Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy

Abstract

Chimeric-antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T-cell – leukemia cell interface, followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA-sequencing and single-cell ATAC-sequencing, we demonstrated that a subset of refractory CD19low cells sustained decreased CD19 expression through transcriptional programs of physiological B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the BTK inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity.

An anti-CD19/CTLA-4 switch improves efficacy and selectivity of CAR T cells targeting CD80/86-upregulated DLBCL

“…Improved selectivity could also allow new CAR/CCR constructs to be designed with increased reactivity against the primary target antigen, protecting against escape by antigen downregulation. 57 , 58 …”

Harnessing the Transcriptional Signatures of CAR-T-Cells and Leukemia/Lymphoma Using Single-Cell Sequencing Technologies

“…A common reason for relapse after CAR-T-cell therapy is the loss of target antigen or loss of antigen expression [115] (Figure 2A). Possible mechanisms include the pre-exiting of antigen negative clones, such as CD34+CD19-CD22+ B-cell progenitors [97,113], gene mutation [116], splicing events [117], the transcriptional plasticity of leukemia cell [114], a reversible antigen loss through trogocytosis [118], the instant introduction of the CAR-T gene to the leukemia cell [119], and lineage switch-induced antigen loss [120].…”

Deciphering and advancing CAR T-cell therapy with single-cell sequencing technologies

“…The recognized mechanisms include the selection of pre-existing antigen-negative tumor cells, mutation, splicing variation, lineage switching-mediated target antigen loss and other factors affecting the presentation and expression of target antigens [ 99 ]. Several single-cell sequencing studies have validated the natural selection theory of the presence of antigen-negative tumor subclones before CAR T-cell treatment [ 100 , 101 ] and unveiled a new mechanism for mediating negative relapse [ 102 ] (Fig. 4 C).…”

“…scRNA-seq and scATAC-seq of surviving leukemic cells co-cultured with CAR T-cells showed that the leukemic cell subsets with the lowest CD19 expression were significantly enriched for gene expression and regulators correlated with B-cell activation signatures and germinal center reaction. Bruton’s tyrosine kinase (BTK) inhibitors can inhibit this process and enhance CAR T-cell killing ability [ 102 ].…”

“… [ 101 ] scRNA-seq B-ALL CD19 CAR T-cells, leukemic cells * Cell 1,039 scRNA-seq was performed on the surviving leukemic cells cocultured with different methods (CAR T-cells and T cells) for 24 h. Existing CD19 low leukemic cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction, which helped achieve immune escape. [ 102 ] Bulk RNA-seq, scRNA-seq, flow cytometry B-ALL CD19 HSPCs 2 second-trimester fetus Human 30,000 Bulk RNA-seq, scRNA-seq and flow cytometry were performed on specific HSPC populations from FBM. CD34 + CD19 − CD22 + cells were detected in BM form B-ALL patients who relapsed or achieved CR.…”