Published in Frontiers in Medical Technology: Characterizing Scalable T Cell Manufacturing Method Using Single-Cell Functional Analysis

Chimeric antigen receptor (CAR) T cell therapies are a type of adoptive cell therapy that have demonstrated success in a variety of cancers and other disorders. Autologous CAR-T cells are created from a patient’s own T cells, alleviating the risk of immune rejection. However, the time needed to isolate, prepare, and expand cells delays treatment, and the final cell product quality may be suboptimal if the patient’s cells lose key characteristics.

Allogeneic, or “off-the-shelf,” CAR-T products are made from healthy donor cells and can be optimized and expanded so they are ready when a patient needs them. In clinical trials, allogeneic cells have demonstrated success in a number of cancers but current methods of production are not sufficient to create the estimated number of cells needed to treat all of the patients who could benefit from CAR-T therapies.

As new manufacturing techniques to culture and expand cells are developed, researchers need a way to ensure that T cell-based products, including CAR-T cells, maintain their function and key quality attributes. While status quo technologies can help to characterize cell products, only single-cell functional phenotyping through highly multiplexed secretomic analysis can give researchers insight into how individual cells function. These data, gathered using IsoPlexis’ Single-Cell Secretome platform, can help to guide manufacturing processes and verify product quality.

Analyzing Function of Expanded Cells with Single-Cell Polyfunctionality Metrics

Researchers recently developed a scalable method for expanding PBMC-derived T cells and described their novel manufacturing technique in a paper that was recently published in Frontiers in Medical Technology. Traditional expansion takes place in two-dimensional flasks, but the authors used bioreactors to culture the cells in a three-dimensional suspension as a scalable method. Agitating the suspension can help to evenly distribute gases and critical cell nutrients, so the authors determined optimal conditions and allowed cells to grow with continuous media perfusion for 14 days, which resulted in a 15-fold increase in the number of T cells. Cells were collected at various time points for analysis and characterization.

The researchers used IsoPlexis’ Single-Cell Adaptive Immune panel to compare PBMC-derived CD4+ and CD8+ T cell polyfunctionality, before (day 0) and after expansion (day 14) in response to PMA/IONO stimulation determined if cells maintained function. As the authors stated, “Polyfunctional T cells are capable of producing 2+ cytokines upon stimulation with antigen and are considered a vital functional characteristic.”

With IsoPlexis technology, they observed that the percentage of polyfunctional cells increased in both the CD4+ and CD8+ groups on day 14 compared to day 0 in response to PMA/IONO stimulation, especially cells secreting 5 or more cytokines. Cells at day 14 also produced a wider array of cytokine combinations, with some CD4+ T cells secreting 11 cytokines and CD8+ T cells secreting 9 cytokines. To further characterize these cytokine secretions, researchers compared the polyfunctional strength index (PSI), or the product of polyfunctionality and intensity, of each cytokine signal. Both CD4+ and CD8+ T cells exhibited an increase in effector PSI from day 0 to day 14. An increase in stimulatory PSI was also observed, particularly for CD8+ T cells. These findings indicate that the expansion did not diminish cell functionality but instead increased the number of polyfunctional cells and variety of cytokines they secrete.

Single-Cell Analysis Provides Key Quality Attributes

Processes that establish novel manufacturing techniques and scalability are important to meet the need for quality T cell-based products, but function must be verified to ensure that manufacturing processes yield quality cells. Single-cell functional analysis of T cells can provide important insights into product quality and potency, giving researchers confidence in their development and expansion techniques. Because increased polyfunctionality has been associated with improved clinical outcomes, this is a key product quality attribute that can help to guide product development and manufacturing. IsoPlexis’ single-cell analysis platforms provide a powerful tool to gain deeper insight into cell behavior and the proteome, all in a benchtop system with a streamlined workflow.

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