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Cell Therapy Overview

CAR-T therapies have quickly shifted the direction of treatments for aggressive diseases, such as blood cancers, where previous treatments were limited. There are currently over 1000 cell and gene therapy trials and two approved cell therapies, axicabtagene ciloleucel and tisagenlecleucel, but determining cellular fitness is still a top need in moving the next generation of cell therapy treatments forward.

Both autologous and allogeneic options are among the cutting-edge therapies of today, which also comprise T cells, NK cells, and more, while using technologies like CRISPR and TALEN to address blood cancers and solid tumor. CRISPR, for example, has paved the way for new possibilities in the cell therapy space, including providing a way for researchers to develop a renewable source of NK cells to be optimized for cell therapies,1 as well as enabling more specific and targeted edits to cells in general.

Challenges in the Field

Because cell therapy relies on immune cells from patients, or healthy donors in the case of allogeneic therapies, development and production is more complicated. To ensure that these cell products are potent and effective, knowing how to engineer potency and durability throughout the development and bioprocessing stages is crucial.

Knowledge of the powerful functional T cell drivers can give complex engineered immune cell therapies the edge that they need in this competitive and fast-paced environment. Legacy technologies, such as bulk ELISA or flow cytometry, can give estimates of a sample’s cytokine secretions, but miss the highly functional cell subsets that correlate to in vivo response. Bulk analysis is unable to analyze single cell function or identify which cells are secreting specific cytokines. Flow cytometry involves fixing and permeabilizing cells and can only provide estimates of cellular function. RNA-Seq can estimate function as well, but only shows a 0.4 correlation from RNA to protein.2 These limitations from legacy technologies remain challenges in determining function of cell products.

Single-Cell Multiplexed Solution

To address this challenge and need, IsoPlexis’ functional T cell biology is rooted in the ability to directly identify what each T cell secretes in a highly multiplexed manner, for the first time. High multiplexing of simultaneous true cytokines reveals most consistently intense and potent T cells. The IsoPlexis system can discover true function in single cell subsets, or highly polyfunctional cells, which correlates to response and reveals highly differentiated T cell insights.

Clinical Research Applied

In the initial phase of approved cell therapies, researchers published data using IsoPlexis’ single-cell functional cytokine detection system to demonstrate the enhanced ability to detect underlying cell therapy product heterogeneity. IsoPlexis’ single-cell cytokine based PCA visualizations (Figure 1) of the CAR-T product, published in JITC,3 revealed the potential to visualize donor differences.

Figure 1. Visualizing CAR-T cell functional groups and donor differences.

In an additional study, researchers used the IsoPlexis single-cell platform to correlate pre-infusion cell product data, using a metric termed Polyfunctional Strength Index (PSITM), with objective response in vivo for the first time. Importantly, existing technologies like flow cytometry and bulk ELISA did not correlate with response (Figure 2), as published in Blood.4 In contrast to flow-based systems, where cells must be fixed and permeabilized, halting biological function and trapping cytokines within the cells, IsoPlexis’ single-cell cytokine system measures true secretions of the full range of functional cytokines, which recruit other immune cells, destroying the tumor.

Figure 2. Polyfunctional Strength Index (PSITM) is uniquely predictive to patient response.

IsoPlexis’ correlative CAR-T cellular fitness metrics are defined by their ability to capture truly released cytokine function of each cell. This knowledge is being applied in advanced bioprocessing with cell product biomarkers (Figure 3) and in donor selection in allogeneic cell therapies by leaders in the cell therapy field.

Figure 3. Novel finding: CAR T cell polyfunctionality associated with outcomes.

References
  1. Zhu H, et al. Notch activation rescues exhaustion in CISH-deleted natural killer cells to promote in vivo persistence and enhance anti-tumor activity. Presented at ASH 2018.
  2. Vogel C, et al. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics, 2012; 13: 227-232.
  3. Xue Q, et al. Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response. Journal for ImmunoTherapy of Cancer, 2017; 5:85.
  4. Rossi, J. et al. Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL, Blood 2018; 132:804-814.

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