cancers ◽  
Car T ◽  
Ifn Γ

CAR-T (chimeric antigen receptor T cells) immunotherapy is effective in many hematological cancers; however, efficacy in solid tumors is disappointing. Doublecortin-like kinase 1 (DCLK1) labels tumor stem cells (TSCs) in genetic mouse models of colorectal cancer (CRC). Here, we describe a novel CAR-T targeting DCLK1 (CBT-511; with our proprietary DCLK1 single-chain antibody variable fragment) as a treatment strategy to eradicate CRC TSCs. The cell surface expression of DCLK1 and cytotoxicity of CBT-511 were assessed in CRC cells (HT29, HCT116, and LoVo). LoVo-derived tumor xenografts in NOD Scid gamma (NSG™) mice were treated with CBT-511 or mock CAR-T cells. Adherent CRC cells express surface DCLK1 (two-dimensional, 2D). A 4.5-fold increase in surface DCLK1 was observed when HT29 cells were grown as spheroids (three-dimensional, 3D). CBT-511 induced cytotoxicity (2D; p < 0.0001), and increased Interferon gamma (IFN-γ) release in CRC cells (2D) compared to mock CAR-T (p < 0.0001). Moreover, an even greater increase in IFN-γ release was observed when cells were grown in 3D. CBT-511 reduced tumor growth by approximately 50 percent compared to mock CAR-T. These data suggest that CRC cells with increased clonogenic capacity express increased surface DCLK1. A DCLK1-targeted CAR-T can induce cytotoxicity in vitro and inhibit xenograft growth in vivo.

scholarly journals Efficient tumor regression by adoptively transferred CEA-specific CAR-T cells associated with symptoms of mild cytokine release syndrome

2016 ◽  
Vol 5 (9) ◽  
pp. e1211218 ◽  
Author(s):  
Linan Wang ◽  
Ning Ma ◽  
Sachiko Okamoto ◽  
Yasunori Amaishi ◽  
Eiichi Sato ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Regression ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T

scholarly journals Pre-Clinical Activity of Allogeneic Anti-CD22 CAR-T Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 808-808
Author(s):  
Julia Wells ◽  
Tianyu Cai ◽  
Cécile Schiffer-Manniou ◽  
Stéphanie Filipe ◽  
Agnès Gouble ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Surface Expression ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Autologous T-cells engineered with chimeric antigen receptors (CARs) against CD19 are proving to be an efficacious immunotherapy for patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). At present, CAR technology is administered through the custom-made manufacturing of therapeutic products from each patient's own T-cells. However, this patient-specific autologous paradigm is a significant limiting factor in the large-scale deployment of CAR technology. In this study, we utilized allogeneic "off-the-shelf" engineered CAR T-cells from third-party healthy donors. The CD22 surface antigen is commonly expressed in B-ALL patients as well as in healthy B-cells. Here, its potential as a CAR target was investigated using allogeneic off-the shelf engineered CAR T-cells against human CD22 (UCART22). UCART22 cells harbor surface expression of an anti-CD22 CAR (CD22 scFv-41BB-CD3z) and the RQR8 ligand, a safety feature rendering the T-cells sensitive to the monoclonal antibody rituximab. To reduce the potential for alloreactivity, the cell surface expression of the T-cell receptor (TCR) is abrogated through the inactivation of the TCRα constant (TRAC) gene using Cellectis' TALEN® gene-editing technology. The level of CD22 cell surface molecules was measured using BD Quantbrite beads for both patient peripheral blood samples and B-ALL cell lines. B-ALL cell lines (n=8) expressed a greater amount of CD22 molecules per cell than patient samples (n=14) (5,028 +/- 1,342 compared to 951 +/-160 molecules/cell, p=0.044), with highest expression of CD22 in two Ph-like B-ALL cell lines (MUTZ5, shown in Figure1A and MHH-CALL4). The in vitro cytotoxic activity of UCART22 cells was evaluated by co-culturing UCART22 or non-transduced CAR(-) TCRαβ(-) control T-cells (NTD) with B-ALL cell lines and primary human samples, at a maximum 10:1 effector to target ratio (represented in Figure1B). Using flow cytometry, significant antigen-specific cytotoxic activity of UCART22 cells was found compared to NTD controls and correlated with CD22 expression factored by the %kolmogorov-smirnov max difference in CD22-PE fluorescence compared to unstained controls (Pearson correlation r-squared for cell lines= 0.6850, p=0.0001 and r-squared for patient samples=0.6204, p=0.0008). Secretion of 13 cytokines was measured after 1:1 co-incubation of effector and target cells. UCART22 cells stimulated by CD22(+) B-ALL, but not NTD cells, secreted high levels of IFNγ, TNFα, IL-5, IL-17A and IL-17F in the culture supernatants, with cytokine levels being proportionate to CD22 abundance (represented in Figure1C). In addition, immune compromised mice engrafted with Daudi cells, a CD22(+) expressing Burkitt's lymphoma cell line, were treated with UCART22 cells. Treatment doses of 1-10x10^6 cells per mouse reduced disease burden (Figure 1D), measured by bioluminescence imaging, and extended survival in a dose-dependent fashion compared to saline or NTD treated controls. Additional PDX studies using B-ALL patient derived xenografts are ongoing and will be presented. Altogether, these results show supporting evidence for the future use of allogenic UCART22 in B-ALL immunotherapy. Disclosures Schiffer-Manniou: Cellectis SA: Employment. Filipe: Cellectis: Employment. Gouble: Cellectis SA: Employment. Galetto: Cellectis SA: Employment. Jain: ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Verastem: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Abbvie: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Smith: Cellectis Inc: Employment.

scholarly journals Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression

2020 ◽  
Vol 218 (2) ◽  
Author(s):  
Evripidis Lanitis ◽  
Giorgia Rota ◽  
Paris Kosti ◽  
Catherine Ronet ◽  
Aodrenn Spill ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Surface Expression ◽  
Fourth Generation ◽  
Cell Phenotype ◽  
Tumor Control ◽  
Retroviral Transduction ◽  
Car T Cells ◽  
Car T

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

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