Immuno-oncology

CAR optimization is critical for effective immunotherapy 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.

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.

The ability of an immunotherapy to potently target specific antigens is critical to its success. The Maestro Z can be used to evaluate the potency of your engineered immune cells.

To separate non-specific killing from specific CAR T-cell killing, non-transduced T cells and donor-matched mock CAR T cells were compared to targeted CAR T cells. The results demonstrate that the Maestro Z in vitro assay platform differentiates between non-specific and specific killing of different effector cell types.

Figure 1HER2-targeted CAR T cells show antigen-specific killing when compared to T cells or mock CAR T cells. (A) HER2-expressing SKOV3 cells were grown over 50 hours. Effector cells were added at 24 hours. (B) Cytolysis over time shows the specific killing of HER2-targeted CAR T cells.

Cancer cells can vary in target antigen expression levels. This can influence CAR T-cell cytolytic activity. In this example, HER2-targeted CAR T cells showed different levels of potency in MCF7 and SKOV3.

The tumor microenvironment can pose a challenge to many immunotherapies. The architecture and composition of tumors can prevent infiltration of immune cells, making the tumor resistant to treatment. Cancer spheroids can be used to recapitulate this biology and assess the potency of immunotherapies against solid tumors.

The Maestro Z can be used to measure tumor growth and immune cell-mediated killing in solid tumors.

Solid tumors can be a challenge for immunotherapies. Cancer spheroids can recapitluate some of the tumor microenvironment in vitro. (A) A cancer spheroid on a CytoView-Z plate. (B) Tumor growth over time as measured by resistance. CAR T cells added at 20 hours show E:T ratio dependent killing. (C) A comparison of resistance at 50 hours post-effector addition.

 

Glioblastoma (GBM) is an aggressive form of brain cancer that has no effective treatments.

Activated human T cells targeting U87MG glioma cell monolayers were evaluated on the Maestro platform. Glioma growth curves were measured continuously. The addition of activated human T-cells resulted in glioma cell lysis. The full time course of in vitro immune cell killing was captured.

Kill time_50%, or the time required to reach 50% cytolysis, was calculated with one plate and experiment.

(A) Impedance was continuously monitored on the Maestro Z for U87 glioma cells seeded on the CytoView-Z plate at three different densities (n=12). After 24 hours, activated human T-cells were added in a 10:1 ratio (n=4 wells for each cell density). The addition of activated human T cells resulted in a decrease of the impedance signal consistent with T-cell-mediated lysis of the U87 cells, while untreated wells continued increasing.

(B) Percent cytolysis was tracked in real time, enabling (C) computation of 50% kill time (KT50) for each density. A higher density of T cells led to faster killing of U87 cells.

Continuous data reveals the kinetics of cancer cell response to T cells for better mechanistic understanding without the time- and cost-intensive process of repeating multiple endpoint assays.  The Maestro Z impedance assay is a sensitive method to continuously monitor cancer cell proliferation and immune cell-mediated cytotoxicity, providing a powerful means of assessing immune cell potency.

T cells and natural killer cells have always targeted cancer but immunotherapies seek to improve specificity and their innate cytotoxicity. Engineered cells like chimeric antigen receptor T cell (CAR T) therapy target tumor cells with greatly increased efficiency.

Assessing potency with endpoint assays are limited to a single snapshot in time. Costly reagents and dyes risk make such assays more expensive and limit the information you can get. The Maestro's impedance assay provides a sensitive, label-free method to continuously monitor cancer cell proliferation and immune cell-mediated cytotoxicity, with automated acquisition and straight-forward analysis.

Applications of immuno-oncology assays:

• >> CAR-T or other immune cell-mediated killing of cancer cells.

• >> Stimulation of the immune cells with checkpoint inhibitors and monoclonal antibodies.

• >> Potency of oncolytic viruses.

• >> Safety and toxicity testing.

Advantages of in vitro immune cell killing assays on the Maestro platform:

• >> No complicated assay protocols, just simple cell culture techniques.

• >> Integrated environmental controls finely regulate temperature and CO₂.

• >> Analyze the kinetics and responses whether they take minutes or weeks.

• >> Research the biology, not the chemistry. No dyes or extra reagents to interfere.

• >> Up to 384-wells, save time, cells, and reagents costs.

• >> Sensitive to small changes in target:effector ratios.

• >> CFR21 Part 11 compliant software available.

• >> Meets the high standards of ISO 20391-2 for GLP/GMP labs.

• >> More data, less work.

With the Maestro platform, quantifying and understanding the dynamics and kinetics of your in vitro cell potency assay has never been easier.