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1.
Blood ; 140(25): 2684-2696, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-35914226

RESUMO

Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.


Assuntos
Neoplasias Hematológicas , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Camundongos , Animais , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores de Antígenos de Linfócitos T , Imunoterapia Adotiva , Neoplasias Hematológicas/terapia , Imunoterapia , Antígenos CD19
2.
Cytotherapy ; 23(9): 787-792, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34119434

RESUMO

BACKGROUND AIMS: Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. METHODS: The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. RESULTS: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. CONCLUSIONS: The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.


Assuntos
Quinase I-kappa B , Estomatite Vesicular , Animais , Vetores Genéticos/genética , Humanos , Quinase I-kappa B/genética , Células Matadoras Naturais , Lentivirus/genética , Leucócitos Mononucleares , Proteínas Serina-Treonina Quinases/genética , Transdução Genética
3.
ACS Appl Bio Mater ; 7(1): 230-237, 2024 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-38133569

RESUMO

This article describes a monolayer-coated gold nanoparticle-based transfection system for the delivery of microRNA (miRNA) into human osteosarcoma (HOS) cells. Two distinct ammonium-terminated adsorbates were used in this study, which provided a platform for ionic bonding of the miRNA onto gold nanoparticles (AuNPs). The custom-designed monolayer-coated gold nanoparticles were characterized by dynamic light scattering, gel mobility shift assay, transmission electron microscopy, ultraviolet-visible spectrometry, zeta potential, and X-ray photoelectron spectroscopy. The miRNA-loaded gold nanoparticles were transfected, and the level of intracellular miRNA delivered and taken up by cells was measured by Taqman qPCR. The overall analysis indicated a successful delivery of miRNA into the HOS cells at an ∼11,000-fold increase compared to nontreated cells.


Assuntos
Nanopartículas Metálicas , MicroRNAs , Humanos , Ouro/química , MicroRNAs/genética , Nanopartículas Metálicas/química , Transfecção , Técnicas de Transferência de Genes
4.
bioRxiv ; 2023 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-37808662

RESUMO

Cell proliferation is a ubiquitous process required for organismal development and homeostasis. However, individuals with partial loss-of-function variants in DNA replicative helicase components often present with immunodeficiency due to specific loss of natural killer (NK) cells. Such lineage-specific disease phenotypes raise questions on how the proliferation is regulated in cell type-specific manner. We aimed to understand NK cell-specific proliferative dynamics and vulnerability to impaired helicase function using iPSCs from individuals with NK cell deficiency (NKD) due to hereditary compound heterozygous GINS4 variants. We observed and characterized heterogeneous cell populations that arise during the iPSC differentiation along with NK cells. While overall cell proliferation decreased with differentiation, early NK cell precursors showed a short burst of cell proliferation. GINS4 deficiency induced replication stress in these early NK cell precursors, which are poised for apoptosis, and ultimately recapitulate the NKD phenotype.

5.
Nat Biomed Eng ; 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38036617

RESUMO

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

6.
Nat Commun ; 13(1): 587, 2022 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-35102167

RESUMO

Developing CAR T cells for acute myeloid leukemia (AML) has been hampered by a paucity of targets that are expressed on AML blasts and not on hematopoietic progenitor cells (HPCs). Here we demonstrate that GRP78 is expressed on the cell surface of primary AML blasts but not HPCs. To target GRP78, we generate T cell expressing a GRP78-specific peptide-based CAR, which show evidence of minimal fratricide post activation/transduction and antigen-dependent T cell differentiation. GRP78-CAR T cells recognize and kill GRP78-positive AML cells without toxicity to HPCs. In vivo, GRP78-CAR T cells have significant anti-AML activity. To prevent antigen-dependent T cell differentiation, we block CAR signaling and GRP78 cell surface expression post activation by using dasatinib during GRP78-CAR T cell manufacturing. This significantly improves their effector function in vitro and in vivo. Thus, targeting cell surface GRP78-positive AML with CAR T cells is feasible, and warrants further active exploration.


Assuntos
Membrana Celular/metabolismo , Chaperona BiP do Retículo Endoplasmático/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Dasatinibe/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Blood Adv ; 6(21): 5737-5749, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-35446934

RESUMO

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.


Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Antígenos CD19 , Linfócitos T CD8-Positivos , Efeitos Psicossociais da Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T
8.
Cancer Discov ; 11(7): 1661-1671, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33563660

RESUMO

The inability of chimeric antigen receptor (CAR) T cells to sustain their effector function after repeated exposure to tumor cells is a major obstacle to their success in patients with solid tumors. To overcome this limitation, we designed a novel chimeric cytokine receptor to create an autocrine loop that links activation-dependent GM-CSF production by CAR T cells to IL18 receptor signaling (GM18). Expression of GM18 in CAR T cells enhanced their effector function in an antigen- and activation-dependent manner. In repeat stimulation assays, which mimic chronic antigen exposure, CAR.GM18 T cells had a significantly greater ability to expand and produce cytokines in comparison with their unmodified counterparts targeting EPHA2 or HER2. In vivo, CAR.GM18 T cells induced tumor regression at cell doses at which standard CAR T cells were ineffective in two solid tumor xenograft models. Thus, our study highlights the potential of hijacking cytokines that are physiologically secreted by T cells to bolster their antitumor activity. SIGNIFICANCE: We designed a chimeric cytokine receptor (GM18) that links CAR T-cell activation to MYD88 signaling. GM18 endows CAR T cells with sustained effector function in the setting of chronic antigen exposure, resulting in potent antitumor activity in preclinical solid tumor models.This article is highlighted in the In This Issue feature, p. 1601.


Assuntos
Imunoterapia Adotiva , Interleucina-18 , Neoplasias/terapia , Receptores de Antígenos Quiméricos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , Animais , Linhagem Celular Tumoral , Humanos , Leucócitos Mononucleares , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Clin Invest ; 130(10): 5272-5286, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32865517

RESUMO

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage-response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.


Assuntos
Células Matadoras Naturais/imunologia , Proteínas de Manutenção de Minicromossomo/genética , Mutação , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Alelos , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Códon sem Sentido , Dano ao DNA/genética , Dano ao DNA/imunologia , Evolução Fatal , Feminino , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lactente , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Imunológicos , Mutação de Sentido Incorreto , Linhagem , Doenças da Imunodeficiência Primária/patologia
10.
Sci Rep ; 9(1): 19089, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31836741

RESUMO

Osteosarcoma (OS) is the most common primary pediatric malignancy of the bone having poor prognosis and long-term survival rates of less than 30% in patients with metastasis. MicroRNA-509 was reported to be downregulated in OS. We and others previously published that miR-509-3p can strongly attenuate cellular migration/invasion and sensitize ovarian cancer to cisplatin. Here, we show that overexpression of miR-509-3p inhibited migration of primary OS cell lines U2OS, HOS, and SaOS2 as well as metastatic derivatives 143B and LM7. miR-509-3p overexpression also inhibited proliferation and invasion of HOS and 143B cells and sensitized cells to cisplatin. Luciferase reporter assays using 3'-UTRs of predicted miR-509-3p targets associated with metastatic phenotypes revealed ARHGAP1 could be one of the downstream effectors of miR-509-3p in HOS. To find the global impact of miR-509-3p overexpression and cisplatin treatment we performed Reverse Phase Protein Analysis (RPPA). AXL, which has been reported to play a critical role in cisplatin resistance and confirmed as direct target of miR-509-3p was downregulated upon miR-509-3p treatment and further down-regulated upon miR-509-3p + cisplatin treatment. We propose that the miR-509-3p/AXL and miR-509-3p/ARHGAP1 axes have the potential to uncover new druggable targets for the treatment of drug resistant metastatic osteosarcoma.


Assuntos
Movimento Celular/genética , Cisplatino/uso terapêutico , MicroRNAs/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Modelos Biológicos , Invasividade Neoplásica , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tirosina Quinase Axl
11.
Cell Rep ; 23(1): 297-312.e12, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617668

RESUMO

Long noncoding RNAs (lncRNAs) are commonly dysregulated in tumors, but only a handful are known to play pathophysiological roles in cancer. We inferred lncRNAs that dysregulate cancer pathways, oncogenes, and tumor suppressors (cancer genes) by modeling their effects on the activity of transcription factors, RNA-binding proteins, and microRNAs in 5,185 TCGA tumors and 1,019 ENCODE assays. Our predictions included hundreds of candidate onco- and tumor-suppressor lncRNAs (cancer lncRNAs) whose somatic alterations account for the dysregulation of dozens of cancer genes and pathways in each of 14 tumor contexts. To demonstrate proof of concept, we showed that perturbations targeting OIP5-AS1 (an inferred tumor suppressor) and TUG1 and WT1-AS (inferred onco-lncRNAs) dysregulated cancer genes and altered proliferation of breast and gynecologic cancer cells. Our analysis indicates that, although most lncRNAs are dysregulated in a tumor-specific manner, some, including OIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergistically dysregulate cancer pathways in multiple tumor contexts.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA Longo não Codificante/genética , Linhagem Celular , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Genes Supressores de Tumor , Humanos , Oncogenes
12.
ACS Appl Mater Interfaces ; 9(28): 23370-23378, 2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28636320

RESUMO

This Article describes the generation and study of surfaces modified with custom-crafted poly(l-lysine) (PLL) coatings for use in the loading and delivery of single-stranded DNA (ssDNA). The experimental strategy utilizes bidentate dithiol adsorbates to generate stably bound azide-terminated self-assembled monolayers (SAMs) on gold possessing an oligo(ethylene glycol) (OEG) spacer. Consequent to the molecular assembly on gold, the azide termini are covalently attached to a maleimide linker moiety via a copper-catalyzed azide-alkyne "click" reaction. Functionalization with maleimide provides a platform for the subsequent attachment of cysteine-terminated poly(l-lysine) (PLL), thus forming a suitable surface for the loading of ssDNA via electrostatic interactions. In efforts to maximize DNA loading, we generate SAMs containing mixtures of short and long PLL segments and explore the DNA-loading capability of the various PLL SAMs. We then use thermal increases to trigger the release of the ssDNA from the surface. By examining the loading and release of ssDNA using these new two-dimensional systems, we gain preliminary insight into the potential efficacy of this approach when using three-dimensional gold nanostructure systems in future gene-delivery and biosensing applications.


Assuntos
Polilisina/química , DNA , Ouro , Polietilenoglicóis , Propriedades de Superfície
13.
Oncotarget ; 7(18): 25930-48, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27036018

RESUMO

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.


Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Biomarcadores Tumorais/análise , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Fosfoproteínas/biossíntese , Esferoides Celulares/patologia , Fatores de Transcrição , Proteínas de Sinalização YAP
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