Published in Nature Communications: Using Single-Cell Secretomics to Characterize the Functional Effects of a Novel CAR-T Expansion Method

Because CAR-T therapies have greatly improved patient outcomes in a variety of cancers, researchers are working to find ways to optimize their efficacy after T cell proliferation. Finding strategies to maintain CAR-T function and enhance expansion before or after CAR-T product infusion can help to improve complete response rates and decrease the occurrence of relapse. Researchers have capitalized on the immune system's innate signaling molecules that potentiate T cell viability, but these endogenous cytokines still need to be improved upon. For example, interleukin-7 (IL-7) is a T cell growth and survival factor that plays a role in the maintenance and proliferation of T cells. Using recombinant human IL-7 (rhIL-7) can improve CAR-T expansion, but the effects are short-lived and can cause unwanted inflammation.

Genetic engineering has been harnessed to improve the activity of endogenous molecules. By combining rhIL-7 with a stable hybrid Fc domain, researchers created a more long-lasting form of IL-7 (rhIL-7-hyFc ) that is less likely to produce complement activation. This form of IL-7 was shown to increase the proportion of central memory T cells. As such, researchers wanted to characterize the functional effects of rhIL-7-hyFc on CAR-T cells, using IsoPlexis' single-cell functional proteomics.

Using Single-Cell Functional Proteomics to Characterize the Effects of CAR-T Cell Expansion

In the study recently published in Nature Communications, researchers assessed how rhIL-7-hyFc administration enhanced CAR-T proliferation and potency for B cell lymphoma. Using UCART19 cells, CD19-targeted CAR-T cells modified to not express T cell receptors to prevent the potential graft versus host disease (GVHD), researchers characterized how rhIL-7-hyFc affects CAR-T cells. When UCART19 cells were co-cultured with a CD19+ B lymphoma cell line with vehicle, rhIL-7, or different concentrations of rhIL-7-hyFc, expansion with rhIL-7 and rhIL-7-hyFc was equivalent and significantly higher than the vehicle, indicating that the modified IL-7 was still capable of potentiating CAR-T cell expansion.

To determine whether the rhIL-7-hyFc-stimulated expansion potentiated cell function, researchers used IsoPlexis' Single-Cell Secretome platform to analyze UCART19 cells and determine the polyfunctional strength index (PSI) as defined by the percentage of cells secreting two or more cytokines multiplied by the mean signal intensity of the measured secreted proteins. Despite robust expansion, PSI of the CAR-T cells was maintained relative similar to the input cells from the start of the experiment on day 0. Single-cell analysis also demonstrated that effector molecules, including granzyme B, IFNγ, MIP-1 α, TNF- α, and TNF- β, drove the observed polyfunctionality. Because polyfunctionality and PSI have been associated with increased potency and improved clinical outcomes, the retention of PSI after rhIL-7-hyFc-mediated expansion suggests that the UCART19 cells will still be effective in vivo.

The researchers confirmed this to be the case in subsequent experiments. The rhIL-7-hyFc-expanded UCART19 cells were more persistent, demonstrated increased cytotoxicity, and improved survival in a mouse model of B cell lymphoma. Further testing showed that rhIL-7-hyFc expansion is also effective at potentiating the effects of other types of CAR-T products in other models, revealing a novel strategy that can improve both the quality and quantity of CAR-T cells.

PSI as a Biomarker of Cell Potency and Cytotoxicity

Using single-cell functional proteomics to assess the polyfunctionality of CAR-T cells can help to reveal deeper insights into cell characteristics including potency and cytotoxicity. In the Nature Communications study, the authors identified PSI as "a quantitative marker of CAR-T functionality" and employed it to characterize their CAR-T cells. PSI and polyfunctionality can provide important biological information about CAR-T cells' functionality and their expansion methods before moving into in vivo experiments, giving researchers a better tool to characterize and understand critical CAR-T cell attributes in the earlier research process. In doing so, IsoPlexis' single-cell platform helps to accelerate immune-oncology research and improve development of novel therapies.