Published in Science Advances: IsoPlexis’ Single-Cell Functional Proteomics Helps Identify Biomarkers of CAR-T Response and Relapse

Clinical outcomes for resistant and/or refractory acute lymphoblastic leukemia (r/r ALL) have been greatly improved with the introduction of chimeric antigen receptor (CAR) T cellular therapy. Though CAR-T therapies that target CD19, a cell surface protein found on B cells, have resulted in complete response in many patients with B-ALL, relapse is common and the response rate of patients with other types of B cell malignancies remains low. Researchers have discovered several factors that are associated with treatment success, including characteristics of cells in CAR-T products, CAR construct design, and pretreatment disease burden. Even with optimal conditions, however, patients can acquire disease resistance and stop responding to therapy.

Disease resistance occurs in a variety of ways. On the one hand, cancer cells may adapt to therapies and downregulate CD19, reducing the efficacy of CD19-targeted CAR-T therapies. On the other, some patients’ cancer cells continue to express CD19 but still experience relapse. The mechanism for this CD19-positive relapse is still poorly understood, but powerful analytical techniques can help researchers to better characterize immune cells and understand the mechanisms underlying treatment response. IsoPlexis’ single-cell analysis platform provides a unique functional data layer allowing researchers to gain deeper insights into immune system’s function.

Single-Cell Functional Phenotyping Identifies Biomarkers of Clinical Response

A new study published in Science Advances, researchers sought to characterize CAR-T infusion products and identify the key drivers associated with clinical response. The researchers took a multi-omic approach by using multiple technologies including IsoPlexis’ multiplexed Single-Cell Secretome solution to investigate potential biomarkers in CAR-T products for predicting and characterizing CAR-T therapy response.

Cells were grouped by co-expressed cytokines and compared between patients who showed a complete response to CAR-T therapy (CR), patients who relapsed (RL), and patients who did not respond at all (NR). CAR-T cells from CR patients were highly enriched with cells secreting cytokines associated with the T-helper type 2 (TH2) phenotype, suggesting that this particular type of cell may help to maintain long-term response.

To validate these findings, the authors investigated a separate set of CAR-T products from CHP959 trial’s B-ALL patients. CD19-targeted CAR-T infusion products were stimulated with CD19-3T3 cells and then functional heterogeneity was assessed using IsoPlexis’ Single-Cell Secretome solution. The researchers calculated a TH2 functional strength index (FSI) by multiplying the frequency of cells secreting a specific cytokine by the average signal intensity of the cytokine. TH2 FSI includes the measurement of IL-4, IL-5 and IL-13. Notably, CR patients were found to have a significantly higher TH2 FSI than RL patients and the FSI of IL-5 was even more predictive of clinical response. These data highlight the role of TH2-type cells in CD19-positive CAR-T efficacy and were incorporated into a prognostic model to predict durable response. The researchers were able to predict response with 70% sensitivity and a false discovery rate of less than 5%, indicating that long-term patient response or relapse could be predicted using these newly identified immune cell biomarkers.

Uncovering Mechanisms of Response and Relapse with the Single-Cell Secretome

The data obtained using IsoPlexis technology provides detailed insights into the functional biological mechanisms that distinguish responder patients from relapse patients. Eventually, this information may be applied towards predicting response or towards directing CAR-T manufacturing strategies to improve overall efficacy.

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