Not all patients respond well to chimeric antigen receptor (CAR) T cell therapies, which reprogram a patient’s own immune cells to recognize and attack cancer. A way to optimize these treatments is to develop a better understanding of how various T-cell features relate to patient outcomes. Now, scientists from the Keck School of Medicine at the University of Southern California (USC) have developed a spectral flow cytometry panel for profiling CAR T cells. It gives scientists a tool for assessing how these cells evolve during manufacturing and which ones are most effective at killing cancer.
Full details are published in a paper titled, “High-Dimensional Temporal Mapping of CAR T Cells Reveals Phenotypic and Functional Remodeling During Manufacturing.” The paper is part of the 25th anniversary special issue of Molecular Therapy, the flagship journal of the American Society of Gene & Cell Therapy.
The cellular analysis platform that the scientists developed uses a spectral flow cytometer, a tool that analyzes the physical and chemical properties of individual cells. “Designed as an integrated single assay, it simultaneously captures high-dimensional immunophenotyping and in vitro cytotoxicity, generating a detailed fingerprint of the CAR T cell product,” the researchers wrote. “This approach enables us to reveal key biological transitions that may inform manufacturing optimization and guide product design.”
“Just as every person has a fingerprint that identifies them, T cells also have fingerprints,” explained Mohamed Abou-el-Enein, MD, PhD, the study’s senior author and executive director of the cell therapy program at USC/Children’s Hospital of Los Angeles. “By measuring the expression of markers on a cell’s surface, we can learn more about what distinguishes one CAR T cell therapy from another,” Abou-el-Enein said.
In their approach, cells are first tagged with fluorescent dyes that can bind to specific molecules that make up a cell’s fingerprint. Tagged cells are passed through the cytometer, where lasers cause the fluorescent tags to emit light that can be detected and measured. This indicates whether a specific molecule is present and how strongly it is expressed. Compared to standard tools, which can only measure about 10 markers at a time, the spectral flow cytometer provides a more comprehensive picture of each cell.
For their panel, Abou-el-Enein and colleagues selected 36 markers that capture T cell characteristics related to their ability to effectively identify and kill cancer cells. This includes markers for things like activation, metabolism, memory, and cytotoxicity. After pairing each marker with a fluorescent tag, they used sophisticated mathematical modeling to ensure that each could be detected separately.
Once they had a panel of markers, the researchers experimented with CAR T cells. They collected data on days five and ten of the manufacturing process. They found that on day five, cells more closely resembled stem-like cells and had higher metabolic activity than those tested on day 10. Both cell types can kill cancer, but their results indicated that day-five cells had qualities that have previously been linked to better long-term outcomes in patients. It suggests that CAR T cells may be better equipped to fight cancer after a five-day expansion process rather than at the 10-day mark.
“This work fills a critical gap in our understanding of how manufacturing conditions shape the therapeutic potential of CAR T cells,” Abou-el-Enein said. “By pinpointing when CAR T cells acquire—or lose—functional fitness, we can now tailor the timing of cell manufacturing.” Furthermore, the platform can help scientists optimize the manufacturing process in other ways. For example, they could compare the impact of using viral vectors with other ways of engineering CAR T cells.
Beyond manufacturing, the developers believe that scientists could use the platform to study the behavior of other cell types, to compare different gene editing technologies and production platforms, and to identify predictive biomarkers that link cell characteristics to patient outcomes.
The post Cell Surface Marker Profiling with Spectral Cytometry Optimizes CAR T Manufacturing appeared first on GEN - Genetic Engineering and Biotechnology News.
Full details are published in a paper titled, “High-Dimensional Temporal Mapping of CAR T Cells Reveals Phenotypic and Functional Remodeling During Manufacturing.” The paper is part of the 25th anniversary special issue of Molecular Therapy, the flagship journal of the American Society of Gene & Cell Therapy.
The cellular analysis platform that the scientists developed uses a spectral flow cytometer, a tool that analyzes the physical and chemical properties of individual cells. “Designed as an integrated single assay, it simultaneously captures high-dimensional immunophenotyping and in vitro cytotoxicity, generating a detailed fingerprint of the CAR T cell product,” the researchers wrote. “This approach enables us to reveal key biological transitions that may inform manufacturing optimization and guide product design.”
“Just as every person has a fingerprint that identifies them, T cells also have fingerprints,” explained Mohamed Abou-el-Enein, MD, PhD, the study’s senior author and executive director of the cell therapy program at USC/Children’s Hospital of Los Angeles. “By measuring the expression of markers on a cell’s surface, we can learn more about what distinguishes one CAR T cell therapy from another,” Abou-el-Enein said.
In their approach, cells are first tagged with fluorescent dyes that can bind to specific molecules that make up a cell’s fingerprint. Tagged cells are passed through the cytometer, where lasers cause the fluorescent tags to emit light that can be detected and measured. This indicates whether a specific molecule is present and how strongly it is expressed. Compared to standard tools, which can only measure about 10 markers at a time, the spectral flow cytometer provides a more comprehensive picture of each cell.
For their panel, Abou-el-Enein and colleagues selected 36 markers that capture T cell characteristics related to their ability to effectively identify and kill cancer cells. This includes markers for things like activation, metabolism, memory, and cytotoxicity. After pairing each marker with a fluorescent tag, they used sophisticated mathematical modeling to ensure that each could be detected separately.
Once they had a panel of markers, the researchers experimented with CAR T cells. They collected data on days five and ten of the manufacturing process. They found that on day five, cells more closely resembled stem-like cells and had higher metabolic activity than those tested on day 10. Both cell types can kill cancer, but their results indicated that day-five cells had qualities that have previously been linked to better long-term outcomes in patients. It suggests that CAR T cells may be better equipped to fight cancer after a five-day expansion process rather than at the 10-day mark.
“This work fills a critical gap in our understanding of how manufacturing conditions shape the therapeutic potential of CAR T cells,” Abou-el-Enein said. “By pinpointing when CAR T cells acquire—or lose—functional fitness, we can now tailor the timing of cell manufacturing.” Furthermore, the platform can help scientists optimize the manufacturing process in other ways. For example, they could compare the impact of using viral vectors with other ways of engineering CAR T cells.
Beyond manufacturing, the developers believe that scientists could use the platform to study the behavior of other cell types, to compare different gene editing technologies and production platforms, and to identify predictive biomarkers that link cell characteristics to patient outcomes.
The post Cell Surface Marker Profiling with Spectral Cytometry Optimizes CAR T Manufacturing appeared first on GEN - Genetic Engineering and Biotechnology News.