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1.
J Vis Exp ; (200)2023 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-37955379

RESUMO

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.


Assuntos
Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Humanos , Receptores de Antígenos Quiméricos/genética , Leucócitos Mononucleares , Linhagem Celular Tumoral , Células Jurkat , Anticorpos de Cadeia Única/genética , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética
2.
Breast Cancer Res Treat ; 197(1): 57-69, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36318382

RESUMO

PURPOSE: There is currently no curative treatment for patients diagnosed with triple-negative breast cancer brain metastases (TNBC-BM). CAR T cells hold potential for curative treatment given they retain the cytolytic activity of a T cell combined with the specificity of an antibody. In this proposal we evaluated the potential of EGFR re-directed CAR T cells as a therapeutic treatment against TNBC cells in vitro and in vivo. METHODS: We leveraged a TNBC-BM tissue microarray and a large panel of TNBC cell lines and identified elevated epidermal growth factor receptor (EGFR) expression. Next, we designed a second-generation anti-EGFR CAR T construct incorporating a clinically relevant mAb806 tumor specific single-chain variable fragment (scFv) and intracellular 4-1BB costimulatory domain and CD3ζ using a lentivirus system and evaluated in vitro and in vivo anti-tumor activity. RESULTS: We demonstrate EGFR is enriched in TNBC-BM patient tissue after neurosurgical resection, with six of 13 brain metastases demonstrating both membranous and cytoplasmic EGFR. Eleven of 13 TNBC cell lines have EGFR surface expression ≥ 85% by flow cytometry. EGFR806 CAR T treated mice effectively eradicated TNBC-BM and enhanced mouse survival (log rank p < 0.004). CONCLUSION: Our results demonstrates anti-tumor activity of EGFR806 CAR T cells against TNBC cells in vitro and in vivo. Given EGFR806 CAR T cells are currently undergoing clinical trials in primary brain tumor patients without obvious toxicity, our results are immediately actionable against the TNBC-BM patient population.


Assuntos
Neoplasias Encefálicas , Receptores de Antígenos Quiméricos , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/uso terapêutico , Neoplasias de Mama Triplo Negativas/terapia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/secundário
3.
Cancer Discov ; 11(9): 2248-2265, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837065

RESUMO

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.


Assuntos
Glioblastoma/tratamento farmacológico , Imunoterapia Adotiva , Interferon gama/metabolismo , Células Mieloides/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Glioblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Methods ; 16(2): 191-198, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30700902

RESUMO

CD8+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery.


Assuntos
Apresentação de Antígeno , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Células Apresentadoras de Antígenos/citologia , Antígenos/química , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD8-Positivos/citologia , Clonagem Molecular , Técnicas de Cocultura , Epitopos/química , Reações Falso-Positivas , Biblioteca Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunoterapia/métodos , Células Jurkat , Células K562 , Lectinas Tipo C/metabolismo , Complexo Principal de Histocompatibilidade , Oligonucleotídeos/genética , Peptídeos/química
5.
Proc Natl Acad Sci U S A ; 115(8): 1877-1882, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29437954

RESUMO

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Epitopos de Linfócito T/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Receptores de Antígenos de Linfócitos T/genética , Clonagem Molecular , Regulação da Expressão Gênica/imunologia , Células HEK293 , Antígeno HLA-B27 , Humanos , Células Jurkat
6.
Sci Rep ; 7(1): 13898, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29066835

RESUMO

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.


Assuntos
Precursor de Proteína beta-Amiloide/genética , Hormese , Hidroxicolesteróis/farmacologia , Espaço Intracelular/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular Tumoral , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espaço Intracelular/metabolismo , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Ratos
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