CAR T Cell Killing

CAR T Cell Killing Application
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Chimeric antigen receptor (CAR) T cell therapy involves the introduction of genetically modified T cells to a patient with the purpose of identifying and targeting cancer cells. Development of successful CAR T cell therapies requires: 

  • >> Appropriate antigen targeting 
    >> CAR optimization 
    >> Predictive potency assessment 
    >> Immune cell exhaustion/persistence evaluation
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  • To address these limitations and develop a safe and robust immune cell product, our live-cell analysis platforms can be used to measure phenotype, potency, and persistence of CAR T cells. 

T Cells and CAR T Cells

T cell receptors (TCRs) in unmodified T cells detect antigens presented by MHC while chimeric antigen receptors (CARs) in CAR T cells recognize antigens expressed by the target cell. 

Structure of a CAR​

  • Signaling domain — stimulates the activation and proliferation of the T cell​
  • Transmembrane domain — anchors the CAR to the membrane​
  • Antigen recognition domain — recognizes and binds to the target cell antigen​
Chimeric antigen receptors (CARs) in CAR T cells recognize antigens expressed by the target cell and can be used to determine cell killing potency.

CAR T Immunotherapy Assays

CAR Optimization with Imaging
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CAR Construct Killing Video

 

CAR optimization is critical for effective in vitro immuno-oncology assay development. The Omni live-cell imaging system was used to compare the killing of different CAR constructs and showed that cell death was observed in a dose-dependent manner.

T cell killing vs time
T cell killing legend

Confluency of RFP-labeled cancer cells after the addition of CAR T cells is measured with the Omni platform. Two different CAR constructs were compared across E:T ratios and Construct 1 showed a greater potency across all ratios.

 

Assess specificity of a CAR T cell-mediated response
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Purpose: To differentiate between non-specific and specific killing of target cells by CAR T cells. High specificity of CAR T cell-mediated killing ensures safety and effectiveness of a CAR T cell therapy. 

Resistance and cytolysis of target SKOV3 cancer cells by HER2-targetting CAR T cells.
Resistance and cytolysis of target SKOV3 cancer cells by HER2-targetting CAR T cells.

HER2-overexpressing cancer cells were treated with CAR T cells with or without the tumor antigen-recognizing domain. CAR T cell-mediated killing of cancer cells was analyzed using the Maestro Z platform

Result: CAR T cells possessing the antigen-recognizing domain demonstrated targeted killing of HER2-cancer cells. 

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Measure efficacy against solid tumor spheroids
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Purpose: To track CAR T cell killing in 3D spheroids. Solid tumors can pose additional challenges to immunotherapies compared to liquid tumors or 2D monolayer cultures. 

Cytolysis over time of the CAR T cell-treated SKOV3 spheroid.

 

A cancer spheroid was treated with CAR T cells. Cancer spheroid cytolysis was measured using the Maestro Z platform

Result: A dose-dependent decrease in cancer spheroid size was observed following CAR T cell treatment. 

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Evaluate CAR T cell exhaustion
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Purpose: To evaluate the persistence of CAR T cell killing. T cell exhaustion is a common hurdle that impedes complete tumor eradication in a patient. 

Repeated stimulation of CAR T cells with SKOV3 cancer cells.
Repeated stimulation of CAR T cells with SKOV3 cancer cells.

Effector CAR T cells were added to target cancer cells. Every 72 hours they were transferred, at the same ratio, to new target cells. Target cell killing was assessed using the Maestro Z platform

Result: After each sequential transfer, cytolysis decreased as the CAR T cells entered a less potent state. 

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Measure CAR T cell-mediated cytotoxicity
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Purpose: To measure the potency of CAR T cell-mediated killing of cancer cells expressing different levels of HER2 antigen. Antigen receptor levels have been shown to influence CAR T cytolytic activity. 

CAR T cell-mediated killing of SKOV3 and MCF7 cells expressing different levels of HER2 antigen.
CAR T cell-mediated killing of SKOV3 and MCF7 cells expressing different levels of HER2 antigen.
CAR T cell-mediated killing of SKOV3 and MCF7 cells expressing different levels of HER2 antigen.

 

CAR T cells targeting HER2 antigen were added to two cancer cell lines and the magnitude of cell killing was compared using the Maestro Z platform

Result: CAR T cell-mediated killing was observed against both target cell lines and across different effector-to-target ratios. The rates of cytolysis were directly proportional to the levels of target antigen expression. 

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Application Note: Development of a functional cardiotoxicity assay for evaluating the safety of cell therapies
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CAR T App Note: Development of a functional cardiotoxicity assay for evaluating the safety of cell therapies

 

In this app note you will learn how:

>> HER2-targeted CAR T cells kill iPSC-derived cardiomyocytes in vitro

>> Non-transduced T cells show no toxicity toward iPSC-CMs, and HER2-CAR T cells do not kill iPSC-derived neurons

>> HER2-CAR T induce electrophysiological changes in iPSC-CMs, often before changes in viability are observed

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Click here to download the Axion BioSystems Immuno-ongcology Brochure

 

5 star review

Great results!


Quantitative data with a convenient mobile app. The Maestro Z provides a quantitative measurement of CAR-T cell-mediated cytotoxicity. The system is very easy to use, and it's powerful to see CAR-T cell killing profiles over time. The number of experiments you can run continuously with majority hands-off time is incredible. The mobile app is also great for making decisions about when to come into the lab when to add effectors, and so on.

- Heather Lin. Emory University, Georgia, USA

Maestro Z