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E - Disease Models and Clinical Applications -> Cancer – Immunotherapy, Cancer Vaccines

Single-cell Antigen-specific Activation Profile of CAR-T Infusion Product Identifies Th2 Deficiency in CD19-Positive Relapsed ALL Patients

Type: Oral Abstract Session

Presentation Details
Session Title: Late-Breaking Abstracts I
Location: Salon H
Start Time: 5/17/2022 8:30
End Time: 5/17/2022 8:45

The remissions in a significant fraction of Chimeric antigen receptor-modified (CAR) T treated subjects are short-lived and 30-60% of patients relapse within one year. Although antigen loss could explain a majority of CD19-negative relapse, the mechanisms of CD19-positive relapse remain elusive. We hypothesized that the functional capacity of CAR T cells in the infusion product could be an essential factor determining long-term therapeutic response. Herein we present 101,326 single cell transcriptomic landscape from the CAR T infusion products of 12 pediatric ALL patients upon CAR antigen-specific stimulation. Ten responders were subdivided into those who had a very durable complete remission (>54 months, CR) and those who had a CD19-positive relapse during the trial (RL, median relapse-free remission duration = 9.6 months). Two patients did not show an objective response to the therapy (NR). We performed an unsupervised analysis to identify modules of co-expressed cytokines (Fig. 1a). In the landscape of responsive states, CAR+ cells from CR, RL, and NR patients were localized in the representation (Fig. 1b), suggesting a distinct cytokine module expression profile for these cells. Notably, the Th2 module was found to be enriched in a region containing mostly cells from CR patients (Fig. 1c), implying that Th2 function might be indispensable for maintaining a long-term remission in CAR T therapy. A quantitative comparison identified a significant depletion of CAR+ cells expressing the Th2 module in RL compared to CR patients (Fig. 1d), whereas other functional modules remained comparable (data not shown). We performed differential expression analyses between CR and RL and found the up-regulation of genes associated with Th2 helper related cytokine production, such as IL4, IL5 and IL13, in CR patients (Fig. 1e). Other immune pathways like Th1 cytokine production, T helper differentiation, and ICOS-ICOSL signaling were comparable between the two groups (Fig. 1f). The assessment of IL13, IL5, IL4 and GATA3 expressions in each of the patient showed uniform enrichment of the four genes in CR compared with RL subjects (Fig. 1g,h). Clustering analysis identified 8 transcriptionally distinct subpopulations, and CAR T cells in cluster 4 mainly functioned as Th2 helpers (Fig. 1i). The cell proportion of cluster 4 was significantly elevated in CR patients and no fraction difference was observed for the combination of clusters 2+3 (Fig. 1j), suggesting that the lack of Th2 function rather than Th1 response could induce CD19-positive relapse. We performed an independent functional validation of these findings in two cohorts comprising 49 patients by means of intracellular flow cytometry and a multiplexed secretomic assay. The combined proportions of IL-4+, IL-5+, and IL-13+ CAR T cells (Th2+) were significantly higher in CR group, with consistent discrimination observed in CD4+ or CD8+ subpopulation (Fig. 1k). In the multiplexed secretomic data set, we used the functional strength index (FSI) to describe the specific functionality profile of CAR T cells, which was defined as the frequency of cells secreting a cytokine multiplied by the average signal intensity of the cytokine. We found a significantly higher Th2 FSI in CR patients as compared with RL patients (Fig. 1l). In aggregate, these findings suggest a potential strategy to improve the therapeutic outcome by re-engineering the CAR T product with augmented type-2 signaling or by boosting Th2 functions post infusion to maintain long term remission.

Zhiliang Bai

Yale University, New Haven, CT
 Z. Bai: None.

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