Published in Cytotherapy: Using Single-Cell Secretomics to Understand Function in Dual-Targeted CAR-T Therapies

Relapsed/refractory B cell lymphoma (R/R BCL) can be aggressive and difficult to treat, but the use of chimeric antigen receptor (CAR) T cell therapies has begun to yield improved outcomes for patients. Despite the success of CAR-T therapies, some R/R BCL patients still experience disease progression or relapse, a challenge that cancer researchers are seeking to overcome. Traditional CAR-T therapies are often single-antigen targeted towards CD19, an antigen expressed by B cells. Targeting a single antigen can result in selective pressure with CD19 downregulation, ultimately leading to resistance or relapse. To combat this, researchers have created dual-targeted CAR-T cells that can target both CD19 and CD20, another B cell antigen.

Polyfunctionality as a Measure of CAR-T Efficacy

In a new study published in Cytotherapy, researchers used IsoPlexis’ Single-Cell Secretome platform to characterize autologous dual-targeting LV20.19 CAR-T cells that target CD19 and CD20, engineered from B cell lymphoma patients. Preliminary studies have demonstrated that these LV20.19 CAR-T cells were safe and effective in a Phase 1 clinical trial. 75% of patients had a complete response, although some patients relapsed 1 year after treatment. To better understand how the CAR-T cells’ function is associated with clinical outcomes, researchers used IsoPlexis’ single-cell functional phenotyping technology to gain insights into polyfunctionality, or the ability of immune cells to secrete more than one cytokine.

Cells from patients were stimulated with either CD19 or CD20 to investigate how LV20.19 cells respond to the different antigens. Cells were then separated into either CD4+ or CD8+ populations and loaded onto IsoCode Single-Cell Adaptive Immune chips for analysis. Researchers measured 32 of the cytokines secreted by the LV20.19 cells, all while using IsoPlexis’ fully automated platform, IsoLight. In addition to polyfunctionality researchers assessed polyfunctional strength index (PSI), a metric combining the percentage of polyfunctional cells with the intensity of the cytokines measured, to compare cell function with clinical outcomes.

When stimulated by CD19, LV20.19 CAR-T cells secreted mostly effector cytokines, including TNF- α,  TNF-β, IFN- , IL-8, MIP-1 α, MIP-1 β, perforin, and granzyme B. When CD20 was used to stimulate the cells, PSI levels were comparable to CD19 stimulation, but the cytokine profile differed slightly. Notably, CD4+ LV20.19 cells stimulated with CD20 had a higher stimulatory and chemoattractive PSI, whereas CD8+ LV20.19 cells had lower inflammatory but higher stimulatory PSI when stimulated with CD20 as compared to CD19. Other dual-targeted CAR-T cells do not show equivalent PSI in response to antigen stimulation, but the similar levels of PSI between CD19 and CD20 stimulation conditions in this study indicates that the LV20.19 cells can still maintain efficacy and prevent relapse if CD19 expression becomes downregulated on lymphoma cells.

The authors compared LV20.19 cell polyfunctionality with clinical outcomes. There was a trend toward higher CD4+ PSI in patients with day 28 complete response (CR) versus partial response (PR)/progressive disease (PD) but no difference in median CD8+ PSI. Among CD4+ cells, patients with CR versus PR/PD at day 28 had a higher median IL-17A PSI and TNF-β PSI. Higher levels of IL-17A in polyfunctional CAR T cells have been previously associated with response to CAR T-cell therapy. The authors also compared LV20.19 cell polyfunctionality with cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), adverse events associated with CAR-T therapies. While the PSI of the cells was not associated with severity of ICANS observed in patients, the PSI of CD4+ cells trended towards association with grade 2 or higher CRS which was associated with higher levels of the chemokines IL-8 and MCP-1. These findings help researchers better understand mechanisms underlying patient responses to CAR-T therapies, highlighting how IsoPlexis’ proteomic platform can be used to assess clinical efficacy and safety profiles of CAR-T products.

Using Single-Cell Functional Phenotyping to Characterize CAR-T Products

The authors of this study stated that they used PSI and polyfunctionality to characterize their dual-targeted CAR-T cells because it has previously been shown to be associated with anti-tumor responses. The utility of this metric underscores the need for platforms that can easily and effectively perform single-cell highly multiplexed proteomic analyses. IsoPlexis’ Single-Cell solution provides researchers with a streamlined, flexible platform to characterize individual cells and better understand the drivers of immune function and response. This functional layer of data can accelerate the development of more effective cell therapies and ultimately improve patient outcomes.

Superpowered Functional Proteomics is Critical for Accelerating Immune Medicines

IsoPlexis’ Proteomic solutions are the only way to measure the true function of each cell and identify the rare subsets of superpowered cells driving response. Using IsoPlexis’ platform, both single-cell and multiplexed bulk proteomic experiments are fully automated, with data sent directly to IsoSpeak software for analysis. With the IsoLight or IsoSpark system, a process that would traditionally require multiple instruments and steps is accomplished in one instrument with a variety of chip options to suit a wide range of research needs.

Want to learn more? Speak to an expert to see for yourself how IsoPlexis can accelerate your research.

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