Improving CAR-T Cell Precision Bioprocessing with Functional Proteomics

Combining automated cell-manufacturing platforms and single-cell functional proteomics to characterize next generation cell therapy products


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Some of the most cutting-edge approaches to treating cancer over the past decade arose from personalized immunotherapy. Chimeric antigen receptor (CAR) T cells can be engineered to specifically target a patient’s cancer using immune cells, but there are many challenges to overcome. Whereas the production of conventional therapies can be uniformly scaled, cell therapies, such as CAR-T cell therapies, can vary in product quality and potency. With the right combination of technology, however, the engineered cell product can be characterized to make the most effective therapy.

CAR-T cell therapies can be created from a patient’s own cells—autologous therapies—or from donor cells—allogeneic therapies. In either approach, heterogeneous cell populations contribute to varying qualities of the final cell product, which can lead to differences in patient response.

Here, we explore how automated cell-manufacturing platforms, such as the Miltenyi Biotec CliniMACS Prodigy®, and single-cell technology from IsoPlexis can be used to produce and assess the functional biology of CAR-T cells. With a deeper understanding of cellular function by characterizing the functional cytokines from each single cell, scientists can analyze how the CAR-T cells are likely to impact the immune system, which can translate to how the therapy would affect patient outcome.

Touring the platform

After the CAR-T cells are produced, there are still challenges in determining effectiveness. The single-cell functional proteomics platform from IsoPlexis, however, helps a researcher assess the true function of their engineered cell products. Specifically, it reveals polyfunctional T cells, which produce two or more cytokines simultaneously. These signaling molecules stimulate the immune system, and polyfunctional T cells produce a more powerful immune response than T cells that release one or no cytokines.

Analysis of the cytokines secreted by a T cell, however, can be difficult. To simplify the analysis, IsoPlexis’ Polyfunctional Strength Index (PSI™) puts all of that information in one metric, which represents the overall immune fitness of the cell product. This metric does that with two crucial pieces of information:

  1. A sample’s percentage of polyfunctional T cells.
  2. The intensity of the cytokines that the T cell secretes.

Using this information, a researcher can identify the most potent and effective cells within a CAR-T cell product. PSI is the most novel and revealing metric for measuring the potency of different immune cell types, helping top researchers accelerate their immunotherapy programs from discovery to predicting response. In a study published in Blood,1 researchers from Kite Pharma and the NCI demonstrated a significant association between the functionality of anti-CD19 CAR-T cell product before treatment, as defined by IsoPlexis’ PSI and the objective response in patients with non-Hodgkin lymphoma. Developers of CAR-T cell therapies can use these tools to compare the results of different methods of preparing therapeutic T cells. Then, a method can be optimized to produce CAR-T cells that exhibit the desired properties.

Producing PSI

To apply IsoPlexis’ technology, a researcher starts with CAR-T cells, enriched and separated into CD4+ and CD8+ subsets, then stimulates them to release cytokines. These cells are then loaded onto the IsoCode Chips that are inserted into IsoPlexis’ IsoLight system.

IsoLight System
Single-cell cytokine profiling of functional quality and potency
of the engineered cell product with the IsoLight System.

In the IsoCode chip, CAR-T cells are incubated, and an ELISA-based assay is used to collect secreted proteins from single-cells, which are analyzed by the automated IsoSpeak software. A scientist can review the raw data or look at derived metrics, such as the PSI and other advanced visualizations like polyfunctional heatmaps and Polyfunctional Activation Topology (PAT-PCA).

Cytokine expert Robert Alan Seder, MD, chief of the cellular immunology section of the U.S. National Institute of Allergy and Infectious Diseases’ Vaccine Research Center, and his colleagues showed that CD4+ T cells that secrete more than one cytokine stimulate a more effective immune response.2 The scientists reported that this information “should be useful for improving the design of preventive and therapeutic vaccines against infections and cancer to elicit qualitatively better T cell responses.” With a therapy based on CAR-T cells, the results depend entirely on the T cell response and the resulting effects.

PSI and performance

In order to produce the most potent and effective therapy, different bioprocessing methods should be explored. Scientists from the U.S. National Institutes of Health used the CliniMACS Prodigy® to produce CAR-T cells in three days or five days. Researchers wanted to understand the changes in function of the cell products based on these two methods.3

As described in a technical note, the researchers found that the shorter method produced a higher percentage of polyfunctional CAR-T cells, which exhibited higher PSIs.

The CAR T-cell products manufactured by the shorter method have much higher secretions in multiple secreted proteins and the enhanced PSIs were mainly driven by effector and stimulatory cytokine secretions. The results also show that CAR-T cells from the shorter method produced more chemoattractive and regulatory cytokine secretions, which means that the shorter method improves the quality of CAR-T cell product functionality in multiple dimensions.

These improvements in developing CAR-T cells can benefit every stage of making a new therapy, from discovery through research and development to production. The improvements in efficiency could trigger economic benefits and reduce the time it takes to get a therapy to the market. Most important of all, this technology can lead to more effective treatments for cancer—ones that can be shown to be quantitatively more effective at an earlier stage of development.


1. Rossi J, Paczkowski P, Shen Y-W, et al. Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL. 2018; Blood 132: 804–814.
2. Darrah PA, Patel DT, De Luca PM, et al. Multifunctional TH1 cells define a correlate of
vaccine-mediated protection against Leishmania major. 2007; Nat. Med. 13: 843–850.
3. Srivastava SK, Panch SR, Jin J, et al. Abbreviated T-cell activation on the automated CliniMACs prodigy device enhances bispecific CD19/22 chimeric antigen receptor T-cell viability and fold expansion, reducing total culture duration. 2018; Blood 132: 4551.


Download our Tech Note, “Getting the most out of your CAR-T bioprocessing with the CliniMACS Prodigy® & IsoCode Single-Cell Chip” to learn more about optimizing cell therapy bioprocessing workflows.

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