Treatment-Free Intervals during CD19xCD3 BiTE ^® Construct-Mediated T-Cell Stimulation Induce Functional Reinvigoration and Transcriptional Reprogramming of Exhausted T Cells

Blinatumomab is a bispecific T-cell engager (BiTE ^®) construct approved for treatment of relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL). It is applied as continuous infusion over 28 days and induces remissions in 43 % of r/r patients. Responses correlated to T-cell expansion (Topp et al. 2011, Zugmaier et al. 2015). Mimicking the clinical application in an in vitro model system, we showed previously that continuous stimulation (CONT) with AMG 562, a half-life extended CD19xCD3 BiTE ^® construct, induces T-cell exhaustion, as seen in chronic infections. Also, we could enhance T-cell function in vitro by treatment-free intervals (TFI) (Zieger et al. ASH 2020). To identify genetic drivers of enhanced T-cell function that could provide anti-exhaustion targets for clinical use, we aimed to characterize the transcriptome of exhausted vs rested T cells by bulk RNA sequencing of CONT and TFI T cells.

To simulate CONT vs TFI AMG 562 stimulation, cocultures of healthy donor T cells and CD19 ⁺ OCI-Ly1 cells were set up for 28 days under CONT or TFI (7 days on/7 days off) AMG 562 exposure. On day 0, 7, 14 and 21, we sorted 5x10 ⁵ CD3 ⁺ T cells for transcriptome assessment (n=3). In parallel, function of TFI vs CONT T cells was analyzed in vitro: (1) AMG 562-mediated killing was evaluated as specific lysis of CD19 ⁺ Ba/F3 cells after 72h, (2) T-cell expansion during the killing assay was calculated as fold change (FC) of CD2 ⁺ counts, (3) AMG 562-mediated cytokine secretion was evaluated via intracellular staining.

We could confirm that function of Day 14 TFI vs CONT T cells was significantly enhanced (% specific lysis: TFI=99±2.2, CONT=34±4.2, p<0.0001; T-cell expansion as FC: TFI=4±0.8, CONT=1±0.6, p<0.01; Granzyme B MFI ratio of CD8 ⁺: TFI=451±168, CONT=144±33, p<0.0001). RNA sequencing and differentially expressed gene (DEG) analysis of Day 14 TFI vs CONT T cells identified 1902 significantly up- and 2603 downregulated genes (p _adj<0.05). Unsupervised clustering of the top 100 DEG showed striking similarity in gene expression patterns in unstimulated (Day 0) and Day 14 TFI vs CONT T cells. Intriguingly, genes related to memory and stemness were highly enriched on Day 0 and Day 14 TFI (TCF7, IL7R, SELL). Among the top downregulated genes in Day 14 TFI vs CONT T cells, we identified genes related to cell cycle (CCNB1, CDK1) and activation (IL2RA). Exhaustion-associated genes were significantly downregulated in Day 14 TFI vs CONT T cells (LAG-3, PDCD1, NR4A3, IRF4). Pathway analysis of Day 14 TFI vs CONT T cells confirmed downregulation of cell cycle (G2M checkpoint, normalized enrichment score (NES)=-2.47, E2F Targets, NES=-2.64; p _adj=6.3E ^-10) and metabolism (MTORC1 signaling, NES=-2.27, OXPHOS, NES=-2.03; p _adj=6.3E ^-10). Gene set enrichment analysis (GSEA) also showed reduction of effector compared to memory-related genes in Day 14 TFI vs CONT (GSE9650, NES=-1.95, FDR q=0.0).

After restimulation of TFI T cells with AMG 562 (Day 21 TFI) we observed higher effector function in TFI vs CONT T cells (% specific lysis, TFI=51±8, CONT=23±7, p<0.0001). DEG analysis of Day 21 TFI vs CONT identified 1417 significantly up- and 1821 downregulated genes (p _adj<0.05). Unsupervised clustering of the top 100 DEG revealed a unique gene set in Day 21 CONT T cells enriched in apoptosis-related genes (TRAF1, ELAPOR1, BMF). Among the top upregulated genes in Day 21 TFI T cells were genes involved in activation and growth (DPP4, SLC3A2) and cell cycle (CDK1, PLK1), induced by AMG 562 restimulation after TFI. Exhaustion-related genes were downregulated in Day 21 TFI vs CONT T cells (LAG-3, BTLA, NFATC1). Remarkably, identical pathways downregulated on Day 14 TFI were enriched in Day 21 TFI T cells (G2M checkpoint, NES=2.63, MTORC1 signaling, NES=2.36, OXPHOS, NES=2.42; p _adj=7.1E ^-10). Accordingly, GSEA showed enrichment of effector- rather than memory-related genes on Day 21 TFI vs CONT (GSE9650, NES=1.75, FDR q=0.0).

Together, our data suggest that TFI functionally and transcriptionally rejuvenates T cells. Upon restimulation (Day 21 TFI), T cells reengage an effector program and are less exhausted compared to CONT T cells. In future analyses we will correlate RNA expression levels to functional traits using whole genome co-expression network analysis (WGCNA). Thereby we aim to identify gene clusters critical for persistent T-cell function that might serve as targets to improve efficacy of T-cell based immunotherapies.

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