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T cells engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) have enabled a new generation of targeted cellular therapeutics. Philip Greenberg, MD, an internationally recognized expert in this field, is part of the first group to demonstrate that adoptive transfer of antigen-specific T cells can eradicate disseminated cancer cells. Dr. Greenberg’s lab at the Fred Hutchinson Cancer Center in Seattle has made seminal advances in methods that are now widely employed for the identification and cloning of antigen-specific TCRs, including high-affinity TCRs, and for the rapid expansion of selected T cells in vitro.
A member of the Greenberg team and a recipient of the Ann and Sol Schreiber Mentored Investigator Award, Kristin Anderson, PhD, focuses on identifying treatments for ovarian cancer, specifically on developing molecular engineering strategies that improve the function, persistence, and migration of genetically engineered antitumor cells to better target cancer-associated proteins.
When she had just turned 28 years old, Anderson was identified with triple-negative breast cancer. After successful treatment she made a huge career pivot. “When I went through cancer, I realized I was only alive because researchers had been in the lab late nights, long weekends, trying to create this drug that saved my life,” Anderson said. “I realized I had an opportunity to pay it forward.”
Targeting Ovarian Cancer
Most women diagnosed with advanced-stage, high-grade serous ovarian cancer have an overall five-year survival rate of less than 50%. In a recent webinar titled, “Engineering Adoptive T Cell Therapy to Overcome Immune Suppression in Ovarian Cancer,” Anderson discussed her lab’s progress in preclinical trials along with strategies to improve the efficacy of T-cell therapies.
The first step was to demonstrate the safety and efficacy of the approach since many tumor antigens such as mesothelin (MSLN), a cell surface protein normally expressed on the mesothelin lining of the pleural and peritoneal cavities, are also expressed at low levels in healthy tissues.
MSLN is overexpressed in 75% of high-grade serous ovarian cancers and often observed on aggressively growing cells. Anderson hypothesized that CD8 T cells engineered to target MSLN with a high-affinity TCR could slow tumor growth and prolong survival.
Assessing Potency of Engineered T Cells
Potency assays are commonly used to measure the specific ability of a cellular therapy to elicit a response at a certain dose. “For our purposes, the most crucial features of a potency assay are sensitivity and efficiency,” said Anderson. “We often wish to compare multiple effector cell populations head-to-head when measuring cancer cell cytotoxicity. The Agilent xCELLigence Real Time Cell Analyzer (RTCA) is both sensitive and high throughput, which allows us to more quickly and accurately determine which therapeutics to advance to the clinic.”
Potency of the engineered T cells was evaluated using the Agilent xCELLigence RTCA platform, which uses impedance measurements to calculate cytotoxicity. As target tumor cells replicate and adhere to a greater surface area on the specialized plate, the impedance of the signal increases. Effector T cells are added at increasing E:T ratios, and target cell death is calculated based on the loss of impedance signal. “We observed tumor cell death in a dose dependent manner but only when the T cells expressed the MSLN-specific TCR,” Anderson said.
In the first set of murine experiments, MSLN-specific T cells preferentially infiltrated the tumors but did not persist, a critical issue for cellular therapy especially in solid tumors. Evaluation of the antitumor cytokine expression showed that the T cells had reduced function after prolonged antigen experiences, another obstacle for cellular therapies. A second set of murine experiments with added strategies and redosing, excitingly, did prolong survival, suggesting the approach could be a translationally relevant strategy to benefit individuals with ovarian cancer.
“We tried several cytotoxicity assays to evaluate the ability of these T cells to lyse human ovarian tumor cells,” Anderson said. “We first evaluated cytotoxicity assays that use chromium release, flow cytometry, and colorimetric readouts. Unfortunately, each of these had limitations. We found that the target cells we were using were poor at retaining chromium and consequently had high background in chromium release assays. In the colorimetric assay, we could not distinguish between death of the target and effector cells, greatly reducing the sensitivity of our analysis. Flow cytometry was sensitive and specific, but it was also labor intensive if we wished to evaluate multiple time points.”
So, the research team again turned to the xCELLigence RTCA to evaluate two new MSLN epitopes. Once the cells reached a steady growth phase, new effector T cells that targeted the newly identified epitopes were added. The co-cultures were monitored for a drop in cell index, or percent cytolysis.
These results continued to suggest that T-cell therapies have the potential to help individuals with ovarian cancer, even late-stage disease, but also indicated there was potential to improve the efficacy. “Persistence was still an issue, and we know that signals from the tumor can affect infiltration and persistence,” Anderson added.
These refined T cells indeed showed enhanced cytokine production after repeated stimulations, suggesting that these cells may be more effective at lysing tumor cells in vivo. Mice receiving the refined engineered T cells exhibited an even greater prolonged survival suggesting that rationally modifying engineered T cells to overcome suppressive signals has the potential to significantly improve the efficacy of adoptive cell therapy in solid tumors.
The Impact of the xCELLigence RTCA for T Cell Therapy Development
The xCELLigence RTCA played an important role in this research dedicated to developing adoptive T-cell therapies to address high-grade serous ovarian cancer. “We have identified three major advantages to this technology compared with other widely available methods. The first is that the platform does not require labeling of target or effector cells to measure tumor-specific lysis. This has saved time and allowed us to evaluate T-cell effector function using either commercial cell lines or primary tumor cells as targets,” Anderson said. “Another benefit has been the real-time kinetic readout of data during the experiment, which has revealed insights we would have missed by performing end-point assays at defined time points. Third, the platform allows investigators to run orthogonal assays with the effector cells, such as serial killing assays or flow cytometry to evaluate phenotypic differences.“
In addition to providing critical cytolytic data for manuscripts, the xCELLigence RTCA increases the rate at which the team can evaluate novel T-cell receptor candidates and engineering strategies for translation to the clinic.
Watch the Webinar On Demand
“Engineering Adoptive T Cell Therapy for Solid Tumors”
by Kristin G. Anderson, PhD, Research Associate, Greenberg Lab, Fred Hutchinson Cancer Center
About the Greenberg Lab at Fred Hutch
The Philip Greenberg Lab at the Fred Hutchinson Cancer Center, a part of the Program on Immunology within the Clinical Research Division, focuses on both basic immunology and cancer immunobiology, and on the development and assessment of adoptive therapies with antigen-specific T cells targeting human malignancies and chronic infections.
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, which also supports FDA 21 CFR Part 11 compliance.
For Research Use Only. Not for use in diagnostic procedures.