Evaluating CAR T Cells: In Vitro Potency Assays for Production, Quality Control, Efficacy, and Safety

The Agilent NovoCyte Quanteon flow cytometer and xCELLigence Real-Time Cell Analysis platform were used to develop a series of in vitro preclinical CAR T-cell safety and efficacy assays

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Cancer immunotherapy, an ever-evolving field, harnesses intrinsic features of the immune system to combat various forms of the disease. An increasingly popular therapeutic approach includes genetically engineering T cells with Chimeric Antigen Receptors (CARs) that recognize and kill target tumor cells.

While the cutting-edge CAR T-cell modality has six FDA-approved therapies for B-cell malignancies, it still faces substantial hurdles, particularly for unpredictable, difficult-to-treat solid tumors.

To meet the clinical need, Charles River Laboratories— the first CDMO in North America to receive EMA approval to commercially produce an allogeneic cell therapy drug product—has invested in developing integrated solutions for cell therapy development. In a recent webinar titled “Utilizing flow cytometry and xCELLigence in cell therapy in vitro efficacy and safety studies,” Sabrina Manuela de Munnik, PhD, senior scientist at Charles River, discussed their Cell and Gene Therapy 360° Solutions platform, which supports the entire development life cycle of cellular immunotherapies.

Targeting HER2 in Solid Tumors

Unlike liquid tumors, there is no good universally expressed target for solid tumors, as cancer targets are often expressed in healthy cells and can mediate off-target effects. One example is the HER2 (human epidermal growth factor receptor 2) receptor,  which plays a role in cell growth and differentiation, but when overexpressed is correlated to several  human cancers—notably breast cancer.

Additionally, treatments must be trafficked to the tumor site, and overcome an immunosuppressive microenvironment. Given the difficulties, it is imperative to develop robust, physiologically relevant assays that aid from discovery to production.

Assessing the Safety and Efficacy of HER2 CAR T Cells

The work de Munnik presented focused on a HER2-targeting CAR T-cell model, but emphasized the workflow shown can be readily adapted: “Although I use CAR T cells as an example, the assays, NovoCyte Quanteon flow cytometer, and xCELLigence platform are modality agnostic and can be applied to other cancer immunotherapy approaches.”

Ensuring proper quality of CAR T cells is crucial in the early stages of development to derisk the cell treatment and save overall production costs. The first phase of assays focused on ensuring proper quality control (QC) of the newly produced HER2 CAR T cells on the NovoCyte. “To me it is very user-friendly. I’m quite pleased with the software,” de Munnik said of the GMP-compliant instrument—meaning it can be used for manufacturing release standards.

Charles River employee workign with the NovoCyte
Credit: Charles River

For the second phase, two different approaches were employed to monitor efficacy and specificity of the CAR T cells: end-point assays and kinetic measurements. To achieve this goal, use of the xCELLigence was incorporated along with flow cytometry.

The xCELLigence platform seeks to overcome the shortcomings of traditional end-point assays. “The nice thing is that the xCELLigence allows for kinetic measurements and evaluates the viability of the co-cultured cells throughout the course of the experiment,” de Munnik says. “This automated, label-free technology provides a continuous flow of information that provides insight that a traditional end-point assay cannot.”

Both the kinetic xCELLigence results and NovoCyte end-point data showed HER2-targeting CAR T cells induced cytotoxicity in HER2-positive target cells in an Effector (E) to target (T) ratio-dependent manner, but not in HER2-negative cells, indicating the CAR T cells were effectively finding and killing their intended targets.

The final phase of the project focused on assessing the safety of the CAR T cells by monitoring unwanted reactivity. Safety assays were designed to ensure engineered T cells specifically kill their targets, not healthy cells. Again, both the NovoCyte flow cytometer and xCELLigence were utilized to evaluate endpoint and kinetic measurements. The results correlated and showed the HER2 CAR T cells of this specific design killed healthy cells in a time- and E:T ratio-dependent manner—a potential safety risk, clearly demonstrating the safety assay’s importance.

In Summary

As cancer immunotherapy grows and flourishes, so does the need for simple, robust workflows to develop safe, effective cellular therapies. In particular, high-quality in vitro potency assays predictive of in vivo outcomes are crucial to spur the development of these novel, and potentially curative, cell therapies.

Charles River incorporated the 21 CFR Part 11 compliant Agilent NovoCyte Quanteon flow cytometer and xCELLigence RTCA platform into a solutions workflow to address this important need. The team developed a series of in vitro assays utilizing the Agilent Cell Analysis portfolio to encompass CAR T-cell development, production, and QC, through functionality tests, and safety assessments. These assays can be applied to a wide range of cancer immunotherapy modalities to meet the needs of individual customers.

About Charles River

The cell therapy team at the Leiden site is one arm of the multi-site end-to-end solution that Charles River provides for customers in the CGT field. With 100 sites worldwide, Charles River offers support from discovery and design, in vitro discovery and in vivo studies, to CDMO activities, clinical trials, and commercialization.

Charles River team
Credit: Charles River

About Agilent xCELLigence RTCA Instruments

The Agilent xCELLigence Real-Time Cell Analysis (RTCA) instruments use label-free cellular impedance to continuously monitor cell behavior with high sensitivity and reproducibility. The instruments operate in a standard CO2 cell culture incubator. Simply plate cells and obtain real-time kinetic data. Data acquisition and analysis are easy to perform using RTCA Software Pro. The xCELLigence RTCA eSight model
combines impedance with live-cell imaging for increased confidence in cell analysis and conclusions.

About Agilent NovoCyte Flow Cytometers

Agilent NovoCyte flow cytometers offer excellent capabilities, high-quality data, and an easy-to-use platform to save researchers time when acquiring and analyzing experiments. They provide an expanded set of capabilities that accommodate today’s high-end and increasingly sophisticated multicolor flow cytometry assays. Choose from 1 to 5 lasers with up to 30 state-of-the-art fluorescence detection channels. The intuitive and industry-leading NovoExpress software has been further advanced, providing an exceptional user
experience in data acquisition, analysis, and reporting.


Watch the webinar: bit.ly/3UA1MIh
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