eoe sub articles

EoE Sub Articles

1. Generation of CAR-encoding lentivirus
NOTE: Once the specific chimeric antigen receptor (CAR) T-cell plasmid construction is completed (CD47 and others), lentiviral CARs are generated by the standard procedure, using 293 FT cells, a lentiviral packaging mix, and transfection agents (see the&#160;Table of Materials) as described. Subsequently, use a quantitative reverse transcription polymerase chain reaction (RT-PCR) kit and a thermal cycler ...

a real-time in vitro cytolysis assay to evaluate the potency of car t cells

Source: Xi, B. et al., <a href="https://app.jove.com/v/59033">A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for monitoring CAR T cell activity against cancer cells through real-time electrical impedance measurements. Tracking changes in impedance assesses the effectiveness of CAR T cells in inducing cancer cell cytolysis, providing insights into their cytotoxic capabilities.

A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

1. Generation of CAR-encoding lentivirus
NOTE: Once the specific chimeric antigen receptor (CAR) T-cell plasmid construction is completed (CD47 and ...

Begin with a cell culture medium-containing E-Plate &#8212; a plate with gold microelectrodes in each well&#39;s bottom.
Seed the plate with target cancer cells, adhering to the gold microelectrodes, forming a physical barrier between the electrodes.
Transfer the plate to a cell analyzer and apply an electrical signal.
Adhered cells disrupt the electron flow between the electrodes, resulting in an impeded flow of electrons, measured as impedance.&#160;&#160;
As these cells proliferate and adhere to the electrodes, electrical impedance increases proportionally with the adherent cell number, enabling real-time measurement.
Introduce chimeric antigen receptor T cells or CAR T cells expressing chimeric antigen receptors. These receptors interact with cancer cells&#39; antigens, activating T cells, and forming an immunological synapse.
Activated CAR T cells release cytotoxic substances, including perforins and granzymes.
Perforins form pores in the cancer cell membrane, facilitating granzymes to enter the cells and induce cytolysis.
This promotes cell detachment, decreasing electrical impedance, correlating with CAR T cells&#39; cytolysis potential.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

JoVE Journal

Cancer Research

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

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