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

Program: Oral and Poster Abstracts
Type: Oral
Session: 614. Acute Lymphoblastic Leukemias: Therapies, Excluding Transplantation and Cellular Immunotherapies II
Hematology Disease Topics & Pathways:
Biological, Lymphoid Leukemias, ALL, Bispecific Antibody Therapy, Translational Research, Clinically Relevant, Diseases, Immunotherapy, Therapies, Lymphoid Malignancies
Sunday, December 12, 2021: 5:00 PM

Nora Zieger1,2*, Maryam Kazerani Pasikhani, PhD1,2*, Tobias Straub3*, Alyssa Nicholls1,2*, Gerulf Hänel1,2*, Jan Wulf1,2*, Michaela Scheurer1,2*, Daniel Nixdorf1,2*, Monika Sponheimer1,2*, Sonja M Lacher1,2*, Bettina Brauchle1,2*, Anetta Marcinek1,2*, Lisa Rohrbacher1,2*, Alexandra Leutbecher1,2*, Michael von Bergwelt, MD, PhD1*, Karsten Spiekermann, MD1,4,5, Oliver Weigert, MD1,5,6, Sebastian Theurich, MD1*, Veit L Buecklein, MD1,2*, Roman Kischel, MD7* and Marion Subklewe, MD1,2,4

1Department of Medicine III, University Hospital, LMU Munich, Munich, Germany
2Laboratory for Translational Cancer Immunology, Gene Center, LMU Munich, Munich, Germany
3Bioinformatics Unit, Biomedical Center, LMU Munich, Martinsried, Germany
4German Cancer Research Center (DKFZ), Heidelberg, Germany
5Experimental Leukemia and Lymphoma Research (ELLF), University Hospital, LMU Munich, Munich, Germany
6German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
7Amgen Research (Munich) GmbH, Munich, Germany

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 5x105 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 (padj<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; padj=6.3E-10) and metabolism (MTORC1 signaling, NES=-2.27, OXPHOS, NES=-2.03; padj=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 (padj<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; padj=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.

Disclosures: Lacher: Roche: Research Funding. Brauchle: Adivo: Current Employment. von Bergwelt: Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding. Kischel: Amgen GmbH Munich: Current Employment. Subklewe: Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

*signifies non-member of ASH